Biochemistry

Amino Acids & Protein Structure

Frequently tested: amino acid carbon counting, polar vs nonpolar classification, isoelectric point (pI), bonds stabilizing protein structure, haemoglobin & the oxygen curve. Collagen and elastin now live in section 2.

🔤 The Basic Structure

Every amino acid has a central alpha-carbon bonded to: –NH₂, –COOH, –H, and an R-group. All human protein amino acids are in the L-configuration. Glycine is the only amino acid with no chiral carbon.

🧬 All 20 Amino Acids in 3D

Every amino acid is the same backbone plus one side chain. In the models below the shared parts — the α-amino group and the α-carboxyl group — are drawn faded, so what stands out on each tile is the only thing that actually differs: the R group. Tap any amino acid to open it full size, spin it with your finger or mouse, and read its properties.

CarbonOxygenNitrogenSulphurHydrogenFaded = backbone shared by all 20

How to read these models

Each structure is drawn as the zwitterion — the form that exists at physiological pH, with the amino group protonated to –NH₃⁺ and the carboxyl group deprotonated to –COO⁻. Tile colour marks the side-chain class, and geometry is built from standard bond lengths and tetrahedral or trigonal angles, so the shapes are chemically faithful rather than decorative.

📊 Classification of Side Chains

Category	Amino Acids	Key Feature
Nonpolar	Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met	Hydrophobic; buried inside protein
Uncharged Polar	Ser, Thr, Tyr, Asn, Gln, Cys	H-bonds; Ser/Thr/Tyr can be phosphorylated
Acidic (–)	Asp, Glu	Negatively charged at physiological pH
Basic (+)	Lys, Arg, His	Positively charged; His is key buffer

Common Mistake

Methionine is nonpolar. Asparagine and Threonine are uncharged polar, not nonpolar.

🧮 Amino Acid Carbon Counting (Exam Tested)

Some exams ask which amino acids do NOT have 5 carbons. Count the alpha-carbon plus all side-chain carbons:

Amino Acid	Total Carbons	Note
Glycine	2	Smallest
Alanine	3
Serine	3	NOT 5 — commonly tested
Proline	5	Has 5 carbons
Valine	5	Has 5 carbons
Lysine	6	NOT 5
Glutamate	5	Has 5 carbons

Exam Q: "Which does NOT have 5 carbons?"

Answer: Serine (3C) and Lysine (6C) do not have 5 carbons. Valine, Proline, and Glutamate all have exactly 5.

🔀 Glucogenic, Ketogenic & Essential Amino Acids

What do these terms mean?

Glucogenic: degraded to pyruvate, OAA, α-ketoglutarate, succinyl-CoA, or fumarate → can enter gluconeogenesis → make glucose.
Ketogenic: degraded to acetyl-CoA or acetoacetate → can only form ketone bodies or fatty acids → CANNOT make glucose.
Both (glucogenic + ketogenic): degraded to products that enter BOTH pathways.

Category	Amino Acids	Exam Tip
Purely Ketogenic	Leu, Lys	The ONLY two that are purely ketogenic. Cannot make glucose. Remember: Leu & Lys = Ketogenic.
Both	Ile, Phe, Tyr, Trp, Thr	These 5 are both glucogenic AND ketogenic. Their catabolism produces precursors for both pathways.
Purely Glucogenic	All remaining amino acids (Ala, Arg, Asp, Asn, Cys, Glu, Gln, Gly, His, Met, Pro, Ser, Val)	The majority — can make glucose. Degraded to pyruvate or Krebs intermediates.

Essential Amino Acids (cannot be synthesized — must come from diet)

PVT TIM HaLL (mnemonic):
Phenylalanine · Valine · Threonine · Tryptophan · Isoleucine · Methionine · Histidine · Leucine · Lysine

Conditionally essential: Tyrosine (if Phe deficient — PKU), Cysteine (if Met deficient), Arginine (in growth/illness).

Non-Essential Amino Acids (body can synthesize)

Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, Pro, Ser, Tyr

Key synthesis links: Tyr from Phe (needs PAH + BH4). Cys from Met (needs CBS + B6). Arg in kidney (from citrulline).

Exam Traps

• Methionine is NONPOLAR (despite containing sulfur) — among the sulfur-containing amino acids, only Cysteine is polar
• Leu & Lys are the ONLY purely ketogenic — cannot make glucose
• In PKU: Tyr becomes conditionally essential (PAH is blocked)

⚡ Isoelectric Point (pI)

pH = pI

Net charge = 0 (zwitterion). Cannot move in electric field. Least soluble.

pH < pI

Net positive charge → moves toward cathode (–).

pH > pI

Net negative charge → moves toward anode (+).

Still Ionized at pI!

–NH₃⁺ and –COO⁻ both present but charges cancel. NOT unionized.

🏗️ Four Levels of Protein Structure

1
Primary structure — the sequence of amino acids linked by peptide bonds (covalent). This is directly encoded by DNA. Denaturation does NOT destroy this level — peptide bonds survive.
2
Secondary structure — local regular folding patterns: α-helix or β-sheet, stabilized by hydrogen bonds between backbone –C=O and –N-H groups. Proline and Glycine destabilize the α-helix (proline has no NH for H-bonding; glycine is too flexible).
3
Tertiary structure — the overall 3D shape of a single polypeptide chain. Stabilized by: hydrogen bonds, hydrophobic interactions (nonpolar side chains buried inside), ionic bonds (electrostatic interactions), and disulfide bridges (Cys–Cys, the ONLY covalent bonds in tertiary structure).
4
Quaternary structure — two or more polypeptide chains (subunits) assembled together. Example: hemoglobin has 4 subunits (2α + 2β). Stabilized by the same forces as tertiary structure plus possible inter-chain disulfide bonds.

Cysteine — Heavily Emphasized by Teacher

Cysteine is unique: it contains a sulfur atom in its thiol (–SH) side chain. Two cysteine residues can be oxidized to form a disulfide bridge (–S–S–) — a covalent bond that stabilizes tertiary (and quaternary) protein structure. This is the only covalent bond within/between protein chains other than the peptide bond. Cysteine is also the active component of glutathione. The teacher specifically emphasized Cysteine and disulfide bonds.

Exam Q: "Tertiary structure is stabilized by all EXCEPT?"

Answer: Peptide bonds. Peptide bonds maintain the PRIMARY structure (the sequence). Tertiary structure is stabilized by disulfide bonds, hydrogen bonds, hydrophobic interactions, and ionic bonds — all non-peptide forces.

🧠 Prions & Amyloid — Disease From Shape Alone ⚪ Never appeared in past exams

A disease of protein conformation: no change in the amino acid sequence, and no nucleic acid involved. The same gene product, folded two ways.

PrP^C — the normal protein

α-helix rich, soluble, and digested by proteases. Present in healthy neurons.

PrP^Sc — the disease form

Identical sequence, but β-sheet rich, insoluble and protease-resistant. It acts as a template, converting PrP^C into more of itself — which is why an infectious agent can exist with no genome.

Consequence

The aggregates build up extracellularly in the brain, producing spongiform change and a rapidly progressive, invariably fatal dementia — Creutzfeldt–Jakob disease in humans. It is a change of fold, not of sequence: that is the whole point of the example.

The same trick elsewhere

Amyloid-β forms neurotoxic β-sheet aggregates in Alzheimer's disease, and amyloid deposits of other proteins occur in the systemic amyloidoses. Once a protein finds the β-sheet aggregate state, it is thermodynamically hard to leave.

Kept for completeness — this topic has not been tested in any of the available past papers, so treat it as low priority.

PrP^sc: β-sheet rich, insoluble, resistant to proteolysis. Causes CJD. Normal PrP^c is α-helical. Amyloid-β: neurotoxic β-sheet aggregates in Alzheimer's disease.

Kept for completeness — this topic has not been tested in any of the available past papers, so treat it as low priority.

🫁 How to Read the Oxygen Curve

The graph answers one question: with this much oxygen around, how full is the haemoglobin? Nothing more.

→ Across the bottom: pO₂

How much oxygen is in the surrounding blood, in mmHg. Three places matter: the lung ≈ 100, a resting tissue ≈ 40, and hard-working muscle ≈ 20. Reading right to left is a red cell leaving the lung and arriving where oxygen is needed.

↑ Up the side: saturation

What percentage of haemoglobin's four oxygen seats are occupied. 100% means all four are full, 50% means half. It is a proportion, never an amount — a severely anaemic patient can still read 100% saturated.

Why the haemoglobin line is S-shaped and the myoglobin line is not

Haemoglobin has four binding sites that talk to each other: the first O₂ is hard to load, and each one that binds makes the next easier — cooperativity. That gives the slow start, the steep middle and the flat top of an S (sigmoid). Myoglobin has one site with nobody to talk to, so it fills smoothly from the start — a plain hyperbola, already nearly full at the pO₂ where haemoglobin is still handing oxygen over. That is the point of the comparison: myoglobin is a store, haemoglobin is a delivery system.

The one number and the one movement

P₅₀ is the pO₂ that fills exactly half the seats — normally ≈26 mmHg, marked on the curve. When the curve shifts right, P₅₀ rises: haemoglobin holds on less tightly and gives oxygen away to the tissue. Shift left, P₅₀ falls: it holds on. Everything else in this topic is a reason for one of those two movements.

📈 Oxygen dissociation — sigmoidal versus hyperbolic

Haemoglobin — normalShifted left / rightMyoglobin — hyperbolic

Haemoglobin is sigmoidal because binding is cooperative: each O₂ that binds makes the next easier, so the molecule loads fully in the lung and unloads steeply in the tissues. Myoglobin has one site, no cooperativity, and a hyperbolic curve — it holds oxygen until the tissue is genuinely hypoxic. Right shift = unload more: ↑CO₂, ↑H⁺ (Bohr), ↑temperature, ↑2,3-BPG. Left shift = hold on: the opposite, plus HbF and carbon monoxide.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧩 What Peptides and Proteins Are For Past Q

A peptide is a short chain of amino acids; a protein is a long one that folds into a working shape. Two classic list questions come from here — name five functions of peptides and name the roles of proteins.

🔹 Five (and more) functions of peptides

Hormones
Insulin, glucagon, ADH, oxytocin, calcitonin, ACTH.

Hormone-releasing factors
The hypothalamic tier — TRH, GnRH, CRH, GHRH.

Neurotransmitters
Substance P, enkephalins, endorphins.

Neuromodulators
They tune the response of a neurone to another transmitter rather than firing it themselves.

They are polyelectrolytes
Many ionisable groups on one molecule: peptides therefore buffer, carry a net charge that depends on pH, migrate in an electric field and each has its own pI.

Protective and transport peptides
Glutathione (antioxidant), peptide antibiotics, and the di-/tripeptides absorbed intact by PepT1.

🔹 Roles of proteins

Catalysis & regulation

Enzymes catalyse every metabolic reaction; receptors and transcription factors read and pass on signals.

Structure & movement

Collagen, elastin, keratin and the cytoskeleton give tissues their mechanical integrity; actin and myosin convert chemical energy into motion.

Transport & storage

Albumin (fatty acids, bilirubin, drugs), haemoglobin (O₂), transferrin (iron), ferritin (iron store), and the membrane carriers and channels.

Defence & homeostasis

Immunoglobulins and complement; fibrinogen and the clotting factors; plasma proteins hold the oncotic pressure, buffer the blood and form a mobilisable amino acid reserve.

📐 The Peptide Bond and the Two Secondary Structures Past Q

Two amino acids condense with the loss of a water molecule: the carbonyl carbon of one joins the α-nitrogen of the next. The chain is written and read N-terminus → C-terminus — the dipeptide Gly–Ala means glycine's carboxyl is bonded to alanine's amino group.

Why the peptide bond cannot rotate

Resonance gives it partial double-bond character, so the six atoms — the α-carbon, the carbonyl C and O, the N, its H and the next α-carbon — are coplanar, and rotation about C–N would mean breaking that bond. The bond is uncharged but polar and almost always trans.

Where the chain can bend

Of the three backbone bonds per residue, only two rotate freely: N–Cα (angle φ) and Cα–C (angle ψ). Every fold a protein can adopt is built from those two rotations.

α-Helix

Right-handed, with 3.6 residues per turn. Stabilised by hydrogen bonds between the C=O of residue n and the N–H of residue n+4, running parallel to the axis, plus van der Waals contacts. R groups point outwards. Proline and glycine break it — proline has no amide H and its ring kinks the chain, glycine is too flexible. An amphipathic helix has hydrophobic side chains on one face and hydrophilic on the other, so it sits neatly at the boundary between a polar and a nonpolar region.

β-Sheet

Extended strands lying side by side in a zigzag, pleated arrangement, with R groups of neighbouring residues pointing in opposite directions (alternately above and below the sheet). Hydrogen bonds run between strands, from carbonyl oxygens to amide hydrogens. Parallel = neighbouring strands run the same way N→C; antiparallel = opposite ways (and slightly more stable). Real sheets are not flat — they carry a right-handed twist.

Loops, turns and bends — the third kind of secondary structure

Turns and bends are short segments that join two units of secondary structure, typically reversing the chain between two antiparallel β-strands; they are rich in glycine and proline. Loops are longer and irregular, sit on the surface, and are far from decorative: in many enzymes the loop that bridges two domains carries the residues that bind the substrate and perform the catalysis.

The four levels in one line each

Primary — the sequence of amino acids, held by peptide bonds. Secondary — geometrically ordered folding of short (3–30 residue) stretches: α-helix, β-sheet, loops. Tertiary — the whole three-dimensional conformation of one chain, i.e. how the helices, sheets, bends and loops assemble into domains and how the domains sit relative to each other. Quaternary — the number, type and spatial arrangement of the subunits of an oligomeric protein.

🧶 Getting Folded — Chaperones, Disulphide Isomerase, and Coming Undone Past Q

The sequence contains all the information needed for the fold, but in a crowded cell a half-made chain would aggregate long before it finished. Over half of all mammalian proteins are folded with help.

🅰️ hsp70 family — while the chain is still being made

Molecular chaperones · ATP-driven

Job: prevent aggregation, buy time

Binds hydrophobic stretches

It grips the short hydrophobic sequences that emerge from the ribosome and shields them from water, so they cannot stick to each other.

Releases in cycles

Repeated ATP-driven binding and release let the chain form its secondary structure and collapse into a molten globule — compact, but not yet the final fold.

▼

🅱️ hsp60 family (chaperonins) — the finishing chamber

Different in sequence and structure from hsp70 · acts later, often together with it

Job: complete the fold in isolation

A barrel with a lid

The molten globule is taken inside a closed cavity where it can finish folding without meeting any other protein, then is released.

Protein sulfhydryl oxidase — makes the bridges

Oxidises the –SH groups of two cysteines into a disulphide bond; the reaction consumes oxygen. It is not specific — any accessible cysteine can be paired with any other, so wrong pairs form routinely.

Protein disulphide isomerase — corrects them Past Q

Catalyses disulphide exchange: it breaks an S–S bond and re-forms it with a different partner cysteine, over and over, until the arrangement that gives the native, most stable conformation is reached. That is its whole contribution to stabilising tertiary and quaternary structure.

Denaturation — what is really lost Past Q

Loss of secondary, tertiary and quaternary structure with the primary sequence intact: peptide bonds survive, non-covalent bonds do not. Caused by heat, strong acid or base, concentrated inorganic salts, organic solvents, urea, detergents, heavy metals and radiation. Two points that get examined: a denatured protein is not a random coil — native-like contacts are favoured and patches of native structure persist — and denaturation is not necessarily irreversible, since a protein can refold (renature) once the stress is removed. Inside a cell, however, the exposed hydrophobic surfaces make denatured chains clump into insoluble aggregates, and cell function fails.

🛡️ Acute-Phase Proteins and How an Immunoglobulin Is Built Past Q

The acute-phase proteins

Made by the liver and rising during inflammation and after tissue damage, sometimes 50-fold and occasionally a thousandfold: C-reactive protein (it activates the classical complement pathway), α₁-antitrypsin (α₁-antiproteinase), which neutralises the proteases released by activated neutrophils, haptoglobin, α₁-acid glycoprotein, fibrinogen and ceruloplasmin. They are what raise the α₁ and α₂ bands of the proteinogram, and they are why ferritin and ESR are unreliable in inflammation. Albumin behaves as a negative acute-phase protein and falls.

Chains, and what decides the class

Every immunoglobulin is two identical light chains + two identical heavy chains, held together by disulphide bonds. Light chains are either kappa (κ) or lambda (λ) — one molecule carries two of one type, never a mixture (κ is the commoner in humans). The heavy chain decides the class: γ = IgG, α = IgA, δ = IgD, ε = IgE, μ = IgM. Heavy chains have one variable domain (V_H) plus three constant domains (γ, α, δ) or four (ε, μ).

The parts with names

The hinge region, between C_H1 and C_H2, gives flexibility so the two arms can move independently. The Fab fragment is the arm that binds antigen; the Fc fragment is the stem that binds receptors and complement. Within the variable domains, three hypervariable regions in V_L and four in V_H form the antigen-binding site — the rest of the variable domain is the framework region.

The two extra pieces

IgM and IgA both carry a J chain, which joins the monomers (IgM as a pentamer, IgA as a dimer). Only secretory IgA also carries the secretory component, added by the epithelial cell, which protects it from proteolysis in mucus, saliva, tears and milk.

The oncotic numbers, since they belong with the plasma proteins

Total plasma protein 7.0–7.5 g/dL exerts a colloid osmotic pressure of about 25 mmHg. At the arteriolar end capillary hydrostatic pressure is roughly 37 mmHg and interstitial pressure 1 mmHg, so about 11 mmHg pushes fluid out; at the venular end hydrostatic pressure has fallen to about 17 mmHg, so roughly 9 mmHg draws it back. Lose the protein — severe protein malnutrition, nephrotic syndrome, liver failure — and the return limb fails: fluid stays in the interstitium as oedema.

🧪 Classifying the Side Chains by Their Chemistry

🔹 The fourth classification — by the chemistry of the side chain

🛢️Aliphatic — plain hydrocarbon

Glycine · Alanine · Valine · Leucine · Isoleucine — hydrophobic, buried in the core.

💧Hydroxylic — carry an –OH

Serine · Threonine · Tyrosine — the answer to "which amino acids have a hydroxyl group in the side chain?", and the three residues that get phosphorylated.

🟡Sulfur-containing

Cysteine (–SH, makes disulphide bridges) and methionine (thioether, the start codon and the methyl donor as SAM).

➖Acidic — and their amides

Aspartate and glutamate carry a second –COOH; asparagine and glutamine are the same skeletons with an amide instead — uncharged, polar, and the body's nitrogen carriers.

➕Basic

Lysine (ε-amino) · Arginine (guanidinium, the most basic) · Histidine (imidazole, the only one that buffers at pH 7.4).

⬡Aromatic — a ring

Phenylalanine · Tyrosine · Tryptophan — they absorb ultraviolet light at 280 nm, which is how protein concentration is measured.

🔁Imino acid

Proline — its side chain loops back onto the α-nitrogen, making a secondary amine. It has no amide hydrogen, so it cannot donate a hydrogen bond, and it kinks helices.

Three more ways the same twenty get sorted

1 · By configuration — only L-α-amino acids are used in proteins (D-forms exist in bacterial cell walls and some antibiotics), and glycine is the only one with no chiral carbon, because its R group is a second hydrogen. 2 · By polarity — hydrophilic (polar, charged) versus hydrophobic (nonpolar). 3 · Protein versus non-protein amino acids — about 300 amino acids exist in nature but only 20 are coded for. The non-protein ones still matter: ornithine and citrulline (urea cycle), GABA (transmitter), taurine (bile salts), homocysteine (methionine metabolism) and homoarginine.

Homoarginine — the one obscure name on the list

An L-α-amino acid that is not found in proteins. Arginase cleaves it to L-lysine + urea, exactly as it cleaves arginine to ornithine + urea. It has been implicated in the human neurological disorder neurolathyrism — the spastic paraparesis caused by eating large amounts of the grass pea Lathyrus sativus.

⚗️ Measuring the Isoelectric Point at the Bench

The bench version — the casein experiment Past Q

Nine tubes are filled with increasing amounts of acetic acid diluted with water, so each holds a different pH; 1 mL of 0.5% casein in sodium acetate is added to each, shaken, and left about 30 minutes. The tube that turns cloudy — the maximum precipitate — marks the isoelectric point of casein (≈ pH 4.6): there the protein carries no net charge, does not move in an electric field, and is at its lowest solubility, so it aggregates. Below that pH the protein is net positive, above it net negative, and in both cases it redissolves.

Two things the pI is actually used for, and one ranking question

Knowing the pI tells you which pH to run an electrophoresis at — two amino acids that differ only slightly in pK₁ or pK₂ can be separated by choosing an acidic or a basic buffer — and it predicts binding to ion-exchange supports such as DEAE-cellulose. Rank by increasing pI: aspartic acid ≈ 3.0 (acidic) < leucine ≈ 6.0 (neutral) < lysine ≈ 9.7–10 (basic).

Name five acidic (proton-donating) functional groups

1 · the α-carboxyl –COOH · 2 · the side-chain carboxyl of aspartate and glutamate · 3 · the thiol –SH of cysteine · 4 · the phenolic –OH of tyrosine · 5 · the imidazolium of histidine — and, strictly, the protonated amino groups too (α-NH₃⁺, the ε-NH₃⁺ of lysine and the guanidinium of arginine), since every one of them can hand off a proton.

🧠 Prion Disease — the Detail Behind the Diagram

The protein and its gene

The human prion-related protein (PrP) is a glycoprotein encoded on the short arm of chromosome 20. Normal PrP^C is monomeric and rich in α-helix; PrP^Sc is rich in β-sheet, with hydrophobic side chains exposed to solvent, which is why it aggregates and resists proteases, heat and formalin.

One disease, three ways of starting

The same illness may be infectious (transmitted by the abnormal protein alone — no DNA or RNA involved), genetic (a PRNP mutation) or sporadic. Animal forms: scrapie in sheep and bovine spongiform encephalopathy in cattle. Human forms: Creutzfeldt–Jakob disease, its variant in younger patients presenting with psychiatric and behavioural change, and kuru.

What the brain looks like

The transmissible spongiform encephalopathies share spongiform vacuolation, astrocytic gliosis and neuronal loss from deposits of insoluble protein — with no inflammation, because nothing foreign is recognised.

The mechanism in one sentence

PrP^Sc acts as a template that forces PrP^C to refold into more PrP^Sc — a conformational chain reaction whose products coalesce into protease-resistant aggregates.

Protein Degradation

Frequently tested: ubiquitin–proteasome system (ATP-dependent, selective, cytosolic), partial proteolysis, amelogenin (TRAP)

♻️ Ubiquitin–Proteasome System

This is the main pathway for selective protein degradation in the cytosol. A chain of at least 4 ubiquitin molecules (a small protein) is attached to the target protein → the 26S proteasome recognizes the polyubiquitin tag → the protein is unfolded and degraded into small peptides → ubiquitin is recycled.

Three enzymes work in sequence: E1 (ubiquitin-activating enzyme) → E2 (ubiquitin-conjugating enzyme) → E3 (Ubiquitin Ligase). E3 determines which protein gets degraded (substrate specificity).

Key Properties — All Tested

The ubiquitin-proteasome system: (1) is located in the cytosol, (2) requires ATP (for E1 activation and proteasome unfolding), and (3) is selective (only proteins tagged with ubiquitin are degraded). All three properties are directly tested together.

✂️ Partial Proteolysis — Zymogen Activation

Partial proteolysis is a covalent, irreversible modification. A peptide bond is cleaved, removing an inhibitory portion of the protein → the enzyme (or hormone) becomes active. Examples: pepsinogen → pepsin, trypsinogen → trypsin, proinsulin → insulin (active hormone + C-peptide). This is also how prohormones are converted to active hormones — partial proteolysis converts a prohormone to its active hormone form.

Protein Denaturation — Why No Precipitation?

A protein may be denatured (lose its 3D structure) without precipitating if: (1) the pH is far from the isoelectric point (pI) — the protein carries a strong net charge and repels other molecules, keeping it soluble even when unfolded, or (2) after cooling, renaturation is possible — the protein refolds back to its native structure (showing denaturation was reversible). At the pI, net charge = 0 → no electrostatic repulsion → proteins aggregate and precipitate.

🧬 Key Aminoacidopathies — PKU, Homocystinuria, MSUD, Alkaptonuria

Disease	Deficient Enzyme	Accumulates	Key Clinical Features	Treatment
PKU	Phenylalanine hydroxylase (PAH) — requires BH₄	Phenylalanine + phenylketones	Intellectual disability (untreated), musty urine, fair skin & hair (↓melanin), eczema. Tyr becomes essential.	Low-Phe diet, BH₄ (sapropterin) for some
Homocystinuria	Cystathionine β-Synthase (CBS) — requires PLP/B6	Homocysteine + methionine	Marfanoid body, lens dislocation DOWNWARD, thromboembolism, intellectual disability, osteoporosis	B6 (if responsive), folate + B12, low-Met diet, betaine
MSUD	BCKAD (Branched-chain α-ketoacid dehydrogenase) — requires TPP	Leu, Ile, Val + branched-chain keto acids	Maple syrup urine/sweat odour, encephalopathy, feeding difficulty. Leucine is most neurotoxic.	Low-BCAA diet, thiamine (B1) supplementation
Alkaptonuria	Homogentisate oxidase	Homogentisic acid	Dark/black urine on air exposure, ochronosis (dark cartilage), arthritis. First "inborn error" (Garrod).	High-dose Vitamin C (reduces polymerisation); nitisinone

PAH Requires Tetrahydrobiopterin (BH₄)

Phenylalanine hydroxylase needs BH₄ as cofactor. Some PKU cases are caused by BH₄ deficiency (GTP cyclohydrolase I deficiency), not PAH itself. These are worse because BH₄ is also required by tyrosine hydroxylase and tryptophan hydroxylase → also affects catecholamine and serotonin synthesis.

🦷 Amelogenin & Dental Biochemistry ★ asked 2022, 2025

Amelogenin is the main protein of tooth enamel matrix (~90% of enamel matrix proteins). It is secreted by ameloblasts and guides the formation of hydroxyapatite crystals during enamel mineralisation.

Direct Exam Question: "What is the main amino acid component of amelogenin?" — 2022 & 2025

Answer: Tyrosine. The full name of the major amelogenin is TRAP = Tyrosine-Rich Amelogenin Protein. Its unusually high tyrosine content is what gives it the name "TRAP." This was tested directly in the 2022 and 2025 exams.

Remember: TRAP = Tyrosine Rich Amelogenin Protein → main amino acid = Tyrosine.

Enamel matrix proteins (primarily amelogenin ~90%) are later degraded by proteases — matrix metalloproteinases (MMPs) and kallikrein-4 — as enamel matures. The organic matrix is almost entirely removed in mature enamel, leaving almost pure inorganic hydroxyapatite with virtually no organic matrix remaining.

Nucleotides & Purine Metabolism

Frequently tested: purines vs pyrimidines, AMP deaminase releasing ammonia, gout & allopurinol, SCID/ADA deficiency, Lesch–Nyhan

🧬 Purines vs Pyrimidines

Purines (double ring)

Adenine, Guanine. Catabolized to uric acid by Xanthine Oxidase.

Pyrimidines (single ring)

Cytosine, Thymine, Uracil. Catabolized to β-alanine, CO₂, NH₃.

Gout

↑ uric acid → crystal deposits in joints. Allopurinol inhibits Xanthine Oxidase. Lesch-Nyhan: HGPRT deficiency → severe gout + neurological symptoms (X-linked).

⭐ EXAM PRIORITY — Teacher Marked

🔄 Purine Synthesis & AMP Deaminase

Purine synthesis de novo builds the purine ring on ribose-5-phosphate (from PPP). The cycle involves multiple steps using glutamine, glycine, aspartate, CO₂, and THF.

🏗️ De Novo Purine Synthesis (building blocks) ⚪ beyond exam scope

Ribose-5-Pfrom PPP → PRPP → IMPfirst nucleotide → AMP & GMP

Ring atoms donated by: Glutamine · Glycine · Aspartate · CO₂ · THF (N₁₀-formyl)

AMP Deaminase — Teacher Specifically Listed

AMP Deaminase converts AMP → IMP + NH₃. It is active in:
• Muscle: during intense exercise, AMP deaminase releases NH₃ from AMP → IMP → this NH₃ is exported as alanine or glutamine (nitrogen disposal from working muscle)
• Liver: part of purine catabolism pathway
Key exam: "Which enzyme releases ammonia from purines?" → AMP Deaminase. Diseases of AMP deaminase: exercise-induced myopathy (rare).

Diseases — Know ONLY These for Purines

Exam-required purine diseases:
• Gout (xanthine oxidase → ↑uric acid)
• Lesch-Nyhan (HGPRT deficiency — X-linked)
• ADA deficiency (Adenosine Deaminase (ADA) — SCID (Severe Combined Immunodeficiency))
Teacher said: "Diseases → ONLY" for purine synthesis section.

🦴 Gout & Lesch-Nyhan — Purine Catabolism Diseases

⚗️ Purine Catabolism Pathway

AMP → IMP → Hypoxanthine Xanthine Oxidase→ Xanthine Xanthine Oxidase→ Uric Acid

Guanine branch merges in at xanthine:

GMP → Guanine → Xanthine Xanthine Oxidase→ Uric Acid

Gout

Cause: Hyperuricaemia → monosodium urate (MSU) crystals deposit in joints.
Hallmark joint: First metatarsophalangeal joint (podagra).
Crystal appearance: Needle-shaped, negatively birefringent under polarised light.
Triggers: High purine diet (red meat, shellfish), alcohol, diuretics, dehydration.
Treatment: Allopurinol (xanthine oxidase inhibitor — structural analog of hypoxanthine; competitive inhibitor → ↑Km, unchanged Vmax).

👆 Tap for full disease card

Lesch-Nyhan Syndrome — HGPRT Deficiency

Enzyme: HGPRT (Hypoxanthine-Guanine Phosphoribosyltransferase) — salvages Hypoxanthine + Guanine back to IMP/GMP. X-linked recessive (affects males).

Without HGPRT: Hypoxanthine and guanine cannot be salvaged → ALL degraded to uric acid → massive hyperuricaemia.

Clinical features:

Severe gout and urate nephropathy (from birth)
Intellectual disability
Choreoathetosis (involuntary movements)
Compulsive self-mutilation (lip/finger biting — pathognomonic)

Treatment: Allopurinol controls gout but does NOT improve the neurological symptoms.

👆 Tap for full disease card

Allopurinol — Mechanism (Exam Favourite)

Allopurinol is a structural analog of hypoxanthine. It is a competitive inhibitor of xanthine oxidase → ↑Km (apparent), unchanged Vmax. It is also oxidised by xanthine oxidase to alloxanthine (oxipurinol), which then becomes a tight-binding inhibitor. Result: ↓uric acid production, ↑xanthine and hypoxanthine (which are more soluble than uric acid).

🧬 Mutations & Molecular Biology ⚪ beyond purine exam scope

Point mutation types

Transition = purine↔purine or pyrimidine↔pyrimidine.
Transversion = purine↔pyrimidine (e.g. adenine → cytosine).
Effects: silent (same AA, code is degenerate), missense (wrong AA — may be tolerated), nonsense (→ stop codon: UAA, UAG, UGA).

Missense — acceptable?

Can be acceptable: the genetic code is degenerate, and swapping one amino acid does not always destroy protein function.

Kozak sequence

A nucleotide sequence in eukaryotic mRNA around the start (AUG) codon — marks where translation begins (the ribosome-recognition context).

Translation needs energy

Amino-acid activation uses ATP (aminoacyl-tRNA synthetase → aminoacyl-tRNA). Elongation/translocation on the ribosome uses GTP.

Ricin — exam fact

Ricin (castor bean toxin) inactivates the 28S rRNA of the eukaryotic 60S subunit (cleaves a single adenine) → halts protein synthesis. Neurolathyrism (from certain plant amino acids) is a separate plant-toxin syndrome causing spastic paresis.

🧬 Pyrimidine Biosynthesis, Degradation & Regulation

Pyrimidines are built ring-first, then attached to ribose — the opposite of purines (which are built directly on ribose-5-phosphate). The whole ring starts from glutamine + CO₂ + aspartate.

⚗️ De Novo Pyrimidine Synthesis (cytosol)

Gln + CO₂ CPS-II→ Carbamoyl-P ATCase→ Carbamoyl aspartate → Dihydroorotate DHO-DH→ Orotate → UMP → UTP · CTP · dTMP

Feature	Detail (high-yield)
Rate-limiting enzyme	CPS-II — cytosolic, uses glutamine as nitrogen source, activated by ATP + PRPP, inhibited by UTP
Ring source atoms	Glutamine (N), CO₂ (C), and aspartate (the rest of the ring)
Only mitochondrial step	Dihydroorotate → orotate (dihydroorotate dehydrogenase, links to CoQ)
Sugar added as	PRPP (ribose-5-P donor) — added after the ring is made
dTMP formation	dUMP → dTMP by thymidylate synthase (needs methylene-THF)
Deoxy forms	Ribonucleotides → deoxyribonucleotides by ribonucleotide reductase
Degradation	Ring is opened and fully broken down → β-alanine (from C), β-aminoisobutyrate (from T), CO₂ + NH₃. Unlike purines, this leaves no insoluble product — so there is no "pyrimidine gout".

Purine vs Pyrimidine synthesis — the one-line contrast

Purines: ring built on ribose-5-P (IMP first). Pyrimidines: ring built first, sugar added later (UMP first). Purine catabolism ends in uric acid (insoluble → gout); pyrimidine catabolism ends in soluble, harmless products.

Hereditary Orotic Aciduria

UMP synthase deficiency → orotic acid accumulates in urine + megaloblastic anaemia that does not respond to B12/folate. Key contrast: orotic aciduria with high ammonia = OTC deficiency; without high ammonia = this disease. Treated with uridine.

👆 Tap for full disease card

💊 Synthetic Nucleotide Analogues — Clinical Use & Mechanism exam-listed

These drugs are fake building blocks: they resemble normal nucleotides/bases and jam the enzymes of DNA/RNA synthesis. Fast-dividing cells (tumour, immune, viral-infected) are hit hardest.

Drug	Target / mechanism	Clinical use
5-Fluorouracil (5-FU)	→ FdUMP → irreversibly inhibits thymidylate synthase → "thymineless death"	Colon, breast cancers
Methotrexate	Inhibits dihydrofolate reductase → ↓THF → ↓purine + dTMP synthesis	Cancer, RA, psoriasis (rescue with folinic acid)
6-Mercaptopurine / Azathioprine	Purine analogue → inhibits de novo purine synthesis (PRPP amidotransferase)	Leukaemia, immunosuppression. Toxicity ↑ with allopurinol (both use xanthine oxidase)
Hydroxyurea	Inhibits ribonucleotide reductase → ↓dNTPs	Sickle cell (↑HbF), myeloproliferative disorders
Cytarabine (Ara-C)	Arabinose cytidine analogue → blocks DNA polymerase / chain termination	Acute myeloid leukaemia
Acyclovir	Guanosine analogue; activated by viral thymidine kinase → chain terminator of viral DNA polymerase	Herpes / varicella (selective for infected cells)
Zidovudine (AZT)	Thymidine analogue → inhibits reverse transcriptase (chain terminator)	HIV
Allopurinol	Hypoxanthine analogue → inhibits xanthine oxidase	Gout, tumour-lysis prophylaxis

Selective toxicity principle

Antivirals (acyclovir) and antibacterials (trimethoprim) exploit species differences in the target enzyme; anticancer analogues exploit the high division rate of tumour cells — which is also why they harm marrow, gut, and hair.

🧾 Compounds That Contain Ribose (or Ribose-Derived) "name 6"

A frequent exam ask: "name compounds containing ribose." Ribose (or its phosphate) is the sugar backbone of every nucleotide-based molecule.

Energy currency

ATP, GTP, UTP, CTP (and their di-/mono-phosphates)

Redox coenzymes

NAD⁺ / NADH, NADP⁺ / NADPH, FAD / FMN

Acyl carrier

Coenzyme A (CoA) — contains an adenosine (ribose) unit

Second messenger

cAMP, cGMP

Methyl donor

S-adenosylmethionine (SAM) — adenosine core

Nucleic acids

RNA (ribose) — DNA uses deoxyribose

Fast answer

A safe "6": ATP, NAD⁺, FAD, Coenzyme A, cAMP, SAM — all built around a ribose-containing adenosine. (FAD's flavin sits on ribitol, but the nucleotide half is AMP-ribose.)

🧬 DNA vs RNA · Genetic Code · Frameshift Mutations

	DNA	RNA
Sugar	Deoxyribose (no 2′-OH)	Ribose (2′-OH present)
Bases	A, G, C, T	A, G, C, U (uracil)
Strands	Double-stranded helix	Usually single-stranded
Stability	Stable (no 2′-OH → resists hydrolysis)	Less stable (2′-OH → alkaline hydrolysis)
Location / role	Nucleus — long-term information store	Cytoplasm — working copy (mRNA, tRNA, rRNA)

Features of the genetic code: it is a triplet code (3 bases = 1 codon), degenerate (most amino acids have several codons — the "wobble" is in the 3rd base), unambiguous (each codon = only one amino acid), non-overlapping and comma-less (read continuously), and nearly universal (same in almost all organisms). Start = AUG (Met); stops = UAA, UAG, UGA.

Point mutations (single base)

Silent — same amino acid (wobble). Missense — different amino acid (may be tolerated). Nonsense — creates a premature stop codon → truncated protein.

Frameshift mutation

Insertion or deletion of a number of bases not divisible by 3 → the entire downstream reading frame shifts → completely different amino acids and usually an early stop. Far more damaging than most point mutations. (Insert/delete in multiples of 3 = in-frame, milder.)

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧵 From Nucleotide to Nucleic Acid — Structure, Polarity, Melting Past Q

The base is joined to carbon 1 of the sugar by a β-N-glycosidic bond; the sugars are then strung together by 3′,5′-phosphodiester bonds. One end of the chain carries a free 5′ phosphate or hydroxyl, the other a free 3′ hydroxyl — so every nucleic acid has a direction.

The double helix

Right-handed, with the two strands antiparallel: one runs 5′→3′ while the other runs 3′→5′. Held together by hydrogen bonds between purine and pyrimidine — A=T (two bonds), G≡C (three) — and, just as importantly, by base stacking: van der Waals and hydrophobic interactions between adjacent pairs. Chargaff's rule follows: A = T and G = C.

Melting temperature (T_m)

Heating separates the strands over a range of temperature; the midpoint is T_m. It depends on base composition and on salt concentration: DNA rich in G–C pairs melts at a higher temperature because each pair has three hydrogen bonds instead of two. Cooling lets the strands re-anneal — the basis of hybridisation and of every probe-based technique.

Template, coding strand, transcript Past Q

The template (antisense, non-coding) strand is the one actually read by RNA polymerase. The coding (sense) strand is its partner, and it has the same sequence as the transcript — except that RNA carries uracil where DNA carries thymine. The RNA transcript is built antiparallel to the template, 5′→3′.

DNA versus RNA — six contrasts Past Q

1 · 2′-deoxyribose vs ribose · 2 · double-stranded antiparallel helix vs single strand that folds back on itself (hairpins) · 3 · thymine vs uracil · 4 · nucleus and mitochondria vs nucleus and cytoplasm · 5 · DNA resists alkali because it has no 2′-OH, whereas RNA is hydrolysed by alkali to 2′,3′-cyclic mononucleotides — RNA is the less stable molecule · 6 · DNA obeys A=T and G=C; in RNA the base amounts need not be equal.

Deoxyribonucleotides, and where the ribose in everything else comes from

Deoxyribonucleotides are not made from scratch: ribonucleotide reductase reduces the ribose of a ribonucleoside diphosphate, using thioredoxin and NADPH, and its allosteric sites keep the four dNTPs balanced — the importance and the regulation in one enzyme. Ribose itself, from the pentose phosphate pathway, also appears in ATP, GTP, NAD(P)⁺, FAD, coenzyme A, cAMP, cGMP, SAM, UDP-glucose and RNA.

📮 The RNA Species — and the Anatomy of tRNA Past Q

✉️mRNA — the messenger

Carries the information of a gene to the protein-synthesising machinery, where it serves as the template on which a specific amino acid sequence is polymerised. Least abundant, shortest-lived, most varied in length.

🔌tRNA — the adaptor

About 20% of cellular RNA; at least one species for each of the 20 amino acids; 74–95 nucleotides long, and made by nuclear processing of a larger precursor. The primary structure is common to all species; the cloverleaf has four arms.

🏭rRNA — the machine

The most abundant RNA; it is the structural and catalytic core of the ribosome, i.e. the machinery that makes protein from the mRNA template.

🔎The small RNAs

Small nuclear RNAs (snRNA) — rRNA and mRNA processing, and splicing. The non-coding regulatory RNAs: microRNA (miRNA) and small interfering RNA (siRNA), which silence messages, and long non-coding RNA (lncRNA).

Acceptor arm

Ends in –CCA at the 3′-hydroxyl adenosyl terminal. This is the site of attachment of the amino acid, esterified there by an aminoacyl-tRNA synthetase at the cost of ATP — the step where the fidelity of translation is actually decided.

Anticodon arm

Seven nucleotides, three of which are the anticodon that base-pairs with the mRNA codon. It reads the codon antiparallel to the message.

D arm

Named for its dihydrouridine. It is the part recognised by the aminoacyl-tRNA synthetase, the enzyme that ensures the correct amino acid is loaded.

TψC arm

Contains the unusual base pseudouridine (ψ). It binds the tRNA to the ribosomal surface at the site of protein synthesis. (A fifth, variable loop sits between the anticodon and TψC arms.)

Ribozymes — RNA that behaves like an enzyme Past Q

Some RNA molecules have intrinsic catalytic activity. Their reactions usually involve cleaving a nucleic acid. Two must be known: the peptidyl transferase of the large ribosomal subunit, which forms the peptide bond — so the ribosome is a ribozyme — and the RNA component of the spliceosome, which removes introns.

🧰 Replication, Transcription and What Happens to the Transcript Past Q

The six proteins of replication Past Q

1 · DNA polymerase — polymerises deoxyribonucleotides, 5′→3′ only. 2 · Helicase — processively unwinds the double helix. 3 · Topoisomerase — relieves the torsional strain that unwinding creates ahead of the fork. 4 · DNA primase — lays down the RNA primers that polymerase needs to start. 5 · Single-strand binding (SSB) proteins — keep the separated strands from re-annealing. 6 · DNA ligase — seals the nick between the growing chain and the Okazaki fragments of the lagging strand.

Nucleases — how nucleic acids are degraded Past Q

Classified by substrate: deoxyribonucleases (DNA), ribonucleases (RNA); and by where they cut: endonucleases cut internal bonds, exonucleases remove one nucleotide at a time from an end, and work in one direction only, either 3′→5′ or 5′→3′. Some cut both strands of a duplex, others only single strands. Digestion runs nucleic acid → nucleotides → nucleosides → free base + sugar.

Exons and introns Past Q

The primary transcript is much larger than the mature mRNA. Exons are the sequences that are expressed and end up in the mature message; introns are the non-coding intervening sequences that separate them, and in most genes they make up far more of the transcript than the exons do. In the nucleus the introns are excised and the exons spliced together; the mature mRNA (capped and polyadenylated) is then exported to the cytoplasm for translation. tRNA precursors can contain introns too.

Retroviruses run the arrow backwards Past Q

Their genome is RNA, not DNA. Reverse transcriptase — a viral RNA-dependent DNA polymerase — copies it into double-stranded DNA, which is then integrated into the host genome. From that "proviral" DNA the cell transcribes new viral genomes and viral mRNAs, and translates the viral proteins. Insertion is itself mutagenic: it can inactivate a host gene or deregulate its expression. HIV is the example, and the reason reverse transcriptase is a drug target.

🔠 The Genetic Code and the End of Translation Past Q

Triplet
Three bases per codon; 64 codons for 20 amino acids.

Degenerate
Most amino acids have several codons — some many, some only one. The third base matters least, which is where nearly all the degeneracy sits.

Unambiguous
Any given codon specifies one and only one amino acid.

Non-overlapping and unpunctuated
No codon shares a base with its neighbour, and once reading starts at the start codon there are no commas — the message is read as a continuous run of triplets until a stop codon.

Almost universal
The same triplets mean the same amino acids in all species and tissues — the exception being mitochondrial tRNAs, which read four codons differently.

Start and stop
Start AUG (methionine); stop UAA, UAG, UGA, which code for no amino acid at all.

Termination, step by step Past Q

Protein synthesis has three phases — initiation, elongation, termination. After many rounds of elongation, a stop codon arrives in the A site. No tRNA recognises it; instead releasing factor RF1 does, bound to a complex of RF3 with GTP. Together with peptidyl transferase this complex hydrolyses the bond between the finished peptide and the tRNA in the P site — a water molecule is added instead of an amino acid. The protein and the tRNA are released, then the mRNA leaves and the ribosome dissociates into its 40S and 60S subunits, ready for another cycle. The products of termination are therefore: the new protein, the mRNA, the tRNA, the two subunits, RF1, GDP-bound RF3 and inorganic phosphate.

Puromycin — the inhibitor to be able to explain Past Q

It is a structural analogue of tyrosinyl-tRNA. It enters the A site and is incorporated into the carboxyl-terminal position of the growing peptide, but it cannot be extended — so the chain is released prematurely. Because it exploits a feature common to both ribosome types, it inhibits protein synthesis on 70S (prokaryotic) and 80S (eukaryotic) ribosomes alike, which is exactly why it is useless as an antibiotic and useful in the laboratory.

🧬 Mutations — Every Type and Its Consequence Past Q

A mutation is a change in the nucleotide sequence. Point mutations are single-base changes, and they come in two chemical flavours: a transition swaps a purine for a purine or a pyrimidine for a pyrimidine; a transversion swaps a purine for a pyrimidine or the reverse.

🟢 Silent — no detectable effect

The new codon still specifies the same amino acid

Why: the code is degenerate

Usually the third base

Because of wobble, translation is least sensitive to a change in the third position of the codon — which is where most silent mutations sit.

▼

🟡 Missense — a different amino acid goes in

Effect depends entirely on which residue, and where

Three grades, and the exam wants the names

Acceptable

The substitute behaves like the original — the protein is indistinguishable from normal.

Partially acceptable

The protein works, but abnormally — partial function.

Unacceptable

The protein cannot function. Sickle-cell disease — Glu→Val in β-globin — is the standard example.

▼

🔴 Nonsense and frameshift — the severe ones

Either the message stops early, or everything after the change is nonsense

Both usually destroy the protein

Nonsense

The codon becomes a stop codon: premature termination, so only a fragment of the intended protein is made and it almost never works.

Frameshift — 1 or 2 bases

Insertion or deletion of a number of bases not divisible by three shifts the reading frame. Since the message is unpunctuated, the machinery does not notice: everything distal to the change is garbled, and a premature stop codon usually appears (or a normal one is read through). A later insertion can restore the frame, leaving only the stretch between the two changes scrambled.

Frameshift — 3 bases

Deletion or insertion of three bases adds or removes one amino acid and leaves the reading frame intact downstream — usually far milder.

📡 The Cyclic Nucleotides as Second Messengers Past Q

cAMP

Made from ATP by adenylyl cyclase when a Gs-coupled receptor is occupied — glucagon, adrenaline, ACTH, TSH — and destroyed by phosphodiesterase (which insulin activates, and caffeine and theophylline inhibit). It activates protein kinase A, and everything downstream follows: phosphorylase kinase → glycogen phosphorylase on, glycogen synthase off, hormone-sensitive lipase on, and — through phosphofructokinase-2/fructose-2,6-bisphosphatase — glycolysis down and gluconeogenesis up.

cGMP

Made by guanylyl cyclase. The soluble form is activated by nitric oxide, the membrane form by atrial natriuretic peptide. cGMP activates protein kinase G and causes relaxation of vascular smooth muscle — the mechanism of nitrates, and the reason a phosphodiesterase-5 inhibitor such as sildenafil works. In the retina, cGMP holds the sodium channels of the photoreceptor open in the dark.

And GTP, which is not a messenger but a currency

GTP drives protein synthesis (initiation, elongation and termination all spend it), is the product of the one substrate-level phosphorylation of the Krebs cycle (succinyl-CoA synthetase), pays for the argininosuccinate step's cousin reactions in gluconeogenesis (PEP carboxykinase uses GTP), and is the switch of every G protein: bound GTP = on, hydrolysis to GDP = off.

🧰 Initiation and Elongation of Protein Synthesis

Initiation — four steps

1 · The 80S ribosome dissociates into 40S and 60S subunits. 2 · The ternary complex of initiator Met-tRNA + GTP + eIF-2 binds the 40S subunit → 43S pre-initiation complex. 3 · The capped mRNA binds and is scanned to the first AUG → 48S initiation complex. 4 · The 60S subunit joins with GTP hydrolysis → 80S initiation complex, Met-tRNA in the P site.

Elongation — four steps, repeated

1 · EF-1 with GTP delivers the next aminoacyl-tRNA to the A site. 2 · Peptidyl transferase (an rRNA activity of the 60S subunit — a ribozyme) forms the peptide bond. 3 · EF-2 with GTP translocates the ribosome three bases. 4 · The deacylated tRNA is expelled from the P site through the E site. The message is read 5′→3′, the protein grows N-terminus first, and each residue costs four high-energy bonds (two to charge the tRNA, two GTP).

Enzymes – Properties & Kinetics

Frequently tested: enzyme classes, Km/Vmax, competitive vs non-competitive inhibition, coenzyme requirements, plasma enzymes in diagnosis

📐 The Seven Classes of Enzymes

Class	Number	What They Do	Example
Oxidoreductases	1	Transfer electrons (oxidation-reduction reactions). Require electron carriers like NAD⁺ or FAD.	Lactate Dehydrogenase, G6PD, Malic enzyme
Transferases	2	Transfer a chemical group from one molecule to another	Kinases (transfer phosphate), Aminotransferases (transfer –NH₂), Glycogen Synthase, Citrate Synthase
Hydrolases	3	Cleave bonds using water (hydrolysis)	Proteases, Lipases, Phosphatases
Lyases	4	Add or remove groups at double bonds (without water)	Aldolase, Pyruvate decarboxylase, PEPCK
Isomerases	5	Rearrange atoms within a single molecule (structural or geometric changes)	Phosphoglucose Isomerase, Triose phosphate isomerase
Ligases	6	Join two molecules together using energy from ATP	Pyruvate Carboxylase, Acetyl-CoA Carboxylase, DNA Ligase, Aminoacyl-tRNA Synthetase
Translocases	7	Catalyze the movement of ions or molecules across membranes (membrane transport coupled to energy)	Na⁺/K⁺-ATPase, ABC transporters, mitochondrial carrier proteins, H⁺/K⁺-ATPase

⚠️ Correction: Citrate Synthase is a Transferase (Class 2), NOT a Lyase

Citrate Synthase is classified as EC 2.3.3.1 — an acyltransferase (Transferase, class 2). It transfers the acetyl group from acetyl-CoA to oxaloacetate in an aldol condensation. A common exam trap is to classify it as a Lyase because it forms a C–C bond, but Lyases only break bonds; Citrate Synthase uses CoA as a leaving group and is officially a Transferase per IUBMB.

Na⁺/K⁺-ATPase — Enzyme Classification ★ asked 2023

Na⁺/K⁺-ATPase (the sodium-potassium pump) belongs to the class of Translocases (class 7) — a subclass of transporters. In some classifications it is grouped under transferases (it transfers ions), but officially it is classified as a translocase (moves substances across membranes). This was asked in the 2023 exam: "Na/K ATPase belongs to which class?" → Translocases.

Glycogen Synthase Classification

Glycogen Synthase belongs to class 2 (Transferases) — it transfers a glucose unit from UDP-glucose to the growing glycogen chain. This is directly tested.

🏷️ Enzyme Naming Convention — Synthetase vs Synthase vs Ligase

Exam Trap (Q34): What class is a "Synthetase"?

Enzyme names ending in "-synthetase" or "-synthase" can be confusing because they both mean "makes something" — but they belong to different classes:

Name ending	Enzyme class	ATP used?	Example
-synthetase	Ligase (class 6)	YES — joins two molecules using ATP	Aminoacyl-tRNA synthetase, Argininosuccinate synthetase, Acetyl-CoA synthetase
-synthase	No fixed class — class VARIES (often Transferase, sometimes Lyase)	NO — forms a bond WITHOUT using ATP	Citrate synthase & HMG-CoA synthase = Transferases (2); Tryptophan synthase = Lyase (4); ATP synthase = Translocase (7)
-ligase	Ligase (class 6)	YES	DNA ligase, Pyruvate carboxylase

The Simple Rule

Synthetase = Ligase (class 6) — makes a bond WITH ATP. This is the reliable, testable rule.
Synthase = no ATP needed — but the EC class is NOT fixed; it must be learned per enzyme. A "-synthase" is not automatically a lyase.

Memory trick: SynthetASE has an extra "A" (for ATP) → Ligase. SynthASE has no extra letter (no ATP) → class varies.
Key examples to memorise: Citrate synthase & HMG-CoA synthase are Transferases (class 2); Glycogen synthase is a Transferase (class 2); Tryptophan synthase is a Lyase (class 4); ATP synthase is a Translocase (class 7) (uses a proton gradient).
Common trap: classifying Citrate synthase as a lyase because it forms a C–C bond — it is officially a Transferase (EC 2.3.3.1), not a lyase.

🏷️ Enzyme Naming — Phosphatase vs Phosphorylase vs Kinase

Easily confused — they do opposite/different things

All three involve phosphate groups, but the mechanism and enzyme class differ. Watch the distinction carefully.

Enzyme type	What it does	Class	Example
Phosphatase	Removes a phosphate group using water (hydrolysis): substrate-P + H₂O → substrate + Pi	Hydrolase (class 3)	Glucose-6-phosphatase, alkaline phosphatase, protein phosphatase
Phosphorylase	Adds inorganic phosphate (Pi) to cleave a bond — phosphorolysis, NOT hydrolysis and NOT using ATP: (glucose)ₙ + Pi → (glucose)ₙ₋₁ + glucose-1-P	Transferase (class 2) — specifically a glycosyltransferase	Glycogen phosphorylase, purine nucleoside phosphorylase
Kinase	Adds a phosphate from ATP (phosphorylation): substrate + ATP → substrate-P + ADP	Transferase (class 2) — a phosphotransferase	Hexokinase, protein kinase A, pyruvate kinase

The Simple Rule

Phosphatase = takes phosphate OFF using water (hydrolase).
Phosphorylase = breaks a bond by adding free Pi (phosphorolysis) — classic example: glycogen phosphorylase releasing glucose-1-phosphate from glycogen.
Kinase = puts phosphate ON using ATP.

Key trap: a phosphorylase does NOT use ATP and does NOT use water — it uses inorganic phosphate to split a bond. That makes glycogen phosphorylase a transferase, while glucose-6-phosphatase (water, removes Pi) is a hydrolase.

📈 Michaelis-Menten Kinetics

Km = the substrate concentration at which the reaction runs at exactly half its maximum speed (Vmax/2). A low Km means the enzyme reaches half-maximum speed with very little substrate = high affinity. A high Km means the enzyme needs lots of substrate to reach half-maximum speed = low affinity. Km does NOT depend on enzyme concentration.

▪ Michaelis–Menten saturation curve

Velocity climbs steeply when substrate is scarce, then flattens as active sites saturate. K_m is the [S] that gives exactly ½V_max — a low K_m = high affinity. The curve approaches V_max but never quite reaches it.

Type of Inhibition	Effect on Km	Effect on Vmax	Mechanism
Competitive	↑ Km (apparent)	Unchanged	Inhibitor competes for the active site. Effect can be overcome by increasing substrate concentration. Example: statins vs HMG-CoA Reductase.
Non-competitive	Unchanged	↓ Vmax	Inhibitor binds a separate (allosteric) site. Reduces enzyme efficiency regardless of substrate concentration. Cannot be overcome by adding more substrate.
Irreversible	—	↓ Vmax (permanently)	Inhibitor forms a covalent bond with the enzyme — permanent inactivation. Example: organophosphates on acetylcholinesterase, aspirin on COX.

Competitive Inhibition — Key Exam Statement ★ asked 2018

"The effect of a competitive inhibitor can be overcome by increasing the substrate concentration" → TRUE. At very high substrate concentrations, the substrate out-competes the inhibitor for the active site, and Vmax is eventually reached.

🎛️ Covalent Regulation — Hormones as Effectors

Covalent modification (most commonly phosphorylation/dephosphorylation) is the most important type of enzyme regulation in the body. The effector molecules that trigger covalent regulation are hormones — they bind receptors and activate downstream kinases (e.g., glucagon → cAMP → PKA → phosphorylates target enzymes). This is distinct from allosteric regulation (which involves non-covalent binding of small molecules).

🔑 Enzyme–Substrate Interaction — The Two Models

An enzyme speeds a reaction by binding the substrate in its active site and stabilising the transition state (the highest-energy point). It lowers the activation energy — it never changes the reaction's equilibrium or ΔG.

▪ Activation energy — why enzymes accelerate reactions

Uncatalysed — tall barrier With enzyme — lower barrier

The enzyme stabilises the transition state, lowering E_a so vastly more molecules cross the barrier each second. Reactant (S) and product (P) levels are identical on both paths — ΔG and the equilibrium are unchanged.

🔒 Lock-and-Key (Fischer)

The active site is a rigid, pre-formed shape that exactly fits one substrate. Explains specificity, but not how binding is optimised.

🤝 Induced Fit (Koshland)

The active site is flexible — the substrate induces a conformational change that wraps the enzyme around it. This is the accepted model; hexokinase closing over glucose is the classic example.

How enzymes lower activation energy	Mechanism
Proximity & orientation	Hold substrates together in the correct alignment to react
Transition-state stabilisation	The active site binds the transition state more tightly than the substrate — the key catalytic trick
Acid–base / covalent catalysis	Side chains (His, Ser, Cys) donate/accept protons or form transient covalent intermediates
Metal-ion catalysis	Cofactors (Zn²⁺, Mg²⁺) polarise bonds or stabilise charge

Exam statement

An enzyme does not change ΔG or the equilibrium constant — it only lowers the activation energy, speeding both forward and reverse rates equally.

📉 Lineweaver–Burk (Double-Reciprocal) Plot

Plotting 1/V against 1/[S] turns the Michaelis–Menten curve into a straight line, making Km and Vmax easy to read and inhibitor types easy to tell apart.

▪ Lineweaver–Burk — competitive vs non-competitive

No inhibitor Competitive (↑K_m, same V_max) Non-competitive (↓V_max, same K_m)

Competitive lines meet on the Y-axis (1/V_max unchanged) — the X-intercept slides right (K_m rises). Non-competitive lines meet on the X-axis (−1/K_m unchanged) — the Y-intercept rises (V_max falls).

Y-intercept

= 1/Vmax

X-intercept

= −1/Km

Slope

= Km/Vmax

Reads best

Small changes at high [S] become clear at low 1/[S]

Inhibitor	On the plot	Meaning
Competitive	Lines meet on the Y-axis (same Vmax); X-intercept moves right (↑Km)	Vmax unchanged, Km ↑ — overcome by more substrate (e.g. statins)
Non-competitive	Lines meet on the X-axis (same Km); Y-intercept rises (↓Vmax)	Km unchanged, Vmax ↓ — not overcome by substrate
Uncompetitive	Parallel lines (both Km and Vmax fall)	Inhibitor binds only the ES complex

Memory hook

Competitive inhibitors "compete" to reach the same finish line → same Vmax → lines meet on the Y-axis. Non-competitive lowers the ceiling → lines meet on the X-axis (Km unchanged).

📈 Optimum pH — each enzyme is tuned to its compartment

Pepsin — stomach (pH 2)Salivary amylase — mouth (pH 7)Trypsin — small intestine (pH 8)

Activity falls away on both sides of the optimum because pH changes the ionisation of catalytic residues and, further out, denatures the protein. The optimum is not a property of enzymes in general but of where that enzyme has to work — which is why pepsin is irreversibly inactivated the moment it reaches the duodenum.

🔬 The Active Site, the Allosteric Site & Ogston's Third Model

The seminar gives three models of enzyme–substrate interaction, not two. Fischer and Koshland explain specificity; Ogston explains stereospecificity — why an enzyme can tell apart two chemically identical arms of a symmetrical molecule.

🔒 Fischer — lock and key

Historical (end of the 19th century). Explains the specificity of the enzyme–substrate interaction, but does not account for the effect of substrate binding on the enzyme's conformation.

🤝 Koshland — induced fit

The current model. The approach of the substrate induces a conformational change in the enzyme — and the enzyme in turn induces reciprocal changes in the substrate.

🧲 Ogston — three-point attachment

The substrate attaches to the surface of the enzyme at three points. The position of the substrate relative to the enzyme is therefore fixed, and the reaction takes place at only one of the three possible attachment points. This is why a symmetrical substrate can still be handled asymmetrically.

Where the active site is. The active (catalytic) centre lies in a pocket or cleft of the apoenzyme. It is made of a group of amino acids that are close together in three dimensions but often far apart in the linear sequence. Examples: hexokinase, carboxypeptidase A.

Features of the active site

1 · It occupies a relatively small part of the total volume of the enzyme — most residues never touch the substrate.
2 · It is a spatial arrangement of chemical groups drawn from different positions in the sequence.
3 · It sits in a depression that is essentially inaccessible to water; the non-polar character strengthens substrate binding, while a few polar residues in the pocket do the actual catalysis.

The allosteric site

A separate region of some enzymes, in a different part of the molecule from the active site, that binds an allosteric effector. Effector binding causes conformational changes within the active site, which change enzyme activity. Complexes formed: E, ES, EI and ESI.

Mono- vs oligomeric enzymes

Monomeric — built from one polypeptide chain (or several joined by covalent bonds); they do not form a quaternary structure. Oligomeric — built from two or more subunits joined non-covalently; they form a strictly defined, symmetrical spatial arrangement.

🧪 How Catalysis Actually Happens — Four Mechanisms, Three Stages

Before any reaction can occur: molecules must collide, there is an energy barrier that has to be overcome, and the colliding molecules must hold enough free energy to clear it. Activation energy is the smallest energy substrate molecules must possess to become reactive.

1 · Catalysis by proximity

Bringing reactants together raises the local concentration, so there is a greater chance of interaction and a faster reaction.

2 · Acid–base catalysis

Ionisable side-chain groups of amino acids, and prosthetic groups, act as acids or bases — donating or accepting protons during the reaction.

3 · Catalysis by substrate strain

The enzyme distorts the substrate molecule, breaking covalent bonds and pushing it towards the transition state.

4 · Covalent catalysis

The enzyme itself becomes a reactant, creating a new reaction pathway with a lower activation energy.

The three stages of an enzymatic reaction

1 · Contact of the substrate molecules with the surface of the enzyme. 2 · Interaction of substrate with enzyme, producing the excited form of the enzyme–substrate complex. 3 · The catalytic reaction itself — ES is converted into an enzyme–product complex, which then breaks down into product and free enzyme.

Temperature and pH

↑ temperature → ↑ frequency of collisions → ↑ reaction rate; but excessive temperature causes gradual denaturation and the rate falls. The optimum temperature is a compromise between the energy the molecules need and the thermal stability of the enzyme. Q₁₀ ≈ 2 — the factor by which the rate of a biological process rises for every 10 °C increase (provided the enzyme is not denatured). pH matters for the ionisation of the substrate and of the active-site side chains, and for the conformation of the enzyme — it is decisive in general acid–base catalysis.

🧬 Isoenzymes — and the Five Forms of LDH

Isoenzymes are coded by genes that occupy different loci, usually arising from gene duplication and divergence. They catalyse the same reaction but differ in molecular structure, catalytic properties, affinity for the substrate and the way their activity is regulated. Examples: lactate dehydrogenase, creatine kinase, hexokinase and glucokinase.

Lactate dehydrogenase (LDH), EC 1.1.1.27 — an oxidoreductase found in liver, muscle and intestine. It catalyses the last step of the glycolytic pathway, the conversion of pyruvate to lactate and back. LDH is a tetramer built from two different subunits — H ("heart") and M ("muscle") — which gives five isoenzymes.

LDH1 — HHHH

Mainly cardiac muscle cells.

LDH2 — HHHM

Heart, erythrocytes, brain.

LDH3 — HHMM

Brain, kidney, leukocytes.

LDH4 — HMMM

Liver and muscle.

LDH5 — MMMM

Mainly skeletal muscle, but also liver — which is why a rise in LDH5 points to liver disease.

🧷 Apoenzyme, Holoenzyme, Prosthetic Group, Coenzyme, Cofactor

apoenzyme (inactive) + non-protein component ⇄ holoenzyme (active)

Prosthetic group — strongly bound

Non-protein components permanently bound to the protein, often by covalent or coordination bonds; they do not leave their binding site during the reaction. They may be organic or inorganic (metal ions and small inorganic molecules). Many organic prosthetic groups are vitamins or their derivatives — which is exactly why those vitamins are indispensable.

Coenzyme — weakly bound

A renewable shuttle. Coenzymes carry functional groups (methyl, acyl, oligosaccharide) and transport substrates from where they are made to where they are used; they also stabilise substrates that would be unstable in the aqueous cell interior (hydrogen atoms, hydrogen ions). Examples: CoA, NAD/NADH.

Cofactor — transient

Its function resembles a prosthetic group, but it attaches reversibly to the protein part or to the substrate. It must be present in the reaction environment for catalysis to occur. The commonest cofactors are metal ions.

Metalloenzyme vs metal-activated

Metalloenzymes — enzymes whose prosthetic group is a metal ion (Fe²⁺, Fe³⁺, Co³⁺, Cu²⁺, Mn²⁺, Zn²⁺). They make up about one third of all enzymes. Metal-activated enzymes bind metal ions loosely from solution — most often Na⁺, K⁺, Mg²⁺, Ca²⁺.

Which metal, which enzyme

Copper (Cu) — superoxide dismutase, cytochrome oxidase, dopamine hydroxylase, lysyl oxidase. Molybdenum (Mo) — xanthine oxidase. Zinc (Zn) — several dozen enzymes, e.g. alcohol dehydrogenase.

What metal ions actually do

They give the substrate its proper structure, they mediate oxidation and reduction reactions, and they stabilise the enzyme–substrate complex.

Vitamin-derived coenzymes — the seminar's list

Riboflavin (B2) → component of FMN and FAD. Pantothenic acid (B5) → component of coenzyme A. Thiamine (B1) → as thiamine pyrophosphate, component of keto-acid decarboxylases. Folic acid (B9) → one-carbon metabolism. Niacin (B3) → its derivatives are NAD and NADH. Pyridoxine (B6) → as pyridoxal phosphate in aminotransferases and decarboxylases. Adenine and ribose residues also occur as coenzyme components.

🏥 Functional versus Non-Functional Plasma Enzymes

The seminar splits plasma enzymes into two groups, and the whole of enzyme diagnostics follows from the split.

Functional plasma enzymes

Present constantly in the circulation of a healthy person and performing strictly defined functions there: pseudocholinesterase (breakdown of acetylcholine), hepatic lipase (lipoprotein metabolism), proenzymes of coagulation and fibrinolysis. They are most often synthesised in the liver — so a fall in their activity may indicate liver damage.

Non-functional plasma enzymes

Normally present in blood in small amounts and performing no function there. Their activity in plasma is up to a million times lower than in tissues. Under physiological conditions they come from the natural breakdown of cells (erythrocytes, leukocytes). Increased activity indicates destruction of a tissue rich in that enzyme.

↑ activity → which tissue

AspAT (AST) — myocardial infarction · AlAT (ALT) — liver dysfunction · Amylase — acute pancreatitis · Creatine kinase — myocardial infarction, muscle disease · LDH isoenzyme 5 — liver disease · Lipase — acute pancreatitis · Alkaline phosphatase — bone disease, liver disease with impaired bile outflow · Acid phosphatase — inflammation or cancer of the prostate.

Diagnosing myocardial infarction

After an infarction the blood shows a rise in the creatine kinase isoenzyme CPK-MB and in cardiac troponins (both T and I). CK isoenzymes are separated by electrophoresis — CK1 (BB), CK2 (MB) and CK3 (MM) migrate differently because they carry different charges.

☠️ Enzyme Poisons, and Methanol Poisoning as Therapy

Enzyme poisons

These are irreversible, non-competitive inhibitors: heavy metal ions (Pb, Hg) and oxidising agents. They cause chemical modification of amino acids that play an essential part in catalysis, show no structural similarity to the substrate, and the modification is not easily reversed — neither by removing the inhibitor nor by raising [S].

Methanol poisoning

Methanol is very dangerous because its oxidation products — formaldehyde and formic acid — are strongly toxic. Oxidation is catalysed by alcohol dehydrogenase, the same enzyme that oxidises ethanol to the much less toxic acetaldehyde.

Why we give ethanol — competitive inhibition used deliberately

First aid in suspected methanol poisoning is the fastest possible administration of about 100 mL of ethyl alcohol. Both alcohols have a similar structure and both bind the active centre of alcohol dehydrogenase; ethanol therefore limits the attachment of methanol and slows the production of formaldehyde and formic acid. Competitive inhibition here is protective.

Competitive inhibition — the other worked example

Pravastatin competes with β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) for the active site of HMG-CoA reductase.

🔢 Reading an EC Number — and the Sub-Types Inside Each Class

Classification is by the type of reaction catalysed, never by the substrate. Every enzyme carries an EC number of four digits, and the first digit is the class — so EC 7.1.2.2 (ATP synthase) is a translocase before you read anything else.

Class	Reaction	Sub-types you are expected to recognise
1 · Oxidoreductases	Transfer of electrons, hydrogen or oxygen	Dehydrogenases, oxidases, reductases, peroxidases, catalase, hydroxylases
2 · Transferases	Move a functional group between molecules	Aminotransferases, kinases, transmethylases, transaldolase, transketolase
3 · Hydrolases	Cleave a bond with the participation of water	Esterases, phosphatases, peptidases, glycosidases, lipases
4 · Lyases	Remove a group without water and without oxidation, usually leaving a double bond — or add across one	Decarboxylases, hydratases, deaminases, aldolases
5 · Isomerases	Intramolecular rearrangement	Epimerases, racemases, mutases (phosphoglucomutase, triose-phosphate isomerase)
6 · Ligases	Join two molecules, coupled to ATP hydrolysis	Synthetases, carboxylases (pyruvate carboxylase, glutamine synthetase)
7 · Translocases	Move ions or molecules across a membrane, or separate them within one	The seventh class added by the IUBMB — ATP synthase, EC 7.1.2.2

The classification trap

An enzyme is filed by what it does to the bond, not by what it acts on: aldolase splits a C–C bond without water, so it is a lyase, not a hydrolase; phosphoglucomutase moves a phosphate within one molecule, so it is an isomerase, not a transferase.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧨 How an Enzyme Actually Speeds a Reaction Up Topic 5Past Q

Lowering the activation energy is what an enzyme does; these four mechanisms are how it does it. Most enzymes use more than one at once.

1 · Catalysis by proximity
Binding both substrates at the active site creates a region of very high local concentration in ideal mutual orientation. That alone accelerates a reaction about a thousandfold.

2 · Acid–base catalysis
Ionisable side chains (His, Glu, Asp, Cys, Lys) and prosthetic groups donate or accept protons during the reaction. Specific acid–base catalysis uses only H⁺ or OH⁻ from the solvent; general acid–base catalysis uses any proton donor or acceptor present — which is why the rate then depends on the buffer as well as the pH.

3 · Catalysis by strain
Lytic enzymes bind the substrate in a shape that is unfavourable for the bond about to be broken, mimicking the transition state. The bond is stretched, weakened and easier to cleave — this is transition-state stabilisation, and it is why transition-state analogues make the most potent inhibitors.

4 · Covalent catalysis
The enzyme forms a transient covalent bond with the substrate — usually through a serine, cysteine or histidine residue — opening a new route with a lower activation energy. The enzyme is chemically modified only for an instant and comes back unchanged, so it is still a catalyst. Common in group-transfer reactions, and typically follows a ping-pong mechanism: the first substrate binds and its product leaves before the second substrate binds.

Turnover number (k_cat)

The number of substrate molecules converted to product per enzyme molecule per second when the enzyme is saturated — typically 10²–10⁴ s⁻¹ (carbonic anhydrase reaches 10⁶). k_cat = Vmax / [E]_total, and k_cat/Km is the measure of catalytic efficiency. Enzyme-catalysed reactions run roughly 10³ to 10⁸ times faster than the uncatalysed ones.

Compartmentation is itself regulation

Enzymes are localised: mitochondria (Krebs cycle, β-oxidation, pyruvate oxidation), cytosol (glycolysis, pentose phosphate pathway, fatty acid synthesis), nucleus (DNA and RNA synthesis), lysosome (degradation of complex macromolecules), smooth ER (cytochrome P450). Separating opposing pathways prevents futile cycling and lets each have its own optimal environment.

Enzyme Regulation

Frequently tested: allosteric regulation, phosphorylation, rate-limiting enzymes of each pathway, synthase vs synthetase classes

🎛️ Mechanisms of Regulation

Mechanism	Covalent?	Reversible?	Key Facts
Allosteric	No	Yes	Effector binds allosteric site; changes shape; can change Km &/or Vmax
Phosphorylation	Yes	Yes (phosphatase)	Kinase adds PO₄ (uses ATP); can activate OR inactivate
Partial proteolysis	Yes	NO	Zymogen activation; irreversible
Induction/Repression	—	Yes	Changes AMOUNT of enzyme; slow response

🔄 Key Phosphorylation Examples

Glycogen Phosphorylase

Glycogen Phosphorylase — Phosphorylated = ACTIVE → breaks down glycogen

Glycogen Synthase

Glycogen Synthase — Phosphorylated = INACTIVE → makes glycogen when dephosphorylated

HMG-CoA Reductase

HMG-CoA Reductase — Phosphorylated = INACTIVE → rate-limiting for cholesterol synthesis

Pyruvate Kinase (liver)

Pyruvate Kinase — Phosphorylated = INACTIVE → glucagon → cAMP → PKA

🔢 Two Ways to Regulate an Enzyme — Amount versus Efficiency

The body controls enzymatic reactions precisely, in response to stimuli, in order to maintain homeostasis. There are exactly two ways to do it: 1 · change the amount of enzyme, or 2 · change the catalytic efficiency of the enzyme without changing its amount.

1 · Changing the amount — regulation at the level of gene expression

The classic example is lactose metabolism by E. coli. Lactose is the inducer of the gene for β-galactosidase (lactose → galactose + glucose); its presence makes mRNA synthesis possible. Cells grown on lactose contain several thousand molecules of the enzyme; when another carbon source such as glycerol is used instead, the amount of β-galactosidase falls dramatically. The change happens at the level of gene expression.

The operon

Operon = operator + the structural genes adjacent to it. Regulatory genes are responsible for the amount of enzyme; structural genes for its quality. The operator lies at the transcription start site and decides whether transcription begins. The regulatory gene sits in a completely different place in the DNA, controls the work of the operon, and is responsible for synthesising the repressor.

Repressor and inducer

The repressor can bind the operator and block gene expression. Lactose acts as the inducer: it binds the repressor and, by changing its conformation, inactivates it — so the repressor can no longer sit on the operator, transcription proceeds, and β-galactosidase, permease and transacetylase are expressed.

2 · Changing catalytic efficiency — allosteric regulation

An effector attaches to the allosteric site (distinct from the active centre) and causes a change in the tertiary structure of the enzyme — which alters the enzyme's ability to bind substrate. Example: feedback inhibitors — inhibition by the end product of a pathway. Note that such an inhibitor generally shows no structural similarity to the substrate of the enzyme it inhibits.

🎚️ Covalent Modification versus Allosteric Regulation

Glycosylation, hydroxylation and acylation are modifications that build a defined structure in a newly synthesised protein. Phosphorylation–dephosphorylation is different: it is a reversible covalent modification that changes the function of proteins, enzymes included, and it is the commonest regulatory mechanism because the two forms interconvert so easily. It lets a given enzyme change its functional properties only when there is a physiological demand.

Kinase

Transfers the γ-phosphate group of ATP onto the hydroxyl residues of serine, threonine or tyrosine.

Phosphatase

Hydrolyses phosphomonoester bonds — removing the phosphate again.

Phosphorylation does not mean "on"

Some enzymes LOSE activity after phosphorylation: acetyl-CoA carboxylase, glycogen synthase, pyruvate dehydrogenase.
Others INCREASE their activity after phosphorylation: glycogen phosphorylase, citrate lyase, phosphorylase b kinase.

Four differences between covalent and allosteric modification

• Covalent modification changes the chemical nature of the enzyme.
• Covalent modification requires the hydrolysis of ATP.
• Covalent modification is regulated by the hormonal system.
• Separate enzymes (a kinase and a phosphatase) are required to restore the original form of the enzyme.

📋 The Five Regulatory Mechanisms and How Fast Each One Works

The seminar table, read as one line each: way of regulation → typical effector → result → reaction time.

Substrate availability

Effector: the substrate · Result: change in rate (v) · Time: immediate

Product inhibition

Effector: the product of the reaction · Result: change in V_max or K_m · Time: immediate

Allosteric control

Effector: the end product of the pathway · Result: change in V_max or K_0.5 · Time: immediate

Covalent modification

Effector: another enzyme · Result: change in V_max or K_m · Time: immediate to minutes

Synthesis or degradation of the enzyme

Effector: a hormone or a metabolite · Result: change in the amount of enzyme · Time: hours to days. Note that inhibition by the end product of a metabolic pathway is also called feedback inhibition.

Limited proteolysis — the irreversible one

Limited proteolysis is the irreversible activation of certain enzymes synthesised as inactive proenzymes. It consists in hydrolysis of one or a few peptide bonds, which uncovers the active centre of the molecule. It occurs in: insulin (its proprotein is proinsulin) · digestive enzymes (pepsinogen→pepsin, trypsinogen→trypsin, chymotrypsinogen→chymotrypsin) · blood clotting and fibrinolysis factors · connective-tissue proteins (procollagen→collagen). The purpose is twofold: protection against unwanted activation at the site of synthesis (digestive enzymes), and the possibility of very rapid activation at the moment the protein is needed (clotting factors).

🚦 Classifying Inhibition — Three Different Questions

The seminar classifies inhibitors on three separate axes. Getting an exam question right usually means noticing which axis is being asked about.

Axis 1 — how the inhibitor binds

Reversible or irreversible.

Axis 2 — where it binds

At the catalytic site → isosteric. At the allosteric site → allosteric.

Axis 3 — does ↑[S] overcome it?

Yes, it is overcome → competitive. No, it is not → non-competitive.

Competitive — the fingerprint

Usually a substrate analogue; binds the catalytic site isosterically. EnzS and EnzI form, but not EnzSI. No product is formed from EnzI. At a high enough [S], v_i tends to V_max as if there were no inhibitor. K_m increases · V_max unchanged.

Non-competitive — the fingerprint

The inhibitor may have a completely different structure from the substrate, and binds domains of the enzyme other than the catalytic one (allosterically). EnzS, EnzI and EnzSI all form. Despite a high [S], v_i never reaches the uninhibited V_max. V_max falls · K_m unchanged.

📈 Reaction Rate and K_m — What the Seminar Actually Says

v = −d[S]/dt = d[P]/dt = k[S] — rate is the measure of substrate disappearance or product appearance over time. Measurement is normally done under conditions corresponding to the initial velocity v_i: at that point only trace amounts of product have accumulated, so the reverse reaction can be ignored. A large molar excess of substrate is used, to saturate the enzyme completely. Measuring v_i allows the amount of enzyme in a biological sample to be assessed.

[S] < K_m

Initial rate is directly proportional to [S].

[S] > K_m

Rate is maximal; further increases in substrate concentration have no effect.

[S] = K_m

Initial rate is half the maximum rate.

Michaelis–Menten model

E + S ⇄ ES → E + P. If [S] is high enough for all enzyme molecules to be converted into ES, then the second reaction — the breakdown of that complex into E + P — is the one that decides the overall rate.

The meaning of K_m — five statements from the slide

• Its value equals the [S] at which v_i = V_max/2.
• Its reciprocal is the measure of the enzyme's affinity for the substrate — the lower the K_m, the higher the affinity.
• Its value does not depend on the enzyme concentration.
• It says nothing about V_max.
• It characterises the first phase of the enzymatic reaction.
Use: to compare the affinity of different enzymes for the same substrate, or of one enzyme for several substrates.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🎚️ Homotropic and Heterotropic Effectors

Homotropic versus heterotropic effectors Past Q

Homotropic — the substrate itself is the effector. One molecule binding at one site raises the affinity of the remaining sites: cooperativity, which is what produces the sigmoidal v–[S] curve instead of the Michaelis–Menten hyperbola. Oxygen on haemoglobin is the classic example. Heterotropic — the effector is a different molecule: citrate inhibiting phosphofructokinase-1, fructose-2,6-bisphosphate activating it, acetyl-CoA activating pyruvate carboxylase. For a sigmoidal enzyme the term Km is replaced by K₀.₅ (S₀.₅), the substrate concentration giving half-maximal velocity; positive and negative effectors can change K₀.₅, Vmax, or both.

Carbohydrate Digestion

Frequently tested: α-amylase & α(1→4) bonds, reducing sugars & Benedict/iodine tests, starch digestion, ketoses vs aldoses

🔑 Where Each Dietary Hexose Goes — Phosphorylation and Entry

Every dietary hexose is phosphorylated immediately on entering a cell. The phosphate is charged, so the sugar can no longer cross the membrane back out — it is trapped and committed to metabolism.

Sugar	Enzyme	Product	Where it goes / exam point
Glucose	Hexokinase (all cells) / glucokinase (liver, β cells)	Glucose-6-P	Glycolysis, glycogen or the PPP. Hexokinase has a low K_m and is inhibited by its product; glucokinase has a high K_m and acts as the glucose sensor
Fructose	Fructokinase (liver) / hexokinase (elsewhere)	Fructose-1-P (liver) or fructose-6-P	Bypasses PFK-1 — enters glycolysis faster than glucose, and unregulated
Galactose	Galactokinase	Galactose-1-P	Defect → galactokinase deficiency (cataracts)
Mannose	Hexokinase (low affinity)	Mannose-6-P	→ fructose-6-P → glycolysis. Most body mannose actually comes from fructose metabolism

Why phosphorylation matters

Three reasons: it traps the sugar inside the cell; it keeps the intracellular free-sugar concentration low so more can enter down its gradient; and it activates the sugar for the next reaction. It costs 1 ATP — the "investment" of glycolysis.

Epimers ★ asked 2023

Glucose and mannose are C-2 epimers — the 2023 paper asked this directly. Glucose and galactose are C-4 epimers, interconverted by UDP-hexose-4-epimerase.

Sorbitol pathway & diabetic complications

In hyperglycaemia, glucose is reduced to sorbitol (a polyol) by aldose reductase, then oxidised to fructose by sorbitol dehydrogenase. Sorbitol crosses membranes poorly, so it accumulates in the insulin-independent tissues — lens, retina, Schwann cells, kidney — and draws in water: cataracts, retinopathy, peripheral neuropathy, nephropathy.

🧬 Glycoproteins, Proteoglycans & Oligosaccharide Synthesis / Degradation

Many proteins carry sugar chains (glycosylation). These "glycoconjugates" do structural, recognition, and lubrication jobs — and their faulty breakdown causes lysosomal storage diseases.

Class	What it is	Role
Glycoproteins	Protein + short branched oligosaccharides (protein-dominant)	Enzymes, hormones (FSH, TSH), antibodies, membrane receptors, mucus (mucins)
Proteoglycans	Core protein + long glycosaminoglycan (GAG) chains (sugar-dominant, highly negative)	Extracellular matrix, cartilage, joint lubrication — hold water (turgor)
GAGs (examples)	Hyaluronic acid, chondroitin sulfate, heparin, keratan/dermatan sulfate	Repeating disaccharides of an amino sugar + uronic acid

N-linked glycosylation

Sugar added to asparagine (Asn). Built on a dolichol phosphate carrier, begun in the ER, trimmed in the Golgi.

O-linked glycosylation

Sugar added to serine / threonine –OH. Built directly in the Golgi, sugar-by-sugar (no dolichol).

Degradation → storage diseases

Glycoconjugates are broken down by lysosomal enzymes. Missing enzymes cause accumulation: Gaucher (glucocerebroside), mucopolysaccharidoses (GAGs — Hurler, Hunter), and I-cell disease (failure of mannose-6-phosphate tagging → enzymes not delivered to lysosomes). Mannose-6-phosphate is the "address label" that sends enzymes to the lysosome.

🔤 Naming Carbohydrates — The Vocabulary

Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield them on hydrolysis. Every name in this topic is built from three questions: how many carbons, aldehyde or ketone, and how many units joined together.

By carbonyl group

Aldose — the carbonyl is an aldehyde at C1 (glucose, galactose, ribose).
Ketose — the carbonyl is a ketone, usually at C2 (fructose, ribulose, xylulose).

By chain length

Triose 3C · Tetrose 4C · Pentose 5C · Hexose 6C · Heptose 7C. Combine the two: glucose is an aldohexose, fructose a ketohexose, ribulose a ketopentose.

By number of units

Monosaccharide — one unit. Disaccharide — two. Oligosaccharide — 3 to about 10. Polysaccharide — many.

Isomer vocabulary

Epimers differ at one carbon only — glucose and galactose at C4, glucose and mannose at C2. Enantiomers are complete mirror images (D- and L-glucose). Anomers differ only at the new asymmetric carbon created on ring closure — α and β.

Mutarotation

In solution the ring opens and closes continuously, so α and β forms interconvert through the open-chain aldehyde until an equilibrium is reached. The measured optical rotation drifts to a fixed value — that drift is mutarotation. It also explains why the anomeric carbon can react: it is transiently a free aldehyde.

🔗 Glycosidic Bonds — Reading the Notation

A glycosidic bond forms between the anomeric carbon of one sugar and a hydroxyl of another, with loss of water. The notation tells you three things: the anomer (α or β), the carbon on the first sugar, and the carbon on the second.

Disaccharide	Components	Bond	Reducing?
Maltose	Glucose + glucose	α(1→4)	✅ Yes
Isomaltose	Glucose + glucose	α(1→6)	✅ Yes
Lactose	Galactose + glucose	β(1→4)	✅ Yes
Sucrose	Glucose + fructose	α(1→2)β	❌ No
Trehalose	Glucose + glucose	α(1→1)α	❌ No
Cellobiose	Glucose + glucose	β(1→4)	✅ Yes

🎯 Why sucrose and trehalose are NOT reducing sugars

A sugar is reducing if it has a free anomeric carbon that can open to an aldehyde. In sucrose the bond joins both anomeric carbons (C1 of glucose to C2 of fructose); in trehalose it joins C1 to C1. With no free anomeric carbon, neither can open, neither can reduce, and neither undergoes mutarotation. Every other common disaccharide is reducing.

🧪 Benedict test — what a positive result actually means

Benedict reagent contains Cu²⁺ in alkaline citrate. A reducing sugar donates electrons, reducing Cu²⁺ to Cu⁺, which precipitates as brick-red Cu₂O. The colour scales with concentration: blue (negative) → green → yellow → orange → brick red. Because it detects any reducing sugar, a positive test in urine does not prove glucose — galactose and fructose also give a positive result, which is exactly how galactosaemia and fructosuria were historically detected. Modern glucose-oxidase dipsticks are specific for glucose only, so the two tests are used together.

🌾 Digestible versus Indigestible Polysaccharides

✅ DIGESTIBLE — α bonds

Starch — amylose: unbranched, α(1→4) only
Starch — amylopectin: α(1→4) chains with α(1→6) branches roughly every 24–30 residues
Glycogen: the same design as amylopectin but more highly branched — branches every 8–10 residues. More branches means more non-reducing ends, so it can be mobilised faster

❌ INDIGESTIBLE — β bonds

Cellulose: β(1→4) glucose. Humans have no β-glucosidase, so it passes through as dietary fibre
Fibre still matters: it adds bulk, slows gastric emptying, blunts the glucose spike, and is fermented by colonic bacteria to short-chain fatty acids

The whole rule in one line

Human digestive enzymes cleave α bonds and cannot touch β bonds — with exactly one exception: lactase cleaves the β(1→4) bond of lactose. That single exception is why lactose is the only β-linked sugar we can absorb, and why losing lactase leaves the sugar in the lumen. See lactose intolerance.

🍬 Digestion — From Starch to Monosaccharides

Two amylases work in the lumen and cut only internal α(1→4) bonds; everything else is finished by the brush-border enzymes on the intestinal wall, which release the free monosaccharides that are actually absorbed.

Salivary α-amylase

Starts starch digestion in the mouth; inactivated by stomach acid, so its contribution is small.

Pancreatic α-amylase

The main starch enzyme. Cleaves internal α(1→4) bonds only — leaving maltose, maltotriose and α-limit dextrins (the branch points it cannot cut).

Maltase

Maltose → 2 glucose. Also cleaves maltotriose, the 3-glucose fragment amylase leaves behind.

Isomaltase

The only enzyme that cuts the α(1→6) branch points in limit dextrins.

Sucrase

Sucrose → glucose + fructose. Part of the same sucrase–isomaltase complex — see sucrase–isomaltase deficiency.

Lactase

Lactose → galactose + glucose. The only β-glycosidase we possess, and the first to be lost in any mucosal injury.

Trehalase

Trehalose → 2 glucose. Trehalose is the sugar of mushrooms and insects; deficiency causes symptoms only after eating them.

Absorption — three transporters, two mechanisms

SGLT-1 takes glucose and galactose across the apical membrane against their gradient, powered by the sodium gradient maintained by the basolateral Na⁺/K⁺-ATPase — secondary active transport. GLUT-5 takes fructose in by facilitated diffusion only. GLUT-2 then moves all three out of the cell into the blood at the basolateral membrane. This is why oral rehydration solutions contain both glucose and salt: SGLT-1 needs the sodium, and the water follows.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🍭 The Sugar Family Album — Which Ones Matter and Why Topic 19Past Q

A monosaccharide cannot be hydrolysed into anything simpler; the exam asks for the physiologically important ones grouped by the number of carbons.

3️⃣Trioses — glyceraldehyde (glycerose) · dihydroxyacetone

The two triose phosphates of glycolysis, and the backbone of every triacylglycerol.

4️⃣Tetroses — erythrose · erythrulose

Erythrose-4-phosphate is the pentose phosphate pathway's link to aromatic compounds.

5️⃣Pentoses — ribose · ribulose · xylose · xylulose · arabinose

Ribose builds RNA and every nucleotide coenzyme; ribulose and xylulose are pentose phosphate pathway intermediates; xylulose is the end product of the uronic acid pathway (essential pentosuria).

6️⃣Hexoses — glucose · galactose · fructose · mannose

The only four that reach the portal blood in quantity. Mannose is a constituent of glycoproteins.

7️⃣Heptose — sedoheptulose

Sedoheptulose-7-phosphate, the transketolase product in the non-oxidative phase of the pentose phosphate pathway.

🔹 The disaccharides — composition and linkage

Disaccharide	Made of	Bond	Note
Maltose	Glucose + glucose	α(1→4)	From starch digestion; reducing
Isomaltose	Glucose + glucose	α(1→6)	The branch point; needs isomaltase
Lactose	Galactose + glucose	β(1→4)	Milk sugar; reducing
Sucrose	Glucose + fructose	α1→2β	Both anomeric carbons used, so non-reducing
Trehalose	Glucose + glucose	α1→1α	Insects, yeast, mushrooms; non-reducing
Lactulose	Galactose + fructose	β(1→4)	Synthetic, not digested by human enzymes — used as an osmotic laxative and in hepatic encephalopathy, where colonic fermentation traps ammonia as NH₄⁺

Oligosaccharides — building and breaking

Three to ten monosaccharides joined by glycosidic bonds, which are formed by glycosyltransferases using activated nucleotide sugars (UDP-glucose, UDP-galactose, GDP-mannose, CMP-sialic acid). They are broken by glycosidases: salivary and pancreatic α-amylase on starch, then the brush-border disaccharidases and oligosaccharidases that release the free monosaccharides which are actually absorbed.

Amino sugars — where the –OH becomes –NH₂ Past Q

D-glucosamine, D-galactosamine and D-mannosamine, usually N-acetylated. Their nitrogen comes from the amide of glutamine. They are the repeating units of the glycosaminoglycans — hyaluronic acid is glucuronic acid + N-acetylglucosamine — and of proteoglycans, glycoproteins and the ABO blood-group antigens; several antibiotics (streptomycin, erythromycin) also contain amino sugars.

Protein Digestion & Absorption

Frequently tested: digestive proteases & their activation (trypsin activates the rest), enterokinase, chymotrypsin specificity, metabolites formed in the intestine

🍽️ Proteases — Grouped by Where They're Activated

The cleanest way to memorize the proteases is by where each one gets switched on as food travels down the GI tract. There are three activation stations:

🟥 1 · Stomach

Acidic environment (HCl from parietal cells)

Activated by: HCl / autocatalysis at pH < 5

Pepsin

Secreted as pepsinogen by chief cells. Cleaves after aromatic residues (Phe, Trp, Tyr).

▼

🟨 2 · Duodenal Brush Border

The master switch that turns on the pancreatic enzymes

Activated by: nothing upstream — it is the FIRST activator

Enterokinase → activates Trypsin

Brush-border enzyme (NOT pancreatic). Cleaves trypsinogen → trypsin, which then triggers everything below. Trypsin cleaves after Arg/Lys (basic residues).

▼

🟩 3 · Pancreatic Zymogens (in the duodenal lumen)

All secreted by the pancreas as inactive zymogens

Activated by: TRYPSIN (one trypsin switches on all of these)

Chymotrypsin

Cleaves after Phe/Trp/Tyr (aromatic / large hydrophobic).

Elastase

Cleaves after small residues (Ala, Gly, Ser) → releases Leu, Ile, Gly, Ser.

Carboxypeptidase A

Trims C-terminal aliphatic residues (Ala, Ile, Leu, Val). Needs Zn²⁺.

Carboxypeptidase B

Trims C-terminal basic residues (Arg, Lys). Needs Zn²⁺.

💡 The logic in one line

Acid wakes Pepsin → Enterokinase wakes Trypsin → Trypsin wakes everyone else. Memorize the chain of command, not six separate facts.

Enterokinase (Enteropeptidase) — Key Facts

Enterokinase is a brush-border enzyme (not a pancreatic enzyme). It is the first activator: it cleaves trypsinogen → active trypsin. Trypsin then activates ALL remaining pancreatic zymogens (chymotrypsinogen, proelastase, procarboxypeptidase). Enterokinase itself is NOT activated by trypsin — it goes in one direction only. This is frequently asked.

Exam Q: "Which enzyme releases Leu, Ile, Gly, Ser?"

Answer: Elastase — it cleaves after small neutral residues, releasing larger ones from the chain end. Carboxypeptidase A releases aliphatic C-terminal amino acids (Ala, Ile, Leu, Val).

▸ Show the same information as a reference table

Enzyme	Secreted by	Activated by	Cleaves after / Releases
Pepsin	Stomach chief cells (as pepsinogen)	HCl / autocatalytic at pH <5	Aromatic residues (Phe, Trp, Tyr)
Trypsin	Pancreas (as trypsinogen)	Enterokinase at brush border	After Arg/Lys (basic residues)
Chymotrypsin	Pancreas	Trypsin	After Phe/Trp/Tyr (aromatic/large hydrophobic)
Elastase	Pancreas	Trypsin	After small residues: Ala, Gly, Ser — releases Leu, Ile, Gly, Ser
Carboxypeptidase A	Pancreas	Trypsin	C-terminal aliphatic residues (Ala, Ile, Leu, Val); needs Zn²⁺
Carboxypeptidase B	Pancreas	Trypsin	C-terminal basic residues (Arg, Lys); needs Zn²⁺

🔗 Haptocorrin, Intrinsic Factor & Vitamin B12 Absorption

Vitamin B12 (cobalamin) absorption requires a carefully coordinated sequence — each step has clinical relevance:

🔗 Vitamin B12 Absorption — location by location

B12 in food Pepsin→stomach B12 + HaptocorrinR-protein, saliva →duodenum · proteases free B12 B12 + Intrinsic Factorparietal cells →terminal ileum · cubilin B12 + Transcobalamin IIinto blood

1
Mouth/Stomach: B12 released from food by pepsin → immediately binds Haptocorrin (R-protein / transcobalamin I) from salivary glands. Haptocorrin protects B12 from stomach acid.
2
Duodenum: Pancreatic proteases degrade haptocorrin → B12 released → binds Intrinsic Factor (IF), secreted by gastric parietal cells.
3
Terminal Ileum: IF-B12 complex binds cubilin receptors → receptor-mediated endocytosis → B12 enters blood bound to transcobalamin II.

Pernicious Anaemia

Autoimmune destruction of gastric parietal cells → no IF → B12 cannot be absorbed → megaloblastic anaemia + subacute combined degeneration of spinal cord. Treat with intramuscular B12 injections.

Exam Trap: Haptocorrin vs Intrinsic Factor ★ asked 2022

Haptocorrin = R-protein, from SALIVA — first binder, protects B12 in stomach.
Intrinsic Factor = from GASTRIC PARIETAL CELLS — second binder, required for ileal absorption.

✂️ Endopeptidases versus Exopeptidases — and How the Products Are Absorbed

No protein digestion happens in the mouth. It begins in the stomach and is completed in the small intestine by pancreatic and brush-border enzymes.

Endopeptidases — cut inside the chain

Hydrolyse internal peptide bonds, producing shorter peptides. Pepsin (aromatic residues), trypsin (after Lys and Arg), chymotrypsin (after aromatic residues), elastase (after small neutral residues). Their significance: they cut one large protein into many fragments, multiplying the number of ends for the next group.

Exopeptidases — trim from the ends

Carboxypeptidase A and B (pancreatic) remove residues from the C-terminus; aminopeptidases of the brush border work from the N-terminus; di- and tripeptidases finish the job inside the enterocyte.

Absorption — two routes into the enterocyte

Free amino acids enter by Na⁺-dependent secondary active transport through several carriers, each serving a group (neutral, basic, acidic, imino). Di- and tripeptides enter on the H⁺-coupled PepT1 transporter and are hydrolysed inside the cell. Everything then leaves into the portal vein to the liver. A defect in the basic-amino-acid carrier of intestine and kidney is cystinuria.

Measuring the stomach: BAO and MAO

BAO (basal acid output) is acid secreted per hour without stimulation — normally about 0–5 mmol/h. MAO (maximal acid output) follows maximal stimulation with pentagastrin or histamine — normally about 5–20 mmol/h. A high BAO with a BAO/MAO ratio above ~0.6 suggests a gastrin-secreting tumour (Zollinger–Ellison); a low or absent output suggests atrophic gastritis or pernicious anaemia.

⚖️ Complete versus Incomplete Proteins — Biological Value

Complete — high biological value

Contain all the essential amino acids in adequate proportions: egg, milk, meat, fish. Egg protein is the reference standard.

Incomplete — low biological value

Deficient in one or more essential amino acids — most plant proteins. Cereals are limiting in lysine; legumes are limiting in methionine; gelatin lacks tryptophan entirely.

Biological value and the limiting amino acid

Biological value is the proportion of absorbed nitrogen that is retained by the body. It is set by the limiting amino acid — the essential one present in the smallest amount relative to need. Protein synthesis stops the moment any single one runs out, and the rest of the intake is deaminated and burned.

Complementation

Combine two incomplete proteins whose limiting amino acids differ — cereals (low lysine) with legumes (low methionine) — and the mixture has a high biological value. That is the biochemistry behind rice and beans.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

📋 The Protease Data Sheet — Proenzyme, Activator, pH, Specificity Topic 8Past Q

Endopeptidases hydrolyse internal peptide bonds; exopeptidases remove residues from an end. Every one of them is secreted as an inactive proenzyme — that is what protects the pancreas, and premature activation inside it is acute pancreatitis.

Enzyme	Proenzyme	Activated by	Optimum pH	Cuts	Products
Pepsin	Pepsinogen	HCl, then autocatalysis	1–2	Internal bonds beside aromatic and dicarboxylic residues, and between Leu and Glu	Large peptides — only about 15% of the protein is cut here
Trypsin	Trypsinogen	Enteropeptidase, then autocatalysis	7–9	Internal bonds after basic residues	Peptides ending in Lys or Arg
Chymotrypsin	Chymotrypsinogen	Trypsin	7–9	Internal bonds after aromatic residues	Peptides ending in Phe, Trp, Tyr
Elastase	Proelastase	Trypsin	7–9	Internal bonds after small aliphatic residues	Peptides ending in Val, Leu, Ser, Ala
Carboxypeptidase A	Procarboxypeptidase A	Trypsin	7–9	C-terminal aromatic and aliphatic residues — i.e. the ends chymotrypsin and elastase have just made	Free Phe, Trp, Tyr, Val, Leu, Ser, Ala
Carboxypeptidase B	Procarboxypeptidase B	Trypsin	7–9	C-terminal basic residues — the ends trypsin has just made	Free Lys and Arg
Aminopeptidases	—	Brush border enzymes	7–9	N-terminal residues	Free amino acids + shorter peptides
Dipeptidases and tripeptidases	—	Inside the enterocyte	~7	The last two or three residues	Free amino acids for the portal blood

Why the two classes need each other

Endopeptidases multiply the number of free ends; exopeptidases can then only work fast because those ends exist. Pepsin's contribution is small in quantity but essential in kind — it is the one that works in acid.

Lipid Digestion & Absorption

Frequently tested: order of events (emulsification→hydrolysis→micelles), bile acids/salts & conjugation, primary vs secondary, what is NOT made in liver

🧈 Order of Lipid Digestion

The correct sequence — tested directly on exams: Emulsification → Hydrolysis → Micelle formation → Absorption

🧈 Lipid Digestion — order of events

1 · Emulsificationbile salts → 2 · Hydrolysispancreatic lipase → 3 · Micelle formationFFA + 2-MAG + bile → 4 · Absorptionenterocyte → chylomicron

1
Emulsification: bile salts (made in liver, stored in gallbladder) break large fat globules into tiny droplets — this massively increases the surface area available for enzymes to act on.
2
Hydrolysis: Pancreatic Lipase (requires colipase as a helper protein) cleaves triglycerides at the sn-1 and sn-3 positions → produces 2-monoacylglycerol (2-MAG) + 2 free fatty acids (FFAs).
3
Micelle formation: the digestion products (FFAs + 2-MAG) combine with bile salts to form mixed micelles — tiny water-soluble aggregates that can travel through the watery intestinal fluid to reach the intestinal cells.
4
Absorption: FFAs and 2-MAG diffuse into enterocytes (intestinal cells) → re-esterified back to triglycerides → packaged into chylomicrons (which carry ApoB-48) → secreted into lymph → eventually enter the bloodstream.

CCK vs Secretin — Must Distinguish

🟢 CCK (Cholecystokinin)

Released when fat & protein enter the duodenum. Stimulates gallbladder contraction (releases bile) + pancreatic enzyme secretion. Also an anorexigenic hormone — suppresses appetite.

🔵 Secretin

Released when acid enters the duodenum. Stimulates pancreatic bicarbonate (HCO₃⁻) secretion to neutralize stomach acid — NOT enzyme secretion.

HCl Effects on Blood Chemistry

When the stomach secretes HCl (hydrogen + chloride into the lumen), bicarbonate is simultaneously released into the blood — this is called the "alkaline tide." Result: ↑ plasma HCO₃⁻ and ↓ plasma Cl⁻ during active acid secretion. Directly tested on exams.

🧴 Saliva Composition (Exam Tested)

Saliva contains: amylase, water, mucus, lactoferrin (iron-binding antimicrobial protein), potassium (saliva is rich in K⁺), cysteine protease inhibitors (cystatins — protect tissues from proteolytic damage), IgA, and bicarbonate. All three of the bolded items have been asked together in one question.

💛 Bile Salts vs Bile Acids — Teacher Required

Bile acids (unconjugated) are cholesterol derivatives. When conjugated with glycine or taurine, they become bile salts.

Unconjugated bile acids: pKa ~6 → partially dissociated at bile pH (half ionized)
Conjugated with glycine or taurine: pKa lowered → fully dissociated at bile pH → cannot donate protons → they are salts
Conjugation makes them more amphiphilic → better detergents → better at forming micelles

Primary Bile Acids (examples)

Synthesized in liver from cholesterol: Cholic acid and Chenodeoxycholic acid. Conjugated with glycine/taurine before secretion.

Secondary Bile Acids (examples)

Formed in colon by bacterial action (deconjugation + dehydroxylation): Deoxycholic acid (from cholic) and Lithocholic acid (from chenodeoxycholic). Teacher requires knowing both primary AND secondary examples.

Enterohepatic Circulation (Teacher Referenced Fig 18.11)

Most bile salts (conjugated + unconjugated) are reabsorbed in the terminal ileum → portal blood → liver → re-secreted into bile. Only ~5% excreted in feces. This conserves the bile salt pool.

🧬 Why Lipids Matter — and What Counts as a Dietary Lipid

Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules. Four functions from the slide: they are the major source of energy for the body; they provide the hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures; some fat-soluble vitamins have regulatory or coenzyme functions; and the prostaglandins and steroid hormones play major roles in the control of homeostasis.

Why this is a clinical topic

Deficiencies or imbalances of lipid metabolism lead to some of the major clinical problems encountered by physicians: atherosclerosis, diabetes and obesity.

Triacylglycerol (TAG)

Three fatty acids esterified to a glycerol backbone — the dominant dietary lipid.

The remainder of dietary lipid

Cholesterol · cholesteryl esters · phospholipids · nonesterified (free) fatty acids (FFA).

🍽️ Digestion of Dietary Lipids — Mouth to Small Intestine

Lipid digestion starts before the stomach and finishes in the duodenum. Each compartment has its own enzyme, and each enzyme has its own preferred substrate.

Mouth and stomach — the acid lipases

Lingual lipase and gastric lipase, with optimal pH values of 4 to 6. These acid lipases hydrolyse fatty acids from TAG, particularly those containing short- or medium-chain length (≤12 carbons) — which makes them the important lipases for milk fat in the neonate.

Duodenum — emulsification

Hydrophobic lipids are broken into fine lipid droplets by bile salts acting as a detergent plus mechanical mixing due to peristalsis. Bile salts are made in the liver, stored in the gallbladder, and are amphipathic derivatives of cholesterol.

Pancreatic lipase + colipase

TAG → 2-monoacylglycerol (2-MAG) + free fatty acids. Colipase is activated in the intestine by trypsin and restores the activity of lipase in the presence of inhibitory substances — including the bile salts that coat the droplet.

The other two pancreatic enzymes

Cholesteryl ester hydrolase (cholesterol esterase) → cholesterol + FFA; its activity is greatly increased in the presence of bile salts. Phospholipase A₂ — activated by trypsin, requires bile salts, and removes the fatty acid from carbon 2, leaving a lysophospholipid, which lysophospholipase then converts to a glycerylphosphoryl base for excretion or absorption.

Hormonal control — two peptide hormones

Cholecystokinin (CCK) — from cells in the mucosa of the lower duodenum and jejunum, released in response to the presence of lipids and partially digested proteins. It makes the gallbladder contract and release bile (a mixture of bile salts, phospholipids and free cholesterol), makes the exocrine pancreas release digestive enzymes, and decreases gastric motility so that stomach contents enter the small intestine more slowly.
Secretin — released in response to the low pH of chyme entering the intestine; it causes the pancreas to release a solution rich in bicarbonate that neutralises the pH so the pancreatic enzymes can work.

💊 Orlistat

An anti-obesity drug that inhibits gastric and pancreatic lipases, thereby decreasing fat absorption and resulting in weight loss.

🫧 Absorption by Enterocytes — Mixed Micelles and the Unstirred Water Layer

The primary products of lipid digestion are FFA, free cholesterol and 2-MAG. Together with bile salts and fat-soluble vitamins they form mixed micelles.

What a mixed micelle is

Disc-shaped clusters of a mixture of amphipathic lipids that coalesce with their hydrophobic groups on the inside and their hydrophilic groups on the outside — which is what makes them soluble in the aqueous environment of the intestinal lumen.

The unstirred water layer

The brush-border membrane is separated from the liquid contents of the lumen by an unstirred water layer that mixes poorly with the bulk fluid. The hydrophilic surface of the micelle is what carries the hydrophobic lipids across that layer to the membrane, where they are absorbed.

Four absorption facts worth memorising

• Short- and medium-chain fatty acids are water soluble and do NOT require mixed micelles.
• Bile salts are absorbed in the terminal ileum, with <5% lost in the faeces.
• Relative to other dietary lipids, cholesterol is only poorly absorbed by enterocytes.
• Ezetimibe further reduces cholesterol absorption in the small intestine.

📦 Resynthesis, Chylomicron Assembly and the Route into the Blood

Inside the enterocyte the products are put back together in the smooth endoplasmic reticulum — and the fate of a fatty acid depends entirely on its chain length.

Long-chain fatty acids

All long-chain FA entering the enterocyte are used to form TAG, phospholipids and cholesteryl esters. Long-chain FA are first activated by fatty acyl-CoA synthetase (thiokinase) using ATP; lysophospholipids are reacylated to phospholipids by acyltransferases.

Short- and medium-chain fatty acids

Not converted into their CoA derivatives — they are released to the portal circulation and carried with albumin to the liver.

How the chylomicron is built and where it goes

Newly resynthesised TAG and cholesteryl esters are very hydrophobic and would aggregate, so they are packaged as lipid droplets surrounded by a thin layer of phospholipids, nonesterified cholesterol and apolipoprotein B-48. That layer stabilises the particle and prevents the particles coalescing. They leave the enterocyte by exocytosis into the lacteals — the lymphatic vessels of the intestinal villi. Lymph after a lipid-rich meal looks milky and is called chyle; the particles are the chylomicrons. They follow the lymphatic system to the thoracic duct and are conveyed to the left subclavian vein, where they enter the blood.

🎯 Use by the Tissues — LPL, Free Fatty Acids, Glycerol and Remnants

Lipoprotein lipase (LPL)

Most chylomicron TAG is broken down in the capillary beds of skeletal and cardiac muscle and adipose tissue. LPL is synthesised and secreted primarily by adipocytes and muscle cells, then anchored to the luminal surface of endothelial cells in those capillaries. Products: FFA + glycerol.

Fate of free fatty acids

FFA may directly enter adjacent muscle cells and adipocytes, or be transported in the blood in association with serum albumin until taken up by cells. Most cells can oxidise FA to produce energy; adipocytes can also re-esterify FFA to TAG for storage.

Fate of glycerol

Taken up from the blood and phosphorylated by hepatic glycerol kinase to glycerol 3-phosphate, which can enter glycolysis or gluconeogenesis after oxidation to DHAP, or be used in TAG synthesis.

Fate of chylomicron remnants

After most TAG has been removed, the remnants — containing cholesteryl esters, phospholipids, apolipoproteins, fat-soluble vitamins and a small amount of TAG — bind receptors on the liver (apo E is the ligand) and are endocytosed. Intracellular remnants are hydrolysed; cholesterol and the nitrogenous bases of phospholipids (e.g. choline) can be recycled.

Functions of plasma FFA

Fatty acids are structural components of membrane lipids (phospholipids, glycolipids); attached to certain proteins they enhance the ability of those proteins to associate with membranes; they are precursors of the hormone-like prostaglandins; and esterified as TAG in white adipose tissue (WAT) they form the major energy reserve of the body.

🧫 When Lipid Digestion Fails — Cystic Fibrosis and Steatorrhoea

Cystic fibrosis (CF)

An autosomal-recessive disorder caused by mutations in the gene for the CF transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel on the epithelium of the pancreas, lungs, testes and sweat glands. Defective CFTR results in decreased secretion of chloride and increased uptake of sodium and water. In the pancreas, depletion of water at the cell surface produces thickened mucus that clogs the pancreatic ducts, preventing pancreatic enzymes from reaching the intestine — hence pancreatic insufficiency. Treatment: replacement of these enzymes and supplementation with fat-soluble vitamins.

Steatorrhoea

Lipid malabsorption — increased lipid in the faeces. It can be caused by disturbances in lipid digestion and/or absorption: for example CF (causing poor digestion) and short bowel syndrome (causing decreased absorption). Because short- and medium-chain FA can be taken up by enterocytes without the aid of mixed micelles, they are important in the medical nutrition therapy of malabsorption.

📏 Fatty Acid Structure, Saturation and the ω / n Numbering

A fatty acid is a hydrophobic hydrocarbon chain with a terminal carboxyl group whose pKa is about 4.8. At physiological pH the –COOH ionises to –COO⁻; this anionic group has an affinity for water, giving the fatty acid its amphipathic nature. For long-chain fatty acids (LCFA) the hydrophobic portion predominates, so they are highly water insoluble and must be transported in association with protein.

How they travel

More than 90% of the fatty acids found in plasma are in the form of fatty acid esters — primarily TAG, cholesteryl esters and phospholipids — contained in circulating lipoprotein particles. Free FFA travel bound to albumin, the most abundant protein in serum.

Saturation

Chains may contain no double bonds (saturated) or one or more (mono- or polyunsaturated). In humans, most are saturated or monounsaturated. Double bonds are nearly always cis, and a cis double bond makes the chain bend or kink. With two or more double bonds they are always spaced at three-carbon intervals.

Melting temperature

Adding double bonds decreases the melting temperature (T_m); increasing chain length increases it. Because membrane lipids typically contain LCFA, the presence of double bonds is what keeps those lipids fluid.

Numbering the carbons

Numbering starts at the carbonyl carbon = carbon 1. Carbon 2 is the α-carbon, carbon 3 the β-carbon, carbon 4 the γ-carbon. The carbon of the terminal methyl group is the ω-carbon, whatever the chain length. In humans, fatty acids with an even number of carbons (16, 18, 20) predominate; longer ones (>22 C) are found in the brain.

