Comprehensive Biochemistry Answers

QUESTION 1: Biochemical Basis

a) Hyperthyroidism causes imbalance in blood sugar levels

• Increased glycogenolysis - T3 upregulates phosphorylase activity, breaking down hepatic glycogen rapidly, raising blood glucose.
• Increased gluconeogenesis - T3 stimulates hepatic gluconeogenesis by increasing expression of PEPCK and glucose-6-phosphatase.
• Enhanced intestinal glucose absorption - T3 upregulates SGLT1 (sodium-glucose cotransporter) in the intestinal mucosa, increasing glucose uptake post-meal, causing transient hyperglycaemia.
• Increased insulin degradation - Elevated metabolic rate accelerates insulin catabolism, reducing effective insulin action.
• Counter-regulatory effect - T3 enhances sensitivity to catecholamines (upregulates beta-adrenergic receptors), further promoting glycogenolysis and lipolysis.
• Peripheral glucose utilization - At the same time, T3 increases GLUT4 expression in muscle, promoting glucose uptake.

b) RNA editing is involved in ApoB48 synthesis

• The gene for ApoB encodes a single mRNA (ApoB100 mRNA), 14,000 nucleotides long.
• In the intestinal enterocytes, an enzyme called APOBEC-1 (apolipoprotein B mRNA editing catalytic polypeptide 1), along with its cofactor A1CF (APOBEC-1 complementation factor), acts as a cytidine deaminase.
• APOBEC-1 deaminates cytosine at codon 2153 (CAA = Glutamine) → converts it to uracil (UAA = Stop codon).
• This introduces a premature stop codon, producing a truncated protein - ApoB48 (48% of the full-length ApoB100).
• ApoB48 lacks the LDL-receptor binding domain (present in the C-terminal half of ApoB100), so ApoB48-containing chylomicrons are cleared by a different receptor (ApoE-mediated).
• In the liver, APOBEC-1 is absent, so the full-length ApoB100 is produced, which is the structural protein of VLDL and LDL.

c) Some mushrooms cause diarrhoea

• Alpha-amanitin is a bicyclic octapeptide that inhibits RNA Polymerase II irreversibly.
• This blocks mRNA synthesis → protein synthesis stops → rapid cell death in intestinal epithelium → diarrhoea, followed by liver/kidney failure.

• Acts as a muscarinic acetylcholine receptor agonist (parasympathomimetic).
• Stimulates M3 receptors in the gut → increased intestinal smooth muscle contraction and secretion → diarrhoea, along with SLUDGE syndrome (Salivation, Lacrimation, Urination, Defecation, GI distress, Emesis).

• Ibotenic acid is a glutamate receptor agonist; muscimol acts on GABA-A receptors - CNS effects predominate but GI irritation also occurs.

• Contain ill-defined irritant compounds (e.g., illuden S in jack-o'-lantern mushrooms) that directly irritate intestinal mucosa, increasing secretion and motility.

• Bind to F-actin, stabilizing it and disrupting intestinal epithelial cell turnover.

d) Xeroderma pigmentosa patients succumb to multiple cancers

• UV radiation induces two types of DNA lesions: cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts - both are bulky helix-distorting lesions.
• NER normally: (1) recognizes the distortion (XPC-RAD23B or CSA/CSB in transcription-coupled NER), (2) unwinds the helix (TFIIH complex with XPB/XPD helicases), (3) excises a ~25-30 nucleotide oligomer (XPG and XPF-ERCC1 nucleases), (4) resynthesizes the gap, (5) ligates.
• In XP, mutations in any of the 8 XP genes (XPA-XPG + XPV) impair this process.
• XPV (variant XP) is a defect in DNA Polymerase eta (η), which normally performs error-free translesion synthesis past CPDs.
• Unrepaired lesions cause replication errors → C→T or CC→TT transition mutations (UV signature mutations).
• These mutations accumulate in tumour suppressor genes (p53, PTCH) and proto-oncogenes (RAS), leading to multiple skin cancers (basal cell carcinoma, squamous cell carcinoma, melanoma) at a rate 1000x higher than normal.
• Internal cancers also increase because NER repairs lesions from endogenous oxidative damage and chemical carcinogens in addition to UV.