Reading 20:4(5,8,11,14) — arachidonic acid

The number before the colon is the number of carbons; the numbers after it are the positions of the double bonds relative to the carboxyl end. Arachidonic acid is 20 carbons long with four double bonds, between carbons 5–6, 8–9, 11–12 and 14–15. It is called an ω-6 fatty acid because the first double bond from the ω end is 6 carbons from that end, and equally an n-6 fatty acid because 20 − 14 = 6 = n. The ω and n designations are equivalent.

Essential fatty acids

Linoleic acid (18:2) — the precursor of the ω-6 fatty acid arachidonic acid, which is the substrate for prostaglandin synthesis. α-Linolenic acid (18:3) — the precursor of the ω-3 fatty acids important for growth and development. Both are dietary essentials in humans because we lack the enzymes needed to synthesise them; plants provide them. Arachidonic acid becomes essential if linoleic acid is deficient in the diet.

🗂️ Classification of Lipids

Lipids are defined by solubility, not structure, so they are classified by what they are made of — four groups.

1 · Simple lipids

Esters of a fatty acid with an alcohol. Fats — esters with glycerol (an oil is a fat that is liquid at room temperature). Waxes — esters with a long-chain monohydric alcohol: cerumen, sebum, the surface film of skin and hair.

2 · Complex lipids

Phospholipids (fatty acid + alcohol + phosphate + nitrogenous base), glycolipids (fatty acid + sphingosine + carbohydrate), and the rest — sulfolipids, amino lipids and lipoproteins.

3 · Precursor and derived lipids

Fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, ketone bodies, hydrocarbons, the lipid-soluble vitamins, micronutrients and the steroid hormones.

4 · Neutral lipids

The uncharged ones — acylglycerols, cholesterol, cholesteryl esters. Uncharged lipids form the core of droplets and lipoproteins; charged amphipathic lipids form the surface and the membranes.

Glycolysis & Gluconeogenesis

Frequently tested: irreversible enzymes (HK, PFK-1, PK), PFK-1 regulation by F-2,6-BP, gluconeogenesis ATP/GTP-requiring steps, glucose-6-phosphatase location

🍬 Glycolysis — The Full Picture

Glycolysis occurs in the cytoplasm of every cell. It converts one glucose molecule into two pyruvate molecules. It has two phases: an investment phase (uses 2 ATP) and a payoff phase (generates 4 ATP + 2 NADH). Net yield: 2 ATP + 2 NADH per glucose. Gross ATP yield: 4 ATP.

Three reactions are irreversible (driven strongly forward) — these are the regulatory steps:

1
Hexokinase/Glucokinase: traps glucose inside the cell as glucose-6-phosphate (G6P). Hexokinase is in all cells (inhibited by G6P). Glucokinase is in liver and beta-cells (not inhibited by G6P — acts as a glucose sensor).
2
PFK-1 (Phosphofructokinase-1): the main rate-limiting enzyme. Converts fructose-6-phosphate → fructose-1,6-bisphosphate. Main activator: fructose-2,6-bisphosphate (F-2,6-BP), also activated by AMP and ADP. Inhibited by ATP and citrate.
3
Pyruvate Kinase: converts phosphoenolpyruvate (PEP) → pyruvate, generating ATP. The liver isoform is inactivated by glucagon (via phosphorylation through the cAMP–PKA pathway).

Gross vs Net ATP — Frequently Tested

Glycolysis gross ATP = 4 ATP. Investment phase uses 2 ATP. Net = 2 ATP. In anaerobic glycolysis (no oxygen), the NADH cannot enter the mitochondria, so the overall yield stays at 2 net ATP.

🔃 Gluconeogenesis Bypass Reactions

Glycolysis enzyme (irreversible)	GNG bypass
Pyruvate Kinase	Pyruvate Carboxylase (biotin+ATP) + PEPCK (GTP)
PFK-1	Fructose-1,6-bisphosphatase
Hexokinase	Glucose-6-Phosphatase (liver/kidney only)

Cannot make glucose from fat!

Even-chain fatty acids → only acetyl-CoA → cannot enter gluconeogenesis. Acetyl-CoA cannot be converted to OAA in humans.

🔀 PFK-2 / FBP-2 — The Bifunctional Enzyme (Teacher Highlighted)

PFK-2/FBP-2 is a single protein with two enzyme activities — a kinase domain and a phosphatase domain. This is the enzyme that controls the level of fructose-2,6-bisphosphate (F-2,6-BP), the most potent activator of glycolysis.

PFK-2 (Kinase Domain)

Phosphorylates fructose-6-phosphate → fructose-2,6-bisphosphate (F-2,6-BP)
Active when the kinase domain is dephosphorylated (insulin state).
F-2,6-BP → activates PFK-1 → ↑glycolysis

FBP-2 (Phosphatase Domain)

Cleaves fructose-2,6-bisphosphate → fructose-6-phosphate + Pi
Active when phosphorylated by PKA (glucagon/fasting state). When kinase domain is ⊖ (phosphorylated), the phosphatase domain is active → ↓F-2,6-BP → promotes gluconeogenesis.

Key Regulation Logic

Fed state (insulin): PFK-2 kinase active → ↑F-2,6-BP → activates PFK-1 → ↑glycolysis, ⊖ FBP-1 → ↓gluconeogenesis
Fasting (glucagon): PKA phosphorylates PFK-2/FBP-2 → kinase domain ⊖ → FBP-2 phosphatase active → ↓F-2,6-BP → ⊖ PFK-1, activates FBP-1 → ↑gluconeogenesis
Only the kinase domain is regulated (by phosphorylation). The phosphatase domain activity is the inverse consequence.

Fructose-2,6-Bisphosphate Action

F-2,6-BP is the strongest known activator of PFK-1. It also inhibits FBP-1 (fructose-1,6-bisphosphatase). So the same molecule promotes glycolysis AND inhibits gluconeogenesis simultaneously.

🏃 Anaerobic Bypass — Cori Cycle / Lactate Shuttle (Teacher: "Know Both Names")

In anaerobic conditions (RBCs, exercising muscle, hypoxic tissue), pyruvate cannot enter the Krebs cycle. Instead, Lactate Dehydrogenase converts pyruvate → lactate, regenerating NAD⁺ to keep glycolysis running.

🏃 Cori Cycle (Lactic Acid Cycle)

Muscle: Glucose →glycolysis (+2 ATP) Pyruvate Lactate Dehydrogenase→ Lactate →blood Liver →gluconeogenesis (−6 ATP) Glucose →blood → muscle Muscle

↩ Net: muscle gains 2 ATP, liver spends 6 ATP — the metabolic burden is shifted from muscle to liver.

This transfers the metabolic burden from muscle to liver — muscle gets energy from glycolysis, liver handles the lactate.

Net Result of Cori Cycle

Net energy transfer: 2 ATP gained by muscle per glucose. 6 ATP spent by liver to convert 2 lactate → 1 glucose. The liver uses energy from fat oxidation to "subsidize" muscle glycolysis. Teacher noted: know the actual pathway name AND the common (Cori cycle) name.

GLUT Transporters — Insulin Independence

GLUT-1, GLUT-2, GLUT-3 are constitutively expressed and insulin-independent. GLUT-4 (muscle and adipose) is insulin-dependent — it moves to the cell surface only in response to insulin. This is why muscle cannot use glucose efficiently without insulin in Type 1 DM.

🔀 Glucose-6-Phosphate — The Central Metabolic Crossroads

Once glucose is phosphorylated, glucose-6-phosphate (G6P) stands at the junction of five pathways. Which way it flows depends on the tissue and the fed/fasting state.

⚗️ The Five Fates of G6P

G6P → Glycolysis → pyruvate + ATP → Glycogenesis → glycogen → Pentose phosphate pathway → Glucose-6-phosphatase → free glucose → Uronic acid (glucuronate) pathway

Why G6P is special

It is the one molecule shared by glycolysis, glycogen metabolism, the PPP, and the uronic acid pathway — and only liver and kidney can strip its phosphate (glucose-6-phosphatase) to release free glucose into the blood. Muscle lacks this enzyme, so muscle G6P can never leave the cell as glucose.

🚪 The GLUT Family — All Five

Transporter	Where	Km / behaviour	Why it matters
GLUT-1	Erythrocytes, brain, blood–brain barrier, cornea, placenta	Low Km — saturated at normal glucose	Guarantees a constant supply to tissues that must never run out. Insulin-independent
GLUT-2	Liver, pancreatic β cell, kidney, small intestine (basolateral)	High Km — uptake rises with blood glucose	The glucose sensor partner. Only takes up glucose when there is plenty. Insulin-independent
GLUT-3	Neurons	Very low Km — highest affinity	Neurons win the competition for glucose at low concentrations
GLUT-4	Skeletal and cardiac muscle, adipose tissue	Stored in intracellular vesicles	The only insulin-dependent transporter. Also recruited by exercise via AMPK, independently of insulin
GLUT-5	Small intestine (apical), sperm	Fructose transporter	Fructose absorption — facilitated diffusion, not sodium-coupled

💡 How to remember which is which

1 = always on (basic supply) · 2 = sensor (liver and β cell, the two glucokinase tissues) · 3 = brain (three syllables, neurons) · 4 = insulin (the only one that moves) · 5 = fructose (the only one that is not for glucose).

🔀 Hexokinase versus Glucokinase — And What GKRP Does

	Glucokinase (hexokinase IV)	Hexokinase I–III
Where	Liver and pancreatic β cells only	Most tissues, including muscle
K_m	High (~10 mM) — only active after a meal	Low (~0.1 mM) — works at all glucose levels
V_max	High — huge capacity	Low
Inhibited by	Fructose-6-phosphate via GKRP — not by its own product	Its own product, glucose-6-phosphate
Kinetics	Sigmoidal — maximises responsiveness to changing glucose	Hyperbolic (Michaelis–Menten)
Purpose	Clears the postprandial glucose load; induced by insulin	Guarantees the cell's own supply first

How GKRP works. Glucokinase regulatory protein sits in the hepatocyte nucleus. When fructose-6-phosphate is high — meaning the cell is not short of hexose — GKRP binds glucokinase and holds it inside the nucleus, away from its substrate. When glucose or fructose-1-phosphate rises, glucokinase is released and translocates to the cytosol. This is regulation by location, not by covalent modification — a mechanism worth naming in an exam.

Clinical links from this one enzyme

Heterozygous inactivating mutations → MODY 2: a mild, stable, non-progressive fasting hyperglycaemia, because the β cell simply resets its threshold upward. Activating mutations cause congenital hyperinsulinism instead.

🩸 The Red Cell Detour — 2,3-Bisphosphoglycerate

Erythrocytes have no mitochondria, so glycolysis to lactate is their only source of ATP. They also run a side loop that costs them ATP but serves the whole body.

1,3-Bisphosphoglycerate

→bisphosphoglycerate mutase

2,3-BPG

→2,3-BPG phosphatase

3-Phosphoglycerate

↻ The Rapoport–Luebering shunt — bypasses the phosphoglycerate kinase step, so no ATP is made from that molecule

2,3-BPG binds deoxyhaemoglobin in the central cavity between the β chains, stabilising the T (tense, low-affinity) state. The result is a rightward shift of the oxygen dissociation curve — haemoglobin gives up oxygen more readily to the tissues. It rises in chronic hypoxia, anaemia and at high altitude.

🎯 Two classic consequences

1. Fetal haemoglobin. HbF has γ chains instead of β, and binds 2,3-BPG poorly — so it holds oxygen more tightly and can pull it across the placenta from maternal blood.
2. Pyruvate kinase deficiency. The block is after the shunt, so 2,3-BPG accumulates. Patients tolerate their anaemia surprisingly well, because the raised 2,3-BPG delivers more oxygen per gram of haemoglobin. Contrast G6PD deficiency, where the problem is oxidative, not energetic.

📈 What 2,3-BPG does to the curve — the right shift

Left-shifted — P₅₀ 19 · holds on Normal — P₅₀ 26 Right-shifted by 2,3-BPG — P₅₀ 38 · lets go

Same haemoglobin, same four sites — only the affinity changes. 2,3-BPG locks the T state, so every pO₂ now carries less saturation: at the 40 mmHg of a resting tissue the normal curve is about 77% full and the right-shifted one about 54% — that gap is the extra oxygen handed over. Read it on the dotted half-saturation line: P₅₀ rises from 26 to 38 mmHg. HbF sits on the other side: it binds 2,3-BPG poorly, so it holds oxygen tightly enough to pull it from maternal blood across the placenta.

🧭 Transporter Locations and Transcriptional Control — Slide Detail

GLUT-1

Glucose uptake in red blood cells, choroid plexus (blood–brain barrier), kidneys, large intestine and placenta.

GLUT-2 · GLUT-3

GLUT-2 — fast uptake and efflux of glucose (fasting state): liver, pancreatic β cells, small intestine, kidneys. GLUT-3 — uptake in brain, kidneys, placenta.

GLUT-4 · GLUT-5

GLUT-4 — insulin-stimulated uptake: heart and skeletal muscle, adipose tissue. GLUT-5 — the primary fructose transporter: small intestine, testes.

SGLT

Na⁺- and ATP-dependent secondary active cotransport that moves glucose up its gradient while Na⁺ moves down; the gradient is created by Na⁺/K⁺-ATPase. Found in small intestine, renal tubules and choroid plexus.

Regulation of enzyme synthesis — the slow lever

Regular carbohydrate-rich meals or insulin administration increase transcription of the regulatory enzymes of the liver — glucokinase, PFK-1 and pyruvate kinase — by 10- to 20-fold, over hours to days. Insulin acts through the transcription factor SREBP-1c; glucose itself acts through the carbohydrate response element-binding protein (ChREBP), which is adaptively expressed in the liver in response to high dietary carbohydrate loads. High plasma glucagon exerts the opposite effect — during fasting, or in diabetes mellitus.

☠️ Arsenate — The Poison That Steals Glycolysis' First ATP

Arsenate (AsO₄³⁻) is close enough to phosphate that glyceraldehyde-3-phosphate dehydrogenase accepts it in place of Pi. The 1-arseno-3-phosphoglycerate produced hydrolyses spontaneously instead of handing its high-energy bond to ADP.

The consequence, and the contrast with arsenite

Glycolysis keeps running and NADH is still made, but the substrate-level phosphorylation at phosphoglycerate kinase is abolished — the pathway is uncoupled from its own ATP. Do not confuse it with arsenite, which binds the –SH groups of lipoic acid and so poisons pyruvate dehydrogenase and α-ketoglutarate dehydrogenase instead.

Glycogen Metabolism

Frequently tested: glycogen phosphorylase (AMP-activated, ATP-inhibited, PLP cofactor), hormonal cascade, glycogen storage diseases (von Gierke, McArdle, Hers, Cori, Andersen)

🍡 Synthesis & Breakdown

Glycogen Synthase: active when dephosphorylated (insulin promotes dephosphorylation via protein phosphatase-1). Glycogen Phosphorylase: active when phosphorylated (glucagon and epinephrine trigger cAMP → PKA → phosphorylation).

Liver Glucose-6-Phosphatase releases free glucose into the blood — muscle lacks this enzyme and therefore cannot directly raise blood glucose. Muscle glycogen is used only for the muscle itself.

Muscle Glycogen — Exam-Tested Distinctions

Muscle glycogen phosphorylase is activated by calcium ions (Ca²⁺) released during muscle contraction and by AMP — NOT by glucagon (glucagon receptors are absent on muscle). Muscle glycogen breakdown does not directly increase plasma glucose. These three facts are tested together.

🏥 Glycogen Storage Diseases

Disease	Enzyme Missing	Key Features
Von Gierke (I)	Glucose-6-Phosphatase	Severe hypoglycemia, hepatomegaly, lactic acidosis
Pompe (II)	Lysosomal α-Glucosidase	Glycogen in lysosomes; cardiomegaly
Anderson (IV)	Branching Enzyme	Liver cirrhosis (amylopectin-like glycogen accumulates). Rare but directly tested in 2022.
McArdle (V)	Muscle Glycogen Phosphorylase	Cramping on exercise

🔋 Glycogenolysis — Full Detail (Teacher Notes)

Glycogenolysis: breakdown of glycogen → releases glucose. Hormones that trigger it:

Liver: Glucagon and Epinephrine → GCPR → adenylate cyclase → ↑cAMP → PKA → phosphorylates glycogen phosphorylase kinase → activates Glycogen Phosphorylase
Muscle: Epinephrine (via cAMP/PKA) AND Ca²⁺ (released from SR during muscle contraction) → Calmodulin → phosphorylase kinase → Glycogen Phosphorylase

Glycogen phosphorylase cleaves α-1,4 glycosidic bonds from the non-reducing end, releasing glucose-1-phosphate (which → glucose-6-phosphate). It cannot cross α-1,6 branch points — a separate debranching enzyme (two activities: transferase + glucosidase) handles branch points, releasing free glucose at the branch.

PKA Simultaneously Activates Glycogenolysis AND Inhibits Glycogen Synthesis

When glucagon/epinephrine → PKA → simultaneously:
1. Phosphorylates glycogen phosphorylase kinase → activates Glycogen Phosphorylase (↑breakdown)
2. Phosphorylates Glycogen Synthase → INACTIVATES it (↓synthesis)
This coordinates: during fasting → break down glycogen AND stop making it.

🌱 Glycogenin — Where the Very First Glucose Goes

Glycogen synthase can only extend an existing chain — it cannot start one from nothing. The problem of the first residue is solved by a protein that acts as its own primer.

UDP-glucose

→glycogenin · autoglucosylation

Glucose attached to a tyrosine residue of glycogenin

→glycogenin extends the chain itself

~8 glucose primer

→

Glycogen synthase takes over

↻ Glycogenin stays buried at the core of every finished glycogen granule — each granule has one

Two exam-ready details

The attachment is to a tyrosine hydroxyl, not serine or threonine. And glycogenin is both the enzyme and the substrate — a rare case of autocatalysis you can be asked to name.

📞 Three Signals, One Enzyme — Calcium, cAMP and AMP

Glycogen phosphorylase is switched on by three quite different messages, each meaning something different to the body. Understanding which signal reaches which tissue answers a whole family of questions.

🧠 Glucagon → cAMP → PKA
Liver only — muscle has no glucagon receptor. Means "blood sugar is low, release glucose for everyone else"

😰 Epinephrine → cAMP → PKA
Liver and muscle. Means "emergency" — liver exports glucose, muscle keeps it for itself

💪 Ca²⁺–calmodulin
Muscle contraction itself activates phosphorylase kinase directly, because calmodulin is the δ subunit of phosphorylase kinase. Means "I am contracting, I need fuel now" — no hormone required

⚡ AMP allosteric
Muscle only. Activates dephosphorylated phosphorylase b directly. Means "ATP is nearly gone". ATP and glucose-6-phosphate oppose it

🎯 The liver/muscle contrast that gets examined

Liver phosphorylase a is inhibited by glucose — free glucose binds and makes the enzyme a better substrate for protein phosphatase 1, switching it off. The liver stops breaking down glycogen the moment blood glucose is adequate. Muscle phosphorylase is not inhibited by glucose at all, but is inhibited by ATP and glucose-6-phosphate: muscle responds to its own energy state, not to the blood.

📦 How Much Glycogen, and Where — The Numbers

Liver glycogen

Stabilises blood glucose, especially in the early periods of fasting. It can keep blood glucose up for less than 24 hours. About 100 g — a maximum of 10% of the fresh weight of the adult liver in a well-nourished state.

Muscle glycogen

A fuel reserve for ATP synthesis during contraction. About 400 g — which is only 1–2% of muscle mass at rest. Most of the body's glycogen is in muscle, simply because muscle mass far exceeds liver mass.

Structure in numbers

A branched polysaccharide built solely from α-D-glucose: α(1→4) chains with α(1→6) branch points every 8–14 glucosyl residues. A single molecule can contain up to 55,000 glucosyl residues. It exists as large spherical cytoplasmic granules that also contain the enzymes of glycogen synthesis and degradation.

How the stores move

Hepatic glycogen rises in the well-fed state and falls during fasting. Muscle glycogen does not change over several days of fasting, decreases only slightly with prolonged (weeks) fasting, and is resynthesised to replace what intense exercise depleted. Synthesis and degradation run continuously; the amount stored is set by the difference in rate between the two.

Why branching at all

The branched form predominates (the unbranched equivalent is amylose in starch) for two reasons: better solubility, and more non-reducing ends — which means both faster synthesis and faster degradation.

Pentose Phosphate Pathway (PPP)

Frequently tested: NADPH for anabolism, ribose-5-P for nucleotides, G6PD deficiency & favism, transketolase vs transaldolase

🔋 What the PPP Does

The Pentose Phosphate Pathway (PPP) runs in the cytoplasm and serves two main purposes: (1) producing NADPH — the cell's main reducing agent for biosynthesis and antioxidant defense, and (2) producing ribose-5-phosphate — needed to build nucleotides (DNA and RNA building blocks).

Rate-Limiting Enzyme of the PPP = G6PD — Exam Tested

G6PD (Glucose-6-Phosphate Dehydrogenase) is the first and rate-limiting enzyme of the PPP. It catalyses Glucose-6-phosphate + NADP⁺ → 6-Phosphogluconolactone + NADPH. This is the only source of NADPH in red blood cells (which have no mitochondria).

Correct pairing (Q29c): PPP — Cytoplasm — Rate-limiting enzyme: G6PD

G6PD Deficiency (Favism)

X-linked recessive. Most common enzyme defect worldwide (~400 million affected). Without G6PD, cells cannot make NADPH → cannot regenerate reduced glutathione (GSH). Glutathione's role in red blood cells is to neutralize hydrogen peroxide (H₂O₂) — without it, H₂O₂ oxidizes hemoglobin → Heinz bodies form → red blood cells are destroyed → hemolytic anemia. Triggered by fava beans, primaquine, dapsone, and infections.

Glutathione Composition — Exam Tested

Glutathione is a tripeptide made of three amino acids: Glutamate + Cysteine + Glycine (remember: GCG or "gamma-Glu-Cys-Gly"). Cysteine's thiol group (–SH) is the active reducing part. This composition is directly asked in exams.

🔀 Transketolase — Exam Detail

Transketolase is a key enzyme of the non-oxidative branch of the PPP. It requires TPP (thiamine pyrophosphate, from vitamin B1) as its cofactor. Transketolase transfers 2-carbon units between sugar phosphates, interconverting 5-carbon, 7-carbon, 4-carbon, and 6-carbon sugars. This is a common exam trap: students confuse transketolase with transaldolase. Transaldolase does NOT require any coenzyme — it works without a cofactor. Only transketolase needs TPP.

Exam Trap

"Transaldolase requires TPP" → FALSE. Transaldolase requires no coenzyme. Transketolase requires TPP (vitamin B1). This distinction appears directly in exam questions.

💨 Nitric Oxide (NO) & NADPH Oxidase

NO Synthesis
Enzyme: NO Synthase (NOS)
Cofactors: NADPH, FMN, FAD, heme, tetrahydrobiopterin (BH₄)
Note: NOS uses BH₄ directly as cofactor — it does NOT require tetrahydropterin reductase (2023 exam)

NOS isoforms:

eNOS (endothelial): Ca²⁺/calmodulin-dependent → vasodilation
nNOS (neuronal): Ca²⁺/calmodulin-dependent → neurotransmitter
iNOS (inducible): Ca²⁺-independent; induced by TNF-α, endotoxins → antimicrobial (large amounts)

NADPH Oxidase & Respiratory Burst
Located in leukocyte membrane. Contains flavocytochrome (FAD + heme). Uses NADPH to reduce O₂ → superoxide (O₂⁻) → respiratory burst → kills bacteria.

Chronic Granulomatous Disease (CGD)

Genetic NADPH oxidase deficiency → phagocytes cannot produce superoxide → cannot kill catalase-positive bacteria (Staphylococcus, Aspergillus) → severe recurrent infections + granuloma formation. X-linked (most common form).

NO Functions

Vasodilator (eNOS) · Neurotransmitter (nNOS) · Inhibits platelet aggregation · Antimicrobial (iNOS in macrophages). Half-life: 3–10 seconds. Reacts with O₂ and superoxide → nitrates/nitrites.

Krebs Cycle & Respiratory Chain

Frequently tested: reversible vs irreversible reactions, malonate inhibits succinate dehydrogenase, anaplerotic reactions, redox steps, respiratory chain complex order, inhibitors & uncouplers

🔢 Full Krebs Cycle — All 8 Steps, Enzymes, Substrates, Reaction Types

Step	Substrate → Product	Enzyme	Cofactors	Reaction Type	Energy yield
1	Acetyl-CoA + OAA → Citrate	Citrate Synthase	None	Condensation (Transferase)	—
2	Citrate → Isocitrate	Aconitase	Fe–S cluster	Isomerization (via cis-aconitate)	—
3 ★	Isocitrate → α-Ketoglutarate + CO₂	Isocitrate Dehydrogenase	NAD⁺, Mn²⁺	Oxidative decarboxylation (rate-limiting ★)	NADH
4 ★	α-Ketoglutarate → Succinyl-CoA + CO₂	α-Ketoglutarate DH	TPP, lipoic acid, CoA, FAD, NAD⁺	Oxidative decarboxylation (rate-limiting ★)	NADH
5	Succinyl-CoA → Succinate	Succinyl-CoA Synthetase	GDP/ADP + Pi	Substrate-level phosphorylation	GTP (or ATP)
6	Succinate → Fumarate	Succinate Dehydrogenase	FAD	Oxidation (dehydrogenation)	FADH₂
7	Fumarate → Malate	Fumarase	None	Hydration	—
8	Malate → OAA	Malate Dehydrogenase	NAD⁺	Oxidation	NADH

Per Turn Summary: 3 NADH + 1 FADH₂ + 1 GTP + 2 CO₂

Irreversible steps (★): Citrate Synthase (step 1), Isocitrate DH (step 3), α-KG DH (step 4).
Inhibited by fluoroacetate: Fluoroacetate → fluorocitrate → inhibits Aconitase → citrate accumulates.

🔗 Anaplerotic Reactions — Replenishing Krebs Intermediates

Anaplerotic reactions add intermediates back into the Krebs cycle to keep it running when intermediates are withdrawn for biosynthesis. They are the opposite of cataplerotic reactions.

Reaction	Source	Krebs Intermediate Added	Enzyme
Pyruvate + CO₂ → OAA	Pyruvate (from glucose)	Oxaloacetate (OAA)	Pyruvate Carboxylase (biotin, ATP)
Glutamate → α-Ketoglutarate	Pro, His, Arg, Gln (via Glu)	α-Ketoglutarate	Glutamate Dehydrogenase
Propionyl-CoA → Succinyl-CoA	Met, Thr, Val, Ile (and odd-chain FAs)	Succinyl-CoA	Propionyl-CoA carboxylase + methylmalonyl-CoA mutase (B12)
Fumarate from urea cycle	Argininosuccinate → Arginine + Fumarate	Fumarate	Arginase / Argininosuccinate lyase
Phenylalanine/Tyrosine → Fumarate	Phe, Tyr catabolism	Fumarate	Fumarylacetoacetase

Amino Acids Entering Krebs — Teacher Highlighted

Pro · His · Arg · Gln →via Glutamate α-Ketoglutarate

Met · Thr · Val · Ile →via propionyl-CoA Succinyl-CoA

Phe · Tyr → Fumarate

Asp · Asn →transamination OAA

OAA → PEP (Gluconeogenesis) — The Key Connection

When the Krebs cycle runs LOW on OAA (e.g. during fasting), OAA is diverted to gluconeogenesis via PEPCK (OAA → PEP). This is why: whole cycle must be intact to withdraw intermediates for GNG. High acetyl-CoA (from FA oxidation) activates Pyruvate Carboxylase → replenishes OAA.

⚙️ Complex V (ATP Synthase) — Matrix + Membrane Proteins (Teacher Noted)

Complex V (ATP Synthase) consists of two major domains — this is NOT part of the ETC electron carriers, but is part of oxidative phosphorylation:

F₀ (membrane domain)

Embedded in the inner mitochondrial membrane. Contains the proton channel — H⁺ flow through F₀ rotates the c-ring. Inhibited by oligomycin (blocks the proton channel).

F₁ (matrix domain)

Projects into the mitochondrial matrix. Contains the catalytic β-subunits that synthesize ATP from ADP + Pi. The α₃β₃ hexamer rotates → mechanical force → ATP synthesis. Also called the "stalk".

⊖ Malonate — Competitive Inhibitor of Complex II ★ asked 2018, 2025

Malonate is a structural analog of succinate → competitive inhibitor of Succinate Dehydrogenase (Complex II). Teacher specifically mentioned this. Unlike Complex I (rotenone), Complex III (antimycin A), and Complex IV (cyanide/CO), malonate specifically targets Complex II. Malonate is NOT a physiological inhibitor — it's used experimentally.

Oxidative Phosphorylation — Essential Understanding

Coupling: ETC pumps H⁺ → builds electrochemical gradient → H⁺ flows back through Complex V → ATP synthesized. Uncouplers (DNP, UCP-1) dissipate gradient → O₂ still consumed, no ATP made → heat. Oligomycin blocks F₀ → gradient cannot be discharged → ETC slows (backed up).

🔄 The Krebs Cycle (Tricarboxylic Acid / TCA Cycle)

The Krebs cycle occurs in the mitochondrial matrix. Each turn processes one acetyl-CoA (2 carbons) and produces: 3 NADH, 1 FADH₂, 1 GTP (via Succinyl-CoA Synthetase), and 2 CO₂. The CO₂ is the carbon waste released at the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase steps.

Three irreversible steps (the regulatory checkpoints):

Citrate Synthase: first step, condenses acetyl-CoA + oxaloacetate (OAA) → citrate.
Isocitrate Dehydrogenase: primary rate-limiting enzyme; produces first NADH + first CO₂. Activated by ADP and Ca²⁺; inhibited by ATP and NADH.
α-Ketoglutarate Dehydrogenase: also rate-limiting; produces second NADH + second CO₂; requires TPP, lipoic acid, CoA, FAD, NAD⁺ (same cofactors as PDC). Activated by Ca²⁺; inhibited by NADH and succinyl-CoA.

Fluoroacetate — Aconitase Inhibitor

Fluoroacetate (a poison found in some plants) is converted inside cells to fluorocitrate, which inhibits Aconitase — the enzyme that converts citrate → isocitrate. This blocks the entire Krebs cycle. Citrate accumulates. This is a directly tested exam fact.

When Does Acetyl-CoA Oxidation Increase?

The cycle runs faster when the cell needs more energy — specifically when the ATP/ADP ratio decreases (ADP increases). High ADP signals low energy → activates isocitrate dehydrogenase → more Krebs cycle activity → more acetyl-CoA oxidized.

⚡ Respiratory Chain (Electron Transport Chain) ★ asked 2018

Complex I (NADH Dehydrogenase, contains FMN) → CoQ → Complex II (Succinate Dehydrogenase, contains FAD) → CoQ → Complex III (Cytochrome bc1) → Cytochrome c → Complex IV (Cytochrome c Oxidase) → O₂ → H₂O

Complex V (ATP Synthase): F₀ subunit is embedded in the inner mitochondrial membrane and acts as the proton channel; F₁ subunit sits in the matrix and synthesizes ATP.

Cytochrome c — Electrons Only ★ asked 2025

Cytochrome c transfers electrons ONLY — it does NOT transfer protons/hydrogen ions. This was explicitly tested in the 2025 exam. Cytochrome c moves between Complex III and Complex IV carrying only electrons via its heme iron (Fe³⁺ ↔ Fe²⁺). Coenzyme Q (ubiquinone) transfers both electrons and protons.

Inhibitor	Blocks	Consequence
Rotenone / Amytal	Complex I	NADH cannot donate electrons; ETC stops
Antimycin A	Complex III	Electron flow blocked at Q cycle
Cyanide (CN⁻), CO, Azide	Complex IV	Cannot transfer electrons to O₂; rapid cellular death
Oligomycin	F₀ of ATP Synthase	Proton channel blocked; ATP synthesis stops

🌡️ Uncoupling — Energy as Heat Instead of ATP

Normally, the proton gradient built by the ETC drives ATP synthesis through Complex V. Uncouplers short-circuit this by allowing protons to cross the inner mitochondrial membrane through an alternative route — dissipating the gradient as heat instead of ATP.

2,4-DNP (dinitrophenol): an artificial chemical uncoupler. It is a lipid-soluble weak acid that carries H⁺ across the inner membrane. Was historically misused as a weight-loss drug — extremely dangerous.
UCP-1 (Thermogenin): the natural uncoupling protein found in brown adipose tissue. Activated by fatty acids; generates body heat. Especially important in newborns (who lack shivering reflex) and in hibernating animals.

🔀 The Amphibolic Role of the Citric Acid Cycle

"Amphibolic" = the cycle works in both directions of metabolism at once: it is catabolic (oxidises acetyl-CoA to make NADH/FADH₂/ATP) and anabolic (its intermediates are pulled out as building blocks for biosynthesis).

Intermediate withdrawn	Used to build (anabolic role)
Citrate	Exported to cytosol → acetyl-CoA → fatty acid & cholesterol synthesis
α-Ketoglutarate	→ Glutamate → other amino acids, GABA, purines
Succinyl-CoA	→ Heme (porphyrin synthesis)
Oxaloacetate	→ Aspartate (→ pyrimidines, urea cycle) and → glucose (gluconeogenesis via PEPCK)

Cataplerosis = withdrawing intermediates for biosynthesis. Anaplerosis = refilling them so the cycle keeps turning (chiefly pyruvate carboxylase: pyruvate + CO₂ → OAA). The balance of the two is what "amphibolic" captures.

One-line answer

The Krebs cycle is amphibolic because it both generates energy (catabolic) and supplies carbon skeletons for fat, amino acid, heme, and glucose synthesis (anabolic) — so its intermediates must be constantly topped up by anaplerotic reactions.

🎡 The Pyruvate Dehydrogenase Complex — Five Cofactors, Three Enzymes

The link between glycolysis and the Krebs cycle is a single, irreversible, tightly regulated step: pyruvate + CoA + NAD⁺ → acetyl-CoA + CO₂ + NADH. Because it is irreversible, no carbon that passes it can ever return to glucose. That one fact explains why fatty acids cannot be converted to glucose.

Pyruvate

PDH complex⟶TPP · lipoate · CoA · FAD · NAD⁺

Acetyl-CoA + CO₂ + NADH

Subunit	Cofactor	From which vitamin
E1 — pyruvate decarboxylase	Thiamine pyrophosphate (TPP)	B1 — thiamine
E2 — dihydrolipoyl transacetylase	Lipoic acid and Coenzyme A	— and B5 (pantothenate)
E3 — dihydrolipoyl dehydrogenase	FAD and NAD⁺	B2 (riboflavin) and B3 (niacin)

💡 "Tender Loving Care For Nancy"

TPP · Lipoic acid · CoA · FAD · NAD⁺ — and these are exactly the same five cofactors used by α-ketoglutarate dehydrogenase and by branched-chain α-keto acid dehydrogenase. All three are α-keto acid dehydrogenase complexes with the same architecture, which is why thiamine deficiency hits all three at once (see beriberi) and why one MSUD variant is thiamine-responsive.

Regulation — covalent plus allosteric

▲ PDH ACTIVE (dephosphorylated)

PDH phosphatase is activated by Ca²⁺ (muscle contraction) and by insulin in adipose tissue. High NAD⁺, CoA, ADP and pyruvate favour the active state.

▼ PDH INACTIVE (phosphorylated)

PDH kinase is activated by the products — acetyl-CoA and NADH — and by ATP. This is why fatty acid oxidation shuts down glucose oxidation: the acetyl-CoA it generates switches PDH off. Also allosterically inhibited directly by acetyl-CoA and NADH.

☠️ Arsenic and the lipoate trap

Arsenite binds the two adjacent thiol groups of lipoic acid, locking it in a stable ring and inactivating every lipoate-dependent enzyme at once — PDH, α-ketoglutarate dehydrogenase and BCKD. The block is identical in effect to severe thiamine deficiency, which is why arsenic poisoning mimics beriberi. Details in arsenic poisoning, and see PDH deficiency for the inherited version.

🔌 Chemiosmotic Theory, Uncouplers and Inhibitors

Peter Mitchell's chemiosmotic hypothesis: the energy of electron transport is not stored in a chemical intermediate but in an electrochemical proton gradient across the inner mitochondrial membrane. Protons pumped into the intermembrane space return through ATP synthase, and it is that flow — the proton-motive force — that drives ATP synthesis.

Why the inner membrane must be impermeable

The gradient only exists if protons cannot leak back any other way. Anything that puts a hole in that membrane destroys ATP synthesis while electron transport continues — the energy comes out as heat instead. That single sentence defines an uncoupler.

🔥 Uncouplers

2,4-Dinitrophenol and aspirin in overdose — lipid-soluble weak acids that ferry protons across the membrane. Oxygen consumption rises, ATP falls, temperature rises. Historically used for weight loss, and lethally dangerous.

Physiological uncoupler: thermogenin (UCP-1) in brown adipose tissue — deliberate heat production in the newborn.

🛑 Electron transport inhibitors

Complex I: rotenone, amytal, piericidin.
Complex II: malonate (competitive with succinate).
Complex III: antimycin A.
Complex IV: cyanide, carbon monoxide, azide, H₂S.
ATP synthase: oligomycin.
ANT translocase: atractyloside.

These stop oxygen consumption AND ATP synthesis together — the opposite pattern to an uncoupler.

🎯 How to tell them apart in one question

Ask what happens to oxygen consumption. Inhibitor → O₂ use falls, ATP falls. Uncoupler → O₂ use rises, ATP falls, heat rises. Oligomycin blocks ATP synthase, so O₂ use falls too — but adding an uncoupler after oligomycin restores oxygen consumption, because electrons no longer need ATP synthase to complete the circuit. That experiment is a favourite.

🚌 The Two Shuttles — Why NADH Is Worth Different Amounts

The inner mitochondrial membrane is impermeable to NADH. Cytosolic NADH made in glycolysis therefore has to hand its electrons across indirectly, and the route chosen determines the ATP yield.

🐴 MALATE–ASPARTATE SHUTTLE

Liver, kidney, heart. Oxaloacetate is reduced to malate in the cytosol, carried in, and re-oxidised to regenerate NADH inside the mitochondrion. Aspartate carries the amino group back out.

Yield: 2.5 ATP per cytosolic NADH. Fully reversible, so it also works in reverse when needed.

🦋 GLYCEROL-3-PHOSPHATE SHUTTLE

Skeletal muscle and brain. Cytosolic NADH reduces DHAP to glycerol-3-phosphate; a FAD-linked enzyme on the outer face of the inner membrane re-oxidises it, delivering electrons to coenzyme Q and bypassing complex I.

Yield: 1.5 ATP per cytosolic NADH. Irreversible, but faster — which suits tissues that need speed over efficiency.

Putting the numbers together

Complete oxidation of one glucose yields 32 ATP when the malate–aspartate shuttle is used and 30 ATP with the glycerol-3-phosphate shuttle — the difference is exactly 2 × 1 ATP from the two cytosolic NADH of glycolysis. Per molecule: NADH = 2.5 ATP, FADH₂ = 1.5 ATP, because FADH₂ enters at coenzyme Q and skips the complex I proton pump.

⚙️ Inside the Pyruvate Dehydrogenase Complex

The five cofactors act as a fixed relay, and the intermediate never leaves the complex — which is why there are no side products and the rate is so high.

1
TPP decarboxylates pyruvate, giving hydroxyethyl-TPP and CO₂ (E1).
2
The two-carbon unit is transferred and oxidised onto lipoamide → acetyl-lipoamide.
3
E2 (dihydrolipoyl transacetylase) hands the acetyl group to CoA → acetyl-CoA, leaving dihydrolipoamide.
4
E3 (dihydrolipoyl dehydrogenase) re-oxidises it through FAD and passes the electrons to NAD⁺, which delivers them to the respiratory chain.

One drug worth naming

Dichloroacetate inhibits PDH kinase, so the complex stays dephosphorylated and active — which is why it has been used in lactic acidosis. Arsenite does the opposite kind of damage, binding the –SH groups of lipoic acid.

Biochemical Logic & Redox

Frequently tested: biochemical reasoning over memorization, redox logic, pathway locations, why specific reactions occur — emphasized by the teacher

⚡ Redox Logic

Oxidation = loss of electrons. Reduction = gain of electrons.

NAD⁺ is the OXIDIZED form → accepts electrons → becomes NADH (reduced).
NADH is oxidized back to NAD⁺ in the ETC.

Exam trap: "The enzyme reduces NADH" → FALSE. NADH is already reduced.

Classic False Statements

• "NADH is the oxidized form" → FALSE (NAD⁺ is oxidized)
• "Cytochrome c transfers protons" → FALSE (transfers only electrons — tested 2025)
• "Alcohol changes NADPH/NADP⁺ ratio" → FALSE (changes NADH/NAD⁺)

📊 Pathway Locations — Quick Reference

Process	Location	Rate-limiting enzyme
Glycolysis	Cytosol	PFK-1
Gluconeogenesis	Cytosol + mitochondria	PEPCK / Pyruvate Carboxylase
Krebs cycle	Mitochondrial matrix	Isocitrate DH / α-KG DH
β-oxidation (fatty acid breakdown)	Mitochondrial matrix	Acyl-CoA Dehydrogenase
Fatty acid synthesis (lipogenesis)	Cytosol — mainly liver, adipose, lactating mammary gland. NOT muscle.	Acetyl-CoA Carboxylase (ACC)
Pentose Phosphate Pathway (PPP)	Cytosol	G6PD
Urea cycle	Mitochondria (CPS-I, OTC) + Cytosol (rest)	CPS-I
Heme synthesis	Mitochondria (first + last steps) + Cytosol (middle steps). NOT in mature red blood cells.	ALA Synthase
Cholesterol synthesis	Cytosol + ER (endoplasmic reticulum)	HMG-CoA Reductase

Lipogenesis Tissues — Exam Tested

Fatty acid synthesis does NOT occur in skeletal muscle (muscle lacks the necessary enzyme levels). It occurs mainly in: liver, adipose tissue, and lactating mammary gland. This is a common exam question.

🧪 Lipogenesis Cofactors (What Is and Is NOT Required)

Fatty acid synthesis requires: ATP (for acetyl-CoA carboxylase reaction), NADPH (for reductive steps — provided by the PPP and malic enzyme), Biotin (covalently attached to acetyl-CoA carboxylase), HCO₃⁻ (bicarbonate, as CO₂ source for malonyl-CoA), and Mn²⁺.

⚗️ Lipogenesis Cofactors — What Is and Is NOT Required

Required for lipogenesis?	Molecule	Why
✅ YES	NADPH	Used in every reductive step (2 NADPH consumed per 2-carbon addition). Comes from PPP and malic enzyme.
✅ YES	ATP	Required by Acetyl-CoA Carboxylase (ACC) to make malonyl-CoA.
✅ YES	Biotin	Covalently attached cofactor of ACC — carries CO₂ during malonyl-CoA synthesis.
✅ YES	HCO₃⁻ (bicarbonate)	CO₂ source for carboxylation by ACC.
❌ NO	NADH	NADH is used in β-OXIDATION (breakdown) and the Krebs cycle. It is NOT used in fatty acid SYNTHESIS. This is a common exam trap.
❌ NO	PLP (Vitamin B6)	PLP is the cofactor for aminotransferases and glycogen phosphorylase — not involved in lipogenesis.
❌ NO	FAD	FAD is used in β-oxidation (acyl-CoA dehydrogenase) — not in synthesis.

Exam Trap (Q33): Lipogenesis requires NADPH, NOT NADH

NADH and NADPH look similar but are used in opposite processes:
• NADPH → used in reductive biosynthesis (lipogenesis, steroid synthesis, glutathione reduction)
• NADH → used in energy generation (glycolysis, Krebs cycle, β-oxidation products)
A question listing ATP + Biotin + NADH as lipogenesis requirements → choose NADPH instead of NADH. Only ATP and Biotin (and NADPH) are correct.

Cytosolic Acetyl-CoA — How It Gets There

Acetyl-CoA is made in mitochondria but cannot cross the inner mitochondrial membrane. It is exported as citrate instead. Citrate exits the mitochondria → in the cytosol, citrate lyase cleaves it back to acetyl-CoA + OAA. Conditions promoting this export: high citrate, high ATP, high-carbohydrate diet, inhibition of isocitrate dehydrogenase (which makes citrate accumulate). Citrate also directly activates acetyl-CoA carboxylase (ACC) in the cytosol — a feedforward signal.

Malic Enzyme

Malic enzyme converts malate → pyruvate while reducing NADP⁺ → NADPH. It belongs to the class of Oxidoreductases (class 1). It is an important source of cytosolic NADPH for lipogenesis. This classification is directly tested.

🏥 Tissue Specificity — WHY Each Tissue Performs Its Pathway

Teacher repeatedly stated this is very high-yield: know not just WHERE pathways occur but WHY that tissue performs them.

Tissue	Specialist pathway(s)	Why / Unique feature
Liver	Gluconeogenesis, ketogenesis, urea cycle, cholesterol synthesis, bile acid synthesis, VLDL secretion, albumin, clotting factors	Central hub. Has glucose-6-phosphatase → only organ (with kidney) that releases free glucose to blood. Exclusive ketone body PRODUCER (cannot use them — lacks thiophorase).
Skeletal Muscle	Glycolysis, glycogen (for itself), BCAA catabolism, creatine phosphate	No G-6-Pase → cannot raise blood glucose. No glucagon receptors. No lipogenesis. BCAA (Leu, Ile, Val) metabolised mainly in muscle.
Brain	Glucose oxidation (primary); ketone bodies after 2–3 weeks fasting	GLUT-1 (insulin-independent). Absolute glucose requirement normally. After prolonged fasting adapts to ketones → spares muscle protein.
Adipose Tissue	TAG storage, lipolysis (HSL), FA esterification	No glycerol kinase → releases glycerol (not reused). GLUT-4 (insulin-dependent) → fat storage only when insulin present.
RBCs	Anaerobic glycolysis, PPP (NADPH)	No mitochondria → no TCA, no β-oxidation, no oxidative phosphorylation. Only ATP = glycolysis. Only NADPH = PPP. 2,3-BPG production is unique to RBCs.
Kidney	Gluconeogenesis (prolonged fasting), glutamine catabolism, arginine synthesis, vitamin D activation	Only site of 1α-hydroxylase (25-OH-D₃ → calcitriol). Only site of arginine synthesis (from citrulline + aspartate).
Intestinal Enterocytes	Chylomicron assembly, glutamine as primary fuel	Use glutamine (not glucose) as primary fuel in fasting. SGLT-1 (Na⁺-dependent glucose/galactose), GLUT-5 (fructose), GLUT-2 (portal exit).

📋 Pathway–Location–Rate-Limiting Enzyme Pairings — Direct Exam Format

The exam directly tests whether you can correctly match a pathway with its subcellular location AND its rate-limiting enzyme. This exact format appeared on the 2025 exam (Q29).

Pathway	Location	Rate-Limiting Enzyme	Exam tip
Glycolysis	Cytoplasm	PFK-1	✅ Correct pairing — tested directly in 2025
Gluconeogenesis	Cytoplasm + mitochondria	FBP-1 (or PEPCK as key bypass enzyme)	Bypasses PFK-1; G6Pase final step in ER
β-Oxidation	Mitochondrial matrix	CPT-I (carnitine shuttle, rate-limiting entry step)	❌ ACC is NOT the rate-limiting enzyme of β-oxidation — ACC is the rate-limiting enzyme of LIPOGENESIS (synthesis). Common trap.
Lipogenesis (FA Synthesis)	Cytoplasm	ACC	ACC makes malonyl-CoA — first committed step of FA synthesis
PPP (HMP Shunt)	Cytoplasm	G6PD	✅ Correct pairing — tested directly in 2025
Krebs Cycle (TCA)	Mitochondrial matrix	Isocitrate DH (primary) + α-KG DH	Both are rate-limiting; Isocitrate DH is the main pacemaker
Urea Cycle	Mitochondria (steps 1–2) + Cytoplasm (steps 3–5)	CPS-I	Requires NAG (obligatory activator)
Cholesterol synthesis	ER (smooth)	HMG-CoA Reductase	Target of statins
Heme synthesis	Mitochondria (1st + last steps) + Cytoplasm (middle)	ALA Synthase	Inhibited by heme (feedback); requires PLP (B6)
ETC / Oxidative phosphorylation	Inner mitochondrial membrane	Complex I (NADH entry point)	O₂ = final electron acceptor; ATP synthase = Complex V

The #1 Exam Trap in Pathway Pairings

β-oxidation rate-limiting enzyme = CPT-I (controls entry of fatty acids into mitochondria via the carnitine shuttle).
ACC is the rate-limiting enzyme of LIPOGENESIS — not β-oxidation. These two are commonly swapped in exam questions. If a question pairs β-oxidation with ACC, that pairing is INCORRECT.

🔥 Biological Oxidation — The Four Enzyme Types definitions asked

"Biological oxidation" = controlled removal of electrons/hydrogen from fuel molecules. Different enzyme classes handle oxygen and electrons in different ways — a favourite definition question.

Enzyme type	What it does with O₂ / electrons	Example
Dehydrogenase	Removes H (2e⁻ + H⁺) and passes it to NAD⁺/FAD — no O₂ used directly	Lactate dehydrogenase, succinate dehydrogenase
Oxidase	Transfers electrons directly to O₂, making H₂O or H₂O₂ (does NOT incorporate O into substrate)	Cytochrome c oxidase (→H₂O); xanthine oxidase, MAO (→H₂O₂)
Oxygenase	Incorporates O₂ into the substrate. Monooxygenase adds one O atom (other → H₂O); dioxygenase adds both	CYP450 (monooxygenase); homogentisate oxidase (dioxygenase)
Peroxidase	Uses H₂O₂ as the oxidant (detoxifies it)	Glutathione peroxidase, catalase (2 H₂O₂ → 2 H₂O + O₂)

Key distinction

Oxidase vs oxygenase: an oxidase gives electrons to O₂ but does not put oxygen atoms into the product; an oxygenase actually inserts O atoms into the substrate.

🚚 Transport of Cytosolic (Extramitochondrial) NADH — The Two Shuttles

NADH made in the cytosol (from glycolysis) cannot cross the inner mitochondrial membrane. Two shuttles carry its electrons in — and they yield different amounts of ATP.

🟢 Malate–Aspartate Shuttle

Cytosolic NADH → OAA → malate (crosses membrane) → regenerates NADH inside the matrix.
Tissues: liver, heart, kidney.
Yield: ~2.5 ATP per NADH (enters at Complex I). Reversible.
Enzymes: malate dehydrogenase + AST.

🔵 Glycerol-Phosphate Shuttle

Cytosolic NADH → DHAP → glycerol-3-phosphate → drops electrons onto FAD at the inner membrane.
Tissues: muscle, brain, brown fat.
Yield: ~1.5 ATP per NADH (enters at CoQ, bypasses Complex I). Irreversible, fast.
Enzymes: cytosolic + mitochondrial G3P dehydrogenase.

Why the ATP differs

The malate–aspartate shuttle delivers electrons as NADH (Complex I → 2.5 ATP); the glycerol-phosphate shuttle delivers them as FADH₂ (CoQ → 1.5 ATP). This is why the ATP yield of glycolysis is sometimes quoted as slightly different between tissues.

⚖️ NAD⁺/NADH vs NADP⁺/NADPH · NADPH Generators

Same chemistry, opposite jobs. The single extra phosphate on NADP is a "label" that routes it to biosynthesis instead of energy.

	NAD⁺ / NADH	NADP⁺ / NADPH
Main role	Catabolism — carries electrons to the ETC to make ATP	Anabolism — donates electrons to biosynthesis
Usually found as	Oxidised (NAD⁺) — ready to accept	Reduced (NADPH) — ready to donate
Used in	Glycolysis, Krebs, β-oxidation	Fatty-acid/cholesterol/steroid synthesis, glutathione reduction, CYP450, respiratory burst

Where NADPH comes from (generators)

1. Pentose phosphate pathway — G6PD + 6-phosphogluconate dehydrogenase (the main source). 2. Malic enzyme — malate → pyruvate. 3. Cytosolic isocitrate dehydrogenase. 4. Folate pathway (minor). In RBCs the PPP is the only NADPH source — which is why G6PD deficiency causes haemolysis.

🔋 Endergonic & Exergonic Reactions — Energy Coupling

Exergonic (ΔG < 0)

Releases energy, spontaneous. e.g. ATP hydrolysis, glucose oxidation. Catabolic reactions are typically exergonic.

Endergonic (ΔG > 0)

Requires energy input, non-spontaneous. e.g. building macromolecules, gluconeogenesis. Anabolic reactions are typically endergonic.

The cell drives endergonic reactions by coupling them to a strongly exergonic one — almost always ATP hydrolysis (ΔG ≈ −30 kJ/mol). If the combined ΔG is negative, the coupled reaction proceeds. ΔG (actual) depends on concentrations; ΔG°′ is the standard value. A negative ΔG says a reaction can go, not how fast — that's set by the enzyme (activation energy).

High-energy phosphate compounds

PEP > 1,3-BPG > creatine phosphate > ATP — each can donate its phosphate to the one below it. This is why substrate-level phosphorylation (PEP → pyruvate, 1,3-BPG → 3-PG) can make ATP directly.

Fed/Fasting State & Alcohol

Frequently tested: well-fed vs fasting characteristics, glucose-alanine cycle, alcohol metabolism (alcohol dehydrogenase) — teacher said "do not forget alcohol"

⚠️ Teacher (Kłos) Exam Topics — From Notes

Explicitly listed by the teacher as exam topics:
• Iron metabolism (see Iron & Lab Tests section)
• Alcohol metabolism via dehydrogenase (ADH + ALDH pathway below)
• Conjugated/unconjugated/urobilinogen hyperbilirubinemia — genetic, erythrocyte enzyme (see Heme section)
• GLUT-4 is insulin-dependent (Skeletal muscle + adipose)
• Skeletal muscles can use ketones earlier than brain, then switch to FAs to preserve ketones for brain
• Exceptions from active when phosphorylated — know enzymes that are INACTIVE when phosphorylated

🍽️ Fed vs Fasting — Complete Overview

Parameter	Fed (Insulin dominant)	Fasting (Glucagon dominant)
Glycolysis	↑ Active	↓ Suppressed
Gluconeogenesis	↓ Suppressed	↑ Active
Glycogen synthesis	↑ Active	↓ Off
Glycogenolysis	↓ Off	↑ Active
Lipogenesis	↑ Active	↓ Off
Lipolysis	↓ Off	↑ Active
β-oxidation	↓ (malonyl-CoA blocks carnitine)	↑ Active
Ketogenesis	↓ Off	↑ Active
Protein synthesis	↑ Active	↓ Muscle breakdown ↑

⚡ Phosphorylation Rules — Active vs Inactive (Teacher Tip)

Most metabolic enzymes are active in their dephosphorylated form (fed/insulin state) and become inactive when phosphorylated during fasting. Key exceptions — enzymes that are ACTIVE when phosphorylated (catabolic enzymes turned on during fasting):

Enzyme	Phosphorylated State	Active State
Glycogen Synthase	INACTIVE (↓glycogen synthesis)	Dephosphorylated (fed/insulin)
Pyruvate Kinase (liver)	INACTIVE (↓glycolysis)	Dephosphorylated (fed/insulin)
HMG-CoA Reductase	INACTIVE (↓cholesterol synthesis)	Dephosphorylated (fed)
Acetyl-CoA Carboxylase (ACC)	INACTIVE (↓FA synthesis)	Dephosphorylated (fed/insulin)
PFK-2 (kinase domain)	INACTIVE kinase → FBP-2 active	Dephosphorylated = PFK-2 active (glycolysis)

The Pattern

Glucagon → PKA → phosphorylation. During fasting: anabolic enzymes get phosphorylated = inactive (no synthesis during fasting). Catabolic enzymes get phosphorylated = active (break down stores for energy).

🍺 Alcohol Metabolism

Ethanol → Acetaldehyde (Alcohol Dehydrogenase, cytosol, NAD⁺→NADH) → Acetate (Aldehyde Dehydrogenase, mitochondria, NAD⁺→NADH) → enters blood.

High NADH/NAD⁺ ratio = root cause of all alcohol metabolic effects:
• ↑NADH → pyruvate → lactate → lactic acidosis
• ↑NADH → OAA → malate → OAA depleted → gluconeogenesis blocked → hypoglycemia
• β-oxidation blocked (needs NAD⁺) → fat accumulates → fatty liver (steatosis)
• ↑Acetyl-CoA → ketogenesis → alcoholic ketoacidosis

Exam Trap

Alcohol changes NADH/NAD⁺ ratio (not NADPH/NADP⁺).

🍔 Diet Composition — High-Carbohydrate vs High-Fat

	High-carbohydrate diet	High-fat diet
Dominant hormone	Insulin	Lower insulin, relatively ↑ glucagon
Main pathways ON	Glycolysis, glycogen synthesis, lipogenesis (excess carbs → fat)	β-oxidation, ketogenesis, gluconeogenesis (from protein/glycerol)
Fuel used	Glucose	Fatty acids + ketone bodies
Effect on lipids	↑ VLDL/TAG if excessive (de novo lipogenesis)	Mild ketosis; can ↑ LDL depending on fat type
Cholesterol synthesis	↑ (insulin activates HMG-CoA reductase)	Dietary cholesterol feedback-inhibits synthesis

Key idea

A high-fat, low-carb intake mimics a fasting-like metabolic pattern (fat oxidation + ketones), while excess carbohydrate is converted to fat via lipogenesis. The body's response is set by the insulin:glucagon ratio, not just calories.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🔀 Two Details the Question Papers Keep Returning To Past Q

In the post-absorptive state, phosphorylated means off — with three exceptions

Glucagon and adrenaline phosphorylate a long list of enzymes and inactivate them: glycogen synthase, acetyl-CoA carboxylase, pyruvate dehydrogenase, pyruvate kinase. Three are switched ON by the same phosphorylation — glycogen phosphorylase, phosphorylase kinase and hormone-sensitive lipase. Learn the three exceptions and the rest of the pattern follows: fuel is being released, not stored.

The liver's two-step response, in order

First glycogenolysis — and only the liver can release free glucose, because only the liver (and kidney) has glucose-6-phosphatase. Then gluconeogenesis: glucagon lowers fructose-2,6-bisphosphate, which relieves the inhibition of fructose-1,6-bisphosphatase. Alongside these, β-oxidation and ketone body synthesis rise.

Uncontrolled diabetes = the post-absorptive state, exaggerated Past Q

Insulin is absent or ineffective, so every fasting programme runs at once and unopposed even after a meal: unrestrained lipolysis, high plasma free fatty acids, maximal β-oxidation, maximal gluconeogenesis and glycogenolysis despite hyperglycaemia, and overproduction of ketone bodies. Underused chylomicrons and VLDL add hypertriacylglycerolaemia. This is why the metabolic disturbances of diabetes read like starvation happening in the middle of plenty.

Lactate, fed versus fasting, in one line

Fed: lactate is the end product of glycolysis in red cells, white muscle fibres and the gut, and the liver oxidises it or uses its carbon for lipogenesis. Fasting: the same lactate becomes one of the main substrates for gluconeogenesis. Red cells always finish at lactate because pyruvate oxidation is mitochondrial and they have no mitochondria.

Fatty Acid Metabolism

Frequently tested: β-oxidation (and odd-chain → succinyl-CoA needing B12), fatty acid synthesis enzyme order, ketone bodies, ACC regulation, arachidonic acid & eicosanoids

🏭 Fatty Acid Synthesis

Builds fatty acids (mainly palmitate, 16:0) de novo from acetyl-CoA in the cytoplasm. Rate-limiting enzyme: Acetyl-CoA Carboxylase (acetyl-CoA → malonyl-CoA). Activated by insulin/citrate; inhibited by palmitoyl-CoA + glucagon.

🟢 Synthesis (lipogenesis)

Where: cytoplasm
Carrier: ACP / fatty acid synthase
Reducing power: NADPH (from PPP + malic enzyme)
Needs: ATP, HCO₃⁻, biotin, Mn²⁺
State: fed / high insulin

🔵 β-Oxidation (breakdown)

Where: mitochondrial matrix
Carrier: carnitine shuttle (CPT-I entry)
Electron acceptors: NAD⁺ & FAD
Product: acetyl-CoA + NADH + FADH₂
State: fasting / high glucagon

Key distinction

Synthesis uses NADPH in the cytoplasm; β-oxidation uses NAD⁺/FAD in the mitochondrial matrix. Malonyl-CoA (from ACC) blocks CPT-I, so the two are never on at once.

💡 Ketone Bodies — What & Why

What they are: small, water-soluble fuel molecules made by the liver from acetyl-CoA (from β-oxidation) when carbohydrate is scarce. They are exported in the blood to other tissues.

Their purpose: during fasting/starvation or uncontrolled diabetes, they provide an alternative energy source — crucially for the brain, which cannot oxidise fatty acids directly. Muscle and heart also use them. This spares glucose and limits muscle protein breakdown. Note: the liver makes them but cannot use them (it lacks thiophorase/SCOT).

🍋 Ketone Bodies — The Three Types

Acetoacetate

True ketone; the primary one synthesised; used by brain/muscle/heart.

β-Hydroxybutyrate

Major ketone in blood during starvation (most abundant).

Acetone

From spontaneous decarboxylation of acetoacetate. Volatile; exhaled. No metabolic role.

Diabetic Ketoacidosis (DKA)

High ketones → metabolic acidosis + Kussmaul breathing + ketonuria.

👆 Tap for full disease card

Why β-Hydroxybutyrate Dominates in Blood During Starvation — Teacher Required

β-Oxidation produces large amounts of NADH. High NADH/NAD⁺ ratio drives β-Hydroxybutyrate Dehydrogenase to convert acetoacetate → β-hydroxybutyrate. Therefore, β-hydroxybutyrate is the dominant circulating ketone body in starvation and DKA. Acetone is formed by spontaneous decarboxylation of acetoacetate (not enzymatic) — it is exhaled.

⚖️ Lipolysis vs Lipogenesis vs β-Oxidation — Definitions (Teacher Notes)

Term	Definition	Key Enzyme	Location
Lipolysis	Breakdown of TAGs (triacylglycerols) → Free fatty acids (FFAs) + glycerol	Hormone-sensitive Lipase (HSL) in adipose; LPL in capillaries	Cytoplasm of adipocytes
β-Oxidation	Breakdown of fatty acids → Acetyl-CoA (2-carbon units are progressively cleaved)	Acyl-CoA Dehydrogenase (first step)	Mitochondrial matrix
Lipogenesis	Synthesis of fatty acids de novo from acetyl-CoA (building FAs from scratch)	Acetyl-CoA Carboxylase (ACC) (rate-limiting)	Cytoplasm
TAG Synthesis	Assembly of triglycerides from glycerol-3-phosphate + fatty acyl-CoAs	Acyltransferases	ER (liver, adipose)

Lipolysis and β-Oxidation Are Reciprocal with Lipogenesis (Teacher Note)

Lipolysis/β-oxidation and lipogenesis are reciprocal processes — they are regulated so that one is active while the other is off. ACC produces malonyl-CoA → malonyl-CoA inhibits CAT-I (carnitine acyltransferase I) → blocks FA entry into mitochondria for β-oxidation. So when FA synthesis is ON (fed state), β-oxidation is automatically OFF. Repeatable process: each enzyme set repeats its reaction cycle on successive 2-carbon units.

🌊 Eicosanoids

Eicosanoids are 20-carbon signalling molecules made from arachidonic acid, which is released from membrane phospholipids by Phospholipase A₂.

From there, two pathways branch off:

🔵 COX pathway

Cyclooxygenase (COX) →
Prostaglandins (inflammation, pain),
Thromboxane A₂ (platelet aggregation, vasoconstriction),
Prostacyclin (opposes thromboxane).
Blocked by NSAIDs (e.g. aspirin).

🟠 Lipoxygenase pathway

Lipoxygenase →
Leukotrienes — drive bronchoconstriction in asthma and allergic responses.

Exam note

Prostanoids (prostaglandins + thromboxanes + prostacyclin) and leukotrienes are both subclasses of eicosanoids. Arachidonic acid is an ω-6, tetraenoic (4 double bonds), 20-carbon acid derived from linoleic acid.

📋 Important Fatty Acids — Reference Table

Rows highlighted in yellow are the highest-yield for the exam.

Fatty acid	Notation	Double bonds	Class / note
Stearic acid	18:0	0 (saturated)	★ No C9 double bond — classic exam trap
Oleic acid	18:1	1 (Δ9)	★ ω-9, monounsaturated
Arachidonic acid	20:4	4 (Δ5,8,11,14)	★ ω-6, precursor of eicosanoids
Linoleic acid	18:2	2	★ ω-6, essential (diet)
α-Linolenic acid	18:3	3	★ ω-3, essential (diet)
Palmitic acid	16:0	0 (saturated)	Main product of FA synthesis
Palmitoleic acid	16:1	1 (Δ9)	ω-7, monounsaturated

Quick reminders

Essential (must be eaten): linoleic (ω-6) & α-linolenic (ω-3). Notation 18:1 = 18 carbons, 1 double bond. More double bonds → more fluid, lower melting point.

🧬 Fatty Acid Physical Properties — Teacher Notes

Property	Effect on Fatty Acid
Length of chain	Longer chain → MORE SOLID (more Van der Waals interactions) → higher melting point
Number of double bonds	More double bonds → MORE FLUID, ↓melting point (cis double bonds create kinks that prevent tight packing)
Degree of saturation (more H+)	More saturated → MORE SOLID, ↑melting point (e.g., animal fats are solid, plant oils are liquid)

Exam favourite: "Which fatty acid has NO double bond at C9?" ★ asked 2024/25

Answer: stearic acid (18:0) — fully saturated, so no double bond anywhere. The trap options oleic (18:1) and palmitoleic (16:1) BOTH have their double bond at Δ9 (C9). So the "no C9 double bond" one is the saturated acid.

🧴 Phospholipids — Structure, Types & Function

Phospholipids are amphipathic (water-loving head + fat-loving tails) — the reason they spontaneously form the cell membrane bilayer. Most are built on a glycerol backbone; one class is built on sphingosine.

Phospholipid	Head group / note	Role
Phosphatidylcholine (lecithin)	Choline — most abundant membrane PL	Membranes; bile; lung surfactant
Phosphatidylethanolamine (cephalin)	Ethanolamine	Inner membrane leaflet
Phosphatidylserine	Serine (negative)	Flips to outer leaflet in apoptosis ("eat me" signal); activates clotting
Phosphatidylinositol (PIP₂)	Inositol	Source of second messengers IP₃ + DAG (via phospholipase C)
Cardiolipin	Two PG units — inner mitochondrial membrane	Needed for ETC function; antibody target in antiphospholipid syndrome
Sphingomyelin	Sphingosine backbone (not glycerol)	Myelin sheath; accumulates in Niemann-Pick disease

🫁 Lung surfactant

Mainly dipalmitoyl-phosphatidylcholine (DPPC) — lowers alveolar surface tension. Deficiency in premature babies → neonatal respiratory distress syndrome.

✂️ Phospholipases

PLA₂ releases arachidonic acid (→ eicosanoids); phospholipase C releases IP₃ + DAG (signalling).

💧 Sources of "Active" Glycerol (Glycerol-3-Phosphate)

The active form of glycerol is glycerol-3-phosphate — the backbone onto which fatty acids are loaded to build triacylglycerols and phospholipids. There are two ways to make it, and which one a tissue uses is exam-relevant.

Source	Enzyme	Tissue
Free glycerol + ATP	Glycerol kinase	Liver, kidney (NOT adipose)
DHAP (from glycolysis) + NADH	Glycerol-3-P dehydrogenase	Adipose tissue (its only route)

Why adipose depends on glucose

Adipose tissue lacks glycerol kinase, so it can only make glycerol-3-phosphate from DHAP — which comes from glucose (glycolysis). This is why fat storage needs insulin/glucose, and why the glycerol released during lipolysis goes to the liver (which has glycerol kinase) rather than being reused locally.

🔬 The Other Three Oxidations — Peroxisomal, α and ω

Mitochondrial β-oxidation cannot handle every fatty acid. Three specialised routes deal with the ones that are too long, branched in the wrong place, or blocked at both ends.

🅿️ PEROXISOMAL β-OXIDATION

For very-long-chain fatty acids — 22 carbons and above

The first enzyme is acyl-CoA oxidase, not acyl-CoA dehydrogenase. It is FAD-dependent but passes its electrons directly to O₂, forming H₂O₂ — which catalase destroys
So the energy of that first step is released as heat, not captured as FADH₂. Peroxisomal oxidation is deliberately inefficient
Chain shortening stops at about C8; the shortened acyl group is then conjugated to carnitine and finishes in the mitochondrion
Peroxisomes also make plasmalogens (needed for myelin) and bile acid intermediates

🩺 Failure of the whole organelle → Zellweger syndrome. Failure of just the VLCFA transporter → adrenoleukodystrophy.

▼

🅰️ α-OXIDATION

For β-methyl branched fatty acids — phytanic acid

A methyl group on the β carbon physically blocks β-oxidation — there is nowhere to put the hydroxyl. Peroxisomal phytanoyl-CoA hydroxylase removes one carbon from the α position, shifting the methyl group to the α carbon where it is no longer in the way. No ATP is generated by this step; it is purely enabling. Deficiency causes Refsum disease.

▼

Ω ω-OXIDATION

A minor rescue route in the endoplasmic reticulum

Cytochrome P450 hydroxylates the terminal (ω) methyl carbon, which is then oxidised to a carboxyl group — producing a dicarboxylic acid that can be β-oxidised from either end. Normally minor, it becomes important when β-oxidation is blocked: dicarboxylic aciduria is the laboratory signature of an MCAD or similar defect. See MCAD deficiency.

🔥 Ketone Bodies — Making Them, Using Them, and the One Enzyme the Liver Lacks

Ketone bodies let the liver export the acetyl-CoA it cannot burn. They are made only in liver mitochondria, and used by almost every other tissue — the asymmetry is the whole point.

🏭 KETOGENESIS — liver mitochondria only

2 acetyl-CoA → acetoacetyl-CoA (thiolase)
+ acetyl-CoA → HMG-CoA (HMG-CoA synthase — the rate-limiting step, mitochondrial isoform)
HMG-CoA → acetoacetate + acetyl-CoA (HMG-CoA lyase)
Acetoacetate → β-hydroxybutyrate (reduced, favoured when NADH is high — so this is the predominant ketone body in DKA)
Acetoacetate → acetone spontaneously — volatile, exhaled, gives the fruity breath, and cannot be metabolised

⚡ KETOLYSIS — everywhere except liver

β-hydroxybutyrate → acetoacetate (reversing the dehydrogenase)
Acetoacetate + succinyl-CoA → acetoacetyl-CoA, by thiophorase (SCOT)
Acetoacetyl-CoA → 2 acetyl-CoA → Krebs cycle
The liver has no thiophorase — so it physically cannot take its own product back. This guarantees the ketone bodies reach the tissues that need them

🎯 Cytosolic versus mitochondrial HMG-CoA

The same molecule is an intermediate in two unrelated pathways. Mitochondrial HMG-CoA → ketone bodies. Cytosolic HMG-CoA → cholesterol. Different synthase isoforms, different compartments, completely different fates — and a very common exam question.

Why the nitroprusside urine test underestimates severity

The standard dipstick detects acetoacetate strongly and acetone weakly, but does NOT detect β-hydroxybutyrate at all. In severe ketoacidosis the high NADH/NAD⁺ ratio pushes nearly everything to β-hydroxybutyrate — so the urine test can look mild while the patient is critically acidotic. Worse, as treatment works and the ratio normalises, β-hydroxybutyrate converts back to acetoacetate and the test appears to get worse while the patient improves.

🏗️ Fatty Acid Synthase and the Road to Triacylglycerol

Acetyl-CoA carboxylase sets the rate, but the chain itself is built by one large multienzyme protein, and the finished fatty acid still has to be esterified before it can be stored.

Fatty acid synthase — one protein, seven activities

A dimeric multienzyme complex: each monomer carries all the catalytic activities plus the acyl carrier protein (ACP) domain. ACP holds a phosphopantetheine arm — the same business end as coenzyme A, derived from pantothenic acid (B5) — which swings the growing chain from one active site to the next. Two carbons are added per cycle from malonyl-CoA, and the product released is palmitate (16:0).

Esterification — the last step

Glycerol-3-phosphate is acylated twice to give phosphatidate, the branch point of the whole field: phosphatidate phosphohydrolase removes the phosphate to give diacylglycerol, a third acyl group makes triacylglycerol, while phosphatidate retained as such goes on to the phospholipids.

Why palmitate is where synthesis stops

Fatty acid synthase releases the chain at 16 carbons. Anything longer, and every double bond, is added afterwards by elongases and desaturases of the endoplasmic reticulum — and human desaturases cannot work beyond carbon 9, which is precisely why linoleic and α-linolenic acid must come from the diet.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

💧 The Three Lipases, Perilipin and the Two Lipolyses

The three lipases and the gatekeeper

Stored TAG is dismantled in order: adipose triglyceride lipase (ATGL) → TAG to diacylglycerol; hormone-sensitive lipase → DAG to monoacylglycerol; monoacylglycerol lipase → the last fatty acid + glycerol. The droplet is coated by perilipin: unphosphorylated, it shields the fat from the lipases; once PKA phosphorylates it, it changes shape and lets HSL reach the surface. Perilipin is the reason hormones can switch lipolysis on so sharply.

Everything that turns lipolysis up or down

Up (through cAMP → PKA): adrenaline and noradrenaline, glucagon, ACTH, MSH, TSH, growth hormone, vasopressin. Permissive: glucocorticoids (new lipase protein, a cAMP-independent route) and thyroid hormone. Down: insulin — it activates phosphodiesterase so cAMP falls, and activates a lipase phosphatase — plus nicotinic acid, prostaglandin E₁ and ketone bodies, all of which lower cAMP.

Where LPL sits, and how the tissues compete Past Q

LPL is anchored to the capillary endothelium by heparan sulfate chains — an injection of heparin releases it into the blood. It needs phospholipid and apo C-II as cofactors and is inhibited by apo A-II and apo C-III. Heart LPL has a Km about a tenth of the adipose enzyme, so when plasma TAG falls in fasting the heart still extracts fatty acids while adipose tissue cannot — the delivery is redirected to where it is needed.

What the two lipolyses actually achieve

Intravascular: lipoprotein TAG is degraded so that parenchymal cells can take up the fatty acids; reaction with LPL strips 70–90% of a chylomicron's TAG and returns apo C to HDL while apo E is retained — the remnant is about half the diameter of the parent and relatively enriched in cholesterol. Intracellular: generates free fatty acids and glycerol for release (white adipose tissue) or for use (other tissues). Triacylglycerols cannot cross membranes, which is why both steps must exist.

🍋 Regulation of Ketogenesis — Three Gates

Regulation of ketogenesis — three gates in series Past Q

Gate 1 · in adipose tissue. Ketosis cannot happen unless circulating free fatty acids rise, so everything that controls lipolysis controls ketogenesis first; the liver extracts about 30% of the fatty acids passing through it, fed or fasting. Gate 2 · at the mitochondrial door. Once inside the hepatocyte a fatty acid is either esterified to TAG and phospholipid or β-oxidised, and CPT-I decides which. In the fed state malonyl-CoA (made by acetyl-CoA carboxylase) inhibits CPT-I, oxidation is low and the fat leaves as VLDL. In starvation acyl-CoA inhibits acetyl-CoA carboxylase, malonyl-CoA falls, CPT-I opens and oxidation rises. Gate 3 · inside the mitochondrion. The acetyl-CoA produced is partitioned between the Krebs cycle and ketogenesis (HMG-CoA synthase, then HMG-CoA lyase, which splits HMG-CoA into acetoacetate + acetyl-CoA). As serum fatty acids rise, proportionally more goes to ketones.

The arithmetic that explains why the liver bothers

Complete oxidation of one mole of palmitate in the liver yields about 106 ATP; if the same carbon leaves as acetoacetate the liver keeps only about 26 ATP, and as 3-hydroxybutyrate about 21. Ketogenesis is therefore a device that lets the liver oxidise very large amounts of fatty acid without being limited by its own tightly coupled oxidative phosphorylation — it exports the unburnt energy to everyone else. Falling mitochondrial oxaloacetate (drawn into gluconeogenesis, and pulled toward malate by the high NADH/NAD⁺ ratio of active β-oxidation) is what tips the partition; acetyl-CoA activating pyruvate carboxylase partly offsets it, but in starvation and untreated diabetes the compensation fails and ketone bodies are overproduced.

🧴 The Phospholipid Families

🧱Glycerophospholipids — built on phosphatidic acid

Phosphatidylcholine (lecithin) — the most abundant in membranes and lipoproteins · phosphatidylethanolamine · phosphatidylserine — normally on the inner leaflet; when it flips outward it flags a cell for phagocytosis · phosphatidylinositol — the source of the second messengers IP₃ and DAG · cardiolipin (diphosphatidylglycerol) — found only in the inner mitochondrial membrane, where it is needed for the respiratory-chain complexes.

🌿Plasmalogens — an ether instead of an ester

Carbon 1 carries an ether-linked alkenyl chain. They make up a large share of the phospholipid of heart muscle and myelin, and platelet-activating factor belongs to this family. They are made in peroxisomes — which is why peroxisomal diseases such as Zellweger syndrome are also plasmalogen-deficiency diseases.

🧠Sphingolipids — built on sphingosine, not glycerol

Sphingomyelin is the only phospholipid of this class: one fatty acid amide-linked to sphingosine, plus phosphocholine. It is concentrated in the nervous system and myelin. Replace the phosphocholine with sugars and you get the glycolipids — cerebrosides and gangliosides — whose lysosomal breakdown fails in the sphingolipidoses.

The common design, and one digestion note

Every phospholipid has two long hydrophobic tails and one polar head — in glycerophospholipids both tails are fatty acids, in sphingolipids one is the sphingosine chain. That is what makes them amphipathic and able to form the bilayer. Like all complex lipids they must be emulsified by bile salts and hydrolysed before absorption, and phospholipid is itself an obligatory cofactor for lipoprotein lipase.

💧 Building a Triacylglycerol — and the Third Oxidation Route

The phosphatidate route — liver and adipose tissue

Fatty acids are first activated to acyl-CoA. Then: glycerol-3-phosphate → (glycerol-3-phosphate acyltransferase) lysophosphatidate → (1-acylglycerol-3-phosphate acyltransferase) phosphatidate → (phosphatidate phosphohydrolase) 1,2-diacylglycerol → (diacylglycerol acyltransferase, DGAT) triacylglycerol. Phosphatidate and diacylglycerol are also the branch point to the phospholipids.

The monoacylglycerol route — the enterocyte

The 2-monoacylglycerol left by pancreatic lipase is re-acylated directly: monoacylglycerol acyltransferase → 1,2-diacylglycerol → DGAT → TAG, packaged into chylomicrons. It bypasses glycerol-3-phosphate entirely, so the gut needs no glycerol kinase.

ω-oxidation

A cytochrome P450 of the smooth ER hydroxylates the terminal methyl carbon and oxidises it to a second carboxyl group, giving a dicarboxylic acid that β-oxidation then shortens from either end. Normally negligible, it takes over when β-oxidation is blocked — which is why dicarboxylic aciduria marks a fatty-acid oxidation defect such as MCAD deficiency. (α-oxidation handles branched chains such as phytanic acid; its failure is Refsum disease.)

Cholesterol & Bile Acids

Frequently tested: HMG-CoA reductase (rate-limiting), cholesterol as substrate of 7α-hydroxylase & ACAT, synthesis location (smooth ER + cytosol), isoprenoid units

🧱 Cholesterol Synthesis

Synthesis pathway (all from acetyl-CoA, in cytoplasm + smooth ER):

Acetyl-CoA Thiolase + HMG-CoA synthase→ HMG-CoA HMG-CoA Reductase→rate-limiting · NADPH · statin target Committed stepMevalonate →×3 ATP Isoprenoid units → Squalene → Cholesterol

Rate-limiting enzyme: HMG-CoA Reductase (HMG-CoA → mevalonate), the target of statins (competitive inhibitors). Cholesterol is then a substrate of 7α-hydroxylase (→ bile acids) and ACAT (→ cholesteryl esters for storage).

🚢 Lipoproteins — Full Detail

Lipoprotein	Made in	Main cargo	Key apoprotein	Fate
Chylomicrons	Intestine (absorptive phase only)	Dietary (exogenous) triglycerides	ApoB-48, ApoC-II, ApoE	LPL strips TG → chylomicron remnants → liver (recognized by ApoE)
VLDL	Liver	Endogenous triglycerides	ApoB-100, ApoC-II, ApoE	LPL strips TG → IDL → LDL
IDL	From VLDL	Cholesterol + remaining TG	ApoB-100, ApoE	Further TG removal → LDL
LDL	Derived from IDL	Cholesterol (main carrier)	ApoB-100 only	Taken up by LDL receptors (via ApoB-100)
HDL	Liver + Intestine	Reverse cholesterol transport	ApoA-I (activates LCAT)	Delivers cholesterol to liver via SR-B1 receptor

Chylomicron Remnants — Recognition

After LPL (Lipoprotein Lipase) removes triglycerides from chylomicrons, the resulting remnants are recognized by the liver via ApoE. This is a directly tested exam fact. ApoC-II activates LPL; ApoE targets remnants to the liver.

What Gets Depleted When VLDL → LDL

As VLDL is processed by LPL into IDL and then LDL, it progressively loses: triglycerides (stripped by LPL), ApoC-II, and ApoE. LDL retains only ApoB-100 and is enriched in cholesterol esters. This is asked as "what gets totally or partially depleted in metabolism of VLDL."

SR-B1 Receptor — Hepatic Cholesterol Uptake

The SR-B1 (Scavenger Receptor class B type 1) receptor on hepatocytes mediates the selective uptake of cholesterol from HDL into the liver — the final step of reverse cholesterol transport. Unlike the LDL receptor (which takes up the entire LDL particle), SR-B1 selectively extracts cholesterol while HDL returns to circulation.

⚗️ Glycerol from Adipose After LPL Action

When LPL (Lipoprotein Lipase) cleaves triglycerides from lipoproteins, it releases free fatty acids AND glycerol. Adipose tissue lacks glycerol kinase (the enzyme needed to reuse glycerol) and therefore releases glycerol into the blood. Glycerol is transported to the liver, where it is phosphorylated and used for triglyceride synthesis or enters gluconeogenesis.

💊 Statins — Mechanism

Statins (e.g., atorvastatin, simvastatin) are structural analogs of mevalonate (the product of HMG-CoA Reductase). They act as competitive inhibitors of HMG-CoA Reductase — they compete with the natural substrate HMG-CoA for the active site. As competitive inhibitors: they increase Km (lower apparent affinity) and do not change Vmax (maximum rate unchanged). Their effect can be overcome by increasing substrate concentration — though at therapeutic doses, they are highly effective.

Exam Trap — Statin Classification

Statins are structural analogs of mevalonate (not HMG-CoA itself). They are competitive inhibitors → increase Km, Vmax unchanged. "Statins do not affect Vmax and increase Km" → TRUE.

🔄 Bile Acid Synthesis, Uronic Acid & Enterohepatic Circulation

Bile acid synthesis pathway (from cholesterol, in the liver):

Cholesterol 7α-Hydroxylase→rate-limiting · CYP7A1 · liver ER Committed step7α-OH-cholesterol → Primary bile acids →+ glycine/taurine Conjugated salts gut bacteria→deconjugate + dehydroxylate Secondary bile acids

Uronic acids are formed from glucose by oxidation of C-6. Glucuronic acid (glucuronate) is the most important — it is the activated form used in bilirubin conjugation (as UDP-glucuronate) and drug detoxification. The 2022 exam asked directly: "Uronic acid is formed from ___?" → Answer: glucose (C-6 oxidation of glucose-6-phosphate).

1
Rate-limiting step of bile acid synthesis: 7α-hydroxylase (CYP7A1) in the liver ER. Introduces –OH at C-7 of cholesterol. Inhibited by bile acids returning via enterohepatic circulation (feedback inhibition).
2
Primary bile acids: Cholic acid and chenodeoxycholic acid — produced by the liver.
3
Conjugation: Primary bile acids + glycine or taurine → conjugated bile salts (more amphipathic, better detergents). Ratio glycine:taurine ≈ 3:1.
4
Secondary bile acids: Intestinal bacteria deconjugate and dehydroxylate primary bile acids → deoxycholic acid (from cholic) and lithocholic acid (from chenodeoxycholic). Lithocholic acid is poorly reabsorbed and mostly excreted.

Enterohepatic Circulation

>95% of bile salts are reabsorbed in the terminal ileum (active transport via Na⁺-bile salt cotransporter) → portal vein → liver → re-secreted into bile. This cycle conserves bile salts (body only has ~3–5 g but recycles them 6–10×/day → effective pool = 20–30 g/day).

Cholestyramine (bile salt sequestrant) binds bile salts in the gut → prevents reabsorption → liver must synthesize new bile acids from cholesterol → ↓ plasma cholesterol.

Hypertriacylglycerolemia — Treatment with Niacin ★ asked 2022

Niacin (Vitamin B3) at pharmacological doses inhibits lipolysis in adipose tissue (by inhibiting HSL via an adipocyte G-protein receptor) → ↓ FFA delivery to liver → ↓ VLDL synthesis → ↓ plasma TAG and LDL, ↑ HDL. This was tested in the 2022 exam.

🔗 Bile Acid Conjugation — Mechanism & Significance

Unconjugated (free) bile acids have a pKa of ~6.0 — at physiological pH they are partly protonated and can be passively reabsorbed across the gut wall. Conjugation lowers the pKa to ~2 (glycine conjugates) or ~1.5 (taurine conjugates) — making bile salts fully ionised at gut pH → they cannot diffuse back passively → they stay in the gut lumen as effective detergents.

	Glycine Conjugates	Taurine Conjugates
Examples	Glycocholic acid, Glycochenodeoxycholic acid	Taurocholic acid, Taurochenodeoxycholic acid
Ratio	Glycine : Taurine conjugates ≈ 3 : 1 in humans
pKa	~2.0 (fully dissociated at gut pH)	~1.5 (even more fully dissociated)
Advantage	Both remain ionised in the gut → cannot passively cross membranes → stay in lumen → more effective emulsification than free bile acids

Conjugation Reaction (Liver)

Bile acid + CoA → Bile acyl-CoA (activated) → + Glycine or Taurine → Conjugated bile salt
Enzyme: Bile acid-CoA:amino acid N-acyltransferase (BAAT) — occurs in hepatocyte peroxisomes and cytosol.
The amide bond formed is resistant to pancreatic proteases — conjugated bile salts remain intact through digestion.

Deconjugation by Gut Bacteria

Intestinal bacteria express bile salt hydrolase (BSH) → cleave glycine/taurine from conjugated bile salts → free primary bile acids → then 7α-dehydroxylation → secondary bile acids (deoxycholic, lithocholic). These are less soluble; lithocholic acid is mostly excreted in faeces (hepatotoxic if not eliminated).

🧭 SREBP-2, SCAP and INSIG — How a Cell Counts Its Cholesterol

HMG-CoA reductase is regulated four ways at once. The transcriptional layer is the most elegant: a membrane-bound transcription factor that is only released when sterols run low.

🔒 When cholesterol is HIGH

The sensor holds everything in place

Sterols bind SCAP (SREBP cleavage-activating protein). SCAP changes shape and binds INSIG, which anchors the SCAP–SREBP complex in the endoplasmic reticulum membrane. SREBP never reaches the Golgi, is never cleaved, and no transcription occurs.

▼

🔓 When cholesterol is LOW

Release, transport, cleave, transcribe

SCAP releases INSIG and escorts SREBP-2 to the Golgi, where two proteases (S1P and S2P) cut it in sequence, freeing the N-terminal domain. That fragment travels to the nucleus, binds the sterol regulatory element, and turns on HMG-CoA reductase and the LDL receptor together — the cell simultaneously makes more cholesterol and imports more.

1 · Transcriptional
SREBP-2 / SCAP / INSIG, as above

2 · Proteolytic degradation
Sterols accelerate degradation of the reductase protein itself

3 · Covalent — AMPK
Phosphorylation by AMPK inactivates it. Low energy means no cholesterol synthesis

4 · Hormonal
Insulin and thyroid hormone increase activity; glucagon and cortisol decrease it

💊 Why statins raise LDL receptors — and why PCSK9 inhibitors work

A statin lowers intracellular cholesterol, which activates SREBP-2, which upregulates the LDL receptor — so the liver pulls more LDL out of the blood. That indirect effect, not the synthesis block itself, is most of the clinical benefit. PCSK9 normally targets the LDL receptor for lysosomal destruction after it recycles; inhibiting PCSK9 lets each receptor make many more trips. The two drug classes attack the same variable from opposite directions.

💧 Bile Acids & The Enterohepatic Circulation

Conversion to bile acids is the only quantitatively important route for eliminating cholesterol from the body — the ring system cannot be broken down. Everything else is recycling.

Cholesterol

→7α-hydroxylase · RATE-LIMITING

7α-Hydroxycholesterol

→

Cholic acid
Chenodeoxycholic acid

PRIMARY bile acids

→conjugation with glycine or taurine

Bile SALTS

ionised at duodenal pH — far better detergents

→intestinal bacteria: dehydroxylation

Deoxycholic · Lithocholic

SECONDARY bile acids

↻ About 95% reabsorbed in the terminal ileum and returned to the liver by the portal vein — the pool cycles 6–10 times a day. Only ~5% is lost in faeces

💊 Why bile acid sequestrants lower cholesterol

Resins bind bile salts in the gut and break the enterohepatic circulation. The liver must replace the lost pool, so it converts more cholesterol to bile acids, depletes its own cholesterol, and upregulates LDL receptors — again through SREBP-2. Ileal resection or Crohn disease does the same thing involuntarily, which is one reason those patients form gallstones.

Key numbers and facts

Bile acids are amphipathic — a hydroxylated, hydrophilic face and a hydrophobic face — which is what makes them emulsifiers. Conjugation lowers the pKa so they stay ionised at intestinal pH. 7α-hydroxylase is inhibited by bile acids returning to the liver (feedback via the FXR receptor) and is the step statins do not touch.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧱 Bile Acid Steps, FXR Feedback, and "Good vs Bad" Cholesterol

Two more details on bile acids

The steps: cholesterol → 7α-hydroxycholesterol (7α-hydroxylase — a microsomal cytochrome P450 monooxygenase needing O₂, NADPH and vitamin C) → 12α-hydroxylation → cholyl-CoA and chenodeoxycholyl-CoA → conjugation with glycine or taurine gives glycocholic, taurocholic, glycochenodeoxycholic and taurochenodeoxycholic acid. In the gut, deconjugation and 7α-dehydroxylation turn them into deoxycholic and lithocholic acid; lithocholic is so insoluble that it is barely reabsorbed and is largely lost in faeces. The feedback: when the returning bile acid pool grows, it activates the nuclear receptor FXR (farnesoid X receptor), which suppresses the CYP7A1 gene; chenodeoxycholic acid is the strongest activator. About 3–5 g of bile acids cycle 6–10 times a day, and exactly the amount lost in faeces is resynthesised from cholesterol.

Why you cannot really call cholesterol "good" or "bad" Past Q

There is only one cholesterol molecule. What differs is the particle carrying it: LDL delivers cholesterol to the artery wall, HDL removes it (reverse transport). And cholesterol is indispensable — membranes, all five steroid hormone classes, bile acids and vitamin D. It becomes harmful only in excess and in the wrong vehicle. Hereditary factors set most of an individual's plasma level; diet, exercise, smoking, alcohol and stress adjust it around that setting.

🧱 SCAP–Insig, and What Everyday Life Does to Plasma Cholesterol

How SREBP-2 is actually held back

SREBP-2 sits in the ER membrane bound to SCAP. When sterols are high, SCAP is clamped by Insig and nothing moves. When cholesterol falls, Insig releases SCAP, which escorts SREBP to the Golgi, where two proteases cut the transcription factor free; it then switches on HMG-CoA reductase and the LDL receptor together. Statins work by driving exactly this switch.

Lifestyle and blood cholesterol

Coffee and emotional stress raise plasma free fatty acids, so the liver exports more TAG and cholesterol as VLDL. Moderate alcohol lowers coronary risk by raising HDL, through more apo A-I synthesis and altered cholesteryl ester transfer protein activity — lost with heavy drinking, which raises TAG. Regular exercise lowers LDL and raises HDL. Unsaturated fats up-regulate LDL receptors and lower cholesterol; saturated fats do the reverse and make smaller, cholesterol-richer VLDL. Other risk factors travelling with it: smoking, male sex, obesity, inactivity, sugar-sweetened drinks, hypertension.

“Good” and “bad” cholesterol

There is only one cholesterol molecule — what differs is the particle carrying it: LDL delivers it to the artery wall, HDL removes it. And it is indispensable: membranes, all five classes of steroid hormone, bile acids and vitamin D.

Lipoproteins Deep Dive

Frequently tested: apolipoproteins (which are purely structural), which particle carries most cholesterol (LDL), LCAT, SR-B1, LPL

⚗️ Key Lipoprotein Enzymes

LPL (Lipoprotein Lipase)

Sits on capillary endothelium. Cleaves triglycerides from chylomicrons and VLDL. Activated by ApoC-II (without ApoC-II, LPL is inactive). Releases free fatty acids to tissues and glycerol to blood (glycerol → liver).

👆 Tap for enzyme details

LCAT (Lecithin-Cholesterol Acyltransferase)

An extracellular enzyme (works in plasma). Converts lecithin + free cholesterol → cholesteryl esters + lyso-PC. Activated by ApoA-I (main HDL apoprotein). Makes HDL spherical. Often confused with ACAT (which is intracellular).

👆 Tap for enzyme details

ACAT (Acyl-CoA:Cholesterol Acyltransferase)

An intracellular enzyme (in the ER). Esterifies cholesterol within cells for storage. NOT activated by ApoA-I (that is LCAT). LCAT = extracellular/plasma; ACAT = intracellular/ER.

👆 Tap for enzyme details

SR-B1 (Scavenger Receptor)

Hepatic receptor. Mediates selective uptake of cholesterol from HDL into liver — the final step of reverse cholesterol transport. HDL docks → cholesterol extracted → HDL returns to circulation (unlike the LDL receptor, which internalizes the whole particle).

👆 Tap for enzyme details

LCAT vs ACAT — Directly Tested

LCAT: extracellular, plasma, activated by ApoA-I, works on HDL. ACAT: intracellular (ER), esterifies cholesterol inside cells for storage. Exam question: "ACAT is an extracellular enzyme activated by Apo-A1" → FALSE (that describes LCAT).

🏥 Familial Hypercholesterolemia — LDL Receptor Defect

Caused by deficiency or dysfunction of LDL receptors on cell surfaces. LDL cannot be cleared from blood → very high LDL-cholesterol → cholesterol deposits in tendons (xanthomas) and arteries (premature atherosclerosis, heart attacks in young adults). This is NOT a lipoprotein enzyme problem — it is a receptor problem.

👆 Tap for the full disease card

📊 The Complete Lipoprotein Table

Class	Density	Main lipid	Made by	Main apolipoproteins	Job
Chylomicron	Lowest	Dietary TAG (~85%)	Intestine	B-48, A-I, then C-II and E from HDL	Carry dietary fat to peripheral tissues
VLDL	Very low	Endogenous TAG	Liver	B-100, C-II, E	Export hepatic TAG
IDL	Intermediate	TAG + cholesterol	From VLDL	B-100, E	Transient — taken up by liver or converted to LDL
LDL	Low	Cholesteryl ester	From IDL	B-100 only	Deliver cholesterol to tissues — the atherogenic particle
HDL	Highest	Cholesteryl ester + protein	Liver and intestine	A-I, C-II, E	Reverse cholesterol transport — and the reservoir that lends C-II and E to the others

The one rule for density

More protein means higher density; more lipid means lower density. Chylomicrons are almost pure fat and float; HDL is roughly half protein and sinks. Particle size runs the other way — chylomicrons are the biggest, HDL the smallest.

🔑 Apolipoproteins — What Each One Actually Does

Apo B-48

Chylomicrons only. The intestinal form — 48% of the B-100 sequence, produced by RNA editing that creates a premature stop codon. Structural; it does not bind the LDL receptor.

Apo B-100

VLDL, IDL, LDL. The ligand for the LDL receptor. One molecule per particle, never transferred. Defects in it cause familial defective apoB, clinically like familial hypercholesterolaemia.

Apo C-II

The obligatory activator of lipoprotein lipase. Lent by HDL. Without it, triacylglycerol-rich particles cannot be unloaded — see familial chylomicronaemia.

Apo C-III

The brake — inhibits lipoprotein lipase and hepatic remnant uptake. High levels raise triacylglycerol.

Apo E

The ligand for remnant uptake by the liver. The E2/E2 genotype clears remnants poorly → type III dysbetalipoproteinaemia. The E4 allele is a risk factor for Alzheimer disease.

Apo A-I

The main HDL protein and the activator of LCAT. Also the acceptor for ABCA1-mediated efflux.

Apo(a)

Attached to apoB-100 to form Lp(a). Structurally resembles plasminogen, so it competes with it and is prothrombotic as well as atherogenic. Genetically determined and largely unresponsive to statins.

⚙️ The Four Enzymes and Two Transfer Proteins

Protein	Where	What it does	Activated / needed by
Lipoprotein lipase	Capillary endothelium of muscle and adipose	Hydrolyses TAG in chylomicrons and VLDL → free fatty acids + glycerol	Apo C-II; induced by insulin in adipose
Hepatic lipase	Liver sinusoids	Completes IDL → LDL conversion; remodels HDL	—
LCAT	Plasma, on HDL	Transfers a fatty acid from lecithin to cholesterol → cholesteryl ester, which moves into the particle core and lets HDL mature from discoid to spherical	Apo A-I
ACAT	Inside cells	Esterifies cholesterol for intracellular storage	Substrate availability
CETP	Plasma	Swaps cholesteryl ester from HDL for triacylglycerol from VLDL. This is why high triacylglycerol drives HDL down — the more VLDL there is, the more cholesterol is pulled out of HDL	—
MTP	Intestinal and hepatic ER	Loads lipid onto apoB during particle assembly	—

🎯 Two "no particle at all" diseases

MTP deficiency → abetalipoproteinaemia: no chylomicrons and no VLDL can be assembled, so fat accumulates in enterocytes and hepatocytes, and the fat-soluble vitamins are not absorbed — the neurological damage is essentially vitamin E deficiency. ABCA1 defect → Tangier disease: cholesterol cannot be effluxed onto nascent HDL, so HDL is almost absent and cholesterol accumulates in macrophages — the orange tonsils.

🔄 Reverse Cholesterol Transport, Step by Step

▸ How cholesterol gets out of a peripheral cell

Liver / intestine

nascent discoid HDL, apoA-I

→ABCA1 effluxes free cholesterol from the cell

Free cholesterol on HDL surface

→LCAT · activated by apoA-I

Cholesteryl ester moves into the core

HDL matures: discoid → spherical HDL₃ → HDL₂

→

Liver via SR-B1

selective uptake — HDL is not destroyed

↻ Alternative route: CETP trades the cholesteryl ester to VLDL/LDL, which the liver then takes up by the LDL receptor — an indirect return path

💡 Why HDL is "good" — the mechanism, not the slogan

HDL does three separate things: it removes cholesterol from peripheral cells including macrophages in the arterial wall, it lends apoC-II and apoE to chylomicrons and VLDL so they can be metabolised at all, and it is antioxidant and anti-inflammatory, protecting LDL from the oxidation that makes it atherogenic. SR-B1 uptake is selective — the liver takes the lipid and returns the particle, so one HDL can make many trips.

🩸 oxLDL, the Scavenger Route and Lp(a)

LDL is a primary cause of atherosclerosis. The reason is that once LDL is oxidised (oxLDL) it stops being handled by the regulated LDL receptor and is taken up instead through the scavenger receptor SR-A on macrophages — an unregulated endocytosis that has no feedback shut-off, so the cell keeps loading until it becomes a foam cell and an atherosclerotic plaque forms.

Lp(a)

Almost identical to LDL, but it carries an additional apolipoprotein — apo(a) — covalently linked to apo B-100. Apo(a) is structurally homologous to plasminogen and competes with it for binding fibrin, which (on the current hypothesis) slows the breakdown of blood clots. Large quantities of Lp(a) in plasma = increased risk of coronary heart disease.

Reading a lipid panel

Cholesterol is measured in fasting blood. Total C = LDL-C + HDL-C + VLDL-C, and VLDL-C = TAG ÷ 5 (the TAG:cholesterol ratio in VLDL is 5:1). Total cholesterol should be <200 mg/dL.

Three storage/handling defects on the LDL route

Familial hypercholesterolaemia (type IIa) — defects in a functional LDL receptor, defects in apo B-100, or increased activity of PCSK9. Wolman disease — inability to hydrolyse cholesteryl esters. Niemann–Pick type C — failure to transport free cholesterol out of lysosomes.

Tangier disease

Deficiency of ABCA1, the transporter that effluxes nonesterified cholesterol from peripheral tissues — resulting in the virtual absence of HDL.

SR-B1 — the one that does not swallow the particle

SR-B1 binds apo A-I on HDL2 and takes up only the cholesteryl esters; the HDL particle itself is not taken up. Compare with the LDL receptor (ligands apo B-100 and apo E; handles chylomicron remnants, IDL, LDL), LRP (ligand apo E; chylomicron remnants), and SR-A (broad ligand range; oxLDL).

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🎯 The LDL Receptor Cycle and How a Cell Turns It Down Topic 26Past Q

The LDL (apo B-100, E) receptor is expressed by the liver and by most extrahepatic tissues. About 70% of LDL is cleared by the liver and 30% elsewhere. It binds apo B-100 — and also, with higher affinity, particles rich in apo E, which is how remnants are removed.

LDL binds the receptorin a clathrin-coated pit on the cell surface

↓receptor-mediated endocytosisthe whole particle is taken in intact

Endosome → lysosomeapoprotein hydrolysed to amino acids; cholesteryl ester hydrolysed to free cholesterol

↓receptor recyclingthe receptor returns to the surface and is used again — unless PCSK9 has tagged it, in which case it is degraded instead

Free cholesterol in the cellnow acts as its own signal

Signal 1 — switch synthesis off

The incoming cholesterol inhibits transcription of the genes for HMG-CoA synthase, HMG-CoA reductase, the other cholesterol-synthesis enzymes and the LDL receptor itself, through the SREBP pathway. Synthesis and uptake are suppressed together.

Signal 2 — store the surplus

ACAT is stimulated, so free cholesterol is esterified and parked as cholesteryl ester droplets. Between the two signals the free cholesterol content of the cell is held within very narrow limits, matched to its needs for membranes, steroid hormones and bile acids.

Why diet changes receptor numbers Past Q

Polyunsaturated and monounsaturated fatty acids up-regulate LDL receptors, so LDL is cleared faster and plasma cholesterol falls. Saturated fatty acids do the opposite and also push cholesterol into smaller, cholesterol-richer VLDL particles that are taken up slowly by the tissues — the more atherogenic pattern.

When the receptor fails

Familial hypercholesterolaemia: a defective LDL receptor, so LDL cholesterol stays in the plasma — tendon xanthomas, corneal arcus and premature atherosclerosis. The same logic explains the two drug classes: statins lower intracellular cholesterol so SREBP makes more receptors; PCSK9 inhibitors stop the receptors being destroyed.

📉 When Lipoprotein Metabolism Goes Wrong Past Q

Two families: too little (hypo-) and too much (hyper-lipoproteinaemia). The primary forms are inherited; by far the commonest cause overall is secondary — diabetes mellitus, where insulin deficiency mobilises fatty acids and leaves chylomicrons and VLDL uncleared, giving hypertriacylglycerolaemia.

Disorder	Defect	Picture
Abetalipoproteinaemia	No apo B (MTP defect) — no chylomicrons, no VLDL	Fat malabsorption, fat-laden enterocytes, acanthocytes, deficiency of fat-soluble vitamins (especially E) with ataxia and retinitis
Tangier disease	Familial α-lipoprotein (HDL) deficiency — ABCA1	Almost no HDL, orange tonsils, cholesterol ester deposits, neuropathy
Fish-eye disease · LCAT deficiency	Cholesterol cannot be esterified in HDL	Corneal opacity, low HDL, abnormal lipoprotein particles
Type I — familial LPL (or apo C-II) deficiency	Chylomicrons cannot be cleared	Milky serum, eruptive xanthomas, pancreatitis; no excess atherosclerosis
Type IIa — familial hypercholesterolaemia	LDL receptor	↑↑ LDL, tendon xanthomas, early coronary disease
Type III — familial dysbetalipoproteinaemia	Abnormal or deficient apo E, so remnants are not taken up	Remnant accumulation, palmar xanthomas, xanthomatosis, premature coronary and peripheral vascular disease
Type IV — familial hypertriacylglycerolaemia	Overproduction of VLDL	Raised TAG, often with obesity, insulin resistance and low HDL
Familial hyperalphalipoproteinaemia	High HDL	Longevity — the one "hyper" that is good news. Hepatic lipase deficiency sits in the same list, with accumulated remnants.

🚢 VLDL Secretion — What Turns It Up and Down, and the Two HDL Exporters

Factors enhancing TAG synthesis and VLDL secretion

The fed state rather than the starved one; a high-carbohydrate (especially high-fructose) diet; a high concentration of circulating free fatty acids; ethanol; and high insulin with low glucagon — though acutely insulin suppresses apo B-100 secretion, so it is chronic hyperinsulinaemia with insulin resistance that raises output.

Factors inhibiting or preventing VLDL assembly

ω-3 fatty acids, orotic acid, the sulfonylurea tolbutamide, puromycin, and experimentally brefeldin A. The inherited equivalent — no apo B at all — is abetalipoproteinaemia, with fat-laden enterocytes and fat-soluble vitamin deficiency.

ABCA1 and ABCG1

Reverse cholesterol transport uses two ATP-binding cassette transporters: ABCA1 exports free cholesterol onto lipid-poor pre-β-HDL and apo A-I (its failure is Tangier disease), while ABCG1 exports it onto the mature HDL particle. LCAT then esterifies it, and SR-B1 delivers the esters to the liver.

Nitrogen Disposal & the Urea Cycle

Frequently tested: which reactions release ammonia, NAG activating CPS-1, urea cycle ATP cost (4 high-energy bonds), CO₂+NH₃+aspartate entering, ornithine/citrulline/arginine, glucose-alanine cycle

🔀 Transamination

Amino acid + α-Ketoglutarate Aminotransferase ⇌ PLP (B6) Keto acid + Glutamate

The key idea: the amino group (–NH₂) is transferred from an amino acid onto α-ketoglutarate, collecting nitrogen into glutamate (the central nitrogen carrier). No ammonia is released here — it's just moved. The reaction is freely reversible and needs PLP (vitamin B6).

Glutamate then hands its nitrogen to the urea cycle: Glutamate Dehydrogenase releases NH₃ from glutamate. Clinically, ALT is liver-specific, while AST is found in liver + heart.

🔵 The Urea Cycle — Full Detail

The urea cycle converts toxic ammonia (NH₃) into urea, which is water-soluble and excreted in urine. It uses 4 high-energy phosphate bonds per cycle (2 ATP → 2 ADP + Pi at CPS-I, and 1 ATP → AMP + PPi at argininosuccinate synthetase, which equals 2 high-energy bonds = total 4). The cycle runs partly in mitochondria and partly in the cytosol.

1
NH₃ + CO₂ → Carbamoyl phosphate — CPS-I, mitochondria, requires 2 ATP and is absolutely dependent on N-Acetylglutamate (NAG) as allosteric activator. NAG is the main regulator of the entire urea cycle.
2
Carbamoyl-P + Ornithine → Citrulline — OTC, still in mitochondria. Most common urea cycle defect (X-linked).
3
Citrulline + Aspartate → Argininosuccinate — in the cytoplasm. Aspartate donates the second nitrogen atom. This step requires 1 ATP (→ AMP + PPi = 2 high-energy bonds).
4
Argininosuccinate → Arginine + Fumarate (Fumarase converts Fumarate → Malate in Krebs, and Malate Dehydrogenase → OAA). Fumarate links the urea cycle to the Krebs cycle.
5
Arginine → Urea + Ornithine — Arginase, cytoplasm. Ornithine re-enters mitochondria to continue the cycle.

NAG — Main Regulator

N-Acetylglutamate (NAG) is synthesized from acetyl-CoA + glutamate. It is the obligatory allosteric activator of CPS-I — without NAG, CPS-I is completely inactive and the entire urea cycle stops. High arginine stimulates NAG synthesis (feedforward). This is the most important regulatory point of the urea cycle and is directly tested.

Arginine Synthesis in the Kidney

The kidney can synthesize arginine using citrulline (from intestine) + aspartate (or from glutamine) as substrates. The kidney therefore plays a role in providing arginine for body needs — this is asked directly in exams.

Ultimate Fate of Ammonia in the Body

Most ammonia is ultimately converted to urea (in liver, via urea cycle) and excreted in urine. A smaller portion is incorporated into glutamine (via glutamine synthetase) for transport. On exam, "ultimate fate of ammonia" = urea.

🔋 Urea Cycle Energy & Regulation — Teacher Emphasized

Energy Cost

3 ATP consumed per cycle → generating 4 high-energy bond equivalents lost:
• CPS-I: 2 ATP → 2 ADP + 2 Pi (2 bonds)
• Argininosuccinate Synthetase: 1 ATP → AMP + PPi (2 bonds, since PPi hydrolysis adds 1 more)
Total = 4 high-energy phosphate bonds

Regulation — Enzyme Induction/Repression

Urea cycle enzymes are regulated by induction and repression (changing amount of enzyme — slow response) AND by substrate availability. High protein diet → ↑all urea cycle enzyme amounts. Starvation/catabolism → also ↑enzyme amounts. Key: regulation is ↑/↓ activity, not rendering enzyme "inactive" per se.

NAG — The Master Regulator (Teacher Highlighted)

N-Acetylglutamate (NAG) is the allosteric activator of CPS-I. Without NAG, CPS-I is completely inactive. NAG synthesis: acetyl-CoA + glutamate → NAG (NAG Synthase, in mitochondria). Arginine stimulates NAG synthase (feedforward activation → more arginine drives more urea synthesis). CPS-II (in pyrimidine synthesis, cytosol) does NOT need NAG.

Urea Cycle "No Beginning, No End" — Teacher Noted

The urea cycle is often described as having "no beginning and no end" because ornithine, citrulline, and arginine are continuously recycled — the cycle is fed by NH₃ (from CPS-I) and aspartate (from transamination) at any entry point.

Ammonia Disposal — Enzymes That Release Ammonia (Teacher Listed)

Enzymes that release ammonia (NH₃):
• Glutamate Dehydrogenase (major): Glutamate → α-KG + NH₃
• AMP Deaminase: AMP → IMP + NH₃ (purine catabolism)
• Adenosine Deaminase: Adenosine → Inosine + NH₃
• Glutaminase: Glutamine → Glutamate + NH₃ (in kidney/liver)
• Asparagine synthetase (reverse): hydrolyzes asparagine → aspartate + NH₃

🚴 Glucose-Alanine Cycle — Teacher Required

During muscle activity, amino groups from muscle amino acid catabolism are shuttled to the liver for urea synthesis via the glucose-alanine cycle:

1
Muscle: amino acids → transamination with pyruvate → Alanine (carries NH₂) + α-keto acids. ALT (requires PLP/B6) catalyzes this.
2
Alanine is exported from muscle and travels in blood to the liver.
3
Liver: ALT converts Alanine + α-KG → Pyruvate + Glutamate.
4
Pyruvate → gluconeogenesis → Glucose → exported to blood → used by muscle (cycle completes).
5
Glutamate → Glutamate Dehydrogenase → NH₃ → enters urea cycle.

Why It Matters

This cycle allows muscle to export nitrogen safely (as non-toxic alanine) while simultaneously supplying the liver with carbon for gluconeogenesis. It is fundamentally linked to both amino acid catabolism and gluconeogenesis.

🗺️ Liver Zonation — Why Ammonia Is Handled Twice

The liver does not treat ammonia the same way everywhere. Hepatocytes are functionally divided along the sinusoid, and the two zones use enzymes with very different affinities — a deliberate two-stage filter.

🟠 PERIPORTAL — first to see portal blood

Urea cycle enzymes — carbamoyl phosphate synthetase I and the rest. High capacity but LOW affinity for ammonia. These cells clear the bulk of the load arriving from the gut, converting it to urea for excretion.

🔵 PERIVENOUS — the last chance

Glutamine synthetase. Low capacity but HIGH affinity. These cells scavenge whatever ammonia escaped the periportal zone, trapping it as glutamine before the blood leaves the liver.

💡 Why the design works

A high-capacity, low-affinity system handles the load efficiently but leaks at low concentrations; a high-affinity system catches the remainder but would saturate if used alone. Putting them in series along the blood flow gives both throughput and a low final concentration. The same logic appears in glucokinase versus hexokinase — capacity first, affinity last.

🔁 Interorgan Nitrogen Transport — Two Carriers, Two Cycles

Peripheral tissues cannot make urea, so nitrogen must be shipped to the liver in a non-toxic form. Two amino acids do the job, and each has its own reason.

▸ Glucose–alanine cycle

Muscle: amino acid + pyruvate

→ALT

Alanine

→blood

Liver: alanine → pyruvate + NH₃

→

Urea excreted
Glucose returned to muscle

↻ Carries one nitrogen and simultaneously returns the carbon skeleton as glucose — it moves fuel and waste in one vehicle

▸ Glutamine transport

Tissue: glutamate + NH₃

→glutamine synthetase · ATP

Glutamine

→blood — the most abundant plasma amino acid

Liver, kidney, gut: glutaminase

→

Urea (liver) · NH₄⁺ (kidney)

↻ Carries two nitrogens per molecule and is the main ammonia carrier from the brain

🧠 Why hyperammonaemia damages the brain

Astrocytes are the only brain cells with glutamine synthetase, so they absorb the ammonia — converting glutamate to glutamine, which accumulates osmotically and causes astrocyte swelling and cerebral oedema. At the same time, α-ketoglutarate is consumed to make more glutamate, draining the Krebs cycle, and glutamate depletion impairs neurotransmission. Three mechanisms, one metabolite. See hyperammonaemia.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

⚖️ Nitrogen Balance and the Nitrogen Sources Topic 9Past Q

Nitrogen balance = nitrogen intake − nitrogen output. Amino acids are the body's main nitrogen source, and because protein is never stored — there is only a small free amino acid pool in constant turnover — the balance is read day by day.

Adult requirement

0.5–0.7

g protein / kg / day

Safe intakes are usually quoted a little higher, ~0.8

Child requirement

≈ 4

g protein / kg / day

Growth demands a positive balance

Nitrogen leaves as

Urea

then NH₄⁺, creatinine, uric acid, amino acids

Plus faeces, skin and hair

Biological quality — the three grades

Standard (full) value — every essential amino acid in adequate amount and proportion: egg, milk, meat, fish. Partially defective value — at least one essential amino acid present but in inadequate amount: most cereals (low lysine) and legumes (low methionine). Defective value — at least one essential amino acid missing: gelatin has no tryptophan, maize zein lacks lysine and tryptophan.

Positive balance — intake > output

Nitrogen is being retained because tissue is being built: growth in infants and children, pregnancy, lactation, convalescence after illness or surgery, repair after injury, increased physical training, and anabolic hormone states (growth hormone, insulin, androgens).

Negative balance — output > intake

Inadequate intake (starvation, low-protein diet, proteins of defective value), failure of digestion or of amino acid absorption, excessive tissue breakdown (fever, sepsis, trauma, burns, surgery, advanced cancer, hyperthyroidism, glucocorticoid excess) and loss of protein (proteinuria, haemorrhage, loss in faeces, exudating burns).

Equilibrium

The normal state of a healthy, weight-stable adult: intake exactly matches output. A negative balance can never be corrected by energy alone — if any single essential amino acid runs short, synthesis stops and the rest are deaminated and burned.

🧬 The Named Urea-Cycle Defects

The named urea-cycle defects — one per enzyme Past Q

CPS I → hyperammonaemia type 1 · Ornithine transcarbamoylase → hyperammonaemia type 2, X-linked, with orotic aciduria · Argininosuccinate synthetase → citrullinaemia · Argininosuccinate lyase → argininosuccinic aciduria · Arginase → hyperargininaemia (autosomal recessive) · N-acetylglutamate synthase → the same clinical picture as a CPS I defect, because CPS I cannot be switched on · ORNT1 mitochondrial ornithine transporter → HHH syndrome: hyperornithinaemia, hyperammonaemia and homocitrullinuria, because cytosolic ornithine cannot get back into the mitochondrion.

What the patient looks like

Vomiting, lethargy, irritability, intermittent ataxia, seizures, and in survivors severe intellectual disability. Older children characteristically avoid high-protein food on their own. The earlier the block sits in the cycle (steps 1 and 2), the more severe the disease, because ammonia has not yet been fixed into citrulline.

💨 Where Portal Ammonia Comes From

And the reaction that fixes it back

The mirror image of oxidative deamination is the reductive amination of α-ketoglutarate by glutamate dehydrogenase (α-ketoglutarate + NH₃ + NADPH → glutamate). The reaction is reversible but strongly favours glutamate synthesis, which keeps the cytotoxic ammonium ion low, and it is the origin of the whole "glutamate family" — glutamine, proline and arginine — as well as the entry point for the nitrogen that later leaves as urea.

Where the ammonia in portal blood comes from

The gut is a major producer: enteric bacteria split urea and dietary amines with urease, and enterocytes deaminate glutamine. That ammonia is absorbed straight into the portal vein, and a healthy liver removes it on the first pass, which is why only traces reach the peripheral blood. If portal blood bypasses the liver — cirrhosis with portosystemic shunting, or a surgical shunt — systemic ammonia rises to toxic levels even though no enzyme is missing.

🅶 Glutamine — Mechanism and Inter-Organ Trip

Two details of the glutamine reaction that get asked

The mechanism: glutamine synthetase binds glutamate and ATP in that order, glutamate attacks the γ-phosphate to give the intermediate γ-glutamyl phosphate plus ADP; NH₄⁺ then binds, uncharged NH₃ attacks the intermediate, and release of Pi releases glutamine. The inter-organ trip: in the post-absorptive state glutamine is released from muscle into the circulation and is extracted by the gut and the kidney, which convert a large part of it to alanine — so glutamine carries nitrogen, carbon and energy between organs, and hands the load on to the glucose–alanine cycle. A rare neonatal glutamine synthetase deficiency causes severe brain damage, multi-organ failure and death.

🔁 Transdeamination, and the Sign That Names a Urea-Cycle Defect

Transdeamination

The name for the two reactions working as a pair: transamination funnels the α-amino group of almost every amino acid onto α-ketoglutarate to make glutamate, and then glutamate dehydrogenase deaminates that single molecule. The nitrogen of twenty amino acids therefore leaves as ammonia through one enzyme — which is also why glutamate is the only amino acid oxidatively deaminated at any appreciable rate.

Argininosuccinic aciduria

Argininosuccinate lyase deficiency: argininosuccinate accumulates in blood, CSF and urine, and the patient has characteristically friable, tufted hair — the physical sign the question papers ask for. It sits with the rest of the series: CPS I → hyperammonaemia type 1; OTC → hyperammonaemia type 2 with orotic aciduria; argininosuccinate synthetase → citrullinaemia; arginase → hyperargininaemia; ornithine transporter → HHH syndrome.

Biogenic Amines & Neurotransmitter Synthesis

Frequently tested: catecholamine synthesis (DOPA→dopamine→noradrenaline→adrenaline), Phe/Tyr-derived products, MAO & DAO degradation, serotonin/melatonin from tryptophan

⚡ Catecholamine Synthesis Pathway (Tyr → Adrenaline)

Catecholamines are synthesized from Tyrosine in a stepwise pathway in the adrenal medulla, sympathetic neurons, and brain:

⚡ Catecholamine Synthesis (Tyr → Adrenaline)

Tyrosine Tyrosine Hydroxylase→rate-limiting · BH₄ L-DOPA DOPA Decarboxylase→PLP/B6 Dopamine Dopamine β-Hydroxylase→Vitamin C Noradrenaline PNMT→SAM · adrenal medulla Adrenaline

1
Tyrosine → L-DOPA: Tyrosine hydroxylase (rate-limiting; requires BH₄ tetrahydrobiopterin). This is the committed step.
2
L-DOPA → Dopamine: DOPA decarboxylase (requires PLP/B6). Dopamine is a neurotransmitter in its own right.
3
Dopamine → Noradrenaline (Norepinephrine): Dopamine β-hydroxylase (requires Vitamin C as cofactor). Located in secretory vesicles.
4
Noradrenaline → Adrenaline (Epinephrine): PNMT (phenylethanolamine N-methyltransferase). Transfers a methyl group from SAM (S-adenosylmethionine). Only in adrenal medulla.

Degradation & Clinical

Catecholamines are inactivated by: MAO (oxidative deamination) + COMT (methylation using SAM as methyl donor).
• Urine metabolites: VMA (vanillylmandelic acid) from adrenaline/noradrenaline; HVA (homovanillic acid) from dopamine.
• Phaeochromocytoma: adrenal tumour → excess catecholamines → ↑VMA in urine.

🌙 Serotonin & Melatonin Synthesis (Trp pathway)

🌙 Tryptophan → Serotonin → Melatonin

Tryptophan Tryptophan Hydroxylase→rate-limiting · BH₄ 5-HTP 5-HTP Decarboxylase→PLP/B6 Serotonin (5-HT) AANAT / ASMT→pineal · SAM Melatonin

1
Tryptophan → 5-Hydroxytryptophan (5-HTP): Tryptophan hydroxylase (requires BH₄). Rate-limiting step.
2
5-HTP → Serotonin (5-HT): 5-HTP decarboxylase (requires PLP/B6).
3
Serotonin → N-Acetylserotonin → Melatonin: In the pineal gland via acetylation (AANAT) + methylation (ASMT uses SAM).

Serotonin functions: pain perception, sleep, appetite, temperature, blood pressure, mood (well-being). Degraded by MAO → 5-HIAA (5-hydroxyindoleacetic acid) in urine. Niacin (B3) is also synthesized from Tryptophan (60 mg Trp → 1 mg niacin).

🤧 Histamine Synthesis

Histidine → Histamine by histidine decarboxylase (requires PLP/B6). Stored in mast cells and basophils. Released by allergic reactions or trauma.

Functions: vasodilator, allergic/inflammatory mediator, gastric acid secretion stimulator. Antihistamines block H₁ receptors (allergy) or H₂ receptors (gastric acid — e.g., ranitidine).

⚗️ Amine Bases Metabolism — MAO & DAO Enzymes (Teacher Listed)

Biogenic amines (dopamine, serotonin, histamine, noradrenaline) are degraded by oxidative deamination:

MAO (Monoamine Oxidase)

MAO: oxidatively deaminates monoamines (one amino group).
MAO-A: degrades serotonin, noradrenaline, dopamine, tyramine
MAO-B: mainly degrades phenylethylamine and dopamine
Produces: aldehyde + NH₃ + H₂O₂
MAO inhibitors: antidepressants (e.g., phenelzine, tranylcypromine)

DAO (Diamine Oxidase / Histaminase)

DAO: oxidatively deaminates diamines (two amino groups).
Main substrates: histamine, putrescine, cadaverine
Contains copper (Cu²⁺) and pyridoxal phosphate (PLP)
Active in intestinal mucosa, kidney, placenta
Deficiency → histamine intolerance

Products of Amine Oxidation

Both MAO and DAO produce: Aldehyde + NH₃ + H₂O₂. The H₂O₂ is then detoxified by catalase. The aldehydes are further oxidized by aldehyde dehydrogenase to carboxylic acids (excreted). The NH₃ enters the urea cycle.

🧘 GABA Metabolism & the GABA Shunt

GABA (γ-aminobutyric acid) is the brain's main inhibitory neurotransmitter. It is made from glutamate and broken down through a small bypass of the Krebs cycle called the GABA shunt.

⚗️ GABA Synthesis & Degradation

Glutamate GAD · PLP→ GABA GABA-T · PLP→ SSA → Succinate → Krebs

Step	Enzyme (cofactor)	Note
Glutamate → GABA	Glutamate decarboxylase (PLP/B6)	B6 deficiency → ↓GABA → seizures. Anti-GAD antibodies: type 1 diabetes, stiff-person syndrome
GABA → succinate	GABA transaminase (PLP/B6)	Inhibited by vigabatrin (antiepileptic) → ↑GABA

Why both GABA enzymes need B6

Both steps use PLP (vitamin B6). This is why B6 deficiency (or B6 antagonists like isoniazid) lowers GABA and provokes seizures — GABA is calming, so less of it means over-excitation.

🌾 Full Tryptophan Metabolism — Serotonin, Melatonin, Niacin

Tryptophan is the most metabolically "busy" amino acid: it feeds three important products through two branches.

🌙 Serotonin branch (~small %)

Trp → 5-HTP (tryptophan hydroxylase, BH₄) → serotonin (PLP) → melatonin (pineal). Serotonin is degraded by MAO → 5-HIAA in urine.

🔆 Kynurenine branch (majority)

Trp → kynurenine → ... → niacin (NAD⁺/NADP⁺). About 60 mg tryptophan → 1 mg niacin. Needs B6 — B6 deficiency impairs this route.

Clinical links

Carcinoid tumour diverts huge amounts of tryptophan to serotonin → ↑5-HIAA and can cause pellagra-like signs (niacin "stolen"). Hartnup disease (↓tryptophan absorption) and corn-based diets also cause pellagra because less tryptophan is available to make niacin.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

⚗️ Tryptophan and Asparagine — the Named Routes

The two routes out of tryptophan, written out

Serotonin route: tryptophan → 5-hydroxytryptophan (tryptophan hydroxylase, BH₄ — the same kind of enzyme as phenylalanine hydroxylase) → serotonin (decarboxylation, PLP) → N-acetylserotonin → melatonin in the pineal gland. Niacin route: the kynurenine pathway, needing B6. Two named diseases follow from tryptophan: Hartnup disease — a defect of the membrane transporter for neutral amino acids in intestine and renal tubule, so tryptophan is lost in stool and urine and the patient develops pellagra-like signs; and pellagra itself, from deficiency of niacin and tryptophan, with photosensitive dermatitis, then diarrhoea and dementia, fatal untreated and cured by either tryptophan or niacin.

Asparagine — synthesis, breakdown, and why oncologists care

Asparagine synthetase makes it from aspartate + glutamine + ATP, through the intermediate aspartyl phosphate; the coupled hydrolysis of PPi to 2 Pi makes the reaction effectively irreversible. Asparaginase runs the other way, giving aspartate + NH₃, and a transaminase then delivers all four carbons to oxaloacetate. Leukaemic lymphoblasts make little asparagine synthetase, so injected asparaginase starves them — a standard drug in acute lymphoblastic leukaemia.

🧬 The Polyamines

Spermidine and spermine are made from ornithine by ornithine decarboxylase (putrescine first), which then receives a propylamine group from decarboxylated S-adenosylmethionine — so arginine/ornithine supplies the backbone and methionine the extension.

Why they matter

They are small polycations that bind DNA, RNA and ribosomes and are required wherever cells grow and divide rapidly — which is why ornithine decarboxylase is one of the shortest-lived and most tightly regulated enzymes in the cell, and a drug target in trypanosomiasis (eflornithine).

Creatine & Whole-Body Protein Status

Frequently tested: creatine synthesis (Gly + Arg + Met/SAM), creatinine as muscle-mass marker, nitrogen balance states, marasmus vs kwashiorkor

💪 Creatine & Creatinine Metabolism

Creatine synthesis (2 steps — Glycine + Arginine + Methionine/SAM):

Glycine + Arginine AGAT→kidney · releases ornithine Guanidinoacetate GAMT→liver · + SAM (methyl donor) Creatine

Creatine phosphate (phosphocreatine):
Creatine + ATP ⇌ Creatine phosphate + ADP (by creatine kinase)
Acts as rapid ATP buffer in the first seconds of muscle contraction.

Creatinine:
Creatine and creatine phosphate spontaneously and non-enzymatically cyclize → creatinine at a constant daily rate.

Creatinine excreted in urine is proportional to muscle mass — useful marker.

↑ Serum Creatinine = Kidney Malfunction

Creatinine is normally rapidly cleared; any rise = renal impairment. CK-MB elevation = MI (different isoform).

⚖️ Nitrogen Balance

Positive Nitrogen Balance

Nitrogen intake > nitrogen excretion. Net protein synthesis. Occurs in: growing children, pregnancy, recovery from illness, athletes building muscle.

Negative Nitrogen Balance

Nitrogen excretion > nitrogen intake. Net protein breakdown. Occurs in: starvation, severe burns, trauma, surgery, cancer, high fever, uncontrolled diabetes.

Zero Balance (Equilibrium)

Intake = excretion. Healthy adults in steady state — protein turnover is constant but no net gain/loss.

Measurement

Nitrogen balance = N intake (dietary protein ÷ 6.25) − N excretion (mainly urine urea + stool + skin). Each gram of protein contains ~16% nitrogen.

Clinical Significance

• Thyroid hormones at physiological levels → positive N balance (anabolic). At high levels → negative N balance (protein catabolism).
• Glucocorticoids → negative N balance (promote muscle proteolysis → gluconeogenic substrates).
• Insulin → positive N balance (promotes amino acid uptake and protein synthesis in muscle).

🥣 Protein-Energy Malnutrition: Marasmus & Kwashiorkor

Feature	Marasmus	Kwashiorkor
Cause	Deficiency of BOTH calories AND protein	Adequate calories but severe protein deficiency
Body	Extreme wasting of both fat AND muscle	Muscle wasting but fat preserved; edema (esp. abdomen)
Albumin	Near-normal (body adapts)	Very low → oncotic pressure ↓ → edema
Key signs	Anaemia, loss of adipose, muscle wasting, growth retardation, "skin and bones" appearance	Oedema (ascites, pot belly), skin/hair changes, fatty liver (↓ VLDL synthesis), moon face
Metabolic	Ketosis; body uses fat + muscle as fuel	Insulin ↑ (from carbs) → inhibits lipolysis → fat in liver; no VLDL (no apolipoproteins)

Marasmus — Exam Points ★ asked 2025

Directly tested: symptoms include anaemia, loss of adipose tissue, muscle wasting, growth retardation. The liver is NOT enlarged (unlike kwashiorkor). Marasmus = starvation → the body uses all energy stores.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

💪 Creatinine Clearance

Creatinine clearance — the formula and what it is for Past Q

Clearance is the volume of plasma completely cleared of a substance by the kidney per unit time. C_cr (mL/min) = (U_cr × V) / P_cr — urine creatinine concentration × urine flow rate, divided by plasma creatinine. Because creatinine is filtered and barely reabsorbed, this estimates the glomerular filtration rate and detects early renal failure long before symptoms. And because 24-hour creatinine output is proportional to muscle mass and constant for a given person, it is also used to check that a 24-hour urine collection was complete.

Heme, Porphyrins & Jaundice

Frequently tested: heme synthesis (ALA synthase, order of porphyrinogens), porphyrias & photosensitivity, conjugated vs unconjugated bilirubin, jaundice types & urine findings

🫀 The Liver — Central Metabolic Hub

The liver is the body's main metabolic organ. Understanding why each function belongs to the liver is high-yield.

Function	Why the liver?	Key enzymes/molecules
Gluconeogenesis	Only tissue (with kidney) expressing glucose-6-phosphatase → can release free glucose into blood	PEPCK, PC, F-1,6-BPase, G-6-Pase
Glycogen storage & release	Liver glycogen (~100 g) maintains blood glucose for other tissues. Muscle glycogen serves muscle only.	Glycogen synthase, glycogen phosphorylase
Urea synthesis	Liver is the only organ with the complete urea cycle. All ammonia from amino acid catabolism arrives here via portal blood as alanine or glutamine.	CPS-I, OTC, arginase — all 5 enzymes
Ketogenesis	Liver mitochondria are the exclusive site of ketone body synthesis. Note: liver cannot use its own ketones (lacks thiophorase/succinyl-CoA transferase).	HMG-CoA synthase, HMG-CoA lyase
Cholesterol & bile acid synthesis	Liver is the exclusive site of bile acid synthesis from cholesterol (7α-hydroxylase). All primary bile acids produced here.	HMG-CoA reductase, 7α-hydroxylase
Lipoprotein assembly	VLDL (carries endogenous lipids) and HDL precursors assembled in liver. Chylomicron remnants cleared here via ApoE.	MTP, ApoB-100, VLDL secretion
Protein synthesis	Albumin (main plasma oncotic protein), all clotting factors (II, V, VII, IX, X, fibrinogen), acute-phase proteins (CRP, ferritin).	Rough ER ribosomes
Bilirubin conjugation	Hepatocytes conjugate bilirubin with glucuronate (UGT1A1) → water-soluble → secreted into bile. Failure → jaundice.	UGT1A1, MRP2 (canalicular export)
Drug detoxification	CYP450 enzymes (Phase I) + glucuronidation/sulfation (Phase II) in ER. Requires NADPH and O₂.	CYP3A4, CYP2D6, UGT
Vitamin D activation (first step)	25-hydroxylase converts cholecalciferol → 25-OH-D₃ (calcidiol). Second step (→calcitriol) occurs in kidney.	CYP27A1 (25-hydroxylase)
Iron storage & regulation	Hepcidin produced by liver. Ferritin stores excess iron. Transferrin synthesised here.	Hepcidin, ferritin, transferrin

Liver Failure — What Fails?

Hepatocellular failure → ↓albumin (oedema, ascites) + ↓clotting factors (bleeding, ↑PT/INR) + ↑unconjugated bilirubin (impaired uptake) + ↑conjugated bilirubin (impaired secretion) + hypoglycaemia (↓GNG) + hyperammonaemia (↓urea cycle) + hypertriglyceridaemia (↓VLDL).

🩸 Heme Synthesis

Rate-limiting: ALA Synthase (Gly + Succinyl-CoA → ALA, needs PLP/B6, mitochondria). Final step: Protoporphyrin IX + Fe²⁺ → Heme (Ferrochelatase, mitochondria). Lead inhibits ALA Dehydratase and Ferrochelatase.

⚡ Porphyrias

Porphyria	Enzyme Defect	Key Features
AIP	PBG Deaminase	Neurological symptoms. NO photosensitivity. ALA + PBG accumulate.
PCT	Uroporphyrinogen Decarboxylase	Photosensitivity; dark urine (uroporphyrin).

💛 Types of Jaundice

Type	Bilirubin ↑	Urine bilirubin	Urine urobilinogen	Stool color	Key markers	Cause
Pre-hepatic (hemolytic)	Indirect/unconjugated only	❌ Absent — unconjugated is albumin-bound, not water-soluble	↑ Increased — more bilirubin enters gut → more urobilinogen	Normal/dark brown	↑LDH, ↑reticulocytes, ↓haptoglobin	Excess RBC destruction (G6PD, spherocytosis, sickle cell)
Hepatic (hepatocellular)	Both conjugated AND unconjugated	✅ Present — conjugated leaks into blood	Variable (↓ if severe damage)	Normal or pale	↑↑ALT > AST (viral hepatitis); AST:ALT >2 (alcoholic). ↑PT	Hepatitis (viral, alcoholic, drug-induced), cirrhosis, liver failure
Post-hepatic (obstructive)	Direct/conjugated only	✅ Present (dark urine = bilirubinuria)	❌ Absent — no bile reaches gut	Pale/clay-colored (acholic)	↑ALP, ↑GGT, ↑conjugated bilirubin; no urobilinogen	Bile duct obstruction (gallstones, pancreatic cancer, cholangitis)

Bilirubin and the Blood-Brain Barrier ★ asked 2018, 2019

Only unconjugated (indirect) bilirubin can cross the blood-brain barrier — it is lipid-soluble. Conjugated bilirubin is water-soluble and cannot. This causes kernicterus (brain damage) in neonates with high unconjugated bilirubin. Directly tested: "which form of bilirubin crosses the BBB?" → unconjugated.

Which Disease Causes Bilirubin in Urine?

Only conjugated (direct) bilirubin appears in urine (it is water-soluble). Diseases: Dubin-Johnson syndrome, Rotor syndrome, obstructive jaundice, hepatocellular jaundice. Unconjugated hyperbilirubinemias (Gilbert, Crigler-Najjar, hemolytic jaundice) do NOT cause bilirubinuria.

💛 Conjugated vs Unconjugated Bilirubin & Urobilinogen — Teacher Listed as High Priority

Teacher explicitly listed: "conjugated, unconjugated urobilinogen, hyperbilirubinemia — genetic, erythrocyte enzymes" as a Kłos exam topic.

Compound	Properties	Where Found	Clinical Significance
Unconjugated (indirect) bilirubin	Water-INsoluble, lipid-soluble, bound to albumin in plasma	Blood only (not urine)	Elevated in: hemolysis, Gilbert, Crigler-Najjar. Can cross BBB → kernicterus
Conjugated (direct) bilirubin	Water-SOLUBLE (two glucuronate groups attached)	Bile, blood (if obstructed), urine	Elevated in: cholestasis, Dubin-Johnson, Rotor. Appears in urine → dark urine (bilirubinuria)
Urobilinogen	Colorless, water-soluble. Formed by gut bacteria from bilirubin (diglucuronide → urobilinogen)	Gut, portal blood, urine, bile	↑ in hemolysis (pre-hepatic). Absent in complete obstruction (no bilirubin enters gut). Small amounts in normal urine
Urobilin / Stercobilin	Urobilinogen oxidized → urobilin (brown). Stercobilin = fecal pigment	Feces, some urine	Absent stools = complete bile duct obstruction (pale stools). Brown stool color = normal stercobilin

Genetic Hyperbilirubinemias — Erythrocyte Enzyme Defects

Hemolytic (pre-hepatic) jaundice caused by erythrocyte enzyme defects:
• G6PD deficiency: ↓NADPH → oxidative hemolysis → ↑unconjugated bilirubin
• Pyruvate kinase deficiency: ↓ATP in RBCs → membrane failure → hemolysis → ↑unconjugated
• Hereditary spherocytosis (spectrin/ankyrin defects): fragile RBCs → hemolysis
All erythrocyte enzyme defects cause unconjugated hyperbilirubinemia (pre-hepatic jaundice): no bilirubinuria, ↑urobilinogen.

Alcohol and Bilirubin (Teacher Topic)

Chronic alcohol use → liver cell damage → hepatocellular jaundice with BOTH conjugated and unconjugated bilirubin elevated. Alcohol Dehydrogenase and Aldehyde Dehydrogenase — alcohol metabolism — were specifically listed by Kłos as exam topics.

Bilirubin conjugation is performed by UDP-Glucuronosyltransferase (UGT) in the liver. Heme synthesis does not occur in mature red blood cells (they have no nucleus or mitochondria).

Rotor Syndrome — OATP1B1/OATP1B3 Deficiency ★ asked 2023

Rotor syndrome is caused by deficiency of the hepatic uptake transporters OATP1B1 and OATP1B3. This impairs reuptake of conjugated bilirubin into hepatocytes → conjugated bilirubin spills into blood → dark urine. The liver color is normal (unlike Dubin-Johnson's black liver). This was asked in the 2023 exam: "OATP1B1 and OATP1B3 deficiency causes?" → Rotor syndrome.

🧪 Hepatic Phase II (Conjugation) Reactions

Drug/toxin detoxification has two phases. Phase I (CYP450: oxidation/reduction/hydrolysis) exposes or adds a reactive group. Phase II attaches a large polar group to make the molecule water-soluble for excretion.

Phase II reaction	Donor / enzyme	Example substrate
Glucuronidation	UDP-glucuronate → UGT	Bilirubin, steroids, many drugs (high capacity)
Sulfation	PAPS → sulfotransferase	Steroids, catecholamines, paracetamol (low capacity)
Glutathione conjugation	GSH → glutathione S-transferase	Reactive electrophiles → mercapturic acid; paracetamol NAPQI
Acetylation	Acetyl-CoA → N-acetyltransferase	Isoniazid, sulfonamides (fast/slow acetylators)
Methylation	SAM	Catecholamines (COMT), thiopurines
Glycine conjugation	Glycine	Benzoic acid → hippuric acid; bile acids

Paracetamol clinical link

Normally paracetamol is glucuronidated/sulfated. In overdose these saturate → CYP450 makes toxic NAPQI → depletes glutathione → liver necrosis. N-acetylcysteine restores glutathione. This is the classic Phase-I-makes-it-worse, Phase-II-detoxifies story.

🍯 The Uronic Acid (Glucuronate) Pathway

A minor branch of glucose metabolism that makes glucuronic acid (as UDP-glucuronate). It makes no ATP — its purpose is to supply an activated sugar acid for conjugation and connective-tissue synthesis.

⚗️ Formation of Glucuronic Acid

Glucose-1-P → UDP-glucose UDP-glucose DH→ UDP-glucuronate

Major functions of the pathway

1. Conjugation/detoxification — UDP-glucuronate is the donor for UGT to conjugate bilirubin and drugs (Phase II). 2. Glycosaminoglycan synthesis — glucuronic acid is a building block of hyaluronic acid, heparin, chondroitin. 3. In many animals (not humans) it leads to vitamin C synthesis — humans lack L-gulonolactone oxidase, so we must eat vitamin C. The exam point "uronic acid is formed from ___" → glucose (oxidation at C-6).

🧱 Porphyrin Chemistry — The Side Chains That Name Them

Porphyrins are cyclic planar molecules formed by linking four pyrrole rings through methenyl bridges. The three you must know differ only in which side chains sit on the ring.

Porphyrin	Side chains
Uroporphyrin	Acetate + propionate
Coproporphyrin	Methyl + propionate
Protoporphyrin IX	Vinyl + methyl + propionate — the one that becomes heme

Porphyrinogens versus porphyrins

Porphyrinogens (such as uroporphyrinogen) are the reduced, colourless forms. They are the true intermediates between porphobilinogen and the oxidised, coloured protoporphyrins of heme biosynthesis. When a porphyrinogen accumulates and leaks out, it oxidises spontaneously in air and light to the coloured porphyrin — which is exactly why porphyria urine darkens on standing and why the skin lesions are photosensitive.

🎚️ ALA Synthase — Two Genes, Two Kinds of Regulation

The formation of δ-aminolevulinic acid from glycine and succinyl-CoA, using pyridoxal phosphate as coenzyme, is the committed and rate-limiting step of porphyrin biosynthesis. There are two separate genes, regulated completely differently.

ALAS1 — housekeeping

Found in all tissues. In the liver the rate is variable, responding to demand. Heme represses it in three ways at once: repressing transcription, increasing degradation of the enzyme, and decreasing import of the enzyme into mitochondria.

Drug induction: administration of a large number of drugs causes a significant increase in hepatic ALAS1 activity, because those drugs are metabolised by the microsomal cytochrome P450 monooxygenase system — which consumes heme, lowering the regulatory pool and releasing the brake. This is precisely why barbiturates, sulfonamides and many others precipitate acute porphyria attacks.

ALAS2 — erythroid

Erythroid tissue produces only ALAS2. In bone marrow the rate is constant, matched to the rate of globin synthesis — not to heme feedback.

Its gene is located on the X chromosome. Loss-of-function mutations in ALAS2 cause X-linked sideroblastic anaemia — see sideroblastic anaemia.

☠️ Lead blocks two enzymes, not one

ALA dehydratase and ferrochelatase are both inhibited by lead — they are the zinc-containing enzymes of the pathway. The result is raised ALA in urine and raised free erythrocyte protoporphyrin together, with basophilic stippling of red cells. See lead poisoning.

🗂️ Classifying the Porphyrias

Two questions sort every porphyria: where is the enzyme defect expressed (liver or bone marrow), and what accumulates — because that determines whether the patient gets neurological attacks, skin disease, or both.

🧠 NEUROVISCERAL — early block

Defects before the porphyrinogens are formed accumulate ALA and porphobilinogen, which are neurotoxic. Symptoms: abdominal pain, vomiting, neuropathy, psychiatric disturbance, red-brown urine. No photosensitivity — these precursors are not photoactive. Example: acute intermittent porphyria.

☀️ CUTANEOUS — late block

Defects after the ring has closed accumulate porphyrinogens, which oxidise to coloured porphyrins that absorb light at ~400 nm and generate free radicals in the skin. Symptoms: blistering, fragility, scarring on sun-exposed skin. Examples: porphyria cutanea tarda, congenital erythropoietic porphyria.

Treatment logic follows directly

Acute attacks are treated with glucose and haem infusion — both repress ALAS1 and shut the pathway down at its source. Cutaneous porphyrias are managed with sunlight avoidance, and PCT additionally with phlebotomy, because iron aggravates it. Avoiding precipitating drugs is central to both.

🧬 The Porphyrin Intermediates — Naming Them in Order

Examiners ask which compound is the first tetrapyrrole, and which enzyme sits between two named intermediates. The chain is worth knowing by name, not just by its two ends.

Succinyl-CoA + Glycine

→ALA synthase · PLP · mitochondrion

δ-Aminolevulinic acid

→ALA dehydratase · Zn · cytosol

Porphobilinogen

→PBG deaminase

Hydroxymethylbilane

→uroporphyrinogen III cosynthase

Uroporphyrinogen III

→uroporphyrinogen decarboxylase

Coproporphyrinogen III

→coproporphyrinogen oxidase

Protoporphyrinogen IX

→protoporphyrinogen oxidase

Protoporphyrin IX

→ferrochelatase + Fe²⁺

Haem

The three points that get tested

1 · Four porphobilinogen molecules condense to the linear hydroxymethylbilane, which then cyclises — so the first tetrapyrrole ring compound is uroporphyrinogen III, with hydroxymethylbilane as its immediate linear precursor.
2 · The type I isomers that form when the cosynthase is missing are useless by-products.
3 · The pathway starts and ends in the mitochondrion and does the middle in the cytosol — and bile pigments come from the degradation of this haem, never from cholesterol.

👶 Neonatal Jaundice — Phototherapy and the Phenobarbital Ladder

In the first days of life an immature, not-yet-induced UGT1A1, a shorter red-cell lifespan and active enterohepatic recycling produce a transient unconjugated hyperbilirubinaemia, appearing after day 2–3. The danger is kernicterus.

Why phototherapy works

Blue light converts bilirubin into water-soluble isomers (lumirubin and photoisomers) that can be excreted in bile and urine without being conjugated — it bypasses the missing enzyme rather than inducing it.

Why phenobarbital sometimes works

Phenobarbital induces UGT1A1. It can only help if there is some enzyme to induce — which is exactly what makes it a diagnostic test.

Ranking the three conjugation defects by their response

Gilbert (UGT1A1 reduced to ~30%) — a low dose is enough; benign, unmasked by fasting, illness, alcohol or stress. → Crigler–Najjar type II (Arias) — partial deficiency, responds to a high dose. → Crigler–Najjar type I — enzyme completely absent, no response at all; phototherapy buys time and liver transplant is the only cure. The phenobarbital response is the classic way of separating them.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧫 Classifying the Porphyrias — One Per Enzyme Past Q

Porphyrias are named and sorted on two axes: by the organ that suffers — hepatic or erythropoietic (liver or bone marrow) — and by the clinical picture — acute (neurovisceral) or cutaneous (photosensitive). Most are autosomal dominant with low penetrance; congenital erythropoietic porphyria is recessive.

The rule that makes the whole table predictable

A block before the ring is closed (steps 1–3) piles up ALA and porphobilinogen — colourless, neurotoxic, not photoactive → abdominal pain and neuropsychiatric signs. A block after the ring is closed (steps 4–8) piles up porphyrinogens, which auto-oxidise to porphyrins — coloured, light-absorbing → photosensitivity. Steps 6 and 7 sit on the boundary and give both.

#	Enzyme blocked	Disease · class	Signs	Laboratory
1	ALA synthase (erythroid form)	X-linked sideroblastic anaemia erythropoietic	Anaemia	↓ red cell count and haemoglobin
2	ALA dehydratase	ALA-dehydratase deficiency porphyria hepatic · acute	Abdominal pain, neuropsychiatric	↑ urinary ALA and coproporphyrin III
3	Uroporphyrinogen I synthase (PBG deaminase)	Acute intermittent porphyria hepatic · acute	Abdominal pain, neuropsychiatric, no skin disease	↑ urinary ALA and PBG
4	Uroporphyrinogen III synthase	Congenital erythropoietic porphyria erythropoietic · cutaneous	Severe photosensitivity, disfigurement — the "werewolf" prototype	↑ uroporphyrin I in urine, faeces and red cells
5	Uroporphyrinogen decarboxylase	Porphyria cutanea tarda — the commonest hepatic · cutaneous	Blistering of sun-exposed skin	↑ urinary uroporphyrin I
6	Coproporphyrinogen oxidase	Hereditary coproporphyria hepatic both	Photosensitivity and abdominal pain, neuropsychiatric	↑ ALA, PBG, faecal coproporphyrin III
7	Protoporphyrinogen oxidase	Variegate porphyria hepatic both	The same combination	↑ ALA, PBG, faecal protoporphyrin IX
8	Ferrochelatase	Erythropoietic protoporphyria erythropoietic · cutaneous	Photosensitivity	↑ red cell and faecal protoporphyrin

Making the diagnosis

Three things together: clinical and family history, physical examination (skin, neurological signs), and laboratory tests — which porphyrin precursor is raised, and in which fluid (urine, faeces or red cells). Note that between attacks the biochemistry can be almost normal.

Treatment as it stands

Symptomatic, with gene-level therapy still a hope. Avoid drugs that induce cytochrome P450 (they consume haem and de-repress ALA synthase), alcohol and fasting. Large amounts of carbohydrate, and intravenous haem (haematin), both repress ALAS1 and cut off production of the harmful precursors. For the photosensitive forms: sunlight avoidance, sunscreens and β-carotene, which quenches free radicals and reduces the skin damage.

💛 Retention, Regurgitation and the Conjugated Defects

The two words the question uses Past Q

Retention hyperbilirubinaemia — too much bilirubin is produced (or not conjugated), so the unconjugated fraction rises. It cannot be filtered, so the urine holds no bilirubin: acholuric jaundice, and the only setting in which kernicterus can occur. Regurgitation hyperbilirubinaemia — conjugated bilirubin already made in the liver refluxes into the blood because bile cannot get out; it is water-soluble, so it appears in urine: choluric jaundice, dark urine.

The two benign conjugated defects

Dubin–Johnson syndrome — the canalicular export pump MRP2 is defective, so conjugated bilirubin cannot be pumped into bile; a dark, almost black liver from pigment deposits, but a normal lifespan. Rotor syndrome — a defect of hepatic storage/uptake of conjugated bilirubin, clinically similar but with no pigment in the liver. Both are benign and need no treatment; both are causes of a raised conjugated bilirubin with normal liver enzymes.

The full causal list, split by fraction

Unconjugated: haemolytic anaemias, neonatal physiological jaundice, Crigler–Najjar I and II, Gilbert syndrome, toxic hyperbilirubinaemia. Conjugated: obstruction of the biliary tree, Dubin–Johnson, Rotor, and the hepatitides and other liver diseases (which usually raise both).

Numbers worth quoting

Hyperbilirubinaemia begins above about 1 mg/dL; at 2–2.5 mg/dL bilirubin diffuses into tissues and jaundice becomes visible. Normal direct (conjugated) 0.1–0.4 mg/dL, indirect (unconjugated) 0.2–0.7 mg/dL; urinary urobilinogen 0–4 mg/24 h and faecal urobilinogen 40–280 mg/24 h — increased in haemolysis, absent in complete obstruction.

💛 Bilirubin Inside the Hepatocyte — and the Uronic Acid Pathway

Uptake, ligandin, and the rate-limiting step

The albumin–bilirubin complex docks at a receptor on the hepatocyte membrane; the bilirubin alone is carried into the cytosol and is then bound by ligandin (a glutathione S-transferase), which prevents it flowing back out and delivers it to the smooth ER for conjugation. Aspirin and sulfonamides can displace bilirubin from albumin — the reason they are avoided in the jaundiced newborn. Secretion into bile is by MRP2 and is an active, rate-limiting step, so it fails first in liver disease; about 85% of what reaches the intestine is the diglucuronide, which is not reabsorbed.

Uronic acid pathway — two details

UDP-glucose is oxidised to UDP-glucuronic acid in a two-step reaction catalysed by UDP-glucose dehydrogenase. Glucuronate is the donor for conjugation of bilirubin, steroid hormones and drugs, and a building block of proteoglycans; it is also reduced to L-gulonate, the direct precursor of ascorbate. Most mammals go on to make vitamin C; humans, other primates and guinea pigs cannot, because they lack L-gulonolactone oxidase. The pathway yields no ATP, and its other end product is xylulose.

Iron & Lab Tests

Frequently tested: Fe²⁺ vs Fe³⁺ forms (ferritin, hemin, hemosiderin), daily iron requirement, TIBC in iron-deficiency vs hemochromatosis, transferrin

🔴 Iron Metabolism — Teacher Listed as Kłos Exam Topic

Iron metabolism is explicitly listed by the teacher (Kłos) as a high-priority exam topic. Key aspects:

Concept	Detail
Forms of iron	Fe²⁺ (ferrous) = absorbed form (in diet, in heme). Fe³⁺ (ferric) = stored (ferritin, hemosiderin), transported (transferrin). Vitamin C reduces Fe³⁺ → Fe²⁺ for absorption.
Absorption	Non-heme iron: Fe³⁺ → Fe²⁺ by duodenal cytochrome b (requires vitamin C) → enters enterocyte via DMT-1. Heme iron: absorbed directly as heme → cleaved by heme oxygenase.
Transport	Transferrin: plasma iron transport protein (carries Fe³⁺). In iron deficiency: TIBC ↑ (more empty transferrin). In hemochromatosis: TIBC ↓ (transferrin saturated).
Storage	Ferritin: intracellular storage (soluble). Hemosiderin: insoluble storage (when ferritin overwhelmed). Ferritin is also an acute-phase protein (rises in inflammation regardless of iron status).
Regulation	Hepcidin (liver peptide): master iron regulator. High iron → ↑hepcidin → degrades ferroportin → ↓iron absorption and ↓iron release from stores. Low iron → ↓hepcidin → ↑iron absorption.

Alcohol & Iron Metabolism

Alcohol disrupts iron metabolism: chronic alcohol use → ↑iron absorption, ↓hepcidin → can worsen hemochromatosis. Also: alcohol induces GGT (as noted in lab tests). ALT/AST ratio >2 suggests alcoholic hepatitis.

🧪 Benedict Test & Reducing Sugars (Exam Tested)

The Benedict test detects reducing sugars — sugars that have a free aldehyde or ketone group that can donate electrons. A positive result gives a brick-red or orange precipitate. Monosaccharides are all reducing sugars. Among disaccharides, sucrose is NOT a reducing sugar (its anomeric carbons are both locked in the glycosidic bond).

Sugar	Benedict Test	Why
Glucose, Fructose, Galactose	✅ Positive	Free reducing group
Maltose, Lactose	✅ Positive	Free reducing group on one end
Sucrose	❌ Negative	No free reducing group

Starch Digestion = Complete

Complete digestion of starch produces monosaccharides (glucose). These ARE reducing sugars → positive Benedict test (brick-red/orange color). Starch itself may give a weaker reaction because its reducing ends are few.

🧪 Other Important Biochemical Tests ★ recur every year

Teacher (Kłos) confirmed these WERE asked in past years

Salkowski (cholesterol), Hay's (bile acids in pathological urine — not Pettenkofer's), reducing sugars, Legal's (ketone bodies), OGTT, ALT/AST, alkaline phosphatase, and urobilinogen. Prioritise these.

Test	Detects	Positive Result
Benedict	Reducing sugars (glucose, fructose, lactose, maltose — NOT sucrose)	Brick-red / orange precipitate
Seliwanov	Ketoses (e.g. fructose) — distinguishes ketoses from aldoses	Red color
Ehrlich	Urobilinogen in urine	Cherry-red ring. Colour appears BEFORE heating → ↑urobilinogen (e.g. haemolysis)
Salkowski	Cholesterol	Red color in chloroform layer
Hay's (sulfur powder)	Bile acids/salts in pathological urine	Fine sulfur powder sprinkled on the surface sinks if bile salts are present (they lower surface tension). A physical surface-tension test. Teacher: Hay's was asked, Pettenkofer's was not.
Legal's	Ketone bodies (acetoacetate / acetone)	Red-violet colour with sodium nitroprusside in alkaline solution (Legal's = nitroprusside test for ketones)
Van den Bergh	Bilirubin (direct vs indirect)	Pink/purple color
Biuret	Proteins (peptide bonds — needs ≥2 peptide bonds, alkaline)	Violet color
Kay test (BAO/MAO)	Gastric acid: Basal & Maximal Acid Output	Normal: BAO 0–5, MAO 5–25 mmol/h

🩸 Clinical Function Tests — OGTT, Transaminases & ALP ★ teacher-listed

OGTT (Oral Glucose Tolerance Test)

Give 75 g oral glucose, measure blood glucose at 0 and 2 h. Diabetes: fasting ≥7.0 mmol/L or 2-h ≥11.1 mmol/L. Impaired glucose tolerance: 2-h 7.8–11.0. Used to diagnose diabetes & gestational diabetes.

ALT & AST (transaminases)

ALT = liver-specific. AST = liver + heart + muscle. Both ↑ in hepatocellular damage. AST/ALT (De Ritis) ratio >2 suggests alcoholic liver disease; <1 suggests viral hepatitis. Require PLP (B6).

Alkaline Phosphatase (ALP)

↑ in cholestasis (bile duct obstruction — most specific) and bone disease (Paget, metastases, rickets — osteoblast activity). Normal AST/ALT + high ALP → think bone or bile. GGT rises with ALP confirms a liver source.

Urobilinogen (urine)

Formed from bilirubin by gut bacteria; some is reabsorbed and excreted in urine. ↑ in haemolysis & hepatocellular damage; absent in complete biliary obstruction. Detected by the Ehrlich test.

🧪 Other Named Tests (reference) ⚪ never appeared in past exams

None of these have shown up in any available past paper — included only for completeness, low priority.

Test	Detects
Burstein & Samaille precipitation	Beta-lipoproteins
Potassium permanganate	Double bonds in unsaturated fatty acids
Weltmann–Barrenscheer	Urea in serum and urine
Schiff's	Aldehydes
Fiske–Subbarow	Inorganic phosphate levels
Pettenkofer's	Bile salts (chemical colour reaction — but teacher says this one was not asked; Hay's was)
Obermayer's test	Indican in urine
Reitman–Frankel	Aminotransferases (ALT / AST)
Wohlgemuth	Amylase activity
Lowry test	Protein levels
Feulgen reaction	Chromosomal material / DNA 🧬

🧪 GGT (Gamma-Glutamyl Transferase / GGTP) — Teacher Specifically Mentioned

GGT (γ-GT, GGTP) is a liver enzyme found on the surface of hepatocytes and bile duct cells. It is an important clinical lab marker for:

Liver/biliary disease — elevated in cholestasis, hepatitis, cirrhosis
Alcohol use — GGT is the most sensitive marker of alcohol-related liver disease; it rises with chronic alcohol consumption (alcohol induces GGT production)
Distinguishing liver vs bone source of ALP — if ALP is elevated, a high GGT confirms the source is liver (not bone), since bone does not express GGT

GGT Biochemical Role

GGT transfers a glutamyl group from glutathione (GSH) or other γ-glutamyl compounds to amino acids. It is part of the γ-glutamyl cycle involved in amino acid transport across cell membranes (especially in kidney). Also involved in glutathione metabolism.

Exam: GGT is the alcohol marker

GGT rises early with alcohol use, before ALT/AST. Combination of elevated GGT + elevated MCV (macrocytosis from B12/folate deficiency in alcoholics) → strongly suggests chronic alcohol use. Teacher specifically mentioned "Lab levels important: GGTP."

🩸 Plasma Proteins & the Proteinogram (Electrophoresis)

Serum protein electrophoresis separates blood proteins into 5 bands by charge/size. The pattern (the "proteinogram") is a classic diagnostic tool. Nearly all plasma proteins are made by the liver — except immunoglobulins (plasma cells).

Fraction	Main proteins	Function
Albumin	Albumin (~60% of total)	Main oncotic pressure; carries drugs, bilirubin, fatty acids, Ca²⁺, hormones
α₁-globulin	α₁-antitrypsin, α-fetoprotein	Antiprotease (protects lung); ↓ in AAT deficiency
α₂-globulin	Haptoglobin, ceruloplasmin, α₂-macroglobulin	Bind free Hb (↓ in haemolysis); copper transport
β-globulin	Transferrin, complement, β-lipoprotein (LDL)	Iron transport; immune; lipid transport
γ-globulin	Immunoglobulins (antibodies)	Immune defence — made by plasma cells, NOT liver

Hypoalbuminaemia

Causes: liver failure (↓synthesis), nephrotic syndrome (urinary loss), malnutrition (kwashiorkor), burns, inflammation. Result: oedema (↓oncotic pressure). Albumin is a negative acute-phase reactant (falls in inflammation).

Globulin changes

Polyclonal ↑γ = chronic infection/inflammation. Monoclonal spike (M-band) = multiple myeloma. ↓γ = immunodeficiency. Acute-phase ↑: CRP, fibrinogen, haptoglobin, ferritin, α₁-antitrypsin.

Plasma proteins as transporters

Albumin (bilirubin, FFA, drugs, Ca²⁺), transferrin (iron), ceruloplasmin (copper), transcortin (cortisol), SHBG (sex steroids), retinol-binding protein (vit A), transthyretin (T4). Bound hormone/ion is a reservoir; only the free fraction is active.

💧 Starling Forces — Capillary Fluid Balance & Oedema

Whether fluid leaves or re-enters a capillary is set by four "Starling forces" — the balance of hydrostatic (pushing) and oncotic (pulling) pressures across the capillary wall. Plasma albumin provides the main pull that keeps fluid in.

Push fluid OUT

Capillary hydrostatic pressure (blood pressure); interstitial oncotic pressure

Pull fluid IN

Plasma oncotic pressure (mainly albumin); interstitial hydrostatic pressure

Oedema cause	Starling force disturbed
↓ Albumin (liver failure, nephrotic, kwashiorkor)	↓ plasma oncotic pressure → fluid leaks out
Heart failure / venous obstruction	↑ capillary hydrostatic pressure
Lymphatic blockage	Fluid/protein not drained (lymphoedema)
Inflammation	↑ capillary permeability → protein leaks into tissue

Link to albumin

This is exactly why hypoalbuminaemia causes oedema: less albumin → less oncotic "pull" → net filtration out of capillaries → fluid accumulates in tissues (and as ascites in kwashiorkor/liver failure).

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🔴 Reciprocal Control of Ferritin and the Transferrin Receptor

Dual reciprocal control — how a cell sets its own iron uptake Past Q

The messenger RNAs for ferritin (storage) and for transferrin receptor 1 (uptake) both carry hairpin iron-response elements (IREs) — on the 5′ untranslated region in ferritin, on the 3′ untranslated region in TfR1 — and iron-regulatory proteins (IRPs) bind them only when iron is low. Low iron: IRP on the ferritin 5′ IRE blocks translation (less ferritin) while IRP on the TfR1 3′ IRE stabilises the message (more receptors) → the cell grabs iron and stores none. High iron: the IRPs let go — ferritin is translated and TfR1 mRNA is rapidly degraded. One switch, two opposite outcomes.

The transferrin cycle, and the daily balance sheet

Cycle: transferrin–Fe³⁺ binds TfR1 → the complex is taken in by receptor-mediated endocytosis → the endosome is acidified, which releases the iron → it is reduced and leaves the endosome through DMT1 into the cytoplasm → apotransferrin and receptor are recycled to the surface. Balance: about 12–15 mg of iron is eaten daily and only 1–2 mg absorbed; total body iron is 3–4 g, mostly in haemoglobin; roughly 25 mg/day is recycled from senescent red cells by the spleen; and only 0.5–1.5 mg/day is lost, in shed cells and (in women) menstrual blood. There is no regulated route of iron excretion — which is why absorption has to be the control point, and why transfusional overload has nowhere to go.

🧪 Classical Bench Tests and the Urinalysis Report

Three classical tests on bile and cholesterol Past Q

Hay's sulfur test — flowers of sulfur sprinkled on the surface of urine or bile sink if bile salts are present, because bile salts lower surface tension; they float on normal urine. Pettenkofer's test — bile acids plus sucrose and concentrated sulfuric acid give a red-violet ring. Salkowski's test — cholesterol in chloroform layered under concentrated sulfuric acid turns the upper layer red and the acid layer green with fluorescence. Bile's other examinable property is simply that it emulsifies fat: shake oil with water and it separates, shake it with bile and a stable emulsion forms.

Urinalysis — the three parts of the report Past Q

1 · Physical: volume, colour, odour, clarity, specific gravity and pH. 2 · Chemical: protein, reducing sugar, ketone bodies, blood, bile salts and bile pigments. 3 · Microscopic: white cells, red cells and casts. Read together they separate a glomerular from a tubular from a post-renal problem — and they are where several of the biochemistry findings in this course (glucosuria, ketonuria, bilirubinuria, urobilinogen, proteinuria) are actually detected.

⚖️ Plasma Protein Levels, Osmolarity and Two Bench Tests

Total plasma protein is 7.0–7.5 g/dL. Besides albumin, the specific carriers include hemopexin, which binds free haem — haptoglobin catches whole haemoglobin, hemopexin catches the haem released from it — alongside transferrin (iron), ceruloplasmin (copper), transcortin (cortisol), SHBG, TBG, retinol-binding protein and the lipoproteins.

Hypoproteinaemia — six routes

1 · Inadequate intake — starvation, low-protein diet, protein of defective value. 2 · Failure of digestion or amino acid absorption. 3 · Excessive tissue breakdown — hyperthyroidism, malignancy, infection, radiation. 4 · Genetically determined defect of synthesis — one plasma fraction lacking or deficient. 5 · Loss — proteinuria, haemorrhage, loss in faeces, burns. 6 · Injury to the synthesising tissue — liver insufficiency, failure of the reticuloendothelial system.

Hyperproteinaemia — two kinds

With a qualitative change (paraproteinaemia) — one abnormal clone making one abnormal protein: multiple myeloma (plasma cells), Waldenström macroglobulinaemia (lymphoplasmacytoid cells), cryoglobulinaemia, amyloidosis; the M spike on electrophoresis. Without one — hyperfunction of the reticuloendothelial system in bacterial and viral infection and in allergy, protein released from injured tissue, and haemoconcentration in dehydration.

Osmolarity vs osmolality vs oncotic pressure

Osmolarity = osmoles per litre of solution; osmolality = per kilogram of water, which is what is measured — normally 285–295 mOsm/kg, estimated as 2 × [Na⁺] + glucose + urea. Small solutes, sodium above all, set it. Oncotic (colloid osmotic) pressure is only the protein share, about 25 mmHg, but it is the share that cannot cross the capillary wall — so proteins, not sodium, retain water in the vessel.

Iron deficiency — two extra markers

Besides low serum iron, low ferritin, high TIBC, low transferrin saturation and microcytic hypochromic cells, the soluble transferrin receptor and the red-cell protoporphyrin both rise. They rise only in true iron deficiency, which is how they separate it from the anaemia of chronic disease.

The biuret reaction

Reagents: sample (egg albumin in the practical), NaOH, dilute CuSO₄. In alkali Cu²⁺ chelates the nitrogens of peptide bonds: blue → violet (pink-violet with short peptides); no colour change = no peptide bond. More protein = more peptide bonds = more colour, so it quantifies total protein. It needs at least two peptide bonds, so a dipeptide is negative — carnosine, anserine and free amino acids are the classic negatives. Ninhydrin, by contrast, detects free α-amino groups.

Kay's test — measuring BAO and MAO

Gastric juice is aspirated by tube from the pyloric antrum and collected hourly: basal first, then after histamine or pentagastrin (maximal secretion at about 15 minutes). The volume is measured and a 5 mL sample titrated with 0.1 M NaOH against phenol red until the colour matches the standard. Output (mmol HCl/h) = V(NaOH) × 0.1 × total volume ÷ volume titrated. BAO ≈ 1–5 mmol/h, MAO ≈ 5–25 mmol/h.

Key Diseases & Disorders

Frequently tested: PKU & phenylalanine hydroxylase, glycogen & lysosomal storage diseases, cystic fibrosis, favism, collagen disorders

🧬 Amino Acid Metabolism Disorders

Disease	Defect	Key Features
PKU	Phenylalanine Hydroxylase	Phe accumulates; intellectual disability; musty odor; treat with Phe-restricted diet
Albinism	Tyrosinase	No pigmentation; UV sensitivity; normal intelligence
Alkaptonuria	Homogentisate Oxidase	Dark urine; ochronosis; arthritis
Homocystinuria	Cystathionine β-Synthase	Marfanoid; lens dislocation (downward); thrombosis
MSUD	Branched-Chain α-Ketoacid DH	Leu/Ile/Val accumulate; sweet-smelling urine; neurological damage

🏪 Storage Diseases

Disease	Enzyme Deficient	Features
Gaucher	β-Glucocerebrosidase	Hepatosplenomegaly, bone pain; most common lysosomal SD
Von Gierke	Glucose-6-Phosphatase	Hypoglycemia, hepatomegaly, lactic acidosis
Lesch-Nyhan	HGPRT	Gout + self-mutilation; X-linked

Acid–Base Balance

Frequently tested: the four disturbances, recognizing them from pCO₂ & HCO₃⁻, full vs partial vs no compensation (gasometry calculations), insulin deficiency → metabolic acidosis

🧮 The Essentials — Equation & Normal Values

pH = pKₐ + log [A⁻] / [HA] → For bicarbonate: pH = 6.1 + log [HCO₃⁻] / (0.03 × pCO₂)

⭐ Normal Arterial Blood Values — memorize these three

pH

7.35–7.45

(7.4 average)

<7.35 = acidosis · >7.45 = alkalosis

pCO₂

35–45

mmHg

RESPIRATORY component (lungs)

HCO₃⁻

22–26

mmol/L

METABOLIC component (kidneys)

Plugging in the averages: pH = 6.1 + log(24 / (0.03 × 40)) = 6.1 + log(24 / 1.2) = 6.1 + 1.3 = 7.4 ✓

🧽 Buffer Systems

Buffer System	Where it matters	Key Feature
Bicarbonate (HCO₃⁻/H₂CO₃)	Blood (main plasma buffer)	Most important — open system: CO₂ regulated by lungs, HCO₃⁻ by kidneys, independently. Must be reabsorbed from the glomerular filtrate (all of it is reabsorbed when acid–base balance is normal).
Phosphate (H₂PO₄⁻/HPO₄²⁻)	Urine + inside cells	pKa ≈ 6.8. In normal acid–base balance, phosphate is the most important buffer in urine; effective in the distal nephron where tubular pH approaches its pKa.
Ammonia (NH₃/NH₄⁺)	Urine	NH₃ from glutamine (glutaminase) traps secreted H⁺ as NH₄⁺ for excretion. Becomes the main route for excreting excess H⁺ in acidosis (upregulated when acidotic).
Plasma proteins	Blood + cells	Amino-acid side chains buffer H⁺ (imidazole of His, carboxyl of Glu/Asp, amino of Lys). Haemoglobin is a major intracellular protein buffer.

⚗️ The Four Types of Disturbances

Disorder	Primary Change	Compensation	Common Causes
MET Metabolic Acidosis	HCO₃⁻ ↓	pCO₂ ↓ (Kussmaul breathing)	Lactic acidosis (hypovolemia), ketoacids in DKA (type 1 diabetes), severe renal failure
MET Metabolic Alkalosis	HCO₃⁻ ↑	pCO₂ ↑ (hypoventilation)	Vomiting / nasogastric suction (loss of H⁺), diuretics (furosemide, thiazides)
RESP Respiratory Acidosis	pCO₂ ↑	HCO₃⁻ ↑ (kidney)	Hypoventilation: lung diseases impairing gas exchange, drugs depressing ventilation (barbiturates)
RESP Respiratory Alkalosis	pCO₂ ↓	HCO₃⁻ ↓ (kidney)	Hyperventilation: high-altitude hypoxia, anxiety disorders, CNS disorders, salicylate poisoning

Exam Trap

In metabolic alkalosis, the compensatory pCO₂ INCREASES (not decreases) — the lungs hypoventilate to retain CO₂ and pull the pH back down.

Anion Gap — classifying metabolic acidosis

Anion gap = [Na⁺] − ([Cl⁻] + [HCO₃⁻]) (normal ≈ 8–12 mmol/L).
High anion gap acidosis = added acid (lactic acidosis, ketoacidosis/DKA, renal failure, toxins). Normal anion gap acidosis = HCO₃⁻ loss (diarrhoea, renal tubular acidosis). A quick way to tell why a metabolic acidosis is happening from the electrolytes.

🔍 Reading a Blood Gas — Identify the Disturbance from pCO₂ & HCO₃⁻

You can name any simple disturbance using just two numbers. Here is the exact method:

Look at pHacidosis or alkalosis? → Which value is abnormal?pCO₂ or HCO₃⁻ → Name ite.g. metabolic acidosis

Step 1 — Which value is the problem? (respiratory vs metabolic)

• If pCO₂ is the abnormal value → it is RESPIRATORY (lungs control CO₂).
• If HCO₃⁻ is the abnormal value → it is METABOLIC (kidneys control HCO₃⁻).
(Whichever value shifts in the direction that explains the pH is the primary driver.)

Step 2 — Which direction? (acidosis vs alkalosis)

• pCO₂ behaves like an ACID: pCO₂ ↑ high → acidosis; pCO₂ ↓ low → alkalosis. (moves OPPOSITE to pH)
• HCO₃⁻ behaves like a BASE: HCO₃⁻ ↓ low → acidosis; HCO₃⁻ ↑ high → alkalosis. (moves the SAME way as pH)

🔴 Acidosis (pH < 7.35)

Respiratory pCO₂ ↑ high
Metabolic HCO₃⁻ ↓ low

🟢 Alkalosis (pH > 7.45)

Respiratory pCO₂ ↓ low
Metabolic HCO₃⁻ ↑ high

💡 Memory Trick — "ROME"

Respiratory = Opposite → pCO₂ moves opposite to pH (↑CO₂ → ↓pH = acidosis).
Metabolic = Equal → HCO₃⁻ moves the same way as pH (↓HCO₃⁻ → ↓pH = acidosis).
In short: abnormal pCO₂ = respiratory, abnormal HCO₃⁻ = metabolic → then use ROME for the direction.

⚠️ Watch for compensation: if both pCO₂ and HCO₃⁻ are shifted in the same direction (both up or both down), one is the primary problem and the other is the body compensating. The primary one is the value that matches the pH side (acidosis vs alkalosis) per the rules above.

⚖️ No vs Partial vs Full Compensation

Once a disturbance starts, the body uses the other organ system to push the pH back toward normal — this is compensation. How far it has gone defines three states, judged by looking at whether the second parameter has changed and whether the pH has returned to normal:

Uncompensated
(no compensation) Only the primary parameter has changed. The second component is still normal — the body hasn't started compensating yet (acute disturbance). e.g. respiratory acidosis: ↑pCO₂, HCO₃⁻ still normal. pH still abnormal

Partially
compensated The second component has now shifted to oppose the primary disturbance, but not enough yet. e.g. respiratory acidosis: ↑pCO₂ AND ↑HCO₃⁻ (kidneys responding) — but pH hasn't made it back. pH still abnormal

Fully
compensated The second component has shifted enough to bring pH all the way back into the normal range. Both parameters are abnormal, but they balance out. e.g. respiratory acidosis: ↑pCO₂ AND ↑HCO₃⁻, pH = 7.36. pH 7.35–7.45 ✓

See it in the equation — pH = pKₐ + log [HCO₃⁻] / (0.03 × pCO₂)

Using respiratory acidosis (primary ↑pCO₂) as the example:
• Uncompensated: ↓pH = pKₐ + log([HCO₃⁻] / ↑pCO₂) — only the denominator rose, so the ratio (and pH) fell.
• Partially compensated: ↓pH = pKₐ + log(↑[HCO₃⁻] / ↑pCO₂) — kidneys raise HCO₃⁻ too, pulling the ratio back up, but pH is not yet normal.
• Fully compensated: pH = pKₐ + log(↑[HCO₃⁻] / ↑pCO₂) — HCO₃⁻ has risen enough that the ratio is restored and pH sits back in range.

How to tell them apart in one glance

1. Is the second parameter still normal? → Uncompensated.
2. Has the second parameter shifted but pH is still outside 7.35–7.45? → Partially compensated.
3. Has the second parameter shifted AND pH is back within 7.35–7.45? → Fully compensated.
Note: full compensation brings pH into range but rarely to exactly 7.40 — the pH usually stays on the side of the original disturbance (e.g. 7.36 in a compensated acidosis), which still tells you which problem came first.

Compensation vs Correction — keep them separate

Compensation changes the second parameter (the lungs adjust pCO₂ for a metabolic problem, or the kidneys adjust HCO₃⁻ for a respiratory problem) — it pushes pH toward normal but does NOT fix the underlying cause.
Correction fixes the primary parameter itself. Per the lesson, correction of a metabolic disturbance is a job for the kidneys (restoring HCO₃⁻), not the lungs.

🫘 Renal Handling, Compensation & Correction

Renal HCO₃⁻ Reabsorption & Ammoniagenesis

Proximal tubule: Na⁺/K⁺-ATPase generates Na⁺ gradient → Na⁺-H⁺ antiporter secretes H⁺ → H⁺ + HCO₃⁻ → H₂CO₃ → CO₂ (carbonic anhydrase at brush border) → CO₂ re-enters cell → H₂CO₃ → H⁺ + HCO₃⁻ → HCO₃⁻ exits to blood. Net: HCO₃⁻ is reabsorbed, H⁺ is recycled.

Ammoniagenesis: Glutamine → NH₃ (glutaminase) + glutamate → NH₃ + α-KG (GDH). NH₃ diffuses into tubule lumen → H⁺ + NH₃ → NH₄⁺ (trapped, excreted). Upregulated in acidosis (↑ glutaminase activity). Main mechanism to excrete excess acid.

Titratable acid (phosphate buffer): secreted H⁺ also binds filtered HPO₄²⁻ → H₂PO₄⁻ (excreted). For each H⁺ excreted this way, a new HCO₃⁻ is generated and returned to blood. So the kidney has three jobs: (1) reabsorb filtered HCO₃⁻, (2) excrete H⁺ as titratable acid, (3) excrete H⁺ as NH₄⁺ — together regenerating bicarbonate and removing the daily fixed-acid load.

Correction vs Compensation — don't confuse them

Compensation: Secondary response (e.g. lungs ↓pCO₂ in metabolic acidosis) — brings pH TOWARD normal but does NOT fix the primary problem.
Correction: Fixing the primary parameter (e.g. kidneys restore HCO₃⁻) — returns pH fully to normal.

🧪 The Buffer Systems — Ranked by Where They Work

A buffer is a weak acid with its conjugate base. It resists pH change best when pH equals its pKa, where the two forms are equal. The body's buffers are chosen for the compartment they serve, not for chemical perfection.

Buffer	pKa	Compartment	Share of total buffering
Bicarbonate / CO₂	6.1	Extracellular fluid	The most important — because it is an OPEN system
Haemoglobin	~7.3 (histidine imidazole)	Erythrocytes	The main intracellular blood buffer; deoxyhaemoglobin is the better buffer
Phosphate	6.8	Intracellular and urine	Minor in plasma, major in urine — it is the titratable acid
Proteins	Various	Intracellular, plasma	Substantial in aggregate
Bone	—	Skeleton	Long-term reservoir; chronic acidosis demineralises bone

🎯 Why a pKa of 6.1 is still the best buffer at pH 7.4

On paper it is a poor choice — nearly 1.3 units away from physiological pH. It works because it is an open system: the lungs continuously remove CO₂ and the kidneys regenerate HCO₃⁻, so neither component is ever exhausted. A closed buffer at the perfect pKa would still run out. This is the single most commonly examined idea in acid–base chemistry.

The Henderson–Hasselbalch equation in words

pH = 6.1 + log([HCO₃⁻] / 0.03 × pCO₂). The numerator is metabolic and kidney-controlled; the denominator is respiratory and lung-controlled. Every acid–base disorder is a change in one, and every compensation is a change in the other in the same direction, to hold the ratio near 20:1. If both move in opposite directions, it is a mixed disorder, not compensation.

🫘 Renal Handling — Three Distinct Jobs

1️⃣ Reclaiming filtered bicarbonate

Proximal tubule — about 85–90% · this conserves, it does not create

H⁺ is secreted into the lumen by the Na⁺/H⁺ exchanger and combines with filtered HCO₃⁻ to form H₂CO₃, which luminal carbonic anhydrase IV splits into CO₂ and water. CO₂ diffuses into the cell, where carbonic anhydrase II reforms H₂CO₃ → H⁺ + HCO₃⁻; the bicarbonate leaves basolaterally. No net acid is excreted — the same bicarbonate simply comes back. Acetazolamide blocks this, causing bicarbonate loss and a metabolic acidosis.

▼

2️⃣ Titratable acidity

Distal tubule — buffering H⁺ on phosphate

Secreted H⁺ binds HPO₄²⁻ to form H₂PO₄⁻. Because the proton is now buffered, urine pH does not fall below the limiting value of about 4.4, and a new bicarbonate is generated and returned to the blood. The capacity is fixed by how much phosphate is filtered, so it cannot expand much.

▼

3️⃣ Ammoniagenesis — the adaptable one

Proximal tubule, from glutamine — this is the system that scales up in chronic acidosis

Glutamine → glutamate → α-ketoglutarate, catalysed by glutaminase and glutamate dehydrogenase, releasing two NH₃ per glutamine. NH₃ diffuses into the lumen, traps a proton as NH₄⁺, and is excreted — while the α-ketoglutarate is metabolised to yield two new bicarbonate ions. Chronic acidosis induces glutaminase, so this route can increase several-fold over days. It is also how the kidney disposes of nitrogen during prolonged fasting without making urea.

🎯 The distinction that gets tested

Reclaiming filtered bicarbonate excretes no acid — it merely prevents a loss. Only titratable acidity and ammonium excretion generate NEW bicarbonate and therefore represent true net acid excretion. Of the two, ammonium is the one that adapts: it accounts for the majority of net acid excretion in chronic acidosis. A kidney that cannot make ammonium (renal failure, type 4 RTA) develops acidosis even with normal filtration.

🫁 Respiratory Regulation & The Chloride Shift

The lung is the fastest responder — minutes, against the kidney's days — but it can only adjust the CO₂ side of the equation.

1
Central chemoreceptors in the medulla sense the pH of cerebrospinal fluid. CO₂ crosses the blood–brain barrier freely; H⁺ and HCO₃⁻ do not — so the CNS responds to CO₂, not directly to plasma acid.
2
Peripheral chemoreceptors in the carotid and aortic bodies sense pO₂, pCO₂ and pH directly. Hypoxia acts only through these.
3
Increased ventilation blows off CO₂ → pCO₂ falls → the ratio in Henderson–Hasselbalch rises → pH rises. Maximum respiratory compensation for a metabolic acidosis is a pCO₂ of about 10–15 mmHg.

The chloride shift — CO₂ transport in three forms

CO₂ travels in blood three ways: ~70% as bicarbonate, ~20–25% bound to haemoglobin as carbamino compounds, and ~5–10% dissolved. In the tissue capillary, CO₂ enters the erythrocyte, carbonic anhydrase makes HCO₃⁻, which leaves the cell in exchange for Cl⁻ entering — the chloride shift, or Hamburger phenomenon. The whole process reverses in the lung.

Bohr effect: H⁺ and CO₂ lower haemoglobin's oxygen affinity, so oxygen is released where it is needed. Haldane effect: deoxygenated haemoglobin carries CO₂ and buffers H⁺ better — the two effects are reciprocal halves of the same mechanism.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧽 What Decides a Buffer's Capacity

What decides a buffer's capacity Past Q

Three things. 1 · The concentration of the buffer — the more of it, the more acid or base it can absorb. 2 · The ratio of its two components, i.e. how close the pH is to the pK: capacity is greatest when [A⁻] = [HA], and the useful range is pK ± 1. 3 · Dilution — diluting a buffer lowers its capacity even though the ratio, and therefore the pH, hardly changes. A weak acid is most effective as a buffer when the pH of the solution stays close to its own pK.

🧂 Hypochloraemic Alkalosis

Prolonged vomiting or nasogastric suction removes H⁺-rich gastric fluid, so [HCO₃⁻] and pH rise — and because Cl⁻ and K⁺ leave with it, the named variety is a hypochloraemic, hypokalaemic metabolic alkalosis, one of the commonest causes of metabolic alkalosis.

Why it persists

Volume depletion and hypokalaemia keep the kidney reabsorbing bicarbonate. It only corrects once chloride and potassium are replaced, after which the kidney can excrete the excess bicarbonate.

Hormones & Blood Sugar Regulation

Frequently tested: insulin (GLUT-4, stimulates uptake into muscle/adipose), glucagon targets, untreated diabetes (glucosuria, ketonuria, acidosis, hyperkalemia), hypoglycemia causes

🔑 Insulin Secretion Mechanism & Oral Hypoglycaemics

How glucose triggers insulin secretion:
1. Glucose enters β-cell via GLUT-2 (high Km — proportional sensing)
2. Glucokinase phosphorylates glucose → G-6-P → ATP generation
3. ↑ATP closes ATP-sensitive K⁺ channels → membrane depolarisation
4. Voltage-gated Ca²⁺ channels open → Ca²⁺ influx
5. Ca²⁺ triggers insulin vesicle exocytosis

Sulfonylureas (e.g. glibenclamide) treat Type 2 DM by closing ATP-sensitive K⁺ channels directly → stimulate insulin secretion regardless of blood glucose.

Incretins (GLP-1 & GIP):
Released from small intestine after food → sensitise β-cells to glucose → enhance insulin secretion.
Explain the "incretin effect": oral glucose → much greater insulin response than IV glucose (same amount).

Incretin-based drugs (2025 tips)

GLP-1 receptor agonists (liraglutide) and DPP-4 inhibitors used in Type 2 DM. Liraglutide also used for obesity (↓ appetite).

🏃 Counterregulatory Hormones & Hypoglycaemia

When blood glucose falls <50 mg/dL, the body activates counterregulatory hormones that oppose insulin:

Hormone	Source	Main anti-hypoglycaemic action
Glucagon	Pancreatic α-cells	↑ hepatic glycogenolysis, ↑ gluconeogenesis — most important acute response
Epinephrine	Adrenal medulla	↑ glycogenolysis, ↑ lipolysis, inhibits insulin secretion — critical when glucagon deficient
Cortisol	Adrenal cortex	↑ gluconeogenesis (long-term), ↑ muscle proteolysis → amino acids for GNG
Growth hormone	Anterior pituitary	↓ glucose uptake, ↑ lipolysis (long-term)

Type 1 DM — Hypoglycaemia Unawareness

In long-standing T1DM, both glucagon secretion AND epinephrine response are impaired → patient cannot detect or correct hypoglycaemia → risk of coma. Combined glucagon + epinephrine deficiency = "hypoglycaemia unawareness."

HbA1c — What It Measures

Haemoglobin non-enzymatically glycated by glucose. Rate of formation is proportional to average blood glucose over the previous 3 months. Normal <6%. Target in treated DM <7%. Used to monitor long-term glycaemic control.

💉 Insulin — Mechanism and Effects

Insulin is secreted by beta-cells of the pancreatic islets of Langerhans in response to high blood glucose. Its receptor has a unique structure: 2 alpha-subunits (extracellular, bind insulin) + 2 beta-subunits (transmembrane, contain intrinsic tyrosine kinase activity). Unlike most hormone receptors, the insulin receptor phosphorylates tyrosine residues — it is its own enzyme.

Insulin promotes: glycolysis, glycogen synthesis, lipogenesis, protein synthesis, GLUT-4 translocation to cell surface (in muscle and adipose). Insulin inhibits: glycogenolysis, gluconeogenesis, lipolysis, ketogenesis.

Insulin and Leptin — Anorexigenic Hormones

Anorexigenic hormones suppress appetite. Both insulin and leptin are anorexigenic — they signal to the hypothalamus that the body has enough energy, suppressing food intake. In contrast, ghrelin is an orexigenic hormone (stimulates hunger). CCK (Cholecystokinin) is also anorexigenic — it signals satiety after a meal. This is a directly tested exam point.

🩺 Diabetes — Acute and Chronic Complications

Acute Complications

Diabetic Ketoacidosis (DKA): mainly Type 1. Insulin absence → lipolysis → ketones → metabolic acidosis. Kussmaul breathing, fruity breath, glucosuria, ketonuria, hyperkalemia.

Hypoglycemia: excess insulin or oral hypoglycemic agents → blood glucose too low → brain symptoms (confusion, seizure, coma).

Chronic Complications

Caused by prolonged hyperglycemia damaging blood vessels and nerves:
Nephropathy (kidney damage — glomerulosclerosis)
Neuropathy (nerve damage — peripheral and autonomic)
Retinopathy (retinal blood vessel damage → blindness)
Atherosclerosis (macrovascular — heart disease, stroke)

Oral Hypoglycemics in Type 2 DM

"Oral hypoglycaemics are NOT used in the treatment of Type 2 DM" → FALSE. Oral agents like metformin, sulfonylureas, and DPP-4 inhibitors ARE used in Type 2 DM. They are NOT appropriate for Type 1 (which requires insulin). This distinction was directly tested.

Insulin Deficiency → Metabolic Acidosis ★ asked 2018, 2019

Insulin deficiency causes metabolic acidosis through two routes: (1) DKA — ketone bodies are weak acids. (2) Lactic acidosis — poor tissue perfusion impairs aerobic metabolism. Both result in decreased blood pH and decreased bicarbonate.

⚖️ Type 1 vs Type 2 Diabetes — Key Differences

🔵 Type 1 (T1DM)

Cause: autoimmune destruction of pancreatic β-cells
Insulin: absolute deficiency (little/none produced)
Blood insulin: very low
Onset: usually childhood / young, sudden
Body weight: typically lean / weight loss
Ketosis: common & prone to DKA
Treatment: insulin (lifelong, essential)
Insulin resistance: no

🟠 Type 2 (T2DM)

Cause: insulin resistance + relative insulin deficiency
Insulin: present but ineffective (resistance)
Blood insulin: high (early) → falls later — ★ asked 2024/25
Onset: usually adult, gradual
Body weight: typically overweight / obese
Ketosis: rare (enough insulin to suppress lipolysis)
Treatment: diet, exercise, oral drugs (metformin), ± insulin later
Insulin resistance: yes (key feature)

Shared features & exam traps

Both cause hyperglycaemia, glucosuria, polyuria, polydipsia. Ketosis is NOT common in both — it is typical of T1 (a frequent trap). T2 has higher blood insulin than T1 because the problem is resistance, not lack of production.

Steroid & Adrenal Hormones

Frequently tested: steroid synthesis & hydroxylase deficiencies, aldosterone, thyroid hormones, hypothyroidism, PTH vs calcitriol

🦴 PTH & Calcium

PTH released when plasma Ca²⁺ is LOW. Effects: kidney ↑Ca²⁺ reabsorption, ↓phosphate reabsorption, stimulates 1α-Hydroxylase → calcitriol. Net result: ↑Ca²⁺, ↓phosphate in blood.

🧬 Steroidogenesis — From Cholesterol to All Steroid Hormones

Every steroid hormone is made from cholesterol. The first step — cholesterol → pregnenolone — is the rate-limiting step for all of them, and pregnenolone is the common branch point.

⚗️ Master Steroid Pathway

Cholesterol Desmolase (CYP11A1) · rate-limiting→ Pregnenolone → Aldosterone → Cortisol → Androgens (testosterone) Aromatase→ Estrogens (estradiol)

Class (site)	Main hormone	Key enzyme(s) & regulation
Mineralocorticoids — zona glomerulosa	Aldosterone	Aldosterone synthase; driven by angiotensin II + high K⁺ → ↑Na⁺/water retention, ↑K⁺/H⁺ loss
Glucocorticoids — zona fasciculata	Cortisol	Needs 21-hydroxylase + 17α-hydroxylase; driven by ACTH; ↑gluconeogenesis, anti-inflammatory
Androgens — zona reticularis / testis (Leydig)	Testosterone → DHT	LH drives Leydig cells; 5α-reductase → potent DHT (prostate, skin)
Estrogens — ovary (granulosa), placenta, adipose	Estradiol	Two-cell model: theca makes androgen (LH), granulosa aromatises it (FSH)

Steroids of the male gonad

Leydig cells make testosterone under LH control (via cAMP → ↑desmolase). Testosterone → DHT (by 5α-reductase, more potent, acts on prostate/genitalia) or → estradiol (by aromatase). Sertoli cells (FSH) support sperm and make inhibin + androgen-binding protein.

Congenital Adrenal Hyperplasia

A block in cortisol synthesis (most often 21-hydroxylase) → ↓cortisol → ↑ACTH → adrenal hyperplasia, and precursors are pushed into androgens. 21-OH deficiency = salt-wasting + virilisation (↑17-OH-progesterone). 17α-OH deficiency = hypertension without virilisation.

👆 Tap for full disease card

🧠 Steroid Hormone Signalling & Transport

Steroids are lipophilic — they cross the cell membrane and bind intracellular (nuclear) receptors, then act as transcription factors. Their effects are slow (hours) but long-lasting, unlike fast peptide-hormone signalling.

Transport in blood

Carried by plasma proteins: cortisol on CBG (transcortin), sex steroids on SHBG, plus albumin. Only free hormone is active.

Contrast: peptide/catecholamine

Water-soluble → bind surface receptors → second messengers (cAMP, IP₃) → fast response.

🪜 The Common Start — Cholesterol → Pregnenolone

All mammalian steroids are synthesised from cholesterol via pregnenolone. In the adrenal, cholesterol is mostly esterified and stored in cytoplasmic lipid droplets. ACTH stimulates an esterase, which frees the cholesterol; free cholesterol is then transported into the mitochondrion.

StAR — the rate-limiting step

Steroidogenic acute regulatory (StAR) protein is an ACTH-dependent protein that transports cholesterol to the inner mitochondrial membrane, where P450scc sits. Delivery of cholesterol to the inner mitochondrial membrane is the rate-limiting step of the whole pathway — in adrenal, testis and ovary alike.

Side-chain cleavage

In the mitochondrion, cytochrome P450 side-chain cleavage enzyme (P450scc) converts cholesterol into pregnenolone. The cleavage involves sequential hydroxylations at C22 and C20 and the removal of a six-carbon fragment, isocaproaldehyde. The product is a 21-carbon steroid.

Three rules that govern every branch

• Hydroxylases require molecular oxygen and NADPH. Isomerases and lyases also take part.
• The conversion of cholesterol to pregnenolone is identical in adrenal, testis and ovary — but in the gonads it is driven by LH, not ACTH.
• There is cellular specificity: 18-hydroxylase and 18-hydroxysteroid dehydrogenase, needed for aldosterone, are found only in zona glomerulosa cells; 17α-hydroxylase is absent from the zona glomerulosa.

🧂 Mineralocorticoid Synthesis — Zona Glomerulosa

Aldosterone is made in the zona glomerulosa, in four steps from pregnenolone.

1 · Pregnenolone → progesterone

3β-hydroxysteroid dehydrogenase with Δ5,4-isomerase.

2 · Progesterone → 11-deoxycorticosterone (DOC)

Hydroxylation at C21 by 21-hydroxylase. DOC is itself an active mineralocorticoid with Na⁺-retaining properties.

3 · DOC → corticosterone

Hydroxylation at C11 by 11β-hydroxylase, a mitochondrial enzyme. Corticosterone has glucocorticoid activity and weak mineralocorticoid activity.

4 · Corticosterone → aldosterone

Via 18-hydroxycorticosterone, by 18-hydroxylase and 18-hydroxydehydrogenase — enzymes found exclusively in the zona glomerulosa.

Regulation is different here

The zona glomerulosa is under special regulation by K⁺ and angiotensin II — not primarily by ACTH. And because it lacks 17α-hydroxylase, it cannot make cortisol or androgens.

🛡️ Glucocorticoid Synthesis — Zona Fasciculata & Reticularis

Cortisol synthesis requires hydroxylases located in both the zona fasciculata and the zona reticularis. Hydroxylations at the 17 and 21 positions are fast, while hydroxylation at the 11 position is relatively slow — and the sequence of hydroxylations matters.

1 · 17α-hydroxylase (SER)

Acts on both progesterone and pregnenolone — more commonly on pregnenolone. 17α-hydroxylase and 17,20-lyase are two activities of one enzyme, P450C17, located in the SER and not present in the zona glomerulosa.

2 · 21-hydroxylase (SER)

17α-hydroxyprogesterone → 11-deoxycortisol by hydroxylation at C21.

3 · 11β-hydroxylase (mitochondrion)

11-deoxycortisol → cortisol.

The headline

Cortisol is the most potent natural glucocorticoid in humans.

♂️ Adrenal Androgen Synthesis — DHEA and Androstenedione

DHEA (dehydroepiandrosterone) is the major androgen — or androgen precursor — produced by the adrenal cortex.

Where DHEA comes from

A small fraction of 17-hydroxypregnenolone undergoes oxidative fission and removal of the two-carbon side chain, catalysed by 17,20-lyase.

DHEA is a prohormone

As a weak androgen, DHEA is converted to the more potent androstenedione by 3β-hydroxysteroid dehydrogenase and Δ5,4-isomerase. Small amounts of androstenedione also come from the action of the lyase on 17α-hydroxyprogesterone.

And on to testosterone

Reduction of androstenedione at C17 gives testosterone — the most potent adrenal androgen. Only small amounts are made this way in the adrenal; this conversion occurs mostly in the testes.

Clinical link

The fraction subjected to lyase action is increased in adrenogenital syndrome — where one of the hydroxylases is missing.

🧔 Testicular Steroidogenesis and DHT

Testicular androgens are produced by Leydig cells in the interstitial tissue, stimulated by LH. There are two pathways of testosterone synthesis — the progesterone (Δ4) pathway and the dehydroepiandrosterone (Δ5) pathway. The Δ5 pathway predominates in human testes.

Metabolism route 1 — oxidation at C17

Occurs in many tissues including the liver and produces 17-ketosteroids, which are less active or inactive compared with testosterone.

Metabolism route 2 — reduction of the A-ring

Less efficient, but it produces DHT (dihydrotestosterone), the potent metabolite, by 5α-reductase.

DHT — the numbers and the targets

DHT is the most potent metabolic product of testosterone in many tissues — prostate, external genitalia and some regions of the skin. In the adult male, plasma DHT is about one tenth of testosterone. About 50–100 µg is secreted by the testes; the rest is produced peripherally by 5α-reductase from testosterone. Testosterone can therefore be regarded as a prohormone — the one steroid not secreted in its final active form.

♀️ Ovarian Steroidogenesis and Aromatisation

Who makes what

Theca cells of the follicle are the source of androstenedione and testosterone. Aromatisation of those androgens happens in the granulosa cells. The corpus luteum synthesises and secretes progesterone as an end product — it does not possess the enzymes needed to convert progesterone to other steroid hormones.

Aromatisation

Oestrogens are produced by aromatisation of androgens — a multi-step process including three hydroxylations, each requiring O₂ and NADPH. The aromatase complex is said to include a P450 monooxygenase and sits in the SER. Estradiol comes from testosterone; estrone is a product of the aromatisation of androstenedione.

Which oestrogen, when

17β-estradiol is the primary oestrogen of ovarian origin (premenopausal). In pregnancy, production of estriol increases — the placenta is the source. In postmenopausal women, the major source of oestrogen is the conversion of androstenedione into estrone.

Peripheral aromatisation — the percentages

In males, 80% of estradiol is a product of the peripheral aromatisation of testosterone. In pregnancy, 50% of E2 comes from aromatisation of (adrenal) androgens. Adrenal androgens are therefore important substrates for E2 production in women.

Aromatase outside the gonad — "estrogenization"

Aromatase activity is present in adipose tissue, liver, skin and elsewhere. Increased aromatase activity can produce "estrogenization", a clinical feature accompanying hepatic cirrhosis, hyperthyroidism, ageing and obesity. Aromatase inhibitors are therapeutic agents in breast cancer and other malignancies of the female reproductive tract.

🚌 Transport in Plasma — CBG, SHBG and the Free Fraction

Steroid hormones are lipophilic, so in most cases they need plasma transport proteins. Bound hormone gives a long plasma half-life (hours to days); only the free hormone is biologically active. Binding proteins provide a circulating reservoir and buffer against sudden changes in plasma hormone concentration.

CBG (transcortin)

An α-globulin synthesised in the liver; oestrogens increase its production. It is the main plasma-binding protein for cortisol when cortisol is in the normal range. Much smaller amounts of cortisol are bound to albumin. About 8% of total plasma cortisol is free — the biologically active fraction. Cortisol binds CBG more tightly than corticosterone does (t½ ≈ 1.5–2 h vs <1 h). Deoxycorticosterone and progesterone compete for cortisol binding.

SHBG (TEBG)

A β-globulin synthesised in the liver that binds testosterone with specificity, relatively high affinity and limited capacity. With albumin it binds 97–99% of circulating testosterone; only 1–3% is free and active. It binds DHT slightly more strongly than testosterone and estradiol about five times more weakly. Progesterone and cortisol have little affinity for SHBG. Women have twice the serum SHBG concentration of men.

What moves SHBG — and why it matters

Increased by oestrogens, certain liver diseases and hyperthyroidism. Decreased by androgens, advanced age and hypothyroidism. Because testosterone binds SHBG more avidly than estradiol does, a change in SHBG causes a greater change in free testosterone than in free estradiol. Metabolic clearance is inversely related to SHBG affinity: estrone is cleared faster than estradiol, which is cleared faster than testosterone or DHT.

The exception to remember

Aldosterone does not have a specific plasma transport protein.

🧠 Mechanism of Action and Trophic Control

Hormones are mobile signalling molecules secreted directly into the blood and carried to distal organs to regulate physiology and behaviour. They are classified by mechanism of action: those binding intracellular receptors (the steroids), and those binding cell-surface receptors — with cAMP (β-adrenergic catecholamines, glucagon), cGMP (NO), calcium/phosphatidylinositides (acetylcholine), or a kinase/phosphatase cascade (insulin) as second messenger.

How a steroid acts

It binds an intracellular (cytosolic or nuclear) receptor; the hormone–receptor complexes accumulate in the nucleus, dimerise and bind specific regulatory DNA sequences — hormone response elements (HRE). The effect is increased transcription of the target genes.

Made and released, not stored

Steroids are synthesised in their final form and secreted immediately. The exception is DHT, which is secreted as its precursor, testosterone, and made active in the target tissue.

Which pituitary hormone drives which gland

ACTH — stimulates synthesis and secretion of adrenal steroid hormones. LH — induces testosterone synthesis in Leydig cells and stimulates oestrogen and progesterone synthesis in the corpus luteum. FSH — stimulates secretion of oestrogens in females and androgens in males.

What each hormone does

Aldosterone — renal reabsorption of Na⁺, excretion of K⁺; ↑ blood volume and arterial pressure. Cortisol — ↑ gluconeogenesis, anti-inflammatory and anti-allergic, stimulates muscle protein breakdown and adipose remodelling. Oestrogens — control the menstrual cycle, female secondary sex characteristics. Progesterone — secretory phase of uterus and mammary gland, implantation and maturation of the fertilised ovum. Testosterone — spermatogenesis, male secondary sex characteristics, anabolism, masculinisation of the fetus.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🧬 Δ4 and Δ5 — the Two Routes to Testosterone

The two routes from pregnenolone to testosterone Past Q

Five enzyme activities in three proteins get you there: 3β-hydroxysteroid dehydrogenase with Δ5,4-isomerase, 17α-hydroxylase and 17,20-lyase (one protein, two activities), and 17β-hydroxysteroid dehydrogenase. They can act in either order, which is why there are two named pathways. Δ4, the progesterone pathway: pregnenolone → progesterone → 17α-hydroxyprogesterone → androstenedione → testosterone. Δ5, the dehydroepiandrosterone pathway: pregnenolone → 17α-hydroxypregnenolone → DHEA → Δ5-androstenediol → testosterone — the route the human testis mainly uses. The rate-limiting step in either case is upstream and LH-dependent: StAR delivering cholesterol to the inner mitochondrial membrane for P450scc. Downstream, 5α-reductase converts testosterone into dihydrotestosterone in prostate, external genitalia and skin — about 400 µg a day against 5 mg of testosterone, but far more potent locally.

🧪 Cis and Trans Isomers of Steroids

The steroid nucleus can be fused two ways at the A/B ring junction, and the difference is functional, not cosmetic.

5α — trans

Rings A and B lie in essentially one flat plane. This is cholesterol, testosterone, dihydrotestosterone and the adrenal steroids — flat, rigid molecules that slot into membranes and receptors.

5β — cis

Ring A is bent up almost at a right angle. This is the bile acids: the kink puts all the hydroxyl groups on one face and the hydrocarbon on the other, which is precisely what makes them detergents. Individual substituents follow the same convention — β drawn solid toward the reader, α dashed away.

Vitamins & Cofactors

Frequently tested: vitamin → coenzyme pairings (B-vitamins), fat-soluble vitamin deficiencies (A, D, E, K), vitamin C in collagen/catecholamines, antioxidants

📋 Vitamins — Quick Reference Summary Table

Vitamin	Type	Active Form / Coenzyme	Deficiency Disease	Key Pathway
A (Retinol)	Fat-soluble	Retinal (vision), Retinoic acid (gene expression)	Night blindness, xerophthalmia	Vision cycle, epithelial integrity
D (Cholecalciferol)	Fat-soluble	Calcitriol (1,25-(OH)₂D₃)	Rickets (children), Osteomalacia (adults)	Ca²⁺/PO₄ absorption, bone mineralisation
E (Tocopherol)	Fat-soluble	α-Tocopherol	Haemolytic anaemia, neuropathy	Lipid antioxidant — protects PUFA in membranes
K (Phylloquinone)	Fat-soluble	Vitamin K hydroquinone	Impaired coagulation, bleeding	γ-Carboxylation of Glu in clotting factors II,VII,IX,X
B1 (Thiamine)	Water-soluble	TPP (Thiamine Pyrophosphate)	Beriberi, Wernicke-Korsakoff	PDC, α-KG DH, Transketolase (PPP), BCKAD
B2 (Riboflavin)	Water-soluble	FAD, FMN	Ariboflavinosis (cheilitis, glossitis)	Succinate DH (Complex II), β-oxidation, PDC
B3 (Niacin)	Water-soluble	NAD⁺, NADP⁺	Pellagra (4 Ds: Dermatitis, Diarrhoea, Dementia, Death)	~500 redox reactions; synthesized from Tryptophan (60:1)
B5 (Pantothenate)	Water-soluble	Coenzyme A (CoA)	Rare (burning feet)	Acetyl-CoA formation, Krebs cycle entry
B6 (Pyridoxine)	Water-soluble	PLP (Pyridoxal Phosphate)	Peripheral neuropathy, sideroblastic anaemia	ALL aminotransferases, ALA synthase (heme), glycogen phosphorylase
B7 (Biotin)	Water-soluble	Biotin (covalently bound)	Dermatitis, alopecia (avidin/raw egg white)	Pyruvate carboxylase, ACC, Propionyl-CoA carboxylase
B9 (Folate)	Water-soluble	THF (Tetrahydrofolate)	Megaloblastic anaemia, neural tube defects	One-carbon transfers: nucleotide synthesis, DNA methylation
B12 (Cobalamin)	Water-soluble	Methylcobalamin, Adenosylcobalamin	Megaloblastic anaemia + subacute combined degeneration (spinal cord)	Methionine synthesis; Methylmalonyl-CoA mutase (myelin)
C (Ascorbate)	Water-soluble	Ascorbic acid itself	Scurvy (bleeding gums, poor wound healing)	Prolyl/lysyl hydroxylase (collagen), iron absorption, antioxidant

Is Hepcidin an Enzyme? — Exam Trap (Q14b)

No — hepcidin is NOT an enzyme. It is a 25-amino acid peptide hormone (antimicrobial peptide) synthesised by the liver. It has no catalytic activity — it does not speed up any chemical reaction. Instead, it works by binding to ferroportin (an iron exporter on cell surfaces) and targeting it for degradation. This is a signalling/regulatory function, not an enzymatic one.

Classification: Hepcidin = peptide hormone / acute-phase reactant. Synthesised by hepatocytes. Regulated by iron stores, inflammation (IL-6), and erythropoietic activity.

Key Rule: Fat-Soluble (A, D, E, K) vs Water-Soluble

Fat-soluble vitamins: stored in adipose tissue → can accumulate → toxicity possible (especially A and D). Absorbed with dietary fat — malabsorption syndromes (coeliac, CF, cholestasis) → fat-soluble vitamin deficiency. Water-soluble vitamins: not stored in large amounts → excreted in urine → require regular dietary intake → deficiency develops faster.

⚗️ Water-Soluble B Vitamins — Full Table with Context

Vitamin	Active Coenzyme Form	Key Reactions It Participates In	Deficiency Disease
B1 (Thiamine)	TPP (Thiamine Pyrophosphate)	PDH (pyruvate → acetyl-CoA), α-KG dehydrogenase (Krebs), Transketolase (PPP), BCKAD (MSUD enzyme)	Beriberi (peripheral neuropathy, heart failure), Wernicke-Korsakoff (confusion, ataxia, ophthalmoplegia, amnesia)
B2 (Riboflavin)	FAD, FMN	Succinate dehydrogenase (Krebs/ETC Complex II), fatty acid oxidation, PDH	Cheilitis (cracked lips), glossitis, ariboflavinosis
B3 (Niacin)	NAD⁺, NADP⁺	~500 oxidation-reduction reactions; glycolysis, Krebs, fatty acid oxidation, PPP	Pellagra (Dermatitis, Diarrhea, Dementia, Death — the "4 Ds")
B5 (Pantothenate)	Coenzyme A (CoA)	Acetyl-CoA formation, fatty acid synthesis, Krebs cycle entry	Rare (burning feet syndrome)
B6 (Pyridoxine)	PLP (Pyridoxal Phosphate)	ALL aminotransferases (transamination), ALA Synthase (heme), glycogen phosphorylase, decarboxylases	Peripheral neuropathy, sideroblastic anemia, seborrheic dermatitis
B7 (Biotin)	Biotin (covalently bound)	Pyruvate Carboxylase (GNG), Acetyl-CoA Carboxylase (lipogenesis), Propionyl-CoA Carboxylase	Dermatitis, alopecia, neurological symptoms (from raw egg white consumption — avidin binds biotin)
B9 (Folate)	THF (Tetrahydrofolate)	One-carbon transfers: nucleotide synthesis, DNA methylation, amino acid interconversion	Megaloblastic anemia (large immature red blood cells), neural tube defects in pregnancy
B12 (Cobalamin)	Methylcobalamin, Adenosylcobalamin	Methionine synthesis (methylcobalamin), odd-chain fatty acid metabolism (adenosylcobalamin)	Megaloblastic anemia + Subacute combined degeneration of the spinal cord (dorsal column + corticospinal tract damage). Pernicious anemia = B12 deficiency due to lack of intrinsic factor.
C (Ascorbate)	Ascorbic acid itself	Cofactor for prolyl and lysyl hydroxylase (collagen), antioxidant, enhances iron absorption (reduces Fe³⁺ → Fe²⁺)	Scurvy (bleeding gums, perifollicular hemorrhages, poor wound healing)

Pernicious Anemia — B12 Deficiency

Pernicious anemia is a specific form of B12 deficiency caused by lack of intrinsic factor (a protein made by gastric parietal cells needed to absorb B12 in the ileum). Treatment: intramuscular B12 injections (bypasses the gut absorption problem).

Lab marker: methylmalonic acid ★ asked 2019

↑ Methylmalonic acid in urine/blood = vitamin B12 deficiency. Without adenosylcobalamin, methylmalonyl-CoA mutase fails → methylmalonyl-CoA builds up → methylmalonic acid. (Folate deficiency does NOT raise it — this distinguishes B12 from folate deficiency, since both cause megaloblastic anaemia.)

Pellagra and Tryptophan/Methionine

Niacin (B3) can be synthesized from tryptophan in the body (60 mg Trp → 1 mg niacin). Deficiency of tryptophan (e.g., Hartnup disease, corn-based diets) causes pellagra-like symptoms even with adequate dietary niacin. Methionine deficiency impairs one-carbon metabolism and can reduce the availability of certain niacin precursors. Both tryptophan and methionine deficiency together = niacin/pyridoxal phosphate deficiency risk.

⚠️ Niacin Overload & Thiamine Deficiency — A Paired Question

Niacin (B3) overload / pharmacological dose

At high (drug) doses niacin causes: flushing and warmth (prostaglandin/PGD₂-mediated — reduced by aspirin), itching, hepatotoxicity (especially slow-release forms), hyperglycaemia (insulin resistance), and hyperuricaemia / gout (competes with urate for excretion). Niacin is used to lower TAG/LDL and raise HDL — but these side effects limit it.

Thiamine (B1) deficiency

TPP is needed by pyruvate dehydrogenase, α-KG dehydrogenase, transketolase, and BCKAD. Deficiency → Beriberi (wet = high-output heart failure; dry = peripheral neuropathy) and Wernicke–Korsakoff (confusion, ataxia, ophthalmoplegia → amnesia + confabulation), classically in alcoholism. Give thiamine before glucose to avoid precipitating Wernicke.

Exam framing

The pairing tests two opposite ideas at once: a water-soluble vitamin causing harm from excess (niacin flush/hepatotoxicity) versus one causing a classic deficiency syndrome (thiamine → beriberi/Wernicke).

🍊 Vitamin C — Beyond Scurvy

Ascorbic acid is a reducing agent. Every one of its functions comes from that single property — keeping a metal ion or a substrate in its reduced state.

Function	Mechanism
Collagen hydroxylation	Keeps the Fe²⁺ of prolyl and lysyl hydroxylase reduced. Without it, the triple helix cannot cross-link properly — the basis of scurvy
Iron absorption	Reduces dietary Fe³⁺ to Fe²⁺ in the gut lumen, the only form DMT-1 transports. Doubles or triples absorption of non-haem iron
Antioxidant	Works in the aqueous phase and regenerates vitamin E from its radical form — the two vitamins are a team
Catecholamine synthesis	Cofactor for dopamine β-hydroxylase (dopamine → norepinephrine), a copper enzyme
Carnitine synthesis	Required by two hydroxylases in the pathway from lysine — which is one reason scurvy causes fatigue
Bile acid synthesis	Supports 7α-hydroxylase activity

Excess

Water-soluble and largely excreted, but very high doses cause gastrointestinal upset and, importantly, increase the risk of oxalate kidney stones — ascorbate is metabolised to oxalate.

🅱️ The B Vitamins — One Table, All the Cofactors

Vitamin	Active cofactor	Reaction type	Deficiency
B1 Thiamine	Thiamine pyrophosphate (TPP)	Oxidative decarboxylation of α-keto acids; transketolase	Beriberi, Wernicke–Korsakoff. Erythrocyte transketolase activity is the diagnostic test
B2 Riboflavin	FAD, FMN	Redox — two-electron and one-electron transfers	Angular stomatitis, cheilosis, glossitis, seborrhoeic dermatitis
B3 Niacin	NAD⁺, NADP⁺	Redox — hydride transfer	Pellagra. Can be made from tryptophan (60 mg → 1 mg niacin), which needs B6
B5 Pantothenate	Coenzyme A, ACP	Acyl group transfer	Rare — burning feet syndrome
B6 Pyridoxine	Pyridoxal phosphate (PLP)	Almost all amino acid chemistry: transamination, decarboxylation, deamination, ALA synthase, glycogen phosphorylase, cystathionine β-synthase	Sideroblastic anaemia, seizures (↓GABA), peripheral neuropathy — isoniazid is the classic cause
B7 Biotin	Biocytin	Carboxylation — pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase	Rare; caused by raw egg white (avidin) or biotinidase deficiency
B9 Folate	Tetrahydrofolate (THF)	One-carbon transfer at several oxidation levels	Megaloblastic anaemia; neural tube defects if deficient periconceptionally
B12 Cobalamin	Methylcobalamin, adenosylcobalamin	Only two reactions in humans: methionine synthase and methylmalonyl-CoA mutase	Megaloblastic anaemia plus subacute combined degeneration of the cord

🎯 The B12 / folate distinction — and why it matters clinically

Both cause megaloblastic anaemia, because both converge on thymidylate synthesis. Only B12 deficiency causes neurological damage, through the methylmalonyl-CoA mutase reaction. Methylmalonic acid is raised in B12 deficiency only; homocysteine is raised in both. Giving folate to a B12-deficient patient corrects the anaemia but lets the neurological disease progress silently — which is why you must never treat megaloblastic anaemia with folate before excluding B12 deficiency.

The mechanism is the methyl-folate trap: without B12, methionine synthase cannot run, so folate is stuck as 5-methyl-THF and is functionally unavailable — a B12 deficiency causes a functional folate deficiency.

☀️ The Fat-Soluble Four — Activation, Action, Toxicity

Vitamin D — a hormone, not a vitamin

Skin: 7-dehydrocholesterol + UVB → cholecalciferol (D3). Liver: 25-hydroxylase → 25-OH-D — the storage form and the one measured. Kidney: 1α-hydroxylase → 1,25-(OH)₂-D (calcitriol) — the active form, stimulated by PTH and low phosphate.

Actions: ↑intestinal calcium and phosphate absorption, ↑bone mineralisation, ↑renal calcium reabsorption. Deficiency → rickets and osteomalacia. Renal failure causes deficiency of the active form despite normal intake, because the last hydroxylation cannot occur. Smith–Lemli–Opitz syndrome removes the precursor itself.

Vitamin A — three active forms

Retinal — vision, as 11-cis-retinal in rhodopsin. Retinoic acid — gene transcription via RAR/RXR nuclear receptors, controlling epithelial differentiation. Retinol — the transport and storage form.

Deficiency → night blindness, then xerophthalmia and keratinisation. Excess is teratogenic — a critical warning for retinoid acne drugs in pregnancy — and causes raised intracranial pressure and liver damage.

Vitamin K — the carboxylation vitamin

Cofactor for γ-glutamyl carboxylase, which converts glutamate to γ-carboxyglutamate — creating the calcium-binding sites of factors II, VII, IX, X, protein C and protein S, plus osteocalcin in bone.

Warfarin blocks vitamin K epoxide reductase, preventing regeneration of the reduced cofactor. Newborns are given vitamin K because the gut is sterile and placental transfer is poor — see vitamin K deficiency.

Vitamin E — the membrane antioxidant

α-Tocopherol is the most active of the eight naturally occurring tocopherols, found mainly in vegetable oils. It terminates lipid peroxidation chain reactions inside membranes and prevents non-enzymic oxidation of LDL.

Deficiency → haemolysis and retinopathy, especially in very-low-birth-weight infants and in any fat malabsorption state. It is the least toxic of the fat-soluble vitamins. See vitamin E deficiency.

The shared vulnerability

All four require bile salts and micelle formation for absorption, and all four travel in chylomicrons. Any fat malabsorption — cystic fibrosis, coeliac disease, cholestasis, abetalipoproteinaemia, ileal resection — causes deficiency of all four together, and all four can accumulate to toxicity (except E, which is safest).

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

💊 Vitamin A Metabolism · Riboflavin Signs · Two Toxicity Notes

Vitamin A — the two families, and the one-way street Past Q

Two groups have vitamin A activity: the retinoids (retinol, retinaldehyde, retinoic acid) from animal food, and the carotenoids (α-, β-, γ-carotene, cryptoxanthin) from plants. In the intestinal mucosa, carotene dioxygenase cleaves β-carotene to retinaldehyde, which is reduced to retinol, esterified and packed into chylomicrons; the enzyme's activity is low, so much β-carotene reaches the circulation unchanged. Inside the body: retinol ⇄ retinaldehyde is reversible, but retinaldehyde → retinoic acid is not — which is why retinoic acid supports growth and differentiation but cannot restore vision.

Riboflavin deficiency, and two toxicity notes

B2 deficiency is widespread but never fatal, because riboflavin released from degraded enzymes is efficiently re-used. It shows as cheilosis (cracked lips), angular stomatitis, desquamation, a sore inflamed tongue (glossitis) and seborrhoeic dermatitis. Vitamin D toxicity: some infants react to as little as 50 µg/day with hypercalcaemia, vasoconstriction, hypertension and calcinosis — yet sunlight can never cause it, because the supply of 7-dehydrocholesterol is limited and prolonged ultraviolet exposure converts previtamin D into inactive compounds. Thiamine has a second job beyond TPP: thiamine triphosphate has a role in nerve conduction.

🔗 Folate — How Carbons Enter the Pool, and Folinic Acid

Entry into the one-carbon pool

The donors are serine, glycine, histidine (FIGLU) and choline, and serine is the most important: serine hydroxymethyltransferase, a PLP enzyme, transfers its β-carbon straight onto tetrahydrofolate as N5,N10-methylene-THF — the main point of entry, and the form used to make dTMP. The activity of the enzyme is itself regulated by how much folate is available and how substituted it is.

Folinic acid (leucovorin)

5-formyl-THF is far more stable than folate itself and is the form used pharmaceutically. Because it enters the pool below dihydrofolate reductase, it rescues normal tissue after methotrexate. Folate supplements lower homocysteine and prevent neural-tube defects; deficiency strikes rapidly dividing cells first, because they have the largest thymidine requirement — hence megaloblastic anaemia from the marrow.

Minerals & Trace Elements

Frequently tested: zinc transport & deficiency, copper, molybdenum, trace-element associated enzymes

⚗️ Mineral Categories

Category	Minerals
Major (macrominerals)	Ca, P, Mg, Na, K, Cl, S
Trace elements	Fe (iron), Zn (zinc), Cu (copper), Mn (manganese), I (iodine), Se (selenium), Cr (chromium), F (fluoride)
Ultratrace elements	Molybdenum (Mo), Co (cobalt), Ni (nickel), V (vanadium), Si (silicon)

Exam Q: "Which is an ultratrace mineral?" ★ asked 2023

Answer: Molybdenum (Mo). Zinc is a trace element. Manganese is a trace element. Magnesium is a major mineral. This exact question appeared in the 2023 exam. Ultratrace = needed in tiny amounts (<1 mg/day), distinct from trace elements which are needed in small but slightly larger amounts.

Acrodermatitis Enteropathica (Brandt's Disease)

Caused by a deficiency of zinc (Zn), specifically due to a mutation in the intestinal zinc transporter SLC39A4. Features: perioral and acral (hands/feet) dermatitis, diarrhea, alopecia (hair loss), immune dysfunction. Also known as Brandt's disease in some texts. Zinc is also required by ALA Dehydratase (heme synthesis) and Carboxypeptidase — lead toxicity displaces Zn²⁺ and inactivates these enzymes.

📋 Macroelements & Trace Elements — Full Reference Table

Element	Category	Key Functions	Deficiency	Excess / Notes
Ca (Calcium)	Macromineral	Bone/teeth mineralisation, muscle contraction, nerve signalling, clotting (cofactor), second messenger	Tetany, osteoporosis, rickets	Regulated by PTH + calcitriol + calcitonin
P (Phosphorus)	Macromineral	Bone (hydroxyapatite), ATP, nucleic acids, phospholipids, 2,3-BPG, signal transduction	Rickets (with Ca), hypophosphataemia → muscle weakness	Refeeding syndrome → acute hypophosphataemia
Mg (Magnesium)	Macromineral	Cofactor for >300 enzymes (ATP-Mg²⁺ complex, hexokinase, PFK-1, ACC, DNA polymerase), muscle relaxation	Arrhythmia, muscle cramps, tremor	Required for proper Ca²⁺ and K⁺ regulation
Na, K, Cl	Macromineral (electrolytes)	Osmolarity, membrane potential, Na⁺/K⁺-ATPase (Na⁺ out/K⁺ in), acid-base balance (Cl⁻ exchange)	Hyponatraemia (cerebral oedema), hypokalaemia (arrhythmia)	Na⁺ excess → hypertension; regulated by aldosterone
Fe (Iron)	Trace element	Haemoglobin/myoglobin O₂ transport, heme proteins (cytochromes, catalase, peroxidase), Fe-S clusters in ETC	Iron deficiency anaemia (microcytic, hypochromic)	Haemochromatosis (HFE mutation → ↑absorption); liver damage
Zn (Zinc)	Trace element	Cofactor for ADH, carbonic anhydrase, ALA dehydratase, carboxypeptidases, DNA polymerase, SOD. Zinc-finger transcription factors.	Acrodermatitis enteropathica (SLC39A4 mutation), poor wound healing, alopecia, impaired immunity, hypogonadism	Zn²⁺ displaced by Pb²⁺ in lead poisoning → enzyme inhibition
Cu (Copper)	Trace element	Cytochrome c oxidase (Complex IV), SOD, ceruloplasmin (Fe mobilisation), lysyl oxidase (collagen cross-linking), tyrosinase (melanin)	Menkes disease (ATP7A defect — X-linked): neurodegeneration, kinky hair, ↓ceruloplasmin	Wilson disease (ATP7B defect): Cu accumulates → liver + brain + Kayser-Fleischer rings
Se (Selenium)	Trace element	Selenocysteine in glutathione peroxidase (antioxidant) and 5'-deiodinase (T4→T3 conversion)	Keshan disease (cardiomyopathy); impaired T4→T3 conversion	Toxicity: hair/nail loss, garlic breath, neuropathy
I (Iodine)	Trace element	Essential component of thyroid hormones (T3, T4) — organified by TPO onto Tg tyrosyl residues	Goitre, hypothyroidism, cretinism (congenital)	Excess iodine → Wolff-Chaikoff effect (transient ↓thyroid function)
Mo (Molybdenum)	Ultratrace element	Cofactor for xanthine oxidase (purine catabolism → uric acid), aldehyde oxidase, sulphite oxidase	Rare; sulphite accumulation if sulphite oxidase impaired	2023 exam: "Which is an ultratrace mineral?" → Molybdenum
Mn (Manganese)	Trace element	Arginase cofactor (urea cycle), pyruvate carboxylase (with biotin), SOD (mitochondrial Mn-SOD), glycosyltransferases	Rare; impaired bone formation	Excess → parkinsonism ("manganism")
Cr (Chromium)	Trace element	Potentiates insulin action (part of Glucose Tolerance Factor)	Impaired glucose tolerance (rare)	Cr(VI) is carcinogenic (industrial, not dietary)
F (Fluoride)	Trace element	Hardens tooth enamel (fluorapatite replaces hydroxyapatite), strengthens bone	Dental caries	Excess → dental/skeletal fluorosis

⚖️ Macrominerals — The Ones Measured in Grams

Mineral	Main roles	Regulation / notes
Calcium	Bone mineral (hydroxyapatite); muscle contraction; second messenger; clotting cascade cofactor; exocytosis	Controlled by PTH (↑), calcitriol (↑) and calcitonin (↓). Plasma calcium is ~50% ionised, ~40% albumin-bound — correct for albumin before calling it low. Alkalosis lowers ionised calcium and causes tetany at a normal total
Phosphorus	Bone; every nucleotide and phospholipid; ATP; 2,3-BPG; the phosphorylation switch on every regulated enzyme; the urinary titratable acid buffer	PTH lowers plasma phosphate by reducing renal reabsorption — the opposite of its calcium effect. Rises in renal failure. Falls dangerously in refeeding syndrome
Magnesium	Cofactor for every ATP-using enzyme — the true substrate is Mg-ATP. Stabilises ribosomes and DNA; needed for PTH secretion and action	Deficiency causes hypocalcaemia that will not correct until magnesium is replaced, because PTH release itself fails. Also causes arrhythmia and tetany
Sodium	Main extracellular cation; osmotic control of volume; the gradient that powers SGLT-1, NHE and every secondary active transporter	Regulated by aldosterone, ADH and natriuretic peptides. Plasma sodium reports water balance, not sodium content
Potassium	Main intracellular cation; sets the resting membrane potential	Insulin and β-agonists drive it into cells; acidosis drives it out. This is why potassium falls sharply during DKA treatment even when it looked normal or high at presentation
Chloride	Main extracellular anion; gastric HCl; the chloride shift in CO₂ transport	Follows sodium. The anion gap (Na⁺ − Cl⁻ − HCO₃⁻) separates the causes of metabolic acidosis
Sulphur	In cysteine and methionine; disulfide bonds; PAPS, the universal sulphate donor for glycosaminoglycans and detoxification	Supplied entirely as the sulphur amino acids

🔬 Trace Elements — Each One Tied to Its Enzyme

Iron (Fe)

Haem in haemoglobin, myoglobin, cytochromes, catalase, peroxidase; iron–sulfur clusters in complexes I–III and aconitase. Deficiency → microcytic hypochromic anaemia. Excess → haemochromatosis.