e) Prolonged use of methotrexate causes drug resistance

1. Gene amplification - The most common mechanism. Cells amplify the DHFR gene (up to 100-fold), producing massively increased amounts of DHFR enzyme that overwhelms the drug. Seen as double minutes (extrachromosomal) or homogeneously staining regions (HSRs) on chromosomes.
2. Decreased drug transport - MTX enters cells via the reduced folate carrier (RFC/SLC19A1). Mutations or downregulation of RFC reduce intracellular drug accumulation.
3. Impaired polyglutamation - Inside cells, FPGS (folylpolyglutamate synthase) adds glutamate residues to MTX, trapping it intracellularly and enhancing its potency. Loss of FPGS activity reduces intracellular retention.
4. Increased efflux - Upregulation of MRP1/ABCC1 and MRP2/ABCC2 (ABC transporters) pumps MTX out of the cell.
5. Altered DHFR binding - Point mutations in the DHFR active site (especially at Leu22, Phe31, Phe34) reduce MTX binding affinity while retaining catalytic function.

QUESTION 2: Why is folate essential for rapidly dividing cells?

• Thymidylate synthesis: 5,10-methylene-THF donates a methylene group to dUMP → dTMP (catalysed by thymidylate synthase). This is the only de novo source of thymidine. In this reaction, DHF is regenerated (not THF), so DHFR must reduce it back to THF (this is where MTX acts).
• Purine synthesis: 10-formyl-THF donates formyl groups at two steps in the de novo purine synthesis pathway (C2 and C8 of the purine ring - catalysed by GART and AICAR transformylase).
• Rapidly dividing cells (e.g., bone marrow, gut epithelium, embryonic cells, cancer cells) have a high demand for both DNA synthesis (dTMP, purines for dATP, dGTP) and RNA synthesis.
• Without adequate folate:
  • dTMP levels fall → uracil is misincorporated into DNA → strand breaks and apoptosis.
  • Purine synthesis fails → cells cannot replicate DNA.
• This is why folate deficiency causes megaloblastic anaemia (bone marrow precursors can't divide properly - nucleus stays large while cytoplasm matures = megaloblast).
• This is also why folic acid supplementation is critical in early pregnancy - rapid neural tube cell division requires folate, and deficiency causes neural tube defects (spina bifida, anencephaly).

QUESTION 3: DNA replication is semi-conservative - Justify

1. Semi-conservative: each daughter DNA has one original strand + one new strand.
2. Conservative: original double helix stays intact; a completely new copy is made.
3. Dispersive: both strands of both daughters contain a mixture of old and new DNA.

• E. coli grown in ¹⁵N (heavy nitrogen) medium for many generations → all DNA was "heavy" (¹⁵N/¹⁵N).
• Transferred to ¹⁴N (light nitrogen) medium.
• After 1 generation: ALL DNA was intermediate density (hybrid ¹⁵N/¹⁴N).
  • Ruled out conservative (which would give 50% heavy + 50% light).
• After 2 generations: 50% intermediate + 50% light (¹⁴N/¹⁴N).
  • Ruled out dispersive (which would give a gradual shift with no pure light band until much later).
  • Consistent ONLY with semi-conservative.

• The two parental strands are antiparallel and complementary.
• During replication, helicase unwinds the double helix at the replication fork.
• Each parental strand serves as a template for a new complementary strand synthesized by DNA polymerase III (5'→3' direction).
• The new strand is built by base pairing (A-T, G-C) against the template.
• Result: two identical daughter duplexes, each retaining one parental strand (semi) and gaining one newly synthesized strand (conservative).

QUESTION 4: Significance of the Purine Salvage Pathway

• De novo synthesis of purines is a multi-step, energy-expensive process (requires 5 ATP per purine ring).
• The salvage pathway recycles free purine bases (hypoxanthine, guanine, adenine) and nucleosides released from nucleic acid degradation.