Zinc (Zn)

Over 300 enzymes: carbonic anhydrase, alcohol dehydrogenase, alkaline phosphatase, carboxypeptidase, superoxide dismutase (Cu/Zn), matrix metalloproteinases. Structural in zinc fingers — the commonest DNA-binding motif. Deficiency: poor wound healing, hypogonadism, impaired taste and smell, dermatitis, immune failure.

Copper (Cu)

Cytochrome c oxidase, lysyl oxidase, tyrosinase, dopamine β-hydroxylase, Cu/Zn-SOD, ceruloplasmin (ferroxidase). Note the pattern: copper enzymes handle oxygen and cross-linking. Deficiency → Menkes disease; overload → Wilson disease.

Selenium (Se)

Present as selenocysteine in glutathione peroxidase, thioredoxin reductase and the T4 → T3 deiodinase. Deficiency → cardiomyopathy (Keshan disease) and impaired thyroid hormone activation.

Iodine (I)

Used for one purpose only — thyroid hormone synthesis. Deficiency → goitre and, in the fetus, cretinism. The thyroid is the only tissue that oxidises iodide.

Manganese (Mn)

Mn-SOD — the mitochondrial superoxide dismutase; also pyruvate carboxylase and arginase. Chronic excess (welders, liver failure) causes a parkinsonian syndrome.

Molybdenum (Mo)

Xanthine oxidase, sulfite oxidase, aldehyde oxidase. Cofactor deficiency causes severe neurological disease from sulfite accumulation.

Chromium (Cr)

Part of the glucose tolerance factor — potentiates insulin action. Deficiency impairs glucose tolerance.

Cobalt (Co)

Required only as the metal centre of vitamin B12. There is no free cobalt requirement.

Fluoride (F)

Incorporated into fluorapatite, which resists acid better than hydroxyapatite. Excess causes dental and skeletal fluorosis.

💡 The pattern worth memorising

Trace elements are almost always at the catalytic centre of an enzyme, and the deficiency syndrome is simply the sum of those enzymes failing. Work backwards: copper deficiency → lysyl oxidase fails → collagen and elastin cannot cross-link → the vascular and connective-tissue signs of Menkes disease. You never need to memorise a symptom list if you know the enzyme.

🦴 Calcium Homeostasis — Three Hormones, Three Organs

Plasma calcium is held at 2.2–2.6 mmol/L (about 9–10.5 mg/dL). Roughly half is free ionised Ca²⁺ — the biologically active fraction — and most of the rest is bound to albumin. Everything below acts on bone, kidney and intestine.

Hormone	Trigger & source	Bone	Kidney	Intestine	Net
PTH	Chief cells, when ionised Ca²⁺ falls — sensed by the calcium-sensing receptor	Stimulates osteoclastic resorption, indirectly through osteoblasts	↑Ca²⁺ reabsorption (distal tubule), ↓phosphate reabsorption (phosphaturic), activates 1α-hydroxylase	Indirect, via calcitriol	↑Ca²⁺, ↓phosphate
Calcitriol 1,25-dihydroxycholecalciferol	The active hormone, made in the kidney under PTH	Permits mineralisation by supplying both ions	↑reabsorption of both	Its principal action: ↑absorption of Ca²⁺ and phosphate by inducing calbindin	↑Ca²⁺, ↑phosphate
Calcitonin	Parafollicular C cells of the thyroid, when calcium is high	Inhibits osteoclasts	↑urinary calcium	—	↓Ca²⁺ — a minor player in adults

Calcium and phosphate move together in bone, and oppositely in plasma

The two are deposited as hydroxyapatite, and their product in plasma is nearly constant — so under PTH they move reciprocally. Phosphate is also needed for ATP, nucleic acids, phospholipids, 2,3-BPG and the phosphate buffer. Acute hypophosphataemia is the hazard of the refeeding syndrome: give carbohydrate to a malnourished patient and phosphate is consumed by a burst of phosphorylation.

Reading a calcium result

Hyperparathyroidism — ↑Ca²⁺, ↓phosphate, ↑PTH.
Hypoparathyroidism — ↓Ca²⁺, ↑phosphate, tetany, with Chvostek and Trousseau signs.
Vitamin D deficiency — ↓Ca²⁺ and ↓phosphate with a secondary ↑PTH: rickets in children, osteomalacia in adults.
Two caveats that change the interpretation: alkalosis lowers ionised calcium (more binds albumin) and causes tetany at a normal total calcium, and a low albumin lowers total calcium without touching the ionised fraction.

Fibrous Proteins — Collagen & Elastin

Collagen is the body's rope; elastin is its rubber band. Almost every connective-tissue disease in the exam is a fault in one of them — or in the copper, vitamin C or protease inhibitor they depend on.

🪢 Collagen — The Body's Structural Rope

Collagen is a fibrillar protein that forms the main component of the intercellular substance of animal tissues. It makes up 25–35% of all body protein — more than any other single protein — and its job is tensile strength: resistance to being stretched.

Share of body protein

25–35%

the most abundant protein

Chains per molecule

3

α-chains → triple helix

Repeating motif

Gly–X–Y

X = Pro · Y = Hyp

The strength comes from a repeating tripeptide sequence in all three chains, which lets them wind around one another into a long triple helix — the collagen helix. Because the helix is so tightly wound, only the smallest amino acid can sit at the inside of the coil.

🔹 Glycine — every third residue

Glycine has no side chain at all. It occupies every third position precisely because it is small enough to fit where the three chains press against each other. Replace it with anything bulkier and the helix cannot form — which is exactly what happens in osteogenesis imperfecta.

🔹 Proline — the kink

Proline contains a ring structure that allows bends to form in the peptide chain. It favours the helical conformation of the α-chain — but note carefully: this is not an α-helix. It is a distinct, more extended collagen helix.

The sequence in one line

(Gly–X–Y)_n — where X is most often proline and Y is most often hydroxyproline (or hydroxylysine). The amino acid side chains point outward, away from the crowded core of the triple helix.

🧬 The Collagen Types — Sorted by What They Do

There are dozens of collagen types, but the exam cares about a handful, and they sort cleanly into three functional families.

🟥 Family 1 · Collagens that form FIBRES

Rope-like bundles built for tensile strength

Type I

Structures with high tensile strength — bone, skin, tendon, dentin. The most abundant collagen. Defective in osteogenesis imperfecta.

Type II

Present only in cartilage. A clean, easily-tested one-liner.

Type III

Found in expanding (reticular) fibres — blood vessels, granulation tissue, gut. Its mutations are the most clinically dangerous form of Ehlers–Danlos syndrome.

▼

🟨 Family 2 · Collagens that form NETWORKS

Three-dimensional meshes, not bundles

Types IV and VIII

Form network systems — three-dimensional structures. Type IV is the collagen of basement membranes: kidney glomerulus, cochlea, eye. Mutated in Alport syndrome.

▼

🟩 Family 3 · Collagens that CONNECT

Molecular staples between fibres and the matrix

Types IX and XII

Enable the connection of collagen fibres with each other, or with other components of the extracellular matrix.

💡 Three families, three diseases

Fibres → Type I → osteogenesis imperfecta. Networks → Type IV → Alport syndrome. Type III → vascular Ehlers–Danlos. Learn the family and the disease follows.

What the paper actually asks ★ asked 2024/25

Type IV = basement membranes (a mesh, not fibrils) and type I = bone and skin are the two that keep coming back; the usual distractor is “type IV forms fibrils” — it does not. The other recurring point sits in the biosynthesis card below: glycosylation is added onto hydroxylysine, after hydroxylation but before the triple helix is assembled.

🏭 Collagen Biosynthesis — Six Stations, Inside then Outside

Collagen is one of the few proteins with major processing on both sides of the cell membrane. Everything that keeps it soluble happens inside; everything that makes it strong happens outside. Every collagen disease can be placed at one of these six stations.

1️⃣ Synthesis — in the fibroblast

Rough endoplasmic reticulum

Product: pro-α chain

Fibroblasts are the site of collagen precursor synthesis. Special sequences at the N-terminus of the prepro-α chain signal for secretion; the chain is transported to the rough endoplasmic reticulum and the N-terminal signal sequences are cleaved off, leaving the pro-α chain.

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2️⃣ Hydroxylation — the vitamin C step

Rough endoplasmic reticulum

Enzymes: prolyl hydroxylase + lysyl hydroxylase

Proline and lysine residues are hydroxylated after they have been built into the chain — a post-translational modification — producing hydroxyproline and hydroxylysine.

Proline / Lysine

prolyl / lysyl hydroxylase⟶O₂ · Fe²⁺ · vitamin C

Hydroxyproline / Hydroxylysine

The extra –OH groups increase the number of hydrogen bonds, stabilising the triple helix. The enzyme is a Fe²⁺-dependent oxidoreductase, and Fe²⁺ must be kept reduced — that is vitamin C's job.

▼

3️⃣ Glycosylation

Before the triple helix forms

Sugar added: glucose and/or galactose

Glucose and/or galactose are attached enzymatically to the –OH groups of hydroxylysine. This affects the size of the final structure and reduces susceptibility to collagenase. Timing matters — it occurs before the triple helix is assembled.

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4️⃣ Assembly & secretion

Golgi apparatus → secretory vesicles

Product: procollagen

Three pro-α chains associate. Interchain disulphide bonds form between their C-terminal extension peptides, which aligns the three chains correctly like a zip guide. The molecule matures in the Golgi, is packed into secretory vesicles, and released into the extracellular space.

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5️⃣ Extracellular processing

Outside the cell

Enzyme: procollagen peptidase

The N- and C-terminal propeptides are removed, converting procollagen into tropocollagen. Tropocollagen molecules then align into parallel fibrils, each shifted by ¾ of its length relative to the last — the staggered pattern that gives collagen its banded appearance.

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6️⃣ Cross-linking — the copper step

Maturation into a load-bearing fibre

Enzyme: lysyl oxidase (contains Cu²⁺)

Lysyl oxidase catalyses oxidative deamination of certain lysine and hydroxylysine residues, producing reactive aldehydes (allysine and hydroxyallysine). These condense spontaneously with lysine residues on neighbouring molecules, forming covalent cross-links — and the fibre finally matures.

⚠️ Two micronutrients, two different steps

Vitamin C is needed at station 2 (hydroxylation) → deficiency = scurvy. Copper is needed at station 6 (cross-linking) → deficiency = Menkes disease. Both weaken collagen, but by different mechanisms and with different clinical pictures.

♻️ Collagen Degradation

Collagen has a very long half-life — up to several years. But connective tissue is a dynamic structure that is constantly remodelled, usually in response to tissue growth or injury.

▸ How a type I collagen fibre is taken apart

Type I collagen

intact fibre

collagenase (MMP)→

¾ fragment

¼ fragment

other MMPs→

Amino acids

Matrix metalloproteinases (MMPs) are a family of enzymes that include the collagenases. Cleavage of type I collagen happens at one specific site, producing fragments of exactly ¾ and ¼ of the original length; other MMPs then finish the job down to amino acids.

🎈 Elastin — The Body's Rubber Band

Where collagen resists stretching, elastin allows it — and then pulls back. It gives lung, skin and large arteries the ability to stretch and bend in any direction under tension and return to shape.

🪢 COLLAGEN

Rich in proline and glycine; large amounts of hydroxyproline and hydroxylysine
Glycine at every third position
Forms an ordered triple helix
Cross-link: from allysine + lysine
Function: tensile strength

🎈 ELASTIN

Precursor tropoelastin, ~700 amino acids, mostly small and nonpolar (glycine, alanine, valine)
Rich in proline and lysine, but very little hydroxyproline and hydroxylysine
Not a regular helix — a random-coil network
Cross-link: desmosine
Function: elasticity / recoil

How elastin is built

Tropoelastin is secreted into the extracellular space.
It interacts with specific glycoprotein microfibrils — notably fibrillin — which serve as a scaffold for tropoelastin deposition.
Some lysyl side chains are oxidatively deaminated by lysyl oxidase, forming allysine residues.
Three allysine side chains plus one unmodified lysine — from the same or neighbouring polypeptides — form a desmosine cross-link.

💡 The result

A highly interconnected, elastic network capable of stretching and bending in any direction under tension — which is what gives connective tissue its elasticity. Note that lysyl oxidase serves both proteins: it is the single copper enzyme that cross-links collagen and elastin.

Why Marfan syndrome is not a collagen disease

Marfan syndrome is a mutation in fibrillin-1 — the microfibril scaffold that tropoelastin is laid down on, not collagen and not elastin itself. The abnormal fibrillin is incorporated alongside the normal protein, which disrupts the formation of functional microfibrils. This is why Marfan gives aortic aneurysm and lens dislocation, not brittle bones.

🛡️ α₁-Antitrypsin — The Lung's Bodyguard

The alveolar wall is under constant low-level attack from neutrophil elastase, released by activated and dying neutrophils. α₁-Antitrypsin (α₁-AT, A1AT) is the inhibitor that neutralises it. Because lung tissue cannot regenerate, losing that protection means permanent destruction.

Property	Detail
Also called	α₁-antiproteinase
Share of plasma α₁-globulin	Over 90% of the normal α₁-globulin fraction
Made by	Mostly the liver; smaller amounts by monocytes and alveolar macrophages (important for local protection)
Main target	Neutrophil elastase — which degrades elastin in alveolar walls
Critical residue	A specific methionine is required for the inhibitor to bind its target protease

Two ways the shield fails

🧬 Genetic — the PiZ mutation

The commonest clinically relevant mutation is a purine base substitution (GAG → AAG), replacing glutamic acid with lysine at position 342. The mutant protein polymerises inside the hepatocyte endoplasmic reticulum, so less α₁-AT is secreted — and the accumulated polymer can itself cause liver cirrhosis.

Two defective alleles must be inherited to be at risk of emphysema.

🚬 Acquired — smoking

Smoking causes oxidation and inactivation of the critical methionine residue, making the inhibitor useless against elastase even when present in normal amounts.

Smokers with α₁-AT deficiency therefore have a significantly higher rate of lung destruction and worse survival than non-smokers with the same deficiency.

▸ The protease–antiprotease balance

Neutrophils

activated / degenerating

→

Neutrophil elastase

blocked by α₁-AT⊣

Alveolar elastin

protected

if α₁-AT deficient or oxidised→

Emphysema

💊 Treatment

The deficiency of elastase inhibitor can be reversed by replacement therapy — weekly intravenous infusions of α₁-AT, which diffuses from the blood into the lungs and reaches therapeutic levels in the fluid surrounding lung epithelial cells. Open the full disease card →

🔬 Connective Tissue Diseases — All Six in One Frame

Every disease below is a fault at one of the stations you have just read. Tap any card for the full clinical picture.

🍊 Scurvy

Vitamin C deficiency → hydroxylation fails (station 2). Fewer hydrogen bonds, unstable helix, no cross-linking of fibrils. Spontaneous bleeding, swollen bleeding gums, loose teeth, poor wound healing, pathological fractures.

▸ Open full card

🤸 Ehlers–Danlos

Either an enzyme deficiency (lysyl hydroxylase or procollagen peptidase) or a mutation in type I, III or V collagen. The most clinically significant mutations are in type III — potentially fatal vascular complications.

▸ Open full card

🦴 Osteogenesis Imperfecta

Mutations in the pro-α1 or pro-α2 chains of type I collagen. Most commonly glycine is replaced by a bulkier residue, preventing triple helix formation. Type I = tarda (childhood fractures); type II = congenita (fatal, pulmonary hypoplasia); type III = enough collagen, wrong structure.

▸ Open full card

👂 Alport Syndrome

Type IV collagen — basement membranes of kidney, cochlea and eye. About 1 in 5000, usually X-linked dominant. Glomerulonephritis, haematuria, proteinuria, hypertension, end-stage renal failure, and hearing loss.

▸ Open full card

🧍 Marfan Syndrome

Fibrillin-1 — an elastic-fibre glycoprotein, NOT collagen. Long limbs and arachnodactyly, tall slender build with short torso, scoliosis and chest deformity, lens dislocation and myopia, aortic aneurysm and valve defects.

▸ Open full card

🧵 Copper Disorders

Copper deficiency (Menkes, X-linked) or overload (Wilson) both disrupt function. Lysyl oxidase is one of several copper enzymes — the others are cytochrome oxidase, dopamine hydroxylase, superoxide dismutase and tyrosinase.

▸ Open full card

🎯 The five copper enzymes — a guaranteed exam list

Lysyl oxidase (collagen/elastin cross-links) · Cytochrome c oxidase (Complex IV) · Dopamine β-hydroxylase (noradrenaline synthesis) · Superoxide dismutase (antioxidant) · Tyrosinase (melanin). Add ceruloplasmin (ferroxidase) and you have every copper protein worth knowing.

📜 Retake Essay-Paper Additions

The cards below cover the points that the retake essay list asks for explicitly. They are the same material carried by Resit Nitro, kept here so the full curriculum always contains everything the shortened modes do.

🪢 Collagen — Structure, Vitamin C, and What Makes It Unique Past Q

The most abundant protein in the body and the exam's model of a fibrous protein. Three polypeptides of about 1000 amino acids each wind together into the collagen triple helix.

The repeating sequence

(Gly–X–Y)ₙ, where X is often proline and Y often hydroxyproline. Every third residue must be glycine: the three chains pack so closely that only a hydrogen atom fits at the axis of the helix.

The geometry

Each chain is a left-handed helix with 3.3 residues per turn; the three chains then wrap around each other in a right-handed superhelix. A mature fibre is a rod with an axial ratio of about 200 — extremely long and thin.

What holds it together

Hydrogen bonds between the chains (the glycine N–H and the hydroxyproline –OH), then permanent covalent cross-links between lysine and hydroxylysine side chains made by lysyl oxidase, a copper enzyme.

Why vitamin C is essential Past Q

Collagen is made first as the larger precursor procollagen. Many of its prolyl and lysyl residues must be hydroxylated by prolyl and lysyl hydroxylase — enzymes that need O₂, α-ketoglutarate, Fe²⁺, and ascorbate as the reducing agent that keeps the iron in its active state. No vitamin C means too few hydroxyproline and hydroxylysine residues, so the triple helix is unstable.

Scurvy — the deficiency in one line

Unstable, under-hydroxylated collagen: bleeding gums, loose teeth, perifollicular haemorrhage, swollen painful joints, poor wound healing, and death if untreated.

Characteristics that make collagen different from other proteins Past Q

1 · glycine at every third position · 2 · exceptionally rich in proline · 3 · contains hydroxyproline and hydroxylysine, which are almost absent elsewhere · 4 · sugars (glucose, galactose) are attached to hydroxylysine, so it is a glycoprotein · 5 · it has no α-helix and no β-sheet — the triple helix is its own class of structure · 6 · it is extracellular, insoluble, cross-linked and very long-lived · 7 · it contains no tryptophan and almost no cysteine, which is why gelatin is a protein of defective nutritional value.

Bioenergetics — Free Energy & Coupling

The rules that decide whether a reaction can happen at all. Bioenergetics predicts whether a process is possible; kinetics estimates how fast. Everything about ΔG, ΔG°, K_eq and ATP coupling lives here.

⚖️ What Bioenergetics Is — and Is Not

Bioenergetics describes the transfer and use of energy in biological systems. It concerns only the initial and final energy states of the reaction components — it says nothing about the reaction mechanism, and nothing about how long the transformation takes.

⚖️ BIOENERGETICS answers

Can this happen? Based on free energy change. Predicts direction and feasibility.

⏱️ KINETICS answers

How fast? Based on activation energy and enzymes. A reaction can be highly favourable and still take a million years without a catalyst.

The three thermodynamic quantities

Symbol	Name	What it measures	Enough on its own?
ΔH	Enthalpy	Change in heat content of substrates and products	No
ΔS	Entropy	Change in randomness or disorder	No
ΔG	Free energy	Combines both — the true predictor of direction	Yes

The key statement

Neither enthalpy nor entropy alone is sufficient to determine the direction of a reaction. Only the change in free energy, ΔG, predicts it.

➡️ Reading the Sign of ΔG

Negative ΔG

A → B

EXERGONIC

Spontaneous · net energy released

Positive ΔG

B → A

ENDERGONIC

Not spontaneous · energy must be supplied

ΔG = 0

A ⇌ B

EQUILIBRIUM

No net change in either direction

Spontaneous reactions proceed until ΔG reaches zero — that is the definition of equilibrium. And the forward and reverse reactions always have equal magnitude but opposite sign: if A → B is −5 kcal/mol, then B → A is +5 kcal/mol.

ΔG depends on concentrations — ΔG° does not

ΔG is the real free energy change at whatever concentrations actually exist in the cell. ΔG° (standard free energy change) is measured with all substrates and products at 1 mol/L and pH 7 — it consists only of the constants R, T and K_eq, so it never changes with concentration.

This has a practical consequence: a reaction with a positive ΔG° can still run forward if the product/substrate ratio is small enough — that is, if there is a large excess of substrate. Cells exploit this constantly.

Relationship to the equilibrium constant. At equilibrium, the rate of A→B equals the rate of B→A, the ratio [B]/[A] becomes constant regardless of the absolute concentrations, that ratio is K_eq, and the overall ΔG = 0. This lets ΔG° be calculated directly from a measurement of K_eq.

🔗 Energetic Coupling — How Cells Break the Rules

A metabolic pathway does not need every step to be favourable. If the sum of the ΔG values of the individual reactions remains negative, the whole pathway can proceed — even though some individual steps have a positive ΔG.

The simplest form of energetic coupling occurs when the energy-requiring reaction and the energy-providing reaction share a common metabolite. Processes with a large positive ΔG are coupled to the strongly exergonic hydrolysis of ATP.

▸ ATP as the universal energy currency

ATP

hydrolysis→ΔG° ≈ −7.3 kcal/mol

ADP + P_i

or → AMP + PP_i

2 ADP ⇌ ATP + AMP

adenylate kinase

💡 Why ATP does not simply fall apart

ATP is very stable, and its hydrolysis has a high activation energy. Enzymes are therefore required to release its energy — which is exactly what makes ATP a controllable currency rather than a leak. Each of the two terminal phosphate groups carries a ΔG° of about −7.3 kcal/mol, which is why ATP is called a high-energy phosphate compound.

🔢 The Energy Arithmetic of the Respiratory Chain

Quantity	Value	Meaning
ΔG° for ADP → ATP	7.3 kcal/mol	The cost of making one ATP
Energy from NADH → O₂	52.6 kcal	Released by transporting one pair of electrons all the way down the chain
Cost of 3 ATP	3 × 7.3 = 21.9 kcal	Comfortably within the 52.6 kcal available
P/O ratio for NADH	3 : 1	ATP made per atom of oxygen reduced
P/O ratio for FADH₂	2 : 1	Lower because Complex I is bypassed

The crucial caveat

Electron flow through the chain is energetically favoured — NADH is a strong electron donor and O₂ a strong acceptor. But the flow of electrons does not lead directly to the synthesis of ATP. The link is the proton gradient, described by the chemiosmotic hypothesis.

🔋 Redox Pairs & Standard Reduction Potential (E°)

Oxidation of one metabolite (the electron donor) is always accompanied by reduction of another (the electron acceptor). Different redox pairs have different tendencies to lose electrons, and that tendency is quantified as E°, the standard reduction potential, measured in volts.

⬅️ MORE NEGATIVE E°

The greater the tendency to LOSE electrons — a strong reducing agent, a good electron donor. NADH sits here.

➡️ MORE POSITIVE E°

The greater the tendency to TAKE electrons — a strong oxidising agent, a good electron acceptor. Oxygen sits here.

💡 The one rule

Electrons flow from the pair with the more negative E° to the pair with the more positive E° — and the components of the electron transport chain are arranged in rising order of E°. That is the entire logic of the respiratory chain in one sentence.

Electrons travel in different chemical forms along the way: as hydride ions on NAD, as hydrogen atoms on FAD, FMN and coenzyme Q, and as bare electrons on cytochrome c. Free energy is released at each transfer.

🔋 Macroergic Compounds and the Three Levels of Energetic Metabolism

🔹 Macroergic “high-energy” compounds — hydrolysis releases more than about 25 kJ/mol

Phosphoanhydrides
ATP, GTP, UTP, CTP, ADP.

Acyl phosphates
1,3-bisphosphoglycerate, carbamoyl phosphate, acetyl phosphate.

Enol phosphate
Phosphoenolpyruvate — the most energetic of all.

Guanidinium phosphate
Creatine phosphate — the muscle ATP buffer.

Thioesters
Acetyl-CoA, succinyl-CoA, acyl-CoA — energy in a C–S bond.

Sulfonium / nucleotide sugars
S-adenosylmethionine, UDP-glucose, PAPS.

Why ATP is a currency

ATP sits in the middle of the energy scale: PEP, creatine phosphate and 1,3-BPG are above it and can phosphorylate ADP; glucose-6-phosphate and glycerol-3-phosphate are below it and are phosphorylated by ATP. Because ΔG values are additive, coupling an endergonic reaction to ATP hydrolysis through a shared enzyme or intermediate makes the overall ΔG negative.

🔹 The three levels

Level 1 · conversion

Lipids → fatty acids + glycerol; sugars → glucose; proteins → amino acids. Almost no energy captured — the purpose is to reach a few common currencies.

Level 2 · convergence

β-oxidation, glycolysis + pyruvate dehydrogenase and amino acid catabolism all end at acetyl-CoA (with ketone bodies as its transport form). A small amount of ATP and reduced coenzyme appears here.

Level 3 · the final common pathway

Krebs cycle → NADH/FADH₂ → respiratory chain → oxidative phosphorylation. Nearly all the ATP is made here, whatever the fuel was.

Fructose & Galactose Metabolism

Two dietary sugars that enter glycolysis by side doors — and four inherited diseases that follow directly from where each door is blocked. Also the polyol pathway, which explains why diabetes damages the eye, nerve and kidney.

🍏 Fructose — Entering Glycolysis Below the Gate

Fructose is supplied in the diet as sucrose (hydrolysed in the intestine to glucose and fructose) and as a free monosaccharide in fruit, honey and products sweetened with glucose–fructose syrup.

▸ The hepatic fructose pathway

Fructose

fructokinase→ATP · liver, kidney, intestine

Fructose-1-P

aldolase B→

DHAP

Glyceraldehyde

→triose kinase

Glycolysis

below PFK-1

Substrate Product Destination

⚡ Why fructose is metabolised faster than glucose

The pathway bypasses phosphofructokinase-1 — the major rate-limiting step of glycolysis. There is no brake. High dietary fructose therefore produces a high flow of metabolites through the pathway, and consequently:
• an increase in synthesis and esterification of fatty acids
• a deterioration of the plasma lipid profile (raised triacylglycerols and LDL)

The two kinases that can touch fructose

Enzyme	Product	Km for fructose	Where	Insulin-sensitive?
Fructokinase	Fructose-1-P	Low Km, high Vmax — fructose is its only substrate	Liver, kidney, intestine	No (but PFK-1 is)
Hexokinase	Fructose-6-P	High Km — low affinity; acts only when fructose is unusually high	Most cells	—

The three aldolase isoforms

Aldolase A

Present in most tissues

Aldolase B

Liver, kidney, small intestine — and the only isoform that cleaves fructose-1-phosphate

Aldolase C

Present in the brain

Shared substrate

All three act on fructose-1,6-bisphosphate of the glycolytic pathway

🍭 Disorders of Fructose Metabolism

😌 Essential Fructosuria — BENIGN

Fructokinase deficiency. Fructose simply accumulates and appears in the urine. No phosphorylated intermediate forms, so nothing toxic builds up. No treatment needed.

▸ Open full card

🚨 Hereditary Fructose Intolerance — SEVERE

Aldolase B deficiency. Fructose-1-phosphate accumulates, trapping inorganic phosphate and depleting ATP. Causes hyperuricaemia, lactic acidaemia, hypoglycaemia with vomiting, jaundice, haemorrhage, hepatomegaly and renal dysfunction. Sucrose, sorbitol and fructose must be removed from the diet to prevent liver failure and possible death.

▸ Open full card

💡 The one-line discriminator

Fructokinase makes fructose-1-phosphate; aldolase B breaks it down. Losing the maker is harmless. Losing the breaker is dangerous — because the intermediate itself is the poison.

👁️ The Polyol Pathway — Why Diabetes Damages Specific Organs

An alternative way of handling a monosaccharide is to convert it into a polyol (a sugar alcohol) by reducing its aldehyde group. Glucose → sorbitol → fructose. The danger lies in the fact that the two enzymes involved are not present in the same tissues.

Glucose

aldose reductase⟶NADPH · high Km

Sorbitol

sorbitol dehydrogenase⟶NAD⁺

Fructose

🟦 ALDOSE REDUCTASE — widely present

Expressed in lens, retina, Schwann cells of peripheral nerves, kidney, placenta, ovaries and seminal vesicles. Has a high Km for glucose — so it only becomes busy when glucose is high.

🟩 SORBITOL DEHYDROGENASE — restricted

Expressed in liver, ovaries and seminal vesicles only. It is low or absent in lens, retina, nerve and kidney — precisely the tissues damaged in diabetes.

▸ The four-step mechanism of diabetic complications

Hyperglycaemia

1 →insulin-independent uptake

Glucose floods lens, nerve, kidney, retina

2 →aldose reductase

Sorbitol

3 ⊣no sorbitol dehydrogenase · cannot cross membranes

TRAPPED in the cell

4 →osmotic effect

Cell swelling

🎯 The four classic diabetic complications — all from this one mechanism

Cataract (lens) · Peripheral neuropathy (Schwann cells) · Nephropathy (kidney) · Retinopathy (retina). Every one of them is a tissue with aldose reductase but without sorbitol dehydrogenase.

A useful aside

Both enzymes are present in the seminal vesicles — where they cooperate to supply fructose, the major carbohydrate energy source for sperm cells. The same pathway, used constructively.

🥛 Galactose — Three Enzymes, Two Diseases

Galactose is an essential energetic and structural material. It is supplied as lactose (hydrolysed in the intestine to galactose and glucose), and some is obtained by lysosomal degradation of complex carbohydrates such as glycoproteins and glycolipids.

▸ The Leloir pathway

Galactose

galactokinase→present in many cells

Galactose-1-P

GALT→uses UDP-glucose

UDP-galactose

UDP-hexose 4-epimerase⇌

UDP-glucose → glycolysis / gluconeogenesis

Enzyme	Reaction	Note
Galactokinase	Galactose → galactose-1-phosphate	Present in many cells
GALT (galactose-1-P uridyltransferase)	Galactose-1-P + UDP-glucose → UDP-galactose + glucose-1-P	Uses UDP-glucose as a second substrate
UDP-hexose 4-epimerase	UDP-galactose ⇌ UDP-glucose	Reversible — so we can also make galactose when the diet contains none

🟡 Galactokinase Deficiency — MILDER

Causes galactosaemia and galactosuria. Galactitol accumulates if galactose is present in the diet → cataracts only. Treatment is dietary restriction.

▸ Open full card

🔴 Classic Galactosaemia — SEVERE

GALT deficiency. Galactose-1-phosphate and galactitol accumulate in brain, lens, liver and kidney → liver damage, severe intellectual disability and cataracts. Remove galactose and lactose from the diet immediately.

▸ Open full card

💡 Why both get cataracts but only one gets brain damage

Galactitol (made by aldose reductase, exactly as sorbitol is) accumulates in the lens in both conditions → cataracts in both. But only GALT deficiency lets galactose-1-phosphate accumulate — and that is the directly toxic metabolite responsible for liver, brain and kidney damage.

🍼 UDP-Galactose as a Building Block — and How Milk Sugar Is Made

UDP-galactose is the donor of galactose units in biosynthesis: lactose, glycoproteins, glycolipids and glycosaminoglycans.

Lactose synthase is one of the most elegant regulatory tricks in biochemistry — a helper protein that does not change an enzyme's activity but changes what it makes.

🅰️ PROTEIN A — β-D-galactosyltransferase

Expressed in many tissues. Transfers galactose from UDP-galactose to N-acetyl-D-glucosamine, forming the β(1→4) linkage and producing N-acetyllactosamine — a component of glycoproteins.

🅱️ PROTEIN B — α-lactalbumin

Expressed only in lactating mammary glands. Its synthesis is stimulated by prolactin. It forms a complex with protein A and changes its substrate specificity — so that lactose, rather than N-acetyllactosamine, is produced.

🎯 Why this is worth remembering

It is the textbook demonstration that a regulatory subunit can alter an enzyme's substrate specificity rather than merely its rate — and it explains why lactose appears in milk only after delivery, when prolactin rises. Open the enzyme card →

NADPH, Antioxidants & Detoxification

NADPH is not just "the other NAD". It is the cell's reducing power for building things and for defence. This section covers every one of its six jobs — and the diseases that appear when the supply fails.

⚡ NAD⁺ vs NADP⁺ — Two Coenzymes, Opposite Jobs

🔵 NAD⁺ / NADH — CATABOLISM

The main coenzyme of dehydrogenases
Cytosolic ratio in hepatocytes ≈ 1000 : 1 (mostly oxidised, ready to accept electrons)
Participates in oxidation reactions — Krebs cycle, glycolysis

🟢 NADP⁺ / NADPH — ANABOLISM

The main coenzyme of monooxygenases and reductases
Ratio ≈ 1 : 10 (mostly reduced, ready to donate electrons)
Participates in reduction reactions — biosynthesis and defence

💡 The whole idea in one line

The cell deliberately keeps NAD⁺ oxidised so it can collect electrons from fuel, and NADP⁺ reduced so it can hand electrons out for building and defence. Same chemistry, opposite redox state, kept in two separate pools.

🎯 The Six Jobs of NADPH

1 · Reductive biosynthesis
Fatty acids, cholesterol, bile acids, steroid hormones

2 · Reduction of H₂O₂
Via glutathione — the red cell's survival

3 · Vitamin D
Synthesis and metabolism

4 · Detoxification
Cytochrome P450 monooxygenase system

5 · Killing microbes
NADPH oxidase in phagocytes

6 · Nitric oxide
NO synthase

Where NADPH comes from

The pentose phosphate pathway (oxidative phase — G6PD and 6-phosphogluconate dehydrogenase) and the malic enzyme. In erythrocytes the pentose phosphate pathway is the ONLY source — which is the single fact that makes G6PD deficiency a disease.

🛡️ Job 2 — The Glutathione Antioxidant System

▸ How a red cell survives an oxidative attack

Glucose-6-P

G6PD→

NADPH

glutathione reductase→FAD (B2)

GSH

reduced glutathione

glutathione peroxidase→selenium

H₂O₂ → 2 H₂O

threat neutralised

Glutathione peroxidase catalyses the reduction of hydrogen peroxide. It contains selenium and requires reduced glutathione — a tripeptide, γ-glutamylcysteinylglycine.
NADPH is necessary to reconstitute the reduced form of glutathione, via glutathione reductase.
The pentose phosphate pathway is the only source of NADPH in erythrocytes.

The other two antioxidant enzymes

🧪 Catalase

Converts hydrogen peroxide to oxygen and water: 2 H₂O₂ → 2 H₂O + O₂. Peroxisomal, heme-containing, and one of the fastest enzymes known.

💨 Superoxide dismutase

Converts superoxide to oxygen and H₂O₂. Cu/Zn form in cytosol, Mn form in mitochondria. Note it creates peroxide — so it must work in series with the other two.

🧪 Job 4 — Cytochrome P450: Two Systems, Two Addresses

Monooxygenases are hemoproteins that incorporate one atom from molecular oxygen into a substrate (creating a hydroxyl group), with the other atom reduced to water. NADPH is the coenzyme. They operate in two different subcellular locations with entirely different jobs.

🔋 MITOCHONDRIAL system — the builder

Associated with the inner mitochondrial membrane

Job: biosynthesis

Steroid hormones — placenta, ovaries, testes, adrenal cortex
Bile acids — liver
Vitamin D₃: hydroxylation of cholecalciferol to 25-hydroxycholecalciferol in the liver, then to the biologically active 1,25-dihydroxy form in the kidney

▼

🧹 MICROSOMAL system — the cleaner

Membranes of the smooth endoplasmic reticulum, particularly in the liver

Job: detoxification of xenobiotics

Handles drugs and pollutants such as petroleum products and pesticides. Hydroxylation serves three purposes:

Activation or inactivation of drugs
Makes xenobiotics more soluble, facilitating excretion in urine or faeces
Creates a new site for conjugation with a polar molecule such as glucuronic acid, which increases solubility further

🔗 The link to porphyria

Administering many drugs induces the CYP system → more CYP protein is made → more heme is consumed → hepatic heme falls → ALA synthase 1 is de-repressed → ALA and porphyrin precursors accumulate. This is precisely why barbiturates and ethanol precipitate acute porphyria attacks.

💥 Job 5 — The Respiratory Burst

NADPH is used in the oxygen-dependent mechanisms by which white blood cells kill bacteria.

NADPH + 2 O₂

NADPH oxidase⟶flavocytochrome: FAD + heme

NADP⁺ + 2 O₂•⁻

Located in the leukocyte cell membrane.
Contains flavocytochrome (FAD and heme).
Reduces molecular oxygen from the surrounding tissue to superoxide — a free radical. The sudden surge in oxygen consumption is called the respiratory burst.

🩺 Genetic deficiency

Causes chronic granulomatous disease (CGD) — characterised by severe, persistent infections and the formation of granulomas. Phagocytes can engulf organisms but cannot kill them.

💨 Job 6 — Nitric Oxide

Nitric oxide is a gas that acts as a signalling molecule. Its half-life in tissues is only 3–10 seconds — it reacts with oxygen and superoxide and is converted into nitrates and nitrites. That extreme brevity is what makes it a precise, local signal.

Function 1

Endothelium-derived vasodilator

Function 2

Neurotransmitter in the central nervous system

Function 3

Inhibitor of platelet aggregation

Function 4

Mediates tumoricidal and bactericidal actions of macrophages

The three NOS isoforms

Isoform	Where	Ca²⁺ dependence	Output
eNOS (constitutive)	Endothelium	Ca²⁺–calmodulin dependent	Constant low levels
nNOS (constitutive)	Neural tissue	Ca²⁺–calmodulin dependent	Constant low levels
iNOS (inducible)	Hepatocytes, macrophages, monocytes, neutrophils	Ca²⁺-independent	Large amounts after induction

iNOS is induced by tumour necrosis factor-α, bacterial endotoxins and inflammatory cytokines — which is why septic shock produces profound, treatment-resistant vasodilation.

Cofactors of NO synthase

NADPH, FMN, FAD, heme and tetrahydrobiopterin. Note that tetrahydrobiopterin is the same cofactor used by phenylalanine, tyrosine and tryptophan hydroxylases — which links NO synthesis to the biogenic amine pathways. Open the enzyme card →

🩸 G6PD Deficiency — When the NADPH Supply Fails

An X-linked inherited disease characterised by haemolytic anaemia caused by the inability to detoxify oxidising agents. It is the most common disease caused by an enzyme abnormality in humans — more than 400 different mutations are known in the G6PD gene.

Mutations disrupt the structure of the active site and change kinetic properties: reduced catalytic activity and stability, or altered binding affinity for NADP⁺ or glucose-6-phosphate.

Where it is common

Highest prevalence in the Middle East, tropical Africa, Asia and parts of the Mediterranean. In Poland the prevalence is low, around 0.1%. Female carriers show increased resistance to Plasmodium falciparum — the selective pressure that kept the mutation common.

The three-step mechanism of the anaemia

1
G6PD deficiency impairs the ability of the cell to form NADPH, which is essential for maintaining the reduced glutathione pool.
2
Detoxification of free radicals and peroxides falls, with two consequences: (a) oxidation of haemoglobin sulfhydryl groups → insoluble masses called Heinz bodies that attach to the erythrocyte membrane; (b) oxidation of membrane proteins makes the red cells rigid and less deformable.
3
Erythrocyte lifespan is reduced — the cells are removed from the circulation by macrophages in the spleen and liver.

What triggers a crisis

Trigger	Examples	Note the exceptions
Oxidant drugs	Sulfamethoxazole, chloramphenicol (antibiotics); primaquine (antimalarial); acetanilid (antipyretic)	Primaquine but NOT quinine. Acetanilid but NOT acetaminophen.
Fava beans	In some forms of the deficiency — hence "favism"	Not all variants react
Infections	Free radicals generated in macrophages	The commonest trigger in practice

Common symptoms

Haemolytic anaemia, jaundice, splenomegaly. Open the full disease card →

🛡️ The Natural Antioxidants — and the Three Items That Are Not

Two lines of defence: enzymes that destroy the reactive species, and small molecules that absorb the unpaired electron and become stable radicals themselves.

Naturally occurring in the body

Vitamin E — the most important lipid-soluble antioxidant, the one that protects membrane PUFA.
Vitamin C — water-soluble, and it regenerates vitamin E.
Uric acid / urate — a major plasma antioxidant.
Glutathione — the cell's internal buffer of reducing power.
Enzymes: superoxide dismutase, catalase, glutathione peroxidase.

The three that get offered and are wrong

β-Carotene — provitamin A, taken from the diet; it is not made in the body, which is why it is the odd one out in a "naturally occurring" list.
Isoprostanes — products of the non-enzymatic auto-oxidation of arachidonic acid. They are a marker of oxidative stress, not a defence against it.
Vitamin K — not an antioxidant at all: it is the cofactor for the γ-carboxylation of glutamate.

Catalase — the detail that is asked

Catalase is a haemoprotein with four haem groups (not one) and decomposes 2 H₂O₂ → 2 H₂O + O₂. Together with the peroxidases it is classed as a hydroperoxidase. Glutathione peroxidase differs in using H₂O₂ as an oxidant of glutathione, and in being a selenoenzyme.

Insulin & Glucagon — Mechanism in Depth

Two hormones from two neighbouring cell types, pulling in opposite directions. Everything from how a β cell senses glucose, through the receptor cascade, to the four kinds of hypoglycaemia.

💉 Insulin — Structure & The C-Peptide

A peptide hormone produced by the β cells of the islets of Langerhans. It is the most important hormone coordinating the use of fuels by tissues, and all of its metabolic effects are anabolic.

Total length

51

amino acids

A chain 21 + B chain 30

Disulfide bonds

3

2 interchain + 1 intrachain

the intrachain bond is within the A chain

Plasma half-life

~6

minutes

degraded in liver & kidney

Insulin favours the synthesis of glycogen, triacylglycerol and protein. It is degraded by insulin-degrading enzyme, present in liver and kidneys — mostly the liver. This short duration of action permits rapid changes in circulating levels of the hormone.

Why C-peptide matters clinically

The C-peptide is essential for proper insulin folding, and because it has a long half-life in plasma it is a good indicator of insulin production and secretion. Injected insulin contains no C-peptide — so a low C-peptide with high insulin means the insulin came from outside the body.

🎛️ How a β Cell Senses Glucose — Six Steps

The β cells are the most important glucose-sensing cells in the body. Like the liver, they contain GLUT-2 transporters and express glucokinase (hexokinase IV) — that pairing is what makes glucose sensing possible.

1
Glucose enters through GLUT-2 — insulin-independent, high Km, so uptake is proportional to blood glucose.
2
At blood glucose above 45 mg/dL, glucokinase phosphorylates glucose in amounts proportional to the glucose concentration — because glucokinase is not inhibited by its product glucose-6-phosphate.
3
The sigmoidal relationship between reaction velocity and substrate concentration maximises the enzyme's responsiveness to changes in blood glucose.
4
Metabolism of glucose-6-phosphate generates ATP.
5
ATP-sensitive K⁺ channels close → the plasma membrane depolarises → voltage-gated Ca²⁺ channels activate → calcium floods in.
6
Calcium causes vesicles containing insulin to be released from the β cell.

💊 Where sulfonylureas act

Sulfonylureas — oral agents for type 2 diabetes — increase insulin secretion by closing ATP-sensitive potassium channels, jumping straight to step 5 without needing glucose at all. That is exactly why they can cause hypoglycaemia.

🎯 The three-kinase inhibition trap

Muscle hexokinase is allosterically inhibited by its own product, glucose-6-phosphate. Liver glucokinase is inhibited NOT by its product but by its isomer, fructose-6-phosphate (acting through GKRP). This is a classic exam distinction. A glucokinase mutation produces MODY 2.

📈 What Raises and Lowers Insulin Secretion

▲ INCREASED BY

Glucose — the primary stimulus, after a carbohydrate-rich meal
Amino acids — protein ingestion causes a transient rise that enhances glucose-stimulated secretion (arginine is a potent example). Fatty acids have a similar effect.
Gastrointestinal peptide hormones — the incretins

▼ DECREASED BY

Scarcity of dietary fuels
Stress — fever, infection, hypoxia, vigorous exercise, trauma
Mediated primarily by epinephrine from the adrenal medulla

The incretin effect. The intestinal peptides GLP-1 (glucagon-like peptide-1) and GIP (gastric inhibitory polypeptide, also called glucose-dependent insulinotropic peptide) increase the sensitivity of β cells to glucose. They are released from the small intestine after food, causing an anticipatory rise in insulin — which is why the same amount of glucose given orally induces much greater insulin secretion than if given intravenously. Incretin-mimetic drugs exploit exactly this.

⚠️ The emergency override

In emergency situations the sympathetic nervous system replaces plasma glucose as the controlling influence over β-cell secretion. Epinephrine can override the normal glucose-stimulated release of insulin — mobilising glucose from the liver and fatty acids from adipose tissue, and preventing hypoglycaemia during a crisis.

🔬 The Insulin Receptor & Signal Transduction

🧩 Receptor structure

A tyrosine kinase receptor

Synthesised as a single polypeptide that is glycosylated and cleaved into α and β subunits, then assembled into a tetramer linked by disulphide bonds. The extracellular α subunits contain the insulin-binding site; a hydrophobic domain in each β subunit spans the membrane; and the cytosolic domain of the β subunit is a tyrosine kinase.

▼

⚡ Signal transduction

Autophosphorylation → IRS → downstream cascades

Insulin binding to the α subunits induces conformational changes transmitted to the β subunits → rapid autophosphorylation of specific tyrosine residues on each β subunit → phosphorylation of insulin receptor substrates (IRS) → phosphorylated IRS proteins interact with other signalling molecules through SH2 domains, activating pathways that affect gene expression, cell metabolism and growth. At least four IRS proteins are known, with similar structures but different tissue distributions.

▼

🚪 Membrane effect — moving GLUT-4

The step that actually lowers blood glucose

IRS binds and activates phosphoinositide 3-kinase → phosphorylation of PIP₂ to PIP₃ → PIP₃ binds and activates phosphoinositide-dependent kinase 1 → which activates Akt (protein kinase B) → GLUT-4 vesicles move to the cell membrane.

Which tissues do NOT need insulin for glucose uptake

Hepatocytes, erythrocytes, nervous system, intestinal mucosa, renal tubules and cornea. Their glucose transport is insulin-insensitive — which is exactly why hyperglycaemia damages nerve, kidney, retina and lens: glucose floods in unchecked.

Three timescales of insulin action

Timescale	What happens	Mechanism
Seconds	Increased glucose transport into adipocytes, skeletal and cardiac muscle	GLUT-4 translocation
Minutes to hours	Changes in enzymatic activity	Phosphorylation states of existing proteins
Hours to days	Increased AMOUNT of glucokinase, liver pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase	Increased transcription (via SREBP-1c) and translation

Receptor regulation — why insulin resistance is self-reinforcing

Binding is followed by internalisation of the hormone–receptor complex; insulin is degraded in lysosomes while most receptors are recycled. But elevated levels of insulin promote the degradation of receptors, decreasing the number of surface receptors — so hyperinsulinaemia itself worsens resistance.

🔥 Glucagon — The Counterweight

A peptide hormone of 29 amino acids secreted by the α cells of the pancreatic islets. It is synthesised as a large precursor (preproglucagon) converted by selective proteolytic cleavages — and, unlike insulin, it is processed to different products in different tissues: in intestinal L cells the same precursor yields GLP-1.

Glucagon, along with epinephrine, norepinephrine, cortisol and growth hormone — the counterregulatory hormones — opposes many of the actions of insulin. Its primary target is the liver, and it has a short half-life like insulin.

Effect on…	What glucagon does
Carbohydrate	Immediate rise in blood glucose from increased liver glycogen degradation and increased hepatic gluconeogenesis
Lipid	Primary effect is inhibition of fatty acid synthesis through phosphorylation and inactivation of acetyl-CoA carboxylase by AMPK. The resulting decrease in malonyl-CoA removes the inhibition on CPT-I, so long-chain fatty acid β-oxidation can proceed.
Protein	Increases hepatic uptake of amino acids supplied by muscle → more carbon skeletons for gluconeogenesis → plasma amino acid levels fall

▸ The glucagon signalling cascade

Glucagon

→G-protein-coupled receptor

Adenylyl cyclase

→

cAMP ↑

→

Protein kinase A

→phosphorylates key enzymes

Glycogenolysis ↑
Gluconeogenesis ↑

Two details worth remembering

The glucagon receptor is a GPCR distinct from the one that binds epinephrine. And glucagon receptors are NOT found on skeletal muscle — which is why muscle glycogen can never be mobilised for blood glucose, no matter how low the sugar. Glucagon also affects gene transcription: it induces expression of PEPCK via CREB.

📉 Hypoglycaemia — Definition, Symptoms, Four Types

Hypoglycaemia is defined by three simultaneous features: (1) central nervous system symptoms including confusion, aberrant behaviour or coma; (2) a simultaneous blood glucose ≤50 mg/dL; and (3) resolution of symptoms within minutes of glucose administration. It is a medical emergency, because the CNS has an absolute requirement for a continuous supply of bloodborne glucose.

😰 ADRENERGIC (neurogenic) symptoms

Anxiety, palpitations, tremor, sweating. Mediated by catecholamine release (primarily epinephrine), regulated by the hypothalamus. Typically occur when blood glucose falls abruptly.

🧠 NEUROGLYCOPENIC symptoms

Impaired delivery of glucose to the brain → impairment of brain function. Often result from a gradual decline in blood glucose, often to levels below 50 mg/dL. The slow fall deprives the CNS of fuel but fails to trigger an adequate adrenergic response — which is why the warning signs can be missing.

The two glucose-regulating systems

Pancreatic α cells, which release glucagon.
Receptors in the hypothalamus, which respond to abnormally low blood glucose. These trigger both catecholamine secretion (via the sympathetic nervous system) and release of ACTH and growth hormone from the anterior pituitary; ACTH then increases cortisol.

Glucagon and epinephrine are most important in acute, short-term regulation; cortisol and growth hormone act in the long-term, transcriptional management of glucose metabolism.

The four types of hypoglycaemia

1️⃣ Insulin-induced

Frequent in diabetic patients on insulin, particularly those striving for tight control. Mild cases in conscious patients are treated with oral carbohydrate; unconscious patients are given glucagon subcutaneously or intramuscularly.

2️⃣ Postprandial (reactive)

The second most common form. Caused by an exaggerated insulin release following a meal, giving transient hypoglycaemia with mild adrenergic symptoms. Glucose returns to normal even if the patient is not fed. Treatment: frequent small meals rather than three large ones.

3️⃣ Fasting

Neuroglycopenic symptoms from reduced glucose production (hepatocellular damage, adrenal insufficiency, ethanol) or increased glucose use (rare insulin-producing pancreatic tumours). Certain inborn errors, such as defects in fatty acid oxidation, also cause it.

4️⃣ Alcohol-related

See the mechanism below. Especially dangerous in those who are fasted or have exercised strenuously — and alcohol also increases hypoglycaemia risk in insulin-treated patients, generally many hours after ingestion.

🍷 The Alcohol Mechanism — One Ratio Explains Everything

Ethanol

alcohol dehydrogenase⟶Zn²⁺ · NAD⁺→NADH

Acetaldehyde

aldehyde dehydrogenase⟶NAD⁺→NADH

Acetate

In each reaction electrons are transferred to NAD⁺, so the NADH/NAD⁺ ratio rises sharply. The abundance of NADH then favours two reductions:

↑ NADH/NAD⁺

→

Pyruvate → Lactate

Oxaloacetate → Malate

→both are gluconeogenic precursors

↓ Glucose synthesis → HYPOGLYCAEMIA

↻ Decreased oxaloacetate also diverts acetyl-CoA into ketone bodies → alcoholic ketosis → ketoacidosis

Two clinical corollaries

1. Hypoglycaemia produces many behaviours associated with alcohol intoxication — agitation, impaired judgement, combativeness. 2. ALDH is inhibited by disulfiram, used in chronic alcoholism: the resulting rise in acetaldehyde causes flushing, tachycardia, hyperventilation and nausea. The smell of acetoacetate or acetone on the breath is a common feature of ketosis.

📈 Biphasic insulin release

Healthy β cellType 2 diabetes

The first phase is the immediate discharge of insulin already packaged in vesicles docked at the membrane — it lasts only minutes but it is what stops the post-meal glucose spike before it happens. The second phase is newly synthesised insulin, sustained for as long as glucose stays high. Loss of the first phase is one of the earliest detectable defects in type 2 diabetes, appearing long before the fasting glucose becomes abnormal.

🩺 Naming It — Whipple's Triad, Insulinoma and the C-Peptide Test

Whipple's triad

The formal definition of a true hypoglycaemia, and the name the exam uses: 1 · a low plasma glucose, 2 · typical symptoms at that moment, 3 · relief when glucose is given. All three, or it is not hypoglycaemia.

Hypoglycaemia unawareness

Repeated episodes blunt the adrenergic warning, so the patient goes straight to neuroglycopenia without the sweating and tremor that would normally prompt them to eat. It is the reason tight control carries risk.

Who made the insulin? The C-peptide answers it

An insulinoma — an insulin-secreting islet tumour — and a sulfonylurea both drive the β cell, so proinsulin is cleaved and C-peptide is high. Injected insulin contains no C-peptide and suppresses the β cell, so C-peptide is low while insulin is high. That single measurement separates a tumour from factitious or accidental insulin administration; a sulfonylurea screen then separates the drug from the tumour.

The Feed–Fast Cycle, Tissue by Tissue

The same four regulatory mechanisms run in every tissue — but each organ uses them for its own purpose. Read this as a grid: absorptive versus fasted, down the four tissues that matter.

🎚️ The Four Levers of Metabolic Control

Whatever the tissue and whatever the state, metabolism is switched by exactly four mechanisms. Learn these once and every specific example below becomes an application of them.

1 · Substrate availability
Fed: glucose, amino acids, chylomicron TAG. Fasted: glucose from glycogenolysis, fatty acids and glycerol from lipolysis, amino acids from muscle proteolysis

2 · Induction/repression of enzyme synthesis
↑insulin → ↑ACC and FAS. ↑glucagon → ↑PEPCK. Takes hours to days

3 · Allosteric regulation
Fructose-2,6-bisphosphate activates PFK-1 and inhibits fructose-1,6-bisphosphatase. Ca²⁺–calmodulin activates liver phosphorylase kinase

4 · Covalent modification
By PKA, AMPK and phosphatases. Fast and reversible

🎯 The single most examinable rule — and its four exceptions

In the well-fed state most enzymes are active in the DEPHOSPHORYLATED form. In the fasted state, enzymes that were active when fed are now phosphorylated and inactive.

The exceptions — active when PHOSPHORYLATED: glycogen phosphorylase kinase · glycogen phosphorylase · hormone-sensitive lipase · the phosphatase domain of PFK-2 in the liver. Every one of them serves fuel release — which is why they run backwards to the rule.

🍽️ The Absorptive (Well-Fed) State — 2–4 Hours After a Meal

↑ Glucose

↑ Amino acids

↑ TAG (chylomicrons)

→

↑ INSULIN (β cells)
↓ glucagon

→

ANABOLIC STATE

↑TAG · ↑glycogen · ↑protein synthesis

🫀 LIVER — carbohydrates

Everything from the gut and pancreas passes here first, via the hepatic portal vein, before entering the general circulation

Retains ~60 g of every 100 g of glucose delivered by the portal vein

GLUT-2 — insulin-independent uptake with a high Km, so it only takes up glucose when blood glucose is high
↑ Phosphorylation to glucose-6-phosphate by glucokinase (high Km)
↑ Glycogenesis — glycogen synthase activated by dephosphorylation (↑phosphodiesterase activity, ↓cAMP, activation of protein phosphatase 1) and by allosteric activation by glucose-6-phosphate
↑ Pentose phosphate pathway — typically up to 10% of glucose; stimulated by the combined availability of glucose-6-phosphate and active use of NADPH in hepatic lipogenesis
↑ Glycolysis — only in the absorptive state: more glucokinase, PFK-1 and pyruvate kinase; allosteric activation of PFK-1 by fructose-2,6-bisphosphate; covalent activation (dephosphorylation) of pyruvate kinase and of PDH
↓ Glucose production — low acetyl-CoA inhibits pyruvate carboxylase and inactivates fructose-1,6-bisphosphatase; glycogen phosphorylase and its kinase are dephosphorylated and inactivated

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🧈 LIVER — fats and amino acids

Building and exporting

↑ Fatty acid synthesis from increased acetyl-CoA (from glucose and amino acid metabolism) and NADPH (pentose phosphate pathway and malic enzyme)
ACC activated by dephosphorylation (no AMPK activity) and by its allosteric activator citrate — citrate is made in mitochondria, transferred to cytoplasm when isocitrate dehydrogenase is inhibited by ATP, and cleaved by citrate lyase back to acetyl-CoA
Fat breakdown inhibited — malonyl-CoA (the ACC product) inhibits CPT-I
↑ TAG synthesis and packaging into VLDL; glycerol-3-phosphate comes from glycolysis
↑ Amino acid catabolism — excess released to blood for other tissues; deamination sends carbon skeletons to pyruvate, acetyl-CoA or Krebs intermediates. Branched-chain amino acids are metabolised in MUSCLE, not liver. ↑ liver protein synthesis

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🥓 ADIPOSE TISSUE

Storing — but mostly not making its own fat

↑ GLUT-4 in the plasma membrane → ↑ glucose transport and phosphorylation (by hexokinase)
↑ Glycolysis — supplies glycerol-3-phosphate for TAG synthesis
↑ Pentose phosphate pathway — supplies NADPH
Key point: de novo fatty acid synthesis is NOT the main source of fat in adipose tissue. Most fatty acids come from chylomicron and VLDL triacylglycerols, hydrolysed by lipoprotein lipase, which is induced by insulin

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💪 RESTING SKELETAL MUSCLE

Glucose is the main fuel here in the fed state

Glucose transport mediated by GLUT-4; phosphorylation by hexokinase; used for energy
↑ Glycogenesis driven by rising glucose-6-phosphate
Lipoprotein lipase releases fatty acids from chylomicrons and VLDL, but the main energy source is glucose
↑ Amino acid uptake and protein synthesis after a protein-rich meal; ↑ branched-chain amino acid uptake and catabolism — used both to synthesise protein and to produce energy

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🧠 BRAIN

The privileged consumer

Consumes about 20% of the oxygen used by the body at rest
In the absorptive state the only fuel is glucose, transported by insulin-independent GLUT-1
About 140 g of glucose per 24 h oxidised to CO₂ and H₂O
Totally dependent on delivery in the blood — there is no significant glycogen reserve
Function is impaired below about 50 mg/dL

🌙 The Fasted (Post-Absorptive) State

The fasted state begins when no food is ingested after the absorptive period. It may result from lack of food, planned weight reduction, or from trauma, surgery, cancer or burns. Three priorities govern everything: keep plasma glucose adequate for brain and red cells; mobilise fatty acids for ketone body synthesis in the liver; and save protein — only about one third of body protein can be used for energy without harm.

↓ Glucose

↓ Amino acids

↓ TAG

→

↓ INSULIN
↑ glucagon · ↑ epinephrine · ↑ cortisol

→

CATABOLIC STATE

↑TAG breakdown · ↑glycogen breakdown · ↑protein breakdown

↻ Glucose stays within a narrow range while fatty acids and ketone bodies rise

🫀 LIVER — the glucose factory

Two phases, in sequence

↑ Glycogenolysis from the rising glucagon/insulin ratio. Fast mobilisation, but reserves run out in 24 hours. Phosphorylase kinase and then glycogen phosphorylase are phosphorylated and activated; glycogen synthase is phosphorylated and inactivated
↑ Gluconeogenesis — starts 4–6 hours after the last meal and is fully active once glycogen runs out. Substrates: glucogenic amino acids and lactate from muscle, glycerol from adipose tissue. Fructose-2,6-bisphosphate falls (PFK-2 phosphorylated, phosphatase domain activated), simultaneously inhibiting glycolysis. PEPCK is induced by glucagon
↑ Fatty acid oxidation — the liver's main energy source now. Malonyl-CoA falls because AMPK phosphorylates and inactivates ACC, releasing CPT-I. This provides: NADH (inhibits the Krebs cycle and favours oxaloacetate → malate, freeing acetyl-CoA for ketogenesis); acetyl-CoA (allosteric activator of pyruvate carboxylase, allosteric inhibitor of PDH — pushing pyruvate into gluconeogenesis); and energy from FADH₂ and NADH for the reactions of gluconeogenesis
↑ Ketogenesis in the first days of fasting. The liver cannot use ketone bodies itself — it lacks thiophorase. Ketogenesis provides CoA for further fatty acid oxidation, supplies many organs including the brain with energy (concentration rises from ~50 µM to ~6 mM), and spares protein. In prolonged fasting glucose falls and stays at a lower level, around 65–70 mg/dL

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🥓 ADIPOSE TISSUE

Releasing

Reduced GLUT-4 transport (↓insulin) → less TAG synthesis
↑ Lipolysis — HSL phosphorylated and activated by PKA in response to epinephrine and norepinephrine
Fatty acids released and transported with albumin to tissues that can oxidise them
Glycerol goes to the liver as a gluconeogenic substrate — adipose tissue cannot use it (no glycerol kinase)
Some fatty acids are oxidised in the adipocyte itself, or re-esterified with glycerol-3-phosphate made by glyceroneogenesis
↓ LPL activity in adipose → less uptake from circulating TAG → more available to muscle

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💪 SKELETAL MUSCLE

Switching fuel, then donating protein

A switch from glucose to fatty acids: ↑AMP → AMPK → phosphorylates and inactivates muscle ACC → ↓malonyl-CoA → fatty acid oxidation proceeds
Exercising muscle first uses creatine phosphate and glycogen; once depleted, fatty acids dominate
Early fasting: fatty acids from adipose plus ketone bodies from liver. Prolonged fasting: fatty acids almost exclusively — ketone bodies are saved for the brain
Fatty acid oxidation → ↑acetyl-CoA → inhibits PDH (activating PDH kinase) → pyruvate is transaminated to alanine → substrate for hepatic gluconeogenesis (the glucose–alanine cycle)
Protein: ↓insulin and ↑glucocorticoids drive proteolysis, delivering glucogenic amino acids — especially alanine and glutamine from branched-chain amino acid catabolism. Muscle has no glucagon receptor. Glutamine is used by enterocytes, which send out alanine for the liver
Second week of fasting: proteolysis falls and the need for glucose falls, because the brain has started using ketone bodies

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🧠 BRAIN

The great adaptation

Beginning of fasting: glucose is the only energy substrate, maintained by liver gluconeogenesis
Prolonged fasting (> 2–3 weeks): ketone bodies take over as the plasma level rises — which spares both glucose and muscle protein

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🫘 KIDNEY — the forgotten organ

Long-term fasting only

The renal cortex increases expression of gluconeogenic enzymes, including glucose-6-phosphatase
Produces about 50% of glucose in late fasting, part of which it uses itself
Compensates for the acidosis caused by ketone bodies
Takes up glutamine from muscle and, via glutaminase and glutamate dehydrogenase, converts it to α-ketoglutarate — a gluconeogenic substrate. Simultaneously produces NH₃, which accepts the protons released by dissociating ketone bodies and is excreted as NH₄⁺: nitrogen leaves as ammonium rather than urea
Rising ketone bodies are consumed by enterocytes instead of glutamine — saving glutamine for the kidney

📈 The fasting timeline — which fuel, when

Glucose · ketone bodiesFree fatty acidsLiver glycogen

Liver glycogen carries the first few hours and is exhausted by about 24 hours. Gluconeogenesis begins at 4–6 hours and takes over. Fatty acids rise steadily from the start. Ketone bodies rise late but climb furthest — by two to three weeks they are the brain’s main fuel, which is what finally spares muscle protein. Glucose never collapses: it settles around 65–70 mg/dL and stays there.

Thyroid Hormones

The only hormones that require a trace element for biological activity. Synthesis happens outside the cell, on a giant storage protein — and almost every exam question comes from one of the four stages below.

🦋 The Molecules

T4 — Thyroxine

4

iodine atoms

3,5,3′,5′-tetraiodothyronine · PRO-hormone

T3 — Triiodothyronine

3

iodine atoms

3,5,3′ · the ACTIVE form

rT3 — Reverse T3

3

iodine atoms

3,3′,5′ · biologically INACTIVE

Why T3 is the active one

T3 binds to receptors in target cells with higher affinity than T4, and is therefore the preponderant metabolically active form. Thyroid hormones are unique in that they contain the trace element iodine for biological activity. Reverse T3 differs only in which ring loses its iodine — and that alone makes it inactive.

🏭 Thyroglobulin — The Storage Scaffold

Thyroid hormone synthesis is unusual: it happens outside the cell, on a giant protein stored in the follicular colloid. The hormones stay attached to it until they are needed.

Property	Detail
Size	Large protein, two subunits, about 5000 amino acids, with 115 tyrosine residues
Role	Precursor of T3 and T4 — a prohormone
Glycosylation	Carbohydrate accounts for 8–10% of mass
Iodination	0.2–1% by weight, depending on dietary iodine
Iodine distribution	70% as the inactive precursors MIT and DIT; 30% as T4 and T3 in a ratio of about 7:1
Route	Synthesised at the basal part of the cell → transported to the luminal part → stored extracellularly in the colloid. To be hydrolysed it must re-enter the cell

In iodine deficiency

The ratios T4:T3 and DIT:MIT both FALL — the gland economises, making proportionally more of the form that uses less iodine.

⚗️ Synthesis — Two Stages, Four Steps

1️⃣ STAGE ONE · Iodination ("organification")

Trapping → oxidation → iodination → second iodination

Enzyme throughout: thyroid peroxidase — a heme-containing tetramer requiring H₂O₂

Trapping: iodide is taken up from plasma by a pump (Na⁺/iodide symporter) or by passive diffusion, and concentrated. The ratio of iodide in the gland to iodide in the blood reflects how hard the pump is working
Oxidation: the thyroid is the only tissue that oxidises iodide. Thyroid peroxidase uses hydrogen peroxide as the oxidising agent — produced by an NADPH-dependent enzyme resembling cytochrome c reductase
Iodination: oxidised iodine reacts with tyrosyl residues of thyroglobulin at the luminal surface, forming MIT (3-monoiodotyrosine), then a second iodination forms DIT (3,5-diiodotyrosine)
Both MIT and DIT remain attached to thyroglobulin. Once incorporated, iodine does not leave the thyroid easily

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2️⃣ STAGE TWO · Coupling

Also catalysed by thyroid peroxidase — occurs WITHIN the thyroglobulin molecule

Mechanism: stimulation of iodotyrosine free radical formation

DIT + DIT → T4

The major coupling reaction. The thyroid secretes mostly T4.

MIT + DIT → T3

The active hormone, made in smaller amounts.

DIT + MIT → reverse T3

The inactive isomer — same atoms, wrong arrangement.

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3️⃣ Release — hydrolysis

Stimulated by TSH via cAMP · inhibited by iodide

TSH and cAMP drive microvilli growth and thyroglobulin uptake. The colloid is endocytosed into phagosomes, where acid proteases and peptidases hydrolyse thyroglobulin and free T4 and T3 into the plasma. Iodine that was not incorporated leaves the thyroid by diffusion (also handled by kidney and liver). There is a lower ratio of T4 to T3 in the blood leaving the thyroid than in thyroglobulin itself.

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4️⃣ Peripheral activation

Where T4 becomes T3 — and where selenium matters

Liver and kidneys contain deiodinase that produces about two-thirds of plasma T3. The deiodinase catalysing T4 → T3 requires selenium, because it contains selenocysteine. A separate 5′-deiodinase that does NOT require selenium catalyses T4 → reverse T3. By modulating the relative production of T3 and rT3, tissues fine-tune their local thyroid status.

🎯 Selenium deficiency — a two-sided effect

It decreases conversion of T4 to T3 while simultaneously increasing conversion of T4 to reverse T3 — because the enzyme making rT3 has no selenocysteine to lose. See selenium deficiency.

🚫 Blocking the Thyroid — Three Drug Classes, Three Steps

🅰️ Block iodide TRANSPORT

Perchlorate (ClO₄⁻), perrhenate (ReO₄⁻), pertechnetate (TcO₄⁻) — similar anions that compete with iodide for the carrier and are concentrated by the gland. A radioisotope of pertechnetate is used clinically to check iodide transport.

Thiocyanate (SCN⁻) is also a competitive inhibitor of iodide transport, but is NOT concentrated by the gland — the key distinction.

🅱️ Block OXIDATION & organification

Thiourea derivatives — the "anti-thyroid drugs" (propylthiouracil, methimazole, carbimazole). They inhibit thyroid hormone biosynthesis at the peroxidase step.

🅾️ Block peripheral CONVERSION

Propylthiouracil and propranolol inhibit the conversion of T4 to T3 in the periphery — which is why both are used in thyroid storm. Propylthiouracil therefore acts at two points.

🚌 Transport in Plasma

Two-thirds of T3 and T4 are outside the thyroid gland, bound to plasma proteins.

Carrier	Detail
TBG — thyroxine-binding globulin	The most important carrier — 100× the affinity of the others. Produced in the liver. Under physiological conditions it binds almost all T3 and T4 non-covalently. Binds about 70% of T4 and 80% of T3
TBPA — thyroxine-binding prealbumin (transthyretin)	Secondary carrier
Albumin	Low affinity, high capacity
Free fractions (FT3, FT4)	Only about 0.05% of T4 and 0.2% of T3 are free — but only the free fractions cross the cell membrane, affect intracellular metabolism, and exert feedback. FT4 has the longer half-life

⚠️ Two laboratory traps

1. Estrogens increase TBG (pregnancy, oral contraceptives); androgens, glucocorticoids and liver disease decrease it. This changes total T3 and T4 but not the free fraction — the patient is euthyroid.
2. Phenytoin and salicylic acid displace T3 and T4 from TBG. The overall pool of hormone falls, but free T3 and T4 remain normal. Both must be considered when interpreting a thyroid function test.

Further metabolism

Beyond deiodination, thyroid hormones undergo deamination, decarboxylation, glucuronidation and sulfation. Glucuronidation produces a more hydrophilic molecule excreted into bile, reabsorbed in the gut, deiodinated in the kidney and excreted in urine as the glucuronide conjugate.

🔄 The HPT Axis & Cellular Actions

▸ Hypothalamic–pituitary–thyroid axis

Hypothalamus

TRH

→

Anterior pituitary

TSH

→

Thyroid

T4 + T3

→

Target tissues

↻ Excess FT4 and FT3 block TSH and TRH by long-loop feedback · TSH blocks TRH by short-loop feedback

💡 The one rule that answers most thyroid questions

If the thyroid is producing too much, circulating TSH will be suppressed. If it is producing too little, TSH will be very high as the pituitary tries to stimulate it. Read the TSH first, then the free T4.

Non-thyroidal illness: a number of agents inhibit TSH release — dopamine, somatostatin, glucocorticoids and interleukins. Factors that decrease T4 → T3 conversion (raising the rT3/T3 ratio) include pregnancy or oral contraceptives, fasting, stress, high plasma cortisol, catabolic diseases, hepatic and renal disease, and thiouracil drugs.

How the hormone acts inside a cell

Thyroid hormones bind weakly to cytoplasmic protein but bind high-affinity receptors in the cell nucleus, where T3 has both higher affinity and higher activity. Thyroid hormone receptors act as transcription factors: unliganded TR, often as a heterodimer with the retinoid X receptor, binds a thyroid hormone response element on DNA and recruits co-repressors and histone deacetylases, repressing gene expression. T3 binding to the ligand-binding domain promotes a conformational change that releases co-repressors and recruits co-activators with histone acetyltransferases, allowing transcription to begin.

🔥 What Thyroid Hormone Does to the Whole Body

⚡ ENERGY & HEAT

Increase basal metabolic rate and oxygen consumption
Intensify Na⁺/K⁺-ATPase activity → ↑ATP use → ↑O₂ consumption
Stimulate heat production by increasing uncoupling protein activity in mitochondria
Increase the number of mitochondria in cells

🍞 FUEL HANDLING

Increase glycolysis, glycogenolysis and gluconeogenesis
Increase lipolysis and protein degradation
Stimulate protein synthesis → positive nitrogen balance — but high concentrations of T3 give a NEGATIVE nitrogen balance
Decrease muscle mass and adipose tissue

Increase β-adrenergic receptors → increased cardiac output; increase systolic blood pressure only; increase ventilation rate
Required for maturation of ovary and testis
Required for the actions of growth hormone to promote linear growth and bone formation — T3 and glucocorticoids increase transcription of the growth hormone gene
Required for development of the CNS in the fetus

🩺 Developmental consequences

Congenital or intrauterine hypothyroidism, if severe and untreated, results in cretinism — irreversible intellectual disability with multiple defects. Hypothyroid children have delayed skeletal maturation, short stature and delayed puberty. If hypothyroidism appears later in childhood, the result is short stature but no intellectual disability, because brain development is already complete.

Some hypothyroid patients have high serum cholesterol — thyroid hormone normally upregulates hepatic LDL receptors, so its loss impairs cholesterol clearance and sterol excretion via the gut.

🩺 Thyroid Disease & Autoantibodies

🔥 Graves Disease

Anti-TSHR — antibodies against the TSH receptor that mimic TSH. High free T4, suppressed TSH, exophthalmos, diffuse goitre.

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❄️ Hashimoto Thyroiditis

Anti-TPO and anti-TG — the antibodies of autoimmune thyroiditis. Low free T4, high TSH.

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🎈 Goitre

Enlargement without necessarily any hormonal overactivity. Iodide deficiency, iodide excess in a damaged gland, or hereditary defects of biosynthesis. Raised TSH is common to all.

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The full antibody list

Antibody	Target	Disease
anti-TSHR	TSH receptor	Graves–Basedow disease
anti-TPO	Thyroid peroxidase	Hashimoto thyroiditis
anti-TG	Thyroglobulin	Autoimmune thyroiditis (Hashimoto)
anti-NIS	Sodium–iodide symporter of the thyrocyte membrane	Disturbs transport of iodide and sodium into thyrocytes
anti-megalin	Megalin — receptor on the luminal surface of some epithelial cells	Less well documented role

Treatment options

Hypothyroidism: exogenous thyroid hormone replacement; iodide supplementation or restriction for some goitres. Hyperthyroidism: drugs that block hormone biosynthesis, radioactive iodine, or surgical removal of the gland.

🩻 Reading a Thyroid Panel — and What Hypothyroidism Looks Like

TSH	Free T4	Interpretation
High	Low	Primary hypothyroidism — the gland has failed and the pituitary is shouting at it. By far the commonest pattern (Hashimoto, iodine deficiency, post-ablation)
Low	Low	Secondary (central) hypothyroidism — the pituitary or hypothalamus has failed, so TSH is inappropriately normal or low for a low T4. A low TSH does not by itself mean an overactive thyroid
Low	High	Hyperthyroidism — Graves, toxic nodule, thyroiditis
High	Normal	Subclinical hypothyroidism — the gland is compensating

The clinical picture — everything runs slow

Low thyroid hormone lowers the basal metabolic rate, and the symptoms follow directly from that: cold intolerance, weight gain, sleepiness and fatigue, bradycardia, constipation, dry skin and hair loss, slow-relaxing (delayed) reflexes, and in severe long-standing disease the non-pitting myxoedema of the skin — mucopolysaccharide deposited in the dermis, which is what the disease is named after. Treatment is levothyroxine (synthetic T4), which the body deiodinates to T3 as required.

Amino Acid Catabolism & Synthesis

Once the nitrogen has been stripped off, twenty different carbon skeletons have to go somewhere. They funnel into just seven molecules — and knowing which amino acid goes where answers most of the questions in this topic.

🧭 Two Classifications You Must Not Mix Up

By dietary need

Essential — no synthesis, or insufficient synthesis, in the human body. Must be supplied with the diet.

Non-essential — synthesised in the human body from ordinary metabolic intermediates.

Question: can I make it?

By metabolic fate

Glucogenic — catabolised to pyruvate or to one of the intermediates of the Krebs cycle. These can become glucose.

Ketogenic — catabolised to acetoacetate, acetyl-CoA or acetoacetyl-CoA. These can never become glucose.

Question: where do the carbons land?

🎯 The two purely ketogenic amino acids

Leucine and lysine — and only these two. Everything else is glucogenic, or both. Remember them as the pair with L: Leucine and Lysine are purely Lipogenic. Five amino acids are both glucogenic and ketogenic: phenylalanine, tyrosine, tryptophan, isoleucine and threonine.

🎯 The Seven Destinations

Every amino acid carbon skeleton ends up as one of seven compounds. Six of them are glucogenic entry points; two are the ketogenic ones. Learn the groups, not the twenty individual pathways.

▸ Where the carbon skeletons go

20 amino acids

→transamination / deamination

Pyruvate

α-Ketoglutarate

Succinyl-CoA

Fumarate

Oxaloacetate

Acetyl-CoA

Acetoacetyl-CoA

glucogenic entryketogenic entry

🅿️ → PYRUVATE

Alanine · Serine · Glycine · Cysteine · Threonine

Glycine

Oxidised to CO₂ and NH₃ by the glycine cleavage system. Alternatively deaminated to glyoxalate by D-amino acid oxidase, then oxidised to oxalate. Glyoxalate is normally transaminated back to glycine — and deficiency of that liver peroxisomal transaminase causes oxalate stones and kidney damage (primary hyperoxaluria).

Cysteine

Desulfurisation to pyruvate. The released sulphate is used for PAPS synthesis (the universal sulphate donor). Cysteine can also be oxidised to cystine — the poorly soluble dimer behind cystinuria stones.

Threonine

Converted to pyruvate only to a slight degree in humans — its main route is dehydration to α-ketobutyrate → propionyl-CoA → succinyl-CoA.

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🅰️ → α-KETOGLUTARATE (all via glutamate)

Glutamine · Glutamate · Proline · Arginine · Histidine

Proline

Oxidation → glutamate → α-ketoglutarate.

Arginine

Hydrolysis by arginase → ornithine (+ urea) → glutamate semialdehyde → α-ketoglutarate.

Histidine

Oxidative deamination to urocanate → … → α-ketoglutarate.

🧪 FIGlu excretion in urine — formiminoglutamate accumulates in the histidine pathway when folate is short. Its urinary excretion is a test used in the diagnosis of folic acid deficiency.

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🅾️ → OXALOACETATE

Asparagine · Aspartate

Asparagine is hydrolysed by asparaginase to aspartate, which is transaminated to oxaloacetate.

💊 Asparagine is essential for cell growth in leukaemia — leukaemic cells cannot make their own. Asparaginase is used to treat leukaemia: it strips asparagine from the plasma and starves the tumour, while normal cells simply synthesise more.

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🅵 → FUMARATE

Phenylalanine · Tyrosine — one shared pathway

Phenylalanine and tyrosine share a common pathway that ends in fumarate and acetoacetate — which is exactly why both amino acids are glucogenic and ketogenic at the same time. Four inherited diseases sit on this one pathway.

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🆂 → SUCCINYL-CoA

Methionine · Valine · Isoleucine · Threonine — all via propionyl-CoA

These undergo oxidative decarboxylation → propionyl-CoA → succinyl-CoA. This is the same three-enzyme run used by oxidation of fatty acids with an odd number of carbon atoms — so a defect here hits both routes at once (see methylmalonic acidaemia). Threonine reaches it by dehydration to α-ketobutyrate.

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🅺 → ACETYL-CoA / ACETOACETYL-CoA

Leucine · Lysine (pure) · Tryptophan · Isoleucine · Phenylalanine · Tyrosine (mixed)

These carbons can only be burned or turned into ketone bodies — they can never be converted to glucose, because the pyruvate dehydrogenase step is irreversible.

🫀 Homocysteine — Why Everyone Measures It

Homocysteine sits at the junction of methionine catabolism and cysteine synthesis. Raised plasma concentrations are linked to oxidative damage, inflammation and epithelial dysfunction, and to increased risk of vascular disease, particularly ischaemic heart disease.

Methionine

→

SAM

methyl donor

→

Homocysteine

→cystathionine β-synthase · B6

Cystathionine → Cysteine

↻ Remethylation back to methionine by methionine synthase — requires B12 and folate

The vitamin relationship — and the twist

Homocysteine concentrations are inversely related to plasma folate, vitamin B12 and vitamin B6. Supplementation with these vitamins reduces plasma homocysteine but does not reduce the number of cardiovascular events — suggesting homocysteine may be only a marker, not the cause of endothelial damage. High concentrations are found in cystathionine β-synthase deficiency (classic homocystinuria) and in defects of the remethylation reaction.

🔗 Folic Acid & The One-Carbon Pool

The pool of single-carbon fragments is carried by THF and SAM — but not biotin. Biotin carries CO₂, which is a one-carbon unit at the fully oxidised level and is handled separately. This distinction is a classic exam trap.

Folate

folate reductase⟶

Dihydrofolate (DHF)

dihydrofolate reductase⟶NADPH

Tetrahydrofolate (THF)

THF is the active form of folic acid. The single carbon unit is attached to N5, or N10, or bridged across both THF nitrogens, and can be carried at several oxidation levels — methyl, methylene, formyl.

🩸 Why folate deficiency causes megaloblastic anaemia

Deficiency reduces the availability of purines and thymidylate for DNA synthesis. The cell can still make RNA and protein, so it keeps growing — but it cannot divide. The result is a large, immature nucleated cell: the megaloblast. This is also why methotrexate (a DHFR inhibitor) works as a chemotherapeutic and why it is toxic to bone marrow.

🏗️ Making the Non-Essential Amino Acids

Route	Products
From α-keto acid intermediates of metabolism — α-ketoglutarate, pyruvate, oxaloacetate — by transamination	Glu, Ala, Asp
From essential amino acids	Phe → Tyr (phenylalanine hydroxylase) · Met → Cys (via homocysteine and cystathionine)
By amidation	Glu → Gln · Asp → Asn
From glutamate	Proline
From glycolytic intermediate 3-phosphoglycerate	Serine → Glycine → Cysteine
By post-translational modification only — never incorporated as such	Hydroxyproline and hydroxylysine

🔬 Selenocysteine — the 21st amino acid

Synthesised from serine and selenium while the serine is already attached to its tRNA — not free in the cytosol, and not by modifying a finished protein. It is inserted co-translationally at a recoded UGA codon. Two enzymes that depend on it: glutathione peroxidase and thioredoxin reductase. Note the contrast with hydroxyproline, which is made after translation.

🧬 Inborn Errors of Amino Acid Metabolism

These are monogenic diseases — congenital errors of metabolism caused by mutations producing abnormal proteins, most often enzymes. The result is complete or partial loss of catalytic activity and accumulation of toxic metabolites. Without treatment they cause intellectual disability and developmental disorders. About 50 diseases of this type are known; individually they are rare — sometimes 1 case in 250,000.

🍼 Phenylketonuria

The most common inborn error of amino acid metabolism — 1:15,000. Phenylalanine hydroxylase deficiency → 10× higher phenylalanine in plasma, tissues and urine.

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🍁 Maple Syrup Urine Disease

Branched-chain α-keto acid dehydrogenase deficiency. Three types: classic, intermediate, and thiamine-responsive.

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⬜ Albinism

Errors in tyrosine metabolism → insufficient melanin. Type 1 (ophthalmic) is partial or complete absence of copper-dependent tyrosinase — the heaviest type.

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👁️ Homocystinuria

Cystathionine β-synthase deficiency. ↑Hcy and ↑Met, ↓cysteine. Lens dislocation, long limbs, thrombosis is the leading cause of early death.

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⚫ Alkaptonuria

Homogentisate oxidase deficiency. Urine darkens on standing in air, early large-joint inflammation, ochronosis — black pigment in cartilage and collagen.

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🥬 Tyrosinaemia Type I

Fumarylacetoacetate hydrolase deficiency — the end enzyme of tyrosine metabolism. The urine smells like cabbage.

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PKU in more detail — the parts that get asked

Why the symptoms happen

The block causes tyrosine deficiency, and tyrosine is the precursor of adrenaline, thyroid hormones and melanin. Hence: intellectual disability (IQ < 50), developmental delay, microcephaly, epileptic seizures, and pigmentation disorders — light hair, pale skin, blue eyes from absent melanin.

Neonatal screening — the timing trap

Newborns have no symptoms. Phenylalanine levels may remain normal until the newborn has had 24–48 hours of protein feeding, so screening is deliberately done after this time to avoid false negatives.

Maternal PKU

Not following a low-phenylalanine diet affects the offspring — maternal hyperphenylalaninaemia is teratogenic to the fetus. Dietary control must be started before conception and maintained through pregnancy.

Treatment & genetics

Most dietary proteins contain phenylalanine (it is essential), so the limit is easily exceeded. Synthetic amino acid preparations without phenylalanine supplement natural dietary ingredients. Prenatal diagnosis is possible despite 100+ PAH mutations and frequent compound heterozygosity.

MSUD in more detail

The α-keto acids and their corresponding amino acids build up in blood, toxic especially to the brain: feeding problems, vomiting, ketoacidosis, changes in muscle tone, coma from raised leucine, and urine smelling of maple syrup from raised isoleucine. Untreated it is fatal; delayed treatment leaves intellectual disability.

Type	Features
1 · Classic	Begins in the perinatal period, the most common form; little or no BCKD activity in leukocytes and fibroblasts; undiagnosed and untreated → death within the first weeks of life
2 · Intermediate	Higher enzyme activity — up to 30% of normal; milder symptoms from infancy to adolescence
3 · Thiamine-dependent	Patients respond to treatment with high doses of thiamine

Treatment

Synthetic BCAA-free preparations supplemented with limited amounts of leucine, isoleucine and valine. Increased leucine is neurotoxic, so dietary leucine must be closely monitored. Most patients are mixed heterozygotes, but screening is possible prenatally and neonatally.

Diabetes Mellitus & Obesity

The two conditions where everything in this course converges. Type 1 is a failure of supply; type 2 is a failure of listening. Both end at the same complications by different routes.

📖 Definition & Diagnosis

Diabetes mellitus is a group of metabolic diseases characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action, or both. It is the most common endocrine disease, and chronic hyperglycaemia is associated with long-term damage, dysfunction and failure of eyes, kidneys, nerves, heart and blood vessels.

Fasting plasma glucose

≥126

mg/dL

on two occasions · normal < 100

2-h OGTT

≥200

mg/dL

after 75 g oral glucose

HbA1c

≥6.5

%

reflects ~3 months · target < 7% in treated patients

Why HbA1c works — and why 3 months

Glucose attaches non-enzymatically to the N-terminal valine of the haemoglobin β chain. The reaction is irreversible and proportional to average glucose, and the label persists for the life of the red cell — about 120 days. That is why HbA1c reports the preceding two to three months and cannot be faked by fasting the morning of the test. Any condition that shortens red cell lifespan falsely lowers HbA1c — haemolysis, recent transfusion, bleeding.

⚖️ Type 1 versus Type 2 — Side by Side

🅰️ TYPE 1 — absolute deficiency

Cause	Autoimmune destruction of β cells; genetic predisposition + environmental trigger
Insulin	Absolute deficiency — C-peptide absent or very low
Onset	Usually childhood or puberty, abrupt; symptoms appear when most β cells are already destroyed
Body habitus	Weight loss despite normal or increased appetite
Acute crisis	Ketoacidosis — the characteristic emergency
Treatment	Exogenous insulin is obligatory

🅱️ TYPE 2 — resistance + relative deficiency

Cause	Insulin resistance plus β-cell dysfunction; strong genetic component, stronger than type 1
Insulin	Normal or high initially — the tissues do not respond. Later, secretion fails too
Onset	Usually adult, gradual, often asymptomatic for years — frequently discovered at a routine test or already with complications
Body habitus	Obesity, especially visceral, in the large majority
Acute crisis	Hyperosmolar hyperglycaemic state — ketosis is rare because residual insulin still restrains lipolysis
Treatment	Diet, exercise, oral agents; insulin later if needed

🅰️ Type 1 Diabetes

Full disease card — autoantibodies, honeymoon phase, management.

▸ Open full card

🅱️ Type 2 Diabetes

Full disease card — resistance mechanisms, drug classes, natural history.

▸ Open full card

🧬 MODY 2

Monogenic, glucokinase mutation — mild stable fasting hyperglycaemia, often needs no treatment.

▸ Open full card

🎯 Metabolic Syndrome

The cluster that precedes type 2 — the diagnostic criteria in full.

▸ Open full card

🚨 The Metabolic Picture of Uncontrolled Diabetes

Without insulin, the body reads a fed state as a fast — and runs the fasting programme on top of a bloodstream already full of glucose. Every symptom follows from that single contradiction.

▸ From missing insulin to coma

↓ Insulin
↑ Glucagon

→

↑ Glycogenolysis
↑ Gluconeogenesis
↓ Glucose uptake

→

HYPERGLYCAEMIA

→exceeds renal threshold ~180 mg/dL

Glucosuria

osmotic diuresis

→

Polyuria → dehydration → polydipsia

↓ Insulin

→HSL disinhibited

↑ Lipolysis → free fatty acids

→CPT-I released, oxaloacetate drained by gluconeogenesis

↑ Ketogenesis

→

KETOACIDOSIS

Kussmaul breathing · acetone breath

🎯 The classic triad — and why "polyphagia with weight loss"

Polyuria, polydipsia, polyphagia. The patient eats more because the cells are starving — glucose cannot enter muscle and adipose tissue without insulin — yet loses weight because protein and fat are being catabolised to make fuel that also cannot be used. Hunger and starvation coexist with hyperglycaemia. Compensatory hyperventilation (Kussmaul respiration) blows off CO₂ to counter the metabolic acidosis; see DKA.

🕰️ Chronic Complications — Four Molecular Mechanisms

The tissues that get damaged are exactly the ones whose glucose uptake is insulin-independent: retina, lens, kidney, nerve, endothelium. They cannot shut the door, so intracellular glucose tracks plasma glucose exactly.

1 · Non-enzymatic glycation → AGEs
Glucose reacts with protein amino groups → Schiff base → Amadori product → irreversible advanced glycation end products. These cross-link collagen, stiffen basement membranes and vessels, and bind RAGE receptors to trigger inflammation and oxidative stress

2 · Polyol pathway
Aldose reductase converts glucose to sorbitol, consuming NADPH. Sorbitol is trapped intracellularly → osmotic swelling (cataract) — and the NADPH loss depletes reduced glutathione, leaving the cell defenceless against oxidative stress

3 · Protein kinase C activation
Via increased diacylglycerol — alters vascular permeability, blood flow and growth factor signalling

4 · Hexosamine pathway
Excess fructose-6-phosphate diverted to UDP-GlcNAc → altered gene transcription and protein O-glycosylation

Microvascular

Retinopathy — leading cause of acquired blindness. Nephropathy — microalbuminuria progressing to renal failure. Neuropathy — distal symmetrical sensory loss, autonomic dysfunction, and the diabetic foot.

Directly glucose-driven — tight control helps most here

Macrovascular

Accelerated atherosclerosis → myocardial infarction, stroke, peripheral arterial disease. Driven by the diabetic dyslipidaemia: high triacylglycerol, low HDL, and small dense LDL particles that are more easily oxidised.

Needs blood pressure and lipid control too

🍔 Obesity — Measurement & Fat Distribution

Obesity is an excess of body fat, resulting from a sustained imbalance between energy intake and energy expenditure. It is the single strongest modifiable risk factor for type 2 diabetes.

BMI (kg/m²)	Category
< 18.5	Underweight
18.5 – 24.9	Normal
25.0 – 29.9	Overweight
≥ 30.0	Obese (class I 30–34.9 · class II 35–39.9 · class III ≥ 40)

⚠️ Why BMI alone is not enough

BMI does not distinguish fat from muscle, and it does not say where the fat is. Distribution matters more than total mass.

🍎 VISCERAL (android, "apple")

Intra-abdominal fat, drained by the portal vein straight to the liver. Metabolically active, lipolytically sensitive, and secretes inflammatory cytokines. Strongly associated with insulin resistance, dyslipidaemia, hypertension and cardiovascular disease. More typical of men.

🍐 SUBCUTANEOUS (gynoid, "pear")

Hip and thigh fat, drained into the systemic circulation. Metabolically much less harmful — it may even be protective, acting as a safe sink for excess lipid. More typical of women.

Why visceral fat is so damaging: free fatty acids released from visceral adipocytes go directly to the liver, where they promote gluconeogenesis, VLDL production and hepatic insulin resistance — the mechanism linking central obesity to fatty liver and type 2 diabetes.

📡 Adipose Tissue as an Endocrine Organ

Adipose tissue is not inert storage. It secretes hormones that report on energy stores and modify insulin sensitivity throughout the body.

Leptin

Secreted by adipocytes in proportion to fat mass. Acts on the hypothalamus to suppress appetite and increase energy expenditure. In common obesity levels are high, not low — the problem is leptin resistance, not leptin deficiency. True congenital leptin deficiency is rare and causes severe early obesity that responds to replacement.

Adiponectin

The one that goes the other way: it falls as fat mass rises. Increases insulin sensitivity, promotes fatty acid oxidation via AMPK, and is anti-inflammatory and anti-atherogenic. Low adiponectin is a marker of metabolic risk.

Ghrelin

Not from adipose — from the stomach. The hunger hormone: rises before meals, falls after eating. The only known orexigenic gut peptide.

TNF-α · IL-6 · resistin

Pro-inflammatory adipokines from expanded, hypoxic adipose tissue. They interfere with insulin receptor signalling — obesity therefore causes a chronic, low-grade inflammatory state that drives insulin resistance.

💡 The unifying idea

Obesity → adipocyte hypertrophy → hypoxia and inflammation → ↑TNF-α, IL-6, free fatty acids and ↓adiponectin → serine phosphorylation of IRS proteins instead of the normal tyrosine phosphorylation → the insulin signal is blocked at the very first step. That single switch — serine instead of tyrosine — is insulin resistance at the molecular level.

💊 Treatment Logic

1️⃣ Lifestyle — always first, never optional

Weight loss of 5–10% measurably improves glycaemic control

Exercise increases GLUT-4 translocation by an insulin-independent, AMPK-mediated route — which is why muscle takes up glucose during exercise even in a diabetic patient.

▼

2️⃣ Oral agents — matched to the defect

Each class attacks a different step you have already studied

Metformin

Activates AMPK → decreases hepatic gluconeogenesis, increases peripheral glucose uptake. First-line, does not cause hypoglycaemia.

Sulfonylureas

Close ATP-sensitive K⁺ channels on the β cell → insulin release independent of glucose → can cause hypoglycaemia.

Incretin-based (GLP-1 agonists, DPP-4 inhibitors)

Amplify the incretin effect — glucose-dependent insulin release, so much lower hypoglycaemia risk.

SGLT2 inhibitors

Block renal glucose reabsorption → deliberate glucosuria. Lower the renal threshold rather than the production.

Thiazolidinediones

PPAR-γ agonists → promote fat storage in subcutaneous rather than visceral depots and raise adiponectin.

α-Glucosidase inhibitors

Slow brush-border carbohydrate digestion → blunt the post-meal glucose spike.

▼

3️⃣ Insulin

Obligatory in type 1 from diagnosis · added in type 2 when β-cell function fails

Monitoring combines HbA1c for long-term control with self-monitored glucose for day-to-day adjustment. The trade-off is permanent: tighter control reduces microvascular complications but increases the risk of hypoglycaemia.

📈 The oral glucose tolerance test

NormalImpaired glucose toleranceDiabetes mellitus

A normal person peaks below 140 mg/dL and is back to baseline within two hours — the insulin response is fast enough to keep up. In impaired tolerance the peak is higher and clearance is slow. In diabetes the curve starts high, climbs higher and never comes down: the two-hour value stays at or above 200 mg/dL, which is the diagnostic criterion.

Enzyme Reference

142 enzymes. Filter by category, class, or search by name, pathway, keyword.

Legend: Activator Inhibitor Cofactor

Disease Reference

54 biochemical diseases. Click any card for full details, pathway, and exam tricks.

Practice Quiz

8 part bundles · 37 sections · 810 exam-style questions · Question order is fixed; the answer options are reshuffled every time you open a bundle

Amino Acids & Protein Structure

Resit topics 1–3 · classification and pI, the peptide bond and the four levels of structure, folding and denaturation, collagen, plasma proteins and immunoglobulins, prions

◆ What to revise before the exam

The revision list sent to the year group before the retake. Every card that answers one of these is marked Lecturer’s priority throughout Resit Nitro.

Enzymes — regulation.
Lipoproteins — composition and function, in general terms.
Urea cycle and ammonia metabolism.
Acid–base balance — the disturbances.
Glycolysis / gluconeogenesis / Krebs cycle — regulatory enzymes and reactions, and the bridge between them.
For every main process — glycolysis, gluconeogenesis, lipolysis, lipogenesis, β-oxidation, the urea cycle and the rest — location, location, location; regulatory enzymes; hormones (fed / fasting) where they apply; and what the pathway is actually for.
Diseases — the obvious ones: diabetes, jaundice and their kind. Not the ones named after somebody.

Answer every pathway question in the same four beats: where it happens (organ, then compartment) · what it starts from and produces · which enzyme is rate-limiting and how it is switched · why the cell bothers. That is the shape the mark scheme is looking for.

LocationSubstrate → productRegulatory enzymeHormone: fed / fastPurpose

🧩 What Peptides and Proteins Are For Past Q

A peptide is a short chain of amino acids; a protein is a long one that folds into a working shape. Two classic list questions come from here — name five functions of peptides and name the roles of proteins.

🔹 Five (and more) functions of peptides

Hormones
Insulin, glucagon, ADH, oxytocin, calcitonin, ACTH.

Hormone-releasing factors
The hypothalamic tier — TRH, GnRH, CRH, GHRH.

Neurotransmitters
Substance P, enkephalins, endorphins.

Neuromodulators
They tune the response of a neurone to another transmitter rather than firing it themselves.

They are polyelectrolytes
Many ionisable groups on one molecule: peptides therefore buffer, carry a net charge that depends on pH, migrate in an electric field and each has its own pI.

Protective and transport peptides
Glutathione (antioxidant), peptide antibiotics, and the di-/tripeptides absorbed intact by PepT1.

🔹 Roles of proteins

Catalysis & regulation

Enzymes catalyse every metabolic reaction; receptors and transcription factors read and pass on signals.

Structure & movement

Collagen, elastin, keratin and the cytoskeleton give tissues their mechanical integrity; actin and myosin convert chemical energy into motion.

Transport & storage

Albumin (fatty acids, bilirubin, drugs), haemoglobin (O₂), transferrin (iron), ferritin (iron store), and the membrane carriers and channels.

Defence & homeostasis

Immunoglobulins and complement; fibrinogen and the clotting factors; plasma proteins hold the oncotic pressure, buffer the blood and form a mobilisable amino acid reserve.

🔤 Classification of Amino Acids Topic 1

Three classifications get examined, and they are independent of one another — an amino acid can be essential and ketogenic and nonpolar at the same time.

Essential — must come from the diet

Phe · Val · Thr · Trp · Ile · Met · His · Arg · Leu · Lys ("PVT TIM HALL"). The body cannot synthesise them, or cannot synthesise them fast enough. His and Arg are the conditionally essential pair — required in growth, pregnancy and recovery.

Non-essential — made from metabolic intermediates

Built from α-keto acids and other intermediates: Ala (from pyruvate), Asp/Asn (from oxaloacetate), Glu/Gln/Pro/Arg (from α-ketoglutarate), Ser/Gly/Cys (from 3-phosphoglycerate; Cys also needs the sulfur of methionine), Tyr (from phenylalanine).

Glucogenic — can become glucose

Degraded to pyruvate or to a Krebs intermediate (α-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate), all of which reach oxaloacetate → PEP → glucose. This is the large majority of amino acids.

Ketogenic — cannot become glucose

Degraded only to acetyl-CoA or acetoacetate. Purely ketogenic: leucine and lysine ("the only two L's"). The reaction catalysed by pyruvate dehydrogenase is irreversible, so acetyl-CoA can never give a net yield of glucose.

Both at once — the five mixed amino acids

Phe · Tyr · Trp · Ile · Thr are glucogenic and ketogenic: part of the carbon skeleton reaches a glucogenic intermediate, part reaches acetyl-CoA or acetoacetate.

Two conditionally essential cases worth naming

Tyrosine becomes essential in phenylketonuria — the enzyme that makes it from phenylalanine is missing. Cysteine becomes essential in homocystinuria, because the block sits upstream of its synthesis.

🔹 The fourth classification — by the chemistry of the side chain

🛢️Aliphatic — plain hydrocarbon

Glycine · Alanine · Valine · Leucine · Isoleucine — hydrophobic, buried in the core.

💧Hydroxylic — carry an –OH

Serine · Threonine · Tyrosine — the answer to "which amino acids have a hydroxyl group in the side chain?", and the three residues that get phosphorylated.

🟡Sulfur-containing

Cysteine (–SH, makes disulphide bridges) and methionine (thioether, the start codon and the methyl donor as SAM).

➖Acidic — and their amides

Aspartate and glutamate carry a second –COOH; asparagine and glutamine are the same skeletons with an amide instead — uncharged, polar, and the body's nitrogen carriers.

➕Basic

Lysine (ε-amino) · Arginine (guanidinium, the most basic) · Histidine (imidazole, the only one that buffers at pH 7.4).

⬡Aromatic — a ring

Phenylalanine · Tyrosine · Tryptophan — they absorb ultraviolet light at 280 nm, which is how protein concentration is measured.

🔁Imino acid

Proline — its side chain loops back onto the α-nitrogen, making a secondary amine. It has no amide hydrogen, so it cannot donate a hydrogen bond, and it kinks helices.

Three more ways the same twenty get sorted

1 · By configuration — only L-α-amino acids are used in proteins (D-forms exist in bacterial cell walls and some antibiotics), and glycine is the only one with no chiral carbon, because its R group is a second hydrogen. 2 · By polarity — hydrophilic (polar, charged) versus hydrophobic (nonpolar). 3 · Protein versus non-protein amino acids — about 300 amino acids exist in nature but only 20 are coded for. The non-protein ones still matter: ornithine and citrulline (urea cycle), GABA (transmitter), taurine (bile salts), homocysteine (methionine metabolism) and homoarginine.

Homoarginine — the one obscure name on the list

An L-α-amino acid that is not found in proteins. Arginase cleaves it to L-lysine + urea, exactly as it cleaves arginine to ornithine + urea. It has been implicated in the human neurological disorder neurolathyrism — the spastic paraparesis caused by eating large amounts of the grass pea Lathyrus sativus.

⚡ Buffering Capacity and the Isoelectric Point Topic 2Past Q

Every amino acid carries at least two ionisable groups — the α-carboxyl (pK ≈ 2) and the α-amino (pK ≈ 9–10) — and seven of them carry a third, ionisable side chain. A group buffers best at its own pK, where it is half dissociated, and its useful buffering range is pK ± 1.

Henderson–Hasselbalch: pH = pK + log([A⁻]/[HA]). When [A⁻] = [HA], the log term is zero and pH = pK — the point of maximum buffering capacity.

The ionisable side chains

Acidic: Asp (pK ≈ 3.9), Glu (≈ 4.3). Basic: His (≈ 6.0), Cys (≈ 8.3), Tyr (≈ 10.1), Lys (≈ 10.5), Arg (≈ 12.5).

Why histidine matters

Its imidazole pK ≈ 6.0 is the only side chain close to blood pH 7.4 — which is why histidine residues make haemoglobin the main protein buffer of blood.

pI — the definition

The pH at which the molecule carries no net charge (it exists as the zwitterion). At pH < pI the molecule is net positive; at pH > pI it is net negative.

Calculating pI

Average the two pK values on either side of the neutral species. Neutral amino acid: pI = (pK₁ + pK₂)/2. Acidic (Asp, Glu): average the two carboxyl pKs → low pI. Basic (Lys, Arg, His): average the two amino-group pKs → high pI.

Measuring pI experimentally Past Q

By electrophoresis at a series of pH values: the pI is the pH at which the molecule does not migrate to either electrode. Isoelectric focusing does this in one run — the protein migrates through an immobilised pH gradient and stops where the pH equals its pI. A titration curve gives the same answer: pI is read off the inflection between the two relevant pK plateaus.

Why solubility is lowest at the pI Past Q

At the pI the net charge is zero, so there is no electrostatic repulsion between molecules and the ordered shell of water around the charged groups is lost. Protein molecules therefore aggregate and precipitate. Away from the pI every molecule carries the same sign of charge, they repel each other, and they stay in solution.

The bench version — the casein experiment Past Q

Nine tubes are filled with increasing amounts of acetic acid diluted with water, so each holds a different pH; 1 mL of 0.5% casein in sodium acetate is added to each, shaken, and left about 30 minutes. The tube that turns cloudy — the maximum precipitate — marks the isoelectric point of casein (≈ pH 4.6). Below that pH the protein is net positive, above it net negative, and in both cases it redissolves.

Two things the pI is actually used for, and one ranking question

Knowing the pI tells you which pH to run an electrophoresis at — two amino acids that differ only slightly in pK₁ or pK₂ can be separated by choosing an acidic or a basic buffer — and it predicts binding to ion-exchange supports such as DEAE-cellulose. Rank by increasing pI: aspartic acid ≈ 3.0 (acidic) < leucine ≈ 6.0 (neutral) < lysine ≈ 9.7–10 (basic).

Name five acidic (proton-donating) functional groups

1 · the α-carboxyl –COOH · 2 · the side-chain carboxyl of aspartate and glutamate · 3 · the thiol –SH of cysteine · 4 · the phenolic –OH of tyrosine · 5 · the imidazolium of histidine — and, strictly, the protonated amino groups too (α-NH₃⁺, the ε-NH₃⁺ of lysine and the guanidinium of arginine), since every one of them can hand off a proton.

📐 The Peptide Bond and the Two Secondary Structures Past Q

Two amino acids condense with the loss of a water molecule: the carbonyl carbon of one joins the α-nitrogen of the next. The chain is written and read N-terminus → C-terminus — the dipeptide Gly–Ala means glycine's carboxyl is bonded to alanine's amino group.

Why the peptide bond cannot rotate

Resonance gives it partial double-bond character, so the six atoms — the α-carbon, the carbonyl C and O, the N, its H and the next α-carbon — are coplanar, and rotation about C–N would mean breaking that bond. The bond is uncharged but polar and almost always trans.

Where the chain can bend

Of the three backbone bonds per residue, only two rotate freely: N–Cα (angle φ) and Cα–C (angle ψ). Every fold a protein can adopt is built from those two rotations.

α-Helix

Right-handed, with 3.6 residues per turn. Stabilised by hydrogen bonds between the C=O of residue n and the N–H of residue n+4, running parallel to the axis, plus van der Waals contacts. R groups point outwards. Proline and glycine break it — proline has no amide H and its ring kinks the chain, glycine is too flexible. An amphipathic helix has hydrophobic side chains on one face and hydrophilic on the other, so it sits neatly at the boundary between a polar and a nonpolar region.

β-Sheet

Extended strands lying side by side in a zigzag, pleated arrangement, with R groups of neighbouring residues pointing in opposite directions (alternately above and below the sheet). Hydrogen bonds run between strands, from carbonyl oxygens to amide hydrogens. Parallel = neighbouring strands run the same way N→C; antiparallel = opposite ways (and slightly more stable). Real sheets are not flat — they carry a right-handed twist.

Loops, turns and bends — the third kind of secondary structure

Turns and bends are short segments that join two units of secondary structure, typically reversing the chain between two antiparallel β-strands; they are rich in glycine and proline. Loops are longer and irregular, sit on the surface, and are far from decorative: in many enzymes the loop that bridges two domains carries the residues that bind the substrate and perform the catalysis.

🏗️ The Bonds That Stabilise Each Level of Structure Topic 3Past Q

Primary

The amino acid sequence, held by the peptide (amide) bond — a covalent bond with partial double-bond character, planar and normally trans. Disulphide bonds are also covalent and are sometimes counted here.

Secondary

Geometrically ordered folding of short 3–30 residue stretches — α-helix, β-sheet, loops — held by hydrogen bonds between backbone C=O and N–H groups only; side chains are not involved. (Geometry in the card above.)

Tertiary Past Q

The whole three-dimensional conformation of one chain — how its helices, sheets, bends and loops assemble into domains and how the domains sit relative to each other. Held by side-chain interactions: hydrophobic interactions (the dominant force — nonpolar side chains buried away from water), hydrogen bonds, ionic/electrostatic bonds (salt bridges), van der Waals forces, and the only covalent one — disulphide bridges between two cysteines.

Quaternary Past Q

The number, type and spatial arrangement of two or more separate polypeptide chains (subunits) in one functional protein — haemoglobin's α₂β₂, lactate dehydrogenase's tetramer. Held by the same non-covalent forces as tertiary structure (hydrophobic, hydrogen, ionic, van der Waals), and occasionally by interchain disulphides. A single-chain protein such as myoglobin has no quaternary structure.

🧶 Getting Folded — Chaperones, Disulphide Isomerase, and Coming Undone Past Q

The sequence contains all the information needed for the fold, but in a crowded cell a half-made chain would aggregate long before it finished. Over half of all mammalian proteins are folded with help.

🅰️ hsp70 family — while the chain is still being made

Molecular chaperones · ATP-driven

Job: prevent aggregation, buy time

Binds hydrophobic stretches

It grips the short hydrophobic sequences that emerge from the ribosome and shields them from water, so they cannot stick to each other.

Releases in cycles

Repeated ATP-driven binding and release let the chain form its secondary structure and collapse into a molten globule — compact, but not yet the final fold.

▼

🅱️ hsp60 family (chaperonins) — the finishing chamber

Different in sequence and structure from hsp70 · acts later, often together with it

Job: complete the fold in isolation

A barrel with a lid

The molten globule is taken inside a closed cavity where it can finish folding without meeting any other protein, then is released.

Protein sulfhydryl oxidase — makes the bridges

Oxidises the –SH groups of two cysteines into a disulphide bond; the reaction consumes oxygen. It is not specific — any accessible cysteine can be paired with any other, so wrong pairs form routinely.

Protein disulphide isomerase — corrects them Past Q

Catalyses disulphide exchange: it breaks an S–S bond and re-forms it with a different partner cysteine, over and over, until the arrangement that gives the native, most stable conformation is reached. That is its whole contribution to stabilising tertiary and quaternary structure.

Denaturation — what is really lost Past Q

Loss of secondary, tertiary and quaternary structure with the primary sequence intact: peptide bonds survive, non-covalent bonds do not (and, with reducing agents, the disulphides go too). Caused by heat, strong acid or base, concentrated inorganic salts, organic solvents, urea, detergents, heavy metals and radiation. Two points that get examined: a denatured protein is not a random coil — native-like contacts are favoured and patches of native structure persist — and denaturation is not necessarily irreversible, since a protein can refold (renature) once the stress is removed. Inside a cell, however, the exposed hydrophobic surfaces make denatured chains clump into insoluble aggregates, and cell function fails.

🪢 Collagen — Structure, Vitamin C, and What Makes It Unique Past Q

The most abundant protein in the body and the exam's model of a fibrous protein. Three polypeptides of about 1000 amino acids each wind together into the collagen triple helix.

The repeating sequence

(Gly–X–Y)ₙ, where X is often proline and Y often hydroxyproline. Every third residue must be glycine: the three chains pack so closely that only a hydrogen atom fits at the axis of the helix.

The geometry

Each chain is a left-handed helix with 3.3 residues per turn; the three chains then wrap around each other in a right-handed superhelix. A mature fibre is a rod with an axial ratio of about 200 — extremely long and thin.

What holds it together

Hydrogen bonds between the chains (the glycine N–H and the hydroxyproline –OH), then permanent covalent cross-links between lysine and hydroxylysine side chains made by lysyl oxidase, a copper enzyme.

Why vitamin C is essential Past Q

Collagen is made first as the larger precursor procollagen. Many of its prolyl and lysyl residues must be hydroxylated by prolyl and lysyl hydroxylase — enzymes that need O₂, α-ketoglutarate, Fe²⁺, and ascorbate as the reducing agent that keeps the iron in its active state. No vitamin C means too few hydroxyproline and hydroxylysine residues, so the triple helix is unstable.

Scurvy — the deficiency in one line

Unstable, under-hydroxylated collagen: bleeding gums, loose teeth, perifollicular haemorrhage, swollen painful joints, poor wound healing, and death if untreated.

Characteristics that make collagen different from other proteins Past Q

1 · glycine at every third position · 2 · exceptionally rich in proline · 3 · contains hydroxyproline and hydroxylysine, which are almost absent elsewhere · 4 · sugars (glucose, galactose) are attached to hydroxylysine, so it is a glycoprotein · 5 · it has no α-helix and no β-sheet — the triple helix is its own class of structure · 6 · it is extracellular, insoluble, cross-linked and very long-lived · 7 · it contains no tryptophan and almost no cysteine, which is why gelatin is a protein of defective nutritional value.

🧬 Post-Translational Modification of Proteins Past Q

Covalent changes made to a polypeptide after it leaves the ribosome. Examples that appear in this course:

Hydroxylation

Proline and lysine → hydroxyproline and hydroxylysine in collagen — the vitamin-C-dependent step on the card above.

Glycosylation

Sugars added to Asn (N-linked) or to Ser/Thr/hydroxylysine (O-linked) — makes glycoproteins.

Phosphorylation

Reversible addition of phosphate to Ser, Thr or Tyr by a kinase, removed by a phosphatase — the main switch of covalent enzyme regulation.

Limited proteolysis

Irreversible cleavage of one or a few peptide bonds that uncovers the active site: proinsulin → insulin, trypsinogen → trypsin, procollagen → collagen, clotting factors.

γ-Carboxylation

Glutamate → γ-carboxyglutamate in clotting factors II, VII, IX, X, protein C and S — vitamin K dependent; it creates the Ca²⁺-binding sites.

Carboxylation, acylation, ubiquitination

Biotin-dependent carboxylation of carboxylases; lipid anchors that attach a protein to membranes; and ubiquitin tagging, which marks a protein for ATP-dependent destruction in the proteasome.

🩸 Plasma Proteins and the Proteinogram Past Q

Almost all plasma proteins except the immunoglobulins are made in the liver. Electrophoresis separates them into five bands: albumin, α₁, α₂, β and γ.

Functions Past Q

1 · Colloid osmotic (oncotic) pressure — mainly albumin. 2 · Transport of insoluble ligands. 3 · Buffering. 4 · Defence — immunoglobulins, complement. 5 · Haemostasis — clotting and fibrinolytic factors. 6 · Enzyme inhibition — α₁-antitrypsin. 7 · A protein reserve.

Plasma proteins as transporters Past Q

Albumin — free fatty acids, unconjugated bilirubin, calcium, many drugs. Transferrin — iron. Ceruloplasmin — copper. Haptoglobin — free haemoglobin. Transcortin (CBG) — cortisol. SHBG — testosterone and oestradiol. TBG — thyroid hormones. Retinol-binding protein — vitamin A. Hemopexin — free haem: haptoglobin catches whole haemoglobin, hemopexin catches the haem that escapes from it. Lipoproteins — the lipids themselves.

Hypoalbuminaemia Past Q

From reduced synthesis (liver failure, malnutrition — kwashiorkor), loss (nephrotic syndrome, burns, protein-losing enteropathy) or dilution. Falling oncotic pressure causes oedema and ascites.

Reading the globulin bands

↑ α₁ and α₂ — acute-phase response (inflammation). Absent α₁ band — α₁-antitrypsin deficiency. β–γ bridging — cirrhosis. Diffuse ↑ γ — chronic infection, autoimmune disease. Sharp single γ spike (M band) — monoclonal gammopathy such as myeloma. ↓ γ — immunodeficiency.

🛡️ Acute-Phase Proteins and How an Immunoglobulin Is Built Past Q

The acute-phase proteins

Made by the liver and rising during inflammation and after tissue damage, sometimes 50-fold and occasionally a thousandfold: C-reactive protein (it activates the classical complement pathway), α₁-antitrypsin (α₁-antiproteinase), which neutralises the proteases released by activated neutrophils, haptoglobin, α₁-acid glycoprotein, fibrinogen and ceruloplasmin. They are what raise the α₁ and α₂ bands of the proteinogram, and they are why ferritin and ESR are unreliable in inflammation. Albumin behaves as a negative acute-phase protein and falls.

Chains, and what decides the class

Every immunoglobulin is two identical light chains + two identical heavy chains, held together by disulphide bonds. Light chains are either kappa (κ) or lambda (λ) — one molecule carries two of one type, never a mixture (κ is the commoner in humans). The heavy chain decides the class: γ = IgG, α = IgA, δ = IgD, ε = IgE, μ = IgM. Heavy chains have one variable domain (V_H) plus three constant domains (γ, α, δ) or four (ε, μ).

The parts with names

The hinge region, between C_H1 and C_H2, gives flexibility so the two arms can move independently. The Fab fragment is the arm that binds antigen; the Fc fragment is the stem that binds receptors and complement. Within the variable domains, three hypervariable regions in V_L and four in V_H form the antigen-binding site — the rest of the variable domain is the framework region.

The two extra pieces

IgM and IgA both carry a J chain, which joins the monomers (IgM as a pentamer, IgA as a dimer). Only secretory IgA also carries the secretory component, added by the epithelial cell, which protects it from proteolysis in mucus, saliva, tears and milk.

⚖️ Too Little Protein, Too Much Protein — and the Word “Osmolarity” Past Q

Both directions get asked, and each answer is a short list of routes, not of diseases.

Hypoproteinaemia — six routes

1 · Too little in — starvation, a low-protein diet, protein of defective biological value. 2 · Not absorbed — failure of digestion or of amino acid absorption. 3 · Excessive tissue breakdown — hyperthyroidism, malignancy, chronic infection, radiation injury. 4 · Genetic failure of synthesis — one fraction missing or deficient (agammaglobulinaemia, analbuminaemia). 5 · Loss — proteinuria, haemorrhage, loss in the faeces, exuding burns. 6 · Injury to the tissue that synthesises — liver insufficiency, or failure of the reticuloendothelial system.

Hyperproteinaemia — two kinds

With a qualitative change — paraproteinaemia. One abnormal clone floods the plasma with one abnormal protein: multiple myeloma (neoplastic plasma cells), Waldenström macroglobulinaemia (lymphoplasmacytoid cells), cryoglobulinaemia and amyloidosis. On the proteinogram this is the sharp M spike. Without one — more of a normal protein: hyperfunction of the reticuloendothelial system in bacterial and viral infection and in allergy, protein released from injured tissue, and plain haemoconcentration in dehydration.

The oncotic numbers

Total plasma protein 7.0–7.5 g/dL exerts a colloid osmotic pressure of about 25 mmHg. At the arteriolar end capillary hydrostatic pressure is roughly 37 mmHg and interstitial pressure 1 mmHg, so about 11 mmHg pushes fluid out; at the venular end hydrostatic pressure has fallen to about 17 mmHg, so roughly 9 mmHg draws it back. Lose the protein — severe protein malnutrition, nephrotic syndrome, liver failure — and the return limb fails: fluid stays in the interstitium as oedema.

Osmolarity, osmolality, oncotic pressure — which is which?Osmolarity counts osmoles per litre of solution; osmolality counts them per kilogram of water — that is what the laboratory measures, normally 285–295 mOsm/kg, and it can be estimated as 2 × [Na⁺] + glucose + urea. It is set almost entirely by small solutes, sodium above all, because number of particles is what counts and proteins are few and huge. Oncotic (colloid osmotic) pressure is the protein‑generated sliver of that total, and it is the only part that cannot cross the capillary wall, which is why albumin and not sodium is what holds water inside the vessel.

🧠 Prion Disease Past Q

A disease of protein conformation, with no change in amino acid sequence and no nucleic acid involved. The normal cellular protein PrP^C is α-helix rich, soluble and digested by proteases. The disease form PrP^Sc has the same sequence but is β-sheet rich, insoluble and protease-resistant, and it acts as a template that converts PrP^C into more of itself.

Consequence

The aggregates accumulate extracellularly in the brain, producing spongiform change and a rapidly progressive, invariably fatal dementia — Creutzfeldt–Jakob disease in humans. It is a change of fold, not of sequence: the whole point of the example.

The protein and its gene

The human prion-related protein (PrP) is a glycoprotein encoded on the short arm of chromosome 20. PrP^C is monomeric; in PrP^Sc the hydrophobic side chains are exposed to solvent, which is why it aggregates and resists proteases, heat and formalin.

One disease, three ways of starting

The same illness may be infectious (transmitted by the abnormal protein alone), genetic (a PRNP mutation) or sporadic. Animal forms: scrapie in sheep and bovine spongiform encephalopathy in cattle. Human forms: Creutzfeldt–Jakob disease, its variant in younger patients presenting with psychiatric and behavioural change, and kuru.

What the brain looks like

The transmissible spongiform encephalopathies share spongiform vacuolation, astrocytic gliosis and neuronal loss from deposits of insoluble protein — with no inflammation, because nothing foreign is recognised.

Enzymes — Classes, Kinetics, Regulation, Inhibitors

Resit topics 4–7 · the single densest block of past questions

📐 Classification of Enzymes Topic 4Past Q

Classification is by the type of reaction catalysed, and every enzyme has an EC number of four digits in which the first digit is the class.

1 · Oxidoreductases

Oxidation–reduction — transfer of electrons, hydrogen or oxygen. Dehydrogenases, oxidases, reductases, peroxidases, catalase, hydroxylases. Example: lactate dehydrogenase.

2 · Transferases

Transfer of a functional group between molecules. Aminotransferases, kinases, transmethylases, transaldolase, transketolase. Example: ALT, hexokinase.

3 · Hydrolases

Cleavage of a bond with the participation of water. Esterases, phosphatases, peptidases, glycosidases, lipases. Example: trypsin.

4 · Lyases

Removal of a group without water and without oxidation, usually leaving a double bond — or the reverse addition. Decarboxylases, hydratases, deaminases, aldolases. Example: fumarase, aldolase.

5 · Isomerases

Intramolecular rearrangements. Epimerases, racemases, mutases. Example: phosphoglucomutase, triose-phosphate isomerase.

6 · Ligases (synthetases)

Formation of a new bond between two molecules, coupled to hydrolysis of ATP. Synthetases, carboxylases. Example: pyruvate carboxylase, glutamine synthetase.

Class 7 · Translocases

The seventh class added by the IUBMB: they catalyse the movement of ions or molecules across membranes, or their separation within a membrane. Example: ATP synthase, EC 7.1.2.2.

Naming traps Past Q

Synthetase = ligase, uses ATP; synthase does not require ATP. Kinase transfers phosphate from ATP; phosphorylase uses inorganic phosphate to cleave a bond (phosphorolysis); phosphatase removes phosphate by hydrolysis.

🧷 Enzyme Structure, Coenzyme and Cofactor Past Q

apoenzyme (protein, inactive) + non-protein component ⇄ holoenzyme (active)

Prosthetic group

Tightly, often covalently bound and does not leave the enzyme during the reaction — e.g. haem in catalase, FAD in succinate dehydrogenase, biotin in carboxylases.

Coenzyme

Loosely bound, dissociable organic molecule that acts as a co-substrate shuttle: NAD⁺, FAD, CoA, TPP, PLP, THF. Most are derivatives of B vitamins, which is why those vitamins are essential.

Cofactor

Usually an inorganic ion that must be present for catalysis — Mg²⁺ (all kinases), Zn²⁺ (carbonic anhydrase, alcohol dehydrogenase), Fe²⁺, Cu²⁺, Mn²⁺, Se (glutathione peroxidase).

Active site

A cleft or pocket formed by residues that are far apart in the sequence but close in three dimensions. It is mostly non-polar (which strengthens substrate binding) with a few polar residues that carry out the catalysis.

🔑 Enzyme–Substrate Interaction — The Models Past Q

Fischer — lock and key

Historical. The active site is a rigid, pre-formed complement of the substrate. It explains specificity but ignores the effect of substrate binding on the enzyme's conformation.

Koshland — induced fit

The accepted model. The approach of the substrate induces a conformational change in the enzyme, and the enzyme in turn induces reciprocal changes in the substrate, straining it towards the transition state.

Ogston — three-point attachment

The substrate binds at three points, which fixes its orientation, and the reaction proceeds at only one of the three. This is how an enzyme distinguishes two chemically identical groups of a symmetrical molecule — the basis of stereospecificity.

🧨 How an Enzyme Actually Speeds a Reaction Up Topic 5Past Q

An enzyme lowers the activation energy by stabilising the transition state; it does not change ΔG, the equilibrium position or the direction of the reaction — it only makes equilibrium arrive sooner. These four mechanisms are how it lowers that barrier. Most enzymes use more than one at once. Most enzymes use more than one at once.

1 · Catalysis by proximity
Binding both substrates at the active site creates a region of very high local concentration in ideal mutual orientation. That alone accelerates a reaction about a thousandfold.

2 · Acid–base catalysis
Ionisable side chains (His, Glu, Asp, Cys, Lys) and prosthetic groups donate or accept protons during the reaction. Specific acid–base catalysis uses only H⁺ or OH⁻ from the solvent; general acid–base catalysis uses any proton donor or acceptor present — which is why the rate then depends on the buffer as well as the pH.

3 · Catalysis by strain
Lytic enzymes bind the substrate in a shape that is unfavourable for the bond about to be broken, mimicking the transition state. The bond is stretched, weakened and easier to cleave — this is transition-state stabilisation, and it is why transition-state analogues make the most potent inhibitors.

4 · Covalent catalysis
The enzyme forms a transient covalent bond with the substrate — usually through a serine, cysteine or histidine residue — opening a new route with a lower activation energy. The enzyme is chemically modified only for an instant and comes back unchanged, so it is still a catalyst. Common in group-transfer reactions, and typically follows a ping-pong mechanism: the first substrate binds and its product leaves before the second substrate binds.

Turnover number (k_cat)

The number of substrate molecules converted to product per enzyme molecule per second when the enzyme is saturated — typically 10²–10⁴ s⁻¹ (carbonic anhydrase reaches 10⁶). k_cat = Vmax / [E]_total, and k_cat/Km is the measure of catalytic efficiency. Enzyme-catalysed reactions run roughly 10³ to 10⁸ times faster than the uncatalysed ones.

📈 Factors Affecting Enzyme Kinetics Topic 5Past Q

Substrate concentration

At [S] < Km the rate is nearly proportional to [S] (first order). At [S] = Km, v = ½Vmax. At [S] ≫ Km the enzyme is saturated and the rate is Vmax, independent of [S] (zero order).

Enzyme concentration

Rate is directly proportional to [E] as long as substrate is in excess.

Temperature

Rate rises with temperature (more collisions, more molecules above the energy barrier) up to an optimum — about 37 °C for human enzymes — then falls steeply as the enzyme denatures. Q₁₀ ≈ 2: the rate roughly doubles per 10 °C rise before denaturation.

pH

Each enzyme has a pH optimum (pepsin ≈ 2, most cytosolic enzymes ≈ 7, pancreatic enzymes ≈ 8, alkaline phosphatase ≈ 10). pH affects the ionisation of the substrate and of active-site side chains, and the conformation of the protein.

Inhibitors and activators

Competitive, non-competitive and irreversible inhibitors; allosteric effectors; covalent modification. See the two cards below.

Vmax and Km — the definitions Past Q

Vmax — the maximal rate when the enzyme is fully saturated; proportional to the amount of enzyme. Km (Michaelis constant) — the substrate concentration at which v = ½Vmax. Km is inversely related to affinity: low Km = high affinity. Vmax is proportional to [E]; Km is not, and says nothing about Vmax.

Michaelis–Menten and the double-reciprocal plot Past Q

v = Vmax·[S] / (Km + [S]) gives a hyperbola, from which Vmax can only be estimated. Taking reciprocals gives the Lineweaver–Burk (double-reciprocal) plot, 1/v against 1/[S], a straight line with y-intercept 1/Vmax, x-intercept −1/Km and slope Km/Vmax — which is why it is used to read both constants and to identify the type of inhibition.

🚦 Competitive versus Non-Competitive Inhibition Topic 7Past Q

Competitive

The inhibitor is usually a structural analogue of the substrate and binds the active site (isosterically). EI forms, ESI does not. The inhibition is overcome by raising [S].
Km ↑ (apparent) · Vmax unchanged.
Lineweaver–Burk: lines meet on the y-axis.

Non-competitive

The inhibitor is structurally unrelated to the substrate and binds a site other than the active site (allosterically). ES, EI and ESI all form. Raising [S] does not relieve it — the inhibitor effectively removes enzyme from the pool.
Vmax ↓ · Km unchanged.
Lineweaver–Burk: lines meet on the x-axis.

Worked examples

Competitive: malonate on succinate dehydrogenase; statins on HMG-CoA reductase; allopurinol on xanthine oxidase; methotrexate on dihydrofolate reductase; ethanol given deliberately in methanol poisoning, because both alcohols compete for alcohol dehydrogenase and ethanol slows the formation of the toxic products formaldehyde and formic acid.
Non-competitive / irreversible: heavy metals (Pb²⁺, Hg²⁺) and oxidising agents, which chemically modify residues essential for catalysis; cyanide on cytochrome c oxidase.

Uncompetitive — the third pattern

The inhibitor binds only to the ES complex. Both Vmax and Km fall, so their ratio is unchanged and the Lineweaver–Burk lines are parallel.

🎛️ Mechanisms of Regulation of Enzyme Activity Topic 6Past QLecturer’s priority

There are only two strategies — change the amount of enzyme, or change the catalytic efficiency of the enzyme already present — and five named mechanisms across them.

1 · Substrate availability

Effector: the substrate. Result: change in rate. Immediate.

2 · Product inhibition

Effector: the product. Result: change in Vmax or Km. Immediate.

3 · Allosteric regulation Past Q

An effector binds a site distinct from the active site and changes the tertiary/quaternary structure, altering substrate binding. Allosteric enzymes are usually oligomeric, sit at the rate-limiting step, and give a sigmoidal v–[S] curve rather than a hyperbola. Inhibition by the end product of the pathway is feedback inhibition, and the effector generally bears no structural resemblance to the substrate.

Homotropic versus heterotropic effectors Past Q

Homotropic — the substrate itself is the effector. One molecule binding at one site raises the affinity of the remaining sites: cooperativity, which is what produces the sigmoidal v–[S] curve instead of the Michaelis–Menten hyperbola. Oxygen on haemoglobin is the classic example. Heterotropic — the effector is a different molecule: citrate inhibiting phosphofructokinase-1, fructose-2,6-bisphosphate activating it, acetyl-CoA activating pyruvate carboxylase. For a sigmoidal enzyme the term Km is replaced by K₀.₅ (S₀.₅), the substrate concentration giving half-maximal velocity; positive and negative effectors can change K₀.₅, Vmax, or both.

4 · Covalent modification

Phosphorylation/dephosphorylation by a kinase and a phosphatase — reversible, hormonally driven, requires ATP hydrolysis, and needs separate enzymes to reverse it. Takes seconds to minutes.

5 · Change in enzyme amount

Induction or repression of gene transcription, or altered degradation. Effector: a hormone or a metabolite. Hours to days.

Plus: limited proteolysis

Irreversible activation of a zymogen by cleaving one or a few peptide bonds — pepsinogen, trypsinogen, chymotrypsinogen, proinsulin, procollagen, clotting factors.

Compartmentation is itself regulation

Enzymes are localised: mitochondria (Krebs cycle, β-oxidation, pyruvate oxidation), cytosol (glycolysis, pentose phosphate pathway, fatty acid synthesis), nucleus (DNA and RNA synthesis), lysosome (degradation of complex macromolecules), smooth ER (cytochrome P450). Separating opposing pathways prevents futile cycling and lets each have its own optimal environment.

Phosphorylation does not mean "on" Past Q

Inactivated by phosphorylation: glycogen synthase, acetyl-CoA carboxylase, pyruvate dehydrogenase, pyruvate kinase (liver). Activated by phosphorylation: glycogen phosphorylase, phosphorylase b kinase, hormone-sensitive lipase, citrate lyase. A useful shortcut: glucagon/adrenaline phosphorylate, and the catabolic enzymes switch on.

🏥 Isoenzymes, LDH and Biological Oxidation Past Q

Isoenzymes

Different proteins, coded at different gene loci, that catalyse the same reaction but differ in structure, kinetics (Km) and regulation — hexokinase vs glucokinase, creatine kinase, LDH.

Lactate dehydrogenase Past Q

An oxidoreductase (EC 1.1.1.27) that interconverts pyruvate and lactate using NADH/NAD⁺. A tetramer of H ("heart") and M ("muscle") subunits, giving five isoenzymes: LDH1 (HHHH) heart · LDH2 (HHHM) heart, red cells, brain · LDH3 (HHMM) brain, kidney, leukocytes · LDH4 (HMMM) liver, muscle · LDH5 (MMMM) muscle and liver.

Why LDH matters in anaerobic conditions Past Q

Glycolysis consumes NAD⁺ at glyceraldehyde-3-phosphate dehydrogenase. Without mitochondria or oxygen there is no electron transport chain to re-oxidise NADH, so glycolysis would stop within seconds. LDH regenerates NAD⁺ by reducing pyruvate to lactate — which is the only way red cells, and hard-working muscle, can keep making ATP.

Biological oxidation — the four enzyme types Past Q

Dehydrogenases remove two hydrogens onto NAD⁺/FAD; oxygen is not involved. Oxidases use O₂ as the electron acceptor, reducing it to H₂O or H₂O₂. Oxygenases incorporate oxygen into the substrate — monooxygenases insert one atom (the other becomes water; cytochrome P450), dioxygenases insert both. Peroxidases use H₂O₂ as the oxidant (glutathione peroxidase); catalase is the special case that converts H₂O₂ into water and O₂.

Enzymes in diagnosis

↑ AST and ALT — hepatocellular damage (ALT more liver-specific; AST also heart and muscle). ↑ ALP — cholestasis and bone disease. ↑ GGT — cholestasis and alcohol; it confirms that a raised ALP is hepatic. ↑ amylase and lipase — acute pancreatitis. ↑ CK-MB and troponins — myocardial infarction. ↑ acid phosphatase — prostate.

Protein Digestion & Nitrogen Metabolism

Resit topics 8–12 · digestion, transamination, ammonia, the urea cycle and glutamine

🍽️ Protein Digestion and Amino Acid Absorption Topic 8Past Q

No protein digestion happens in the mouth. It begins in the stomach and is completed in the small intestine by pancreatic and brush-border enzymes.

Stomach — role of gastric HCl Topic 8

HCl is secreted by the parietal cells; carbonic anhydrase supplies H⁺ from CO₂ + H₂O, the H⁺/K⁺-ATPase pumps it into the lumen, and Cl⁻ follows (the HCO₃⁻ released into blood is the "alkaline tide"). Its three jobs: denature dietary protein so peptide bonds are exposed; convert pepsinogen to pepsin (autocatalytically, below pH 5); and kill ingested microorganisms. Pepsin is an endopeptidase with an optimum near pH 2.

Pancreas — activation cascade

Alkaline pancreatic juice neutralises the chyme. Enteropeptidase (enterokinase) of the duodenal brush border converts trypsinogen → trypsin, and trypsin then activates everything else including more trypsinogen: chymotrypsinogen, proelastase, procarboxypeptidases.

Endopeptidases Past Q

Hydrolyse internal peptide bonds. Their significance: they cut a large protein into many fragments, multiplying the number of ends the exopeptidases can then work on. Pepsin, trypsin, chymotrypsin and elastase — specificities tabulated on the next card.

Exopeptidases Past Q

Remove amino acids from the ends: carboxypeptidases A and B from the C-terminus, brush-border aminopeptidases from the N-terminus, then di- and tripeptidases inside the enterocyte.

Absorption

Free amino acids are taken up by Na⁺-dependent secondary active transport through several carriers, each serving a group of amino acids (neutral, basic, acidic, imino). Di- and tripeptides are absorbed by the H⁺-coupled PepT1 transporter and hydrolysed inside the cell. Amino acids then leave into the portal vein to the liver. A defect in the basic-amino-acid carrier of the intestine and kidney is cystinuria.

Basal and maximal gastric acid output Past Q

BAO — basal acid output: acid secreted per hour without stimulation, normally about 0–5 mmol/h. MAO — maximal acid output: acid per hour after maximal stimulation (pentagastrin or histamine), normally about 5–20 mmol/h. A high BAO with a BAO/MAO ratio above ~0.6 suggests a gastrin-secreting tumour (Zollinger–Ellison); a low or absent output suggests atrophic gastritis or pernicious anaemia.

📋 The Protease Data Sheet — Proenzyme, Activator, pH, Specificity Topic 8Past Q

Every one of them is secreted as an inactive proenzyme — that is what protects the pancreas, and premature activation inside it is acute pancreatitis. Pepsin’s contribution is small in quantity but essential in kind: it is the one that works in acid.

Enzyme	Proenzyme	Activated by	Optimum pH	Cuts	Products
Pepsin	Pepsinogen	HCl, then autocatalysis	1–2	Internal bonds beside aromatic and dicarboxylic residues, and between Leu and Glu	Large peptides — only about 15% of the protein is cut here
Trypsin	Trypsinogen	Enteropeptidase, then autocatalysis	7–9	Internal bonds after basic residues	Peptides ending in Lys or Arg
Chymotrypsin	Chymotrypsinogen	Trypsin	7–9	Internal bonds after aromatic residues	Peptides ending in Phe, Trp, Tyr
Elastase	Proelastase	Trypsin	7–9	Internal bonds after small aliphatic residues	Peptides ending in Val, Leu, Ser, Ala
Carboxypeptidase A	Procarboxypeptidase A	Trypsin	7–9	C-terminal aromatic and aliphatic residues — i.e. the ends chymotrypsin and elastase have just made	Free Phe, Trp, Tyr, Val, Leu, Ser, Ala
Carboxypeptidase B	Procarboxypeptidase B	Trypsin	7–9	C-terminal basic residues — the ends trypsin has just made	Free Lys and Arg
Aminopeptidases	—	Brush border enzymes	7–9	N-terminal residues	Free amino acids + shorter peptides
Dipeptidases and tripeptidases	—	Inside the enterocyte	~7	The last two or three residues	Free amino acids for the portal blood

⚖️ Complete versus Incomplete Proteins Topic 9

Complete (high biological value)

Contain all the essential amino acids in adequate proportions: egg, milk, meat, fish. Egg protein is the reference standard.

Incomplete (low biological value)

Deficient in one or more essential amino acids — most plant proteins. Cereals are limiting in lysine; legumes are limiting in methionine. Gelatin lacks tryptophan.

Biological value

The proportion of absorbed nitrogen that is retained by the body. It is set by the limiting amino acid — the essential amino acid present in the smallest amount relative to need. Protein synthesis stops when any one runs out, and the rest is deaminated and burned.

Complementation

Combining two incomplete proteins whose limiting amino acids differ — cereals + legumes — gives a mixture of high biological value. This is why "rice and beans" works nutritionally.

⚖️ Nitrogen Balance and the Nitrogen Sources Topic 9Past Q

Nitrogen balance = nitrogen intake − nitrogen output. Amino acids are the body's main nitrogen source, and because protein is never stored — there is only a small free amino acid pool in constant turnover — the balance is read day by day.

Adult requirement

0.5–0.7

g protein / kg / day

Safe intakes are usually quoted a little higher, ~0.8

Child requirement

≈ 4

g protein / kg / day

Growth demands a positive balance

Nitrogen leaves as

Urea

then NH₄⁺, creatinine, uric acid, amino acids

Plus faeces, skin and hair

Biological quality — the three grades

Standard (full) value — every essential amino acid in adequate amount and proportion: egg, milk, meat, fish. Partially defective value — at least one essential amino acid present but in inadequate amount: most cereals (low lysine) and legumes (low methionine). Defective value — at least one essential amino acid missing: gelatin has no tryptophan, maize zein lacks lysine and tryptophan.

Positive balance — intake > output

Nitrogen is being retained because tissue is being built: growth in infants and children, pregnancy, lactation, convalescence after illness or surgery, repair after injury, increased physical training, and anabolic hormone states (growth hormone, insulin, androgens).

Negative balance — output > intake

Inadequate intake (starvation, low-protein diet, proteins of defective value), failure of digestion or of amino acid absorption, excessive tissue breakdown (fever, sepsis, trauma, burns, surgery, advanced cancer, hyperthyroidism, glucocorticoid excess) and loss of protein (proteinuria, haemorrhage, loss in faeces, exudating burns).

Equilibrium

The normal state of a healthy, weight-stable adult: intake exactly matches output. A negative balance can never be corrected by energy alone — if any single essential amino acid runs short, synthesis stops and the rest are deaminated and burned.

🔀 Aminotransferases and the Alanine Cycle Topic 10Past Q

Transamination transfers the α-amino group of an amino acid onto an α-keto acid — almost always onto α-ketoglutarate, forming glutamate. It is fully reversible, so it serves both degradation and synthesis, and it releases no ammonia: nitrogen is collected, not liberated.

Mechanism & cofactor

A ping-pong mechanism through the cofactor pyridoxal phosphate (PLP), the active form of vitamin B6. PLP accepts the amino group to become pyridoxamine phosphate, then hands it to the acceptor keto acid.

The two examined examples

ALT (AlAT, GPT): alanine + α-ketoglutarate ⇄ pyruvate + glutamate. AST (AspAT, GOT): aspartate + α-ketoglutarate ⇄ oxaloacetate + glutamate.

Diagnostic value Topic 10

ALT is more liver-specific (mainly cytosolic, mainly hepatic). AST is also abundant in heart, skeletal muscle and red cells, and has a mitochondrial form. ALT > AST suggests viral hepatitis; AST > ALT (ratio > 2) suggests alcoholic liver disease; very high values of both indicate acute hepatocellular necrosis.

Two exceptions

Lysine and threonine do not undergo transamination. All other amino acids do.

The glucose–alanine (alanine) cycle Topic 10Past Q

In muscle, amino acid breakdown and glycolysis meet: the amino group is transferred onto pyruvate to make alanine, which travels in the blood to the liver. There ALT runs in reverse — alanine gives back pyruvate (used for gluconeogenesis) and the nitrogen enters glutamate and then the urea cycle. The new glucose returns to muscle. The cycle transports nitrogen to the liver in a non-toxic form and simultaneously supplies a gluconeogenic substrate — at the cost, to the liver, of the ATP of gluconeogenesis. Compare the Cori cycle, which carries lactate (carbon only, no nitrogen) on the same route.

💨 Reactions That Release and Incorporate Ammonia Topic 11Past QLecturer’s priority

Releasing ammonia

Oxidative deamination of glutamate by glutamate dehydrogenase (mitochondria, uses NAD⁺ or NADP⁺) — the main route. Glutaminase (glutamine → glutamate + NH₃; kidney and liver). Asparaginase. AMP deaminase in the purine nucleotide cycle of muscle. Serine and threonine dehydratases (PLP-dependent). Bacterial urease in the gut acting on urea. Amine oxidases (MAO, DAO). Transdeamination is the name for the pairing that does most of the work: transamination first funnels the nitrogen of almost every amino acid onto glutamate, then glutamate dehydrogenase deaminates that one molecule — so the nitrogen of twenty amino acids leaves as ammonia through a single enzyme.

Incorporating ammonia

Glutamate dehydrogenase in reverse — the reductive amination of α-ketoglutarate (α-ketoglutarate + NH₃ + NADPH → glutamate), the reaction that builds the whole "glutamate family" of amino acids and keeps cytotoxic ammonium low. Glutamine synthetase (glutamate + NH₃ + ATP → glutamine) — the main ammonia-trapping reaction of brain and muscle. Carbamoyl phosphate synthetase I, the entry step of the urea cycle. Also CPS II for pyrimidines, which uses the amide nitrogen of glutamine instead.

Glutamate dehydrogenase — regulation

The only enzyme that can both fix and release ammonia. Allosterically activated by ADP and GDP (low energy → degrade amino acids) and inhibited by ATP and GTP (high energy).

Why ammonia is toxic Past Q

NH₃ crosses the blood–brain barrier. In the brain it is trapped by glutamate → glutamine, which drains glutamate and α-ketoglutarate: the Krebs cycle slows, ATP falls, glutamate (a transmitter) is depleted, and glutamine accumulates osmotically, causing astrocyte swelling and cerebral oedema. Clinically: tremor, slurred speech, blurred vision, asterixis, confusion, coma.

Where the ammonia in portal blood comes from

The gut is a major producer: enteric bacteria split urea and dietary amines with urease, and enterocytes deaminate glutamine. That ammonia is absorbed straight into the portal vein, and a healthy liver removes it on the first pass, which is why only traces reach the peripheral blood. If portal blood bypasses the liver, systemic ammonia rises even though no enzyme is missing — the acquired hyperammonaemia below.

🔵 The Urea Cycle Topic 11Past QLecturer’s priority

Where: the liver (periportal hepatocytes) — and only the liver makes urea. Subcellular location: the first two steps are mitochondrial, the last three cytosolic. The kidney excretes the urea; a small amount diffuses into the gut.

1 · CPS I — mitochondrion

NH₃ + HCO₃⁻ + 2 ATP → carbamoyl phosphate. The rate-limiting, committed step; requires the obligatory allosteric activator N-acetylglutamate (NAG).

2 · OTC — mitochondrion

Ornithine transcarbamoylase: carbamoyl phosphate + ornithine → citrulline, which leaves for the cytosol.

3 · Argininosuccinate synthetase — cytosol

Citrulline + aspartate + ATP → argininosuccinate. ATP → AMP + PPi, so two high-energy bonds are spent. This is where the second nitrogen of urea enters — from aspartate, not from free ammonia.

4 · Argininosuccinate lyase — cytosol

Argininosuccinate → arginine + fumarate. The fumarate is the link to the Krebs cycle (fumarate → malate → oxaloacetate → aspartate again): the "aspartate–argininosuccinate shunt".

5 · Arginase — cytosol

Arginine + H₂O → urea + ornithine. Ornithine re-enters the mitochondrion and the cycle turns again. Arginase is essentially liver-specific.

Cost and stoichiometry

One urea per turn consumes 3 ATP but 4 high-energy phosphate bonds. The two nitrogens come from free ammonia (via CPS I) and from aspartate; the carbon comes from bicarbonate.

Regulation Topic 11

Short term — allosteric: a protein-rich meal raises glutamate, which drives N-acetylglutamate synthase (activated by arginine) to make more NAG, which switches on CPS I. Long term — enzyme induction: a sustained high-protein diet, or prolonged starvation (muscle protein is being broken down), raises the amount of all five enzymes over days.

Hyperammonaemia Disorder 3

Acquired — liver failure or cirrhosis with portosystemic shunting; ammonia bypasses the liver. Inherited — a deficiency of any urea-cycle enzyme; OTC deficiency is the commonest and is X-linked, all the others being autosomal recessive. In OTC deficiency the accumulated carbamoyl phosphate spills out of the mitochondrion into pyrimidine synthesis, so the urine contains orotic acid together with the high ammonia — the pairing that distinguishes it from hereditary orotic aciduria, which has orotate but normal ammonia. Management: restrict dietary protein, give benzoate and phenylbutyrate to excrete nitrogen by an alternative route, plus arginine.

The named urea-cycle defects — one per enzyme Past Q

CPS I → hyperammonaemia type 1 · Ornithine transcarbamoylase → hyperammonaemia type 2 (the card above) · Argininosuccinate synthetase → citrullinaemia · Argininosuccinate lyase → argininosuccinic aciduria (argininosuccinate raised in blood, CSF and urine, with the give-away sign of friable, tufted hair) · Arginase → hyperargininaemia (autosomal recessive) · N-acetylglutamate synthase → the same clinical picture as a CPS I defect, because CPS I cannot be switched on · ORNT1 mitochondrial ornithine transporter → HHH syndrome: hyperornithinaemia, hyperammonaemia and homocitrullinuria, because cytosolic ornithine cannot get back into the mitochondrion.

What the patient looks like

Vomiting, lethargy, irritability, intermittent ataxia, seizures, and in survivors severe intellectual disability. Older children characteristically avoid high-protein food on their own. The earlier the block sits in the cycle (steps 1 and 2), the more severe the disease, because ammonia has not yet been fixed into citrulline.

🅶 The Role of Glutamine Topic 12Past Q

Glutamine is the most abundant amino acid in blood and the body's general-purpose nitrogen carrier: it holds two nitrogens — an α-amino group and an amide group — and is electrically neutral and non-toxic.

1 · Non-toxic ammonia transport

Glutamine synthetase traps ammonia in muscle and especially brain; the blood carries glutamine to the liver, where glutaminase releases the ammonia straight into the urea cycle. Note the liver zonation: periportal cells make urea, and the perivenous cells that the blood meets last mop up the residue with glutamine synthetase.

2 · Renal ammoniagenesis

In the kidney, glutaminase and glutamate dehydrogenase release two NH₃ per glutamine, which buffer H⁺ in the tubular lumen as NH₄⁺ and are excreted. Each NH₄⁺ excreted equals one new bicarbonate returned to the blood. The process is strongly induced by chronic metabolic acidosis.

3 · Nitrogen donor for biosynthesis

The amide nitrogen of glutamine supplies: purine ring (N3 and N9), CPS II for pyrimidines, asparagine, glucosamine for glycosaminoglycans, and NAD⁺.

4 · Fuel and carbon source

The preferred fuel of rapidly dividing cells — enterocytes, lymphocytes and other immune cells. Its carbon skeleton enters the Krebs cycle as α-ketoglutarate, so it is also glucogenic and is a major gluconeogenic substrate in the kidney during fasting and acidosis.

Two details of the glutamine reaction that get asked

The mechanism: glutamine synthetase binds glutamate and ATP in that order, glutamate attacks the γ-phosphate to give the intermediate γ-glutamyl phosphate plus ADP; NH₄⁺ then binds, uncharged NH₃ attacks the intermediate, and release of Pi releases glutamine. The inter-organ trip: in the post-absorptive state glutamine is released from muscle into the circulation and is extracted by the gut and the kidney, which convert a large part of it to alanine — so glutamine carries nitrogen, carbon and energy between organs, and hands the load on to the glucose–alanine cycle. A rare neonatal glutamine synthetase deficiency causes severe brain damage, multi-organ failure and death.

Amino Acid Fates & Specialised Products

Past questions 2, 5, 7–12, 15, 23, 33, 39, 81, 86, 113, 125, 156

🎯 The Seven Entry Points into Central Metabolism Past Q 7–12

Every carbon skeleton ends at one of seven compounds. Six are Krebs-cycle intermediates or their precursors; acetyl-CoA and acetoacetate are the ketogenic exits.

→ Pyruvate Past Q 7

Ala, Ser, Gly, Cys, Thr (and Trp in part). Glucogenic. Alanine by transamination; serine by dehydratase; cysteine by desulfuration (its sulfur leaves as sulfate, used for PAPS); glycine ⇄ serine via the one-carbon pool.
Disturbances: non-ketotic hyperglycinaemia; cystinuria — a transport defect, not a catabolic one.

→ Oxaloacetate Past Q 8

Asp and Asn. Asparaginase gives aspartate + NH₃; aspartate transaminates to oxaloacetate. Glucogenic. Aspartate also donates the second urea nitrogen and nitrogens to purines and pyrimidines.
Synthesis: both are made from oxaloacetate, so they are non-essential.

→ α-Ketoglutarate Past Q 10

Glu, Gln, Pro, Arg, His — all converge on glutamate first. Glucogenic. Histidine loses ammonia to urocanate and yields FIGLU, whose disposal needs tetrahydrofolate — the basis of the FIGLU test for folate deficiency.
Synthesis: the whole family is made from α-ketoglutarate, so all are non-essential (Arg conditionally).

→ Succinyl-CoA Past Q 11

Met, Val, Ile, Thr. Glucogenic. The propionyl-CoA route: propionyl-CoA → (biotin, propionyl-CoA carboxylase) → methylmalonyl-CoA → (methylmalonyl-CoA mutase, vitamin B12) → succinyl-CoA. B12 deficiency therefore causes methylmalonic aciduria.
Disturbances: homocystinuria (methionine route); MSUD (Val, Ile — shared with Leu).

→ Fumarate Past Q 12

Phe and Tyr. Phe → Tyr (phenylalanine hydroxylase, BH₄) → … → homogentisate → fumarate + acetoacetate. Both glucogenic and ketogenic.
Disturbances: PKU (phenylalanine hydroxylase), alkaptonuria (homogentisate oxidase), tyrosinaemia, albinism (tyrosinase — a synthesis defect, not catabolic).

→ Acetyl-CoA / acetoacetate Past Q 9

Leu and Lys purely ketogenic; Phe, Tyr, Trp, Ile partly. No net glucose can be made from these carbons, because pyruvate dehydrogenase is irreversible. Leucine catabolism runs through HMG-CoA, the same intermediate as ketogenesis.
Disturbance: MSUD — branched-chain α-ketoacid dehydrogenase, with the same five cofactors as pyruvate dehydrogenase.

Non-essential amino acid synthesis in one line Past Q 113

α-Ketoglutarate → Glu → Gln, Pro, Arg. Oxaloacetate → Asp → Asn. Pyruvate → Ala. 3-Phosphoglycerate → Ser → Gly and Cys (sulfur from methionine). Phenylalanine → Tyr. Every one of these uses transamination or a Krebs intermediate as the carbon skeleton — which is the mechanism the question is asking for.

⚗️ Amino Acids as Precursors of Specialised Products Past Q 2, 5, 15

Tyrosine

Melanin, the thyroid hormones T3/T4, and the catecholamines — written out on the card below.

Tryptophan Past Q 125

Serotonin → melatonin, and — via the kynurenine pathway — niacin (NAD⁺), at roughly 60 mg Trp per 1 mg niacin. Both routes are written out below.

Glutamate

Glutathione, the nitrogen for many transaminations, and GABA — card below.

Glycine

Haem (with succinyl-CoA), purines, creatine, glutathione, bile-salt conjugation.

Histidine · Arginine

Histidine → histamine — card below. Arginine → nitric oxide (NO synthase), creatine, urea, and the polyamines spermidine and spermine — made from ornithine by ornithine decarboxylase using the propylamine of decarboxylated SAM, and needed wherever cells grow and divide fast.

Methionine · Cysteine

Methionine → SAM, the universal methyl donor. Cysteine → taurine (bile-salt conjugation), glutathione, and PAPS, the active sulfate donor.

The two routes out of tryptophan, written out

Serotonin route: tryptophan → 5-hydroxytryptophan (tryptophan hydroxylase, BH₄ — the same kind of enzyme as phenylalanine hydroxylase) → serotonin (decarboxylation, PLP) → N-acetylserotonin → melatonin in the pineal gland. Niacin route: the kynurenine pathway, needing B6. Two named diseases follow from tryptophan: Hartnup disease — a defect of the membrane transporter for neutral amino acids in intestine and renal tubule, so tryptophan is lost in stool and urine and the patient develops pellagra-like signs; and pellagra itself, from deficiency of niacin and tryptophan, with photosensitive dermatitis, then diarrhoea and dementia, fatal untreated and cured by either tryptophan or niacin.

Asparagine — synthesis, breakdown, and why oncologists care

Asparagine synthetase makes it from aspartate + glutamine + ATP, through the intermediate aspartyl phosphate; the coupled hydrolysis of PPi to 2 Pi makes the reaction effectively irreversible. Asparaginase runs the other way, giving aspartate + NH₃, and a transaminase then delivers all four carbons to oxaloacetate. Leukaemic lymphoblasts make little asparagine synthetase, so injected asparaginase starves them — a standard drug in acute lymphoblastic leukaemia.

⚡ Catecholamines and the Biogenic Amines Past Q 23, 33, 19

Tyrosine → DOPA → dopamine → noradrenaline → adrenaline. Step 1 is tyrosine hydroxylase, the rate-limiting enzyme, requiring BH₄. DOPA decarboxylase requires PLP. Dopamine β-hydroxylase is a copper enzyme that needs vitamin C. The final N-methylation to adrenaline uses SAM and happens almost only in the adrenal medulla, driven by cortisol.

Degradation

By MAO (mitochondrial outer membrane) and COMT (catechol-O-methyltransferase, SAM-dependent). End products: VMA from adrenaline and noradrenaline, HVA from dopamine, 5-HIAA from serotonin. Excess VMA and metanephrines in urine = phaeochromocytoma.

Histamine

From histidine by histidine decarboxylase (PLP). Stored in mast cells, basophils and gastric ECL cells. Actions: vasodilation and capillary permeability (allergy, anaphylaxis), gastric acid secretion via H₂ receptors, and neurotransmission. Degraded by DAO and histamine N-methyltransferase.

GABA Past Q 86

The main inhibitory transmitter of the CNS. Glutamate → GABA by glutamate decarboxylase (PLP). Degraded by GABA transaminase to succinic semialdehyde and then to succinate, which enters the Krebs cycle — the GABA shunt. B6 deficiency lowers GABA and causes seizures.

MAO and DAO — abbreviations Past Q 19

MAO = monoamine oxidase, which deaminates monoamines (catecholamines, serotonin); MAO inhibitors are antidepressants and cause the "cheese reaction" with dietary tyramine. DAO = diamine oxidase, which degrades histamine and other diamines, and is abundant in intestinal mucosa, kidney and placenta.

💪 Creatine and Creatinine Past Q 39

Synthesis takes two organs: the kidney combines glycine + arginine to make guanidinoacetate (releasing ornithine), and the liver methylates it with SAM to give creatine. Creatine travels to muscle and brain, where creatine kinase phosphorylates it to phosphocreatine — the immediate, high-energy ATP buffer for the first seconds of contraction.

Creatinine

Phosphocreatine and creatine spontaneously and irreversibly cyclise to creatinine at a nearly constant rate — about 1–2% of the creatine pool per day. Creatinine has no function; it is freely filtered by the glomerulus and essentially not reabsorbed, so its excretion is proportional to muscle mass and its plasma level is a standard index of glomerular filtration rate.

Creatinine clearance — the formula and what it is for Past Q

Clearance is the volume of plasma completely cleared of a substance by the kidney per unit time. C_cr (mL/min) = (U_cr × V) / P_cr — urine creatinine concentration × urine flow rate, divided by plasma creatinine. Because creatinine is filtered and barely reabsorbed, this estimates the glomerular filtration rate and detects early renal failure long before symptoms. And because 24-hour creatinine output is proportional to muscle mass and constant for a given person, it is also used to check that a 24-hour urine collection was complete.

🔗 Folic Acid, THF and SAM — The One-Carbon Pool Past Q 81, 156

Tetrahydrofolate (THF)

Folate is reduced by dihydrofolate reductase to THF, the carrier of one-carbon units at every oxidation level except CO₂ (that is biotin's job). It supplies carbons for: purine ring (C2 and C8), dTMP (via thymidylate synthase, which oxidises THF to DHF), and the remethylation of homocysteine. Main donors of the units: serine, glycine, histidine (FIGLU) and choline — serine is the most important, because serine hydroxymethyltransferase (PLP) hands its β-carbon straight to THF as N5,N10-methylene-THF, the main entry point into the pool. The stable pharmaceutical form is 5-formyl-THF — folinic acid (leucovorin), which enters below dihydrofolate reductase and is therefore used to rescue normal tissue after methotrexate. Supplements lower homocysteine and prevent neural-tube defects, and folate deficiency hits rapidly dividing cells first because they need the most thymidine.

SAM

S-adenosylmethionine, made from methionine + ATP, is the universal methyl donor: creatine, adrenaline, phosphatidylcholine, melatonin, DNA and protein methylation, and COMT. After donating its methyl it becomes SAH → homocysteine.

The junction — and why B12 matters

Homocysteine has two fates: remethylation to methionine by methionine synthase, which needs N5-methyl-THF and vitamin B12; or transsulfuration to cystathionine and cysteine by cystathionine β-synthase, which needs PLP (B6).

The methyl-folate trap

In B12 deficiency methionine synthase stalls, so folate accumulates as N5-methyl-THF and cannot be recycled into the forms needed for DNA synthesis. The result is a megaloblastic anaemia identical to folate deficiency — but B12 deficiency also causes neurological damage, through the separate methylmalonyl-CoA mutase route. Folate alone corrects the anaemia and lets the neurology progress, which is why B12 must be excluded first.

Raised homocysteine

Caused by deficiency of B6, B12 or folate, or by CBS deficiency (homocystinuria). It damages vascular endothelium and is an independent risk factor for thrombosis and atherosclerosis.

Iron, Haem, Bilirubin & Jaundice

Resit topics 13–14 · disorders 1, 2 and 15 · past questions on TIBC, porphobilinogen, phase II and uronic acid

🔴 Iron Absorption, Storage and Transport Topic 13Past Q 102

Absorption — duodenum

Dietary iron is mostly Fe³⁺, which cannot be absorbed. Gastric HCl and vitamin C reduce it to Fe²⁺ (helped by the brush-border reductase DcytB), and DMT1 carries Fe²⁺ into the enterocyte. Haem iron is absorbed separately and far more efficiently. Absorption is inhibited by phytates, oxalates, tannins, antacids and calcium.

Export and the hepcidin switch

Ferroportin is the only exporter out of the enterocyte; hephaestin (a copper ferroxidase) re-oxidises the iron to Fe³⁺ for loading onto transferrin. Hepcidin, made by the liver, blocks ferroportin: high hepcidin (inflammation, iron excess) keeps iron locked in cells; low hepcidin allows absorption. Iron that is not exported stays as ferritin and is lost when the enterocyte is shed — the "mucosal block".

Transport — transferrin

A β-globulin made in the liver that binds two Fe³⁺ ions and delivers them by receptor-mediated endocytosis. Normally about one third saturated.

Storage — ferritin and haemosiderin

Ferritin — the soluble, readily mobilised store in liver, spleen and marrow; serum ferritin reflects total body iron but is also an acute-phase protein and rises in inflammation. Haemosiderin — the insoluble, degraded aggregate found in iron overload.

TIBC and the diagnosis of iron deficiency Past Q 40, 173

TIBC = total iron-binding capacity — the total amount of iron that plasma transferrin could carry, so it is an indirect measure of transferrin concentration.
Iron deficiency anaemia: ↓ serum iron, ↑ TIBC, ↓ transferrin saturation, ↓ ferritin (the earliest and most specific change), microcytic hypochromic red cells, plus a raised soluble transferrin receptor and raised red-cell protoporphyrin — the two that rise only in true iron deficiency, and so separate it from the anaemia of chronic disease. Causes: chronic blood loss (menstrual, gastrointestinal — the one that must be excluded in adults), poor intake, malabsorption, increased demand in pregnancy and growth.
Anaemia of chronic disease: ↓ serum iron but ↓ TIBC and normal-or-high ferritin — hepcidin is high and iron is trapped in stores.
Haemochromatosis: ↑ serum iron, ↓ TIBC, ↑↑ ferritin, high saturation.

Dual reciprocal control — how a cell sets its own iron uptake Past Q

The messenger RNAs for ferritin (storage) and for transferrin receptor 1 (uptake) both carry hairpin iron-response elements (IREs) — on the 5′ untranslated region in ferritin, on the 3′ untranslated region in TfR1 — and iron-regulatory proteins (IRPs) bind them only when iron is low. Low iron: IRP on the ferritin 5′ IRE blocks translation (less ferritin) while IRP on the TfR1 3′ IRE stabilises the message (more receptors) → the cell grabs iron and stores none. High iron: the IRPs let go — ferritin is translated and TfR1 mRNA is rapidly degraded. One switch, two opposite outcomes.

The transferrin cycle, and the daily balance sheet

Cycle: transferrin–Fe³⁺ binds TfR1 → the complex is taken in by receptor-mediated endocytosis → the endosome is acidified, which releases the iron → it is reduced and leaves the endosome through DMT1 into the cytoplasm → apotransferrin and receptor are recycled to the surface. Balance: about 12–15 mg of iron is eaten daily and only 1–2 mg absorbed; total body iron is 3–4 g, mostly in haemoglobin; roughly 25 mg/day is recycled from senescent red cells by the spleen; and only 0.5–1.5 mg/day is lost, in shed cells and (in women) menstrual blood. There is no regulated route of iron excretion — which is why absorption has to be the control point, and why transfusional overload has nowhere to go.

🩸 Haem Synthesis and Porphobilinogen Past Q 27, 142

Eight steps, starting and finishing in the mitochondrion with the middle steps in the cytosol. Main sites: liver and erythroid marrow.

Step 1 — the rate-limiting step

Succinyl-CoA + glycine → δ-aminolevulinic acid (ALA), by ALA synthase in the mitochondrion, with PLP (vitamin B6) as cofactor. Haem inhibits ALA synthase by feedback, and also represses its synthesis. The hepatic isoform is induced by drugs that consume haem through cytochrome P450 — which is what precipitates a porphyria attack.

Step 2 — porphobilinogen

Two ALA condense to porphobilinogen (PBG), catalysed by ALA dehydratase in the cytosol — a zinc enzyme, and the classic target of lead, which is why lead poisoning raises ALA in urine and causes anaemia with basophilic stippling.

Steps 3–4 — the ring

Four PBG condense (PBG deaminase / hydroxymethylbilane synthase) and are cyclised by uroporphyrinogen III synthase to uroporphyrinogen III. Without the cosynthase the useless type I isomer forms.

Steps 5–8 — to haem

Decarboxylations and oxidations give coproporphyrinogen III → protoporphyrinogen IX → protoporphyrin IX; then ferrochelatase inserts Fe²⁺ in the mitochondrion. Ferrochelatase is the second enzyme inhibited by lead, which is why free erythrocyte protoporphyrin rises.

🧫 Classifying the Porphyrias — One Per Enzyme Past Q

Porphyrias are named and sorted on two axes: by the organ that suffers — hepatic or erythropoietic (liver or bone marrow) — and by the clinical picture — acute (neurovisceral) or cutaneous (photosensitive). Most are autosomal dominant with low penetrance; congenital erythropoietic porphyria is recessive.

The rule that makes the whole table predictable

A block before the ring is closed (steps 1–3) piles up ALA and porphobilinogen — colourless, neurotoxic, not photoactive → abdominal pain and neuropsychiatric signs. A block after the ring is closed (steps 4–8) piles up porphyrinogens, which auto-oxidise to porphyrins — coloured, light-absorbing → photosensitivity. Steps 6 and 7 sit on the boundary and give both.

#	Enzyme blocked	Disease · class	Signs	Laboratory
1	ALA synthase (erythroid form)	X-linked sideroblastic anaemia erythropoietic	Anaemia	↓ red cell count and haemoglobin
2	ALA dehydratase	ALA-dehydratase deficiency porphyria hepatic · acute	Abdominal pain, neuropsychiatric	↑ urinary ALA and coproporphyrin III
3	Uroporphyrinogen I synthase (PBG deaminase)	Acute intermittent porphyria hepatic · acute	Abdominal pain, neuropsychiatric, no skin disease	↑ urinary ALA and PBG
4	Uroporphyrinogen III synthase	Congenital erythropoietic porphyria erythropoietic · cutaneous	Severe photosensitivity, disfigurement — the "werewolf" prototype	↑ uroporphyrin I in urine, faeces and red cells
5	Uroporphyrinogen decarboxylase	Porphyria cutanea tarda — the commonest hepatic · cutaneous	Blistering of sun-exposed skin	↑ urinary uroporphyrin I
6	Coproporphyrinogen oxidase	Hereditary coproporphyria hepatic both	Photosensitivity and abdominal pain, neuropsychiatric	↑ ALA, PBG, faecal coproporphyrin III
7	Protoporphyrinogen oxidase	Variegate porphyria hepatic both	The same combination	↑ ALA, PBG, faecal protoporphyrin IX
8	Ferrochelatase	Erythropoietic protoporphyria erythropoietic · cutaneous	Photosensitivity	↑ red cell and faecal protoporphyrin

Making the diagnosis

Three things together: clinical and family history, physical examination (skin, neurological signs), and laboratory tests — which porphyrin precursor is raised, and in which fluid (urine, faeces or red cells). Note that between attacks the biochemistry can be almost normal.

Treatment as it stands

Symptomatic, with gene-level therapy still a hope. Avoid drugs that induce cytochrome P450 (they consume haem and de-repress ALA synthase), alcohol and fasting. Large amounts of carbohydrate, and intravenous haem (haematin), both repress ALAS1 and cut off production of the harmful precursors. For the photosensitive forms: sunlight avoidance, sunscreens and β-carotene, which quenches free radicals and reduces the skin damage.

💛 Haem Degradation and Bilirubin Topic 14Past Q 123

Red cells live about 120 days and are taken up by macrophages of the reticuloendothelial system — spleen, liver, marrow.

1 · Haem → biliverdin

Haem oxygenase (microsomal, needs O₂ and NADPH) opens the ring, releasing Fe²⁺ (recycled onto transferrin) and carbon monoxide — the only endogenous source of CO in the body. The product is green biliverdin.

2 · Biliverdin → bilirubin

Biliverdin reductase (NADPH) gives yellow-orange bilirubin.

3 · Transport to the liver

Bilirubin is hydrophobic, so it travels bound non-covalently to albumin. This is unconjugated (indirect) bilirubin: not water-soluble, never appears in urine, and — if it exceeds albumin's capacity or is displaced by drugs — able to cross the blood–brain barrier and cause kernicterus in newborns.

4 · Uptake and conjugation

At the hepatocyte membrane the albumin–bilirubin complex docks on a receptor, the bilirubin alone is carried in, and inside it is bound by ligandin (a glutathione S-transferase) — which stops it leaking back out and ferries it to the smooth ER. Note that aspirin and sulfonamides displace bilirubin from albumin, which is how they push a jaundiced newborn toward kernicterus. There, UDP-glucuronosyltransferase (UGT1A1) attaches two glucuronic acids to give bilirubin diglucuronide — conjugated (direct) bilirubin, which is water-soluble and therefore can appear in urine.

5 · Secretion and the gut

Conjugated bilirubin is pumped into bile by MRP2 — an active transport step, and the rate-limiting one of the whole hepatic pathway, which is why it fails first in liver disease. About 85% arrives in the gut as the diglucuronide and is not reabsorbed. In the intestine bacteria hydrolyse and reduce it to urobilinogen (colourless). Most is oxidised to stercobilin and gives faeces its colour; a fraction is reabsorbed, most returning to the liver and a little reaching the kidney to be excreted as urobilin, which colours urine.

🟡 Jaundice — Differentiating the Three Types Topic 14 · Disorder 15Past Q 31, 98Lecturer’s priority

Jaundice is yellowing of skin, sclerae and nail beds from excess bilirubin. The whole differentiation rests on which bilirubin is raised and what the urine and stool show.

Prehepatic (haemolytic)

Cause: excessive red-cell breakdown — the liver is overwhelmed but healthy.
Blood: ↑↑ unconjugated bilirubin; conjugated normal.
Urine: no bilirubin (unconjugated cannot be filtered) but ↑ urobilinogen.
Stool: dark — more stercobilin.
Other: anaemia, ↑ LDH, ↓ haptoglobin, reticulocytosis.

Hepatocellular

Cause: hepatitis, cirrhosis — uptake, conjugation and secretion all impaired.
Blood: both fractions raised.
Urine: bilirubin present; urobilinogen variable, often raised early then falling.
Stool: normal or pale.
Other: ↑↑ ALT and AST, ↓ albumin, prolonged prothrombin time.

Posthepatic (obstructive / cholestatic)

Cause: blocked bile duct — gallstone, tumour.
Blood: ↑↑ conjugated bilirubin.
Urine: dark — bilirubin present; urobilinogen absent or very low, because nothing reaches the gut.
Stool: pale, clay-coloured — no stercobilin. Fat malabsorption and steatorrhoea follow.
Other: ↑↑ ALP and GGT, itching from bile salts.

Neonatal (physiological)

An immature, not-yet-induced UGT1A1, a shorter red-cell lifespan and enterohepatic recycling give a transient unconjugated hyperbilirubinaemia after day 2–3. Phototherapy converts bilirubin into water-soluble isomers that can be excreted without conjugation. The danger is kernicterus. (Toxic hyperbilirubinaemia belongs with these unconjugated causes; the conjugated ones are obstruction, Dubin–Johnson and Rotor, while hepatitis raises both.)

The two inherited conjugation defects Disorders 1 & 2Past Q 17

Gilbert syndrome — a mild reduction of UGT1A1 to roughly 30% of normal. Benign, common, and produces a mild unconjugated hyperbilirubinaemia that appears on fasting, illness, alcohol or stress. Liver enzymes are normal, there is no haemolysis and no treatment is needed.
Crigler–Najjar syndrome — type I: UGT1A1 completely absent; extreme unconjugated hyperbilirubinaemia from birth, kernicterus, no response to phenobarbital (there is no enzyme to induce); phototherapy buys time, liver transplant is the only cure. Type II (Arias): partial deficiency, milder, and it does respond to phenobarbital. The phenobarbital response is the classic way of ranking the three: Gilbert (low dose works) → Crigler–Najjar II (high dose works) → Crigler–Najjar I (no response).

The two words the question uses Past Q

Retention hyperbilirubinaemia — too much bilirubin is produced (or not conjugated), so the unconjugated fraction rises. It cannot be filtered, so the urine holds no bilirubin: acholuric jaundice, and the only setting in which kernicterus can occur. Regurgitation hyperbilirubinaemia — conjugated bilirubin already made in the liver refluxes into the blood because bile cannot get out; it is water-soluble, so it appears in urine: choluric jaundice, dark urine.

The two benign conjugated defects

Dubin–Johnson syndrome — the canalicular export pump MRP2 is defective, so conjugated bilirubin cannot be pumped into bile; a dark, almost black liver from pigment deposits, but a normal lifespan. Rotor syndrome — a defect of hepatic storage/uptake of conjugated bilirubin, clinically similar but with no pigment in the liver. Both are benign and need no treatment; both are causes of a raised conjugated bilirubin with normal liver enzymes.

Numbers worth quoting

Hyperbilirubinaemia begins above about 1 mg/dL; at 2–2.5 mg/dL bilirubin diffuses into tissues and jaundice becomes visible. Total bilirubin ≈ 0.2–1.0 mg/dL, mostly unconjugated — direct (conjugated) 0.1–0.4 mg/dL, indirect (unconjugated) 0.2–0.7 mg/dL; urinary urobilinogen 0–4 mg/24 h and faecal urobilinogen 40–280 mg/24 h — increased in haemolysis, absent in complete obstruction.

🧪 Hepatic Phase II Reactions and the Uronic Acid Pathway Past Q 94, 112, 166

Phase I versus phase II

Phase I — cytochrome P450 monooxygenases in the smooth ER introduce or expose a polar group (hydroxylation, oxidation, reduction, hydrolysis), using O₂ and NADPH. Phase II — conjugation of that group with a polar partner, which greatly increases water solubility for excretion in urine or bile.

The conjugations

Glucuronidation (UDP-glucuronosyltransferase; donor UDP-glucuronic acid) — bilirubin, steroids, many drugs. Sulfation (donor PAPS). Glutathione conjugation (glutathione S-transferase) — the route that detoxifies the reactive paracetamol metabolite NAPQI. Acetylation, methylation (SAM), amino acid conjugation with glycine or glutamine.

The uronic acid pathway — what it is for

An alternative oxidation of glucose that starts from UDP-glucose and, by a two-step oxidation catalysed by UDP-glucose dehydrogenase, produces UDP-glucuronic acid. Its major functions: supplying glucuronate for conjugation and detoxification, for bilirubin excretion, and as a building block of glycosaminoglycans and proteoglycans. It does not generate ATP.

The human limitation

Glucuronate is reduced to L-gulonate, the direct precursor of ascorbate, and in most mammals the pathway then continues to ascorbic acid. Humans, other primates and guinea pigs lack L-gulonolactone oxidase, so we cannot make vitamin C and must eat it. The pathway's other end product is xylulose; a block in its handling causes the harmless essential pentosuria.

Krebs Cycle & Respiratory Chain

Resit topics 15–18 · the eight reactions, the amphibolic role, anaplerosis, the pyruvate dehydrogenase bridge, inhibitors and uncouplers

🔄 The Eight Reactions, Their Enzymes and Cofactors Topic 15Lecturer’s priority

Location: the mitochondrial matrix — with the single exception of succinate dehydrogenase, which is embedded in the inner membrane as Complex II. One turn oxidises one acetyl-CoA to 2 CO₂, and regenerates oxaloacetate.

1 · Citrate synthase

Oxaloacetate + acetyl-CoA + H₂O → citrate. An aldol condensation; irreversible and the first committed step. Inhibited by ATP, NADH, succinyl-CoA and citrate.

2 · Aconitase

Citrate ⇄ isocitrate (via cis-aconitate) — an isomerisation. Contains an Fe–S centre; inhibited by fluoroacetate.

3 · Isocitrate dehydrogenase

Isocitrate → α-ketoglutarate + CO₂ + NADH. Oxidative decarboxylation, irreversible, and the rate-limiting step of the cycle. Activated by ADP and Ca²⁺; inhibited by ATP and NADH.

4 · α-Ketoglutarate dehydrogenase complex

α-KG → succinyl-CoA + CO₂ + NADH. Irreversible. Uses the same five cofactors as pyruvate dehydrogenase (listed on the link-reaction card). Inhibited by its products, by ATP and by NADH; activated by Ca²⁺.

5 · Succinyl-CoA synthetase (succinate thiokinase)

Succinyl-CoA → succinate + GTP. The cycle's only substrate-level phosphorylation. Reversible.

6 · Succinate dehydrogenase

Succinate → fumarate + FADH₂. The only membrane-bound enzyme of the cycle = Complex II; feeds electrons directly to coenzyme Q. Competitively inhibited by malonate.

7 · Fumarase

Fumarate + H₂O → malate — a hydration. Reversible.

8 · Malate dehydrogenase

Malate → oxaloacetate + NADH. Reversible; pulled forward because oxaloacetate is immediately consumed by citrate synthase.

Energy yield per turn

3 NADH + 1 FADH₂ + 1 GTP, and 2 CO₂ released. Through the respiratory chain that is 3 × 2.5 + 1 × 1.5 + 1 = 10 ATP per acetyl-CoA (older textbooks, and these seminars, use 3 and 2 to give 12). The cycle itself uses no oxygen — but it stops without it, because NAD⁺ and FAD can only be regenerated by the chain.

Vitamins in the cycle Past Q 177

B1 thiamine (TPP), B2 riboflavin (FAD), B3 niacin (NAD⁺), B5 pantothenate (CoA) — plus lipoic acid, which is not a vitamin. All four are needed by the α-ketoglutarate dehydrogenase complex.

🔀 The Amphibolic Role and Anaplerotic Reactions Topic 15Past Q 3, 16, 29

Amphibolic means the cycle is both catabolic and anabolic: it oxidises acetyl-CoA to CO₂ for energy, and at the same time it is the source of carbon skeletons for biosynthesis. That is why intermediates are constantly withdrawn — and why they must be replaced.

Withdrawals (cataplerosis)

Citrate → exported to the cytosol for fatty acid and cholesterol synthesis. α-Ketoglutarate → glutamate, glutamine, proline, arginine. Succinyl-CoA → haem. Oxaloacetate → aspartate, pyrimidines, and gluconeogenesis via PEP.

Anaplerosis — the definition

"Filling-up" reactions that replenish Krebs intermediates so the cycle can keep turning. Without them, withdrawing oxaloacetate would stall citrate synthase.

The main anaplerotic reaction

Pyruvate carboxylase: pyruvate + CO₂ + ATP → oxaloacetate. Mitochondrial, requires biotin, and is allosterically activated by acetyl-CoA — the perfect signal, since accumulating acetyl-CoA means more oxaloacetate is needed.

The other anaplerotic routes

Transamination of glutamate → α-ketoglutarate and of aspartate → oxaloacetate (reversible, so they work both ways). Glutamate dehydrogenase → α-ketoglutarate. Propionyl-CoA from odd-chain fatty acids and from Met/Val/Ile/Thr → methylmalonyl-CoA → succinyl-CoA (biotin then B12). Malic enzyme in reverse → malate.

🌉 The Link Reaction — Pyruvate Dehydrogenase, the Bridge Topic 20fPast Q 54, 138Lecturer’s priority

Structure: a multienzyme complex of three enzymes — E1 pyruvate decarboxylase, E2 dihydrolipoyl transacetylase, E3 dihydrolipoyl dehydrogenase — using five cofactors: TPP (B1), lipoic acid, CoA (B5), FAD (B2), NAD⁺ (B3). It converts pyruvate to acetyl-CoA + CO₂ + NADH, irreversibly, in the mitochondrial matrix — the one-way bridge from carbohydrate to the Krebs cycle and to fat.
Regulation is double. Allosteric/product: inhibited by its products acetyl-CoA and NADH, activated by pyruvate, CoA, NAD⁺, ADP and Ca²⁺. Covalent: PDH kinase phosphorylates and inactivates the complex (kinase itself stimulated by ATP, acetyl-CoA and NADH — i.e. by plenty), while PDH phosphatase dephosphorylates and activates it (stimulated by Ca²⁺ in contracting muscle, and by insulin in adipose tissue). Deficiency, or thiamine deficiency, causes lactic acidosis and neurological damage; arsenite poisons it by binding the –SH groups of lipoic acid. The five cofactors work in a fixed relay, and nothing leaves the complex until the end — which is why there are no side products and the rate is so high: TPP decarboxylates pyruvate to hydroxyethyl-TPP; that two-carbon unit is transferred and oxidised onto lipoamide to give acetyl-lipoamide; E2 hands the acetyl group to CoA, leaving dihydrolipoamide; E3 re-oxidises it through FAD and passes the electrons to NAD⁺, which delivers them to the respiratory chain. Dichloroacetate inhibits PDH kinase and therefore forces the complex on — the reason it has been tried in lactic acidosis.

⚡ The Respiratory Chain Topic 16Past Q 160

Four complexes plus two mobile carriers, in the inner mitochondrial membrane, arranged in order of increasing standard reduction potential — electrons flow downhill from NADH (E°′ = −0.32 V) to oxygen (+0.82 V), and the energy released pumps protons out.

Complex I — NADH dehydrogenase

NADH → FMN → Fe–S centres → coenzyme Q. Pumps 4 H⁺.

Complex II — succinate dehydrogenase

Succinate → FAD → Fe–S → coenzyme Q. Pumps no protons, which is exactly why FADH₂ yields less ATP than NADH. Glycerol-3-phosphate dehydrogenase and acyl-CoA dehydrogenase (via ETF) also feed in at CoQ.

Complex III — cytochrome bc₁

CoQH₂ → cytochromes b and c₁, Fe–S → cytochrome c. Pumps 4 H⁺.

Complex IV — cytochrome c oxidase

Cytochrome c → cytochromes a and a₃ with two copper centres → O₂ + 4 H⁺ + 4 e⁻ → 2 H₂O. Pumps 2 H⁺. This is the only step that consumes oxygen.

The mobile carriers

Coenzyme Q (ubiquinone) — small, lipid-soluble, moves within the membrane; the collection point for all electron sources. Cytochrome c — a small water-soluble haem protein on the outer face of the inner membrane. Its release into the cytosol triggers apoptosis.

Complex V — ATP synthase Past Q 4

Not part of the electron chain. F₀ is the membrane proton channel; F₁ is the matrix knob (α₃β₃γδε) that carries the catalytic sites. Protons flowing back through F₀ rotate the stalk and drive conformational changes in F₁ that release ATP.

🔌 Substrate-Level vs Oxidative Phosphorylation, Inhibitors and Uncouplers Topics 17–18Past Q 4

Substrate-level phosphorylation

ATP (or GTP) made by direct transfer of a phosphate group from a high-energy substrate to ADP, by a soluble enzyme, without oxygen and without the membrane. The three examples: phosphoglycerate kinase and pyruvate kinase in glycolysis, and succinyl-CoA synthetase in the Krebs cycle. It is the only ATP source in a red cell or in anoxic tissue.

Oxidative phosphorylation

ATP made by ATP synthase, driven by the proton-motive force that electron transport builds across the inner membrane. Requires oxygen, an intact inner membrane and a coupled chain. It supplies the great majority of cellular ATP.

Chemiosmotic theory (Mitchell)

The energy of electron transport is not stored in a chemical intermediate but in an electrochemical proton gradient — a pH difference plus a membrane potential — across the inner membrane. ATP synthesis is powered by the return flow of those protons. This is why the inner membrane must be impermeable to protons.

Uncouplers Topic 18

Lipid-soluble proton carriers that short-circuit the gradient: protons re-enter the matrix without passing through ATP synthase. Consequence: electron transport and oxygen consumption speed up, ATP synthesis falls, and the energy is released as heat. Chemical: 2,4-dinitrophenol, aspirin in overdose. Physiological: thermogenin (UCP-1) in brown adipose tissue — non-shivering thermogenesis in the newborn.

Inhibitors, by site Topic 18

Complex I: rotenone, amytal (barbiturates), piericidin. Complex II: malonate (competitive with succinate). Complex III: antimycin A. Complex IV: cyanide, carbon monoxide, azide, hydrogen sulfide — the lethal ones, because they stop the chain at its final step and oxygen can no longer be used. ATP synthase (Complex V): oligomycin. ADP/ATP translocase: atractyloside.
How to tell an inhibitor from an uncoupler: a chain inhibitor stops oxygen consumption and ATP synthesis; an uncoupler increases oxygen consumption while ATP synthesis stops. Oligomycin blocks ATP synthesis and, because the gradient cannot discharge, also stops respiration — unless an uncoupler is added, which restarts it.

🔋 The Energy Ledger — Macroergic Compounds and the Three Levels Past Q

Two list questions live here: name the macroergic compounds and their role, and describe the levels of energetic metabolism.

🔹 Macroergic “high-energy” compounds — hydrolysis releases more than about 25 kJ/mol

Phosphoanhydrides
ATP, GTP, UTP, CTP — and ADP. Two or three phosphates in a row, repelling each other.

Acyl phosphates
1,3-bisphosphoglycerate, carbamoyl phosphate, acetyl phosphate.

Enol phosphate
Phosphoenolpyruvate — the most energetic of all, which is why pyruvate kinase is irreversible.

Guanidinium phosphate
Creatine phosphate — the instant ATP buffer of muscle and brain.

Thioesters
Acetyl-CoA, succinyl-CoA, acyl-CoA — the energy is in a C–S bond, not in a phosphate.

Sulfonium and nucleotide sugars
S-adenosylmethionine (methyl donor), UDP-glucose (sugar donor), PAPS (sulfate donor).

Why ATP is a currency and not a store

ATP sits in the middle of the scale. PEP, creatine phosphate and 1,3-BPG are above it, so they can phosphorylate ADP; glucose-6-phosphate and glycerol-3-phosphate are below it, so ATP can phosphorylate them. A compound at the top could only give, one at the bottom could only take. And because ΔG values simply add, coupling an endergonic reaction to ATP hydrolysis — through a shared enzyme or a shared intermediate — makes the sum negative and the whole thing go.

🔹 The three levels of energetic metabolism

Level 1

Break down to the common building blocks. Lipids → fatty acids + glycerol · sugars → glucose · proteins → amino acids. Essentially no useful energy is captured here; the point is to reach three currencies the cell can handle.

Level 2

Converge on acetyl-CoA. β-oxidation, glycolysis followed by pyruvate dehydrogenase, and amino acid catabolism all arrive at the same two-carbon unit — with ketone bodies as its transportable form. A small amount of ATP and some reduced coenzyme appear at this stage.

Level 3

The final common pathway. Krebs cycle → NADH and FADH₂ → respiratory chain → oxidative phosphorylation. Almost all of the ATP is made here, whatever the fuel was — which is why one cycle and one chain serve every foodstuff.

🚌 Getting Cytosolic NADH into the Mitochondrion Past Q 133, 136, 50

The inner mitochondrial membrane is impermeable to NADH. The reducing equivalents from glycolysis must therefore be carried across by one of two shuttles — and which one a tissue uses determines the ATP yield of glucose.

Glycerol-3-phosphate shuttle

Cytosolic NADH reduces DHAP to glycerol-3-phosphate (cytosolic glycerol-3-phosphate dehydrogenase); the membrane-bound isoenzyme on the outer face of the inner membrane re-oxidises it, passing the electrons to FAD → coenzyme Q. Bypasses Complex I, so each cytosolic NADH yields only ~1.5 (older: 2) ATP. Used by skeletal muscle and brain; irreversible, but fast.

Malate–aspartate shuttle

Oxaloacetate is reduced to malate in the cytosol, malate enters the matrix and is re-oxidised there, regenerating NADH inside; the oxaloacetate returns as aspartate. Electrons enter at Complex I, so each cytosolic NADH yields ~2.5 (older: 3) ATP. Used by liver, kidney and heart; fully reversible.

Dihydroxyacetone phosphate — the three roles Past Q 50

a · In glycolysis — one of the two triose phosphates from aldolase; isomerised by triose-phosphate isomerase to glyceraldehyde-3-phosphate so that both halves of glucose continue. b · In TAG synthesis — reduced to glycerol-3-phosphate, the backbone onto which fatty acids are esterified. c · In the glycerol-3-phosphate shuttle — the cytosolic acceptor of the electrons of NADH. One enzyme, glycerol-3-phosphate dehydrogenase, does b and c: its two isoenzymes run the same reaction in opposite directions, which puts it at the junction of glycolysis, TAG synthesis and mitochondrial re-oxidation of NADH. Past Q 169

Carbohydrates — Digestion, Glycogen, Glycolysis, Gluconeogenesis

Resit topics 19–20 · localisation, significance, substrates, products, regulation

🍞 Digestion and Absorption of Sugars Topic 19Past Q 45

Mouth and stomach

Salivary α-amylase (ptyalin) hydrolyses internal α(1→4) bonds of starch and glycogen. It cannot touch α(1→6) branch points, and it cannot touch β(1→4) bonds — which is why humans do not digest cellulose. It is inactivated by gastric acid, so its contribution is short.

Small intestine — lumen

Pancreatic α-amylase resumes once bicarbonate has neutralised the chyme. Products: maltose, maltotriose and α-limit dextrins (the branched fragments containing the α(1→6) bonds).

Brush-border disaccharidases

Membrane enzymes of the enterocytes of the duodenum and upper jejunum. Isomaltase — α(1→6) of isomaltose. Maltase — maltose and maltotriose, α(1→4). Lactase (β-galactosidase) — lactose, β(1→4) → galactose + glucose. Sucrase — sucrose, α(1→2) → glucose + fructose. Trehalase — trehalose (mushrooms), α(1→1).

Absorption

Only monosaccharides are absorbed. Glucose and galactose — SGLT-1, Na⁺-dependent secondary active transport (symport), which can move sugar against its gradient using the Na⁺ gradient made by Na⁺/K⁺-ATPase. Fructose — GLUT-5, facilitated diffusion, no ATP, no Na⁺. All three then leave the enterocyte into the portal blood via GLUT-2.

When digestion fails

Undigested sugars are osmotically active and pull water into the lumen, and colonic bacteria ferment them to lactic and acetic acid with CO₂ and H₂ — giving osmotic diarrhoea, cramps and flatulence. Lactose intolerance (adult-type hypolactasia) is the common example, affecting most of the world's adults; congenital sucrase–isomaltase deficiency is the rare one. Diagnosis by oral tolerance testing and the hydrogen breath test.

🍭 The Sugar Family Album — Which Ones Matter and Why Topic 19Past Q

A monosaccharide cannot be hydrolysed into anything simpler; the exam asks for the physiologically important ones grouped by the number of carbons.

3️⃣Trioses — glyceraldehyde (glycerose) · dihydroxyacetone

The two triose phosphates of glycolysis, and the backbone of every triacylglycerol.

4️⃣Tetroses — erythrose · erythrulose

Erythrose-4-phosphate is the pentose phosphate pathway's link to aromatic compounds.

5️⃣Pentoses — ribose · ribulose · xylose · xylulose · arabinose

Ribose builds RNA and every nucleotide coenzyme; ribulose and xylulose are pentose phosphate pathway intermediates; xylulose is the end product of the uronic acid pathway (essential pentosuria).

6️⃣Hexoses — glucose · galactose · fructose · mannose

The only four that reach the portal blood in quantity. Mannose is a constituent of glycoproteins.

7️⃣Heptose — sedoheptulose

Sedoheptulose-7-phosphate, the transketolase product in the non-oxidative phase of the pentose phosphate pathway.

🔹 The disaccharides — composition and linkage

Disaccharide	Made of	Bond	Note
Maltose	Glucose + glucose	α(1→4)	From starch digestion; reducing
Isomaltose	Glucose + glucose	α(1→6)	The branch point; needs isomaltase
Lactose	Galactose + glucose	β(1→4)	Milk sugar; reducing
Sucrose	Glucose + fructose	α1→2β	Both anomeric carbons used, so non-reducing
Trehalose	Glucose + glucose	α1→1α	Insects, yeast, mushrooms; non-reducing
Lactulose	Galactose + fructose	β(1→4)	Synthetic, not digested by human enzymes — used as an osmotic laxative and in hepatic encephalopathy, where colonic fermentation traps ammonia as NH₄⁺

Oligosaccharides — building and breaking

Three to ten monosaccharides joined by glycosidic bonds, which are formed by glycosyltransferases using activated nucleotide sugars (UDP-glucose, UDP-galactose, GDP-mannose, CMP-sialic acid). They are broken by glycosidases: salivary and pancreatic α-amylase on starch, then the brush-border disaccharidases and oligosaccharidases that release the free monosaccharides which are actually absorbed.

Amino sugars — where the –OH becomes –NH₂ Past Q

D-glucosamine, D-galactosamine and D-mannosamine, usually N-acetylated. Their nitrogen comes from the amide of glutamine. They are the repeating units of the glycosaminoglycans — hyaluronic acid is glucuronic acid + N-acetylglucosamine — and of proteoglycans, glycoproteins and the ABO blood-group antigens; several antibiotics (streptomycin, erythromycin) also contain amino sugars.

🚗 GLUT 1–5 and SGLT Topic 19

GLUT-1

Red blood cells, brain and the blood–brain barrier, choroid plexus, kidney, colon, placenta. Basal, constant uptake; low Km, so it works at any blood glucose.

GLUT-2

Liver, pancreatic β cells, small intestine, kidney. High Km, high capacity, bidirectional — it lets the liver take glucose in when blood levels are high and release it when they are low, and it lets the β cell sense glucose concentration.

GLUT-3

Neurons, brain, placenta, kidney. The lowest Km of all, guaranteeing the brain gets glucose even during hypoglycaemia.

GLUT-4

Skeletal muscle, cardiac muscle, adipose tissue. The only insulin-dependent transporter: insulin recruits it from intracellular vesicles to the membrane. Exercise recruits it independently of insulin — the reason exercise lowers glucose in diabetes.

GLUT-5

Small intestine (apical) and testes. The fructose transporter; a poor glucose transporter.

SGLT-1 and SGLT-2

Secondary active, Na⁺-coupled symporters — the only ones that move glucose against its concentration gradient. SGLT-1: intestinal absorption and the late proximal tubule. SGLT-2: the early proximal tubule, where most filtered glucose is reabsorbed — the target of the gliflozin drugs.

🍬 Glycolysis Topic 20Past Q 90Lecturer’s priority

Localisation & significance

Cytosol of every cell. It is the only ATP source for cells with no mitochondria (red cells, lens, cornea) or with no oxygen, it provides pyruvate for the Krebs cycle, and it supplies intermediates for other pathways (DHAP → glycerol-3-phosphate; 1,3-BPG → 2,3-BPG in red cells).

Substrates and products

Glucose + 2 ADP + 2 Pi + 2 NAD⁺ → 2 pyruvate + 2 ATP + 2 NADH (aerobic), or → 2 lactate + 2 ATP with no net NADH (anaerobic). Energy invested: 2 ATP; produced: 4 ATP; net 2 ATP.

The three irreversible enzymes

1 · Hexokinase / glucokinase — glucose → glucose-6-phosphate, first ATP spent. 2 · PFK-1 — fructose-6-P → fructose-1,6-bisP, second ATP spent; the rate-limiting step and the main control point. 3 · Pyruvate kinase — PEP → pyruvate, substrate-level ATP.

Hexokinase vs glucokinase Past Q 162

Hexokinase — all tissues, low Km (high affinity), low Vmax, works at low glucose, inhibited by its product glucose-6-phosphate, accepts several hexoses. Glucokinase (hexokinase IV) — liver and pancreatic β cells, high Km, high Vmax, so it only works after a carbohydrate meal, is not inhibited by G6P, is induced by insulin, and is regulated by GKRP in the nucleus. Mutations cause MODY 2.

Regulation of PFK-1

Inhibited by ATP and citrate (plenty of energy and building blocks). Activated by AMP and above all by fructose-2,6-bisphosphate, the most potent activator, which overrides ATP inhibition. F-2,6-BP is made and destroyed by the bifunctional PFK-2/FBPase-2: glucagon → PKA phosphorylates it → kinase off, phosphatase on → F-2,6-BP falls → glycolysis down, gluconeogenesis up; insulin does the reverse.

Regulation of pyruvate kinase

Activated by fructose-1,6-bisphosphate — feed-forward activation. Inhibited by ATP, acetyl-CoA and alanine. In liver it is also phosphorylated and inactivated by glucagon. PK deficiency is the second commonest cause of hereditary non-spherocytic haemolytic anaemia: the red cell cannot make enough ATP, loses flexibility and is destroyed.

Phosphorylation of hexoses — why Past Q 139

Phosphorylation makes the sugar negatively charged, so it cannot leave the cell (no transporter carries phosphorylated sugars); it keeps the intracellular free-glucose concentration low so uptake continues; and it activates the sugar for the next step. Glucose → G6P (hexokinase/glucokinase), fructose → F1P (fructokinase, liver) or F6P (hexokinase), galactose → Gal-1-P (galactokinase), mannose → mannose-6-P.

🔃 Gluconeogenesis and the Cori Cycle Topic 20Past Q 35, 91, 151Lecturer’s priority

Localisation & significance

Liver (~90%) and kidney cortex (~10%); cytosol plus mitochondria. It maintains blood glucose for the brain and red cells once liver glycogen runs out (after roughly 12–24 hours of fasting). Muscle cannot do it — see bypass 4.

Substrates

Lactate (from red cells and exercising muscle), glucogenic amino acids — above all alanine and glutamine — and glycerol from the hydrolysis of triacylglycerol. Fatty acids cannot be converted into glucose: acetyl-CoA cannot give a net gain of oxaloacetate. Propionyl-CoA from odd-chain fatty acids is the only exception.

The four bypass enzymes

1 · Pyruvate carboxylase (mitochondrion, biotin, activated by acetyl-CoA) → oxaloacetate. 2 · PEP carboxykinase (PEPCK) → PEP, using GTP; induced by glucagon and cortisol. 3 · Fructose-1,6-bisphosphatase — the rate-limiting step; inhibited by AMP and fructose-2,6-bisphosphate. 4 · Glucose-6-phosphatase — in the lumen of the endoplasmic reticulum of liver and kidney only.

Cost and regulation Past Q 151

6 high-energy phosphates per glucose (4 ATP + 2 GTP) plus 2 NADH. Hormonal: glucagon and cortisol stimulate, insulin suppresses. Allosteric: acetyl-CoA activates pyruvate carboxylase, AMP inhibits FBPase-1. Substrate supply and the ATP/NADH from β-oxidation are what make it possible during fasting.

Cori cycle and glucose–alanine cycle Past Q 35, 37

Cori cycle: muscle (or red cell) glycolysis → lactate → blood → liver → gluconeogenesis → glucose → back to the muscle. It moves the metabolic burden of anaerobic work to the liver: the muscle gains 2 ATP, the liver spends 6. Glucose–alanine cycle: the same route, but the carbon travels as alanine and carries nitrogen with it, which the liver feeds into the urea cycle. Cori = carbon only. Alanine = carbon plus nitrogen.

🍡 Glycogenesis and Glycogenolysis Topic 20Past Q 84, 87, 88Lecturer’s priority

Glycogenesis — cytosol

G6P → G1P (phosphoglucomutase) → UDP-glucose (UDP-glucose pyrophosphorylase, using UTP). Glycogen synthase — the rate-limiting enzyme — adds glucose in α(1→4) linkage; it cannot start a chain from free glucose, so it needs the primer protein glycogenin, which glucosylates itself. Branching enzyme (amylo-α(1→4)→α(1→6)-transglycosylase) moves a block of 6–8 residues to create an α(1→6) branch.

Glycogenolysis — cytosol

Glycogen phosphorylase — the rate-limiting enzyme — cleaves α(1→4) bonds by phosphorolysis (inorganic phosphate, not hydrolysis), giving glucose-1-phosphate and requiring pyridoxal phosphate (B6). It stops four residues from a branch. The bifunctional debranching enzyme then transfers three residues (transferase activity) and hydrolyses the last α(1→6) bond (α-1,6-glucosidase activity), releasing free glucose — about 8–10% of the total.

Liver versus muscle — the key difference Topic 20e

Liver has glucose-6-phosphatase, so G6P → free glucose → blood: liver glycogen serves blood glucose. It responds to glucagon and adrenaline. Muscle has no glucose-6-phosphatase, so G6P enters glycolysis and serves the muscle itself; muscle glycogen can never raise blood glucose. Muscle has no glucagon receptors — it responds to adrenaline, Ca²⁺ and AMP.

Regulation

Hormonal (covalent): glucagon/adrenaline → cAMP → PKA → phosphorylase kinase → phosphorylase a (active) and glycogen synthase b (inactive) — one signal, opposite effects. Insulin does the reverse via protein phosphatase-1. Allosteric: in muscle, AMP activates phosphorylase b and ATP and G6P inhibit it; in liver, free glucose inhibits phosphorylase a. Ca²⁺–calmodulin activates phosphorylase kinase directly during contraction, without any phosphorylation.

Glycogen storage diseases Past Q 55

Type I · von Gierke — glucose-6-phosphatase: severe fasting hypoglycaemia, hepatomegaly, lactic acidosis, hyperuricaemia, hyperlipidaemia. Type II · Pompe — lysosomal acid α-glucosidase: the only lysosomal one; cardiomegaly and hypotonia, normal blood glucose. Type III · Cori — debranching enzyme: milder hypoglycaemia, short outer branches. Type IV · Andersen — branching enzyme: long unbranched chains, liver cirrhosis. Type V · McArdle — muscle phosphorylase: exercise cramps, myoglobinuria, no rise in lactate on exercise, second-wind phenomenon, no hypoglycaemia.

🍏 Fructose and Galactose Topic 20Past Q 122, 128, 182

Fructose — liver route

Fructokinase → fructose-1-phosphate → aldolase B → DHAP + glyceraldehyde → (triokinase) glyceraldehyde-3-phosphate. It enters glycolysis below PFK-1, so it bypasses the main regulatory step — which is why a large fructose load is metabolised rapidly and unregulated, driving fatty acid synthesis, hypertriacylglycerolaemia and a worse lipid profile. Fructokinase is insulin-independent.

Fructose disorders Disorders 4 & 5

Essential fructosuria — fructokinase deficiency; benign, fructose simply appears in blood and urine (a reducing substance without glucose). Hereditary fructose intolerance — aldolase B deficiency; fructose-1-phosphate accumulates and traps inorganic phosphate, so ATP falls and both glycogenolysis and gluconeogenesis are blocked → severe hypoglycaemia, vomiting, jaundice, hepatomegaly, renal dysfunction after fructose or sucrose. Children develop an aversion to sweets. Treatment: remove fructose, sucrose and sorbitol from the diet.

Galactose

Galactokinase → galactose-1-phosphate → GALT (galactose-1-phosphate uridyltransferase), using UDP-glucose, → UDP-galactose + glucose-1-phosphate → UDP-hexose 4-epimerase interconverts UDP-galactose and UDP-glucose.

Why galactose is not essential Past Q 182

Because the 4-epimerase works in both directions: when no galactose is eaten, the body makes UDP-galactose from UDP-glucose. So galactose needed for lactose, glycoproteins, glycolipids and glycosaminoglycans is always available from glucose.

Galactosaemia

Galactokinase deficiency — galactose accumulates and is reduced by aldose reductase to galactitol, which draws water into the lens: cataracts, otherwise mild. Classic galactosaemia (GALT deficiency) — galactose-1-phosphate accumulates and is toxic: vomiting, jaundice, hepatomegaly and liver failure, cataracts, intellectual disability, and E. coli sepsis. Treatment: remove galactose and lactose from the diet.

Pentose Phosphate Pathway, NADPH & Glutathione

Resit topics 20 and 21 · past questions 30, 130, 137, 180, 181, 185

🔋 The HMP Shunt — What It Is For Topic 20Past Q 137

Cytosol. An alternative route for glucose-6-phosphate that produces no ATP. Its two products are what matter: NADPH and ribose-5-phosphate.

Oxidative phase — irreversible

G6P → 6-phosphogluconolactone → 6-phosphogluconate → ribulose-5-phosphate + CO₂, producing 2 NADPH. The first enzyme, glucose-6-phosphate dehydrogenase (G6PD), is the rate-limiting step; it is inhibited by NADPH (so the ratio NADPH/NADP⁺ controls the flux) and induced by insulin in the fed state.

Non-oxidative phase — reversible

Transketolase (moves two carbons; needs TPP, vitamin B1, and Mg²⁺) and transaldolase (moves three carbons; no cofactor) interconvert sugars of 3–7 carbons, linking the pathway back to fructose-6-phosphate and glyceraldehyde-3-phosphate of glycolysis. Because it is reversible, a cell that needs only ribose can make it from glycolytic intermediates without running the oxidative phase.

Why it matters in adipose tissue Past Q 181, 185

Adipose tissue and liver are the main sites of fatty acid synthesis, and lipogenesis is reductive: it consumes 14 NADPH per palmitate. The pentose phosphate pathway is the principal supplier — with the malic enzyme providing the remainder.

Why it matters in red cells Past Q 180, 185

The red cell is a bag of oxygen and iron and is under constant oxidative attack, yet it has no mitochondria. The HMP shunt is its only source of NADPH, and NADPH is the only way to keep glutathione reduced. It also produces no ribose need — it is purely about antioxidant defence.

NAD⁺/NADH versus NADP⁺/NADPH Past Q 30

NAD⁺/NADH — the coenzyme of catabolic dehydrogenases; kept oxidised (a cytosolic ratio of about 1000:1 in favour of NAD⁺) so it is always ready to accept electrons; its NADH is spent in the respiratory chain to make ATP. NADP⁺/NADPH — the coenzyme of reductive biosynthesis and of monooxygenases and reductases; kept reduced (about 10:1 in favour of NADPH) so it is always ready to donate electrons; it is not used to make ATP. The only structural difference is a phosphate on the 2′-OH of the adenosine ribose — and that is what lets enzymes tell the two pools apart.

🎯 The Uses of NADPH and Its Generators Past Q 130

Where NADPH comes from

1 · The oxidative phase of the pentose phosphate pathway — G6PD and 6-phosphogluconate dehydrogenase (the major source). 2 · Malic enzyme — malate → pyruvate + CO₂ + NADPH. 3 · Cytosolic isocitrate dehydrogenase.

Reductive biosynthesis

Fatty acids, cholesterol, steroid hormones, bile acids, and amino acids via glutamate dehydrogenase. Also the synthesis and activation of vitamin D.

Antioxidant defence

Regeneration of reduced glutathione — the defence of every cell against peroxides, and the only one a red cell has. The cycle itself is on the glutathione card below.

Detoxification and killing

Cytochrome P450 monooxygenases of the liver — drugs, xenobiotics, steroid and vitamin D hydroxylations. NADPH oxidase of phagocytes — the respiratory burst that makes superoxide to kill bacteria; its deficiency is chronic granulomatous disease. NO synthase — nitric oxide.

🛡️ The Role of Glutathione Topic 21

Glutathione is the tripeptide γ-glutamyl-cysteinyl-glycine. Two features define it: the unusual γ-peptide bond, which protects it from ordinary peptidases, and the –SH group of its cysteine, which does all the work. It cycles between reduced GSH and the disulphide-linked dimer GSSG.

1 · Antioxidant

Glutathione peroxidase — a selenium-containing enzyme — uses 2 GSH to reduce H₂O₂ to water (or a lipid peroxide to an alcohol), producing GSSG. Glutathione reductase then restores GSH using NADPH from the pentose phosphate pathway. This is the pairing that G6PD deficiency destroys.

2 · Detoxification

As a phase II conjugating agent through glutathione S-transferase — the route that neutralises the reactive paracetamol metabolite NAPQI; when glutathione is exhausted, liver necrosis follows (treated with N-acetylcysteine, which restores it).

3 · Maintaining protein and haem

It keeps protein –SH groups reduced and keeps haemoglobin iron in the Fe²⁺ state. When it fails, haemoglobin oxidises and precipitates as Heinz bodies, and the membrane stiffens.

4 · Transport and synthesis

Amino acid transport across membranes via the γ-glutamyl cycle (the enzyme GGT belongs to it). It is also a reservoir of cysteine and a cofactor for some enzymes.

G6PD deficiency (favism) Disorder 6

X-linked, and the commonest enzyme deficiency in the world — highest in the Middle East, tropical Africa, Asia and the Mediterranean, because carriers are protected against Plasmodium falciparum. Without G6PD, the red cell cannot make NADPH, so glutathione cannot be kept reduced. Oxidative stress then produces Heinz bodies, rigid cells and acute haemolytic anaemia with jaundice, sometimes with splenomegaly. Triggers: fava beans, infections, and oxidant drugs — primaquine, sulfonamides such as sulfamethoxazole, chloramphenicol, nitrofurantoin. Between crises the patient is well; management is avoidance of triggers.

Lipid Digestion, Transport & Lipoproteins

Resit topics 22 and 26 · bile, essential fatty acids, apolipoproteins, LCAT

🧈 Digestion and Absorption of Lipids Topic 22Past Q 52

Mouth and stomach

Lingual and gastric lipase — acid lipases with an optimum around pH 4–6 that act on short- and medium-chain triacylglycerols. They matter most in the newborn, for milk fat.

Emulsification — the role of bile Topic 22b

Bile salts, made in the liver and stored in the gallbladder, are amphipathic derivatives of cholesterol. With the mechanical mixing of peristalsis they break the fat into fine droplets, hugely increasing the surface area available to the lipases. Bile is essential for absorbing the fat-soluble vitamins A, D, E and K as well.

Pancreatic enzymes

Pancreatic lipase + colipase (activated by trypsin; it anchors the lipase to the bile-salt-coated droplet) → 2-monoacylglycerol + 2 free fatty acids. Cholesterol esterase → cholesterol + fatty acid. Phospholipase A₂ (activated by trypsin, needs bile salts) removes the fatty acid at carbon 2, leaving a lysophospholipid.

Hormonal control

CCK — released by the duodenal mucosa in response to fat and protein; contracts the gallbladder, triggers pancreatic enzyme release, and slows gastric emptying. Secretin — released in response to acid; makes the pancreas secrete bicarbonate to raise the pH.

Absorption

The products plus bile salts and fat-soluble vitamins form mixed micelles, which ferry them across the unstirred water layer to the enterocyte brush border. Bile salts themselves are reabsorbed in the terminal ileum and recycled. Short- and medium-chain fatty acids do not need micelles: they pass straight into the portal blood bound to albumin.

Resynthesis and export Topic 22a

In the smooth endoplasmic reticulum, long-chain fatty acids are activated to acyl-CoA and re-esterified: 2-MAG → TAG, and cholesterol → cholesteryl esters (ACAT). These are packaged with phospholipid and apolipoprotein B-48 into chylomicrons, released by exocytosis into the lacteals, and carried by the lymph through the thoracic duct into the left subclavian vein.

Cystic fibrosis Disorder 7

Autosomal recessive, caused by mutations in CFTR, a chloride channel of epithelia in the pancreas, lung, testes and sweat glands. Defective CFTR means reduced chloride secretion and increased sodium and water uptake, so secretions are dehydrated and thick. Thickened mucus blocks the pancreatic ducts → the enzymes never reach the intestine → pancreatic insufficiency, fat malabsorption and steatorrhoea, with deficiency of the fat-soluble vitamins. Treatment: pancreatic enzyme replacement plus A, D, E and K supplementation. Diagnosis rests on the raised sweat chloride.

🌿 Essential Fatty Acids and the ω-6 / ω-3 Families Topic 22c–dPast Q 64

Why they are essential

Humans possess Δ9, Δ6 and Δ5 desaturases but cannot introduce a double bond beyond carbon 9 counting from the carboxyl end. So we cannot make the ω-6 and ω-3 parent acids and must eat them; plants supply them.

The two parents

Linoleic acid, 18:2 ω-6 — the precursor of arachidonic acid (20:4), and thus of the eicosanoids: prostaglandins, thromboxanes, leukotrienes. α-Linolenic acid, 18:3 ω-3 — the precursor of EPA and DHA, important for growth, neural development and retina. Arachidonic acid becomes essential if linoleic acid is lacking.

Reading the notation

ω (or n) counts from the terminal methyl carbon; the numbers after the colon in the Δ system count from the carboxyl carbon. Arachidonic acid 20:4(5,8,11,14) is ω-6 because 20 − 14 = 6. Naturally occurring double bonds are cis and are spaced three carbons apart.

Saturated vs mono- vs polyunsaturated Past Q 64

No double bond = saturated, straight chains that pack tightly, solid at room temperature, higher melting point. One = monounsaturated (oleic 18:1). Two or more = polyunsaturated. Each cis double bond puts a kink in the chain, so the molecules pack badly: the melting point falls and membranes stay fluid. Deficiency of essential fatty acids gives dermatitis, poor wound healing and growth failure.

🗂️ Classification of Lipids Past Q

Lipids are defined by a property — insoluble in water, soluble in organic solvents — not by a shared structure. So the classification is by what they are made of, and the answer the paper wants is four groups.

1 · Simple — ester of a fatty acid with an alcoholFats: esters with glycerol (an oil is simply a fat that is liquid at room temperature). Waxes: esters with a long-chain monohydric alcohol — cerumen, sebum, the water-repellent film on skin and hair.

2 · Complex — an ester carrying something extraPhospholipids: fatty acid + alcohol + phosphate + a nitrogenous base. Glycolipids: fatty acid + sphingosine + carbohydrate. The rest: sulfolipids, amino lipids and the lipoproteins.

3 · Precursor and derivedWhat hydrolysis of groups 1 and 2 gives, plus everything built from it: fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, ketone bodies, hydrocarbons, the lipid-soluble vitamins and the steroid hormones.

4 · Neutral — a label that cuts across the other threeThe uncharged lipids: acylglycerols, cholesterol and cholesteryl esters. Being uncharged they are the storage and core forms; the charged, amphipathic lipids are the membrane and surface forms — which is exactly the architecture of a lipoprotein.

🚢 The Four Lipoproteins Topic 26Past Q 108, 117, 170Lecturer’s priority

All have the same architecture — a core of triacylglycerol and cholesteryl esters in a shell of phospholipid, free cholesterol and apolipoproteins. As density rises, protein rises and lipid falls: chylomicron → VLDL → IDL → LDL → HDL.

Chylomicron

Made by the intestine, after a meal. Carries dietary TAG (about 90% of its mass). Structural protein apo B-48; receives apo C-II and apo E from HDL. LPL strips its TAG; the remnant is cleared by the liver via apo E.

VLDL

Made by the liver, in the fed state and whenever the liver has excess fatty acids. Carries endogenous TAG. Structural protein apo B-100. LPL converts it to IDL, and hepatic lipase converts IDL to LDL. What raises VLDL secretion: the fed state rather than the starved one, a high-carbohydrate (especially high-fructose) diet, a high flux of circulating free fatty acids, ethanol, and high insulin with low glucagon — although insulin given acutely suppresses apo B-100 output, so it is chronic hyperinsulinaemia with insulin resistance that drives secretion up. What blocks assembly: ω-3 fatty acids, orotic acid, the sulfonylurea tolbutamide, puromycin and — experimentally — brefeldin A; the inherited version of the same failure is abetalipoproteinaemia.

LDL

The end product of VLDL catabolism — the cholesterol-richest particle, carrying cholesterol to peripheral tissues. Its only protein is apo B-100, the ligand for the LDL receptor. Oxidised LDL is taken up unregulated by macrophage scavenger receptors → foam cells → atherosclerotic plaque. This is why LDL is the "bad" cholesterol.

HDL

Made by liver and intestine as a discoidal, protein-rich particle. Functions: a reservoir of apo C-II and apo E which it lends to chylomicrons and VLDL; and reverse cholesterol transport — picking up free cholesterol from peripheral tissues (via ABCA1 onto lipid-poor pre-β-HDL and apo A-I, and via ABCG1 onto the mature particle), esterifying it with LCAT, and delivering the esters to the liver via SR-B1 (the particle itself is not taken up). Hence the "good" cholesterol.

Apolipoproteins — who does what Topic 26c

apo A-I — structural on HDL; activates LCAT; ligand for SR-B1. apo B-48 — structural on chylomicrons (intestine). apo B-100 — structural on VLDL, IDL and LDL; the ligand for the LDL receptor. apo C-II — the obligatory activator of lipoprotein lipase. apo C-III — inhibits LPL. apo E — the ligand for hepatic uptake of chylomicron remnants and IDL.

LCAT — role, site, activator Topic 26d

Lecithin:cholesterol acyltransferase is synthesised in the liver and works in the plasma, on the surface of HDL. It transfers a fatty acid from lecithin (phosphatidylcholine) to free cholesterol, giving a cholesteryl ester and lysolecithin. Because the ester is hydrophobic it sinks into the core, so the flat discoidal HDL matures into a spherical HDL₂ — and the surface is freed to take up more cholesterol. Activator: apo A-I. Its deficiency leaves cholesterol unesterified and HDL immature. CETP then exchanges those esters for TAG from VLDL. Compare ACAT, which esterifies cholesterol inside cells for storage.

🎯 The LDL Receptor Cycle and How a Cell Turns It Down Topic 26Past Q

The LDL (apo B-100, E) receptor is expressed by the liver and by most extrahepatic tissues. About 70% of LDL is cleared by the liver and 30% elsewhere. It binds apo B-100 — and also, with higher affinity, particles rich in apo E, which is how remnants are removed.

LDL binds the receptorin a clathrin-coated pit on the cell surface

↓receptor-mediated endocytosisthe whole particle is taken in intact

Endosome → lysosomeapoprotein hydrolysed to amino acids; cholesteryl ester hydrolysed to free cholesterol

↓receptor recyclingthe receptor returns to the surface and is used again — unless PCSK9 has tagged it, in which case it is degraded instead

Free cholesterol in the cellnow acts as its own signal

Signal 1 — switch synthesis off

The incoming cholesterol inhibits transcription of the genes for HMG-CoA synthase, HMG-CoA reductase, the other cholesterol-synthesis enzymes and the LDL receptor itself, through the SREBP pathway. Synthesis and uptake are suppressed together.

Signal 2 — store the surplus

ACAT is stimulated, so free cholesterol is esterified and parked as cholesteryl ester droplets. Between the two signals the free cholesterol content of the cell is held within very narrow limits, matched to its needs for membranes, steroid hormones and bile acids.

Why diet changes receptor numbers Past Q

Polyunsaturated and monounsaturated fatty acids up-regulate LDL receptors, so LDL is cleared faster and plasma cholesterol falls. Saturated fatty acids do the opposite and also push cholesterol into smaller, cholesterol-richer VLDL particles that are taken up slowly by the tissues — the more atherogenic pattern.

When the receptor fails

Familial hypercholesterolaemia Past Q 66, 103: loss of the receptor — or of apo B-100, or excessive PCSK9 — so LDL cholesterol stays in the plasma and is very high from birth: tendon xanthomas, xanthelasma, corneal arcus and premature atherosclerosis. The same logic explains the two drug classes: statins lower intracellular cholesterol so SREBP makes more receptors; PCSK9 inhibitors stop the receptors being destroyed.

📉 When Lipoprotein Metabolism Goes Wrong Past Q

Two families: too little (hypo-) and too much (hyper-lipoproteinaemia). The primary forms are inherited; by far the commonest cause overall is secondary — diabetes mellitus, where insulin deficiency mobilises fatty acids and leaves chylomicrons and VLDL uncleared, giving hypertriacylglycerolaemia.

Disorder	Defect	Picture
Abetalipoproteinaemia	No apo B (MTP defect) — no chylomicrons, no VLDL	Fat malabsorption, fat-laden enterocytes, acanthocytes, deficiency of fat-soluble vitamins (especially E) with ataxia and retinitis
Tangier disease	Familial α-lipoprotein (HDL) deficiency — ABCA1	Almost no HDL, orange tonsils, cholesterol ester deposits, neuropathy
Fish-eye disease · LCAT deficiency	Cholesterol cannot be esterified in HDL	Corneal opacity, low HDL, abnormal lipoprotein particles
Type I — familial LPL (or apo C-II) deficiency	Chylomicrons cannot be cleared	Milky serum, eruptive xanthomas, pancreatitis; no excess atherosclerosis
Type IIa — familial hypercholesterolaemia	LDL receptor	↑↑ LDL, tendon xanthomas, early coronary disease
Type III — familial dysbetalipoproteinaemia	Abnormal or deficient apo E, so remnants are not taken up	Remnant accumulation, palmar xanthomas, xanthomatosis, premature coronary and peripheral vascular disease
Type IV — familial hypertriacylglycerolaemia	Overproduction of VLDL	Raised TAG, often with obesity, insulin resistance and low HDL
Familial hyperalphalipoproteinaemia	High HDL	Longevity — the one "hyper" that is good news. Hepatic lipase deficiency sits in the same list, with accumulated remnants.

Lipid Metabolism — Lipogenesis, β-Oxidation, Ketones, Cholesterol

Resit topics 23–28 · localisation, significance, substrates, products, regulation

🏭 Lipogenesis — Fatty Acid Synthesis Topic 23Past Q 13Lecturer’s priority

Localisation & significance

Cytosol of liver, adipose tissue and lactating mammary gland. It converts surplus carbohydrate into a compact, water-free energy store. Active in the fed state, driven by insulin.

Getting acetyl-CoA out of the mitochondrion

Acetyl-CoA is made in the matrix but the membrane is impermeable to it. It condenses with oxaloacetate to citrate, which is exported, and ATP-citrate lyase cleaves it back to acetyl-CoA + oxaloacetate in the cytosol. This is one half of the citrate shuttle and the specific link between the Krebs cycle and lipogenesis.

Acetyl-CoA carboxylase — the regulated step

Acetyl-CoA + CO₂ + ATP → malonyl-CoA, the rate-limiting and committed step. Requires biotin. Allosterically activated by citrate (which polymerises it into the active filament) and inhibited by palmitoyl-CoA. Covalently: inactivated by phosphorylation (glucagon, adrenaline, and AMP-activated protein kinase) and activated by dephosphorylation (insulin). Long term it is induced by a high-carbohydrate diet.

Fatty acid synthase and the role of –SH groups Past Q 41

A dimeric multienzyme with seven activities. Two thiol (–SH) groups hold the growing chain: the phosphopantetheine –SH of ACP (acyl carrier protein, derived from pantothenic acid) and the cysteine –SH of the condensing enzyme. The chain is passed between them at each cycle: condensation → reduction → dehydration → reduction, lengthening by two carbons each turn and consuming 2 NADPH per cycle. The product is palmitate (16:0), released by a thioesterase.

Sources of NADPH · the malic enzyme Topic 23f

Palmitate synthesis needs 14 NADPH. About two thirds come from the oxidative phase of the pentose phosphate pathway; the rest from the malic enzyme, which completes the citrate shuttle: cytosolic oxaloacetate → malate (malate dehydrogenase, using NADH) → pyruvate + CO₂ + NADPH (malic enzyme). The pyruvate returns to the mitochondrion. So the shuttle exports carbon and generates reducing power at the same time.

Fate of palmitate Past Q 43

Elongation in the smooth ER (two carbons at a time) and desaturation by Δ9, Δ6 and Δ5 desaturases — never beyond carbon 9 from the carboxyl end. Then esterification to TAG, or into phospholipids and cholesteryl esters.

💧 TAG Synthesis, Glycerol and the Two Lipolyses Topics 23, 24, 27, 28Past Q 44, 101, 152, 168Lecturer’s priority

Sources of "active glycerol" — glycerol-3-phosphate Topic 28

Fatty acids can only be esterified onto glycerol-3-phosphate. There are two routes, and they are tissue-specific: (1) From DHAP of glycolysis, by glycerol-3-phosphate dehydrogenase — available in both liver and adipose tissue. (2) From free glycerol by glycerol kinase — present in the liver (and kidney) but essentially absent from adipose tissue. Consequence: adipose tissue can only make TAG when glucose is available, which is why insulin and glucose are required for fat storage; and the glycerol released by lipolysis must leave the adipocyte and go to the liver.

The two ways a triacylglycerol is actually built Past Q

The phosphatidate route — liver, adipose tissue, everywhere. Glycerol-3-phosphate + acyl-CoA → (glycerol-3-phosphate acyltransferase) lysophosphatidate → (1-acylglycerol-3-phosphate acyltransferase) phosphatidate → (phosphatidate phosphohydrolase) 1,2-diacylglycerol → (diacylglycerol acyltransferase, DGAT) triacylglycerol. Note that the fatty acids must be activated to acyl-CoA first, and that phosphatidate and diacylglycerol are the branch point to the phospholipids — the same two intermediates serve storage fat and membrane lipid. The monoacylglycerol route — the enterocyte only. The 2-monoacylglycerol that pancreatic lipase left behind is re-acylated directly: monoacylglycerol acyltransferase → 1,2-diacylglycerol → DGAT → TAG, which is packed into chylomicrons. It skips glycerol-3-phosphate altogether, which is why the gut does not need glycerol kinase either.