• HGPRT (Hypoxanthine-Guanine Phosphoribosyl Transferase): converts hypoxanthine → IMP and guanine → GMP using PRPP.
• APRT (Adenine Phosphoribosyl Transferase): converts adenine → AMP.
• Adenosine Kinase: phosphorylates adenosine → AMP.

1. Energy efficiency - Salvage requires only 1 PRPP (vs. 5 ATP in de novo), saving significant energy.
2. Maintains nucleotide pools - Ensures adequate ATP, GTP for energy metabolism, signalling, RNA synthesis.
3. Regulation of de novo synthesis - Salvaged purines (as IMP, AMP, GMP) inhibit de novo synthesis by feedback inhibition.
4. Rapidly dividing tissues - Brain, bone marrow, and lymphocytes rely heavily on salvage because they have limited de novo capacity.

• Lesch-Nyhan Syndrome - X-linked deficiency of HGPRT. Without salvage, hypoxanthine and guanine accumulate → xanthine oxidase converts them to uric acid → hyperuricaemia, gout, nephropathy. Neurologically: self-mutilation, spasticity, intellectual disability (mechanisms not fully understood but involve dopaminergic dysregulation due to altered purine metabolism in basal ganglia).
• Gout - Allopurinol (xanthine oxidase inhibitor) reduces uric acid. Allopurinol itself is salvaged by HGPRT to allopurinol ribonucleotide, which inhibits de novo synthesis - therapeutic benefit.
• Adenosine Deaminase (ADA) deficiency - deoxyadenosine accumulates → converted to dATP → inhibits ribonucleotide reductase → T-cell lymphopenia → SCID (Severe Combined Immunodeficiency).
• Cancer chemotherapy - 6-Mercaptopurine (6-MP) and 6-thioguanine are purine analogues that must be activated by HGPRT to their nucleotide forms to inhibit purine synthesis. Loss of HGPRT = resistance to these drugs.

QUESTION 5: Biochemical Reasons

a) Why liver cannot utilize ketone bodies

• Ketone bodies (acetoacetate, beta-hydroxybutyrate) are synthesized in the liver mitochondria from acetyl-CoA (during fasting/starvation) via HMG-CoA pathway.
• For utilization (oxidation), ketone bodies must be converted back to acetyl-CoA:
  • Beta-hydroxybutyrate → acetoacetate (by beta-hydroxybutyrate dehydrogenase)
  • Acetoacetate + Succinyl-CoA → Acetoacetyl-CoA + Succinate - this reaction is catalysed by Succinyl-CoA:3-ketoacid CoA Transferase (SCOT), also called Thiophorase.
  • Acetoacetyl-CoA → 2 Acetyl-CoA (by thiolase) → enters TCA cycle.
• The liver lacks SCOT (it is absent from hepatocytes). Therefore, the liver cannot activate acetoacetate back to acetoacetyl-CoA.
• This is teleologically important: the liver produces ketone bodies as fuel for other tissues (brain, muscle, heart, kidney). If the liver could use them, it would consume its own product and starve the other organs.
• Extra-hepatic tissues (brain, heart, skeletal muscle, kidney cortex) have abundant SCOT and readily oxidize ketone bodies.

b) Statins are prescribed in hypercholesterolaemia

• HMG-CoA Reductase catalyses the rate-limiting step of cholesterol synthesis: HMG-CoA → Mevalonate.
• Statins (lovastatin, atorvastatin, rosuvastatin) are structural analogues of HMG-CoA. They competitively inhibit HMG-CoA reductase with 1000x higher affinity than the natural substrate.
• Reduced hepatic cholesterol synthesis → decreases intracellular cholesterol in hepatocytes.
• Upregulation of LDL receptors (LDLR) on hepatocyte surface (SREBP-2 pathway is de-repressed) → increased uptake of LDL from blood → reduces plasma LDL-C by 30-50%.
• Also mildly reduces VLDL, raises HDL.
• Additional pleiotropic effects: anti-inflammatory (reduce CRP), improve endothelial function, stabilize atherosclerotic plaques.

c) Zinc is important for reproductive health

• Testosterone synthesis: Zinc is required for the activity of enzymes in the steroidogenesis pathway in Leydig cells (including 17β-hydroxysteroid dehydrogenase).
• Spermatogenesis: Zinc stabilizes the nuclear chromatin of spermatozoa (zinc-protamine interactions), protects sperm from oxidative damage (zinc-SOD), and maintains sperm motility.
• Sperm capacitation and acrosome reaction: Zinc release from sperm is required for the acrosome reaction.
• Zinc deficiency → hypogonadism, low testosterone, oligospermia.