Intracellular lipolysis — adipose tissue Topic 23e

Hydrolysis of stored TAG inside the adipocyte by the three lipases below, hormone-sensitive lipase (HSL) among them, releasing free fatty acids into the blood (bound to albumin) and glycerol to the liver — triacylglycerol itself cannot cross a membrane, which is why both lipolyses have to exist. HSL is activated by phosphorylation and inhibited by insulin: it is the fasting-state enzyme. What raises and lowers lipolysis is listed in full further down this card.

Intravascular lipolysis Topic 23ePast Q 101

Hydrolysis of the TAG carried inside circulating chylomicrons and VLDL by lipoprotein lipase (LPL), which is made by adipocytes and myocytes but anchored to the capillary endothelium (details below). Its significance: it delivers dietary and hepatic fat to the tissues, stripping 70–90% of a chylomicron’s TAG — apo C goes back to HDL, apo E stays, and the remnant leaves about half the diameter of the parent and enriched in cholesterol. VLDL becomes IDL the same way.

LPL vs HSL — the regulation contrast Topics 24 & 27

Adipose LPL is induced by insulin — in the fed state, so fat is captured and stored. Muscle LPL has a lower Km and is not insulin-dependent, so muscle takes fat during fasting. HSL is inhibited by insulin and activated by glucagon and adrenaline. Insulin therefore switches LPL on and HSL off simultaneously: one hormone, storage in both directions.

The three lipases and the gatekeeper

Stored TAG is dismantled in order: adipose triglyceride lipase (ATGL) → TAG to diacylglycerol; hormone-sensitive lipase → DAG to monoacylglycerol; monoacylglycerol lipase → the last fatty acid + glycerol. The droplet is coated by perilipin: unphosphorylated, it shields the fat from the lipases; once PKA phosphorylates it, it changes shape and lets HSL reach the surface. Perilipin is the reason hormones can switch lipolysis on so sharply.

Everything that turns lipolysis up or down

Up (through cAMP → PKA): adrenaline and noradrenaline, glucagon, ACTH, MSH, TSH, growth hormone, vasopressin. Permissive: glucocorticoids (new lipase protein, a cAMP-independent route) and thyroid hormone. Down: insulin — it activates phosphodiesterase so cAMP falls, and activates a lipase phosphatase — plus nicotinic acid, prostaglandin E₁ and ketone bodies, all of which lower cAMP.

Where LPL sits, and how the tissues compete Past Q

LPL is anchored to the capillary endothelium by heparan sulfate chains — an injection of heparin releases it into the blood. It needs phospholipid and apo C-II as cofactors and is inhibited by apo A-II and apo C-III (which is why phospholipid counts as an obligatory LPL cofactor). Heart LPL has a Km about a tenth of the adipose enzyme, so when plasma TAG falls in fasting the heart still extracts fatty acids while adipose tissue cannot — the delivery is redirected to where it is needed.

🔥 β-Oxidation and the Role of Carnitine Topic 23Past Q 26, 80Lecturer’s priority

Localisation & significance

Mitochondrial matrix, in almost every tissue except brain (fatty acids do not cross the blood–brain barrier well) and red cells (no mitochondria). It is the main fuel of heart, resting skeletal muscle and liver during fasting, and it supplies the ATP and NADH that make gluconeogenesis possible.

Activation

In the cytosol, acyl-CoA synthetase (thiokinase) makes fatty acyl-CoA, spending ATP → AMP + PPi = two high-energy bonds.

The carnitine shuttle Topic 23g

Long-chain acyl-CoA cannot cross the inner membrane. CPT-I on the outer membrane swaps CoA for carnitine → acylcarnitine; a translocase carries it in; CPT-II on the inner face restores the acyl-CoA. CPT-I is the rate-limiting step of β-oxidation and is inhibited by malonyl-CoA — the first intermediate of fatty acid synthesis. That single inhibition prevents a futile cycle: whenever the cell is making fat, it cannot burn it. Short- and medium-chain fatty acids do not need carnitine. Carnitine deficiency causes muscle weakness with lipid accumulation and fasting hypoglycaemia without ketosis.

The four repeating steps & the yield

Oxidation (FAD) → hydration → oxidation (NAD⁺) → thiolysis. Each cycle removes two carbons as acetyl-CoA and yields 1 FADH₂ + 1 NADH. Palmitate (16 C) gives 7 cycles, 8 acetyl-CoA, 7 FADH₂ and 7 NADH — about 106 ATP net after subtracting the 2 spent on activation (older accounting: 129 − 2 = 131/106 depending on P/O ratios used). That is why fat yields more than twice as much energy per gram as carbohydrate.

Odd-chain and unsaturated fatty acids

An odd-chain fatty acid ends with propionyl-CoA → (biotin) methylmalonyl-CoA → (vitamin B12) succinyl-CoA — the only part of a fatty acid that is glucogenic. Unsaturated fatty acids need two extra enzymes, an isomerase and a reductase, and yield slightly less ATP because a double bond is already present.

Peroxisomal oxidation

Very-long-chain fatty acids are shortened first in peroxisomes, where the first oxidation passes electrons to O₂ giving H₂O₂ (removed by catalase) instead of making ATP. Branched chains such as phytanic acid require α-oxidation, whose failure is Refsum disease. A third, minor route runs from the far end: in ω-oxidation a cytochrome P450 of the smooth ER hydroxylates the terminal methyl carbon and oxidises it to a second carboxyl group, giving a dicarboxylic acid that β-oxidation then shortens from either end. It is negligible normally but takes over when β-oxidation is blocked — which is why dicarboxylic aciduria is the marker of a fatty-acid oxidation defect such as MCAD deficiency.

🍋 Ketogenesis and Ketolysis Topic 23Past Q 79, 148, 163Lecturer’s priority

Where and when

Ketogenesis: mitochondria of liver cells only. It happens when β-oxidation outruns the Krebs cycle — in prolonged fasting, starvation, a very-low-carbohydrate diet and uncontrolled type 1 diabetes — because oxaloacetate has been drawn off into gluconeogenesis, so acetyl-CoA cannot be condensed into citrate.

The three ketone bodies and the pathway

2 acetyl-CoA → acetoacetyl-CoA → HMG-CoA (HMG-CoA synthase — the rate-limiting step; note the mitochondrial isoform, whereas cholesterol synthesis uses the cytosolic one) → acetoacetate → reduced to β-hydroxybutyrate (the most abundant in blood) or spontaneously decarboxylated to acetone (volatile, exhaled — the fruity breath). Acetoacetate and β-hydroxybutyrate are acids; acetone is not and cannot be metabolised.

Significance

They are water-soluble, transportable equivalents of fatty acids that need no albumin and, crucially, can cross the blood–brain barrier. After a few days of starvation the brain covers most of its needs from ketones, which spares glucose and therefore spares muscle protein. Heart and renal cortex use them preferentially at any time.

Ketolysis — and the enzyme the liver lacks

In extrahepatic tissue: β-hydroxybutyrate → acetoacetate → activated by thiophorase (succinyl-CoA:acetoacetate CoA transferase) → acetoacetyl-CoA → 2 acetyl-CoA → Krebs cycle. The liver has no thiophorase — which is precisely why it makes ketone bodies for everyone else and cannot consume them itself.

When it goes too far

Overproduction gives ketonaemia, ketonuria and ketotic breath, and because two of the three are acids, a metabolic acidosis with an increased anion gap. In diabetic ketoacidosis the absence of insulin removes all restraint on lipolysis and ketogenesis at once — hence Kussmaul breathing as respiratory compensation.

Regulation of ketogenesis — three gates in series Past Q

Gate 1 · in adipose tissue. Ketosis cannot happen unless circulating free fatty acids rise, so everything that controls lipolysis controls ketogenesis first; the liver extracts about 30% of the fatty acids passing through it, fed or fasting. Gate 2 · at the mitochondrial door. Once inside the hepatocyte a fatty acid is either esterified to TAG and phospholipid or β-oxidised, and CPT-I decides which. In the fed state malonyl-CoA (made by acetyl-CoA carboxylase) inhibits CPT-I, oxidation is low and the fat leaves as VLDL. In starvation acyl-CoA inhibits acetyl-CoA carboxylase, malonyl-CoA falls, CPT-I opens and oxidation rises. Gate 3 · inside the mitochondrion. The acetyl-CoA produced is partitioned between the Krebs cycle and ketogenesis (HMG-CoA synthase, then HMG-CoA lyase, which splits HMG-CoA into acetoacetate + acetyl-CoA). As serum fatty acids rise, proportionally more goes to ketones.

The arithmetic that explains why the liver bothers

Complete oxidation of one mole of palmitate in the liver yields about 106 ATP; if the same carbon leaves as acetoacetate the liver keeps only about 26 ATP, and as 3-hydroxybutyrate about 21. Ketogenesis is therefore a device that lets the liver oxidise very large amounts of fatty acid without being limited by its own tightly coupled oxidative phosphorylation — it exports the unburnt energy to everyone else. What tips the partition is the falling mitochondrial oxaloacetate — not only drawn into gluconeogenesis but also pulled toward malate by the high NADH/NAD⁺ ratio of active β-oxidation. Acetyl-CoA activating pyruvate carboxylase partly offsets it, but in starvation and untreated diabetes the compensation fails.

🧱 Cholesterol and Bile Acid Synthesis Topic 25Past Q 20, 24, 150

Cholesterol — localisation and steps

Cytosol and smooth ER, mainly of the liver (also intestine, adrenal cortex, gonads, skin). 2 acetyl-CoA → acetoacetyl-CoA → HMG-CoA → mevalonate → isoprene units → squalene → lanosterol → cholesterol. It needs NADPH and ATP throughout.

HMG-CoA reductase — the key enzyme

HMG-CoA → mevalonate, in the ER membrane, using 2 NADPH. Rate-limiting and irreversible. Regulation on four levels: feedback inhibition by cholesterol; transcriptional control by SREBP-2 — the factor sits in the ER membrane held by SCAP; when sterols are high, SCAP is clamped by Insig and nothing moves, and when cholesterol falls Insig lets go, SCAP escorts SREBP to the Golgi, two proteases cut the transcription factor free, and it switches on the enzyme and the LDL receptor together; covalent — phosphorylated and inactivated by AMP-activated protein kinase (AMPK), dephosphorylated and activated by insulin; and degradation of the enzyme when sterols are high. Statins are competitive inhibitors of this enzyme.

Roles of cholesterol Past Q 20

Membrane component and modulator of fluidity; precursor of the five classes of steroid hormone, of bile acids, and of vitamin D (via 7-dehydrocholesterol in skin).

Bile acids Topic 25Past Q 24

Liver only, from cholesterol. The rate-limiting enzyme is 7α-hydroxylase (CYP7A1), inhibited by bile acids returning to the liver. Primary bile acids: cholic and chenodeoxycholic acid; they are conjugated with glycine or taurine to give bile salts, which are far better detergents at intestinal pH. Intestinal bacteria dehydroxylate them into the secondary bile acids, deoxycholic and lithocholic. Enterohepatic circulation: about 95% are reabsorbed in the terminal ileum and returned to the liver, the pool recycling several times per meal. Significance: the only meaningful route for excreting cholesterol, plus emulsification of dietary fat.

Two more details on bile acids

The steps: cholesterol → 7α-hydroxycholesterol (7α-hydroxylase — a microsomal cytochrome P450 monooxygenase needing O₂, NADPH and vitamin C) → 12α-hydroxylation → cholyl-CoA and chenodeoxycholyl-CoA → conjugation with glycine or taurine gives glycocholic, taurocholic, glycochenodeoxycholic and taurochenodeoxycholic acid. In the gut, deconjugation and 7α-dehydroxylation turn them into deoxycholic and lithocholic acid; lithocholic is so insoluble that it is barely reabsorbed and is largely lost in faeces. The feedback: when the returning bile acid pool grows, it activates the nuclear receptor FXR (farnesoid X receptor), which suppresses the CYP7A1 gene; chenodeoxycholic acid is the strongest activator. About 3–5 g of bile acids cycle 6–10 times a day, and exactly the amount lost in faeces is resynthesised from cholesterol.

Why you cannot really call cholesterol "good" or "bad" Past Q

There is only one cholesterol molecule. What differs is the particle carrying it: LDL delivers cholesterol to the artery wall, HDL removes it (reverse transport). And cholesterol is indispensable — see its roles above. It becomes harmful only in excess and in the wrong vehicle. Hereditary factors set most of an individual's plasma level; diet, exercise, smoking, alcohol and stress adjust it around that setting.

Lifestyle and blood cholesterol Past Q 92

Saturated and trans fats raise LDL, mainly by suppressing LDL-receptor activity; trans fats additionally lower HDL. Polyunsaturated and monounsaturated fats lower LDL. Soluble fibre and plant sterols reduce the reabsorption of bile acids and cholesterol, forcing the liver to consume cholesterol. Aerobic exercise and weight loss raise HDL and lower TAG; smoking lowers HDL; excess alcohol raises TAG. Dietary cholesterol itself has a much smaller effect than saturated fat, because synthesis is down-regulated in response. Three more the paper names explicitly: coffee and emotional stress raise plasma free fatty acids, so the liver exports more triacylglycerol and cholesterol as VLDL; moderate alcohol lowers coronary risk by raising HDL — through increased apo A-I synthesis and a change in cholesteryl ester transfer protein activity — an effect that is lost with heavy drinking, which raises TAG instead; and regular exercise lowers LDL while raising HDL. The company a raised cholesterol keeps: smoking, male sex, obesity, physical inactivity, sugar-sweetened drinks and hypertension.

🧴 Phospholipids Past Q 141

Amphipathic lipids built on glycerol-3-phosphate (glycerophospholipids) or on sphingosine (sphingomyelin). Two long hydrophobic tails — two fatty acids in glycerophospholipids, one of them the sphingosine chain in sphingolipids — and one polar, phosphate-linked head group: choline, ethanolamine, serine or inositol. That is what makes them amphipathic and able to form the bilayer, and, like all complex lipids, they must be emulsified by bile salts and hydrolysed before absorption.

Functions

The structural backbone of every membrane; the surface layer of lipoproteins and bile micelles; lung surfactant (dipalmitoylphosphatidylcholine), whose deficiency causes neonatal respiratory distress syndrome; membrane anchors; and a source of signalling molecules — phosphatidylinositol gives IP₃ and DAG, and phosphatidylcholine gives arachidonic acid when phospholipase A₂ acts on it.

Lecithin and its enzymes

Lecithin = phosphatidylcholine, the commonest phospholipid and the fatty-acid donor for LCAT. Phospholipase A₂ removes the fatty acid at position 2 — releasing arachidonate for eicosanoid synthesis, and leaving lysolecithin. Glucocorticoids inhibit phospholipase A₂, which is how they suppress the whole eicosanoid cascade.

🧱Glycerophospholipids — built on phosphatidic acid

Phosphatidylcholine (lecithin) — the most abundant in membranes and lipoproteins · phosphatidylethanolamine · phosphatidylserine — normally on the inner leaflet; when it flips outward it flags a cell for phagocytosis · phosphatidylinositol — the source of the second messengers IP₃ and DAG · cardiolipin (diphosphatidylglycerol) — found only in the inner mitochondrial membrane, where it is needed for the respiratory-chain complexes.

🌿Plasmalogens — an ether instead of an ester

Carbon 1 carries an ether-linked alkenyl chain. They make up a large share of the phospholipid of heart muscle and myelin, and platelet-activating factor belongs to this family. They are made in peroxisomes — which is why peroxisomal diseases such as Zellweger syndrome are also plasmalogen-deficiency diseases.

🧠Sphingolipids — built on sphingosine, not glycerol

Sphingomyelin is the only phospholipid of this class: one fatty acid amide-linked to sphingosine, plus phosphocholine. It is concentrated in the nervous system and myelin. Replace the phosphocholine with sugars and you get the glycolipids — cerebrosides and gangliosides — whose lysosomal breakdown fails in the sphingolipidoses.

Fed & Post-Absorptive States

Resit topic 29 · past questions 46, 47, 60, 67, 95, 106, 111, 118, 186

🍽️ The Absorptive (Fed) State Topic 29Past Q 46Lecturer’s priority

The two to four hours after a meal. Insulin is high, glucagon is low; the theme is storage, and the dominant fuel is glucose.

Liver

Glucose enters freely through GLUT-2 and is trapped by glucokinase. Glycogenesis, glycolysis, the pentose phosphate pathway and lipogenesis are all up; gluconeogenesis, glycogenolysis and ketogenesis are off. Surplus carbon leaves as VLDL.

Adipose tissue

Insulin recruits GLUT-4, so glucose enters and supplies glycerol-3-phosphate via DHAP (there is no glycerol kinase here). Insulin induces LPL on the capillary endothelium so chylomicron and VLDL fatty acids are captured, and inhibits hormone-sensitive lipase. Net effect: TAG is stored and lipolysis stops.

Muscle

GLUT-4 opens; glucose is used for glycolysis and for replenishing glycogen. Insulin stimulates amino acid uptake and protein synthesis and inhibits proteolysis.

Brain

Uses glucose exclusively and its uptake is insulin-independent (GLUT-1 and GLUT-3), so it takes what it needs at any insulin level. About 120 g/day.

Lactate in the fed state Past Q 111, 118

Red cells produce lactate at all times, and after a large carbohydrate meal the gut and muscle add to it. The liver takes it up and, because gluconeogenesis is suppressed by insulin, largely oxidises it or uses its carbon for lipogenesis. In the post-absorptive state the same lactate becomes a prized gluconeogenic substrate — the Cori cycle. Red cells always finish at lactate because pyruvate oxidation is mitochondrial and they have none.

🌙 The Post-Absorptive and Fasting States Topic 29Past Q 47, 60, 186Lecturer’s priority

Insulin falls, glucagon rises; the theme is mobilisation, and the whole design is to protect the brain's glucose supply while sparing protein.

Hours 0–12 — glycogen

Hepatic glycogenolysis is the first line and is essentially exhausted within about 24 hours. Muscle glycogen cannot contribute to blood glucose.

Hours 12–24 onward — gluconeogenesis

The liver (and, later, the kidney) makes glucose from lactate, glycerol and glucogenic amino acids — chiefly alanine and glutamine from muscle. Cortisol induces the enzymes; glucagon phosphorylates the switches and lowers fructose-2,6-bisphosphate, which relieves the inhibition of fructose-1,6-bisphosphatase. Alongside both steps, β-oxidation and ketone body synthesis rise.

Days — ketone adaptation

Adipose lipolysis floods the liver with fatty acids; oxaloacetate is committed to gluconeogenesis, so acetyl-CoA is diverted to ketone bodies. The brain shifts to ketones, gluconeogenesis slows, and muscle protein breakdown falls — the key survival adaptation of prolonged starvation.

What happens to fatty acids, tissue by tissue Past Q 186

a · Liver — takes them up and β-oxidises them, using the ATP and NADH for gluconeogenesis and converting the surplus acetyl-CoA into ketone bodies. b · Adipose tissue — releases them: HSL is active, LPL is down, and the glycerol goes to the liver because adipocytes lack glycerol kinase. c · Muscle — oxidises them as its preferred fuel, which spares glucose; after a few days it also uses ketone bodies, then reduces even that so the brain can have them.

High-fat / low-carbohydrate diet Past Q 95

Metabolically it mimics fasting: low insulin, high glucagon, active lipolysis and β-oxidation, glucose supplied by gluconeogenesis from glycerol and amino acids, and a mild physiological ketosis. Glycogen stores and their bound water fall, which explains the rapid initial weight loss.

Why ethanol causes fatty liver Past Q 67

Alcohol dehydrogenase and aldehyde dehydrogenase both reduce NAD⁺, so ethanol drives the NADH/NAD⁺ ratio sharply up. High NADH pushes pyruvate → lactate and oxaloacetate → malate, removing both gluconeogenic substrates — hence fasting hypoglycaemia and lactic acidosis. It also inhibits β-oxidation (which itself needs NAD⁺) and pushes DHAP toward glycerol-3-phosphate. Fatty acids therefore accumulate and are esterified rather than burned: triacylglycerol builds up in the hepatocyte — hepatic steatosis. Acetate from ethanol also supplies extra acetyl-CoA for lipogenesis, and acetaldehyde damages the cell directly.

🔀 Two Details the Question Papers Keep Returning To Past Q

In the post-absorptive state, phosphorylated means off — with three exceptions

Glucagon and adrenaline phosphorylate a long list of enzymes and inactivate them: glycogen synthase, acetyl-CoA carboxylase, pyruvate dehydrogenase, pyruvate kinase. Three are switched ON by the same phosphorylation — glycogen phosphorylase, phosphorylase kinase and hormone-sensitive lipase. Learn the three exceptions and the rest of the pattern follows: fuel is being released, not stored.

Uncontrolled diabetes = the post-absorptive state, exaggerated Past Q

Insulin is absent or ineffective, so every fasting programme runs at once and unopposed even after a meal: unrestrained lipolysis, high plasma free fatty acids, maximal β-oxidation, maximal gluconeogenesis and glycogenolysis despite hyperglycaemia, and overproduction of ketone bodies. Underused chylomicrons and VLDL add hypertriacylglycerolaemia. This is why the metabolic disturbances of diabetes read like starvation happening in the middle of plenty.

Acid–Base Balance

Resit topics 30–32 · buffers, renal and respiratory regulation, the four disturbances

🧽 Human Buffering Systems Topic 30Past Q 36, 140

Normal arterial pH 7.35–7.45; pCO₂ 35–45 mmHg; HCO₃⁻ 22–26 mmol/L. A buffer is a weak acid with its conjugate base. Henderson–Hasselbalch for blood: pH = 6.1 + log([HCO₃⁻] / 0.03 × pCO₂).

Bicarbonate — why it is the most important Past Q 140

Its pK is 6.1, which is more than a unit away from 7.4, so on chemistry alone it should be a poor buffer. It is nevertheless the principal one because it is an open system: the lungs continuously blow off CO₂ and the kidneys continuously regenerate HCO₃⁻, so neither component is used up. Both sides are under independent physiological control, and it is present in the largest amount in extracellular fluid.

Phosphate

H₂PO₄⁻/HPO₄²⁻, pK 6.8 — close to 7.4, so chemically excellent, but its concentration in plasma is low. It is important intracellularly and it is the main urinary buffer, where it accounts for most of the titratable acidity.

Protein and haemoglobin

Haemoglobin is the main buffer of whole blood after bicarbonate, buffering through the imidazole side chains of histidine (pK ≈ 6). Deoxyhaemoglobin is a better proton acceptor than oxyhaemoglobin, so unloading oxygen in the tissues makes haemoglobin ready to take up the H⁺ generated there — the isohydric mechanism, the mirror of the Bohr effect. Plasma proteins buffer through histidine too.

Cellular and bone buffering Past Q 36

Over hours, H⁺ moves into cells in exchange for K⁺ (which is why acidosis causes hyperkalaemia) and is buffered there by proteins, phosphates and haemoglobin. In chronic acidosis, bone carbonate is mobilised as a long-term buffer — at the cost of demineralisation and osteoporosis. Ammonia is the other cellular contributor, in the kidney.

The acid load, and the liver's part

Daily metabolism produces roughly 15,000 mmol of CO₂ ("volatile acid", removed by the lungs) and about 50–100 mmol of fixed acid — sulfuric acid from sulfur amino acids, phosphoric acid from phospholipids, plus lactate and ketoacids — which only the kidney can excrete. The liver contributes by choosing between urea synthesis, which consumes bicarbonate, and glutamine synthesis, which spares it and hands the nitrogen to the kidney; in acidosis it shifts toward glutamine.

What decides a buffer's capacity Past Q

Three things. 1 · The concentration of the buffer — the more of it, the more acid or base it can absorb. 2 · The ratio of its two components, i.e. how close the pH is to the pK: capacity is greatest when [A⁻] = [HA], and the useful range is pK ± 1. 3 · Dilution — diluting a buffer lowers its capacity even though the ratio, and therefore the pH, hardly changes. A weak acid is most effective as a buffer when the pH of the solution stays close to its own pK.

🫁 Respiratory and Renal Regulation Topic 31Past Q 42

The lungs — minutes

Central and peripheral chemoreceptors sense pCO₂ and H⁺. Acidosis → hyperventilation → CO₂ blown off → H₂CO₃ falls → pH rises. Alkalosis → hypoventilation → CO₂ retained. It is fast but incomplete: it can only adjust the CO₂ side of the equation and never fully corrects the pH.

The chloride shift

In the tissue capillary, CO₂ enters the red cell, carbonic anhydrase makes H₂CO₃ → H⁺ + HCO₃⁻; the H⁺ is buffered by deoxyhaemoglobin and the HCO₃⁻ leaves in exchange for Cl⁻ (band 3 anion exchanger). The reverse happens in the lung. This is how most CO₂ is carried — as plasma bicarbonate.

The kidney — hours to days

Three jobs. 1 · Reabsorb filtered bicarbonate — about 90% in the proximal tubule, indirectly: secreted H⁺ (Na⁺/H⁺ exchanger) combines with filtered HCO₃⁻, luminal carbonic anhydrase makes CO₂, which diffuses in and is remade into HCO₃⁻ inside the cell. No new bicarbonate is generated by this step. 2 · Excrete fixed acid and 3 · generate new bicarbonate, in the distal nephron and collecting duct, by the two buffer systems below.

Titratable acidity and ammonium Past Q 42

Titratable acidity — H⁺ secreted onto filtered phosphate (HPO₄²⁻ → H₂PO₄⁻); it is limited by how much phosphate is filtered, and is measured as the amount of strong base needed to titrate urine back to pH 7.4. Ammoniagenesis — the adaptable system: the proximal tubule takes up glutamine and, with glutaminase and glutamate dehydrogenase, releases two NH₃ per glutamine, which trap H⁺ in the lumen as NH₄⁺ and are excreted. Each H⁺ excreted by either route generates one new HCO₃⁻ for the blood. Ammoniagenesis is strongly induced by chronic acidosis and is what allows the kidney to increase acid excretion several-fold over days.

⚗️ The Four Disturbances Topic 32Past Q 58, 121, 158, 159Lecturer’s priority

Read the pH first to name acidosis or alkalosis; then see whether pCO₂ or HCO₃⁻ explains it — that one is the primary disorder, and the other moves in the same direction as compensation.

Respiratory acidosis Past Q 158

↓pH, ↑pCO₂ (primary), ↑HCO₃⁻ (renal compensation). Cause: hypoventilation — COPD, asthma, airway obstruction, respiratory-centre depression by opioids, neuromuscular disease, chest wall injury. Compensation is renal and takes days: more H⁺ excretion, more ammoniagenesis, more new bicarbonate.

Respiratory alkalosis Past Q 159

↑pH, ↓pCO₂ (primary), ↓HCO₃⁻ (compensation). Cause: hyperventilation — anxiety, pain, fever, high altitude, hypoxaemia, salicylate poisoning early on, mechanical over-ventilation. Symptoms of paraesthesia and tetany arise because alkalosis increases the binding of Ca²⁺ to albumin and lowers ionised calcium.

Metabolic acidosis Past Q 121

↓pH, ↓HCO₃⁻ (primary), ↓pCO₂ (respiratory compensation — Kussmaul breathing, within minutes). High anion gap (added acid): ketoacidosis, lactic acidosis, renal failure, methanol, ethylene glycol, salicylates. Normal anion gap (bicarbonate lost): severe diarrhoea, renal tubular acidosis, carbonic anhydrase inhibitors. Anion gap = Na⁺ − (Cl⁻ + HCO₃⁻), normally 8–16 mmol/L. Correction is renal: maximal H⁺ and NH₄⁺ excretion with regeneration of bicarbonate.

Metabolic alkalosis

↑pH, ↑HCO₃⁻ (primary), ↑pCO₂ (compensatory hypoventilation, and limited because hypoxia eventually forces breathing). Causes: prolonged vomiting or nasogastric suction (loss of gastric HCl), diuretics, hyperaldosteronism, excessive alkali. Vomiting and nasogastric suction give the variety that has its own name — hypochloraemic (and hypokalaemic) metabolic alkalosis — because Cl⁻ and K⁺ leave with the acid, and losing H⁺‑rich gastric fluid raises [HCO₃⁻] and therefore the pH. It is then maintained by that volume depletion and hypokalaemia; the kidney corrects it by excreting bicarbonate once chloride and potassium are replaced.

Primary versus secondary, and compensation Past Q 58

The primary (secondary = compensatory) change is the one that matches the direction of the pH shift. Compensation never overshoots, so the pH tells you which disorder is primary; if pH is normal but pCO₂ and HCO₃⁻ are both abnormal, the disturbance is fully compensated — or there are two disorders at once. Compensation restores pH by changing the other variable; correction repairs the variable that was primarily wrong.

Nucleotides, Nucleic Acids & Protein Synthesis

Resit topics 33–35 · past questions 21, 22, 100, 114, 129 — plus the DNA/RNA, replication, transcription, translation and mutation block

🧬 Structure and Roles Topic 33Past Q 100, 129

Structure

Nucleoside = nitrogenous base + pentose, joined by an N-glycosidic bond. Nucleotide = nucleoside + one, two or three phosphate groups. Purines: adenine and guanine — two fused rings. Pyrimidines: cytosine, uracil, thymine — one ring.

Roles

Building blocks of DNA and RNA; the energy currency (ATP, GTP); components of coenzymes (NAD⁺, NADP⁺, FAD, coenzyme A); activated intermediates (UDP-glucose, CDP-choline, SAM, PAPS); and second messengers (cAMP, cGMP).

Six compounds containing ribose Past Q 129

ATP · NAD⁺ · NADP⁺ · FAD · coenzyme A · RNA — and, if more are wanted, SAM, cAMP and UDP-glucose. All trace back to ribose-5-phosphate from the pentose phosphate pathway, activated to PRPP.

Purine vs pyrimidine synthesis — the difference Topic 33

Purines are built directly onto ribose: the ring is assembled piece by piece on PRPP, giving IMP, which then branches to AMP and GMP. Pyrimidines are built first and attached afterwards: the ring is made as orotate and only then joined to PRPP to give OMP → UMP. That single contrast is the exam point.

🔄 Purine Synthesis, Salvage and Regulation Topics 33, 35Past Q 22

De novo — substrates

Cytosol, mainly liver. Ribose-5-P → PRPP (PRPP synthetase). The committed, rate-limiting step is glutamine–PRPP amidotransferase. The ring atoms come from glutamine (2 N), aspartate (1 N), glycine (whole molecule, 2 C + 1 N), CO₂, and two one-carbon units from N10-formyl-THF. The first complete nucleotide is IMP; GTP is needed to make AMP and ATP is needed to make GMP — a reciprocal arrangement that keeps the two in balance.

Regulation

Feedback inhibition of PRPP synthetase and of the amidotransferase by AMP, GMP and IMP; PRPP activates the amidotransferase. Also the reciprocal ATP/GTP requirement above.

Salvage — and why it matters Topic 35

HGPRT: hypoxanthine → IMP, guanine → GMP. APRT: adenine → AMP. Both use PRPP. Significance: de novo synthesis is energetically expensive (about 6 ATP per purine), so salvage conserves energy; and brain, erythrocytes and bone marrow lack a full de novo pathway and depend on salvage. Salvage also limits the amount of purine degraded to uric acid.

Deoxyribonucleotides

Ribonucleotide reductase converts NDP → dNDP, using thioredoxin and NADPH. Its allosteric sites keep the four dNTPs balanced, and dATP inhibits it — the mechanism behind ADA deficiency and SCID (card below). dTMP comes from dUMP by thymidylate synthase, using N5,N10-methylene-THF.

🦴 Catabolism, Uric Acid and Gout Topic 34 · Disorder 13

Purine catabolism

Nucleic acids → nucleotides → nucleosides. AMP → IMP → inosine → hypoxanthine → xanthine → uric acid, the last two steps by xanthine oxidase (a molybdenum-containing enzyme that produces H₂O₂). Guanine → xanthine by guanase. Uric acid is the end product in humans — we lack uricase — and it is poorly soluble, which is the whole clinical problem.

Pyrimidine catabolism Past Q 21

The ring is opened and fully degraded to β-alanine (from cytosine and uracil) or β-aminoisobutyrate (from thymine), plus NH₃ and CO₂. The products are water-soluble, so — unlike purines — pyrimidine breakdown causes no clinical problem. Pyrimidine synthesis is regulated at CPS II, which is activated by PRPP and inhibited by UTP.

Gout

Hyperuricaemia with deposition of monosodium urate crystals in joints — classically the first metatarsophalangeal joint (podagra) — giving acute inflammatory arthritis, and tophi and urate stones in chronic disease. Causes: underexcretion (90% — renal impairment, diuretics, alcohol, lactate and ketones competing for tubular secretion) or overproduction (high purine diet, cell lysis in leukaemia and chemotherapy, PRPP synthetase overactivity, HGPRT deficiency, and von Gierke disease).

Treatment and the two named syndromes

Acute attack: NSAIDs, colchicine, glucocorticoids. Long term: allopurinol, a hypoxanthine analogue that inhibits xanthine oxidase, so the more soluble hypoxanthine and xanthine are excreted instead; plus uricosurics. Lesch–Nyhan syndrome — complete HGPRT deficiency, X-linked: no salvage, so purines are all degraded → massive uric acid, gout from childhood, stones, intellectual disability, choreoathetosis and compulsive self-mutilation; allopurinol treats the gout but not the neurology. ADA deficiency — adenosine deaminase, autosomal recessive: dATP accumulates, ribonucleotide reductase is inhibited, lymphocytes cannot divide → severe combined immunodeficiency.

Synthetic nucleotide analogues Past Q 114

They work because they are close enough in shape to be accepted by an enzyme or a polymerase, but wrong enough to stop it. Allopurinol — the gout example above. 5-Fluorouracil — a uracil analogue whose product irreversibly inhibits thymidylate synthase, so no dTMP (cancer). Methotrexate — a folate analogue that blocks dihydrofolate reductase, starving both purine and dTMP synthesis (cancer, autoimmune disease). 6-Mercaptopurine — blocks purine synthesis. Cytarabine, azathioprine — chain terminators and immunosuppressants. Acyclovir, AZT — antiviral chain terminators that are activated selectively in infected cells.

🧵 From Nucleotide to Nucleic Acid — Structure, Polarity, Melting Past Q

The base is joined to carbon 1 of the sugar by a β-N-glycosidic bond; the sugars are then strung together by 3′,5′-phosphodiester bonds. One end of the chain carries a free 5′ phosphate or hydroxyl, the other a free 3′ hydroxyl — so every nucleic acid has a direction.

The double helix

Right-handed, with the two strands antiparallel: one runs 5′→3′ while the other runs 3′→5′. Held together by hydrogen bonds between purine and pyrimidine — A=T (two bonds), G≡C (three) — and, just as importantly, by base stacking: van der Waals and hydrophobic interactions between adjacent pairs. Chargaff's rule follows: A = T and G = C.

Melting temperature (T_m)

Heating separates the strands over a range of temperature; the midpoint is T_m. It depends on base composition and on salt concentration: DNA rich in G–C pairs melts at a higher temperature because each pair has three hydrogen bonds instead of two. Cooling lets the strands re-anneal — the basis of hybridisation and of every probe-based technique.

Template, coding strand, transcript Past Q

The template (antisense, non-coding) strand is the one actually read by RNA polymerase. The coding (sense) strand is its partner, and it has the same sequence as the transcript — except that RNA carries uracil where DNA carries thymine. The RNA transcript is built antiparallel to the template, 5′→3′.

DNA versus RNA — six contrasts Past Q

1 · 2′-deoxyribose vs ribose · 2 · double-stranded antiparallel helix vs single strand that folds back on itself (hairpins) · 3 · thymine vs uracil · 4 · nucleus and mitochondria vs nucleus and cytoplasm · 5 · DNA resists alkali because it has no 2′-OH, whereas RNA is hydrolysed by alkali to 2′,3′-cyclic mononucleotides — RNA is the less stable molecule · 6 · DNA obeys A=T and G=C; in RNA the base amounts need not be equal.

📮 The RNA Species — and the Anatomy of tRNA Past Q

✉️mRNA — the messenger

Carries the information of a gene to the protein-synthesising machinery, where it serves as the template on which a specific amino acid sequence is polymerised. Least abundant, shortest-lived, most varied in length.

🔌tRNA — the adaptor

About 20% of cellular RNA; at least one species for each of the 20 amino acids; 74–95 nucleotides long, and made by nuclear processing of a larger precursor. The primary structure is common to all species; the cloverleaf has four arms.

🏭rRNA — the machine

The most abundant RNA; it is the structural and catalytic core of the ribosome, i.e. the machinery that makes protein from the mRNA template.

🔎The small RNAs

Small nuclear RNAs (snRNA) — rRNA and mRNA processing, and splicing. The non-coding regulatory RNAs: microRNA (miRNA) and small interfering RNA (siRNA), which silence messages, and long non-coding RNA (lncRNA).

Acceptor arm

Ends in –CCA at the 3′-hydroxyl adenosyl terminal. This is the site of attachment of the amino acid, esterified there by an aminoacyl-tRNA synthetase at the cost of ATP — the step where the fidelity of translation is actually decided.

Anticodon arm

Seven nucleotides, three of which are the anticodon that base-pairs with the mRNA codon. It reads the codon antiparallel to the message.

D arm

Named for its dihydrouridine. It is the part recognised by the aminoacyl-tRNA synthetase, the enzyme that ensures the correct amino acid is loaded.

TψC arm

Contains the unusual base pseudouridine (ψ). It binds the tRNA to the ribosomal surface at the site of protein synthesis. (A fifth, variable loop sits between the anticodon and TψC arms.)

Ribozymes — RNA that behaves like an enzyme Past Q

Some RNA molecules have intrinsic catalytic activity. Their reactions usually involve cleaving a nucleic acid. Two must be known: the peptidyl transferase of the large ribosomal subunit, which forms the peptide bond — so the ribosome is a ribozyme — and the RNA component of the spliceosome, which removes introns.

🧰 Replication, Transcription and What Happens to the Transcript Past Q

The six proteins of replication Past Q

1 · DNA polymerase — polymerises deoxyribonucleotides, 5′→3′ only. 2 · Helicase — processively unwinds the double helix. 3 · Topoisomerase — relieves the torsional strain that unwinding creates ahead of the fork. 4 · DNA primase — lays down the RNA primers that polymerase needs to start. 5 · Single-strand binding (SSB) proteins — keep the separated strands from re-annealing. 6 · DNA ligase — seals the nick between the growing chain and the Okazaki fragments of the lagging strand.

Nucleases — how nucleic acids are degraded Past Q

Classified by substrate: deoxyribonucleases (DNA), ribonucleases (RNA); and by where they cut: endonucleases cut internal bonds, exonucleases remove one nucleotide at a time from an end, and work in one direction only, either 3′→5′ or 5′→3′. Some cut both strands of a duplex, others only single strands. Digestion runs nucleic acid → nucleotides → nucleosides → free base + sugar.

Exons and introns Past Q

The primary transcript is much larger than the mature mRNA. Exons are the sequences that are expressed and end up in the mature message; introns are the non-coding intervening sequences that separate them, and in most genes they make up far more of the transcript than the exons do. In the nucleus the introns are excised and the exons spliced together; the mature mRNA (capped and polyadenylated) is then exported to the cytoplasm for translation. tRNA precursors can contain introns too.

Retroviruses run the arrow backwards Past Q

Their genome is RNA, not DNA. Reverse transcriptase — a viral RNA-dependent DNA polymerase — copies it into double-stranded DNA, which is then integrated into the host genome. From that "proviral" DNA the cell transcribes new viral genomes and viral mRNAs, and translates the viral proteins. Insertion is itself mutagenic: it can inactivate a host gene or deregulate its expression. HIV is the example, and the reason reverse transcriptase is a drug target.

🔠 The Genetic Code and the End of Translation Past Q

Triplet
Three bases per codon; 64 codons for 20 amino acids.

Degenerate
Most amino acids have several codons — some many, some only one. The third base matters least, which is where nearly all the degeneracy sits.

Unambiguous
Any given codon specifies one and only one amino acid.

Non-overlapping and unpunctuated
No codon shares a base with its neighbour, and once reading starts at the start codon there are no commas — the message is read as a continuous run of triplets until a stop codon.

Almost universal
The same triplets mean the same amino acids in all species and tissues — the exception being mitochondrial tRNAs, which read four codons differently.

Start and stop
Start AUG (methionine); stop UAA, UAG, UGA, which code for no amino acid at all.

Initiation — four steps Past Q

1 · The 80S ribosome dissociates into its 40S and 60S subunits. 2 · A ternary complex of initiator Met-tRNA + GTP + eIF-2 binds the 40S subunit → the 43S pre-initiation complex. 3 · The capped mRNA binds and is scanned to the first AUG → the 48S initiation complex. 4 · The 60S subunit joins, GTP is hydrolysed → the 80S initiation complex, with Met-tRNA already sitting in the P site.

Elongation — four steps, on a loop Past Q

1 · The next aminoacyl-tRNA is delivered to the A site by EF-1 with GTP — the codon decides which. 2 · Peptide bond formation by peptidyl transferase — the 60S ribozyme above: the chain is handed onto the A-site tRNA. 3 · Translocation — EF-2 with GTP moves the ribosome exactly three bases along the message. 4 · The now-empty deacylated tRNA is expelled from the P site through the E site. The message is read 5′→3′, the protein is built N-terminus first, and each residue costs four high-energy bonds — two to charge the tRNA, two GTP on the ribosome.

Termination, step by step Past Q

After many rounds of elongation, a stop codon arrives in the A site. No tRNA recognises it; instead releasing factor RF1 does, bound to a complex of RF3 with GTP. Together with peptidyl transferase this complex hydrolyses the bond between the finished peptide and the tRNA in the P site — a water molecule is added instead of an amino acid. The protein and the tRNA are released, then the mRNA leaves and the ribosome dissociates into its 40S and 60S subunits, ready for another cycle. The products of termination are therefore: the new protein, the mRNA, the tRNA, the two subunits, RF1, GDP-bound RF3 and inorganic phosphate.

Puromycin — the inhibitor to be able to explain Past Q

It is a structural analogue of tyrosinyl-tRNA. It enters the A site and is incorporated into the carboxyl-terminal position of the growing peptide, but it cannot be extended — so the chain is released prematurely. Because it exploits a feature common to both ribosome types, it inhibits protein synthesis on 70S (prokaryotic) and 80S (eukaryotic) ribosomes alike, which is exactly why it is useless as an antibiotic and useful in the laboratory.

🧬 Mutations — Every Type and Its Consequence Past Q

A mutation is a change in the nucleotide sequence. Point mutations are single-base changes, and they come in two chemical flavours: a transition swaps a purine for a purine or a pyrimidine for a pyrimidine; a transversion swaps a purine for a pyrimidine or the reverse.

🟢 Silent — no detectable effect

The new codon still specifies the same amino acid

Why: the code is degenerate

Usually the third base

Because of wobble the third position is the least sensitive, which is where most silent mutations sit.

▼

🟡 Missense — a different amino acid goes in

Effect depends entirely on which residue, and where

Three grades, and the exam wants the names

Acceptable

The substitute behaves like the original — the protein is indistinguishable from normal.

Partially acceptable

The protein works, but abnormally — partial function.

Unacceptable

The protein cannot function. Sickle-cell disease — Glu→Val in β-globin — is the standard example.

▼

🔴 Nonsense and frameshift — the severe ones

Either the message stops early, or everything after the change is nonsense

Both usually destroy the protein

Nonsense

The codon becomes a stop codon: premature termination, so only a fragment of the intended protein is made and it almost never works.

Frameshift — 1 or 2 bases

Insertion or deletion of a number of bases not divisible by three shifts the reading frame. Since the message is unpunctuated, the machinery does not notice: everything distal to the change is garbled, and a premature stop codon usually appears (or a normal one is read through). A later insertion can restore the frame, leaving only the stretch between the two changes scrambled.

Frameshift — 3 bases

Deletion or insertion of three bases adds or removes one amino acid and leaves the reading frame intact downstream — usually far milder.

📡 The Cyclic Nucleotides as Second Messengers Past Q

cAMP

Made from ATP by adenylyl cyclase when a Gs-coupled receptor is occupied — glucagon, adrenaline, ACTH, TSH — and destroyed by phosphodiesterase (which insulin activates, and caffeine and theophylline inhibit). It activates protein kinase A, and everything downstream follows: phosphorylase kinase → glycogen phosphorylase on, glycogen synthase off, hormone-sensitive lipase on, and — through phosphofructokinase-2/fructose-2,6-bisphosphatase — glycolysis down and gluconeogenesis up.

cGMP

Made by guanylyl cyclase. The soluble form is activated by nitric oxide, the membrane form by atrial natriuretic peptide. cGMP activates protein kinase G and causes relaxation of vascular smooth muscle — the mechanism of nitrates, and the reason a phosphodiesterase-5 inhibitor such as sildenafil works. In the retina, cGMP holds the sodium channels of the photoreceptor open in the dark.

And GTP, which is not a messenger but a currency

GTP drives protein synthesis (initiation, elongation and termination all spend it), is the product of the one substrate-level phosphorylation of the Krebs cycle (succinyl-CoA synthetase), pays for the argininosuccinate step's cousin reactions in gluconeogenesis (PEP carboxykinase uses GTP), and is the switch of every G protein: bound GTP = on, hydrolysis to GDP = off.

Calcium, Minerals & Vitamins

Resit topics 36–38 · disorder 14 · past questions 25, 53, 119, 134, 154, 155, 172, 176, 178

🦴 Calcium Homeostasis Topic 36Past Q 155

Plasma calcium is held at 2.2–2.6 mmol/L (about 9–10.5 mg/dL), of which roughly half is free ionised Ca²⁺ — the biologically active fraction — with most of the rest bound to albumin. Three hormones act on three organs: bone, kidney, intestine.

Parathyroid hormone — raises calcium

Secreted by the chief cells when ionised Ca²⁺ falls (sensed by the calcium-sensing receptor). Bone: stimulates osteoclastic resorption (indirectly, through osteoblasts) → releases Ca²⁺ and phosphate. Kidney: increases Ca²⁺ reabsorption in the distal tubule, decreases phosphate reabsorption (phosphaturic), and activates 1α-hydroxylase. Net: ↑ plasma Ca²⁺, ↓ plasma phosphate.

Calcitriol — raises calcium and phosphate

1,25-dihydroxycholecalciferol is the active hormone. Intestine: increases absorption of both Ca²⁺ and phosphate by inducing calbindin — its principal action. Bone: permits mineralisation by supplying both ions. Kidney: increases reabsorption of both.

Calcitonin — lowers calcium

From the parafollicular C cells of the thyroid when calcium is high; inhibits osteoclasts and increases urinary calcium. Physiologically a minor player in adults.

Phosphate and the bone mineral Topic 37

Calcium and phosphate are deposited as hydroxyapatite. Their product in plasma is nearly constant, so the two move reciprocally under PTH. Phosphate is also essential for ATP, nucleic acids, phospholipids, 2,3-BPG and the phosphate buffer. Acute hypophosphataemia occurs in the refeeding syndrome, when carbohydrate is given to a malnourished patient and phosphate is rapidly consumed by phosphorylation.

Reading a calcium result

Hyperparathyroidism: ↑ Ca²⁺, ↓ phosphate, ↑ PTH. Hypoparathyroidism: ↓ Ca²⁺, ↑ phosphate, tetany, Chvostek and Trousseau signs. Vitamin D deficiency: ↓ Ca²⁺ and ↓ phosphate with secondary ↑ PTH — rickets in children, osteomalacia in adults. Remember also that alkalosis lowers ionised calcium (more binds to albumin) and causes tetany at a normal total calcium, and that a low albumin lowers total calcium without affecting the ionised fraction.

⚗️ Macro- and Microelements Topic 37Past Q 154

Sodium, potassium, chloride

Na⁺ — the main extracellular cation; osmolality, extracellular volume, blood pressure, and the gradient that powers SGLT and other secondary active transport. K⁺ — the main intracellular cation; resting membrane potential and cardiac excitability; it shifts out of cells in acidosis and into cells with insulin. Cl⁻ — the main extracellular anion; gastric HCl, and the chloride shift.

Magnesium Past Q 154

The second most abundant intracellular cation. It is the obligatory cofactor of every reaction that uses ATP — the true substrate of kinases is the Mg-ATP complex — and of over 300 enzymes. It stabilises DNA, RNA and ribosomes, antagonises calcium at the neuromuscular junction, and is required for PTH secretion and action, which is why hypomagnesaemia causes a hypocalcaemia that will not correct until magnesium is replaced. Deficiency: alcoholism, diarrhoea, diuretics; it produces tremor, tetany and arrhythmias.

Trace elements — each tied to its enzyme

Iron — haemoglobin, myoglobin, cytochromes, catalase. Zinc — carbonic anhydrase, alcohol dehydrogenase, ALA dehydratase, zinc-finger transcription factors; deficiency gives poor wound healing, dermatitis, hypogonadism and impaired taste. Copper — cytochrome c oxidase, superoxide dismutase, lysyl oxidase, tyrosinase, dopamine β-hydroxylase, ceruloplasmin. Selenium — glutathione peroxidase and the deiodinases. Iodine — thyroid hormones. Manganese — mitochondrial SOD, pyruvate carboxylase. Molybdenum — xanthine oxidase, sulfite oxidase. Chromium — potentiates insulin action. Fluoride — hardens enamel as fluoroapatite.

The two copper diseases

Wilson disease — ATP7B: copper cannot leave the hepatocyte into bile or be loaded onto ceruloplasmin, so it accumulates in liver, brain and cornea. Serum ceruloplasmin is low despite copper overload; Kayser–Fleischer rings; treated by chelation. Menkes disease — ATP7A, X-linked: copper is trapped in the enterocyte and the tissues are starved, so all the copper enzymes fail — kinky, brittle hair, connective-tissue failure (lysyl oxidase) and neurodegeneration.

💊 Vitamins — Role and the Processes They Serve Topic 38 · Disorder 14Past Q 53, 119, 134, 172, 176, 178

A — retinol Past Q 119, 178

Vision — 11-cis-retinal is the chromophore of rhodopsin; growth and differentiation — retinoic acid acts on nuclear receptors; epithelial integrity and immunity; antioxidant as β-carotene. Transported by retinol-binding protein. Deficiency: night blindness → xerophthalmia → keratomalacia and blindness, plus follicular hyperkeratosis. Excess is teratogenic and causes raised intracranial pressure and liver damage.

D — calciferol Past Q 25, 172

Synthesis: 7-dehydrocholesterol + UVB in skin → cholecalciferol (D3) → 25-hydroxylation in the liver → 1α-hydroxylation in the kidney (stimulated by PTH, low phosphate and low calcium) → calcitriol. Function: intestinal absorption of calcium and phosphate; a steroid-type hormone acting on nuclear receptors. Deficiency: rickets and osteomalacia. Toxicity — it is the most toxic vitamin in overdose: hypercalcaemia, hypercalciuria, nephrocalcinosis, metastatic calcification.

E — tocopherol Past Q 176

The most important natural lipid-soluble antioxidant. It sits in membranes and lipoproteins and terminates the chain reaction of lipid peroxidation by donating a hydrogen to the peroxyl radical, protecting polyunsaturated fatty acids, LDL and the red-cell membrane. It is itself regenerated by vitamin C. Deficiency (rare, in fat malabsorption): haemolysis and neurological damage.

K — phylloquinone

Cofactor for the γ-carboxylation of glutamate in clotting factors II, VII, IX, X and proteins C and S, and in bone osteocalcin — creating the Ca²⁺-binding sites. Supplied partly by gut bacteria. Deficiency: bleeding, prolonged prothrombin time — in the newborn (hence the routine injection), in fat malabsorption and with prolonged antibiotics. Warfarin is its antagonist.

C — ascorbic acid

A reducing agent: keeps Fe²⁺ and Cu⁺ reduced for prolyl and lysyl hydroxylase in collagen synthesis (its central role), for dopamine β-hydroxylase and for carnitine synthesis; enhances non-haem iron absorption; regenerates vitamin E; water-soluble antioxidant. Deficiency = scurvy: bleeding gums, loose teeth, perifollicular haemorrhages, poor wound healing, bone and joint pain.

The B group

B1 thiamine → TPP: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain ketoacid dehydrogenase, transketolase. Deficiency: beri-beri and Wernicke–Korsakoff. B2 riboflavin → FMN/FAD. B3 niacin → NAD⁺/NADP⁺; also made from tryptophan; deficiency pellagra — dermatitis, diarrhoea, dementia. B5 pantothenate → coenzyme A and ACP. B6 pyridoxine → PLP: transaminases, decarboxylases, glycogen phosphorylase, ALA synthase, cystathionine β-synthase. B7 biotin: the carboxylases. B9 folate → THF: one-carbon transfers; deficiency gives megaloblastic anaemia and neural-tube defects. B12 cobalamin: methionine synthase and methylmalonyl-CoA mutase; needs intrinsic factor; deficiency gives megaloblastic anaemia plus subacute combined degeneration.

Niacin overload and thiamine deficiency Past Q 53

Pharmacological doses of niacin (used to lower lipids) cause cutaneous flushing and itching — a prostaglandin-mediated vasodilation — plus hyperglycaemia, hyperuricaemia and hepatotoxicity. Thiamine deficiency blocks the three dehydrogenase complexes and transketolase, so pyruvate and lactate accumulate: dry beri-beri (peripheral neuropathy), wet beri-beri (high-output cardiac failure and oedema) and, in alcoholism, Wernicke encephalopathy — confusion, ophthalmoplegia, ataxia — progressing to Korsakoff psychosis. The classic laboratory sign is a rise in erythrocyte transketolase activity after adding TPP. Never give glucose before thiamine to a suspected case: the glucose load consumes the remaining thiamine and precipitates the encephalopathy.

Vitamin A — the two families, and the one-way street Past Q

Two groups have vitamin A activity: the retinoids (retinol, retinaldehyde, retinoic acid) from animal food, and the carotenoids (α-, β-, γ-carotene, cryptoxanthin) from plants. In the intestinal mucosa, carotene dioxygenase cleaves β-carotene to retinaldehyde, which is reduced to retinol, esterified and packed into chylomicrons; the enzyme's activity is low, so much β-carotene reaches the circulation unchanged. Inside the body: retinol ⇄ retinaldehyde is reversible, but retinaldehyde → retinoic acid is not — which is why retinoic acid supports growth and differentiation but cannot restore vision.

Riboflavin deficiency, and two toxicity notes

B2 deficiency is widespread but never fatal, because riboflavin released from degraded enzymes is efficiently re-used. It shows as cheilosis (cracked lips), angular stomatitis, desquamation, a sore inflamed tongue (glossitis) and seborrhoeic dermatitis. Vitamin D: some infants react to as little as 50 µg/day — yet sunlight can never cause toxicity, because the supply of 7-dehydrocholesterol is limited and prolonged ultraviolet exposure converts previtamin D into inactive compounds. Thiamine has a second job beyond TPP: thiamine triphosphate has a role in nerve conduction.

Thyroid & Steroid Hormones

Resit topics 39–40 · past questions 161, 164, 167

🦋 Thyroid Hormone Synthesis Topic 39

Substrates: iodide and the tyrosine residues of thyroglobulin. Everything happens on the thyroglobulin scaffold, at the apical membrane and in the follicular colloid.

1 · Trapping

Iodide is taken from the blood against a steep gradient by the Na⁺/I⁻ symporter (NIS) — an active, TSH-stimulated step, blocked by thiocyanate and perchlorate.

2 · Oxidation and organification

Thyroid peroxidase (TPO), using H₂O₂, oxidises I⁻ and attaches it to tyrosine residues of thyroglobulin, giving MIT (monoiodotyrosine) and DIT (diiodotyrosine).

3 · Coupling

Also by TPO: DIT + DIT → T4; MIT + DIT → T3. The hormones remain part of thyroglobulin in the colloid, which is the body's only hormone store of any size — enough for weeks.

4 · Release and activation

TSH triggers endocytosis of colloid and proteolysis of thyroglobulin, freeing T4 and T3. About 90% of secretion is T4, which is a prohormone: peripheral 5′-deiodinase — a selenium enzyme — converts it to the far more active T3, or, by removing the other iodine, to inactive reverse T3 in illness and starvation.

Transport, control and actions

In plasma they travel bound to TBG, transthyretin and albumin — over 99% bound, only the free fraction active, and T4's half-life is about a week. Control is the HPT axis: TRH → TSH → thyroid, with negative feedback by free T3/T4 on the pituitary. T3 binds nuclear receptors and changes gene transcription: it raises the basal metabolic rate and thermogenesis (more Na⁺/K⁺-ATPase), stimulates glycolysis, gluconeogenesis, lipolysis and cholesterol turnover, increases the number of β-adrenergic receptors, and is essential for brain and skeletal development. Thioureas (propylthiouracil, methimazole) inhibit TPO; PTU also blocks peripheral deiodination.

Hyper- and hypothyroidism Topic 39

Hyperthyroidism (Graves disease) — TSH-receptor stimulating antibodies. ↑ free T4/T3, ↓ TSH. Weight loss despite good appetite, heat intolerance, sweating, tachycardia and palpitations, tremor, anxiety, diarrhoea, goitre, and in Graves also exophthalmos. Cholesterol is low.
Hypothyroidism (Hashimoto thyroiditis; or iodine deficiency) — ↓ free T4, ↑ TSH in primary disease. Weight gain, cold intolerance, fatigue, bradycardia, constipation, dry skin, hair loss, slowed reflexes, raised cholesterol, and myxoedema. In the newborn, untreated hypothyroidism causes cretinism — irreversible intellectual disability — which is why it is screened for. Iodine deficiency causes goitre because low T4 releases TSH from feedback and TSH is a growth factor for the gland.

🧬 Steroid Hormones — Classification, Synthesis, Function, Transport Topic 40

The two routes from pregnenolone to testosterone Past Q

Five enzyme activities in three proteins get you there: 3β-hydroxysteroid dehydrogenase with Δ5,4-isomerase, 17α-hydroxylase and 17,20-lyase (one protein, two activities), and 17β-hydroxysteroid dehydrogenase. They can act in either order, which is why there are two named pathways. Δ4, the progesterone pathway: pregnenolone → progesterone → 17α-hydroxyprogesterone → androstenedione → testosterone. Δ5, the dehydroepiandrosterone pathway: pregnenolone → 17α-hydroxypregnenolone → DHEA → Δ5-androstenediol → testosterone — the route the human testis mainly uses. The rate-limiting step in either case is the LH-dependent one below. Downstream, 5α-reductase converts testosterone into dihydrotestosterone in prostate, external genitalia and skin — about 400 µg a day against 5 mg of testosterone, but far more potent locally.

Past Q 161, 164, 167

Classification — five classes

Glucocorticoids (cortisol) · mineralocorticoids (aldosterone) · androgens (testosterone, DHEA) · oestrogens (oestradiol) · progestogens (progesterone). Plus calcitriol, a steroid-derived hormone. All are made from cholesterol. The four rings can be welded together two ways, and the exam asks for the names: in the 5α (trans) series rings A and B lie in one flat plane — cholesterol, testosterone, dihydrotestosterone and the adrenal steroids; in the 5β (cis) series ring A is bent up almost at a right angle — the bile acids, and that kink is what puts all their hydroxyls on one face and turns the molecule into a detergent. Individual substituents are labelled the same way: β drawn as a solid bond toward the reader, α dashed away from it.

The general pathway

Cholesterol → pregnenolone is the common, rate-limiting first step: StAR protein carries cholesterol to the inner mitochondrial membrane, where P450scc (desmolase) cleaves the side chain. It is stimulated by ACTH in the adrenal and by LH in the gonads. Everything after that is a series of hydroxylations requiring O₂ and NADPH, plus isomerases and lyases, in mitochondria and smooth ER.

Aldosterone — zona glomerulosa Past Q 167

Pregnenolone → progesterone → (21-hydroxylase) 11-deoxycorticosterone → (11β-hydroxylase) corticosterone → (18-hydroxylase / 18-dehydrogenase, found only here) aldosterone. The zona glomerulosa lacks 17α-hydroxylase, so it cannot make cortisol or androgens, and it is controlled by angiotensin II and K⁺ rather than by ACTH. Action: Na⁺ reabsorption and K⁺/H⁺ excretion in the distal nephron → ↑ blood volume and pressure.

Cortisol — fasciculata and reticularis

Pregnenolone → (17α-hydroxylase) 17-OH-pregnenolone/progesterone → (21-hydroxylase) 11-deoxycortisol → (11β-hydroxylase) cortisol, the most potent natural glucocorticoid. Actions: ↑ gluconeogenesis and hepatic glycogen, ↑ muscle proteolysis and lipolysis, and anti-inflammatory and immunosuppressive effects. Controlled by CRH → ACTH with negative feedback.

Male gonadal steroids Past Q 161

Made by the Leydig cells of the testis under LH; FSH acts on Sertoli cells to support spermatogenesis. Testosterone is made by the Δ5 route on the card above, and aromatase converts a small part of it to oestradiol. Feedback: testosterone inhibits LH, inhibin from Sertoli cells inhibits FSH.

Oestrogen synthesis Past Q 164

A two-cell process: LH drives the theca cells to make androstenedione and testosterone, which cross to the granulosa cells, where FSH-induced aromatase converts them to oestrone and oestradiol. Aromatisation is three hydroxylations, each needing O₂ and NADPH. Oestradiol is the main ovarian oestrogen; oestriol predominates in pregnancy (placenta); after the menopause, oestrogen comes mainly from peripheral aromatisation of androstenedione to oestrone in adipose tissue. The corpus luteum secretes progesterone. Aromatase inhibitors are used in breast cancer.

Transport in the blood, and mechanism Topic 40

Steroids are lipophilic, so they travel bound to plasma proteins — CBG (transcortin) for cortisol and progesterone, SHBG for testosterone and oestradiol, and albumin for the rest. Binding gives them a long half-life and a circulating reservoir, and buffers sudden changes; only the small free fraction is active (about 8% for cortisol, 1–3% for testosterone). Aldosterone has no specific carrier and therefore a short half-life. Being lipophilic, they cross the plasma membrane and bind intracellular receptors; the complex dimerises, enters the nucleus and binds hormone response elements, changing transcription — which is why steroid effects take hours, unlike the minutes of a peptide hormone.

Congenital adrenal hyperplasia

21-hydroxylase deficiency is the commonest form: cortisol and aldosterone cannot be made, precursors are diverted into androgens, and the loss of cortisol feedback drives ACTH up and enlarges the adrenals. Result: salt-wasting with hyponatraemia and hyperkalaemia, plus virilisation; the marker is a raised 17-OH-progesterone.

Diabetes, Obesity & Hypoglycaemia

Disorders 10–12 on the resit list

🩺 Diabetes Mellitus and Its Complications Disorder 10Lecturer’s priority

Diagnosis

Fasting plasma glucose ≥ 7.0 mmol/L (126 mg/dL), or 2-hour OGTT ≥ 11.1 mmol/L (200 mg/dL), or a random glucose ≥ 11.1 with symptoms, or HbA1c ≥ 6.5%. Impaired fasting glucose 5.6–6.9 mmol/L; impaired glucose tolerance 7.8–11.0 at two hours.

Type 1 versus type 2

Type 1 — autoimmune destruction of β cells, absolute insulin deficiency, usually young and lean, prone to ketoacidosis, C-peptide absent, requires insulin. Type 2 — insulin resistance with relative deficiency and progressive β-cell failure, usually older and obese, prone to the hyperosmolar non-ketotic state rather than ketoacidosis (residual insulin restrains lipolysis), C-peptide normal or high.

The metabolic picture when uncontrolled

Insulin is absent or unheard, glucagon dominates: hyperglycaemia (uptake fails, gluconeogenesis and glycogenolysis run unopposed), glucosuria once the renal threshold of about 10 mmol/L is passed, osmotic diuresis with polyuria, polydipsia and dehydration, unchecked lipolysis and ketogenesis, hypertriacylglycerolaemia, and muscle proteolysis with weight loss in spite of a raised appetite (polyphagia). In ketoacidosis: metabolic acidosis with a high anion gap, Kussmaul breathing, acetone on the breath, and hyperkalaemia in the blood despite a depleted total-body potassium.

Chronic complications — the mechanisms

Non-enzymatic glycation of proteins → advanced glycation end-products, which cross-link collagen and damage the basement membrane and vessel wall; HbA1c is the measurable version and reflects the previous 2–3 months. The polyol pathway: aldose reductase converts glucose to sorbitol, which is trapped in lens, retina, nerve and kidney and draws in water — cataract and neuropathy. Plus oxidative stress and protein kinase C activation. Clinically: microvascular — retinopathy, nephropathy, neuropathy; macrovascular — accelerated atherosclerosis, ischaemic heart disease, stroke, diabetic foot.

🍔 Obesity and the Metabolic Syndrome Disorder 11

Measuring it

BMI = weight (kg) / height (m)²: normal 18.5–24.9, overweight 25–29.9, obese ≥ 30. Waist circumference and the distribution matter more than BMI alone: visceral (central, "apple") fat is far more metabolically dangerous than subcutaneous gluteofemoral ("pear") fat, because it is more lipolytic and drains straight into the portal vein, exposing the liver to free fatty acids.

Adipose tissue as an endocrine organ

Leptin — made in proportion to fat mass; acts on the hypothalamus to suppress appetite and increase expenditure. Obesity is a state of leptin resistance, not leptin deficiency. Adiponectin — falls in obesity; it improves insulin sensitivity and is anti-inflammatory. TNF-α, IL-6, resistin — rise and promote insulin resistance. Ghrelin from the stomach stimulates appetite before meals.

Metabolic syndrome

Three or more of: central obesity, fasting glucose ≥ 5.6 mmol/L or diagnosed diabetes, triacylglycerol ≥ 1.7 mmol/L, HDL < 1.0 (men) or < 1.3 mmol/L (women), and blood pressure ≥ 130/85. Its core is insulin resistance, and it carries a high risk of type 2 diabetes and cardiovascular disease.

Why insulin resistance produces this pattern

Insulin fails to suppress hormone-sensitive lipase, so free fatty acids pour into the liver: VLDL output rises → high TAG, and CETP exchange then leaves low HDL and small dense LDL. In the liver the same fatty acids drive steatosis; in muscle they impair glucose uptake further. Weight reduction — caloric restriction plus exercise — reverses much of it.

📉 Hypoglycaemia Disorder 12

Plasma glucose below about 3.9 mmol/L (70 mg/dL), classically defined by Whipple's triad: low glucose, typical symptoms, and relief when glucose is given.

Symptoms

Adrenergic (first, from the counter-regulatory response): sweating, tremor, palpitations, anxiety, hunger, pallor. Neuroglycopenic (as the brain runs out): confusion, difficulty speaking, blurred vision, behavioural change, seizures, coma. Repeated episodes blunt the adrenergic warning — hypoglycaemia unawareness.

The counter-regulatory response

Glucagon first (glycogenolysis and gluconeogenesis) and adrenaline alongside it; then cortisol and growth hormone over hours. Insulin secretion switches off. Together they defend the brain, which cannot make or store glucose.

Types

Insulin-induced — the commonest, from too much insulin or sulfonylurea, a missed meal or unusual exercise. Postprandial (reactive) — an exaggerated insulin response a few hours after a meal, notably after gastric surgery. Fasting — insulinoma, adrenal or pituitary insufficiency, liver failure, some tumours. Distinguish exogenous insulin (C-peptide low) from an insulinoma or sulfonylurea (C-peptide high).

Alcohol-induced hypoglycaemia

Ethanol raises NADH/NAD⁺, diverting pyruvate to lactate and oxaloacetate to malate, so gluconeogenesis is blocked. In someone who has been fasting and has already used up liver glycogen, this produces a dangerous hypoglycaemia — the classic combination of alcohol on an empty stomach.

Named Disorders & Lab Odds and Ends

The remaining items from both lists, gathered in one place

🧬 Phenylketonuria and Cystinuria Disorders 8 & 9

Phenylketonuria

Phenylalanine hydroxylase deficiency (or, rarely, of its cofactor BH₄). Phenylalanine accumulates and is diverted to phenylpyruvate, phenylacetate and phenyllactate — the phenylketones, which give the urine its musty, mousy odour. Tyrosine becomes essential, so melanin falls: fair skin and hair. Untreated it causes severe intellectual disability, seizures and eczema; the infant is normal at birth, which is why newborn screening matters. Treatment: a phenylalanine-restricted diet with tyrosine supplementation, continued for life — and strictly during pregnancy, because maternal PKU damages a fetus that does not itself have the disease.

Cystinuria

Not a metabolic block but a transport defect: the shared carrier for the dibasic amino acids — cystine, ornithine, arginine and lysine ("COAL") — fails in the renal tubule and the intestine. Because cystine is the least soluble of the four, it precipitates and forms recurrent radio-opaque kidney stones; the other three simply appear in urine without harm. Autosomal recessive. Management: high fluid intake, alkalinisation of the urine, and chelating agents such as penicillamine.

🧪 Loose Ends from the Question List

Three classical tests on bile and cholesterol Past Q

Hay's sulfur test — flowers of sulfur sprinkled on the surface of urine or bile sink if bile salts are present, because bile salts lower surface tension; they float on normal urine. Pettenkofer's test — bile acids plus sucrose and concentrated sulfuric acid give a red-violet ring. Salkowski's test — cholesterol in chloroform layered under concentrated sulfuric acid turns the upper layer red and the acid layer green with fluorescence. Bile's other examinable property is simply that it emulsifies fat: shake oil with water and it separates, shake it with bile and a stable emulsion forms.

Urinalysis — the three parts of the report Past Q

1 · Physical: volume, colour, odour, clarity, specific gravity and pH. 2 · Chemical: protein, reducing sugar, ketone bodies, blood, bile salts and bile pigments. 3 · Microscopic: white cells, red cells and casts. Read together they separate a glomerular from a tubular from a post-renal problem — and they are where several of the biochemistry findings in this course (glucosuria, ketonuria, bilirubinuria, urobilinogen, proteinuria) are actually detected.

The biuret reaction — the protein test Past Q

Reagents: the sample (egg albumin in the practical), NaOH and dilute CuSO₄. In alkali Cu²⁺ chelates the nitrogens of peptide bonds and the blue solution turns violet (pink-violet with short peptides); no change of colour = no peptide bond. More protein means more peptide bonds, more chelate and more colour — which is why it is used to measure total protein. The catch the examiner is looking for: it needs at least two peptide bonds, so a dipeptide gives a negative result — carnosine, anserine and free amino acids are the standard negative examples. (Contrast ninhydrin, which detects free α-amino groups instead.)

Kay's test — how BAO and MAO are actually measured Past Q

Gastric juice is aspirated through a tube sitting in the pyloric antrum and collected hour by hour: first unstimulated (basal), then after histamine or pentagastrin, which peaks the secretion at about 15 minutes. The volume is measured, then a 5 mL sample is titrated with 0.1 M NaOH using phenol red until the colour matches the standard, and the NaOH used is noted. Output (mmol HCl/h) = V(NaOH) × 0.1 × total volume of juice ÷ volume titrated. Reference values: BAO ≈ 1–5 mmol/h and MAO ≈ 5–25 mmol/h (sources quote slightly different ceilings).

Starling forces Past Q 165

Fluid movement across a capillary is set by four pressures: capillary hydrostatic pressure (pushes out), interstitial hydrostatic pressure (pushes in), plasma colloid osmotic (oncotic) pressure — mainly albumin — which pulls fluid in, and interstitial oncotic pressure, which pulls out. Filtration dominates at the arterial end, reabsorption at the venous end, and the lymphatics remove the small surplus. Oedema follows a rise in capillary hydrostatic pressure (heart failure, venous obstruction), a fall in plasma oncotic pressure (hypoalbuminaemia — nephrotic syndrome, liver failure, kwashiorkor), increased permeability (inflammation) or blocked lymphatics.

Experimental detection of inorganic phosphate Past Q 71

The classical colorimetric method: phosphate reacts with ammonium molybdate in acid to form phosphomolybdate, which is then reduced (Fiske–Subbarow reagent, ascorbic acid or stannous chloride) to an intensely coloured molybdenum blue. The absorbance is read on a spectrophotometer and is proportional to the phosphate concentration against a standard curve. The same principle is used to follow any reaction that releases Pi — for example ATPase or phosphatase activity.

Formation of hydrochloric acid in the stomach Past Q 78

In the parietal cell: CO₂ + H₂O → H₂CO₃ by carbonic anhydrase, dissociating to H⁺ and HCO₃⁻. The H⁺/K⁺-ATPase (the proton pump, the target of omeprazole) pumps H⁺ into the lumen in exchange for K⁺; Cl⁻ follows through a channel; and the HCO₃⁻ leaves across the basolateral membrane in exchange for Cl⁻ — the alkaline tide in venous blood after a meal. Secretion is stimulated by gastrin, histamine (H₂) and acetylcholine, and inhibited by somatostatin. Functions: denature protein, activate pepsinogen, kill microorganisms, and help solubilise and reduce iron and calcium.

Endergonic and exergonic reactions Past Q 72

Exergonic: ΔG negative — energy released, the reaction proceeds spontaneously (glycolysis, β-oxidation, ATP hydrolysis at about −30 kJ/mol). Endergonic: ΔG positive — energy required, will not proceed alone (gluconeogenesis, urea synthesis, protein synthesis). Cells drive the second with the first by energetic coupling: because ΔG values are additive, linking an endergonic step to ATP hydrolysis in a shared enzyme or shared intermediate makes the overall ΔG negative. That is the entire logic of ATP as the energy currency.

Amino Acids & Protein Structure

Frequently tested: amino acid carbon counting, polar vs nonpolar classification, isoelectric point (pI), bonds stabilizing protein structure, collagen synthesis & types

🔤 The Basic Structure

Every amino acid has a central alpha-carbon bonded to: –NH₂, –COOH, –H, and an R-group. All human protein amino acids are in the L-configuration. Glycine is the only amino acid with no chiral carbon.

📊 Classification of Side Chains

Category	Amino Acids	Key Feature
Nonpolar	Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met	Hydrophobic; buried inside protein
Uncharged Polar	Ser, Thr, Tyr, Asn, Gln, Cys	H-bonds; Ser/Thr/Tyr can be phosphorylated
Acidic (–)	Asp, Glu	Negatively charged at physiological pH
Basic (+)	Lys, Arg, His	Positively charged; His is key buffer

Common Mistake

Methionine is nonpolar. Asparagine and Threonine are uncharged polar, not nonpolar.

🧮 Amino Acid Carbon Counting (Exam Tested)

Some exams ask which amino acids do NOT have 5 carbons. Count the alpha-carbon plus all side-chain carbons:

Amino Acid	Total Carbons	Note
Glycine	2	Smallest
Alanine	3
Serine	3	NOT 5 — commonly tested
Proline	5	Has 5 carbons
Valine	5	Has 5 carbons
Lysine	6	NOT 5
Glutamate	5	Has 5 carbons

Exam Q: "Which does NOT have 5 carbons?"

Answer: Serine (3C) and Lysine (6C) do not have 5 carbons. Valine, Proline, and Glutamate all have exactly 5.

🔀 Glucogenic, Ketogenic & Essential Amino Acids

What do these terms mean?

Glucogenic: degraded to pyruvate, OAA, α-ketoglutarate, succinyl-CoA, or fumarate → can enter gluconeogenesis → make glucose.
Ketogenic: degraded to acetyl-CoA or acetoacetate → can only form ketone bodies or fatty acids → CANNOT make glucose.
Both (glucogenic + ketogenic): degraded to products that enter BOTH pathways.

Category	Amino Acids	Exam Tip
Purely Ketogenic	Leu, Lys	The ONLY two that are purely ketogenic. Cannot make glucose. Remember: Leu & Lys = Ketogenic.
Both	Ile, Phe, Tyr, Trp, Thr	These 5 are both glucogenic AND ketogenic. Their catabolism produces precursors for both pathways.
Purely Glucogenic	All remaining amino acids (Ala, Arg, Asp, Asn, Cys, Glu, Gln, Gly, His, Met, Pro, Ser, Val)	The majority — can make glucose. Degraded to pyruvate or Krebs intermediates.

Essential Amino Acids (cannot be synthesized — must come from diet)

PVT TIM HaLL (mnemonic):
Phenylalanine · Valine · Threonine · Tryptophan · Isoleucine · Methionine · Histidine · Leucine · Lysine

Conditionally essential: Tyrosine (if Phe deficient — PKU), Cysteine (if Met deficient), Arginine (in growth/illness).

Non-Essential Amino Acids (body can synthesize)

Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, Pro, Ser, Tyr

Key synthesis links: Tyr from Phe (needs PAH + BH4). Cys from Met (needs CBS + B6). Arg in kidney (from citrulline).

Exam Traps

• Methionine is NONPOLAR (despite containing sulfur) — among the sulfur-containing amino acids, only Cysteine is polar
• Leu & Lys are the ONLY purely ketogenic — cannot make glucose
• In PKU: Tyr becomes conditionally essential (PAH is blocked)

⚡ Isoelectric Point (pI)

pH = pI

Net charge = 0 (zwitterion). Cannot move in electric field. Least soluble.

pH < pI

Net positive charge → moves toward cathode (–).

pH > pI

Net negative charge → moves toward anode (+).

Still Ionized at pI!

–NH₃⁺ and –COO⁻ both present but charges cancel. NOT unionized.

🏗️ Four Levels of Protein Structure

1
Primary structure — the sequence of amino acids linked by peptide bonds (covalent). This is directly encoded by DNA. Denaturation does NOT destroy this level — peptide bonds survive.
2
Secondary structure — local regular folding patterns: α-helix or β-sheet, stabilized by hydrogen bonds between backbone –C=O and –N-H groups. Proline and Glycine destabilize the α-helix (proline has no NH for H-bonding; glycine is too flexible).
3
Tertiary structure — the overall 3D shape of a single polypeptide chain. Stabilized by: hydrogen bonds, hydrophobic interactions (nonpolar side chains buried inside), ionic bonds (electrostatic interactions), and disulfide bridges (Cys–Cys, the ONLY covalent bonds in tertiary structure).
4
Quaternary structure — two or more polypeptide chains (subunits) assembled together. Example: hemoglobin has 4 subunits (2α + 2β). Stabilized by the same forces as tertiary structure plus possible inter-chain disulfide bonds.

Cysteine — Heavily Emphasized by Teacher

Cysteine is unique: it contains a sulfur atom in its thiol (–SH) side chain. Two cysteine residues can be oxidized to form a disulfide bridge (–S–S–) — a covalent bond that stabilizes tertiary (and quaternary) protein structure. This is the only covalent bond within/between protein chains other than the peptide bond. Cysteine is also the active component of glutathione. The teacher specifically emphasized Cysteine and disulfide bonds.

Exam Q: "Tertiary structure is stabilized by all EXCEPT?"

Answer: Peptide bonds. Peptide bonds maintain the PRIMARY structure (the sequence). Tertiary structure is stabilized by disulfide bonds, hydrogen bonds, hydrophobic interactions, and ionic bonds — all non-peptide forces.

🦷 Collagen

Most abundant protein. Fibrous. Every 3rd residue = Gly. Rich in Pro/Hydroxyproline. Requires Vitamin C for hydroxylation by Prolyl Hydroxylase and Lysyl Hydroxylase.

Two exam-tested specifics ★ asked 2024/25

• Type IV collagen = the collagen of basement membranes (forms a mesh, not fibrils). Type I = bone/skin.
• Glycosylation in collagen synthesis is added onto hydroxylysine residues — and it happens after hydroxylation (order: hydroxylation → then glycosylation).

Collagen Diseases

Ehlers-Danlos: defect in collagen → hyperextensible skin/joints. Osteogenesis Imperfecta: type I collagen defect → brittle bones. Scurvy: Vit C deficiency → impaired hydroxylation → bleeding gums.

🧠 Prions & Amyloid ⚪ Never appeared in past exams

PrP^sc: β-sheet rich, insoluble, resistant to proteolysis. Causes CJD. Normal PrP^c is α-helical. Amyloid-β: neurotoxic β-sheet aggregates in Alzheimer's disease.

Kept for completeness — this topic has not been tested in any of the available past papers, so treat it as low priority.

🔤 Characteristics of Amino Acids — The 2026 Question, Option by Option 2026

The recollection gives three options. Two are true as stated; one is a trap that is only half right.

✅ "Methionine has S in its side chain and is non-polar"

True. Methionine's side chain is –CH₂–CH₂–S–CH₃: a thioether. The sulfur is bonded to carbon on both sides, so it carries no ionisable hydrogen and the whole chain behaves as hydrophobic / non-polar. Cysteine also contains sulfur but as a free –SH (thiol), which is polar, ionises around pH 8.3, and forms disulfide bridges. Methionine and cysteine are the only two sulfur-containing amino acids.

❌ "Glutamate has a hydroxyl group and can therefore be phosphorylated"

False — and this is the trap. Glutamate's side chain is a carboxyl (–COOH), not a hydroxyl; it is acidic and negatively charged at pH 7.4. The amino acids that carry a side-chain –OH and are phosphorylated by kinases are serine, threonine and tyrosine. If an option puts "hydroxyl" and "glutamate" in the same sentence, it is wrong.

✅ "Proline's ring makes it rigid"

True. Proline is the only imino acid: its side chain loops back and bonds to the α-amino nitrogen, forming a five-membered pyrrolidine ring. That ring restricts rotation about the N–Cα bond, so proline is conformationally rigid — it breaks α-helices and β-sheets, creates turns and kinks, and is what makes the collagen helix possible.

The other classics from older papers

Methionine is non-polar, not polar (asked repeatedly). At the isoelectric point an amino acid does not have "no ionisable groups" — it has one negative and one positive charge that cancel. Histidine has an important buffering role (imidazole pK ≈ 6.0, closest to blood pH). Racemases interconvert D- and L-isomers. Threonine and tyrosine both carry –OH.

Carbon counting — asked directly

Arginine has 6 carbons (as does citrulline); ornithine has 5; the four-carbon amino acids are aspartate, asparagine and threonine (and homoserine). Glycine 2 · alanine 3 · serine and cysteine 3 · valine, proline, glutamate, glutamine, methionine 5 · leucine, isoleucine, lysine 6 · histidine 6 · phenylalanine and tyrosine 9 · tryptophan 11.

Ketogenic and essential 2026

Essential (10): Phe, Val, Thr, Trp, Ile, Met, His, Arg, Leu, Lys. Purely ketogenic (2): leucine and lysine. Both glucogenic and ketogenic (5): Phe, Tyr, Trp, Ile, Thr. Everything else is purely glucogenic. Notice the overlap: Leu and Lys are both essential and purely ketogenic — the pair that answers most versions of this question.

🏗️ Tertiary and Quaternary Structure 2026

The 2026 stem offered collagen as one option and two true statements about tertiary structure. Read the definitions carefully — the distinction between "one chain" and "several chains" is the whole question.

✅ "Depends on the effects of amino acid side chains"

True. Tertiary structure is the folding of one polypeptide chain, and it is held together entirely by side-chain (R group) interactions: hydrophobic interactions (the dominant force), hydrogen bonds, ionic bonds / salt bridges, van der Waals forces, and the only covalent one, the disulfide bridge between two cysteines. Contrast secondary structure, which is held by backbone hydrogen bonds only.

✅ "Determines the nature of a monomeric protein"

True. A protein made of a single chain — myoglobin is the standard example — has no quaternary structure at all, so its final functional shape is its tertiary structure.

❌ Why collagen is the wrong option

Collagen is the classic example of quaternary structure: three separate α-chains wound into one triple helix. It is also the classic fibrous protein, whereas tertiary structure questions are about globular folding. Older papers ask the same thing the other way round: quaternary structure "occurs in a monomeric protein" is false, and haemoglobin is tetrameric.

Quaternary structure — the recurring facts

It is mostly stabilised by non-covalent bonds (hydrophobic, hydrogen, ionic), sometimes with disulfides — so "mostly stabilised by covalent bonds" is false. It is sensitive to denaturation. Denaturation destroys secondary, tertiary and quaternary structure while leaving the primary sequence intact.

The peptide bond — characteristics that keep appearing

It is covalent, formed between the α-carboxyl of one residue and the α-amino of the next with loss of water. It has partial double-bond character from resonance, which makes it planar and rigid, with no free rotation; it is almost always trans; it is uncharged but polar; and it does not ionise. A characteristic that does not match it: "allows free rotation" or "is an ionisable group".

Folding helpers and protein turnover

Chaperones prevent incorrect interactions during folding and do not appear in the final product; protein disulfide isomerase shuffles disulfides to the correct pairing; peptidyl-prolyl cis–trans isomerase speeds the slow proline isomerisation step. Degradation runs through the ubiquitin–proteasome system (ATP-dependent) and the lysosome. The PEST sequence — rich in proline, glutamate, serine and threonine — marks a protein for rapid degradation.

🧬 The Genetic Code, Mutations and Translation

Features of the code

Unambiguous · degenerate · non-overlapping · universal · non-punctuating (commaless) · triplet. The one that appears as a false option is "overlapping" — the code is non-overlapping. Start codon AUG (methionine); stop codons UAA, UAG, UGA.

Point mutations — what they can do

A single base change can produce a silent mutation (same amino acid, thanks to degeneracy), a missense mutation (wrong amino acid incorporated), or a nonsense mutation (a stop codon, so translation terminates prematurely). "Transition or transversion" is a point mutation. Frameshift comes from an insertion or deletion not divisible by three and rewrites everything downstream.

Amino acid activation — the traps

The reaction needs aminoacyl-tRNA synthetase (not "aminoacyl-mRNA synthetase") and it uses ATP (not GTP), releasing AMP + PPi. It happens in the cytosol, not in mitochondria. GTP is needed for the initiation, elongation and translocation steps on the ribosome — that is where GTP belongs.

Translation — the rest

The cap sits at the 5′ end of mRNA. The first step is formation of the initiation complex (small subunit + initiator Met-tRNA + mRNA), which requires GTP. RNA polymerase belongs to transcription, not translation — a favourite wrong option. Ribosomes read 5′→3′ and build the protein N-terminus first.

🩸 Plasma Proteins, Immunoglobulins and Transport

Who carries what

Albumin — free fatty acids, unconjugated bilirubin, calcium, many drugs; it is also the main determinant of colloid osmotic pressure. Retinol-binding protein — vitamin A. Transferrin — iron. Ceruloplasmin — copper. Haptoglobin — free haemoglobin. Transcortin/CBG — cortisol. SHBG — testosterone, oestradiol.

The acute-phase set

Fibrinogen, α₁-antiproteinase (= α₁-antitrypsin), orosomucoid, haptoglobin and ceruloplasmin are plasma proteins made by the liver; haemoglobin is not a plasma protein — it is intracellular, inside the red cell, and only appears free in plasma during haemolysis.

Immunoglobulins — the recurring pair

IgG — crosses the placenta, activates complement, has 3 constant domains on the heavy chain. IgM — pentamer, the strongest complement activator, does not cross the placenta. IgE — has 4 constant heavy-chain domains (like IgM), does not fix complement, and does not cross the placenta; it mediates allergy through mast cells. "IgE easily crosses the placenta" is always false.

Partial (limited) proteolysis

It converts inactive precursors into active proteins and is irreversible — an option saying "usually reversible" is false. It does not regulate the quantity of protein made, only its activation. Examples: proinsulin → insulin, trypsinogen → trypsin, procollagen → collagen, the clotting cascade.

Protein Degradation

Frequently tested: ubiquitin–proteasome system (ATP-dependent, selective, cytosolic), partial proteolysis, amelogenin (TRAP)

♻️ Ubiquitin–Proteasome System

This is the main pathway for selective protein degradation in the cytosol. A chain of at least 4 ubiquitin molecules (a small protein) is attached to the target protein → the 26S proteasome recognizes the polyubiquitin tag → the protein is unfolded and degraded into small peptides → ubiquitin is recycled.

Three enzymes work in sequence: E1 (ubiquitin-activating enzyme) → E2 (ubiquitin-conjugating enzyme) → E3 (Ubiquitin Ligase). E3 determines which protein gets degraded (substrate specificity).

Key Properties — All Tested

The ubiquitin-proteasome system: (1) is located in the cytosol, (2) requires ATP (for E1 activation and proteasome unfolding), and (3) is selective (only proteins tagged with ubiquitin are degraded). All three properties are directly tested together.

✂️ Partial Proteolysis — Zymogen Activation

Partial proteolysis is a covalent, irreversible modification. A peptide bond is cleaved, removing an inhibitory portion of the protein → the enzyme (or hormone) becomes active. Examples: pepsinogen → pepsin, trypsinogen → trypsin, proinsulin → insulin (active hormone + C-peptide). This is also how prohormones are converted to active hormones — partial proteolysis converts a prohormone to its active hormone form.

Protein Denaturation — Why No Precipitation?

A protein may be denatured (lose its 3D structure) without precipitating if: (1) the pH is far from the isoelectric point (pI) — the protein carries a strong net charge and repels other molecules, keeping it soluble even when unfolded, or (2) after cooling, renaturation is possible — the protein refolds back to its native structure (showing denaturation was reversible). At the pI, net charge = 0 → no electrostatic repulsion → proteins aggregate and precipitate.

🧬 Key Aminoacidopathies — PKU, Homocystinuria, MSUD, Alkaptonuria

Disease	Deficient Enzyme	Accumulates	Key Clinical Features	Treatment
PKU	Phenylalanine hydroxylase (PAH) — requires BH₄	Phenylalanine + phenylketones	Intellectual disability (untreated), musty urine, fair skin & hair (↓melanin), eczema. Tyr becomes essential.	Low-Phe diet, BH₄ (sapropterin) for some
Homocystinuria	Cystathionine β-Synthase (CBS) — requires PLP/B6	Homocysteine + methionine	Marfanoid body, lens dislocation DOWNWARD, thromboembolism, intellectual disability, osteoporosis	B6 (if responsive), folate + B12, low-Met diet, betaine
MSUD	BCKAD (Branched-chain α-ketoacid dehydrogenase) — requires TPP	Leu, Ile, Val + branched-chain keto acids	Maple syrup urine/sweat odour, encephalopathy, feeding difficulty. Leucine is most neurotoxic.	Low-BCAA diet, thiamine (B1) supplementation
Alkaptonuria	Homogentisate oxidase	Homogentisic acid	Dark/black urine on air exposure, ochronosis (dark cartilage), arthritis. First "inborn error" (Garrod).	High-dose Vitamin C (reduces polymerisation); nitisinone

PAH Requires Tetrahydrobiopterin (BH₄)

Phenylalanine hydroxylase needs BH₄ as cofactor. Some PKU cases are caused by BH₄ deficiency (GTP cyclohydrolase I deficiency), not PAH itself. These are worse because BH₄ is also required by tyrosine hydroxylase and tryptophan hydroxylase → also affects catecholamine and serotonin synthesis.

🦷 Amelogenin & Dental Biochemistry ★ asked 2022, 2025

Amelogenin is the main protein of tooth enamel matrix (~90% of enamel matrix proteins). It is secreted by ameloblasts and guides the formation of hydroxyapatite crystals during enamel mineralisation.

Direct Exam Question: "What is the main amino acid component of amelogenin?" — 2022 & 2025

Answer: Tyrosine. The full name of the major amelogenin is TRAP = Tyrosine-Rich Amelogenin Protein. Its unusually high tyrosine content is what gives it the name "TRAP." This was tested directly in the 2022 and 2025 exams.

Remember: TRAP = Tyrosine Rich Amelogenin Protein → main amino acid = Tyrosine.

Enamel matrix proteins (primarily amelogenin ~90%) are later degraded by proteases — matrix metalloproteinases (MMPs) and kallikrein-4 — as enamel matures. The organic matrix is almost entirely removed in mature enamel, leaving almost pure inorganic hydroxyapatite with virtually no organic matrix remaining.

Nucleotides & Mutations

Frequently tested: purines vs pyrimidines, AMP deaminase releasing ammonia, gout & allopurinol, SCID/ADA deficiency, Lesch–Nyhan

🧬 Purines vs Pyrimidines

Purines (double ring)

Adenine, Guanine. Catabolized to uric acid by Xanthine Oxidase.

Pyrimidines (single ring)

Cytosine, Thymine, Uracil. Catabolized to β-alanine, CO₂, NH₃.

Gout

↑ uric acid → crystal deposits in joints. Allopurinol inhibits Xanthine Oxidase. Lesch-Nyhan: HGPRT deficiency → severe gout + neurological symptoms (X-linked).

⭐ EXAM PRIORITY — Teacher Marked

🔄 Purine Synthesis & AMP Deaminase

Purine synthesis de novo builds the purine ring on ribose-5-phosphate (from PPP). The cycle involves multiple steps using glutamine, glycine, aspartate, CO₂, and THF.

🏗️ De Novo Purine Synthesis (building blocks) ⚪ beyond exam scope

Ribose-5-Pfrom PPP → PRPP → IMPfirst nucleotide → AMP & GMP

Ring atoms donated by: Glutamine · Glycine · Aspartate · CO₂ · THF (N₁₀-formyl)

AMP Deaminase — Teacher Specifically Listed

AMP Deaminase converts AMP → IMP + NH₃. It is active in:
• Muscle: during intense exercise, AMP deaminase releases NH₃ from AMP → IMP → this NH₃ is exported as alanine or glutamine (nitrogen disposal from working muscle)
• Liver: part of purine catabolism pathway
Key exam: "Which enzyme releases ammonia from purines?" → AMP Deaminase. Diseases of AMP deaminase: exercise-induced myopathy (rare).

Diseases — Know ONLY These for Purines

Exam-required purine diseases:
• Gout (xanthine oxidase → ↑uric acid)
• Lesch-Nyhan (HGPRT deficiency — X-linked)
• ADA deficiency (Adenosine Deaminase (ADA) — SCID (Severe Combined Immunodeficiency))
Teacher said: "Diseases → ONLY" for purine synthesis section.

🦴 Gout & Lesch-Nyhan — Purine Catabolism Diseases

⚗️ Purine Catabolism Pathway

AMP → IMP → Hypoxanthine Xanthine Oxidase→ Xanthine Xanthine Oxidase→ Uric Acid

Guanine branch merges in at xanthine:

GMP → Guanine → Xanthine Xanthine Oxidase→ Uric Acid

Gout

Cause: Hyperuricaemia → monosodium urate (MSU) crystals deposit in joints.
Hallmark joint: First metatarsophalangeal joint (podagra).
Crystal appearance: Needle-shaped, negatively birefringent under polarised light.
Triggers: High purine diet (red meat, shellfish), alcohol, diuretics, dehydration.
Treatment: Allopurinol (xanthine oxidase inhibitor — structural analog of hypoxanthine; competitive inhibitor → ↑Km, unchanged Vmax).

👆 Tap for full disease card

Lesch-Nyhan Syndrome — HGPRT Deficiency

Enzyme: HGPRT (Hypoxanthine-Guanine Phosphoribosyltransferase) — salvages Hypoxanthine + Guanine back to IMP/GMP. X-linked recessive (affects males).

Without HGPRT: Hypoxanthine and guanine cannot be salvaged → ALL degraded to uric acid → massive hyperuricaemia.

Clinical features:

Severe gout and urate nephropathy (from birth)
Intellectual disability
Choreoathetosis (involuntary movements)
Compulsive self-mutilation (lip/finger biting — pathognomonic)

Treatment: Allopurinol controls gout but does NOT improve the neurological symptoms.

👆 Tap for full disease card

Allopurinol — Mechanism (Exam Favourite)

Allopurinol is a structural analog of hypoxanthine. It is a competitive inhibitor of xanthine oxidase → ↑Km (apparent), unchanged Vmax. It is also oxidised by xanthine oxidase to alloxanthine (oxipurinol), which then becomes a tight-binding inhibitor. Result: ↓uric acid production, ↑xanthine and hypoxanthine (which are more soluble than uric acid).

🧬 Mutations & Molecular Biology ⚪ beyond purine exam scope

Point mutation types

Transition = purine↔purine or pyrimidine↔pyrimidine.
Transversion = purine↔pyrimidine (e.g. adenine → cytosine).
Effects: silent (same AA, code is degenerate), missense (wrong AA — may be tolerated), nonsense (→ stop codon: UAA, UAG, UGA).

Missense — acceptable?

Can be acceptable: the genetic code is degenerate, and swapping one amino acid does not always destroy protein function.

Kozak sequence

A nucleotide sequence in eukaryotic mRNA around the start (AUG) codon — marks where translation begins (the ribosome-recognition context).

Translation needs energy

Amino-acid activation uses ATP (aminoacyl-tRNA synthetase → aminoacyl-tRNA). Elongation/translocation on the ribosome uses GTP.

Ricin — exam fact

Ricin (castor bean toxin) inactivates the 28S rRNA of the eukaryotic 60S subunit (cleaves a single adenine) → halts protein synthesis. Neurolathyrism (from certain plant amino acids) is a separate plant-toxin syndrome causing spastic paresis.

🧬 Pyrimidine Biosynthesis, Degradation & Regulation

Pyrimidines are built ring-first, then attached to ribose — the opposite of purines (which are built directly on ribose-5-phosphate). The whole ring starts from glutamine + CO₂ + aspartate.

⚗️ De Novo Pyrimidine Synthesis (cytosol)

Gln + CO₂ CPS-II→ Carbamoyl-P ATCase→ Carbamoyl aspartate → Dihydroorotate DHO-DH→ Orotate → UMP → UTP · CTP · dTMP

Feature	Detail (high-yield)
Rate-limiting enzyme	CPS-II — cytosolic, uses glutamine as nitrogen source, activated by ATP + PRPP, inhibited by UTP
Ring source atoms	Glutamine (N), CO₂ (C), and aspartate (the rest of the ring)
Only mitochondrial step	Dihydroorotate → orotate (dihydroorotate dehydrogenase, links to CoQ)
Sugar added as	PRPP (ribose-5-P donor) — added after the ring is made
dTMP formation	dUMP → dTMP by thymidylate synthase (needs methylene-THF)
Deoxy forms	Ribonucleotides → deoxyribonucleotides by ribonucleotide reductase
Degradation	Ring is opened and fully broken down → β-alanine (from C), β-aminoisobutyrate (from T), CO₂ + NH₃. Unlike purines, this leaves no insoluble product — so there is no "pyrimidine gout".

Purine vs Pyrimidine synthesis — the one-line contrast

Purines: ring built on ribose-5-P (IMP first). Pyrimidines: ring built first, sugar added later (UMP first). Purine catabolism ends in uric acid (insoluble → gout); pyrimidine catabolism ends in soluble, harmless products.

Hereditary Orotic Aciduria

UMP synthase deficiency → orotic acid accumulates in urine + megaloblastic anaemia that does not respond to B12/folate. Key contrast: orotic aciduria with high ammonia = OTC deficiency; without high ammonia = this disease. Treated with uridine.

👆 Tap for full disease card

💊 Synthetic Nucleotide Analogues — Clinical Use & Mechanism exam-listed

These drugs are fake building blocks: they resemble normal nucleotides/bases and jam the enzymes of DNA/RNA synthesis. Fast-dividing cells (tumour, immune, viral-infected) are hit hardest.

Drug	Target / mechanism	Clinical use
5-Fluorouracil (5-FU)	→ FdUMP → irreversibly inhibits thymidylate synthase → "thymineless death"	Colon, breast cancers
Methotrexate	Inhibits dihydrofolate reductase → ↓THF → ↓purine + dTMP synthesis	Cancer, RA, psoriasis (rescue with folinic acid)
6-Mercaptopurine / Azathioprine	Purine analogue → inhibits de novo purine synthesis (PRPP amidotransferase)	Leukaemia, immunosuppression. Toxicity ↑ with allopurinol (both use xanthine oxidase)
Hydroxyurea	Inhibits ribonucleotide reductase → ↓dNTPs	Sickle cell (↑HbF), myeloproliferative disorders
Cytarabine (Ara-C)	Arabinose cytidine analogue → blocks DNA polymerase / chain termination	Acute myeloid leukaemia
Acyclovir	Guanosine analogue; activated by viral thymidine kinase → chain terminator of viral DNA polymerase	Herpes / varicella (selective for infected cells)
Zidovudine (AZT)	Thymidine analogue → inhibits reverse transcriptase (chain terminator)	HIV
Allopurinol	Hypoxanthine analogue → inhibits xanthine oxidase	Gout, tumour-lysis prophylaxis

Selective toxicity principle

Antivirals (acyclovir) and antibacterials (trimethoprim) exploit species differences in the target enzyme; anticancer analogues exploit the high division rate of tumour cells — which is also why they harm marrow, gut, and hair.

🧾 Compounds That Contain Ribose (or Ribose-Derived) "name 6"

A frequent exam ask: "name compounds containing ribose." Ribose (or its phosphate) is the sugar backbone of every nucleotide-based molecule.

Energy currency

ATP, GTP, UTP, CTP (and their di-/mono-phosphates)

Redox coenzymes

NAD⁺ / NADH, NADP⁺ / NADPH, FAD / FMN

Acyl carrier

Coenzyme A (CoA) — contains an adenosine (ribose) unit

Second messenger

cAMP, cGMP

Methyl donor

S-adenosylmethionine (SAM) — adenosine core

Nucleic acids

RNA (ribose) — DNA uses deoxyribose

Fast answer

A safe "6": ATP, NAD⁺, FAD, Coenzyme A, cAMP, SAM — all built around a ribose-containing adenosine. (FAD's flavin sits on ribitol, but the nucleotide half is AMP-ribose.)

🧬 DNA vs RNA · Genetic Code · Frameshift Mutations

	DNA	RNA
Sugar	Deoxyribose (no 2′-OH)	Ribose (2′-OH present)
Bases	A, G, C, T	A, G, C, U (uracil)
Strands	Double-stranded helix	Usually single-stranded
Stability	Stable (no 2′-OH → resists hydrolysis)	Less stable (2′-OH → alkaline hydrolysis)
Location / role	Nucleus — long-term information store	Cytoplasm — working copy (mRNA, tRNA, rRNA)

Features of the genetic code: it is a triplet code (3 bases = 1 codon), degenerate (most amino acids have several codons — the "wobble" is in the 3rd base), unambiguous (each codon = only one amino acid), non-overlapping and comma-less (read continuously), and nearly universal (same in almost all organisms). Start = AUG (Met); stops = UAA, UAG, UGA.

Point mutations (single base)

Silent — same amino acid (wobble). Missense — different amino acid (may be tolerated). Nonsense — creates a premature stop codon → truncated protein.

Frameshift mutation

Insertion or deletion of a number of bases not divisible by 3 → the entire downstream reading frame shifts → completely different amino acids and usually an early stop. Far more damaging than most point mutations. (Insert/delete in multiples of 3 = in-frame, milder.)

Enzymes – Properties & Kinetics

Frequently tested: enzyme classes, Km/Vmax, competitive vs non-competitive inhibition, coenzyme requirements, plasma enzymes in diagnosis

📐 The Seven Classes of Enzymes

Class	Number	What They Do	Example
Oxidoreductases	1	Transfer electrons (oxidation-reduction reactions). Require electron carriers like NAD⁺ or FAD.	Lactate Dehydrogenase, G6PD, Malic enzyme
Transferases	2	Transfer a chemical group from one molecule to another	Kinases (transfer phosphate), Aminotransferases (transfer –NH₂), Glycogen Synthase, Citrate Synthase
Hydrolases	3	Cleave bonds using water (hydrolysis)	Proteases, Lipases, Phosphatases
Lyases	4	Add or remove groups at double bonds (without water)	Aldolase, Pyruvate decarboxylase, PEPCK
Isomerases	5	Rearrange atoms within a single molecule (structural or geometric changes)	Phosphoglucose Isomerase, Triose phosphate isomerase
Ligases	6	Join two molecules together using energy from ATP	Pyruvate Carboxylase, Acetyl-CoA Carboxylase, DNA Ligase, Aminoacyl-tRNA Synthetase
Translocases	7	Catalyze the movement of ions or molecules across membranes (membrane transport coupled to energy)	Na⁺/K⁺-ATPase, ABC transporters, mitochondrial carrier proteins, H⁺/K⁺-ATPase

⚠️ Correction: Citrate Synthase is a Transferase (Class 2), NOT a Lyase

Citrate Synthase is classified as EC 2.3.3.1 — an acyltransferase (Transferase, class 2). It transfers the acetyl group from acetyl-CoA to oxaloacetate in an aldol condensation. A common exam trap is to classify it as a Lyase because it forms a C–C bond, but Lyases only break bonds; Citrate Synthase uses CoA as a leaving group and is officially a Transferase per IUBMB.

Na⁺/K⁺-ATPase — Enzyme Classification ★ asked 2023

Na⁺/K⁺-ATPase (the sodium-potassium pump) belongs to the class of Translocases (class 7) — a subclass of transporters. In some classifications it is grouped under transferases (it transfers ions), but officially it is classified as a translocase (moves substances across membranes). This was asked in the 2023 exam: "Na/K ATPase belongs to which class?" → Translocases.

Glycogen Synthase Classification

Glycogen Synthase belongs to class 2 (Transferases) — it transfers a glucose unit from UDP-glucose to the growing glycogen chain. This is directly tested.

🏷️ Enzyme Naming Convention — Synthetase vs Synthase vs Ligase

Exam Trap (Q34): What class is a "Synthetase"?

Enzyme names ending in "-synthetase" or "-synthase" can be confusing because they both mean "makes something" — but they belong to different classes:

Name ending	Enzyme class	ATP used?	Example
-synthetase	Ligase (class 6)	YES — joins two molecules using ATP	Aminoacyl-tRNA synthetase, Argininosuccinate synthetase, Acetyl-CoA synthetase
-synthase	No fixed class — class VARIES (often Transferase, sometimes Lyase)	NO — forms a bond WITHOUT using ATP	Citrate synthase & HMG-CoA synthase = Transferases (2); Tryptophan synthase = Lyase (4); ATP synthase = Translocase (7)
-ligase	Ligase (class 6)	YES	DNA ligase, Pyruvate carboxylase

The Simple Rule

Synthetase = Ligase (class 6) — makes a bond WITH ATP. This is the reliable, testable rule.
Synthase = no ATP needed — but the EC class is NOT fixed; it must be learned per enzyme. A "-synthase" is not automatically a lyase.

Memory trick: SynthetASE has an extra "A" (for ATP) → Ligase. SynthASE has no extra letter (no ATP) → class varies.
Key examples to memorise: Citrate synthase & HMG-CoA synthase are Transferases (class 2); Glycogen synthase is a Transferase (class 2); Tryptophan synthase is a Lyase (class 4); ATP synthase is a Translocase (class 7) (uses a proton gradient).
Common trap: classifying Citrate synthase as a lyase because it forms a C–C bond — it is officially a Transferase (EC 2.3.3.1), not a lyase.

🏷️ Enzyme Naming — Phosphatase vs Phosphorylase vs Kinase

Easily confused — they do opposite/different things

All three involve phosphate groups, but the mechanism and enzyme class differ. Watch the distinction carefully.

Enzyme type	What it does	Class	Example
Phosphatase	Removes a phosphate group using water (hydrolysis): substrate-P + H₂O → substrate + Pi	Hydrolase (class 3)	Glucose-6-phosphatase, alkaline phosphatase, protein phosphatase
Phosphorylase	Adds inorganic phosphate (Pi) to cleave a bond — phosphorolysis, NOT hydrolysis and NOT using ATP: (glucose)ₙ + Pi → (glucose)ₙ₋₁ + glucose-1-P	Transferase (class 2) — specifically a glycosyltransferase	Glycogen phosphorylase, purine nucleoside phosphorylase
Kinase	Adds a phosphate from ATP (phosphorylation): substrate + ATP → substrate-P + ADP	Transferase (class 2) — a phosphotransferase	Hexokinase, protein kinase A, pyruvate kinase

The Simple Rule

Phosphatase = takes phosphate OFF using water (hydrolase).
Phosphorylase = breaks a bond by adding free Pi (phosphorolysis) — classic example: glycogen phosphorylase releasing glucose-1-phosphate from glycogen.
Kinase = puts phosphate ON using ATP.

Key trap: a phosphorylase does NOT use ATP and does NOT use water — it uses inorganic phosphate to split a bond. That makes glycogen phosphorylase a transferase, while glucose-6-phosphatase (water, removes Pi) is a hydrolase.

📈 Michaelis-Menten Kinetics

Km = the substrate concentration at which the reaction runs at exactly half its maximum speed (Vmax/2). A low Km means the enzyme reaches half-maximum speed with very little substrate = high affinity. A high Km means the enzyme needs lots of substrate to reach half-maximum speed = low affinity. Km does NOT depend on enzyme concentration.

Type of Inhibition	Effect on Km	Effect on Vmax	Mechanism
Competitive	↑ Km (apparent)	Unchanged	Inhibitor competes for the active site. Effect can be overcome by increasing substrate concentration. Example: statins vs HMG-CoA Reductase.
Non-competitive	Unchanged	↓ Vmax	Inhibitor binds a separate (allosteric) site. Reduces enzyme efficiency regardless of substrate concentration. Cannot be overcome by adding more substrate.
Irreversible	—	↓ Vmax (permanently)	Inhibitor forms a covalent bond with the enzyme — permanent inactivation. Example: organophosphates on acetylcholinesterase, aspirin on COX.

Competitive Inhibition — Key Exam Statement ★ asked 2018

"The effect of a competitive inhibitor can be overcome by increasing the substrate concentration" → TRUE. At very high substrate concentrations, the substrate out-competes the inhibitor for the active site, and Vmax is eventually reached.

🎛️ Covalent Regulation — Hormones as Effectors

Covalent modification (most commonly phosphorylation/dephosphorylation) is the most important type of enzyme regulation in the body. The effector molecules that trigger covalent regulation are hormones — they bind receptors and activate downstream kinases (e.g., glucagon → cAMP → PKA → phosphorylates target enzymes). This is distinct from allosteric regulation (which involves non-covalent binding of small molecules).

🏥 Clinically Important Plasma Enzymes

Enzyme	Elevated in
AST / ALT	Liver disease; AST also in MI
CK-MB	Myocardial infarction
Amylase & Lipase	Pancreatitis
Alkaline Phosphatase	Liver/bone disease

🧩 Coenzymes & Prosthetic Groups

Apoenzyme = protein alone (inactive). Holoenzyme = apoenzyme + cofactor (active). Coenzyme = transiently associated organic cofactor (NAD⁺, CoA). Prosthetic group = tightly/covalently bound (FAD in Succinate Dehydrogenase, heme in Cytochrome c).

🔑 Enzyme–Substrate Interaction — The Two Models

An enzyme speeds a reaction by binding the substrate in its active site and stabilising the transition state (the highest-energy point). It lowers the activation energy — it never changes the reaction's equilibrium or ΔG.

🔒 Lock-and-Key (Fischer)

The active site is a rigid, pre-formed shape that exactly fits one substrate. Explains specificity, but not how binding is optimised.

🤝 Induced Fit (Koshland)

The active site is flexible — the substrate induces a conformational change that wraps the enzyme around it. This is the accepted model; hexokinase closing over glucose is the classic example.

How enzymes lower activation energy	Mechanism
Proximity & orientation	Hold substrates together in the correct alignment to react
Transition-state stabilisation	The active site binds the transition state more tightly than the substrate — the key catalytic trick
Acid–base / covalent catalysis	Side chains (His, Ser, Cys) donate/accept protons or form transient covalent intermediates
Metal-ion catalysis	Cofactors (Zn²⁺, Mg²⁺) polarise bonds or stabilise charge

Exam statement

An enzyme does not change ΔG or the equilibrium constant — it only lowers the activation energy, speeding both forward and reverse rates equally.

📉 Lineweaver–Burk (Double-Reciprocal) Plot

Plotting 1/V against 1/[S] turns the Michaelis–Menten curve into a straight line, making Km and Vmax easy to read and inhibitor types easy to tell apart.

Y-intercept

= 1/Vmax

X-intercept

= −1/Km

Slope

= Km/Vmax

Reads best

Small changes at high [S] become clear at low 1/[S]

Inhibitor	On the plot	Meaning
Competitive	Lines meet on the Y-axis (same Vmax); X-intercept moves right (↑Km)	Vmax unchanged, Km ↑ — overcome by more substrate (e.g. statins)
Non-competitive	Lines meet on the X-axis (same Km); Y-intercept rises (↓Vmax)	Km unchanged, Vmax ↓ — not overcome by substrate
Uncompetitive	Parallel lines (both Km and Vmax fall)	Inhibitor binds only the ES complex

Memory hook

Competitive inhibitors "compete" to reach the same finish line → same Vmax → lines meet on the Y-axis. Non-competitive lowers the ceiling → lines meet on the X-axis (Km unchanged).

📐 Enzyme Classes — and the Mevalonate Question 2026

The 2026 paper asked what mevalonate is made from and which enzyme class a drug acting there inhibits. The chain of reasoning is short but each link is a separate fact.

Working the mevalonate question

HMG-CoA is reduced to mevalonate by HMG-CoA reductase, using 2 NADPH, in the endoplasmic-reticulum membrane. It is the rate-limiting, irreversible step of cholesterol synthesis and the target of the statins. Because the reaction is a reduction, the enzyme belongs to class 1 — oxidoreductases. That is the answer: oxidoreductase. Note the trap of reading "reductase" as if it were a lyase or a transferase, and the second trap of confusing this HMG-CoA step with HMG-CoA lyase, which is ketogenesis, or HMG-CoA synthase, which is a condensation.

1 Oxidoreductases

Transfer of electrons, hydrogen or oxygen. Dehydrogenases, oxidases, reductases, peroxidases, catalase, hydroxylases. Coenzymes: NAD⁺/NADP⁺, FAD/FMN.

2 Transferases

Move a functional group. Aminotransferases (coenzyme PLP), kinases (ATP), transketolase (TPP), transaldolase, methyltransferases (SAM).

3 Hydrolases

Cleave a bond using water. Peptidases, esterases, phosphatases, glycosidases, lipases — and glutaminase, arginase and urease, which are commonly mis-assigned in options.

4 Lyases

Break a bond without water and without oxidation. Decarboxylases, aldolases, fumarase (hydratase), ATP-citrate lyase, HMG-CoA lyase.

5 Isomerases

Intramolecular rearrangement. Phosphotriose isomerase, phosphohexose isomerase, phosphoglucomutase, epimerases, racemases.

6 Ligases (synthetases)

Join two molecules using ATP. Pyruvate carboxylase and other biotin carboxylases, glutamine synthetase, argininosuccinate synthetase, acyl-CoA synthetase (thiokinase).

Class questions that have appeared verbatim

Pyruvate carboxylase — a ligase (it also gets called a carboxylase; the biotin gives it away). Glutaminase — a hydrolase. Urease — a hydrolase, splitting urea into CO₂ and ammonia. Carbamoyl phosphate synthetase — a ligase. Phosphotriose isomerase — isomerase, and its reaction is reversible. And the cofactor pairings: carboxylase → biotin · dehydrogenase → NAD or NADP · oxidase → FAD, copper, flavins · transaminase → PLP.

🔥 Biological Oxidation — Oxidase, Oxygenase, Peroxidase, Dehydrogenase

Oxidase

Uses O₂ as the electron acceptor, reducing it to H₂O₂ or to water. It does not incorporate oxygen into the substrate. Examples: xanthine oxidase, monoamine oxidase, cytochrome c oxidase (which makes water).

Oxygenase

Incorporates oxygen into the substrate. A monooxygenase (mixed-function oxidase, e.g. cytochrome P450) inserts one atom into the substrate and reduces the other to water, using NADPH; a dioxygenase inserts both.

Peroxidase & catalase

Peroxidase uses H₂O₂ as the oxidant — glutathione peroxidase (a selenoenzyme) reduces it to water. Catalase is a haemoprotein with four haem groups that decomposes 2 H₂O₂ → 2 H₂O + O₂; the option "has 1 haem group" is false. Both are classed as hydroperoxidases.

Dehydrogenase

Removes two hydrogens onto NAD(P)⁺ or FAD; oxygen is not involved. This is the largest group in metabolism.

Natural antioxidants — the exact list that was asked

Naturally occurring: vitamin E (the most important lipid-soluble antioxidant), vitamin C, uric acid/urate, glutathione, and enzymes such as superoxide dismutase and catalase. β-carotene is provitamin A from the diet and is repeatedly marked as the "not naturally occurring in the body" option. Isoprostanes are products of the non-enzymatic auto-oxidation of arachidonic acid — a marker of oxidative stress, not an antioxidant. And note the trap: vitamin K is not the antioxidant — it is the cofactor for the γ-carboxylation of glutamate.

📈 Km, Vmax and Competitive Inhibition 2026

This came up more than once in 2026 and appears in nearly every older paper. The whole thing rests on four numbers moving in known directions.

Km — what it is and is not

The substrate concentration at which v = ½Vmax. It characterises the affinity of the enzyme for its substrate — low Km = high affinity, and it therefore characterises substrate specificity. It is independent of enzyme concentration and says nothing about Vmax. An option saying "Km depends on Vmax of the reaction" is false.

Vmax

The rate when the enzyme is fully saturated. It is proportional to the amount of enzyme present.

✅ Competitive inhibitor

Raises Km, does not affect Vmax. It is usually a substrate analogue binding the active site; it forms EI but not ESI, and it is overcome by raising [S]. On a Lineweaver–Burk plot the lines meet on the y-axis (same 1/Vmax).

❌ The 2026 distractors

"Change Vmax but not Km" is non-competitive. "They can also bind to the enzyme–substrate complex" describes an uncompetitive (or mixed) inhibitor, not a classical competitive one — a competitive inhibitor cannot bind ES, because the site is occupied. "Lower Km" in any form is wrong for a competitive inhibitor.

The other two patterns

Non-competitive: binds a site other than the active site, forms ES, EI and ESI, is not relieved by more substrate — Vmax falls, Km unchanged; lines meet on the x-axis. Uncompetitive: binds only ES — both Vmax and Km fall, giving parallel lines. And the general rule that anchors all of it: enzymes never change the equilibrium constant of a reaction; they only lower the activation energy and reach the same equilibrium sooner. Any option saying "enzymes change the equilibrium constant" is false.

🎛️ Regulation of Enzyme Activity

Covalent modification

Phosphorylation is a covalent and reversible modification, carried out by kinases — not by phosphorylases, which is a recurring wrong option. It happens on the –OH of serine, threonine or tyrosine, needs ATP, and is reversed by a separate phosphatase.

The direction rule

Catabolic enzymes are active when phosphorylated; anabolic enzymes are active when dephosphorylated. Glycogen phosphorylase, hormone-sensitive lipase and phosphorylase b kinase switch on with phosphate; glycogen synthase, acetyl-CoA carboxylase and pyruvate dehydrogenase switch off. One hormone (glucagon or adrenaline) therefore flips a whole panel of enzymes in the same metabolic direction.

Allosteric enzymes

They are oligomeric (so they do have a quaternary structure), they sit at the rate-limiting step, they give a sigmoidal curve, and their effector binds a site distinct from the active site. Allosteric proteins share similar properties and regulatory behaviour, not similar structure or function.

Hydrophobic residues of an enzyme

They face the inside of the molecule, they are next to the active centre, they take part in enzyme–substrate binding, and they protect the enzyme–substrate complex from attack by water — all four statements have been marked true in past papers.

Enzymes induced by insulin, and enzymes inhibited by citrate

Induced by insulin: glucokinase, pyruvate kinase, pyruvate dehydrogenase, acetyl-CoA carboxylase, fatty acid synthase, ATP-citrate lyase, HMG-CoA reductase, glucose-6-phosphate dehydrogenase. Inhibited by citrate: PFK-1 (citrate signals that the Krebs cycle is full) — while the same citrate activates acetyl-CoA carboxylase. That reciprocal pair is a favourite.

Enzyme Regulation

Frequently tested: allosteric regulation, phosphorylation, rate-limiting enzymes of each pathway, synthase vs synthetase classes

🎛️ Mechanisms of Regulation

Mechanism	Covalent?	Reversible?	Key Facts
Allosteric	No	Yes	Effector binds allosteric site; changes shape; can change Km &/or Vmax
Phosphorylation	Yes	Yes (phosphatase)	Kinase adds PO₄ (uses ATP); can activate OR inactivate
Partial proteolysis	Yes	NO	Zymogen activation; irreversible
Induction/Repression	—	Yes	Changes AMOUNT of enzyme; slow response

🔄 Key Phosphorylation Examples

Glycogen Phosphorylase

Glycogen Phosphorylase — Phosphorylated = ACTIVE → breaks down glycogen

Glycogen Synthase

Glycogen Synthase — Phosphorylated = INACTIVE → makes glycogen when dephosphorylated

HMG-CoA Reductase

HMG-CoA Reductase — Phosphorylated = INACTIVE → rate-limiting for cholesterol synthesis

Pyruvate Kinase (liver)

Pyruvate Kinase — Phosphorylated = INACTIVE → glucagon → cAMP → PKA

Carbohydrate Digestion

Frequently tested: α-amylase & α(1→4) bonds, reducing sugars & Benedict/iodine tests, starch digestion, ketoses vs aldoses

🧪 Digestion Steps

1
Salivary Amylase: cleaves α-1→4 bonds → maltose, maltotriose, dextrins. Does NOT cleave α-1→6 (branch points). Works in the mouth, inactivated by stomach acid.
2
Pancreatic Amylase: same action in small intestine; stimulated by CCK (cholecystokinin) and secretin.
3
Brush border enzymes on the intestinal wall: Maltase (cleaves maltose → 2 glucose), Sucrase (cleaves sucrose → glucose + fructose), Lactase (cleaves lactose → glucose + galactose), Isomaltase (cleaves α-1→6 branch points) → complete digestion to monosaccharides.

Reducing Sugars & Benedict Test

Monosaccharides (glucose, fructose, galactose) are ALL reducing sugars → positive Benedict test (brick-red/orange). Maltose and Lactose are also positive. Sucrose is negative (no free reducing group). Seliwanov test = detects fructose (ketoses specifically) — gives red color.

Maltotriose

Maltotriose (a 3-glucose chain from amylase action on starch) is degraded by maltase at the brush border. This is a commonly tested fact.

🩸 RBC Special Metabolism — 2,3-BPG

Red blood cells (RBCs) have no mitochondria → they rely entirely on anaerobic glycolysis for energy. The Rapoport-Luebering shunt diverts 1,3-bisphosphoglycerate (1,3-BPG) into 2,3-BPG instead of making ATP at the phosphoglycerate kinase step — this means one ATP is sacrificed to make 2,3-BPG.

2,3-BPG stabilizes deoxy-hemoglobin (the T/tense state) → decreases oxygen affinity → shifts the oxygen-hemoglobin curve to the right → promotes oxygen release to tissues.

The Bypass Explained

The 2,3-BPG shunt bypasses the phosphoglycerate kinase reaction — this is exactly what is asked in exams. No ATP is generated at this step when the shunt is active.

🔄 Mannose Metabolism & Glucose Epimers

Dietary carbohydrate digestion yields three main monosaccharides: glucose, fructose, and mannose. Each enters metabolism differently.

Monosaccharide	Entry into metabolism	Enzyme	Exam point
Glucose	→ Glucose-6-phosphate → glycolysis/glycogen/PPP	Hexokinase / Glucokinase	Main fuel; GLUT-2 in liver (high Km)
Mannose	→ Mannose-6-phosphate → Fructose-6-phosphate → glycolysis	Hexokinase (phosphorylates mannose; low affinity for it)	Mannose and glucose are C-2 epimers (differ only at carbon 2)
Fructose	→ Fructose-1-phosphate (liver) or Fructose-6-P (other tissues)	Fructokinase (liver) / Hexokinase (others)	Bypasses PFK-1 — faster than glucose through glycolysis

Epimer Relationships ★ asked 2023

Epimers are sugars differing only in the configuration at ONE carbon atom.
• Glucose & Mannose = C-2 epimers (the 2023 exam asked this directly)
• Glucose & Galactose = C-4 epimers (interconverted by UDP-hexose-4-epimerase)
• Most body mannose comes from fructose metabolism (another 2023 exam point)

Sorbitol Pathway & Diabetic Complications

In hyperglycaemia, glucose is converted to sorbitol (a sugar alcohol / polyol) by aldose reductase. Sorbitol is then oxidized to fructose by sorbitol dehydrogenase.

Problem: Sorbitol cannot easily cross cell membranes, so it accumulates in insulin-independent cells (lens, retina, peripheral nerves, kidney) → osmotic swelling → cell damage.

Diabetic complications from sorbitol accumulation:

Cataracts — sorbitol in lens causes osmotic opacity
Peripheral neuropathy — sorbitol in Schwann cells
Nephropathy — sorbitol in kidney cells
Retinopathy — sorbitol in retinal cells

🔑 Phosphorylation of Hexoses — Trapping Sugars in the Cell

Every dietary hexose is phosphorylated immediately on entering a cell. The phosphate group is charged, so the sugar can no longer cross the membrane back out — it is "trapped" and committed to metabolism.

Sugar	Enzyme	Product	Note
Glucose	Hexokinase (all cells) / Glucokinase (liver, β-cells)	Glucose-6-P	Hexokinase: low Km, inhibited by G6P. Glucokinase: high Km, glucose sensor
Fructose	Fructokinase (liver) / hexokinase (other)	Fructose-1-P	Bypasses PFK-1 → enters glycolysis fast
Galactose	Galactokinase	Galactose-1-P	Defect → galactokinase deficiency (cataracts)
Mannose	Hexokinase (low affinity)	Mannose-6-P	→ fructose-6-P → glycolysis

Why phosphorylation matters

Three reasons: (1) traps the sugar inside the cell, (2) keeps the intracellular free-glucose concentration low so more can enter down its gradient, and (3) activates the sugar for the next reaction. It costs 1 ATP — the "investment" of glycolysis.

🧬 Glycoproteins, Proteoglycans & Oligosaccharide Synthesis / Degradation

Many proteins carry sugar chains (glycosylation). These "glycoconjugates" do structural, recognition, and lubrication jobs — and their faulty breakdown causes lysosomal storage diseases.

Class	What it is	Role
Glycoproteins	Protein + short branched oligosaccharides (protein-dominant)	Enzymes, hormones (FSH, TSH), antibodies, membrane receptors, mucus (mucins)
Proteoglycans	Core protein + long glycosaminoglycan (GAG) chains (sugar-dominant, highly negative)	Extracellular matrix, cartilage, joint lubrication — hold water (turgor)
GAGs (examples)	Hyaluronic acid, chondroitin sulfate, heparin, keratan/dermatan sulfate	Repeating disaccharides of an amino sugar + uronic acid

N-linked glycosylation

Sugar added to asparagine (Asn). Built on a dolichol phosphate carrier, begun in the ER, trimmed in the Golgi.

O-linked glycosylation

Sugar added to serine / threonine –OH. Built directly in the Golgi, sugar-by-sugar (no dolichol).

Degradation → storage diseases

Glycoconjugates are broken down by lysosomal enzymes. Missing enzymes cause accumulation: Gaucher (glucocerebroside), mucopolysaccharidoses (GAGs — Hurler, Hunter), and I-cell disease (failure of mannose-6-phosphate tagging → enzymes not delivered to lysosomes). Mannose-6-phosphate is the "address label" that sends enzymes to the lysosome.

Protein Digestion & Absorption

Frequently tested: digestive proteases & their activation (trypsin activates the rest), enterokinase, chymotrypsin specificity, metabolites formed in the intestine

🍽️ Proteases — Grouped by Where They're Activated

The cleanest way to memorize the proteases is by where each one gets switched on as food travels down the GI tract. There are three activation stations:

🟥 1 · Stomach

Acidic environment (HCl from parietal cells)

Activated by: HCl / autocatalysis at pH < 5

Pepsin

Secreted as pepsinogen by chief cells. Cleaves after aromatic residues (Phe, Trp, Tyr).

▼

🟨 2 · Duodenal Brush Border

The master switch that turns on the pancreatic enzymes

Activated by: nothing upstream — it is the FIRST activator

Enterokinase → activates Trypsin

Brush-border enzyme (NOT pancreatic). Cleaves trypsinogen → trypsin, which then triggers everything below. Trypsin cleaves after Arg/Lys (basic residues).

▼

🟩 3 · Pancreatic Zymogens (in the duodenal lumen)

All secreted by the pancreas as inactive zymogens

Activated by: TRYPSIN (one trypsin switches on all of these)

Chymotrypsin

Cleaves after Phe/Trp/Tyr (aromatic / large hydrophobic).

Elastase

Cleaves after small residues (Ala, Gly, Ser) → releases Leu, Ile, Gly, Ser.

Carboxypeptidase A

Trims C-terminal aliphatic residues (Ala, Ile, Leu, Val). Needs Zn²⁺.

Carboxypeptidase B

Trims C-terminal basic residues (Arg, Lys). Needs Zn²⁺.

💡 The logic in one line

Acid wakes Pepsin → Enterokinase wakes Trypsin → Trypsin wakes everyone else. Memorize the chain of command, not six separate facts.

Enterokinase (Enteropeptidase) — Key Facts

Enterokinase is a brush-border enzyme (not a pancreatic enzyme). It is the first activator: it cleaves trypsinogen → active trypsin. Trypsin then activates ALL remaining pancreatic zymogens (chymotrypsinogen, proelastase, procarboxypeptidase). Enterokinase itself is NOT activated by trypsin — it goes in one direction only. This is frequently asked.

Exam Q: "Which enzyme releases Leu, Ile, Gly, Ser?"

Answer: Elastase — it cleaves after small neutral residues, releasing larger ones from the chain end. Carboxypeptidase A releases aliphatic C-terminal amino acids (Ala, Ile, Leu, Val).

▸ Show the same information as a reference table

Enzyme	Secreted by	Activated by	Cleaves after / Releases
Pepsin	Stomach chief cells (as pepsinogen)	HCl / autocatalytic at pH <5	Aromatic residues (Phe, Trp, Tyr)
Trypsin	Pancreas (as trypsinogen)	Enterokinase at brush border	After Arg/Lys (basic residues)
Chymotrypsin	Pancreas	Trypsin	After Phe/Trp/Tyr (aromatic/large hydrophobic)
Elastase	Pancreas	Trypsin	After small residues: Ala, Gly, Ser — releases Leu, Ile, Gly, Ser
Carboxypeptidase A	Pancreas	Trypsin	C-terminal aliphatic residues (Ala, Ile, Leu, Val); needs Zn²⁺
Carboxypeptidase B	Pancreas	Trypsin	C-terminal basic residues (Arg, Lys); needs Zn²⁺

🔗 Haptocorrin, Intrinsic Factor & Vitamin B12 Absorption

Vitamin B12 (cobalamin) absorption requires a carefully coordinated sequence — each step has clinical relevance:

🔗 Vitamin B12 Absorption — location by location

B12 in food Pepsin→stomach B12 + HaptocorrinR-protein, saliva →duodenum · proteases free B12 B12 + Intrinsic Factorparietal cells →terminal ileum · cubilin B12 + Transcobalamin IIinto blood

1
Mouth/Stomach: B12 released from food by pepsin → immediately binds Haptocorrin (R-protein / transcobalamin I) from salivary glands. Haptocorrin protects B12 from stomach acid.
2
Duodenum: Pancreatic proteases degrade haptocorrin → B12 released → binds Intrinsic Factor (IF), secreted by gastric parietal cells.
3
Terminal Ileum: IF-B12 complex binds cubilin receptors → receptor-mediated endocytosis → B12 enters blood bound to transcobalamin II.

Pernicious Anaemia

Autoimmune destruction of gastric parietal cells → no IF → B12 cannot be absorbed → megaloblastic anaemia + subacute combined degeneration of spinal cord. Treat with intramuscular B12 injections.

Exam Trap: Haptocorrin vs Intrinsic Factor ★ asked 2022

Haptocorrin = R-protein, from SALIVA — first binder, protects B12 in stomach.
Intrinsic Factor = from GASTRIC PARIETAL CELLS — second binder, required for ileal absorption.

🍖 Trypsin and Enteropeptidase 2026

The 2026 paper asked "what catalyses trypsinogen → trypsin?" with options CCK, trypsin and enteropeptidase — and the recorded answer was both trypsin and enteropeptidase. That double answer is the whole point of the cascade.

Enteropeptidase (enterokinase)

A brush-border enzyme of the duodenum. It performs the initial activation: trypsinogen → trypsin. Nothing else activates trypsinogen first.

Trypsin — autocatalysis

Once formed, trypsin activates more trypsinogen (hence both options being true) and then activates everything else: chymotrypsinogen → chymotrypsin, proelastase → elastase, procarboxypeptidases → carboxypeptidases A and B, and prophospholipase A₂ → phospholipase A₂, and it also activates procolipase.

CCK is not the answer

Cholecystokinin is a hormone: it makes the gallbladder contract and the pancreas secrete its enzymes. It does not catalyse anything. It acts on stomach (slowing motility), pancreas and gallbladder. Secretin, released in response to acid, drives bicarbonate secretion.

Endo- vs exopeptidases

Endopeptidases cut internal bonds: pepsin (aromatic residues, optimum pH ≈ 2), trypsin (after Lys/Arg), chymotrypsin (after aromatics), elastase (small neutral residues). Exopeptidases trim the ends: carboxypeptidase A and B from the C-terminus; aminopeptidase from the N-terminus. Aminopeptidase works in the brush border, requires a basic pH (≈ 8) — not acidic — and its action follows that of the endopeptidases.

Gastric acid, BAO and MAO

In the parietal (oxyntic) cell, carbonic anhydrase makes H⁺ from CO₂ + H₂O and the H⁺/K⁺-ATPase pumps it into the lumen; Cl⁻ follows; the bicarbonate leaving into blood is the alkaline tide. Secretion is stimulated by gastrin (via cAMP), histamine on H₂ receptors, and acetylcholine. BAO = basal acid output, the acid secreted per hour unstimulated (about 0–5 mmol/h); MAO = maximal acid output after maximal stimulation (about 5–20 mmol/h). A high BAO with a BAO/MAO ratio above ~0.6 suggests a gastrinoma.

🧪 Benedict's Reagent — and the Other Named Tests 2026

The 2026 recollection records the Benedict question with its options, and the correct one is unambiguous.

✅ "It detects all monosaccharides"

Correct. Benedict's reagent is alkaline Cu²⁺ (copper sulfate + citrate); any sugar with a free aldehyde or ketone group — a reducing sugar — reduces it to Cu₂O. All monosaccharides are reducing sugars, aldoses and ketoses alike, because in alkali a ketose tautomerises to an aldose.

❌ "It can distinguish glucose from fructose"

False — and this is the intended trap. Both are reducing sugars and both give the same positive result. The test that does distinguish them is Selivanoff's, which separates ketoses (fructose — rapid cherry-red) from aldoses (glucose — slow, faint).

❌ "The colour is pink"

False. A negative test stays blue; a positive one goes green → yellow → orange → brick-red as the amount of reducing sugar rises. Older papers ask this as "Benedict — orange — reducing sugars".

Which sugars are and are not reducing

Reducing: all monosaccharides (glucose, fructose, galactose, mannose, ribose), plus maltose, isomaltose and lactose — they keep a free anomeric carbon. Non-reducing: sucrose (saccharose), because both anomeric carbons are locked in the glycosidic bond, and trehalose. Sucrose is the standard "does not have reducing properties" answer.

The named colour tests, each with its target

Benedict's — reducing sugars. Selivanoff's — ketoses, i.e. fructose, distinguishing them from aldoses. Bial's (orcinol) — pentoses / ribose. Hay's (sulfur powder sinks) — bile salts in urine. Pettenkofer's — also bile salts. Salkowski's — cholesterol. Van den Bergh — bilirubin; the direct (conjugated) fraction reacts immediately in water, the indirect (unconjugated) fraction only after adding methanol/alcohol — which is exactly why the two fractions are called direct and indirect. Ehrlich's — urobilinogen. Iodine test — starch/glycogen, going blue-black.

🍞 Carbohydrate Digestion, Structure and Absorption

The disaccharides and their enzymes

Lactose = galactose + glucose, β(1→4), split by lactase (β-galactosidase). Sucrose (saccharose) = glucose + fructose, α(1→2), split by sucrase. Maltose = glucose + glucose, α(1→4). Isomaltose = glucose + glucose, α(1→6) — an isomer of maltose, split by isomaltase. Trehalose = glucose + glucose, α(1→1).

Isomer vocabulary — asked directly

Glucose and galactose are epimers (they differ at one carbon, C4); glucose and mannose are epimers at C2. Glucose and fructose are aldose–ketose isomers, not epimers and not anomers. Anomers are α and β forms of the same sugar, differing only at the anomeric carbon. Enantiomers are mirror images (D- and L-); D-glucose and L-glucose. So "fructose is an enantiomer of glucose" is false — a repeated distractor.

Which are aldoses, which ketoses

Aldoses: glucose, galactose, mannose, ribose, glyceraldehyde, erythrose. Ketoses: fructose, ribulose, xylulose, sedoheptulose, erythrulose, dihydroxyacetone. Fructose, mannose and galactose do share the formula C₆H₁₂O₆ with glucose.

Glycosaminoglycans

Polymers of amino sugars and uronic acids, found in mucus and joint cartilage, and the family includes heparin, hyaluronic acid, chondroitin sulfate and keratan sulfate. They are not broken down into oligosaccharides by deamination — that option is false.

Lactose intolerance

Deficiency of brush-border lactase. Undigested lactose is osmotically active and is fermented by colonic bacteria to lactic and acetic acid with H₂ and CO₂ — giving osmotic diarrhoea, bloating, cramps and flatulence after milk. Diagnosed with the hydrogen breath test or a lactose tolerance test. The adult-onset (primary) form is by far the commonest and is normal for most of the world's population.

Lipid Digestion & Absorption

Frequently tested: order of events (emulsification→hydrolysis→micelles), bile acids/salts & conjugation, primary vs secondary, what is NOT made in liver

🧈 Order of Lipid Digestion

The correct sequence — tested directly on exams: Emulsification → Hydrolysis → Micelle formation → Absorption

🧈 Lipid Digestion — order of events

1 · Emulsificationbile salts → 2 · Hydrolysispancreatic lipase → 3 · Micelle formationFFA + 2-MAG + bile → 4 · Absorptionenterocyte → chylomicron

1
Emulsification: bile salts (made in liver, stored in gallbladder) break large fat globules into tiny droplets — this massively increases the surface area available for enzymes to act on.
2
Hydrolysis: Pancreatic Lipase (requires colipase as a helper protein) cleaves triglycerides at the sn-1 and sn-3 positions → produces 2-monoacylglycerol (2-MAG) + 2 free fatty acids (FFAs).
3
Micelle formation: the digestion products (FFAs + 2-MAG) combine with bile salts to form mixed micelles — tiny water-soluble aggregates that can travel through the watery intestinal fluid to reach the intestinal cells.
4
Absorption: FFAs and 2-MAG diffuse into enterocytes (intestinal cells) → re-esterified back to triglycerides → packaged into chylomicrons (which carry ApoB-48) → secreted into lymph → eventually enter the bloodstream.

CCK vs Secretin — Must Distinguish

🟢 CCK (Cholecystokinin)

Released when fat & protein enter the duodenum. Stimulates gallbladder contraction (releases bile) + pancreatic enzyme secretion. Also an anorexigenic hormone — suppresses appetite.

🔵 Secretin

Released when acid enters the duodenum. Stimulates pancreatic bicarbonate (HCO₃⁻) secretion to neutralize stomach acid — NOT enzyme secretion.

HCl Effects on Blood Chemistry

When the stomach secretes HCl (hydrogen + chloride into the lumen), bicarbonate is simultaneously released into the blood — this is called the "alkaline tide." Result: ↑ plasma HCO₃⁻ and ↓ plasma Cl⁻ during active acid secretion. Directly tested on exams.

🧴 Saliva Composition (Exam Tested)

Saliva contains: amylase, water, mucus, lactoferrin (iron-binding antimicrobial protein), potassium (saliva is rich in K⁺), cysteine protease inhibitors (cystatins — protect tissues from proteolytic damage), IgA, and bicarbonate. All three of the bolded items have been asked together in one question.

💛 Bile Salts vs Bile Acids — Teacher Required

Bile acids (unconjugated) are cholesterol derivatives. When conjugated with glycine or taurine, they become bile salts.

Unconjugated bile acids: pKa ~6 → partially dissociated at bile pH (half ionized)
Conjugated with glycine or taurine: pKa lowered → fully dissociated at bile pH → cannot donate protons → they are salts
Conjugation makes them more amphiphilic → better detergents → better at forming micelles

Primary Bile Acids (examples)

Synthesized in liver from cholesterol: Cholic acid and Chenodeoxycholic acid. Conjugated with glycine/taurine before secretion.

Secondary Bile Acids (examples)

Formed in colon by bacterial action (deconjugation + dehydroxylation): Deoxycholic acid (from cholic) and Lithocholic acid (from chenodeoxycholic). Teacher requires knowing both primary AND secondary examples.

Enterohepatic Circulation (Teacher Referenced Fig 18.11)

Most bile salts (conjugated + unconjugated) are reabsorbed in the terminal ileum → portal blood → liver → re-secreted into bile. Only ~5% excreted in feces. This conserves the bile salt pool.

Glycolysis & Gluconeogenesis

Frequently tested: irreversible enzymes (HK, PFK-1, PK), PFK-1 regulation by F-2,6-BP, gluconeogenesis ATP/GTP-requiring steps, glucose-6-phosphatase location

🍬 Glycolysis — The Full Picture

Glycolysis occurs in the cytoplasm of every cell. It converts one glucose molecule into two pyruvate molecules. It has two phases: an investment phase (uses 2 ATP) and a payoff phase (generates 4 ATP + 2 NADH). Net yield: 2 ATP + 2 NADH per glucose. Gross ATP yield: 4 ATP.

Three reactions are irreversible (driven strongly forward) — these are the regulatory steps:

1
Hexokinase/Glucokinase: traps glucose inside the cell as glucose-6-phosphate (G6P). Hexokinase is in all cells (inhibited by G6P). Glucokinase is in liver and beta-cells (not inhibited by G6P — acts as a glucose sensor).
2
PFK-1 (Phosphofructokinase-1): the main rate-limiting enzyme. Converts fructose-6-phosphate → fructose-1,6-bisphosphate. Main activator: fructose-2,6-bisphosphate (F-2,6-BP), also activated by AMP and ADP. Inhibited by ATP and citrate.
3
Pyruvate Kinase: converts phosphoenolpyruvate (PEP) → pyruvate, generating ATP. The liver isoform is inactivated by glucagon (via phosphorylation through the cAMP–PKA pathway).

Gross vs Net ATP — Frequently Tested

Glycolysis gross ATP = 4 ATP. Investment phase uses 2 ATP. Net = 2 ATP. In anaerobic glycolysis (no oxygen), the NADH cannot enter the mitochondria, so the overall yield stays at 2 net ATP.

🔃 Gluconeogenesis Bypass Reactions

Glycolysis enzyme (irreversible)	GNG bypass
Pyruvate Kinase	Pyruvate Carboxylase (biotin+ATP) + PEPCK (GTP)
PFK-1	Fructose-1,6-bisphosphatase
Hexokinase	Glucose-6-Phosphatase (liver/kidney only)

Cannot make glucose from fat!

Even-chain fatty acids → only acetyl-CoA → cannot enter gluconeogenesis. Acetyl-CoA cannot be converted to OAA in humans.

🔀 PFK-2 / FBP-2 — The Bifunctional Enzyme (Teacher Highlighted)

PFK-2/FBP-2 is a single protein with two enzyme activities — a kinase domain and a phosphatase domain. This is the enzyme that controls the level of fructose-2,6-bisphosphate (F-2,6-BP), the most potent activator of glycolysis.

PFK-2 (Kinase Domain)

Phosphorylates fructose-6-phosphate → fructose-2,6-bisphosphate (F-2,6-BP)
Active when the kinase domain is dephosphorylated (insulin state).
F-2,6-BP → activates PFK-1 → ↑glycolysis

FBP-2 (Phosphatase Domain)

Cleaves fructose-2,6-bisphosphate → fructose-6-phosphate + Pi
Active when phosphorylated by PKA (glucagon/fasting state). When kinase domain is ⊖ (phosphorylated), the phosphatase domain is active → ↓F-2,6-BP → promotes gluconeogenesis.

Key Regulation Logic

Fed state (insulin): PFK-2 kinase active → ↑F-2,6-BP → activates PFK-1 → ↑glycolysis, ⊖ FBP-1 → ↓gluconeogenesis
Fasting (glucagon): PKA phosphorylates PFK-2/FBP-2 → kinase domain ⊖ → FBP-2 phosphatase active → ↓F-2,6-BP → ⊖ PFK-1, activates FBP-1 → ↑gluconeogenesis
Only the kinase domain is regulated (by phosphorylation). The phosphatase domain activity is the inverse consequence.

Fructose-2,6-Bisphosphate Action

F-2,6-BP is the strongest known activator of PFK-1. It also inhibits FBP-1 (fructose-1,6-bisphosphatase). So the same molecule promotes glycolysis AND inhibits gluconeogenesis simultaneously.

🏃 Anaerobic Bypass — Cori Cycle / Lactate Shuttle (Teacher: "Know Both Names")

In anaerobic conditions (RBCs, exercising muscle, hypoxic tissue), pyruvate cannot enter the Krebs cycle. Instead, Lactate Dehydrogenase converts pyruvate → lactate, regenerating NAD⁺ to keep glycolysis running.

🏃 Cori Cycle (Lactic Acid Cycle)

Muscle: Glucose →glycolysis (+2 ATP) Pyruvate Lactate Dehydrogenase→ Lactate →blood Liver →gluconeogenesis (−6 ATP) Glucose →blood → muscle Muscle

↩ Net: muscle gains 2 ATP, liver spends 6 ATP — the metabolic burden is shifted from muscle to liver.

This transfers the metabolic burden from muscle to liver — muscle gets energy from glycolysis, liver handles the lactate.

Net Result of Cori Cycle

Net energy transfer: 2 ATP gained by muscle per glucose. 6 ATP spent by liver to convert 2 lactate → 1 glucose. The liver uses energy from fat oxidation to "subsidize" muscle glycolysis. Teacher noted: know the actual pathway name AND the common (Cori cycle) name.

GLUT Transporters — Insulin Independence

GLUT-1, GLUT-2, GLUT-3 are constitutively expressed and insulin-independent. GLUT-4 (muscle and adipose) is insulin-dependent — it moves to the cell surface only in response to insulin. This is why muscle cannot use glucose efficiently without insulin in Type 1 DM.

🔀 Glucose-6-Phosphate — The Central Metabolic Crossroads

Once glucose is phosphorylated, glucose-6-phosphate (G6P) stands at the junction of five pathways. Which way it flows depends on the tissue and the fed/fasting state.

⚗️ The Five Fates of G6P

G6P → Glycolysis → pyruvate + ATP → Glycogenesis → glycogen → Pentose phosphate pathway → Glucose-6-phosphatase → free glucose → Uronic acid (glucuronate) pathway

Why G6P is special

It is the one molecule shared by glycolysis, glycogen metabolism, the PPP, and the uronic acid pathway — and only liver and kidney can strip its phosphate (glucose-6-phosphatase) to release free glucose into the blood. Muscle lacks this enzyme, so muscle G6P can never leave the cell as glucose.

↔️ Which Steps Are Reversible, Which Are Not 2026

The 2026 stem listed hexokinase, pyruvate kinase and phosphoglycerate kinase and asked which catalyses a reversible reaction. Two of the three are one-way.

🔒 Irreversible — the three of glycolysis

Hexokinase / glucokinase (glucose → G6P), PFK-1 (F6P → F1,6BP) and pyruvate kinase (PEP → pyruvate). These are the regulated steps, and each needs its own bypass enzyme in gluconeogenesis.

🔓 Reversible — everything else

Phosphoglycerate kinase is reversible — that is the answer. So are phosphohexose isomerase, aldolase, phosphotriose isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase and enolase. Because they are reversible, gluconeogenesis simply runs them backwards.

The four gluconeogenic bypasses

Pyruvate carboxylase (mitochondrial, biotin, activated by acetyl-CoA) → oxaloacetate; PEP carboxykinase (uses GTP); fructose-1,6-bisphosphatase; glucose-6-phosphatase (in the ER lumen of liver and kidney only — it yields Pi, it does not require ATP).

Reactions that produce ATP

Substrate-level phosphorylation happens at phosphoglycerate kinase and pyruvate kinase in glycolysis, and at succinyl-CoA synthetase (succinate thiokinase) in the Krebs cycle. Glycerol kinase and lactate dehydrogenase consume or produce no ATP — glycerol kinase spends one.

Enzymes that compete for pyruvate

Pyruvate dehydrogenase (→ acetyl-CoA), pyruvate carboxylase (→ oxaloacetate), alanine aminotransferase (→ alanine) and lactate dehydrogenase (→ lactate). Pyruvate kinase does not compete for pyruvate — it produces it. Note also that enolase is specifically inhibited by fluoride, and glyceraldehyde-3-phosphate dehydrogenase by arsenate, which uncouples the ATP-yielding step.

🎚️ PFK-2, Fructose-2,6-Bisphosphate and What It Bypasses 2026

Two 2026 items concern this: what PFK-2 bypasses, and what activates fructose-2,6-bisphosphate and in which state.

What PFK-2 actually does

PFK-2 phosphorylates fructose-6-phosphate at position 2, producing fructose-2,6-bisphosphate — a regulator, not an intermediate of glycolysis. Its side-product route therefore bypasses PFK-1, the main regulatory step, in the sense that F-2,6-BP is made on a branch line and then comes back to control PFK-1 allosterically.

The bifunctional enzyme

PFK-2 and fructose-2,6-bisphosphatase are two activities of one polypeptide. Glucagon → cAMP → PKA phosphorylates it → kinase OFF, phosphatase ON → F-2,6-BP falls. Insulin → dephosphorylation → kinase ON → F-2,6-BP rises.

✅ In which state, and what it does

F-2,6-BP is high in the fed / absorptive state, under insulin. It is the most potent activator of PFK-1 — strong enough to override inhibition by ATP — and simultaneously inhibits fructose-1,6-bisphosphatase. One molecule therefore turns glycolysis on and gluconeogenesis off at the same time. In fasting, glucagon reverses it.

The rest of PFK-1's regulation

Inhibited by ATP and citrate; activated by AMP and by F-2,6-BP. Older papers ask it as a set: "glycolysis in the liver is activated → PFK-2 is active → PFK-1 is activated" — all three true together.

🚗 Transporters, Glucokinase and the β Cell 2026

✅ Insulin induces the production of glucokinase

True — a 2026 item. Insulin acts on transcription, increasing the amount of glucokinase (as well as pyruvate kinase and PFK-1) over hours. Glucokinase is not regulated allosterically by its product; it is regulated by induction and by the nuclear glucokinase regulatory protein.

Glucokinase vs hexokinase

Glucokinase (hexokinase IV): liver and pancreatic β cells, high Km (low affinity), high Vmax, not inhibited by glucose-6-phosphate, specific for glucose, insulin-inducible, paired with GLUT-2. Hexokinase: all tissues, low Km, saturated at normal glycaemia, inhibited by G6P, accepts several hexoses — including fructose, which it phosphorylates at position 6. The option "glucokinase has a high Km for fructose" is a nonsense distractor: glucokinase does not act on fructose.

GLUT family — where each one is

GLUT-1 red cells, brain and blood–brain barrier, placenta, kidney, colon. GLUT-2 liver, β cells, small intestine, kidney — high Km, bidirectional. GLUT-3 neurons, placenta — lowest Km. GLUT-4 muscle, heart, adipose — the insulin-dependent one. GLUT-5 intestine and testis — fructose.

❌ "GLUT-2 needs energy"

False — a 2026 item. Every GLUT is facilitated diffusion: down the gradient, no ATP, no Na⁺. Only SGLT-1 and SGLT-2 use energy, and even then indirectly: they are secondary active Na⁺ symporters, powered by the Na⁺ gradient that Na⁺/K⁺-ATPase maintains. SGLT-2 does the bulk of renal glucose reabsorption in the early proximal tubule; SGLT-1 works in the intestine and late proximal tubule.

Pancreatic β cells 2026

They have GLUT-2 and glucokinase — both true — which together let the cell's rate of glucose phosphorylation track blood glucose across the physiological range: glucokinase is the glucose sensor. Glucose → glycolysis → ↑ATP/ADP → closure of the K_ATP channel → depolarisation → voltage-gated Ca²⁺ entry → insulin exocytosis. The false option is that GLP-1 decreases glucose sensitivity — glucagon-like peptide-1 increases glucose-stimulated insulin secretion; it is an incretin, and incretin drugs work precisely because of it. Sulfonylureas close the same K_ATP channel pharmacologically.

🩸 The Erythrocyte — Bypass, ATP Yield and the Calculation 2026

Two 2026 items are about red cells: which glycolytic step is bypassed, and how much ATP a given amount of glucose yields.

The bypass: the Rapoport–Luebering shunt

In the red cell, 1,3-bisphosphoglycerate can be diverted by bisphosphoglycerate mutase to 2,3-bisphosphoglycerate, which is then hydrolysed by a phosphatase to 3-phosphoglycerate. Because the shunt goes round the phosphoglycerate kinase step, that ATP is lost — the answer the 2026 recollection records. What the cell gains is 2,3-BPG, which binds deoxyhaemoglobin and shifts the oxygen dissociation curve to the right, releasing more O₂ to the tissues. So the red cell trades ATP for oxygen delivery.

Doing the arithmetic

Anaerobic glycolysis gives a net 2 ATP per glucose. 0.1 mmol glucose × 2 = 0.2 mmol ATP = 200 µmol — the recorded answer. The same question appears as "0.3 mol glucose → 0.6 mol ATP". Do not use 38 or 32: the red cell has no mitochondria, so there is no oxidative phosphorylation at all.

Why the red cell is built this way

No mitochondria and no nucleus. Its only ATP source is anaerobic glycolysis, ending at lactate via lactate dehydrogenase — which is also the only way to regenerate the NAD⁺ that glycolysis consumes. Its only source of NADPH is the pentose phosphate pathway, needed to keep glutathione reduced.

The pH conversion 2026

"If pH is 7, what is [H⁺]?" — pH = −log[H⁺], so [H⁺] = 10⁻⁷ mol/L. Converting: 10⁻⁷ mol/L = 10⁻⁴ mmol/L = 10⁻¹ µmol/L = 100 nmol/L. The recorded answer is 100 nmol/L. At the physiological pH 7.4 the same arithmetic gives about 40 nmol/L — worth knowing, because it shows how tiny the absolute concentrations are.

Which tissues cannot use which fuels

Red cells — glucose only, anaerobically. Brain — glucose, and ketone bodies after several days of starvation; it cannot use fatty acids. Lens, cornea and retina — largely glycolytic. Glucose is not the basic fuel of the liver, which in the fed state runs largely on amino acids and in fasting on fatty acids — the answer to "glucose is not a basic energy fuel of…".

🍏 Fructose, Galactose and the Regulatory Bypass 2026

The 2026 stem: which characteristic of fructose metabolism bypasses the major regulatory step of glycolysis? The options given were three different conversions; only one is the fructose route.

✅ Fructose-1-phosphate → DHAP + glyceraldehyde

This is the answer. In the liver, fructokinase makes fructose-1-phosphate and aldolase B splits it into dihydroxyacetone phosphate and glyceraldehyde; triose kinase (glyceraldehyde kinase) then phosphorylates the glyceraldehyde. Both products join glycolysis below PFK-1, so the pathway escapes the main control point entirely — which is why a fructose load is metabolised rapidly and unregulated, feeding lipogenesis and raising triacylglycerol.

❌ The two distractors

"Glucose-6-P → fructose-6-P" is phosphohexose isomerase, an ordinary reversible glycolytic step. "Fructose-6-P → fructose-1,6-BP" is PFK-1 itself — the step being bypassed, not the bypass.

Where each hexose is phosphorylated

Fructose — position 1 by fructokinase in the liver; position 6 by hexokinase in muscle and adipose. Galactose — position 1 by galactokinase, giving galactose-1-phosphate plus ADP. Glucose — position 6.

Galactose and why it is not essential

Galactokinase → Gal-1-P → GALT (using UDP-glucose) → UDP-galactose → 4-epimerase, which is reversible, so UDP-galactose can always be made from UDP-glucose. Galactose is therefore not an essential nutrient. UDP-galactose is the substrate for lactose synthesis.

The two fructose disorders, and galactosaemia

Essential fructosuria — fructokinase deficiency; benign, fructose simply appears in blood and urine. Hereditary fructose intolerance — aldolase B deficiency; fructose-1-phosphate accumulates and sequesters inorganic phosphate, so ATP falls and both glycogenolysis and gluconeogenesis are blocked: severe hypoglycaemia, vomiting, jaundice, hepatomegaly and renal dysfunction after fructose or sucrose, with an acquired aversion to sweet foods. Galactosaemia — GALT deficiency: Gal-1-P is toxic to liver, brain and kidney, while galactitol (from aldose reductase) causes cataracts; galactokinase deficiency alone gives cataracts only.

🔋 The Pentose Phosphate Pathway and NADPH

The two phases

Oxidative, irreversible: G6P → 6-phosphogluconate → ribulose-5-phosphate + CO₂, giving 2 NADPH. The rate-limiting enzyme glucose-6-phosphate dehydrogenase uses NADP⁺ only, never NAD⁺, is inhibited by NADPH, and is induced by insulin in the fed state. Non-oxidative, reversible: transketolase (moves 2 carbons, needs TPP/vitamin B1 and Mg²⁺) and transaldolase (moves 3 carbons, no cofactor) interconvert sugars, linking back to fructose-6-P and glyceraldehyde-3-P.

The sugars that appear as options

Ribulose-5-phosphate (the direct product of the oxidative phase) and its isomers ribose-5-phosphate and xylulose-5-phosphate; then sedoheptulose-7-phosphate, erythrose-4-phosphate, fructose-6-phosphate and glyceraldehyde-3-phosphate. Ribose-5-P feeds nucleotide synthesis; erythrose-4-P feeds aromatic amino acid synthesis in plants.

Where NADPH comes from — the list

Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the PPP; malic enzyme; and cytosolic isocitrate dehydrogenase. Cytosolic malate dehydrogenase uses NADH, not NADPH — the standard wrong option in that list.

Why it matters where

Red cells — the only NADPH source, for glutathione. Adipose tissue and liver — reducing power for lipogenesis (14 NADPH per palmitate). Adrenal cortex and gonads — steroid hydroxylations. Neutrophils — the NADPH oxidase respiratory burst. It produces no ATP.

The uronic acid pathway — asked as a set of statements

It starts from UDP-glucose, occurs in the liver, produces no ATP (it consumes one), and its point is to make UDP-glucuronic acid for conjugation, bilirubin excretion and glycosaminoglycan synthesis. It is not a cycle — its products feed into the pentose phosphate pathway. And its most-tested line: it produces ascorbic acid in plants and most animals but not in humans, because we lack L-gulonolactone oxidase — which is why ascorbic acid is required in the human diet.

Glycogen Metabolism

Frequently tested: glycogen phosphorylase (AMP-activated, ATP-inhibited, PLP cofactor), hormonal cascade, glycogen storage diseases (von Gierke, McArdle, Hers, Cori, Andersen)

🍡 Synthesis & Breakdown

Glycogen Synthase: active when dephosphorylated (insulin promotes dephosphorylation via protein phosphatase-1). Glycogen Phosphorylase: active when phosphorylated (glucagon and epinephrine trigger cAMP → PKA → phosphorylation).

Liver Glucose-6-Phosphatase releases free glucose into the blood — muscle lacks this enzyme and therefore cannot directly raise blood glucose. Muscle glycogen is used only for the muscle itself.

Muscle Glycogen — Exam-Tested Distinctions

Muscle glycogen phosphorylase is activated by calcium ions (Ca²⁺) released during muscle contraction and by AMP — NOT by glucagon (glucagon receptors are absent on muscle). Muscle glycogen breakdown does not directly increase plasma glucose. These three facts are tested together.

🏥 Glycogen Storage Diseases

Disease	Enzyme Missing	Key Features
Von Gierke (I)	Glucose-6-Phosphatase	Severe hypoglycemia, hepatomegaly, lactic acidosis
Pompe (II)	Lysosomal α-Glucosidase	Glycogen in lysosomes; cardiomegaly
Anderson (IV)	Branching Enzyme	Liver cirrhosis (amylopectin-like glycogen accumulates). Rare but directly tested in 2022.
McArdle (V)	Muscle Glycogen Phosphorylase	Cramping on exercise

🔋 Glycogenolysis — Full Detail (Teacher Notes)

Glycogenolysis: breakdown of glycogen → releases glucose. Hormones that trigger it:

Liver: Glucagon and Epinephrine → GCPR → adenylate cyclase → ↑cAMP → PKA → phosphorylates glycogen phosphorylase kinase → activates Glycogen Phosphorylase
Muscle: Epinephrine (via cAMP/PKA) AND Ca²⁺ (released from SR during muscle contraction) → Calmodulin → phosphorylase kinase → Glycogen Phosphorylase

Glycogen phosphorylase cleaves α-1,4 glycosidic bonds from the non-reducing end, releasing glucose-1-phosphate (which → glucose-6-phosphate). It cannot cross α-1,6 branch points — a separate debranching enzyme (two activities: transferase + glucosidase) handles branch points, releasing free glucose at the branch.

PKA Simultaneously Activates Glycogenolysis AND Inhibits Glycogen Synthesis

When glucagon/epinephrine → PKA → simultaneously:
1. Phosphorylates glycogen phosphorylase kinase → activates Glycogen Phosphorylase (↑breakdown)
2. Phosphorylates Glycogen Synthase → INACTIVATES it (↓synthesis)
This coordinates: during fasting → break down glycogen AND stop making it.

Pentose Phosphate Pathway (PPP)

Frequently tested: NADPH for anabolism, ribose-5-P for nucleotides, G6PD deficiency & favism, transketolase vs transaldolase

🔋 What the PPP Does

The Pentose Phosphate Pathway (PPP) runs in the cytoplasm and serves two main purposes: (1) producing NADPH — the cell's main reducing agent for biosynthesis and antioxidant defense, and (2) producing ribose-5-phosphate — needed to build nucleotides (DNA and RNA building blocks).

Rate-Limiting Enzyme of the PPP = G6PD — Exam Tested

G6PD (Glucose-6-Phosphate Dehydrogenase) is the first and rate-limiting enzyme of the PPP. It catalyses Glucose-6-phosphate + NADP⁺ → 6-Phosphogluconolactone + NADPH. This is the only source of NADPH in red blood cells (which have no mitochondria).

Correct pairing (Q29c): PPP — Cytoplasm — Rate-limiting enzyme: G6PD

G6PD Deficiency (Favism)

X-linked recessive. Most common enzyme defect worldwide (~400 million affected). Without G6PD, cells cannot make NADPH → cannot regenerate reduced glutathione (GSH). Glutathione's role in red blood cells is to neutralize hydrogen peroxide (H₂O₂) — without it, H₂O₂ oxidizes hemoglobin → Heinz bodies form → red blood cells are destroyed → hemolytic anemia. Triggered by fava beans, primaquine, dapsone, and infections.

Glutathione Composition — Exam Tested

Glutathione is a tripeptide made of three amino acids: Glutamate + Cysteine + Glycine (remember: GCG or "gamma-Glu-Cys-Gly"). Cysteine's thiol group (–SH) is the active reducing part. This composition is directly asked in exams.

🔀 Transketolase — Exam Detail

Transketolase is a key enzyme of the non-oxidative branch of the PPP. It requires TPP (thiamine pyrophosphate, from vitamin B1) as its cofactor. Transketolase transfers 2-carbon units between sugar phosphates, interconverting 5-carbon, 7-carbon, 4-carbon, and 6-carbon sugars. This is a common exam trap: students confuse transketolase with transaldolase. Transaldolase does NOT require any coenzyme — it works without a cofactor. Only transketolase needs TPP.

Exam Trap

"Transaldolase requires TPP" → FALSE. Transaldolase requires no coenzyme. Transketolase requires TPP (vitamin B1). This distinction appears directly in exam questions.

💨 Nitric Oxide (NO) & NADPH Oxidase

NO Synthesis
Enzyme: NO Synthase (NOS)
Cofactors: NADPH, FMN, FAD, heme, tetrahydrobiopterin (BH₄)
Note: NOS uses BH₄ directly as cofactor — it does NOT require tetrahydropterin reductase (2023 exam)

NOS isoforms:

eNOS (endothelial): Ca²⁺/calmodulin-dependent → vasodilation
nNOS (neuronal): Ca²⁺/calmodulin-dependent → neurotransmitter
iNOS (inducible): Ca²⁺-independent; induced by TNF-α, endotoxins → antimicrobial (large amounts)

NADPH Oxidase & Respiratory Burst
Located in leukocyte membrane. Contains flavocytochrome (FAD + heme). Uses NADPH to reduce O₂ → superoxide (O₂⁻) → respiratory burst → kills bacteria.

Chronic Granulomatous Disease (CGD)

Genetic NADPH oxidase deficiency → phagocytes cannot produce superoxide → cannot kill catalase-positive bacteria (Staphylococcus, Aspergillus) → severe recurrent infections + granuloma formation. X-linked (most common form).

NO Functions

Vasodilator (eNOS) · Neurotransmitter (nNOS) · Inhibits platelet aggregation · Antimicrobial (iNOS in macrophages). Half-life: 3–10 seconds. Reacts with O₂ and superoxide → nitrates/nitrites.

Krebs Cycle & Respiratory Chain

Frequently tested: reversible vs irreversible reactions, malonate inhibits succinate dehydrogenase, anaplerotic reactions, redox steps, respiratory chain complex order, inhibitors & uncouplers

🔢 Full Krebs Cycle — All 8 Steps, Enzymes, Substrates, Reaction Types

Step	Substrate → Product	Enzyme	Cofactors	Reaction Type	Energy yield
1	Acetyl-CoA + OAA → Citrate	Citrate Synthase	None	Condensation (Transferase)	—
2	Citrate → Isocitrate	Aconitase	Fe–S cluster	Isomerization (via cis-aconitate)	—
3 ★	Isocitrate → α-Ketoglutarate + CO₂	Isocitrate Dehydrogenase	NAD⁺, Mn²⁺	Oxidative decarboxylation (rate-limiting ★)	NADH
4 ★	α-Ketoglutarate → Succinyl-CoA + CO₂	α-Ketoglutarate DH	TPP, lipoic acid, CoA, FAD, NAD⁺	Oxidative decarboxylation (rate-limiting ★)	NADH
5	Succinyl-CoA → Succinate	Succinyl-CoA Synthetase	GDP/ADP + Pi	Substrate-level phosphorylation	GTP (or ATP)
6	Succinate → Fumarate	Succinate Dehydrogenase	FAD	Oxidation (dehydrogenation)	FADH₂
7	Fumarate → Malate	Fumarase	None	Hydration	—
8	Malate → OAA	Malate Dehydrogenase	NAD⁺	Oxidation	NADH

Per Turn Summary: 3 NADH + 1 FADH₂ + 1 GTP + 2 CO₂

Irreversible steps (★): Citrate Synthase (step 1), Isocitrate DH (step 3), α-KG DH (step 4).
Inhibited by fluoroacetate: Fluoroacetate → fluorocitrate → inhibits Aconitase → citrate accumulates.

🔗 Anaplerotic Reactions — Replenishing Krebs Intermediates

Anaplerotic reactions add intermediates back into the Krebs cycle to keep it running when intermediates are withdrawn for biosynthesis. They are the opposite of cataplerotic reactions.

Reaction	Source	Krebs Intermediate Added	Enzyme
Pyruvate + CO₂ → OAA	Pyruvate (from glucose)	Oxaloacetate (OAA)	Pyruvate Carboxylase (biotin, ATP)
Glutamate → α-Ketoglutarate	Pro, His, Arg, Gln (via Glu)	α-Ketoglutarate	Glutamate Dehydrogenase
Propionyl-CoA → Succinyl-CoA	Met, Thr, Val, Ile (and odd-chain FAs)	Succinyl-CoA	Propionyl-CoA carboxylase + methylmalonyl-CoA mutase (B12)
Fumarate from urea cycle	Argininosuccinate → Arginine + Fumarate	Fumarate	Arginase / Argininosuccinate lyase
Phenylalanine/Tyrosine → Fumarate	Phe, Tyr catabolism	Fumarate	Fumarylacetoacetase

Amino Acids Entering Krebs — Teacher Highlighted

Pro · His · Arg · Gln →via Glutamate α-Ketoglutarate

Met · Thr · Val · Ile →via propionyl-CoA Succinyl-CoA

Phe · Tyr → Fumarate

Asp · Asn →transamination OAA

OAA → PEP (Gluconeogenesis) — The Key Connection

When the Krebs cycle runs LOW on OAA (e.g. during fasting), OAA is diverted to gluconeogenesis via PEPCK (OAA → PEP). This is why: whole cycle must be intact to withdraw intermediates for GNG. High acetyl-CoA (from FA oxidation) activates Pyruvate Carboxylase → replenishes OAA.

⚙️ Complex V (ATP Synthase) — Matrix + Membrane Proteins (Teacher Noted)

Complex V (ATP Synthase) consists of two major domains — this is NOT part of the ETC electron carriers, but is part of oxidative phosphorylation:

F₀ (membrane domain)

Embedded in the inner mitochondrial membrane. Contains the proton channel — H⁺ flow through F₀ rotates the c-ring. Inhibited by oligomycin (blocks the proton channel).

F₁ (matrix domain)

Projects into the mitochondrial matrix. Contains the catalytic β-subunits that synthesize ATP from ADP + Pi. The α₃β₃ hexamer rotates → mechanical force → ATP synthesis. Also called the "stalk".

⊖ Malonate — Competitive Inhibitor of Complex II ★ asked 2018, 2025

Malonate is a structural analog of succinate → competitive inhibitor of Succinate Dehydrogenase (Complex II). Teacher specifically mentioned this. Unlike Complex I (rotenone), Complex III (antimycin A), and Complex IV (cyanide/CO), malonate specifically targets Complex II. Malonate is NOT a physiological inhibitor — it's used experimentally.

Oxidative Phosphorylation — Essential Understanding

Coupling: ETC pumps H⁺ → builds electrochemical gradient → H⁺ flows back through Complex V → ATP synthesized. Uncouplers (DNP, UCP-1) dissipate gradient → O₂ still consumed, no ATP made → heat. Oligomycin blocks F₀ → gradient cannot be discharged → ETC slows (backed up).

🔄 The Krebs Cycle (Tricarboxylic Acid / TCA Cycle)

The Krebs cycle occurs in the mitochondrial matrix. Each turn processes one acetyl-CoA (2 carbons) and produces: 3 NADH, 1 FADH₂, 1 GTP (via Succinyl-CoA Synthetase), and 2 CO₂. The CO₂ is the carbon waste released at the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase steps.

Three irreversible steps (the regulatory checkpoints):

Citrate Synthase: first step, condenses acetyl-CoA + oxaloacetate (OAA) → citrate.
Isocitrate Dehydrogenase: primary rate-limiting enzyme; produces first NADH + first CO₂. Activated by ADP and Ca²⁺; inhibited by ATP and NADH.
α-Ketoglutarate Dehydrogenase: also rate-limiting; produces second NADH + second CO₂; requires TPP, lipoic acid, CoA, FAD, NAD⁺ (same cofactors as PDC). Activated by Ca²⁺; inhibited by NADH and succinyl-CoA.

Fluoroacetate — Aconitase Inhibitor

Fluoroacetate (a poison found in some plants) is converted inside cells to fluorocitrate, which inhibits Aconitase — the enzyme that converts citrate → isocitrate. This blocks the entire Krebs cycle. Citrate accumulates. This is a directly tested exam fact.

When Does Acetyl-CoA Oxidation Increase?

The cycle runs faster when the cell needs more energy — specifically when the ATP/ADP ratio decreases (ADP increases). High ADP signals low energy → activates isocitrate dehydrogenase → more Krebs cycle activity → more acetyl-CoA oxidized.

⚡ Respiratory Chain (Electron Transport Chain) ★ asked 2018

Complex I (NADH Dehydrogenase, contains FMN) → CoQ → Complex II (Succinate Dehydrogenase, contains FAD) → CoQ → Complex III (Cytochrome bc1) → Cytochrome c → Complex IV (Cytochrome c Oxidase) → O₂ → H₂O

Complex V (ATP Synthase): F₀ subunit is embedded in the inner mitochondrial membrane and acts as the proton channel; F₁ subunit sits in the matrix and synthesizes ATP.

Cytochrome c — Electrons Only ★ asked 2025

Cytochrome c transfers electrons ONLY — it does NOT transfer protons/hydrogen ions. This was explicitly tested in the 2025 exam. Cytochrome c moves between Complex III and Complex IV carrying only electrons via its heme iron (Fe³⁺ ↔ Fe²⁺). Coenzyme Q (ubiquinone) transfers both electrons and protons.

Inhibitor	Blocks	Consequence
Rotenone / Amytal	Complex I	NADH cannot donate electrons; ETC stops
Antimycin A	Complex III	Electron flow blocked at Q cycle
Cyanide (CN⁻), CO, Azide	Complex IV	Cannot transfer electrons to O₂; rapid cellular death
Oligomycin	F₀ of ATP Synthase	Proton channel blocked; ATP synthesis stops

🌡️ Uncoupling — Energy as Heat Instead of ATP

Normally, the proton gradient built by the ETC drives ATP synthesis through Complex V. Uncouplers short-circuit this by allowing protons to cross the inner mitochondrial membrane through an alternative route — dissipating the gradient as heat instead of ATP.

2,4-DNP (dinitrophenol): an artificial chemical uncoupler. It is a lipid-soluble weak acid that carries H⁺ across the inner membrane. Was historically misused as a weight-loss drug — extremely dangerous.
UCP-1 (Thermogenin): the natural uncoupling protein found in brown adipose tissue. Activated by fatty acids; generates body heat. Especially important in newborns (who lack shivering reflex) and in hibernating animals.

🔀 The Amphibolic Role of the Citric Acid Cycle

"Amphibolic" = the cycle works in both directions of metabolism at once: it is catabolic (oxidises acetyl-CoA to make NADH/FADH₂/ATP) and anabolic (its intermediates are pulled out as building blocks for biosynthesis).

Intermediate withdrawn	Used to build (anabolic role)
Citrate	Exported to cytosol → acetyl-CoA → fatty acid & cholesterol synthesis
α-Ketoglutarate	→ Glutamate → other amino acids, GABA, purines
Succinyl-CoA	→ Heme (porphyrin synthesis)
Oxaloacetate	→ Aspartate (→ pyrimidines, urea cycle) and → glucose (gluconeogenesis via PEPCK)

Cataplerosis = withdrawing intermediates for biosynthesis. Anaplerosis = refilling them so the cycle keeps turning (chiefly pyruvate carboxylase: pyruvate + CO₂ → OAA). The balance of the two is what "amphibolic" captures.

One-line answer

The Krebs cycle is amphibolic because it both generates energy (catabolic) and supplies carbon skeletons for fat, amino acid, heme, and glucose synthesis (anabolic) — so its intermediates must be constantly topped up by anaplerotic reactions.

🔄 The Cycle, Its Enzymes and Its Vitamins 2026

The 2026 item: "Krebs cycle… derivative of vitamin B2 — correct answer: succinate." The logic is: vitamin B2 = riboflavin = FAD, and the only FAD-linked enzyme of the cycle is succinate dehydrogenase, whose substrate is succinate.

The eight steps

Citrate synthase (irreversible, condensation) → aconitase (isomerisation; inhibited by fluorocitrate) → isocitrate dehydrogenase (NADH + CO₂; the rate-limiting step; activated by ADP and Ca²⁺, inhibited by ATP and NADH) → α-ketoglutarate dehydrogenase (NADH + CO₂) → succinyl-CoA synthetase (GTP — the only substrate-level phosphorylation) → succinate dehydrogenase (FADH₂) → fumarase → malate dehydrogenase (NADH).

Vitamins in the cycle

B1 thiamine (TPP), B2 riboflavin (FAD), B3 niacin (NAD⁺), B5 pantothenate (CoA) — plus lipoic acid, which is not a vitamin. The α-ketoglutarate dehydrogenase complex needs all of them, exactly like pyruvate dehydrogenase.

Location — the one exception

Everything is in the mitochondrial matrix except succinate dehydrogenase, which is embedded in the inner membrane as Complex II. That is also why succinate dehydrogenase feeds electrons straight to coenzyme Q and yields less ATP.

Which amino acid enters where

Valine, isoleucine, methionine and threonine → succinyl-CoA. Phenylalanine and tyrosine → fumarate (and acetoacetate). Aspartate → oxaloacetate. Glutamate, glutamine, proline, arginine, histidine → α-ketoglutarate. Alanine, serine, glycine, cysteine → pyruvate. Leucine and lysine → acetyl-CoA only.

Amphibolic role and anaplerosis

Amphibolic = both catabolic and anabolic. Intermediates are constantly withdrawn — citrate for lipogenesis, succinyl-CoA for haem, α-ketoglutarate and oxaloacetate for amino acids and gluconeogenesis — and must be replaced by anaplerotic reactions: chiefly pyruvate carboxylase (biotin, activated by acetyl-CoA), plus transamination of glutamate and aspartate, glutamate dehydrogenase, and propionyl-CoA → succinyl-CoA from odd-chain fatty acids. The cycle itself produces CO₂ and, through succinyl-CoA synthetase, GTP.

⚡ Coenzyme Q — Every Option Dissected 2026

The 2026 stem offered three statements about CoQ. Only the first is true, and the other two are the classic misconceptions.

✅ "Takes electrons from succinate dehydrogenase"

True. Coenzyme Q is the collection point of the chain. It accepts electrons from Complex I (NADH, via FMN), from Complex II — succinate dehydrogenase (FADH₂), from the glycerol-3-phosphate dehydrogenase of the shuttle, and from ETF in fatty acid β-oxidation. It therefore accepts hydrogen from both FADH₂ and FMNH₂.

❌ "Is water-soluble like cytochrome c"

False. CoQ is a lipid-soluble quinone with a long isoprenoid tail; it diffuses within the hydrophobic core of the inner membrane. Cytochrome c is the water-soluble one, sitting loosely on the outer face of the inner membrane — and its escape into the cytosol triggers apoptosis.

❌ "Is a protein"

False. CoQ is a small non-protein molecule, the only non-protein carrier of the chain. Cytochromes are haemoproteins; CoQ is not. It is also called ubiquinone — a name that appears in many stems.

The sequence — asked as a matching item

FMN + Fe-S → CoQ ← FAD + Fe-S, then Cyt b + Cyt c₁ → Cyt c → haem a + a₃. Written as the exam writes it: FMN + Fe-S · Cyt b + Cyt c₁ · haem a + a₃, with FAD + Fe-S entering at the CoQ level, not at the start — the option that puts FAD before FMN is wrong.

The complexes and their inhibitors

Complex I NADH dehydrogenase (FMN, Fe-S) — blocked by rotenone and amytal; it oxidises NADH, it does not "reduce FADH₂". Complex II succinate dehydrogenase (FAD) — pumps no protons, blocked by malonate. Complex III cytochromes b and c₁ — antimycin A. Complex IV cytochrome c oxidase, haem a + a₃, contains copper — blocked by cyanide, carbon monoxide, azide and H₂S. Complex V is ATP synthase — blocked by oligomycin. Uncouplers (2,4-dinitrophenol, thermogenin/UCP-1 in brown fat) discharge the gradient: oxygen consumption rises, ATP synthesis falls, heat is produced. The inner mitochondrial membrane is impermeable to protons and to most metabolites; the outer membrane is freely permeable.

🚌 The Glycerol-3-Phosphate Shuttle — Option by Option 2026

The 2026 stem offered three statements. This is a case where all three of the recorded options are defensible, so what matters is understanding each one.

✅ "More widespread than the malate–aspartate shuttle"

The glycerol-3-phosphate shuttle is the general-purpose one, prominent in skeletal muscle, brain, brown fat and insects; the malate–aspartate shuttle is concentrated in liver, kidney and heart. It is also faster but irreversible, whereas malate–aspartate is reversible.

✅ "Allows oxidation of NADH + H⁺"

That is its entire purpose. The inner membrane is impermeable to NADH, so cytosolic NADH reduces DHAP to glycerol-3-phosphate; the membrane-bound isoenzyme on the outer face of the inner membrane re-oxidises it and hands the electrons to FAD → CoQ. Because it enters at CoQ and skips Complex I, each cytosolic NADH yields only about 1.5 ATP (older texts: 2) instead of 2.5 (3).

✅ "Has little importance in cardiac muscle"

Correct — the heart uses the malate–aspartate shuttle, which is why cardiac muscle extracts the full ATP yield from cytosolic NADH. Cardiac muscle also has low glycolytic activity at rest, running mainly on fatty acids and lactate.

The malate–aspartate shuttle

Oxaloacetate → malate in the cytosol; malate crosses and is re-oxidised inside, regenerating NADH in the matrix; the carbon returns as aspartate. Electrons enter at Complex I, so the yield is the full 2.5 (3) ATP. It is fully reversible.

The three faces of dihydroxyacetone phosphate

In glycolysis — one of the two triose phosphates from aldolase, isomerised to glyceraldehyde-3-phosphate by phosphotriose isomerase. In triacylglycerol synthesis — reduced to glycerol-3-phosphate, the backbone for esterification. In the shuttle — the cytosolic acceptor of NADH's electrons. The enzyme sitting at all three junctions is glycerol-3-phosphate dehydrogenase, which exists as a cytosolic NAD-linked form and a mitochondrial FAD-linked form.

⚙️ Mitochondrial and Cytosolic — Which Enzyme Sits Where 2026

Mitochondrial regulatory enzymes

Pyruvate dehydrogenase, pyruvate carboxylase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, citrate synthase, carbamoyl phosphate synthetase I, mitochondrial HMG-CoA synthase (ketogenesis), CPT-I, ALA synthase. If a stem asks for "the mitochondrial regulatory enzyme" in a list that also contains glycolytic or lipogenic enzymes, it is one of these.

Cytosolic

Glycolysis, the pentose phosphate pathway, lipogenesis (fatty acid synthase, acetyl-CoA carboxylase, ATP-citrate lyase), cholesterol synthesis to mevalonate, glycogen synthesis and glycogenolysis, and the last three steps of the urea cycle. Ketogenesis is the exception in that list — it is mitochondrial.

Made inside the mitochondrion

Acetyl-CoA, NADH and FADH₂ from the Krebs cycle, CO₂, carbamoyl phosphate (CPS I), citrulline, δ-aminolevulinic acid, ketone bodies and the bulk of ATP. Note that acetyl-CoA cannot cross the inner membrane — it leaves as citrate and is released again by ATP-citrate lyase in the cytosol.

Energy bookkeeping

Substrate-level phosphorylation — direct phosphate transfer from a high-energy substrate, no oxygen needed: phosphoglycerate kinase, pyruvate kinase, succinyl-CoA synthetase. Oxidative phosphorylation — ATP synthase driven by the proton gradient, requiring oxygen and an intact, coupled inner membrane. High-energy compounds include ATP, phosphocreatine, 1,3-bisphosphoglycerate, PEP, acetyl-CoA and carbamoyl phosphate; dihydroxyacetone phosphate and glucose-6-phosphate are not high-energy compounds.

Biochemical Logic & Redox

Frequently tested: biochemical reasoning over memorization, redox logic, pathway locations, why specific reactions occur — emphasized by the teacher

⚡ Redox Logic

Oxidation = loss of electrons. Reduction = gain of electrons.

NAD⁺ is the OXIDIZED form → accepts electrons → becomes NADH (reduced).
NADH is oxidized back to NAD⁺ in the ETC.

Exam trap: "The enzyme reduces NADH" → FALSE. NADH is already reduced.

Classic False Statements

• "NADH is the oxidized form" → FALSE (NAD⁺ is oxidized)
• "Cytochrome c transfers protons" → FALSE (transfers only electrons — tested 2025)
• "Alcohol changes NADPH/NADP⁺ ratio" → FALSE (changes NADH/NAD⁺)

📊 Pathway Locations — Quick Reference

Process	Location	Rate-limiting enzyme
Glycolysis	Cytosol	PFK-1
Gluconeogenesis	Cytosol + mitochondria	PEPCK / Pyruvate Carboxylase
Krebs cycle	Mitochondrial matrix	Isocitrate DH / α-KG DH
β-oxidation (fatty acid breakdown)	Mitochondrial matrix	Acyl-CoA Dehydrogenase
Fatty acid synthesis (lipogenesis)	Cytosol — mainly liver, adipose, lactating mammary gland. NOT muscle.	Acetyl-CoA Carboxylase (ACC)
Pentose Phosphate Pathway (PPP)	Cytosol	G6PD
Urea cycle	Mitochondria (CPS-I, OTC) + Cytosol (rest)	CPS-I
Heme synthesis	Mitochondria (first + last steps) + Cytosol (middle steps). NOT in mature red blood cells.	ALA Synthase
Cholesterol synthesis	Cytosol + ER (endoplasmic reticulum)	HMG-CoA Reductase

Lipogenesis Tissues — Exam Tested

Fatty acid synthesis does NOT occur in skeletal muscle (muscle lacks the necessary enzyme levels). It occurs mainly in: liver, adipose tissue, and lactating mammary gland. This is a common exam question.

🧪 Lipogenesis Cofactors (What Is and Is NOT Required)

Fatty acid synthesis requires: ATP (for acetyl-CoA carboxylase reaction), NADPH (for reductive steps — provided by the PPP and malic enzyme), Biotin (covalently attached to acetyl-CoA carboxylase), HCO₃⁻ (bicarbonate, as CO₂ source for malonyl-CoA), and Mn²⁺.

⚗️ Lipogenesis Cofactors — What Is and Is NOT Required

Required for lipogenesis?	Molecule	Why
✅ YES	NADPH	Used in every reductive step (2 NADPH consumed per 2-carbon addition). Comes from PPP and malic enzyme.
✅ YES	ATP	Required by Acetyl-CoA Carboxylase (ACC) to make malonyl-CoA.
✅ YES	Biotin	Covalently attached cofactor of ACC — carries CO₂ during malonyl-CoA synthesis.
✅ YES	HCO₃⁻ (bicarbonate)	CO₂ source for carboxylation by ACC.
❌ NO	NADH	NADH is used in β-OXIDATION (breakdown) and the Krebs cycle. It is NOT used in fatty acid SYNTHESIS. This is a common exam trap.
❌ NO	PLP (Vitamin B6)	PLP is the cofactor for aminotransferases and glycogen phosphorylase — not involved in lipogenesis.
❌ NO	FAD	FAD is used in β-oxidation (acyl-CoA dehydrogenase) — not in synthesis.

Exam Trap (Q33): Lipogenesis requires NADPH, NOT NADH

NADH and NADPH look similar but are used in opposite processes:
• NADPH → used in reductive biosynthesis (lipogenesis, steroid synthesis, glutathione reduction)
• NADH → used in energy generation (glycolysis, Krebs cycle, β-oxidation products)
A question listing ATP + Biotin + NADH as lipogenesis requirements → choose NADPH instead of NADH. Only ATP and Biotin (and NADPH) are correct.

Cytosolic Acetyl-CoA — How It Gets There

Acetyl-CoA is made in mitochondria but cannot cross the inner mitochondrial membrane. It is exported as citrate instead. Citrate exits the mitochondria → in the cytosol, citrate lyase cleaves it back to acetyl-CoA + OAA. Conditions promoting this export: high citrate, high ATP, high-carbohydrate diet, inhibition of isocitrate dehydrogenase (which makes citrate accumulate). Citrate also directly activates acetyl-CoA carboxylase (ACC) in the cytosol — a feedforward signal.

Malic Enzyme

Malic enzyme converts malate → pyruvate while reducing NADP⁺ → NADPH. It belongs to the class of Oxidoreductases (class 1). It is an important source of cytosolic NADPH for lipogenesis. This classification is directly tested.

🏥 Tissue Specificity — WHY Each Tissue Performs Its Pathway

Teacher repeatedly stated this is very high-yield: know not just WHERE pathways occur but WHY that tissue performs them.

Tissue	Specialist pathway(s)	Why / Unique feature
Liver	Gluconeogenesis, ketogenesis, urea cycle, cholesterol synthesis, bile acid synthesis, VLDL secretion, albumin, clotting factors	Central hub. Has glucose-6-phosphatase → only organ (with kidney) that releases free glucose to blood. Exclusive ketone body PRODUCER (cannot use them — lacks thiophorase).
Skeletal Muscle	Glycolysis, glycogen (for itself), BCAA catabolism, creatine phosphate	No G-6-Pase → cannot raise blood glucose. No glucagon receptors. No lipogenesis. BCAA (Leu, Ile, Val) metabolised mainly in muscle.
Brain	Glucose oxidation (primary); ketone bodies after 2–3 weeks fasting	GLUT-1 (insulin-independent). Absolute glucose requirement normally. After prolonged fasting adapts to ketones → spares muscle protein.
Adipose Tissue	TAG storage, lipolysis (HSL), FA esterification	No glycerol kinase → releases glycerol (not reused). GLUT-4 (insulin-dependent) → fat storage only when insulin present.
RBCs	Anaerobic glycolysis, PPP (NADPH)	No mitochondria → no TCA, no β-oxidation, no oxidative phosphorylation. Only ATP = glycolysis. Only NADPH = PPP. 2,3-BPG production is unique to RBCs.
Kidney	Gluconeogenesis (prolonged fasting), glutamine catabolism, arginine synthesis, vitamin D activation	Only site of 1α-hydroxylase (25-OH-D₃ → calcitriol). Only site of arginine synthesis (from citrulline + aspartate).
Intestinal Enterocytes	Chylomicron assembly, glutamine as primary fuel	Use glutamine (not glucose) as primary fuel in fasting. SGLT-1 (Na⁺-dependent glucose/galactose), GLUT-5 (fructose), GLUT-2 (portal exit).

📋 Pathway–Location–Rate-Limiting Enzyme Pairings — Direct Exam Format

The exam directly tests whether you can correctly match a pathway with its subcellular location AND its rate-limiting enzyme. This exact format appeared on the 2025 exam (Q29).

Pathway	Location	Rate-Limiting Enzyme	Exam tip
Glycolysis	Cytoplasm	PFK-1	✅ Correct pairing — tested directly in 2025
Gluconeogenesis	Cytoplasm + mitochondria	FBP-1 (or PEPCK as key bypass enzyme)	Bypasses PFK-1; G6Pase final step in ER
β-Oxidation	Mitochondrial matrix	CPT-I (carnitine shuttle, rate-limiting entry step)	❌ ACC is NOT the rate-limiting enzyme of β-oxidation — ACC is the rate-limiting enzyme of LIPOGENESIS (synthesis). Common trap.
Lipogenesis (FA Synthesis)	Cytoplasm	ACC	ACC makes malonyl-CoA — first committed step of FA synthesis
PPP (HMP Shunt)	Cytoplasm	G6PD	✅ Correct pairing — tested directly in 2025
Krebs Cycle (TCA)	Mitochondrial matrix	Isocitrate DH (primary) + α-KG DH	Both are rate-limiting; Isocitrate DH is the main pacemaker
Urea Cycle	Mitochondria (steps 1–2) + Cytoplasm (steps 3–5)	CPS-I	Requires NAG (obligatory activator)
Cholesterol synthesis	ER (smooth)	HMG-CoA Reductase	Target of statins
Heme synthesis	Mitochondria (1st + last steps) + Cytoplasm (middle)	ALA Synthase	Inhibited by heme (feedback); requires PLP (B6)
ETC / Oxidative phosphorylation	Inner mitochondrial membrane	Complex I (NADH entry point)	O₂ = final electron acceptor; ATP synthase = Complex V

The #1 Exam Trap in Pathway Pairings

β-oxidation rate-limiting enzyme = CPT-I (controls entry of fatty acids into mitochondria via the carnitine shuttle).
ACC is the rate-limiting enzyme of LIPOGENESIS — not β-oxidation. These two are commonly swapped in exam questions. If a question pairs β-oxidation with ACC, that pairing is INCORRECT.

🔥 Biological Oxidation — The Four Enzyme Types definitions asked

"Biological oxidation" = controlled removal of electrons/hydrogen from fuel molecules. Different enzyme classes handle oxygen and electrons in different ways — a favourite definition question.

Enzyme type	What it does with O₂ / electrons	Example
Dehydrogenase	Removes H (2e⁻ + H⁺) and passes it to NAD⁺/FAD — no O₂ used directly	Lactate dehydrogenase, succinate dehydrogenase
Oxidase	Transfers electrons directly to O₂, making H₂O or H₂O₂ (does NOT incorporate O into substrate)	Cytochrome c oxidase (→H₂O); xanthine oxidase, MAO (→H₂O₂)
Oxygenase	Incorporates O₂ into the substrate. Monooxygenase adds one O atom (other → H₂O); dioxygenase adds both	CYP450 (monooxygenase); homogentisate oxidase (dioxygenase)
Peroxidase	Uses H₂O₂ as the oxidant (detoxifies it)	Glutathione peroxidase, catalase (2 H₂O₂ → 2 H₂O + O₂)

Key distinction

Oxidase vs oxygenase: an oxidase gives electrons to O₂ but does not put oxygen atoms into the product; an oxygenase actually inserts O atoms into the substrate.

🚚 Transport of Cytosolic (Extramitochondrial) NADH — The Two Shuttles

NADH made in the cytosol (from glycolysis) cannot cross the inner mitochondrial membrane. Two shuttles carry its electrons in — and they yield different amounts of ATP.

🟢 Malate–Aspartate Shuttle

Cytosolic NADH → OAA → malate (crosses membrane) → regenerates NADH inside the matrix.
Tissues: liver, heart, kidney.
Yield: ~2.5 ATP per NADH (enters at Complex I). Reversible.
Enzymes: malate dehydrogenase + AST.

🔵 Glycerol-Phosphate Shuttle

Cytosolic NADH → DHAP → glycerol-3-phosphate → drops electrons onto FAD at the inner membrane.
Tissues: muscle, brain, brown fat.
Yield: ~1.5 ATP per NADH (enters at CoQ, bypasses Complex I). Irreversible, fast.
Enzymes: cytosolic + mitochondrial G3P dehydrogenase.

Why the ATP differs

The malate–aspartate shuttle delivers electrons as NADH (Complex I → 2.5 ATP); the glycerol-phosphate shuttle delivers them as FADH₂ (CoQ → 1.5 ATP). This is why the ATP yield of glycolysis is sometimes quoted as slightly different between tissues.

⚖️ NAD⁺/NADH vs NADP⁺/NADPH · NADPH Generators

Same chemistry, opposite jobs. The single extra phosphate on NADP is a "label" that routes it to biosynthesis instead of energy.

	NAD⁺ / NADH	NADP⁺ / NADPH
Main role	Catabolism — carries electrons to the ETC to make ATP	Anabolism — donates electrons to biosynthesis
Usually found as	Oxidised (NAD⁺) — ready to accept	Reduced (NADPH) — ready to donate
Used in	Glycolysis, Krebs, β-oxidation	Fatty-acid/cholesterol/steroid synthesis, glutathione reduction, CYP450, respiratory burst

Where NADPH comes from (generators)

1. Pentose phosphate pathway — G6PD + 6-phosphogluconate dehydrogenase (the main source). 2. Malic enzyme — malate → pyruvate. 3. Cytosolic isocitrate dehydrogenase. 4. Folate pathway (minor). In RBCs the PPP is the only NADPH source — which is why G6PD deficiency causes haemolysis.

🔋 Endergonic & Exergonic Reactions — Energy Coupling

Exergonic (ΔG < 0)

Releases energy, spontaneous. e.g. ATP hydrolysis, glucose oxidation. Catabolic reactions are typically exergonic.

Endergonic (ΔG > 0)

Requires energy input, non-spontaneous. e.g. building macromolecules, gluconeogenesis. Anabolic reactions are typically endergonic.

The cell drives endergonic reactions by coupling them to a strongly exergonic one — almost always ATP hydrolysis (ΔG ≈ −30 kJ/mol). If the combined ΔG is negative, the coupled reaction proceeds. ΔG (actual) depends on concentrations; ΔG°′ is the standard value. A negative ΔG says a reaction can go, not how fast — that's set by the enzyme (activation energy).

High-energy phosphate compounds

PEP > 1,3-BPG > creatine phosphate > ATP — each can donate its phosphate to the one below it. This is why substrate-level phosphorylation (PEP → pyruvate, 1,3-BPG → 3-PG) can make ATP directly.

Fed/Fasting State & Alcohol

Frequently tested: well-fed vs fasting characteristics, glucose-alanine cycle, alcohol metabolism (alcohol dehydrogenase) — teacher said "do not forget alcohol"

⚠️ Teacher (Kłos) Exam Topics — From Notes

Explicitly listed by the teacher as exam topics:
• Iron metabolism (see Iron & Lab Tests section)
• Alcohol metabolism via dehydrogenase (ADH + ALDH pathway below)
• Conjugated/unconjugated/urobilinogen hyperbilirubinemia — genetic, erythrocyte enzyme (see Heme section)
• GLUT-4 is insulin-dependent (Skeletal muscle + adipose)
• Skeletal muscles can use ketones earlier than brain, then switch to FAs to preserve ketones for brain
• Exceptions from active when phosphorylated — know enzymes that are INACTIVE when phosphorylated

🍽️ Fed vs Fasting — Complete Overview

Parameter	Fed (Insulin dominant)	Fasting (Glucagon dominant)
Glycolysis	↑ Active	↓ Suppressed
Gluconeogenesis	↓ Suppressed	↑ Active
Glycogen synthesis	↑ Active	↓ Off
Glycogenolysis	↓ Off	↑ Active
Lipogenesis	↑ Active	↓ Off
Lipolysis	↓ Off	↑ Active
β-oxidation	↓ (malonyl-CoA blocks carnitine)	↑ Active
Ketogenesis	↓ Off	↑ Active
Protein synthesis	↑ Active	↓ Muscle breakdown ↑

⚡ Phosphorylation Rules — Active vs Inactive (Teacher Tip)

Most metabolic enzymes are active in their dephosphorylated form (fed/insulin state) and become inactive when phosphorylated during fasting. Key exceptions — enzymes that are ACTIVE when phosphorylated (catabolic enzymes turned on during fasting):

Enzyme	Phosphorylated State	Active State
Glycogen Synthase	INACTIVE (↓glycogen synthesis)	Dephosphorylated (fed/insulin)
Pyruvate Kinase (liver)	INACTIVE (↓glycolysis)	Dephosphorylated (fed/insulin)
HMG-CoA Reductase	INACTIVE (↓cholesterol synthesis)	Dephosphorylated (fed)
Acetyl-CoA Carboxylase (ACC)	INACTIVE (↓FA synthesis)	Dephosphorylated (fed/insulin)
PFK-2 (kinase domain)	INACTIVE kinase → FBP-2 active	Dephosphorylated = PFK-2 active (glycolysis)

The Pattern

Glucagon → PKA → phosphorylation. During fasting: anabolic enzymes get phosphorylated = inactive (no synthesis during fasting). Catabolic enzymes get phosphorylated = active (break down stores for energy).

🍺 Alcohol Metabolism

Ethanol → Acetaldehyde (Alcohol Dehydrogenase, cytosol, NAD⁺→NADH) → Acetate (Aldehyde Dehydrogenase, mitochondria, NAD⁺→NADH) → enters blood.

High NADH/NAD⁺ ratio = root cause of all alcohol metabolic effects:
• ↑NADH → pyruvate → lactate → lactic acidosis
• ↑NADH → OAA → malate → OAA depleted → gluconeogenesis blocked → hypoglycemia
• β-oxidation blocked (needs NAD⁺) → fat accumulates → fatty liver (steatosis)
• ↑Acetyl-CoA → ketogenesis → alcoholic ketoacidosis

Exam Trap

Alcohol changes NADH/NAD⁺ ratio (not NADPH/NADP⁺).

🍔 Diet Composition — High-Carbohydrate vs High-Fat

	High-carbohydrate diet	High-fat diet
Dominant hormone	Insulin	Lower insulin, relatively ↑ glucagon
Main pathways ON	Glycolysis, glycogen synthesis, lipogenesis (excess carbs → fat)	β-oxidation, ketogenesis, gluconeogenesis (from protein/glycerol)
Fuel used	Glucose	Fatty acids + ketone bodies
Effect on lipids	↑ VLDL/TAG if excessive (de novo lipogenesis)	Mild ketosis; can ↑ LDL depending on fat type
Cholesterol synthesis	↑ (insulin activates HMG-CoA reductase)	Dietary cholesterol feedback-inhibits synthesis

Key idea

A high-fat, low-carb intake mimics a fasting-like metabolic pattern (fat oxidation + ketones), while excess carbohydrate is converted to fat via lipogenesis. The body's response is set by the insulin:glucagon ratio, not just calories.

Fatty Acid Metabolism

Frequently tested: β-oxidation (and odd-chain → succinyl-CoA needing B12), fatty acid synthesis enzyme order, ketone bodies, ACC regulation, arachidonic acid & eicosanoids

🏭 Fatty Acid Synthesis

Builds fatty acids (mainly palmitate, 16:0) de novo from acetyl-CoA in the cytoplasm. Rate-limiting enzyme: Acetyl-CoA Carboxylase (acetyl-CoA → malonyl-CoA). Activated by insulin/citrate; inhibited by palmitoyl-CoA + glucagon.

🟢 Synthesis (lipogenesis)

Where: cytoplasm
Carrier: ACP / fatty acid synthase
Reducing power: NADPH (from PPP + malic enzyme)
Needs: ATP, HCO₃⁻, biotin, Mn²⁺
State: fed / high insulin

🔵 β-Oxidation (breakdown)

Where: mitochondrial matrix
Carrier: carnitine shuttle (CPT-I entry)
Electron acceptors: NAD⁺ & FAD
Product: acetyl-CoA + NADH + FADH₂
State: fasting / high glucagon

Key distinction

Synthesis uses NADPH in the cytoplasm; β-oxidation uses NAD⁺/FAD in the mitochondrial matrix. Malonyl-CoA (from ACC) blocks CPT-I, so the two are never on at once.

💡 Ketone Bodies — What & Why

What they are: small, water-soluble fuel molecules made by the liver from acetyl-CoA (from β-oxidation) when carbohydrate is scarce. They are exported in the blood to other tissues.

Their purpose: during fasting/starvation or uncontrolled diabetes, they provide an alternative energy source — crucially for the brain, which cannot oxidise fatty acids directly. Muscle and heart also use them. This spares glucose and limits muscle protein breakdown. Note: the liver makes them but cannot use them (it lacks thiophorase/SCOT).

🍋 Ketone Bodies — The Three Types

Acetoacetate

True ketone; the primary one synthesised; used by brain/muscle/heart.

β-Hydroxybutyrate

Major ketone in blood during starvation (most abundant).

Acetone

From spontaneous decarboxylation of acetoacetate. Volatile; exhaled. No metabolic role.

Diabetic Ketoacidosis (DKA)

High ketones → metabolic acidosis + Kussmaul breathing + ketonuria.

👆 Tap for full disease card

Why β-Hydroxybutyrate Dominates in Blood During Starvation — Teacher Required

β-Oxidation produces large amounts of NADH. High NADH/NAD⁺ ratio drives β-Hydroxybutyrate Dehydrogenase to convert acetoacetate → β-hydroxybutyrate. Therefore, β-hydroxybutyrate is the dominant circulating ketone body in starvation and DKA. Acetone is formed by spontaneous decarboxylation of acetoacetate (not enzymatic) — it is exhaled.

⚖️ Lipolysis vs Lipogenesis vs β-Oxidation — Definitions (Teacher Notes)

Term	Definition	Key Enzyme	Location
Lipolysis	Breakdown of TAGs (triacylglycerols) → Free fatty acids (FFAs) + glycerol	Hormone-sensitive Lipase (HSL) in adipose; LPL in capillaries	Cytoplasm of adipocytes
β-Oxidation	Breakdown of fatty acids → Acetyl-CoA (2-carbon units are progressively cleaved)	Acyl-CoA Dehydrogenase (first step)	Mitochondrial matrix
Lipogenesis	Synthesis of fatty acids de novo from acetyl-CoA (building FAs from scratch)	Acetyl-CoA Carboxylase (ACC) (rate-limiting)	Cytoplasm
TAG Synthesis	Assembly of triglycerides from glycerol-3-phosphate + fatty acyl-CoAs	Acyltransferases	ER (liver, adipose)

Lipolysis and β-Oxidation Are Reciprocal with Lipogenesis (Teacher Note)

Lipolysis/β-oxidation and lipogenesis are reciprocal processes — they are regulated so that one is active while the other is off. ACC produces malonyl-CoA → malonyl-CoA inhibits CAT-I (carnitine acyltransferase I) → blocks FA entry into mitochondria for β-oxidation. So when FA synthesis is ON (fed state), β-oxidation is automatically OFF. Repeatable process: each enzyme set repeats its reaction cycle on successive 2-carbon units.

🌊 Eicosanoids

Eicosanoids are 20-carbon signalling molecules made from arachidonic acid, which is released from membrane phospholipids by Phospholipase A₂.

From there, two pathways branch off:

🔵 COX pathway

Cyclooxygenase (COX) →
Prostaglandins (inflammation, pain),
Thromboxane A₂ (platelet aggregation, vasoconstriction),
Prostacyclin (opposes thromboxane).
Blocked by NSAIDs (e.g. aspirin).

🟠 Lipoxygenase pathway

Lipoxygenase →
Leukotrienes — drive bronchoconstriction in asthma and allergic responses.

Exam note

Prostanoids (prostaglandins + thromboxanes + prostacyclin) and leukotrienes are both subclasses of eicosanoids. Arachidonic acid is an ω-6, tetraenoic (4 double bonds), 20-carbon acid derived from linoleic acid.

📋 Important Fatty Acids — Reference Table

Rows highlighted in yellow are the highest-yield for the exam.

Fatty acid	Notation	Double bonds	Class / note
Stearic acid	18:0	0 (saturated)	★ No C9 double bond — classic exam trap
Oleic acid	18:1	1 (Δ9)	★ ω-9, monounsaturated
Arachidonic acid	20:4	4 (Δ5,8,11,14)	★ ω-6, precursor of eicosanoids
Linoleic acid	18:2	2	★ ω-6, essential (diet)
α-Linolenic acid	18:3	3	★ ω-3, essential (diet)
Palmitic acid	16:0	0 (saturated)	Main product of FA synthesis
Palmitoleic acid	16:1	1 (Δ9)	ω-7, monounsaturated

Quick reminders

Essential (must be eaten): linoleic (ω-6) & α-linolenic (ω-3). Notation 18:1 = 18 carbons, 1 double bond. More double bonds → more fluid, lower melting point.

🧬 Fatty Acid Physical Properties — Teacher Notes

Property	Effect on Fatty Acid
Length of chain	Longer chain → MORE SOLID (more Van der Waals interactions) → higher melting point
Number of double bonds	More double bonds → MORE FLUID, ↓melting point (cis double bonds create kinks that prevent tight packing)
Degree of saturation (more H+)	More saturated → MORE SOLID, ↑melting point (e.g., animal fats are solid, plant oils are liquid)

Exam favourite: "Which fatty acid has NO double bond at C9?" ★ asked 2024/25

Answer: stearic acid (18:0) — fully saturated, so no double bond anywhere. The trap options oleic (18:1) and palmitoleic (16:1) BOTH have their double bond at Δ9 (C9). So the "no C9 double bond" one is the saturated acid.

🧴 Phospholipids — Structure, Types & Function

Phospholipids are amphipathic (water-loving head + fat-loving tails) — the reason they spontaneously form the cell membrane bilayer. Most are built on a glycerol backbone; one class is built on sphingosine.

Phospholipid	Head group / note	Role
Phosphatidylcholine (lecithin)	Choline — most abundant membrane PL	Membranes; bile; lung surfactant
Phosphatidylethanolamine (cephalin)	Ethanolamine	Inner membrane leaflet
Phosphatidylserine	Serine (negative)	Flips to outer leaflet in apoptosis ("eat me" signal); activates clotting
Phosphatidylinositol (PIP₂)	Inositol	Source of second messengers IP₃ + DAG (via phospholipase C)
Cardiolipin	Two PG units — inner mitochondrial membrane	Needed for ETC function; antibody target in antiphospholipid syndrome
Sphingomyelin	Sphingosine backbone (not glycerol)	Myelin sheath; accumulates in Niemann-Pick disease

🫁 Lung surfactant

Mainly dipalmitoyl-phosphatidylcholine (DPPC) — lowers alveolar surface tension. Deficiency in premature babies → neonatal respiratory distress syndrome.

✂️ Phospholipases

PLA₂ releases arachidonic acid (→ eicosanoids); phospholipase C releases IP₃ + DAG (signalling).

💧 Sources of "Active" Glycerol (Glycerol-3-Phosphate)

The active form of glycerol is glycerol-3-phosphate — the backbone onto which fatty acids are loaded to build triacylglycerols and phospholipids. There are two ways to make it, and which one a tissue uses is exam-relevant.

Source	Enzyme	Tissue
Free glycerol + ATP	Glycerol kinase	Liver, kidney (NOT adipose)
DHAP (from glycolysis) + NADH	Glycerol-3-P dehydrogenase	Adipose tissue (its only route)

Why adipose depends on glucose

Adipose tissue lacks glycerol kinase, so it can only make glycerol-3-phosphate from DHAP — which comes from glucose (glycolysis). This is why fat storage needs insulin/glucose, and why the glycerol released during lipolysis goes to the liver (which has glycerol kinase) rather than being reused locally.

✂️ Phospholipase A₂ — All Three Recorded Statements 2026

✅ "Cleaves at C2"

True. Phospholipase A₂ hydrolyses the ester bond at the sn-2 position of a glycerophospholipid — the position that characteristically carries an unsaturated fatty acid, which is why this enzyme is the one that liberates arachidonic acid for eicosanoid synthesis.

✅ "Trypsin activates prophospholipase A₂"

True. Like the other pancreatic enzymes it is secreted as a zymogen and activated in the duodenum by trypsin. It also requires bile salts and Ca²⁺.

✅ "The products are a lysophospholipid and a fatty acid"

True. Removing the C2 acyl chain leaves a lysophospholipid (e.g. lysolecithin) plus the free fatty acid. Lysophospholipase then removes the remaining chain to give a glycerylphosphoryl base.

Where else it matters

Glucocorticoids inhibit phospholipase A₂ — which is how they shut down the entire prostaglandin, thromboxane and leukotriene cascade at once, upstream of where NSAIDs act. The same enzyme appears in snake and bee venom.

The other lipases, and which are intracellular

Lingual and gastric lipase — acid lipases, optimum pH 4–6, short- and medium-chain TAG, important in the neonate. Pancreatic lipase + colipase — TAG → 2-monoacylglycerol + 2 fatty acids. Cholesterol esterase — cholesteryl esters. Lipoprotein lipase — extracellular, anchored to capillary endothelium, activated by apo C-II. Hormone-sensitive lipase — intracellular, in the adipocyte. In a list asking which enzymes are intracellular, phospholipase and acyl-CoA synthase are intracellular while lipoprotein lipase is not.

🌿 Essential Fatty Acids — ω-3 versus ω-6 2026

Two 2026 items: whether linoleic acid is ω-3 or ω-6, and what the ω-3 parent is. They are two halves of the same fact.

Linoleic acid — 18:2, ω-6

The ω-6 parent. It is the precursor of arachidonic acid (20:4, ω-6), which is the substrate for prostaglandins, thromboxanes and leukotrienes. Arachidonic acid becomes essential only if linoleic acid is lacking.

α-Linolenic acid — 18:3, ω-3

The ω-3 parent, and the recorded 2026 answer. It gives rise to EPA and DHA, important for growth, neural development and the retina.

Why they are essential

Humans have Δ9, Δ6 and Δ5 desaturases but cannot introduce a double bond beyond carbon 9 counted from the carboxyl end. Plants can, so we must eat them. Deficiency causes dermatitis, poor wound healing and growth failure.

Reading the notation

ω (or n) counts from the terminal methyl carbon. Arachidonic acid 20:4(5,8,11,14) is ω-6 because 20 − 14 = 6. Natural double bonds are cis and are spaced at three-carbon intervals. Adding double bonds lowers the melting point; lengthening the chain raises it — which is what keeps membranes fluid.

Eicosanoids — the family names that appear as options

All are derived from arachidonic acid: prostaglandins, prostacyclin and thromboxanes (cyclooxygenase branch), leukotrienes (lipoxygenase branch), and lipoxins — bioactive autacoid metabolites of arachidonic acid. They are autacoids: local, short-lived signals, not circulating hormones. Isoprostanes come from the same fatty acid but by non-enzymatic auto-oxidation, which is why they are used as markers of oxidative stress.

🔥 β-Oxidation — Location, Enzymes, End Product 2026

The 2026 item asks for exactly these three things.

Location

The mitochondrial matrix — in most tissues, but not the brain (fatty acids cross the blood–brain barrier poorly) and not red cells (no mitochondria). Very-long-chain and branched fatty acids are shortened first in peroxisomes, where the first oxidation gives H₂O₂ instead of ATP.

Activation and entry

Acyl-CoA synthetase (thiokinase) activates the fatty acid in the cytosol, spending ATP → AMP + PPi — two high-energy bonds. Long-chain acyl-CoA then needs the carnitine shuttle: CPT-I (the rate-limiting enzyme, inhibited by malonyl-CoA) → translocase → CPT-II. Short- and medium-chain acids do not need carnitine.

The four repeating steps

Oxidation (acyl-CoA dehydrogenase, FAD) → hydration → oxidation (NAD⁺) → thiolysis. Per cycle: two carbons removed as acetyl-CoA, plus 1 FADH₂ and 1 NADH. The process requires oxygen indirectly — it cannot run anaerobically, because the chain must reoxidise FADH₂ and NADH.

End product

Acetyl-CoA — the answer. Palmitate gives 8 acetyl-CoA, 7 FADH₂, 7 NADH. Odd-chain fatty acids end with propionyl-CoA → (biotin) methylmalonyl-CoA → (vitamin B12) succinyl-CoA, the only glucogenic part of a fatty acid. Branched phytanic acid needs α-oxidation; its failure is Refsum disease.

Fatty acids cannot become glucose

Because pyruvate dehydrogenase is irreversible, acetyl-CoA can never give a net gain of oxaloacetate. The conversion of fatty acids to glucose therefore does not occur — except for the propionyl-CoA of odd-chain fatty acids. Fatty acids also have a higher caloric value (about 9 kcal/g) than carbohydrate or protein, and their oxidation is catabolised by mitochondrial enzymes.

🏭 Lipogenesis, TAG Synthesis and Glycerol

Acetyl-CoA carboxylase — what activates it

Activated by citrate (which polymerises it into the active filament) and by dephosphorylation, i.e. by insulin. Inhibited by palmitoyl-CoA and by phosphorylation — glucagon and adrenaline. It requires biotin and makes malonyl-CoA, the rate-limiting, committed step. Recorded answer to "what activates ACC": dephosphorylation.

Fatty acid synthase — the false options

It uses biotin? No — biotin belongs to the carboxylase; the synthase uses pantothenic acid in the ACP phosphopantetheine arm. It uses acetyl-CoA and malonyl-CoA as substrates, not citrate. Malonyl-CoA is not subsequently oxidised — it is condensed. What is true: it is a multienzyme complex, active in its polymerised (dimeric) form, it needs 2 NADPH per cycle, and its product is palmitate.

Citrate and ATP-citrate lyase

Citrate is the carrier that exports mitochondrial acetyl-CoA: it crosses the membrane on the tricarboxylate carrier — so "acetyl-CoA can freely penetrate the mitochondrial membrane" is false, but "citrate is a substrate for ATP-citrate lyase" is true. The lyase is cytosolic, and its activity falls in starvation, not rises.

TAG synthesis and glycerol kinase

Fatty acids are activated to acyl-CoAs, esterified onto glycerol-3-phosphate, and phosphatidate is converted by phosphatidate phosphohydrolase to diacylglycerol before the last acyl group is added. Glycerol kinase is present in the liver and kidney and essentially absent from adipose tissue — so adipose tissue cannot re-use its own glycerol and depends on DHAP from glycolysis for its glycerol-3-phosphate. That is also why glycerol kinase is not needed for lipolysis; it is needed to use the glycerol lipolysis releases.

Cholesterol & Lipoproteins

Frequently tested: HMG-CoA reductase (rate-limiting), cholesterol as substrate of 7α-hydroxylase & ACAT, synthesis location (smooth ER + cytosol), isoprenoid units

🧱 Cholesterol Synthesis

Synthesis pathway (all from acetyl-CoA, in cytoplasm + smooth ER):

Acetyl-CoA Thiolase + HMG-CoA synthase→ HMG-CoA HMG-CoA Reductase→rate-limiting · NADPH · statin target Committed stepMevalonate →×3 ATP Isoprenoid units → Squalene → Cholesterol

Rate-limiting enzyme: HMG-CoA Reductase (HMG-CoA → mevalonate), the target of statins (competitive inhibitors). Cholesterol is then a substrate of 7α-hydroxylase (→ bile acids) and ACAT (→ cholesteryl esters for storage).

🚢 Lipoproteins — Full Detail

Lipoprotein	Made in	Main cargo	Key apoprotein	Fate
Chylomicrons	Intestine (absorptive phase only)	Dietary (exogenous) triglycerides	ApoB-48, ApoC-II, ApoE	LPL strips TG → chylomicron remnants → liver (recognized by ApoE)
VLDL	Liver	Endogenous triglycerides	ApoB-100, ApoC-II, ApoE	LPL strips TG → IDL → LDL
IDL	From VLDL	Cholesterol + remaining TG	ApoB-100, ApoE	Further TG removal → LDL
LDL	Derived from IDL	Cholesterol (main carrier)	ApoB-100 only	Taken up by LDL receptors (via ApoB-100)
HDL	Liver + Intestine	Reverse cholesterol transport	ApoA-I (activates LCAT)	Delivers cholesterol to liver via SR-B1 receptor

Chylomicron Remnants — Recognition

After LPL (Lipoprotein Lipase) removes triglycerides from chylomicrons, the resulting remnants are recognized by the liver via ApoE. This is a directly tested exam fact. ApoC-II activates LPL; ApoE targets remnants to the liver.

What Gets Depleted When VLDL → LDL

As VLDL is processed by LPL into IDL and then LDL, it progressively loses: triglycerides (stripped by LPL), ApoC-II, and ApoE. LDL retains only ApoB-100 and is enriched in cholesterol esters. This is asked as "what gets totally or partially depleted in metabolism of VLDL."

SR-B1 Receptor — Hepatic Cholesterol Uptake

The SR-B1 (Scavenger Receptor class B type 1) receptor on hepatocytes mediates the selective uptake of cholesterol from HDL into the liver — the final step of reverse cholesterol transport. Unlike the LDL receptor (which takes up the entire LDL particle), SR-B1 selectively extracts cholesterol while HDL returns to circulation.

⚗️ Glycerol from Adipose After LPL Action

When LPL (Lipoprotein Lipase) cleaves triglycerides from lipoproteins, it releases free fatty acids AND glycerol. Adipose tissue lacks glycerol kinase (the enzyme needed to reuse glycerol) and therefore releases glycerol into the blood. Glycerol is transported to the liver, where it is phosphorylated and used for triglyceride synthesis or enters gluconeogenesis.

💊 Statins — Mechanism

Statins (e.g., atorvastatin, simvastatin) are structural analogs of mevalonate (the product of HMG-CoA Reductase). They act as competitive inhibitors of HMG-CoA Reductase — they compete with the natural substrate HMG-CoA for the active site. As competitive inhibitors: they increase Km (lower apparent affinity) and do not change Vmax (maximum rate unchanged). Their effect can be overcome by increasing substrate concentration — though at therapeutic doses, they are highly effective.

Exam Trap — Statin Classification

Statins are structural analogs of mevalonate (not HMG-CoA itself). They are competitive inhibitors → increase Km, Vmax unchanged. "Statins do not affect Vmax and increase Km" → TRUE.

🔄 Bile Acid Synthesis, Uronic Acid & Enterohepatic Circulation

Bile acid synthesis pathway (from cholesterol, in the liver):

Cholesterol 7α-Hydroxylase→rate-limiting · CYP7A1 · liver ER Committed step7α-OH-cholesterol → Primary bile acids →+ glycine/taurine Conjugated salts gut bacteria→deconjugate + dehydroxylate Secondary bile acids

Uronic acids are formed from glucose by oxidation of C-6. Glucuronic acid (glucuronate) is the most important — it is the activated form used in bilirubin conjugation (as UDP-glucuronate) and drug detoxification. The 2022 exam asked directly: "Uronic acid is formed from ___?" → Answer: glucose (C-6 oxidation of glucose-6-phosphate).

1
Rate-limiting step of bile acid synthesis: 7α-hydroxylase (CYP7A1) in the liver ER. Introduces –OH at C-7 of cholesterol. Inhibited by bile acids returning via enterohepatic circulation (feedback inhibition).
2
Primary bile acids: Cholic acid and chenodeoxycholic acid — produced by the liver.
3
Conjugation: Primary bile acids + glycine or taurine → conjugated bile salts (more amphipathic, better detergents). Ratio glycine:taurine ≈ 3:1.
4
Secondary bile acids: Intestinal bacteria deconjugate and dehydroxylate primary bile acids → deoxycholic acid (from cholic) and lithocholic acid (from chenodeoxycholic). Lithocholic acid is poorly reabsorbed and mostly excreted.

Enterohepatic Circulation

>95% of bile salts are reabsorbed in the terminal ileum (active transport via Na⁺-bile salt cotransporter) → portal vein → liver → re-secreted into bile. This cycle conserves bile salts (body only has ~3–5 g but recycles them 6–10×/day → effective pool = 20–30 g/day).

Cholestyramine (bile salt sequestrant) binds bile salts in the gut → prevents reabsorption → liver must synthesize new bile acids from cholesterol → ↓ plasma cholesterol.

Hypertriacylglycerolemia — Treatment with Niacin ★ asked 2022

Niacin (Vitamin B3) at pharmacological doses inhibits lipolysis in adipose tissue (by inhibiting HSL via an adipocyte G-protein receptor) → ↓ FFA delivery to liver → ↓ VLDL synthesis → ↓ plasma TAG and LDL, ↑ HDL. This was tested in the 2022 exam.

🔗 Bile Acid Conjugation — Mechanism & Significance

Unconjugated (free) bile acids have a pKa of ~6.0 — at physiological pH they are partly protonated and can be passively reabsorbed across the gut wall. Conjugation lowers the pKa to ~2 (glycine conjugates) or ~1.5 (taurine conjugates) — making bile salts fully ionised at gut pH → they cannot diffuse back passively → they stay in the gut lumen as effective detergents.

	Glycine Conjugates	Taurine Conjugates
Examples	Glycocholic acid, Glycochenodeoxycholic acid	Taurocholic acid, Taurochenodeoxycholic acid
Ratio	Glycine : Taurine conjugates ≈ 3 : 1 in humans
pKa	~2.0 (fully dissociated at gut pH)	~1.5 (even more fully dissociated)
Advantage	Both remain ionised in the gut → cannot passively cross membranes → stay in lumen → more effective emulsification than free bile acids

Conjugation Reaction (Liver)

Bile acid + CoA → Bile acyl-CoA (activated) → + Glycine or Taurine → Conjugated bile salt
Enzyme: Bile acid-CoA:amino acid N-acyltransferase (BAAT) — occurs in hepatocyte peroxisomes and cytosol.
The amide bond formed is resistant to pancreatic proteases — conjugated bile salts remain intact through digestion.

Deconjugation by Gut Bacteria

Intestinal bacteria express bile salt hydrolase (BSH) → cleave glycine/taurine from conjugated bile salts → free primary bile acids → then 7α-dehydroxylation → secondary bile acids (deoxycholic, lithocholic). These are less soluble; lithocholic acid is mostly excreted in faeces (hepatotoxic if not eliminated).

🚢 The Particles and What Is In Them 2026

All lipoproteins share one architecture: a core of triacylglycerol and cholesteryl esters wrapped in a shell of phospholipid, free cholesterol and apolipoproteins. As density rises, protein rises and TAG falls.

Decreasing TAG content — the ranking

Chylomicron (≈ 90% TAG) → VLDL (≈ 60%) → IDL → LDL → HDL. Density runs the other way. HDL is roughly 40% protein, 30% phospholipid, 25% cholesterol and cholesteryl ester, 5% TAG. Note the trap in an older paper: HDL1 is more dense than HDL3? No — HDL3 is the denser, smaller, protein-richer particle; HDL2 is larger and lipid-richer.

✅ Chylomicrons 2026

Made by the intestine; the structural protein is apo B-48; they acquire apo C-II and apo E from HDL. Apo E is the ligand that lets the liver take up the remnants. They contain triacylglycerol, cholesterol, cholesteryl esters and phospholipids — but not bile salts, which is the recurring "contains all except" answer.

VLDL, IDL, LDL

VLDL — liver-made, endogenous TAG, structural protein apo B-100; it receives apo C-II and apo E from HDL. LPL converts it to IDL, hepatic lipase converts IDL to LDL. LDL is the most cholesterol-rich particle and carries only apo B-100, the ligand for the LDL receptor. Chylomicron remnants, IDL and LDL are the particles taken up by the liver.

Apolipoprotein A 2026

Apo A-I is the main structural protein of HDL. It is the activator of LCAT and the ligand for SR-B1. Apo A-II and A-IV also sit on HDL. Its deficiency (Tangier-type picture) causes very low HDL. Do not confuse apo A-I with apo(a) of Lp(a) — different molecules entirely.

The rest of the apolipoprotein table

apo B-48 chylomicrons only. apo B-100 VLDL, IDL, LDL — LDL-receptor ligand. apo C-II activates lipoprotein lipase. apo C-III inhibits it. apo E ligand for hepatic uptake of chylomicron remnants and IDL. apo D associates with HDL2 and HDL3 and may act as a lipid transfer protein. Choline is a lipotropic factor — it prevents fatty liver by allowing phospholipid, and therefore VLDL, to be made.

🔁 LCAT, CETP and ACAT 2026

The 2026 paper carried two LCAT questions and one on CETP. These three enzymes all esterify or move cholesteryl esters, and telling them apart is the whole exercise.

LCAT

Lecithin:cholesterol acyltransferase. Made in the liver, active in the plasma on the surface of HDL — so "LCAT is intracellular" is false. It transfers a fatty acid from lecithin (phosphatidylcholine) to free cholesterol, giving a cholesteryl ester + lysolecithin. Its activator is apo A-I — the recorded answer to "the main LCAT activator". The ester sinks into the core, so discoidal HDL3 matures into spherical HDL2. It is stimulated by insulin.

CETP 2026

Cholesteryl ester transfer protein. It exchanges cholesteryl esters from HDL for triacylglycerol from VLDL/LDL. That is its key role: it provides an indirect route for cholesteryl esters to reach the liver — through the apo B-containing particles and their receptors — alongside the direct SR-B1 route. It does not hydrolyse cholesteryl esters (that is cholesterol esterase) and it transfers rather than converts; the HDL3 → HDL2 conversion is LCAT's doing, while hepatic lipase converts HDL2 back to HDL3.

ACAT

Acyl-CoA:cholesterol acyltransferase — the intracellular counterpart. It esterifies cholesterol inside cells for storage, using acyl-CoA as the fatty acid donor. So LCAT and ACAT both make cholesteryl esters, but they use different substrates and are in different locations — exactly how older papers phrase the comparison.

The receptors

LDL receptor — ligands apo B-100 and apo E; takes up LDL, IDL and remnants by receptor-mediated endocytosis; the released cholesterol then inhibits HMG-CoA reductase, activates ACAT and suppresses further receptor synthesis. LRP — apo E, remnants. SR-B1 — apo A-I; takes up the cholesteryl esters only, not the HDL particle. SR-A — the unregulated macrophage scavenger route for oxLDL. ABCA1 — effluxes free cholesterol out of peripheral cells; its loss is Tangier disease.

Lp(a) — thrombosis and atherosclerosis 2026

Lp(a) is almost identical to LDL but carries an extra apolipoprotein, apo(a), covalently linked to apo B-100. Apo(a) is structurally homologous to plasminogen and competes with it for fibrin binding, which is thought to slow the breakdown of clots. So Lp(a) is doubly atherogenic: it delivers cholesterol like LDL and it is prothrombotic. A high plasma Lp(a) is an independent risk factor for coronary heart disease.

Reading a lipid panel, and LPL versus HSL

Measured fasting. Total cholesterol = LDL-C + HDL-C + VLDL-C, with VLDL-C = TAG ÷ 5; total should be < 200 mg/dL. LPL and LCAT are both stimulated by insulin; hormone-sensitive lipase is inhibited by insulin and activated by glucagon and adrenaline. Intravascular lipolysis = LPL acting on circulating chylomicrons and VLDL; intracellular lipolysis = HSL acting on stored adipocyte TAG. Both produce free glycerol, both are proteins, both are insulin-regulated — but in opposite directions.

🧱 Cholesterol and Bile Acids

Cholesterol — the three functions

Structural — a component of the cell membrane and modulator of its fluidity. Metabolic — the precursor of steroid hormones, vitamin D₂ and D₃, and bile acids. Regulatory — it activates ACAT and inhibits HMG-CoA reductase. It is the major sterol synthesised by all animals; its side chain has 8 carbons; in membranes it is unesterified, while the esterified form is for transport and storage. Bile pigments are not made from cholesterol — they come from haem, which is the standard wrong option in that list.

Where the liver's cholesterol comes from

Three sources, all asked together: de novo synthesis, degradation of lipoproteins taken up by the liver, and dietary cholesterol arriving in chylomicron remnants — note that dietary lipid arrives by the lymph and systemic circulation, not the portal vein, so "cholesterol from the portal vein" is the doubtful option.

HMG-CoA — one intermediate, two pathways

HMG-CoA synthase catalyses a condensation and exists in two forms: the cytosolic one feeds cholesterol synthesis, the mitochondrial one feeds ketogenesis. HMG-CoA reductase (cholesterol, → mevalonate, 2 NADPH) and HMG-CoA lyase (ketogenesis, → acetoacetate) then diverge. The pathway continues mevalonate → isoprene units → squalene → lanosterol → cholesterol; squalene synthase uses NADPH.

Bile acids and coprostanol

Primary bile acids — cholic and chenodeoxycholic acid — are made in the liver, rate-limited by 7α-hydroxylase, and conjugated with glycine or taurine. Colonic bacteria produce the secondary ones, deoxycholic and lithocholic. About 95% are reabsorbed in the terminal ileum — the enterohepatic circulation — and this is the only meaningful route of cholesterol excretion. Coprostanol is the principal sterol of the faeces, produced by bacterial reduction of cholesterol in the intestine — not synthesised in the liver. Bile itself contains bile salts, phospholipids, free cholesterol, bilirubin and electrolytes.

🍋 Ketone Bodies 2026

Several 2026 items converge here: why the liver cannot use them, whether the brain uses acetone, and that they cause metabolic acidosis.

✅ The liver lacks thiophorase

Ketone bodies are made in the mitochondria of hepatocytes only, yet the liver cannot burn them, because it lacks thiophorase (succinyl-CoA:acetoacetate CoA transferase) — the enzyme that reactivates acetoacetate to acetoacetyl-CoA in peripheral tissue. That is the recorded answer, and it is the reason the liver is a pure exporter.

❌ The brain does not use acetone

The brain uses acetoacetate and β-hydroxybutyrate. Acetone is a dead end: it is formed by the spontaneous decarboxylation of acetoacetate, it is not metabolised in the body, it is not an acid, and it is volatile and exhaled — the fruity breath. Any option saying acetone is used as fuel, or that acetone is the acid causing the acidosis, is wrong.

✅ They cause metabolic acidosis

Acetoacetate and β-hydroxybutyrate are acids, so overproduction gives a metabolic acidosis with a raised anion gap, compensated by Kussmaul hyperventilation. β-Hydroxybutyrate is the most abundant in blood.

When ketosis is seen

Uncontrolled diabetes mellitus, starvation and prolonged fasting, a high-fat / low-carbohydrate diet, severe exercise in the post-absorptive state, and pregnancy. Not on a high-carbohydrate diet. Ketogenesis is increased by glucagon and by high free fatty acid delivery, and its rate-limiting enzyme is mitochondrial HMG-CoA synthase. Ketone bodies cannot be converted back into triacylglycerol or glucose.

Lipoproteins Deep Dive

Frequently tested: apolipoproteins (which are purely structural), which particle carries most cholesterol (LDL), LCAT, SR-B1, LPL

⚗️ Key Lipoprotein Enzymes

LPL (Lipoprotein Lipase)

Sits on capillary endothelium. Cleaves triglycerides from chylomicrons and VLDL. Activated by ApoC-II (without ApoC-II, LPL is inactive). Releases free fatty acids to tissues and glycerol to blood (glycerol → liver).

👆 Tap for enzyme details

LCAT (Lecithin-Cholesterol Acyltransferase)

An extracellular enzyme (works in plasma). Converts lecithin + free cholesterol → cholesteryl esters + lyso-PC. Activated by ApoA-I (main HDL apoprotein). Makes HDL spherical. Often confused with ACAT (which is intracellular).

👆 Tap for enzyme details

ACAT (Acyl-CoA:Cholesterol Acyltransferase)

An intracellular enzyme (in the ER). Esterifies cholesterol within cells for storage. NOT activated by ApoA-I (that is LCAT). LCAT = extracellular/plasma; ACAT = intracellular/ER.

👆 Tap for enzyme details

SR-B1 (Scavenger Receptor)

Hepatic receptor. Mediates selective uptake of cholesterol from HDL into liver — the final step of reverse cholesterol transport. HDL docks → cholesterol extracted → HDL returns to circulation (unlike the LDL receptor, which internalizes the whole particle).

👆 Tap for enzyme details

LCAT vs ACAT — Directly Tested

LCAT: extracellular, plasma, activated by ApoA-I, works on HDL. ACAT: intracellular (ER), esterifies cholesterol inside cells for storage. Exam question: "ACAT is an extracellular enzyme activated by Apo-A1" → FALSE (that describes LCAT).

🏥 Familial Hypercholesterolemia — LDL Receptor Defect

Caused by deficiency or dysfunction of LDL receptors on cell surfaces. LDL cannot be cleared from blood → very high LDL-cholesterol → cholesterol deposits in tendons (xanthomas) and arteries (premature atherosclerosis, heart attacks in young adults). This is NOT a lipoprotein enzyme problem — it is a receptor problem.

👆 Tap for the full disease card

Nitrogen Disposal & the Urea Cycle

Frequently tested: which reactions release ammonia, NAG activating CPS-1, urea cycle ATP cost (4 high-energy bonds), CO₂+NH₃+aspartate entering, ornithine/citrulline/arginine, glucose-alanine cycle

🔀 Transamination

Amino acid + α-Ketoglutarate Aminotransferase ⇌ PLP (B6) Keto acid + Glutamate

The key idea: the amino group (–NH₂) is transferred from an amino acid onto α-ketoglutarate, collecting nitrogen into glutamate (the central nitrogen carrier). No ammonia is released here — it's just moved. The reaction is freely reversible and needs PLP (vitamin B6).

Glutamate then hands its nitrogen to the urea cycle: Glutamate Dehydrogenase releases NH₃ from glutamate. Clinically, ALT is liver-specific, while AST is found in liver + heart.

🔵 The Urea Cycle — Full Detail

The urea cycle converts toxic ammonia (NH₃) into urea, which is water-soluble and excreted in urine. It uses 4 high-energy phosphate bonds per cycle (2 ATP → 2 ADP + Pi at CPS-I, and 1 ATP → AMP + PPi at argininosuccinate synthetase, which equals 2 high-energy bonds = total 4). The cycle runs partly in mitochondria and partly in the cytosol.

1
NH₃ + CO₂ → Carbamoyl phosphate — CPS-I, mitochondria, requires 2 ATP and is absolutely dependent on N-Acetylglutamate (NAG) as allosteric activator. NAG is the main regulator of the entire urea cycle.
2
Carbamoyl-P + Ornithine → Citrulline — OTC, still in mitochondria. Most common urea cycle defect (X-linked).
3
Citrulline + Aspartate → Argininosuccinate — in the cytoplasm. Aspartate donates the second nitrogen atom. This step requires 1 ATP (→ AMP + PPi = 2 high-energy bonds).
4
Argininosuccinate → Arginine + Fumarate (Fumarase converts Fumarate → Malate in Krebs, and Malate Dehydrogenase → OAA). Fumarate links the urea cycle to the Krebs cycle.
5
Arginine → Urea + Ornithine — Arginase, cytoplasm. Ornithine re-enters mitochondria to continue the cycle.

NAG — Main Regulator

N-Acetylglutamate (NAG) is synthesized from acetyl-CoA + glutamate. It is the obligatory allosteric activator of CPS-I — without NAG, CPS-I is completely inactive and the entire urea cycle stops. High arginine stimulates NAG synthesis (feedforward). This is the most important regulatory point of the urea cycle and is directly tested.

Arginine Synthesis in the Kidney

The kidney can synthesize arginine using citrulline (from intestine) + aspartate (or from glutamine) as substrates. The kidney therefore plays a role in providing arginine for body needs — this is asked directly in exams.

Ultimate Fate of Ammonia in the Body

Most ammonia is ultimately converted to urea (in liver, via urea cycle) and excreted in urine. A smaller portion is incorporated into glutamine (via glutamine synthetase) for transport. On exam, "ultimate fate of ammonia" = urea.

🔋 Urea Cycle Energy & Regulation — Teacher Emphasized

Energy Cost

3 ATP consumed per cycle → generating 4 high-energy bond equivalents lost:
• CPS-I: 2 ATP → 2 ADP + 2 Pi (2 bonds)
• Argininosuccinate Synthetase: 1 ATP → AMP + PPi (2 bonds, since PPi hydrolysis adds 1 more)
Total = 4 high-energy phosphate bonds

Regulation — Enzyme Induction/Repression

Urea cycle enzymes are regulated by induction and repression (changing amount of enzyme — slow response) AND by substrate availability. High protein diet → ↑all urea cycle enzyme amounts. Starvation/catabolism → also ↑enzyme amounts. Key: regulation is ↑/↓ activity, not rendering enzyme "inactive" per se.

NAG — The Master Regulator (Teacher Highlighted)

N-Acetylglutamate (NAG) is the allosteric activator of CPS-I. Without NAG, CPS-I is completely inactive. NAG synthesis: acetyl-CoA + glutamate → NAG (NAG Synthase, in mitochondria). Arginine stimulates NAG synthase (feedforward activation → more arginine drives more urea synthesis). CPS-II (in pyrimidine synthesis, cytosol) does NOT need NAG.

Urea Cycle "No Beginning, No End" — Teacher Noted

The urea cycle is often described as having "no beginning and no end" because ornithine, citrulline, and arginine are continuously recycled — the cycle is fed by NH₃ (from CPS-I) and aspartate (from transamination) at any entry point.

Ammonia Disposal — Enzymes That Release Ammonia (Teacher Listed)

Enzymes that release ammonia (NH₃):
• Glutamate Dehydrogenase (major): Glutamate → α-KG + NH₃
• AMP Deaminase: AMP → IMP + NH₃ (purine catabolism)
• Adenosine Deaminase: Adenosine → Inosine + NH₃
• Glutaminase: Glutamine → Glutamate + NH₃ (in kidney/liver)
• Asparagine synthetase (reverse): hydrolyzes asparagine → aspartate + NH₃

🚴 Glucose-Alanine Cycle — Teacher Required

During muscle activity, amino groups from muscle amino acid catabolism are shuttled to the liver for urea synthesis via the glucose-alanine cycle:

1
Muscle: amino acids → transamination with pyruvate → Alanine (carries NH₂) + α-keto acids. ALT (requires PLP/B6) catalyzes this.
2
Alanine is exported from muscle and travels in blood to the liver.
3
Liver: ALT converts Alanine + α-KG → Pyruvate + Glutamate.
4
Pyruvate → gluconeogenesis → Glucose → exported to blood → used by muscle (cycle completes).
5
Glutamate → Glutamate Dehydrogenase → NH₃ → enters urea cycle.

Why It Matters

This cycle allows muscle to export nitrogen safely (as non-toxic alanine) while simultaneously supplying the liver with carbon for gluconeogenesis. It is fundamentally linked to both amino acid catabolism and gluconeogenesis.

🔵 Argininosuccinate — All Three 2026 Sub-Questions 2026

The recollection asks where it is made, whether its cleavage releases a Krebs intermediate, and how many of its nitrogens end up in urea.

✅ Where it is synthesised

In the cytosol, by argininosuccinate synthetase, from citrulline + aspartate + ATP (ATP → AMP + PPi, so two high-energy bonds). Only the first two urea-cycle steps — CPS I and OTC — are mitochondrial; citrulline leaves the mitochondrion and the last three steps are cytosolic. An option placing argininosuccinate synthesis in the mitochondrion is false.

✅ Cleavage releases a Krebs intermediate

Argininosuccinate lyase splits it into arginine + fumarate. The fumarate is the link to the citric acid cycle: fumarate → malate → oxaloacetate → transaminated back to aspartate, which re-enters the urea cycle. This is the aspartate–argininosuccinate shunt, and it is why the two cycles are drawn as interlocking rings.

✅ Only one of its nitrogens reaches urea

Argininosuccinate carries the nitrogen of citrulline (from carbamoyl phosphate) and the nitrogen of aspartate. Both end up in urea — but they arrive at different points: the aspartate nitrogen is the second urea nitrogen, and the aspartate carbon skeleton leaves as fumarate. So of the amino nitrogen carried in on aspartate, one atom is retained in urea.

The whole cycle in one line

1 CPS I (mitochondrion; NH₃ + HCO₃⁻ + 2 ATP; rate-limiting; needs N-acetylglutamate) → 2 ornithine transcarbamoylase (→ citrulline) → 3 argininosuccinate synthetase (cytosol) → 4 argininosuccinate lyase (→ arginine + fumarate) → 5 arginase (→ urea + ornithine). Cost: 3 ATP, 4 high-energy bonds. Urea is synthesised only in the liver and contains nitrogen from amino acid catabolism; the carbon comes from bicarbonate/CO₂. Ornithine and citrulline are not incorporated into proteins. Adenylosuccinate belongs to purine synthesis, not the urea cycle — the standard distractor.

Hyperammonaemia and ammonia intoxication

The commonest congenital cause is OTC deficiency, which is X-linked; all the other urea-cycle defects are autosomal recessive. In OTC deficiency the backed-up carbamoyl phosphate spills into pyrimidine synthesis, so orotic acid appears in urine alongside the high ammonia. Acquired causes: liver failure and portosystemic shunting. Ammonia is toxic because in the brain it is trapped as glutamine, draining glutamate and α-ketoglutarate — the Krebs cycle slows, ATP falls, and glutamine accumulates osmotically causing astrocyte swelling and cerebral oedema: tremor, slurred speech, blurred vision, asterixis, confusion, coma. Arginase can be inhibited by ornithine and by lysine, which compete with arginine.

💨 Reactions That Release Ammonia, and Where It Goes

✅ Reactions that release ammonia

Oxidative deamination of glutamate by glutamate dehydrogenase — the main one, and the only enzyme that can both release and fix ammonia (activated by ADP/GDP, inhibited by ATP/GTP). Glutaminase (deamidation of glutamine). Asparaginase. AMP deaminase (purine nucleotide cycle in muscle). Serine and threonine dehydratases. Bacterial urease in the gut, acting on urea.

❌ Reactions that do not

Transamination releases no ammonia — it moves an amino group from an amino acid to a keto acid and back; that is the point of it. So aspartate aminotransferase and alanine aminotransferase are the wrong answers to "which releases ammonia". Arginase releases urea, not ammonia. In the list of enzymes responsible for renal ammoniagenesis, the odd one out is again the aminotransferase.

Reactions that fix it

Glutamate dehydrogenase in reverse (the reductive amination of α-ketoglutarate), glutamine synthetase (glutamate + NH₄⁺ + ATP → glutamine — the main trap in brain and muscle) and carbamoyl phosphate synthetase I. Glutamine formed in working muscle can be taken to the liver for urea synthesis, used by the kidney for ammoniagenesis, or consumed by enterocytes and lymphocytes as fuel.

Amino acids and their sites of release

From muscle: alanine and glutamine above all. From the gut: alanine. From the kidney: alanine and serine. Amino acids that do not undergo transamination: lysine and threonine. The first reaction in amino acid catabolism is transamination, followed by oxidative deamination of the glutamate that collects the nitrogen.

💪 Creatine and Creatinine 2026

The 2026 items ask about the substrates, the phosphorylation, and whether creatinine production is spontaneous.

✅ Substrates — three amino acids, two organs

Glycine, arginine and methionine. The kidney combines glycine + arginine to make guanidinoacetate (releasing ornithine); the liver methylates it using SAM, from methionine, giving creatine. Options offering "glycine, alanine and histidine" or "methionine, alanine and glycine" are wrong — arginine must be in the list.

✅ Phosphorylation

Creatine travels to muscle and brain, where creatine kinase phosphorylates it to phosphocreatine — a high-energy compound and the immediate ATP buffer for the first seconds of contraction. Creatine kinase in serum rises in myocardial infarction and muscle disease, not in liver disease — a recurring wrong pairing.

✅ Creatinine formation is spontaneous

True. Phosphocreatine and creatine cyclise spontaneously and irreversibly — non-enzymatically — to creatinine, at a nearly constant 1–2% of the pool per day. That constancy is what makes creatinine useful.

Why it is measured

Creatinine has no function, is freely filtered and essentially not reabsorbed, and its production is proportional to muscle mass. Its plasma concentration is therefore a standard index of glomerular filtration rate. In urine it is normally present — unlike glucose (normally 0–0.8 mmol/day), bilirubin (normally absent) and urobilinogen (a trace, about 0.4 mg/day).

Glycine — what else it builds

Haem (with succinyl-CoA, via ALA synthase), purines (the whole glycine molecule is incorporated), creatine, glutathione, bile-salt conjugation, and it is a neurotransmitter. It can be formed from serine (and from choline and threonine). Glycine is a favourite "participates in the formation of" stem, and the answer is usually "all of the above".

Biogenic Amines & Neurotransmitter Synthesis

Frequently tested: catecholamine synthesis (DOPA→dopamine→noradrenaline→adrenaline), Phe/Tyr-derived products, MAO & DAO degradation, serotonin/melatonin from tryptophan

⚡ Catecholamine Synthesis Pathway (Tyr → Adrenaline)

Catecholamines are synthesized from Tyrosine in a stepwise pathway in the adrenal medulla, sympathetic neurons, and brain:

⚡ Catecholamine Synthesis (Tyr → Adrenaline)

Tyrosine Tyrosine Hydroxylase→rate-limiting · BH₄ L-DOPA DOPA Decarboxylase→PLP/B6 Dopamine Dopamine β-Hydroxylase→Vitamin C Noradrenaline PNMT→SAM · adrenal medulla Adrenaline

1
Tyrosine → L-DOPA: Tyrosine hydroxylase (rate-limiting; requires BH₄ tetrahydrobiopterin). This is the committed step.
2
L-DOPA → Dopamine: DOPA decarboxylase (requires PLP/B6). Dopamine is a neurotransmitter in its own right.
3
Dopamine → Noradrenaline (Norepinephrine): Dopamine β-hydroxylase (requires Vitamin C as cofactor). Located in secretory vesicles.
4
Noradrenaline → Adrenaline (Epinephrine): PNMT (phenylethanolamine N-methyltransferase). Transfers a methyl group from SAM (S-adenosylmethionine). Only in adrenal medulla.

Degradation & Clinical

Catecholamines are inactivated by: MAO (oxidative deamination) + COMT (methylation using SAM as methyl donor).
• Urine metabolites: VMA (vanillylmandelic acid) from adrenaline/noradrenaline; HVA (homovanillic acid) from dopamine.
• Phaeochromocytoma: adrenal tumour → excess catecholamines → ↑VMA in urine.

🌙 Serotonin & Melatonin Synthesis (Trp pathway)

🌙 Tryptophan → Serotonin → Melatonin

Tryptophan Tryptophan Hydroxylase→rate-limiting · BH₄ 5-HTP 5-HTP Decarboxylase→PLP/B6 Serotonin (5-HT) AANAT / ASMT→pineal · SAM Melatonin

1
Tryptophan → 5-Hydroxytryptophan (5-HTP): Tryptophan hydroxylase (requires BH₄). Rate-limiting step.
2
5-HTP → Serotonin (5-HT): 5-HTP decarboxylase (requires PLP/B6).
3
Serotonin → N-Acetylserotonin → Melatonin: In the pineal gland via acetylation (AANAT) + methylation (ASMT uses SAM).

Serotonin functions: pain perception, sleep, appetite, temperature, blood pressure, mood (well-being). Degraded by MAO → 5-HIAA (5-hydroxyindoleacetic acid) in urine. Niacin (B3) is also synthesized from Tryptophan (60 mg Trp → 1 mg niacin).

🤧 Histamine Synthesis

Histidine → Histamine by histidine decarboxylase (requires PLP/B6). Stored in mast cells and basophils. Released by allergic reactions or trauma.

Functions: vasodilator, allergic/inflammatory mediator, gastric acid secretion stimulator. Antihistamines block H₁ receptors (allergy) or H₂ receptors (gastric acid — e.g., ranitidine).

⚗️ Amine Bases Metabolism — MAO & DAO Enzymes (Teacher Listed)

Biogenic amines (dopamine, serotonin, histamine, noradrenaline) are degraded by oxidative deamination:

MAO (Monoamine Oxidase)

MAO: oxidatively deaminates monoamines (one amino group).
MAO-A: degrades serotonin, noradrenaline, dopamine, tyramine
MAO-B: mainly degrades phenylethylamine and dopamine
Produces: aldehyde + NH₃ + H₂O₂
MAO inhibitors: antidepressants (e.g., phenelzine, tranylcypromine)

DAO (Diamine Oxidase / Histaminase)

DAO: oxidatively deaminates diamines (two amino groups).
Main substrates: histamine, putrescine, cadaverine
Contains copper (Cu²⁺) and pyridoxal phosphate (PLP)
Active in intestinal mucosa, kidney, placenta
Deficiency → histamine intolerance

Products of Amine Oxidation

Both MAO and DAO produce: Aldehyde + NH₃ + H₂O₂. The H₂O₂ is then detoxified by catalase. The aldehydes are further oxidized by aldehyde dehydrogenase to carboxylic acids (excreted). The NH₃ enters the urea cycle.

🧘 GABA Metabolism & the GABA Shunt

GABA (γ-aminobutyric acid) is the brain's main inhibitory neurotransmitter. It is made from glutamate and broken down through a small bypass of the Krebs cycle called the GABA shunt.

⚗️ GABA Synthesis & Degradation

Glutamate GAD · PLP→ GABA GABA-T · PLP→ SSA → Succinate → Krebs

Step	Enzyme (cofactor)	Note
Glutamate → GABA	Glutamate decarboxylase (PLP/B6)	B6 deficiency → ↓GABA → seizures. Anti-GAD antibodies: type 1 diabetes, stiff-person syndrome
GABA → succinate	GABA transaminase (PLP/B6)	Inhibited by vigabatrin (antiepileptic) → ↑GABA

Why both GABA enzymes need B6

Both steps use PLP (vitamin B6). This is why B6 deficiency (or B6 antagonists like isoniazid) lowers GABA and provokes seizures — GABA is calming, so less of it means over-excitation.

🌾 Full Tryptophan Metabolism — Serotonin, Melatonin, Niacin

Tryptophan is the most metabolically "busy" amino acid: it feeds three important products through two branches.

🌙 Serotonin branch (~small %)

Trp → 5-HTP (tryptophan hydroxylase, BH₄) → serotonin (PLP) → melatonin (pineal). Serotonin is degraded by MAO → 5-HIAA in urine.

🔆 Kynurenine branch (majority)

Trp → kynurenine → ... → niacin (NAD⁺/NADP⁺). About 60 mg tryptophan → 1 mg niacin. Needs B6 — B6 deficiency impairs this route.

Clinical links

Carcinoid tumour diverts huge amounts of tryptophan to serotonin → ↑5-HIAA and can cause pellagra-like signs (niacin "stolen"). Hartnup disease (↓tryptophan absorption) and corn-based diets also cause pellagra because less tryptophan is available to make niacin.

Creatine & Whole-Body Protein Status

Frequently tested: creatine synthesis (Gly + Arg + Met/SAM), creatinine as muscle-mass marker, nitrogen balance states, marasmus vs kwashiorkor

💪 Creatine & Creatinine Metabolism

Creatine synthesis (2 steps — Glycine + Arginine + Methionine/SAM):

Glycine + Arginine AGAT→kidney · releases ornithine Guanidinoacetate GAMT→liver · + SAM (methyl donor) Creatine

Creatine phosphate (phosphocreatine):
Creatine + ATP ⇌ Creatine phosphate + ADP (by creatine kinase)
Acts as rapid ATP buffer in the first seconds of muscle contraction.

Creatinine:
Creatine and creatine phosphate spontaneously and non-enzymatically cyclize → creatinine at a constant daily rate.

Creatinine excreted in urine is proportional to muscle mass — useful marker.

↑ Serum Creatinine = Kidney Malfunction

Creatinine is normally rapidly cleared; any rise = renal impairment. CK-MB elevation = MI (different isoform).

⚖️ Nitrogen Balance

Positive Nitrogen Balance

Nitrogen intake > nitrogen excretion. Net protein synthesis. Occurs in: growing children, pregnancy, recovery from illness, athletes building muscle.

Negative Nitrogen Balance

Nitrogen excretion > nitrogen intake. Net protein breakdown. Occurs in: starvation, severe burns, trauma, surgery, cancer, high fever, uncontrolled diabetes.

Zero Balance (Equilibrium)

Intake = excretion. Healthy adults in steady state — protein turnover is constant but no net gain/loss.

Measurement

Nitrogen balance = N intake (dietary protein ÷ 6.25) − N excretion (mainly urine urea + stool + skin). Each gram of protein contains ~16% nitrogen.

Clinical Significance

• Thyroid hormones at physiological levels → positive N balance (anabolic). At high levels → negative N balance (protein catabolism).
• Glucocorticoids → negative N balance (promote muscle proteolysis → gluconeogenic substrates).
• Insulin → positive N balance (promotes amino acid uptake and protein synthesis in muscle).

🥣 Protein-Energy Malnutrition: Marasmus & Kwashiorkor

Feature	Marasmus	Kwashiorkor
Cause	Deficiency of BOTH calories AND protein	Adequate calories but severe protein deficiency
Body	Extreme wasting of both fat AND muscle	Muscle wasting but fat preserved; edema (esp. abdomen)
Albumin	Near-normal (body adapts)	Very low → oncotic pressure ↓ → edema
Key signs	Anaemia, loss of adipose, muscle wasting, growth retardation, "skin and bones" appearance	Oedema (ascites, pot belly), skin/hair changes, fatty liver (↓ VLDL synthesis), moon face
Metabolic	Ketosis; body uses fat + muscle as fuel	Insulin ↑ (from carbs) → inhibits lipolysis → fat in liver; no VLDL (no apolipoproteins)

Marasmus — Exam Points ★ asked 2025

Directly tested: symptoms include anaemia, loss of adipose tissue, muscle wasting, growth retardation. The liver is NOT enlarged (unlike kwashiorkor). Marasmus = starvation → the body uses all energy stores.

Heme, Porphyrins & Jaundice

Frequently tested: heme synthesis (ALA synthase, order of porphyrinogens), porphyrias & photosensitivity, conjugated vs unconjugated bilirubin, jaundice types & urine findings

🫀 The Liver — Central Metabolic Hub

The liver is the body's main metabolic organ. Understanding why each function belongs to the liver is high-yield.

Function	Why the liver?	Key enzymes/molecules
Gluconeogenesis	Only tissue (with kidney) expressing glucose-6-phosphatase → can release free glucose into blood	PEPCK, PC, F-1,6-BPase, G-6-Pase
Glycogen storage & release	Liver glycogen (~100 g) maintains blood glucose for other tissues. Muscle glycogen serves muscle only.	Glycogen synthase, glycogen phosphorylase
Urea synthesis	Liver is the only organ with the complete urea cycle. All ammonia from amino acid catabolism arrives here via portal blood as alanine or glutamine.	CPS-I, OTC, arginase — all 5 enzymes
Ketogenesis	Liver mitochondria are the exclusive site of ketone body synthesis. Note: liver cannot use its own ketones (lacks thiophorase/succinyl-CoA transferase).	HMG-CoA synthase, HMG-CoA lyase
Cholesterol & bile acid synthesis	Liver is the exclusive site of bile acid synthesis from cholesterol (7α-hydroxylase). All primary bile acids produced here.	HMG-CoA reductase, 7α-hydroxylase
Lipoprotein assembly	VLDL (carries endogenous lipids) and HDL precursors assembled in liver. Chylomicron remnants cleared here via ApoE.	MTP, ApoB-100, VLDL secretion
Protein synthesis	Albumin (main plasma oncotic protein), all clotting factors (II, V, VII, IX, X, fibrinogen), acute-phase proteins (CRP, ferritin).	Rough ER ribosomes
Bilirubin conjugation	Hepatocytes conjugate bilirubin with glucuronate (UGT1A1) → water-soluble → secreted into bile. Failure → jaundice.	UGT1A1, MRP2 (canalicular export)
Drug detoxification	CYP450 enzymes (Phase I) + glucuronidation/sulfation (Phase II) in ER. Requires NADPH and O₂.	CYP3A4, CYP2D6, UGT
Vitamin D activation (first step)	25-hydroxylase converts cholecalciferol → 25-OH-D₃ (calcidiol). Second step (→calcitriol) occurs in kidney.	CYP27A1 (25-hydroxylase)
Iron storage & regulation	Hepcidin produced by liver. Ferritin stores excess iron. Transferrin synthesised here.	Hepcidin, ferritin, transferrin

Liver Failure — What Fails?

Hepatocellular failure → ↓albumin (oedema, ascites) + ↓clotting factors (bleeding, ↑PT/INR) + ↑unconjugated bilirubin (impaired uptake) + ↑conjugated bilirubin (impaired secretion) + hypoglycaemia (↓GNG) + hyperammonaemia (↓urea cycle) + hypertriglyceridaemia (↓VLDL).

🩸 Heme Synthesis

Rate-limiting: ALA Synthase (Gly + Succinyl-CoA → ALA, needs PLP/B6, mitochondria). Final step: Protoporphyrin IX + Fe²⁺ → Heme (Ferrochelatase, mitochondria). Lead inhibits ALA Dehydratase and Ferrochelatase.

⚡ Porphyrias

Porphyria	Enzyme Defect	Key Features
AIP	PBG Deaminase	Neurological symptoms. NO photosensitivity. ALA + PBG accumulate.
PCT	Uroporphyrinogen Decarboxylase	Photosensitivity; dark urine (uroporphyrin).

💛 Types of Jaundice

Type	Bilirubin ↑	Urine bilirubin	Urine urobilinogen	Stool color	Key markers	Cause
Pre-hepatic (hemolytic)	Indirect/unconjugated only	❌ Absent — unconjugated is albumin-bound, not water-soluble	↑ Increased — more bilirubin enters gut → more urobilinogen	Normal/dark brown	↑LDH, ↑reticulocytes, ↓haptoglobin	Excess RBC destruction (G6PD, spherocytosis, sickle cell)
Hepatic (hepatocellular)	Both conjugated AND unconjugated	✅ Present — conjugated leaks into blood	Variable (↓ if severe damage)	Normal or pale	↑↑ALT > AST (viral hepatitis); AST:ALT >2 (alcoholic). ↑PT	Hepatitis (viral, alcoholic, drug-induced), cirrhosis, liver failure
Post-hepatic (obstructive)	Direct/conjugated only	✅ Present (dark urine = bilirubinuria)	❌ Absent — no bile reaches gut	Pale/clay-colored (acholic)	↑ALP, ↑GGT, ↑conjugated bilirubin; no urobilinogen	Bile duct obstruction (gallstones, pancreatic cancer, cholangitis)

Bilirubin and the Blood-Brain Barrier ★ asked 2018, 2019

Only unconjugated (indirect) bilirubin can cross the blood-brain barrier — it is lipid-soluble. Conjugated bilirubin is water-soluble and cannot. This causes kernicterus (brain damage) in neonates with high unconjugated bilirubin. Directly tested: "which form of bilirubin crosses the BBB?" → unconjugated.

Which Disease Causes Bilirubin in Urine?

Only conjugated (direct) bilirubin appears in urine (it is water-soluble). Diseases: Dubin-Johnson syndrome, Rotor syndrome, obstructive jaundice, hepatocellular jaundice. Unconjugated hyperbilirubinemias (Gilbert, Crigler-Najjar, hemolytic jaundice) do NOT cause bilirubinuria.

💛 Conjugated vs Unconjugated Bilirubin & Urobilinogen — Teacher Listed as High Priority

Teacher explicitly listed: "conjugated, unconjugated urobilinogen, hyperbilirubinemia — genetic, erythrocyte enzymes" as a Kłos exam topic.

Compound	Properties	Where Found	Clinical Significance
Unconjugated (indirect) bilirubin	Water-INsoluble, lipid-soluble, bound to albumin in plasma	Blood only (not urine)	Elevated in: hemolysis, Gilbert, Crigler-Najjar. Can cross BBB → kernicterus
Conjugated (direct) bilirubin	Water-SOLUBLE (two glucuronate groups attached)	Bile, blood (if obstructed), urine	Elevated in: cholestasis, Dubin-Johnson, Rotor. Appears in urine → dark urine (bilirubinuria)
Urobilinogen	Colorless, water-soluble. Formed by gut bacteria from bilirubin (diglucuronide → urobilinogen)	Gut, portal blood, urine, bile	↑ in hemolysis (pre-hepatic). Absent in complete obstruction (no bilirubin enters gut). Small amounts in normal urine
Urobilin / Stercobilin	Urobilinogen oxidized → urobilin (brown). Stercobilin = fecal pigment	Feces, some urine	Absent stools = complete bile duct obstruction (pale stools). Brown stool color = normal stercobilin

Genetic Hyperbilirubinemias — Erythrocyte Enzyme Defects

Hemolytic (pre-hepatic) jaundice caused by erythrocyte enzyme defects:
• G6PD deficiency: ↓NADPH → oxidative hemolysis → ↑unconjugated bilirubin
• Pyruvate kinase deficiency: ↓ATP in RBCs → membrane failure → hemolysis → ↑unconjugated
• Hereditary spherocytosis (spectrin/ankyrin defects): fragile RBCs → hemolysis
All erythrocyte enzyme defects cause unconjugated hyperbilirubinemia (pre-hepatic jaundice): no bilirubinuria, ↑urobilinogen.

Alcohol and Bilirubin (Teacher Topic)

Chronic alcohol use → liver cell damage → hepatocellular jaundice with BOTH conjugated and unconjugated bilirubin elevated. Alcohol Dehydrogenase and Aldehyde Dehydrogenase — alcohol metabolism — were specifically listed by Kłos as exam topics.

Bilirubin conjugation is performed by UDP-Glucuronosyltransferase (UGT) in the liver. Heme synthesis does not occur in mature red blood cells (they have no nucleus or mitochondria).

Rotor Syndrome — OATP1B1/OATP1B3 Deficiency ★ asked 2023

Rotor syndrome is caused by deficiency of the hepatic uptake transporters OATP1B1 and OATP1B3. This impairs reuptake of conjugated bilirubin into hepatocytes → conjugated bilirubin spills into blood → dark urine. The liver color is normal (unlike Dubin-Johnson's black liver). This was asked in the 2023 exam: "OATP1B1 and OATP1B3 deficiency causes?" → Rotor syndrome.

🧪 Hepatic Phase II (Conjugation) Reactions

Drug/toxin detoxification has two phases. Phase I (CYP450: oxidation/reduction/hydrolysis) exposes or adds a reactive group. Phase II attaches a large polar group to make the molecule water-soluble for excretion.

Phase II reaction	Donor / enzyme	Example substrate
Glucuronidation	UDP-glucuronate → UGT	Bilirubin, steroids, many drugs (high capacity)
Sulfation	PAPS → sulfotransferase	Steroids, catecholamines, paracetamol (low capacity)
Glutathione conjugation	GSH → glutathione S-transferase	Reactive electrophiles → mercapturic acid; paracetamol NAPQI
Acetylation	Acetyl-CoA → N-acetyltransferase	Isoniazid, sulfonamides (fast/slow acetylators)
Methylation	SAM	Catecholamines (COMT), thiopurines
Glycine conjugation	Glycine	Benzoic acid → hippuric acid; bile acids

Paracetamol clinical link

Normally paracetamol is glucuronidated/sulfated. In overdose these saturate → CYP450 makes toxic NAPQI → depletes glutathione → liver necrosis. N-acetylcysteine restores glutathione. This is the classic Phase-I-makes-it-worse, Phase-II-detoxifies story.

🍯 The Uronic Acid (Glucuronate) Pathway

A minor branch of glucose metabolism that makes glucuronic acid (as UDP-glucuronate). It makes no ATP — its purpose is to supply an activated sugar acid for conjugation and connective-tissue synthesis.

⚗️ Formation of Glucuronic Acid

Glucose-1-P → UDP-glucose UDP-glucose DH→ UDP-glucuronate

Major functions of the pathway

1. Conjugation/detoxification — UDP-glucuronate is the donor for UGT to conjugate bilirubin and drugs (Phase II). 2. Glycosaminoglycan synthesis — glucuronic acid is a building block of hyaluronic acid, heparin, chondroitin. 3. In many animals (not humans) it leads to vitamin C synthesis — humans lack L-gulonolactone oxidase, so we must eat vitamin C. The exam point "uronic acid is formed from ___" → glucose (oxidation at C-6).

🩸 Haem Synthesis and ALA 2026

ALA — where it comes from

δ-Aminolevulinic acid is made from succinyl-CoA + glycine by ALA synthase in the mitochondrion, using pyridoxal phosphate (vitamin B6). It is the rate-limiting step, feedback-inhibited and repressed by haem, and induced by drugs that consume haem through cytochrome P450.

ALA → porphobilinogen

Two ALA condense to porphobilinogen by ALA dehydratase in the cytosol — a zinc enzyme and the classic target of lead, which also inhibits ferrochelatase.

The first tetrapyrrole

Asked directly. Four porphobilinogen molecules condense, catalysed by PBG deaminase (hydroxymethylbilane synthase), and cyclise via uroporphyrinogen III cosynthase. The first tetrapyrrole compound is uroporphyrinogen III (its immediate linear precursor being hydroxymethylbilane). The type I isomers are the useless by-products.

To haem

Uroporphyrinogen III → coproporphyrinogen III → protoporphyrinogen IX → protoporphyrin IX → ferrochelatase inserts Fe²⁺ in the mitochondrion. Bile pigments come from the degradation of this haem, not from cholesterol.

Acute intermittent porphyria — correcting the recollection 2026

The 2026 note lists three claims, and only one of them is right.
• "Is photosensitive" — FALSE. AIP is the porphyria with no skin disease at all, because ALA and PBG are not photoactive pigments. Photosensitivity belongs to porphyria cutanea tarda (uroporphyrinogen decarboxylase), the commonest porphyria.
• "Due to ALA dehydratase" — FALSE. AIP is caused by a defect in PBG deaminase, also called hydroxymethylbilane synthase (or uroporphyrinogen I synthase) — exactly as the older paper states it. ALA dehydratase deficiency is a separate, very rare porphyria.
• "ALA and porphobilinogen accumulate" — TRUE, and they are neurotoxic: attacks of severe abdominal pain, peripheral neuropathy and psychiatric disturbance, with urine that darkens to port-wine on standing. Triggered by barbiturates, sulfonamides, oestrogens, alcohol and fasting; treated with IV glucose and haem, which switch ALA synthase off.

💛 Bilirubin and Obstructive Jaundice 2026

Obstructive jaundice appears at least twice in the 2026 recollection, always with the same three-part answer: what is in the blood, what is in the urine, what is in the stool.

The normal route first

Haem → (haem oxygenase, releasing Fe²⁺ and carbon monoxide) → biliverdin → (biliverdin reductase, a reduction) → bilirubin → carried on albumin → conjugated in the hepatocyte by UDP-glucuronosyltransferase to the diglucuronide → secreted into bile → bacteria make urobilinogen → stercobilin in faeces, a little urobilin in urine.

✅ Obstructive (post-hepatic) jaundice

Blood: conjugated (direct) bilirubin raised. Urine: bilirubin PRESENT — conjugated bilirubin is water-soluble and filterable — and urobilinogen absent or very low, because nothing reaches the gut. Stool: pale, clay-coloured (no stercobilin), with fat malabsorption and steatorrhoea. Enzymes: ↑↑ ALP and GGT. The classic option set "urobilinogen not present in urine / bilirubin present in urine / bilirubin is conjugated" is all true for obstruction.

Haemolytic jaundice

Blood: unconjugated raised. Urine: no bilirubin (unconjugated is albumin-bound and cannot be filtered) but urobilinogen increased. Stool: dark. Also ↑ LDH, ↓ haptoglobin, reticulocytosis, and serum iron rises — which is the answer to "iron in plasma increases with… increased haemolysis". Note that in haemolysis bilirubin and urinary urobilinogen are elevated while iron is released, not lost.

Van den Bergh, and solubility

The direct reaction identifies conjugated bilirubin, which reacts immediately in water; the indirect fraction only reacts after methanol is added — hence the names. And the trap: bilirubin diglucuronide is MORE soluble than free bilirubin, so any option calling the diglucuronide "less soluble" is false.

The inherited hyperbilirubinaemias

Gilbert — mildly reduced UGT1A1 (~30%); unconjugated, benign, worse on fasting. Crigler–Najjar type I — complete absence of hepatic UDP-glucuronosyltransferase: severe congenital jaundice, brain damage (kernicterus), fatal in infancy without transplant, and phenobarbital does not help — phenobarbital works in type II. Dubin–Johnson — a defect in the transporter that moves conjugated bilirubin out of the hepatocyte, giving conjugated hyperbilirubinaemia and a black liver. Rotor — similar but without the pigment. So: Gilbert and Crigler–Najjar = unconjugated; Dubin–Johnson and Rotor = conjugated.

Iron & Lab Tests

Frequently tested: Fe²⁺ vs Fe³⁺ forms (ferritin, hemin, hemosiderin), daily iron requirement, TIBC in iron-deficiency vs hemochromatosis, transferrin

🔴 Iron Metabolism — Teacher Listed as Kłos Exam Topic

Iron metabolism is explicitly listed by the teacher (Kłos) as a high-priority exam topic. Key aspects:

Concept	Detail
Forms of iron	Fe²⁺ (ferrous) = absorbed form (in diet, in heme). Fe³⁺ (ferric) = stored (ferritin, hemosiderin), transported (transferrin). Vitamin C reduces Fe³⁺ → Fe²⁺ for absorption.
Absorption	Non-heme iron: Fe³⁺ → Fe²⁺ by duodenal cytochrome b (requires vitamin C) → enters enterocyte via DMT-1. Heme iron: absorbed directly as heme → cleaved by heme oxygenase.
Transport	Transferrin: plasma iron transport protein (carries Fe³⁺). In iron deficiency: TIBC ↑ (more empty transferrin). In hemochromatosis: TIBC ↓ (transferrin saturated).
Storage	Ferritin: intracellular storage (soluble). Hemosiderin: insoluble storage (when ferritin overwhelmed). Ferritin is also an acute-phase protein (rises in inflammation regardless of iron status).
Regulation	Hepcidin (liver peptide): master iron regulator. High iron → ↑hepcidin → degrades ferroportin → ↓iron absorption and ↓iron release from stores. Low iron → ↓hepcidin → ↑iron absorption.

Alcohol & Iron Metabolism

Alcohol disrupts iron metabolism: chronic alcohol use → ↑iron absorption, ↓hepcidin → can worsen hemochromatosis. Also: alcohol induces GGT (as noted in lab tests). ALT/AST ratio >2 suggests alcoholic hepatitis.

🧪 Benedict Test & Reducing Sugars (Exam Tested)

The Benedict test detects reducing sugars — sugars that have a free aldehyde or ketone group that can donate electrons. A positive result gives a brick-red or orange precipitate. Monosaccharides are all reducing sugars. Among disaccharides, sucrose is NOT a reducing sugar (its anomeric carbons are both locked in the glycosidic bond).

Sugar	Benedict Test	Why
Glucose, Fructose, Galactose	✅ Positive	Free reducing group
Maltose, Lactose	✅ Positive	Free reducing group on one end
Sucrose	❌ Negative	No free reducing group

Starch Digestion = Complete

Complete digestion of starch produces monosaccharides (glucose). These ARE reducing sugars → positive Benedict test (brick-red/orange color). Starch itself may give a weaker reaction because its reducing ends are few.

🧪 Other Important Biochemical Tests ★ recur every year

Teacher (Kłos) confirmed these WERE asked in past years

Salkowski (cholesterol), Hay's (bile acids in pathological urine — not Pettenkofer's), reducing sugars, Legal's (ketone bodies), OGTT, ALT/AST, alkaline phosphatase, and urobilinogen. Prioritise these.

Test	Detects	Positive Result
Benedict	Reducing sugars (glucose, fructose, lactose, maltose — NOT sucrose)	Brick-red / orange precipitate
Seliwanov	Ketoses (e.g. fructose) — distinguishes ketoses from aldoses	Red color
Ehrlich	Urobilinogen in urine	Cherry-red ring. Colour appears BEFORE heating → ↑urobilinogen (e.g. haemolysis)
Salkowski	Cholesterol	Red color in chloroform layer
Hay's (sulfur powder)	Bile acids/salts in pathological urine	Fine sulfur powder sprinkled on the surface sinks if bile salts are present (they lower surface tension). A physical surface-tension test. Teacher: Hay's was asked, Pettenkofer's was not.
Legal's	Ketone bodies (acetoacetate / acetone)	Red-violet colour with sodium nitroprusside in alkaline solution (Legal's = nitroprusside test for ketones)
Van den Bergh	Bilirubin (direct vs indirect)	Pink/purple color
Biuret	Proteins (peptide bonds — needs ≥2 peptide bonds, alkaline)	Violet color
Kay test (BAO/MAO)	Gastric acid: Basal & Maximal Acid Output	Normal: BAO 0–5, MAO 5–25 mmol/h

🩸 Clinical Function Tests — OGTT, Transaminases & ALP ★ teacher-listed

OGTT (Oral Glucose Tolerance Test)

Give 75 g oral glucose, measure blood glucose at 0 and 2 h. Diabetes: fasting ≥7.0 mmol/L or 2-h ≥11.1 mmol/L. Impaired glucose tolerance: 2-h 7.8–11.0. Used to diagnose diabetes & gestational diabetes.

ALT & AST (transaminases)

ALT = liver-specific. AST = liver + heart + muscle. Both ↑ in hepatocellular damage. AST/ALT (De Ritis) ratio >2 suggests alcoholic liver disease; <1 suggests viral hepatitis. Require PLP (B6).

Alkaline Phosphatase (ALP)

↑ in cholestasis (bile duct obstruction — most specific) and bone disease (Paget, metastases, rickets — osteoblast activity). Normal AST/ALT + high ALP → think bone or bile. GGT rises with ALP confirms a liver source.

Urobilinogen (urine)

Formed from bilirubin by gut bacteria; some is reabsorbed and excreted in urine. ↑ in haemolysis & hepatocellular damage; absent in complete biliary obstruction. Detected by the Ehrlich test.

🧪 Other Named Tests (reference) ⚪ never appeared in past exams

None of these have shown up in any available past paper — included only for completeness, low priority.

Test	Detects
Burstein & Samaille precipitation	Beta-lipoproteins
Potassium permanganate	Double bonds in unsaturated fatty acids
Weltmann–Barrenscheer	Urea in serum and urine
Schiff's	Aldehydes
Fiske–Subbarow	Inorganic phosphate levels
Pettenkofer's	Bile salts (chemical colour reaction — but teacher says this one was not asked; Hay's was)
Obermayer's test	Indican in urine
Reitman–Frankel	Aminotransferases (ALT / AST)
Wohlgemuth	Amylase activity
Lowry test	Protein levels
Feulgen reaction	Chromosomal material / DNA 🧬

🧪 GGT (Gamma-Glutamyl Transferase / GGTP) — Teacher Specifically Mentioned

GGT (γ-GT, GGTP) is a liver enzyme found on the surface of hepatocytes and bile duct cells. It is an important clinical lab marker for:

Liver/biliary disease — elevated in cholestasis, hepatitis, cirrhosis
Alcohol use — GGT is the most sensitive marker of alcohol-related liver disease; it rises with chronic alcohol consumption (alcohol induces GGT production)
Distinguishing liver vs bone source of ALP — if ALP is elevated, a high GGT confirms the source is liver (not bone), since bone does not express GGT

GGT Biochemical Role

GGT transfers a glutamyl group from glutathione (GSH) or other γ-glutamyl compounds to amino acids. It is part of the γ-glutamyl cycle involved in amino acid transport across cell membranes (especially in kidney). Also involved in glutathione metabolism.

Exam: GGT is the alcohol marker

GGT rises early with alcohol use, before ALT/AST. Combination of elevated GGT + elevated MCV (macrocytosis from B12/folate deficiency in alcoholics) → strongly suggests chronic alcohol use. Teacher specifically mentioned "Lab levels important: GGTP."

🩸 Plasma Proteins & the Proteinogram (Electrophoresis)

Serum protein electrophoresis separates blood proteins into 5 bands by charge/size. The pattern (the "proteinogram") is a classic diagnostic tool. Nearly all plasma proteins are made by the liver — except immunoglobulins (plasma cells).

Fraction	Main proteins	Function
Albumin	Albumin (~60% of total)	Main oncotic pressure; carries drugs, bilirubin, fatty acids, Ca²⁺, hormones
α₁-globulin	α₁-antitrypsin, α-fetoprotein	Antiprotease (protects lung); ↓ in AAT deficiency
α₂-globulin	Haptoglobin, ceruloplasmin, α₂-macroglobulin	Bind free Hb (↓ in haemolysis); copper transport
β-globulin	Transferrin, complement, β-lipoprotein (LDL)	Iron transport; immune; lipid transport
γ-globulin	Immunoglobulins (antibodies)	Immune defence — made by plasma cells, NOT liver

Hypoalbuminaemia

Causes: liver failure (↓synthesis), nephrotic syndrome (urinary loss), malnutrition (kwashiorkor), burns, inflammation. Result: oedema (↓oncotic pressure). Albumin is a negative acute-phase reactant (falls in inflammation).

Globulin changes

Polyclonal ↑γ = chronic infection/inflammation. Monoclonal spike (M-band) = multiple myeloma. ↓γ = immunodeficiency. Acute-phase ↑: CRP, fibrinogen, haptoglobin, ferritin, α₁-antitrypsin.

Plasma proteins as transporters

Albumin (bilirubin, FFA, drugs, Ca²⁺), transferrin (iron), ceruloplasmin (copper), transcortin (cortisol), SHBG (sex steroids), retinol-binding protein (vit A), transthyretin (T4). Bound hormone/ion is a reservoir; only the free fraction is active.

💧 Starling Forces — Capillary Fluid Balance & Oedema

Whether fluid leaves or re-enters a capillary is set by four "Starling forces" — the balance of hydrostatic (pushing) and oncotic (pulling) pressures across the capillary wall. Plasma albumin provides the main pull that keeps fluid in.

Push fluid OUT

Capillary hydrostatic pressure (blood pressure); interstitial oncotic pressure

Pull fluid IN

Plasma oncotic pressure (mainly albumin); interstitial hydrostatic pressure

Oedema cause	Starling force disturbed
↓ Albumin (liver failure, nephrotic, kwashiorkor)	↓ plasma oncotic pressure → fluid leaks out
Heart failure / venous obstruction	↑ capillary hydrostatic pressure
Lymphatic blockage	Fluid/protein not drained (lymphoedema)
Inflammation	↑ capillary permeability → protein leaks into tissue

Link to albumin

This is exactly why hypoalbuminaemia causes oedema: less albumin → less oncotic "pull" → net filtration out of capillaries → fluid accumulates in tissues (and as ascites in kwashiorkor/liver failure).

🔩 Iron, TIBC and the Regulators 2026

TIBC — what it measures

Total iron-binding capacity = the total iron that plasma transferrin could carry, so it is an indirect measure of transferrin concentration. Transferrin is a β-globulin, made in the liver, normally about one third saturated, and it binds two Fe³⁺.

Which way TIBC moves

Iron deficiency: TIBC increased, plasma iron decreased — the pairing asked as a single option, and also the answer to "increased TIBC is observed in…". Haemochromatosis / iron overload: TIBC decreased, plasma iron increased. Anaemia of chronic disease: both low. Ferritin follows body stores (and rises in inflammation as an acute-phase protein).

Absorption

Dietary Fe³⁺ cannot be transported across membranes — it must first be reduced to Fe²⁺ by gastric acid, vitamin C and DcytB, then carried in by DMT1. Ferritin does store iron in the intestinal wall, and iron that is not exported is lost when the enterocyte is shed. Ferroportin exports it; body iron content is regulated by absorption in the gut, not by the kidneys.

Hepcidin and hemojuvelin

Hepcidin, from the liver, blocks ferroportin and therefore shuts absorption down. Hemojuvelin modulates the expression of hepcidin — that is the single-choice answer; it is not similar to ceruloplasmin, does not contain copper, and is not a degraded ferritin. Mutations cause juvenile haemochromatosis.

Iron-containing compounds, and the Fe²⁺⇄Fe³⁺ oscillation

Fe²⁺ ("ferrous"): haem in haemoglobin and myoglobin. Fe³⁺ ("ferric"): haemin, methaemoglobin, transferrin, ferritin and haemosiderin. The proteins that work by cycling between Fe²⁺ and Fe³⁺ are the cytochromes — including cytochrome oxidase — plus catalase and peroxidase. Haemoglobin and myoglobin do not oscillate: their iron must stay Fe²⁺ to carry oxygen, and if it oxidises they become non-functional methaemoglobin. Transferrin and ferritin merely hold Fe³⁺; they do not cycle catalytically.

Key Diseases & Disorders

Frequently tested: PKU & phenylalanine hydroxylase, glycogen & lysosomal storage diseases, cystic fibrosis, favism, collagen disorders

🧬 Amino Acid Metabolism Disorders

Disease	Defect	Key Features
PKU	Phenylalanine Hydroxylase	Phe accumulates; intellectual disability; musty odor; treat with Phe-restricted diet
Albinism	Tyrosinase	No pigmentation; UV sensitivity; normal intelligence
Alkaptonuria	Homogentisate Oxidase	Dark urine; ochronosis; arthritis
Homocystinuria	Cystathionine β-Synthase	Marfanoid; lens dislocation (downward); thrombosis
MSUD	Branched-Chain α-Ketoacid DH	Leu/Ile/Val accumulate; sweet-smelling urine; neurological damage

🏪 Storage Diseases

Disease	Enzyme Deficient	Features
Gaucher	β-Glucocerebrosidase	Hepatosplenomegaly, bone pain; most common lysosomal SD
Von Gierke	Glucose-6-Phosphatase	Hypoglycemia, hepatomegaly, lactic acidosis
Lesch-Nyhan	HGPRT	Gout + self-mutilation; X-linked

Acid–Base Balance

Frequently tested: the four disturbances, recognizing them from pCO₂ & HCO₃⁻, full vs partial vs no compensation (gasometry calculations), insulin deficiency → metabolic acidosis

🧮 The Essentials — Equation & Normal Values

pH = pKₐ + log [A⁻] / [HA] → For bicarbonate: pH = 6.1 + log [HCO₃⁻] / (0.03 × pCO₂)

⭐ Normal Arterial Blood Values — memorize these three

pH

7.35–7.45

(7.4 average)

<7.35 = acidosis · >7.45 = alkalosis

pCO₂

35–45

mmHg

RESPIRATORY component (lungs)

HCO₃⁻

22–26

mmol/L

METABOLIC component (kidneys)

Plugging in the averages: pH = 6.1 + log(24 / (0.03 × 40)) = 6.1 + log(24 / 1.2) = 6.1 + 1.3 = 7.4 ✓

🧽 Buffer Systems

Buffer System	Where it matters	Key Feature
Bicarbonate (HCO₃⁻/H₂CO₃)	Blood (main plasma buffer)	Most important — open system: CO₂ regulated by lungs, HCO₃⁻ by kidneys, independently. Must be reabsorbed from the glomerular filtrate (all of it is reabsorbed when acid–base balance is normal).
Phosphate (H₂PO₄⁻/HPO₄²⁻)	Urine + inside cells	pKa ≈ 6.8. In normal acid–base balance, phosphate is the most important buffer in urine; effective in the distal nephron where tubular pH approaches its pKa.
Ammonia (NH₃/NH₄⁺)	Urine	NH₃ from glutamine (glutaminase) traps secreted H⁺ as NH₄⁺ for excretion. Becomes the main route for excreting excess H⁺ in acidosis (upregulated when acidotic).
Plasma proteins	Blood + cells	Amino-acid side chains buffer H⁺ (imidazole of His, carboxyl of Glu/Asp, amino of Lys). Haemoglobin is a major intracellular protein buffer.

⚗️ The Four Types of Disturbances

Disorder	Primary Change	Compensation	Common Causes
MET Metabolic Acidosis	HCO₃⁻ ↓	pCO₂ ↓ (Kussmaul breathing)	Lactic acidosis (hypovolemia), ketoacids in DKA (type 1 diabetes), severe renal failure
MET Metabolic Alkalosis	HCO₃⁻ ↑	pCO₂ ↑ (hypoventilation)	Vomiting / nasogastric suction (loss of H⁺), diuretics (furosemide, thiazides)
RESP Respiratory Acidosis	pCO₂ ↑	HCO₃⁻ ↑ (kidney)	Hypoventilation: lung diseases impairing gas exchange, drugs depressing ventilation (barbiturates)
RESP Respiratory Alkalosis	pCO₂ ↓	HCO₃⁻ ↓ (kidney)	Hyperventilation: high-altitude hypoxia, anxiety disorders, CNS disorders, salicylate poisoning

Exam Trap

In metabolic alkalosis, the compensatory pCO₂ INCREASES (not decreases) — the lungs hypoventilate to retain CO₂ and pull the pH back down.

Anion Gap — classifying metabolic acidosis

Anion gap = [Na⁺] − ([Cl⁻] + [HCO₃⁻]) (normal ≈ 8–12 mmol/L).
High anion gap acidosis = added acid (lactic acidosis, ketoacidosis/DKA, renal failure, toxins). Normal anion gap acidosis = HCO₃⁻ loss (diarrhoea, renal tubular acidosis). A quick way to tell why a metabolic acidosis is happening from the electrolytes.

🔍 Reading a Blood Gas — Identify the Disturbance from pCO₂ & HCO₃⁻

You can name any simple disturbance using just two numbers. Here is the exact method:

Look at pHacidosis or alkalosis? → Which value is abnormal?pCO₂ or HCO₃⁻ → Name ite.g. metabolic acidosis

Step 1 — Which value is the problem? (respiratory vs metabolic)

• If pCO₂ is the abnormal value → it is RESPIRATORY (lungs control CO₂).
• If HCO₃⁻ is the abnormal value → it is METABOLIC (kidneys control HCO₃⁻).
(Whichever value shifts in the direction that explains the pH is the primary driver.)

Step 2 — Which direction? (acidosis vs alkalosis)

• pCO₂ behaves like an ACID: pCO₂ ↑ high → acidosis; pCO₂ ↓ low → alkalosis. (moves OPPOSITE to pH)
• HCO₃⁻ behaves like a BASE: HCO₃⁻ ↓ low → acidosis; HCO₃⁻ ↑ high → alkalosis. (moves the SAME way as pH)

🔴 Acidosis (pH < 7.35)

Respiratory pCO₂ ↑ high
Metabolic HCO₃⁻ ↓ low

🟢 Alkalosis (pH > 7.45)

Respiratory pCO₂ ↓ low
Metabolic HCO₃⁻ ↑ high

💡 Memory Trick — "ROME"

Respiratory = Opposite → pCO₂ moves opposite to pH (↑CO₂ → ↓pH = acidosis).
Metabolic = Equal → HCO₃⁻ moves the same way as pH (↓HCO₃⁻ → ↓pH = acidosis).
In short: abnormal pCO₂ = respiratory, abnormal HCO₃⁻ = metabolic → then use ROME for the direction.

⚠️ Watch for compensation: if both pCO₂ and HCO₃⁻ are shifted in the same direction (both up or both down), one is the primary problem and the other is the body compensating. The primary one is the value that matches the pH side (acidosis vs alkalosis) per the rules above.

⚖️ No vs Partial vs Full Compensation

Once a disturbance starts, the body uses the other organ system to push the pH back toward normal — this is compensation. How far it has gone defines three states, judged by looking at whether the second parameter has changed and whether the pH has returned to normal:

Uncompensated
(no compensation) Only the primary parameter has changed. The second component is still normal — the body hasn't started compensating yet (acute disturbance). e.g. respiratory acidosis: ↑pCO₂, HCO₃⁻ still normal. pH still abnormal

Partially
compensated The second component has now shifted to oppose the primary disturbance, but not enough yet. e.g. respiratory acidosis: ↑pCO₂ AND ↑HCO₃⁻ (kidneys responding) — but pH hasn't made it back. pH still abnormal

Fully
compensated The second component has shifted enough to bring pH all the way back into the normal range. Both parameters are abnormal, but they balance out. e.g. respiratory acidosis: ↑pCO₂ AND ↑HCO₃⁻, pH = 7.36. pH 7.35–7.45 ✓

See it in the equation — pH = pKₐ + log [HCO₃⁻] / (0.03 × pCO₂)

Using respiratory acidosis (primary ↑pCO₂) as the example:
• Uncompensated: ↓pH = pKₐ + log([HCO₃⁻] / ↑pCO₂) — only the denominator rose, so the ratio (and pH) fell.
• Partially compensated: ↓pH = pKₐ + log(↑[HCO₃⁻] / ↑pCO₂) — kidneys raise HCO₃⁻ too, pulling the ratio back up, but pH is not yet normal.
• Fully compensated: pH = pKₐ + log(↑[HCO₃⁻] / ↑pCO₂) — HCO₃⁻ has risen enough that the ratio is restored and pH sits back in range.

How to tell them apart in one glance

1. Is the second parameter still normal? → Uncompensated.
2. Has the second parameter shifted but pH is still outside 7.35–7.45? → Partially compensated.
3. Has the second parameter shifted AND pH is back within 7.35–7.45? → Fully compensated.
Note: full compensation brings pH into range but rarely to exactly 7.40 — the pH usually stays on the side of the original disturbance (e.g. 7.36 in a compensated acidosis), which still tells you which problem came first.

Compensation vs Correction — keep them separate

Compensation changes the second parameter (the lungs adjust pCO₂ for a metabolic problem, or the kidneys adjust HCO₃⁻ for a respiratory problem) — it pushes pH toward normal but does NOT fix the underlying cause.
Correction fixes the primary parameter itself. Per the lesson, correction of a metabolic disturbance is a job for the kidneys (restoring HCO₃⁻), not the lungs.

🫘 Renal Handling, Compensation & Correction

Renal HCO₃⁻ Reabsorption & Ammoniagenesis

Proximal tubule: Na⁺/K⁺-ATPase generates Na⁺ gradient → Na⁺-H⁺ antiporter secretes H⁺ → H⁺ + HCO₃⁻ → H₂CO₃ → CO₂ (carbonic anhydrase at brush border) → CO₂ re-enters cell → H₂CO₃ → H⁺ + HCO₃⁻ → HCO₃⁻ exits to blood. Net: HCO₃⁻ is reabsorbed, H⁺ is recycled.

Ammoniagenesis: Glutamine → NH₃ (glutaminase) + glutamate → NH₃ + α-KG (GDH). NH₃ diffuses into tubule lumen → H⁺ + NH₃ → NH₄⁺ (trapped, excreted). Upregulated in acidosis (↑ glutaminase activity). Main mechanism to excrete excess acid.

Titratable acid (phosphate buffer): secreted H⁺ also binds filtered HPO₄²⁻ → H₂PO₄⁻ (excreted). For each H⁺ excreted this way, a new HCO₃⁻ is generated and returned to blood. So the kidney has three jobs: (1) reabsorb filtered HCO₃⁻, (2) excrete H⁺ as titratable acid, (3) excrete H⁺ as NH₄⁺ — together regenerating bicarbonate and removing the daily fixed-acid load.

Correction vs Compensation — don't confuse them

Compensation: Secondary response (e.g. lungs ↓pCO₂ in metabolic acidosis) — brings pH TOWARD normal but does NOT fix the primary problem.
Correction: Fixing the primary parameter (e.g. kidneys restore HCO₃⁻) — returns pH fully to normal.

⚗️ The Three-Day Vomiting Case — Working the Numbers 2026

The recollection gives pH 7.37 · pCO₂ 33 mmHg · HCO₃⁻ 17 mmol/L and records two different answers on two different lines. Rather than guess, work it the way the examiner intends.

Step by step

1 · Look at the pH. 7.37 sits inside the reference range (7.35–7.45) but on the acidic side of 7.40. A pH that is normal while the other two values are abnormal means the disturbance is compensated, and the side of 7.40 it sits on tells you the primary problem: here, acidosis.
2 · Which value explains an acidosis? HCO₃⁻ 17 is low (22–26) — that is a metabolic acidosis. pCO₂ 33 is also low (35–45), but a low pCO₂ would cause an alkalosis, so it cannot be primary: it is the respiratory compensation, moving in the same direction as the bicarbonate.
3 · Name it. A primary metabolic acidosis with appropriate respiratory compensation and a pH brought back inside the range = compensated metabolic acidosis. If you regard 7.37 as fully normalised, "fully compensated"; if you weigh that it has not returned to 7.40, "partially compensated" — which is why both appear in the recollection. The disorder itself is not in doubt: metabolic acidosis.

But doesn't vomiting cause alkalosis?

Yes — loss of gastric HCl classically causes metabolic alkalosis, and that is what a short vomiting history gives. After three days, however, the picture is dominated by what has been happening alongside: starvation ketosis, dehydration with lactic acidosis from poor perfusion, and loss of bicarbonate-rich intestinal fluid. The examiner has chosen numbers that make the point that you must read the values, not the story. If you are told vomiting and given a high HCO₃⁻ with a high pH, it is metabolic alkalosis; here you are given the opposite.

The four patterns

Metabolic acidosis: ↓pH, ↓HCO₃⁻ primary, ↓pCO₂ compensating. Metabolic alkalosis: ↑pH, ↑HCO₃⁻, ↑pCO₂. Respiratory acidosis: ↓pH, ↑pCO₂ primary, ↑HCO₃⁻ compensating. Respiratory alkalosis: ↑pH, ↓pCO₂, ↓HCO₃⁻ — and it results from hyperventilation and a decrease in bicarbonate, not an increase.

Insulin insufficiency

Single-choice, and the answer is metabolic acidosis: no insulin → unrestrained lipolysis → ketogenesis → acetoacetate and β-hydroxybutyrate → high-anion-gap metabolic acidosis with Kussmaul breathing as compensation. Anion gap = Na⁺ − (Cl⁻ + HCO₃⁻), normally 8–16 mmol/L.

Compensation rules

Compensation always moves the other variable in the same direction as the primary one, and it never overshoots past 7.40. Respiratory compensation is minutes; renal compensation is days. If pH is frankly abnormal, compensation is partial; if it is back inside the range, full.

The pH arithmetic 2026

pH 7 → [H⁺] = 10⁻⁷ mol/L = 100 nmol/L. The unit conversion is the whole question: mol → mmol (×10³) → µmol (×10⁶) → nmol (×10⁹). At pH 7.4, [H⁺] ≈ 40 nmol/L.

🫘 Bicarbonate Regeneration in the Kidney 2026

The 2026 stem: "What increases bicarbonate regeneration in the kidneys?" with options ↑pCO₂ · hyperkalaemia · ↓plasma HCO₃⁻.

✅ Increased plasma pCO₂

More CO₂ inside the tubular cell means more carbonic anhydrase substrate, so more H⁺ is generated for secretion and more HCO₃⁻ is returned to blood. This is the renal compensation for respiratory acidosis.

✅ Decreased plasma HCO₃⁻

A low bicarbonate is the stimulus to regenerate it: the metabolic acidosis drives H⁺ secretion, titratable acidity and ammoniagenesis, and every H⁺ excreted returns one new HCO₃⁻ to the blood.

❌ Hyperkalaemia

The wrong option. It is hypokalaemia that increases H⁺ secretion and bicarbonate regeneration — K⁺ leaves cells and H⁺ enters, so the tubular cell becomes acidic and secretes more. Hyperkalaemia does the opposite and promotes acidosis.

How it is actually done

Proximal tubule — reabsorbs about 90% of filtered bicarbonate, indirectly, via secreted H⁺ and carbonic anhydrase; no new bicarbonate is made here. Distal nephron — makes new bicarbonate by two routes: titratable acidity (H⁺ onto filtered phosphate) and ammoniagenesis (glutamine → glutaminase and glutamate dehydrogenase → 2 NH₃, trapped as NH₄⁺). Ammoniagenesis is the adaptable one and is strongly induced by chronic acidosis.

Buffers

Bicarbonate is the most important extracellular buffer despite a pK of 6.1, because it is an open system: the lungs remove CO₂ and the kidneys regenerate HCO₃⁻, so neither side is exhausted, and both are under independent control. Phosphate (pK 6.8) is the main intracellular and urinary buffer. Haemoglobin buffers through histidine imidazole groups and is the main buffer of whole blood after bicarbonate; deoxyhaemoglobin is the better proton acceptor. Cellular buffering shifts H⁺ into cells in exchange for K⁺ — which is why acidosis causes hyperkalaemia — and chronic acidosis draws on bone.

Hormones & Blood Sugar Regulation

Frequently tested: insulin (GLUT-4, stimulates uptake into muscle/adipose), glucagon targets, untreated diabetes (glucosuria, ketonuria, acidosis, hyperkalemia), hypoglycemia causes

🔑 Insulin Secretion Mechanism & Oral Hypoglycaemics

How glucose triggers insulin secretion:
1. Glucose enters β-cell via GLUT-2 (high Km — proportional sensing)
2. Glucokinase phosphorylates glucose → G-6-P → ATP generation
3. ↑ATP closes ATP-sensitive K⁺ channels → membrane depolarisation
4. Voltage-gated Ca²⁺ channels open → Ca²⁺ influx
5. Ca²⁺ triggers insulin vesicle exocytosis

Sulfonylureas (e.g. glibenclamide) treat Type 2 DM by closing ATP-sensitive K⁺ channels directly → stimulate insulin secretion regardless of blood glucose.

Incretins (GLP-1 & GIP):
Released from small intestine after food → sensitise β-cells to glucose → enhance insulin secretion.
Explain the "incretin effect": oral glucose → much greater insulin response than IV glucose (same amount).

Incretin-based drugs (2025 tips)

GLP-1 receptor agonists (liraglutide) and DPP-4 inhibitors used in Type 2 DM. Liraglutide also used for obesity (↓ appetite).

🏃 Counterregulatory Hormones & Hypoglycaemia

When blood glucose falls <50 mg/dL, the body activates counterregulatory hormones that oppose insulin:

Hormone	Source	Main anti-hypoglycaemic action
Glucagon	Pancreatic α-cells	↑ hepatic glycogenolysis, ↑ gluconeogenesis — most important acute response
Epinephrine	Adrenal medulla	↑ glycogenolysis, ↑ lipolysis, inhibits insulin secretion — critical when glucagon deficient
Cortisol	Adrenal cortex	↑ gluconeogenesis (long-term), ↑ muscle proteolysis → amino acids for GNG
Growth hormone	Anterior pituitary	↓ glucose uptake, ↑ lipolysis (long-term)

Type 1 DM — Hypoglycaemia Unawareness

In long-standing T1DM, both glucagon secretion AND epinephrine response are impaired → patient cannot detect or correct hypoglycaemia → risk of coma. Combined glucagon + epinephrine deficiency = "hypoglycaemia unawareness."

HbA1c — What It Measures

Haemoglobin non-enzymatically glycated by glucose. Rate of formation is proportional to average blood glucose over the previous 3 months. Normal <6%. Target in treated DM <7%. Used to monitor long-term glycaemic control.

💉 Insulin — Mechanism and Effects

Insulin is secreted by beta-cells of the pancreatic islets of Langerhans in response to high blood glucose. Its receptor has a unique structure: 2 alpha-subunits (extracellular, bind insulin) + 2 beta-subunits (transmembrane, contain intrinsic tyrosine kinase activity). Unlike most hormone receptors, the insulin receptor phosphorylates tyrosine residues — it is its own enzyme.

Insulin promotes: glycolysis, glycogen synthesis, lipogenesis, protein synthesis, GLUT-4 translocation to cell surface (in muscle and adipose). Insulin inhibits: glycogenolysis, gluconeogenesis, lipolysis, ketogenesis.

Insulin and Leptin — Anorexigenic Hormones

Anorexigenic hormones suppress appetite. Both insulin and leptin are anorexigenic — they signal to the hypothalamus that the body has enough energy, suppressing food intake. In contrast, ghrelin is an orexigenic hormone (stimulates hunger). CCK (Cholecystokinin) is also anorexigenic — it signals satiety after a meal. This is a directly tested exam point.

🩺 Diabetes — Acute and Chronic Complications

Acute Complications

Diabetic Ketoacidosis (DKA): mainly Type 1. Insulin absence → lipolysis → ketones → metabolic acidosis. Kussmaul breathing, fruity breath, glucosuria, ketonuria, hyperkalemia.

Hypoglycemia: excess insulin or oral hypoglycemic agents → blood glucose too low → brain symptoms (confusion, seizure, coma).

Chronic Complications

Caused by prolonged hyperglycemia damaging blood vessels and nerves:
Nephropathy (kidney damage — glomerulosclerosis)
Neuropathy (nerve damage — peripheral and autonomic)
Retinopathy (retinal blood vessel damage → blindness)
Atherosclerosis (macrovascular — heart disease, stroke)

Oral Hypoglycemics in Type 2 DM

"Oral hypoglycaemics are NOT used in the treatment of Type 2 DM" → FALSE. Oral agents like metformin, sulfonylureas, and DPP-4 inhibitors ARE used in Type 2 DM. They are NOT appropriate for Type 1 (which requires insulin). This distinction was directly tested.

Insulin Deficiency → Metabolic Acidosis ★ asked 2018, 2019

Insulin deficiency causes metabolic acidosis through two routes: (1) DKA — ketone bodies are weak acids. (2) Lactic acidosis — poor tissue perfusion impairs aerobic metabolism. Both result in decreased blood pH and decreased bicarbonate.

🔱 Glucagon

From α-cells. Acts via cAMP→PKA. Promotes: glycogenolysis, gluconeogenesis, lipolysis, ketogenesis. Inhibits: glycolysis (inactivates Pyruvate Kinase).

🩺 Diabetes

Untreated T1DM features

Glucosuria (renal threshold ~10 mmol/L), ketonuria, Kussmaul breathing, metabolic acidosis, hyperkalemia (acidosis shifts K⁺ out of cells), osmotic diuresis.

⚖️ Type 1 vs Type 2 Diabetes — Key Differences

🔵 Type 1 (T1DM)

Cause: autoimmune destruction of pancreatic β-cells
Insulin: absolute deficiency (little/none produced)
Blood insulin: very low
Onset: usually childhood / young, sudden
Body weight: typically lean / weight loss
Ketosis: common & prone to DKA
Treatment: insulin (lifelong, essential)
Insulin resistance: no

🟠 Type 2 (T2DM)

Cause: insulin resistance + relative insulin deficiency
Insulin: present but ineffective (resistance)
Blood insulin: high (early) → falls later — ★ asked 2024/25
Onset: usually adult, gradual
Body weight: typically overweight / obese
Ketosis: rare (enough insulin to suppress lipolysis)
Treatment: diet, exercise, oral drugs (metformin), ± insulin later
Insulin resistance: yes (key feature)

Shared features & exam traps

Both cause hyperglycaemia, glucosuria, polyuria, polydipsia. Ketosis is NOT common in both — it is typical of T1 (a frequent trap). T2 has higher blood insulin than T1 because the problem is resistance, not lack of production.

🔑 The Insulin Receptor — Every Option 2026

✅ Autophosphorylation of tyrosines

True. Insulin binds the α subunits outside the cell; the β subunits inside are a receptor tyrosine kinase that autophosphorylates on tyrosine residues. The receptor is composed of two types of subunit (α₂β₂), joined by disulfide bonds, with part of it outside the cell, and it is constantly synthesised and degraded — all statements marked true in older papers.

✅ Phosphorylation of IRS

True. The activated receptor phosphorylates insulin receptor substrate (IRS) proteins, which then recruit PI3-kinase and the rest of the cascade. Insulin's second messenger is therefore a kinase cascade, not cAMP.

❌ "Increased movement of GLUT-4 from membrane to vesicles"

False — the direction is reversed. Insulin moves GLUT-4 from intracellular vesicles TO the membrane. Read that option carefully; it is a pure direction trap.

What insulin then does

↑ glucose uptake in muscle and adipose (GLUT-4), ↑ glycogenesis, glycolysis, lipogenesis and protein synthesis, ↓ gluconeogenesis, glycogenolysis, lipolysis and ketogenesis. It works by dephosphorylation of the key enzymes and by induction of glucokinase, pyruvate kinase, PDH, ACC and G6PD. Glucagon and adrenaline act through adenylate cyclase → cAMP → PKA; insulin inhibits adenylate cyclase, so "insulin stimulates adenylate cyclase" is false.

🩺 Type 1 versus Type 2 2026

✅ Type 1 — what is expressed

The 2026 options were hypertriacylglycerolaemia, increased lipolysis and ketosis — all three are true. Absolute insulin deficiency removes every brake at once: hormone-sensitive lipase runs unopposed (increased lipolysis), the liver exports the surplus as VLDL while lipoprotein lipase is not induced (hypertriacylglycerolaemia), and the fatty acid flood becomes ketone bodies (ketosis, ketonaemia, ketonuria). Add hyperglycaemia, glucosuria above the renal threshold of about 10 mmol/L, osmotic diuresis, dehydration, hyperkalaemia and muscle wasting. Cause: autoimmune destruction of β cells.

✅ Type 2 — the cause

Insulin resistance, with relative deficiency and progressive β-cell failure. The 2026 stem explicitly asks whether it is caused by an immune defect — it is not. Nor is it viral. Autoimmune destruction and viral triggers belong to type 1.

Diabetes versus starvation

Both raise lipolysis and ketogenesis, but in starvation insulin is low and glucose is low, ketogenesis is controlled and the brain adapts; in diabetes insulin is absent while glucose is high, so there is no brake at all and ketoacidosis develops. Starvation ketosis rarely acidifies the blood; diabetic ketoacidosis does.

MODY, obesity and the young

Diabetes in the young that is not type 1 is most often a mutation in the glucokinase gene (MODY 2) — the answer in an older stem whose distractors were a GLUT-4 defect, excess adipose lipase and Leigh syndrome. In obesity, adipose tissue shows increased leptin secretion and decreased adiponectin secretion, and the raised TNF-α and IL-6 drive insulin resistance.

Diagnosis, and the OGTT

Fasting glucose ≥ 7.0 mmol/L (126 mg/dL), or 2-hour OGTT ≥ 11.1 mmol/L (200 mg/dL), or HbA1c ≥ 6.5%. A patient with a normal fasting glucose (e.g. 95 mg/dL) but a 2-hour value above 200 mg/dL is diabetic on the OGTT criterion — the point of the worked case in an older paper. Chronic damage comes from non-enzymatic glycation (AGEs, measured as HbA1c) and the polyol pathway, in which aldose reductase traps sorbitol in lens, nerve and retina.

🌙 Absorptive and Post-Absorptive States 2026

✅ In fasting the brain still uses glucose

True — a 2026 item, and it is true for the first days. The brain uses about 120 g of glucose a day and its uptake is insulin-independent. Only after several days of starvation does it shift most of its supply to ketone bodies, and even then it never uses fatty acids. Without any fuel the brain fails first: coma and death.

Absorptive (fed) state

Insulin high. Active: glycolysis, glycogenesis, the pentose phosphate pathway, lipogenesis, protein synthesis. Inhibited: gluconeogenesis (marked true in the archive), glycogenolysis, lipolysis, ketogenesis. Cholesterol synthesis is increased, not diminished. The liver's own fuel in the fed state is amino acids, not glucose.

Post-absorptive state

Glucagon high. The hepatic pathways are glycogenolysis, gluconeogenesis, ketogenesis, urea synthesis and bile acid synthesis; glycolysis and lipogenesis are off. Gluconeogenic substrates arrive as amino acids from muscle, lactate from muscle and erythrocytes, and glycerol from adipose tissue. In the liver itself the fuel is fatty acids. TCA activity is increased in the fasted, catabolic state.

Insulin-independent processes

Transport of glucose into hepatocytes is via GLUT-2 and is insulin-independent — the recorded answer. So is uptake by brain and red cells. Only GLUT-4 tissues (muscle, heart, adipose) depend on insulin for transport.

Ethanol 2026

Alcohol dehydrogenase has a low Km for ethanol — it is saturated at low blood concentrations, so ethanol is cleared by zero-order kinetics, a fixed amount per hour. Both ADH and aldehyde dehydrogenase use and therefore compete for NAD⁺, which is the answer to the second half of the 2026 item: the NADH/NAD⁺ ratio rises sharply. High NADH pushes pyruvate → lactate and oxaloacetate → malate, so gluconeogenesis is blocked (fasting hypoglycaemia, lactic acidosis), β-oxidation is inhibited and DHAP is pushed to glycerol-3-phosphate — hence fatty liver. Thiamine (B1) deficiency is the classic accompaniment in chronic alcoholism: Wernicke encephalopathy — confusion, ophthalmoplegia, ataxia — and the rule that thiamine must be given before glucose. ALDH is inhibited by disulfiram.

Steroid & Thyroid Hormones

Frequently tested: steroid synthesis & hydroxylase deficiencies, aldosterone, thyroid hormones, hypothyroidism, PTH vs calcitriol

🦋 Thyroid Hormone Synthesis

I⁻ concentrated → Thyroperoxidase (TPO) oxidizes I⁻→I₂ → iodination of Tg tyrosyl residues → MIT + DIT → coupling (TSH stimulates): MIT+DIT→T3, DIT+DIT→T4 → proteolysis of Tg → T3 + T4 released. T4 is pro-hormone; converted to active T3 in peripheral tissues by 5'-Deiodinase.

🦴 PTH & Calcium

PTH released when plasma Ca²⁺ is LOW. Effects: kidney ↑Ca²⁺ reabsorption, ↓phosphate reabsorption, stimulates 1α-Hydroxylase → calcitriol. Net result: ↑Ca²⁺, ↓phosphate in blood.

🧬 Steroidogenesis — From Cholesterol to All Steroid Hormones

Every steroid hormone is made from cholesterol. The first step — cholesterol → pregnenolone — is the rate-limiting step for all of them, and pregnenolone is the common branch point.

⚗️ Master Steroid Pathway

Cholesterol Desmolase (CYP11A1) · rate-limiting→ Pregnenolone → Aldosterone → Cortisol → Androgens (testosterone) Aromatase→ Estrogens (estradiol)

Class (site)	Main hormone	Key enzyme(s) & regulation
Mineralocorticoids — zona glomerulosa	Aldosterone	Aldosterone synthase; driven by angiotensin II + high K⁺ → ↑Na⁺/water retention, ↑K⁺/H⁺ loss
Glucocorticoids — zona fasciculata	Cortisol	Needs 21-hydroxylase + 17α-hydroxylase; driven by ACTH; ↑gluconeogenesis, anti-inflammatory
Androgens — zona reticularis / testis (Leydig)	Testosterone → DHT	LH drives Leydig cells; 5α-reductase → potent DHT (prostate, skin)
Estrogens — ovary (granulosa), placenta, adipose	Estradiol	Two-cell model: theca makes androgen (LH), granulosa aromatises it (FSH)

Steroids of the male gonad

Leydig cells make testosterone under LH control (via cAMP → ↑desmolase). Testosterone → DHT (by 5α-reductase, more potent, acts on prostate/genitalia) or → estradiol (by aromatase). Sertoli cells (FSH) support sperm and make inhibin + androgen-binding protein.

Congenital Adrenal Hyperplasia

A block in cortisol synthesis (most often 21-hydroxylase) → ↓cortisol → ↑ACTH → adrenal hyperplasia, and precursors are pushed into androgens. 21-OH deficiency = salt-wasting + virilisation (↑17-OH-progesterone). 17α-OH deficiency = hypertension without virilisation.

👆 Tap for full disease card

🧠 Steroid Hormone Signalling & Transport

Steroids are lipophilic — they cross the cell membrane and bind intracellular (nuclear) receptors, then act as transcription factors. Their effects are slow (hours) but long-lasting, unlike fast peptide-hormone signalling.

Transport in blood

Carried by plasma proteins: cortisol on CBG (transcortin), sex steroids on SHBG, plus albumin. Only free hormone is active.

Contrast: peptide/catecholamine

Water-soluble → bind surface receptors → second messengers (cAMP, IP₃) → fast response.

🦋 Hypothyroidism — Reading "Low TSH and Low fT4" 2026

The 2026 note records low TSH with low free T4, the symptoms, and treatment. The hormone pattern is the interesting part.

Primary vs secondary

Primary hypothyroidism (the thyroid itself has failed — Hashimoto, iodine deficiency): fT4 low, TSH HIGH, because feedback is lost. Secondary (central) hypothyroidism (the pituitary or hypothalamus has failed): fT4 low and TSH low or inappropriately normal — which is exactly the pattern in the recollection. So "low TSH + low fT4" points to a pituitary/hypothalamic cause, not to Hashimoto.

✅ Symptoms

Sleepiness and fatigue, bradycardia, cold intolerance, weight gain, constipation, dry skin, hair loss, slowed reflexes, raised cholesterol, myxoedema. In the newborn, untreated hypothyroidism causes cretinism.

✅ Treatment — untangling the wording

Treatment is levothyroxine, which is T4 (thyroxine); the body then converts it peripherally by 5′-deiodinase — a selenium enzyme — into the active T3. The recollection's phrasing is garbled: it is not tyrosine that is given. Tyrosine is the amino acid substrate of synthesis, on thyroglobulin.

Synthesis, in four steps

Trapping (Na⁺/I⁻ symporter) → oxidation of iodide and iodination of the tyrosyl residues of thyroglobulin by thyroid peroxidase, giving MIT and DIT → coupling: DIT + DIT → T4, MIT + DIT → T3 → proteolysis of thyroglobulin and release. Thyroglobulin is a prohormone; selenium deficiency reduces the conversion of T4 to T3. Most secretion is T4, and reverse T3 is the inactive product made in illness and starvation.

☀️ Vitamin D — Active Form and Where It Acts 2026

✅ The active form

1,25-dihydroxycholecalciferol (calcitriol). The route: 7-dehydrocholesterol + UVB in skin → cholecalciferol (D3) → 25-hydroxylation in the liver → 1α-hydroxylation in the kidney, the regulated step, stimulated by PTH, low calcium and low phosphate.

✅ Where it acts

Its principal action is on the intestine — increasing the absorption of both calcium and phosphate by inducing calbindin. It also increases renal reabsorption of both and permits bone mineralisation. Being a steroid-type hormone, it acts on nuclear receptors and changes transcription.

Deficiency and toxicity

Deficiency: rickets in children, osteomalacia in adults — vitamin D is essential for the prevention of osteomalacia; osteoporosis is a different, multifactorial disease. Toxicity is real and important: it is the most toxic vitamin in overdose, causing hypercalcaemia, hypercalciuria, nephrocalcinosis and metastatic calcification. Among the water-soluble vitamins, significant toxicity belongs to niacin and pyridoxine (B6).

PTH and calcium

PTH rises when plasma calcium falls and when phosphate is low it is calcitriol that rises. PTH increases bone resorption, increases renal calcium reabsorption, decreases renal phosphate reabsorption (phosphaturic) and activates 1α-hydroxylase. Net: calcium up, phosphate down. Bone mineral is hydroxyapatite; osteocalcin is a Gla protein, γ-carboxylated by a vitamin K-dependent enzyme.

Magnesium 2026

The second most abundant intracellular cation. It is the obligatory partner of ATP — the true kinase substrate is Mg-ATP — and a cofactor of over 300 enzymes; it stabilises DNA, RNA and ribosomes, and it antagonises calcium at the neuromuscular junction. Hypomagnesaemia (alcoholism, diarrhoea, diuretics) causes tremor, tetany, arrhythmias and a hypocalcaemia that will not correct until the magnesium is replaced, because magnesium is required for PTH secretion and action. Hypermagnesaemia (renal failure, magnesium sulfate therapy) causes the opposite: depressed reflexes, muscle weakness, hypotension, bradycardia and respiratory depression.

🪢 Prolyl Hydroxylase and Collagen 2026

The 2026 note lists several claims about prolyl hydroxylase. Two are true, two are traps.

❌ "Needs ascorbate as an oxidising agent"

False as worded. Vitamin C is needed — but as a reducing agent: it keeps the enzyme's Fe²⁺ in the reduced state. The enzyme also requires molecular oxygen and α-ketoglutarate. If an option calls ascorbate an oxidising agent, the wording is wrong even though the vitamin is right.

❌ "Copper is a coenzyme for this reaction"

False. Prolyl and lysyl hydroxylase use iron (Fe²⁺). The copper enzyme of collagen is lysyl oxidase, which acts later and outside the cell, forming the cross-links. Mixing the two up is the intended error — and it is also the difference between scurvy (vitamin C, hydroxylation) and Menkes disease (copper, cross-linking).

✅ "Acts intracellularly"

True. Hydroxylation happens in the rough endoplasmic reticulum, on the pro-α chains, before the triple helix forms. Glycosylation of hydroxylysine follows, also intracellularly. Only the later steps — cleavage of the propeptides by procollagen peptidase and cross-linking by lysyl oxidase — are extracellular.

✅ "Is needed for the linkage"

True in substance: the extra hydroxyl groups increase hydrogen bonding between the chains and so stabilise the triple helix, and hydroxylysine additionally provides the attachment point for sugars and for cross-links. Without hydroxylation the helix is unstable and is degraded — which is scurvy: bleeding gums, perifollicular haemorrhage, poor wound healing.

Collagen — the facts asked around it

Every third residue is glycine; X is usually proline, Y usually hydroxyproline. Type I bone, skin, tendon; type II cartilage, intervertebral disc, vitreous; type III reticular fibres and blood vessels; type IV basement membrane — a network, not fibrils. Genetic disorders of collagen biosynthesis cause osteogenesis imperfecta (type I, brittle bones, blue sclerae) and Ehlers–Danlos. Marfan is fibrillin-1, not collagen.

Vitamins & Cofactors

Frequently tested: vitamin → coenzyme pairings (B-vitamins), fat-soluble vitamin deficiencies (A, D, E, K), vitamin C in collagen/catecholamines, antioxidants

📋 Vitamins — Quick Reference Summary Table

Vitamin	Type	Active Form / Coenzyme	Deficiency Disease	Key Pathway
A (Retinol)	Fat-soluble	Retinal (vision), Retinoic acid (gene expression)	Night blindness, xerophthalmia	Vision cycle, epithelial integrity
D (Cholecalciferol)	Fat-soluble	Calcitriol (1,25-(OH)₂D₃)	Rickets (children), Osteomalacia (adults)	Ca²⁺/PO₄ absorption, bone mineralisation
E (Tocopherol)	Fat-soluble	α-Tocopherol	Haemolytic anaemia, neuropathy	Lipid antioxidant — protects PUFA in membranes
K (Phylloquinone)	Fat-soluble	Vitamin K hydroquinone	Impaired coagulation, bleeding	γ-Carboxylation of Glu in clotting factors II,VII,IX,X
B1 (Thiamine)	Water-soluble	TPP (Thiamine Pyrophosphate)	Beriberi, Wernicke-Korsakoff	PDC, α-KG DH, Transketolase (PPP), BCKAD
B2 (Riboflavin)	Water-soluble	FAD, FMN	Ariboflavinosis (cheilitis, glossitis)	Succinate DH (Complex II), β-oxidation, PDC
B3 (Niacin)	Water-soluble	NAD⁺, NADP⁺	Pellagra (4 Ds: Dermatitis, Diarrhoea, Dementia, Death)	~500 redox reactions; synthesized from Tryptophan (60:1)
B5 (Pantothenate)	Water-soluble	Coenzyme A (CoA)	Rare (burning feet)	Acetyl-CoA formation, Krebs cycle entry
B6 (Pyridoxine)	Water-soluble	PLP (Pyridoxal Phosphate)	Peripheral neuropathy, sideroblastic anaemia	ALL aminotransferases, ALA synthase (heme), glycogen phosphorylase
B7 (Biotin)	Water-soluble	Biotin (covalently bound)	Dermatitis, alopecia (avidin/raw egg white)	Pyruvate carboxylase, ACC, Propionyl-CoA carboxylase
B9 (Folate)	Water-soluble	THF (Tetrahydrofolate)	Megaloblastic anaemia, neural tube defects	One-carbon transfers: nucleotide synthesis, DNA methylation
B12 (Cobalamin)	Water-soluble	Methylcobalamin, Adenosylcobalamin	Megaloblastic anaemia + subacute combined degeneration (spinal cord)	Methionine synthesis; Methylmalonyl-CoA mutase (myelin)
C (Ascorbate)	Water-soluble	Ascorbic acid itself	Scurvy (bleeding gums, poor wound healing)	Prolyl/lysyl hydroxylase (collagen), iron absorption, antioxidant

Is Hepcidin an Enzyme? — Exam Trap (Q14b)

No — hepcidin is NOT an enzyme. It is a 25-amino acid peptide hormone (antimicrobial peptide) synthesised by the liver. It has no catalytic activity — it does not speed up any chemical reaction. Instead, it works by binding to ferroportin (an iron exporter on cell surfaces) and targeting it for degradation. This is a signalling/regulatory function, not an enzymatic one.

Classification: Hepcidin = peptide hormone / acute-phase reactant. Synthesised by hepatocytes. Regulated by iron stores, inflammation (IL-6), and erythropoietic activity.

Key Rule: Fat-Soluble (A, D, E, K) vs Water-Soluble

Fat-soluble vitamins: stored in adipose tissue → can accumulate → toxicity possible (especially A and D). Absorbed with dietary fat — malabsorption syndromes (coeliac, CF, cholestasis) → fat-soluble vitamin deficiency. Water-soluble vitamins: not stored in large amounts → excreted in urine → require regular dietary intake → deficiency develops faster.

⚗️ Water-Soluble B Vitamins — Full Table with Context

Vitamin	Active Coenzyme Form	Key Reactions It Participates In	Deficiency Disease
B1 (Thiamine)	TPP (Thiamine Pyrophosphate)	PDH (pyruvate → acetyl-CoA), α-KG dehydrogenase (Krebs), Transketolase (PPP), BCKAD (MSUD enzyme)	Beriberi (peripheral neuropathy, heart failure), Wernicke-Korsakoff (confusion, ataxia, ophthalmoplegia, amnesia)
B2 (Riboflavin)	FAD, FMN	Succinate dehydrogenase (Krebs/ETC Complex II), fatty acid oxidation, PDH	Cheilitis (cracked lips), glossitis, ariboflavinosis
B3 (Niacin)	NAD⁺, NADP⁺	~500 oxidation-reduction reactions; glycolysis, Krebs, fatty acid oxidation, PPP	Pellagra (Dermatitis, Diarrhea, Dementia, Death — the "4 Ds")
B5 (Pantothenate)	Coenzyme A (CoA)	Acetyl-CoA formation, fatty acid synthesis, Krebs cycle entry	Rare (burning feet syndrome)
B6 (Pyridoxine)	PLP (Pyridoxal Phosphate)	ALL aminotransferases (transamination), ALA Synthase (heme), glycogen phosphorylase, decarboxylases	Peripheral neuropathy, sideroblastic anemia, seborrheic dermatitis
B7 (Biotin)	Biotin (covalently bound)	Pyruvate Carboxylase (GNG), Acetyl-CoA Carboxylase (lipogenesis), Propionyl-CoA Carboxylase	Dermatitis, alopecia, neurological symptoms (from raw egg white consumption — avidin binds biotin)
B9 (Folate)	THF (Tetrahydrofolate)	One-carbon transfers: nucleotide synthesis, DNA methylation, amino acid interconversion	Megaloblastic anemia (large immature red blood cells), neural tube defects in pregnancy
B12 (Cobalamin)	Methylcobalamin, Adenosylcobalamin	Methionine synthesis (methylcobalamin), odd-chain fatty acid metabolism (adenosylcobalamin)	Megaloblastic anemia + Subacute combined degeneration of the spinal cord (dorsal column + corticospinal tract damage). Pernicious anemia = B12 deficiency due to lack of intrinsic factor.
C (Ascorbate)	Ascorbic acid itself	Cofactor for prolyl and lysyl hydroxylase (collagen), antioxidant, enhances iron absorption (reduces Fe³⁺ → Fe²⁺)	Scurvy (bleeding gums, perifollicular hemorrhages, poor wound healing)

Pernicious Anemia — B12 Deficiency

Pernicious anemia is a specific form of B12 deficiency caused by lack of intrinsic factor (a protein made by gastric parietal cells needed to absorb B12 in the ileum). Treatment: intramuscular B12 injections (bypasses the gut absorption problem).

Lab marker: methylmalonic acid ★ asked 2019

↑ Methylmalonic acid in urine/blood = vitamin B12 deficiency. Without adenosylcobalamin, methylmalonyl-CoA mutase fails → methylmalonyl-CoA builds up → methylmalonic acid. (Folate deficiency does NOT raise it — this distinguishes B12 from folate deficiency, since both cause megaloblastic anaemia.)

Pellagra and Tryptophan/Methionine

Niacin (B3) can be synthesized from tryptophan in the body (60 mg Trp → 1 mg niacin). Deficiency of tryptophan (e.g., Hartnup disease, corn-based diets) causes pellagra-like symptoms even with adequate dietary niacin. Methionine deficiency impairs one-carbon metabolism and can reduce the availability of certain niacin precursors. Both tryptophan and methionine deficiency together = niacin/pyridoxal phosphate deficiency risk.

⚠️ Niacin Overload & Thiamine Deficiency — A Paired Question

Niacin (B3) overload / pharmacological dose

At high (drug) doses niacin causes: flushing and warmth (prostaglandin/PGD₂-mediated — reduced by aspirin), itching, hepatotoxicity (especially slow-release forms), hyperglycaemia (insulin resistance), and hyperuricaemia / gout (competes with urate for excretion). Niacin is used to lower TAG/LDL and raise HDL — but these side effects limit it.

Thiamine (B1) deficiency

TPP is needed by pyruvate dehydrogenase, α-KG dehydrogenase, transketolase, and BCKAD. Deficiency → Beriberi (wet = high-output heart failure; dry = peripheral neuropathy) and Wernicke–Korsakoff (confusion, ataxia, ophthalmoplegia → amnesia + confabulation), classically in alcoholism. Give thiamine before glucose to avoid precipitating Wernicke.

Exam framing

The pairing tests two opposite ideas at once: a water-soluble vitamin causing harm from excess (niacin flush/hepatotoxicity) versus one causing a classic deficiency syndrome (thiamine → beriberi/Wernicke).

🧬 Purine Synthesis, Salvage and Gout 2026

De novo — which enzyme

Ribose-5-phosphate → PRPP by PRPP synthetase; then the committed, rate-limiting step is glutamine–PRPP amidotransferase. The purine ring is built directly onto the ribose — the opposite of pyrimidines, where the orotate ring is made first and attached afterwards. Nitrogen and carbon come from glutamine, aspartate, glycine, CO₂ and N10-formyl-THF. The first complete nucleotide is IMP; GTP is needed to make AMP and ATP to make GMP. Adenylosuccinate is the AMP-branch intermediate — not a urea-cycle compound.

✅ A salvage (reutilisation) defect causes gout

True. HGPRT recycles hypoxanthine → IMP and guanine → GMP. Without it, every purine is degraded to uric acid instead: massive hyperuricaemia and gout from childhood, plus intellectual disability and compulsive self-mutilation — Lesch–Nyhan syndrome, X-linked. Salvage matters because de novo synthesis is expensive and because brain, erythrocytes and marrow depend on it.

Which compounds are purines

Adenine, guanine, hypoxanthine, xanthine, uric acid — and caffeine. Uracil, cytosine and thymine are pyrimidines. Purine catabolism ends at uric acid via xanthine oxidase, a molybdenum enzyme; pyrimidine catabolism opens the ring completely to β-alanine, β-aminoisobutyrate, NH₃ and CO₂ — all water-soluble, which is why only purines cause clinical disease.

Compounds containing ribose

ATP, NAD⁺, NADP⁺, FAD, coenzyme A, RNA — plus SAM, cAMP and UDP-sugars. Allopurinol, a hypoxanthine analogue, inhibits xanthine oxidase; 5-fluorouracil blocks thymidylate synthase; methotrexate blocks dihydrofolate reductase. Ribonucleotide reductase makes deoxyribonucleotides using thioredoxin and NADPH.

🫀 The Hepatocyte — What Only the Liver Does 2026

A whole block of archive questions turns on the difference between "only in the liver", "mainly in the liver" and "also elsewhere".

Exclusively hepatic

Urea synthesis (the complete cycle, and arginase). Bilirubin conjugation. Ketogenesis (essentially — astrocytes manage a trace). Primary bile acid synthesis. Fibrinogen and most plasma protein synthesis. Glycerol phosphorylation (glycerol kinase; the kidney has a little).

Not exclusive — the traps

Gluconeogenesis — also the kidney. Lipogenesis — also adipose, kidney, lung, brain. Lipoprotein synthesis — also the intestine. Transamination — every tissue. Porphyrin synthesis — also erythroid marrow. Cholesterol synthesis — most tissues. Secondary bile acids — made by colonic bacteria; urobilinogen — made in the intestine; acetone — also released from other tissues.

Cytosol or mitochondria

In the cytosol: glycolysis, the pentose phosphate pathway, lipogenesis, glycogen metabolism. Ketogenesis is mitochondrial — the exception in that list. Glycogen phosphorylase needs PLP (vitamin B6), which is why B6 is involved in glycogenolysis.

Liver vs muscle glycogen

Concentration is higher in liver (up to ~10% of fresh weight) than in muscle (1–2%); total amount is greater in muscle, because there is so much more muscle. Liver glycogen serves blood glucose (it has glucose-6-phosphatase); muscle glycogen serves only the muscle.

Xenobiotics, P450 and phase II

Phase I — hydroxylation and other oxidations by cytochrome P450 monooxygenases, which are in the smooth endoplasmic reticulum (microsomes), not exclusively in mitochondria, use O₂ and NADPH, make the molecule more polar, and can produce carcinogenic intermediates. Phase II — conjugation: glucuronidation (UDP-glucuronic acid), sulfation (PAPS), acetylation, methylation, and conjugation with glutathione. Together they raise water solubility for excretion in urine or bile.

💊 Vitamins — The Items That Recur

Active forms

Folate → tetrahydrofolate. Biotin → carboxybiotin (carrying CO₂). Niacin → NAD⁺/NADP⁺ — not flavin, which is riboflavin's. Riboflavin → FAD/FMN. Thiamine → TPP. Pyridoxine → pyridoxal phosphate. B12 → methylcobalamin and adenosylcobalamin. Only the water-soluble vitamins act as coenzymes; the fat-soluble ones do not.

Deficiency pictures asked by symptom

Depression, hallucinations and dermatitis — the "3 Ds" of pellagra: niacin deficiency (or tryptophan deficiency, since niacin is made from it; hence pellagra in Hartnup disease, where tryptophan absorption fails). Peripheral neuritis — thiamine, not riboflavin. Megaloblastic anaemia — folate or B12; B12 alone adds neurological damage. Bleeding with a long prothrombin time — vitamin K, the cofactor for γ-carboxylation of glutamate; dicumarol and warfarin are its antagonists. Keshan's disease — a cardiomyopathy prevented by selenium. Isoniazid induces a B6 deficiency; methotrexate is an antifolate.

Fat-soluble

A, D, E and K — absorbed with dietary lipid and bile, so they fail in cholestasis and in cystic fibrosis. A — rhodopsin and vision, growth, epithelium; teratogenic in excess. E — tocopherol, the most important lipid-soluble antioxidant; it reacts with lipid peroxides, and at high concentration it can act as a pro-oxidant. D — see above. K — synthesised by intestinal bacteria, as is a share of biotin.

Which enzymes need which

Biotin — acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase. PLP — all transaminases, decarboxylases, ALA synthase, glycogen phosphorylase, cystathionine β-synthase. TPP — PDH, α-KGDH, branched-chain ketoacid dehydrogenase, transketolase. B12 — methionine synthase (with folate) and methylmalonyl-CoA mutase. Note also: methionine synthase uses B12, not B6 — a stated false option.

🧩 Named Disorders From the Archive

Glycogen storage

Hypoglycaemia, hepatomegaly and hepatic glycogen deposition = von Gierke (glucose-6-phosphatase). Defect in muscle phosphorylase = McArdle — exercise cramps, no lactate rise, no hypoglycaemia. Debranching enzyme = Cori. Branching enzyme = Andersen. Accumulation in lysosomes with muscle hypotonia and dystrophy = Pompe (lysosomal acid α-glucosidase).

Storage and transport

Hepatosplenomegaly with accumulation of polysaccharide/glycolipid — the lysosomal storage diseases, Gaucher being the commonest. Hartnup disease — defective absorption of tryptophan and other neutral amino acids → pellagra-like rash. Refsum disease — accumulation of phytanic acid (failure of α-oxidation). Leigh syndrome — a mitochondrial encephalopathy.

Neonatal hypoglycaemia

Its causes as listed: increased use of glucose by the fetus/neonate and functional immaturity of the gluconeogenic enzymes. A defect in gluconeogenesis is lethal precisely because glucose is the major fuel of the brain.

Two toxicology items

Arsenic (arsenite) binds the –SH groups of lipoic acid, poisoning pyruvate dehydrogenase and α-ketoglutarate dehydrogenase — death is mainly from brain failure. Arsenate separately uncouples the glyceraldehyde-3-phosphate dehydrogenase step, abolishing its ATP. Aconitase is inhibited by fluorocitrate; enolase by fluoride; cytochrome oxidase by cyanide, CO, azide and H₂S.

A note on the oral-cavity questions

The merged archive comes from a dentistry cohort and contains a block on the biochemistry of the oral cavity: saliva (α-amylase, proline-rich proteins, histatins, cystatins — which are protease inhibitors of salivary, not plasma, origin; chloride around 25–33 mmol/L; potassium higher than in plasma), enamel and dentin (mineralised connective tissue; dentin forms the body of the tooth, contains metabolically active cells, is about 70% inorganic by weight and contains more fluoride than enamel at the pulpal side), and osteocalcin as a Gla protein. These items are included for completeness; if you are sitting the medical paper they are unlikely to be examined, and nothing else in this mode depends on them.

Minerals & Trace Elements

Frequently tested: zinc transport & deficiency, copper, molybdenum, trace-element associated enzymes

⚗️ Mineral Categories

Category	Minerals
Major (macrominerals)	Ca, P, Mg, Na, K, Cl, S
Trace elements	Fe (iron), Zn (zinc), Cu (copper), Mn (manganese), I (iodine), Se (selenium), Cr (chromium), F (fluoride)
Ultratrace elements	Molybdenum (Mo), Co (cobalt), Ni (nickel), V (vanadium), Si (silicon)

Exam Q: "Which is an ultratrace mineral?" ★ asked 2023

Answer: Molybdenum (Mo). Zinc is a trace element. Manganese is a trace element. Magnesium is a major mineral. This exact question appeared in the 2023 exam. Ultratrace = needed in tiny amounts (<1 mg/day), distinct from trace elements which are needed in small but slightly larger amounts.

Acrodermatitis Enteropathica (Brandt's Disease)

Caused by a deficiency of zinc (Zn), specifically due to a mutation in the intestinal zinc transporter SLC39A4. Features: perioral and acral (hands/feet) dermatitis, diarrhea, alopecia (hair loss), immune dysfunction. Also known as Brandt's disease in some texts. Zinc is also required by ALA Dehydratase (heme synthesis) and Carboxypeptidase — lead toxicity displaces Zn²⁺ and inactivates these enzymes.

📋 Macroelements & Trace Elements — Full Reference Table

Element	Category	Key Functions	Deficiency	Excess / Notes
Ca (Calcium)	Macromineral	Bone/teeth mineralisation, muscle contraction, nerve signalling, clotting (cofactor), second messenger	Tetany, osteoporosis, rickets	Regulated by PTH + calcitriol + calcitonin
P (Phosphorus)	Macromineral	Bone (hydroxyapatite), ATP, nucleic acids, phospholipids, 2,3-BPG, signal transduction	Rickets (with Ca), hypophosphataemia → muscle weakness	Refeeding syndrome → acute hypophosphataemia
Mg (Magnesium)	Macromineral	Cofactor for >300 enzymes (ATP-Mg²⁺ complex, hexokinase, PFK-1, ACC, DNA polymerase), muscle relaxation	Arrhythmia, muscle cramps, tremor	Required for proper Ca²⁺ and K⁺ regulation
Na, K, Cl	Macromineral (electrolytes)	Osmolarity, membrane potential, Na⁺/K⁺-ATPase (Na⁺ out/K⁺ in), acid-base balance (Cl⁻ exchange)	Hyponatraemia (cerebral oedema), hypokalaemia (arrhythmia)	Na⁺ excess → hypertension; regulated by aldosterone
Fe (Iron)	Trace element	Haemoglobin/myoglobin O₂ transport, heme proteins (cytochromes, catalase, peroxidase), Fe-S clusters in ETC	Iron deficiency anaemia (microcytic, hypochromic)	Haemochromatosis (HFE mutation → ↑absorption); liver damage
Zn (Zinc)	Trace element	Cofactor for ADH, carbonic anhydrase, ALA dehydratase, carboxypeptidases, DNA polymerase, SOD. Zinc-finger transcription factors.	Acrodermatitis enteropathica (SLC39A4 mutation), poor wound healing, alopecia, impaired immunity, hypogonadism	Zn²⁺ displaced by Pb²⁺ in lead poisoning → enzyme inhibition
Cu (Copper)	Trace element	Cytochrome c oxidase (Complex IV), SOD, ceruloplasmin (Fe mobilisation), lysyl oxidase (collagen cross-linking), tyrosinase (melanin)	Menkes disease (ATP7A defect — X-linked): neurodegeneration, kinky hair, ↓ceruloplasmin	Wilson disease (ATP7B defect): Cu accumulates → liver + brain + Kayser-Fleischer rings
Se (Selenium)	Trace element	Selenocysteine in glutathione peroxidase (antioxidant) and 5'-deiodinase (T4→T3 conversion)	Keshan disease (cardiomyopathy); impaired T4→T3 conversion	Toxicity: hair/nail loss, garlic breath, neuropathy
I (Iodine)	Trace element	Essential component of thyroid hormones (T3, T4) — organified by TPO onto Tg tyrosyl residues	Goitre, hypothyroidism, cretinism (congenital)	Excess iodine → Wolff-Chaikoff effect (transient ↓thyroid function)
Mo (Molybdenum)	Ultratrace element	Cofactor for xanthine oxidase (purine catabolism → uric acid), aldehyde oxidase, sulphite oxidase	Rare; sulphite accumulation if sulphite oxidase impaired	2023 exam: "Which is an ultratrace mineral?" → Molybdenum
Mn (Manganese)	Trace element	Arginase cofactor (urea cycle), pyruvate carboxylase (with biotin), SOD (mitochondrial Mn-SOD), glycosyltransferases	Rare; impaired bone formation	Excess → parkinsonism ("manganism")
Cr (Chromium)	Trace element	Potentiates insulin action (part of Glucose Tolerance Factor)	Impaired glucose tolerance (rare)	Cr(VI) is carcinogenic (industrial, not dietary)
F (Fluoride)	Trace element	Hardens tooth enamel (fluorapatite replaces hydroxyapatite), strengthens bone	Dental caries	Excess → dental/skeletal fluorosis

Entry-Test Lessons

Memorization Apps

Final Exam Revision

Practice

By carbonyl group

By chain length

By number of units

Isomer vocabulary

Regulation — covalent plus allosteric

🔥 Uncouplers

🛑 Electron transport inhibitors

Vitamin D — a hormone, not a vitamin

Vitamin A — three active forms

Vitamin K — the carboxylation vitamin

Vitamin E — the membrane antioxidant

How elastin is built

Two ways the shield fails

The three thermodynamic quantities

The two kinases that can touch fructose

The three aldolase isoforms

The other two antioxidant enzymes

The three NOS isoforms

Where it is common

The three-step mechanism of the anaemia

What triggers a crisis

Three timescales of insulin action

The two glucose-regulating systems

The four types of hypoglycaemia

Further metabolism

How the hormone acts inside a cell

The full antibody list

By dietary need

By metabolic fate

PKU in more detail — the parts that get asked

Why the symptoms happen

Neonatal screening — the timing trap

Maternal PKU

Treatment & genetics

MSUD in more detail

Microvascular

Macrovascular