• Required for follicular development and oocyte maturation - zinc is released ("zinc spark") at fertilisation and is involved in meiosis resumption.
• Implantation - uterine zinc is essential for blastocyst implantation.
• Fetal development - zinc-finger transcription factors regulate organogenesis.

• Zinc is a structural component of the androgen receptor (zinc-finger DNA-binding domain).
• Required for IGF-1 signalling, which is important for gonadal development.

d) Depigmented (grey/white) hair is seen in copper deficiency

• Hair colour is determined by melanin produced by melanocytes in the hair follicle bulb.
• Tyrosinase is the key enzyme in melanin synthesis: it catalyses the hydroxylation of tyrosine → DOPA, and DOPA → dopaquinone (the first two steps in melanogenesis).
• Tyrosinase is a copper-dependent enzyme - it contains two copper atoms (CuA and CuB) at its active site, essential for its oxidase activity.
• In copper deficiency: tyrosinase activity is severely reduced → melanin synthesis fails → hair lacks pigment → depigmentation/greying.
• Also, Lysyl oxidase (another copper enzyme) catalyses cross-linking of collagen and elastin; its deficiency causes structural hair abnormalities (kinky, "steely" hair - as seen in Menkes disease, an X-linked disorder of copper transport with absent ATP7A).
• Menkes disease is the classic clinical example: kinky/steely hair + progressive neurodegeneration + connective tissue defects.

QUESTION 6

a) Why creatinine clearance is a better marker of GFR than urea clearance?

b) Normal reference range of GFR and new biochemical marker

• Normal GFR: 90-120 mL/min/1.73 m² (by CKD-EPI or MDRD equations).
• CKD is defined as GFR < 60 mL/min/1.73 m² for >3 months.

• Cystatin C - a cysteine protease inhibitor produced at a constant rate by all nucleated cells, freely filtered by glomerulus, completely reabsorbed and catabolized by tubular cells (not secreted).
• Advantages over creatinine: not affected by muscle mass, age, sex, or diet; better detects early GFR decline (earlier than creatinine rise); more accurate in elderly and sarcopenic patients.
• CKD-EPI Cystatin C equation and CKD-EPI Creatinine-Cystatin C combined equation are now endorsed by KDIGO 2024 guidelines as superior to creatinine-only equations.
• Another emerging marker: NGAL (Neutrophil Gelatinase-Associated Lipocalin) - marker of acute tubular injury (not GFR per se, but early AKI detection).

QUESTION 7: Subluxation of lens in hyperhomocysteinaemia

• Homocysteine is a thiol-containing amino acid formed from methionine demethylation.
• Normally: homocysteine is remethylated back to methionine (requires B12 + folate) or transsulphurated to cystathionine (requires B6, catalysed by cystathionine beta-synthase - CBS).
• In homocystinuria (CBS deficiency): homocysteine accumulates.
• Lens dislocation (subluxation) occurs because:
  • The zonular fibres (suspensory ligaments of the lens) are made of fibrillin (a glycoprotein rich in disulfide bonds).
  • Lysyl oxidase catalyses cross-linking of these fibres by oxidizing lysine residues to allysine, which then form cross-links.
  • Excess homocysteine (being a thiol) reduces disulfide bonds in fibrillin → disrupts cross-linking → weakens zonular fibres.
  • Also: homocysteine interferes with lysyl oxidase activity directly.
  • Weakened zonules cannot maintain the lens in position → downward and inward subluxation (cf. upward and outward in Marfan syndrome where fibrillin-1 is structurally abnormal).
• Additionally: homocysteine promotes oxidative stress and endothelial damage, contributing to thromboembolism and premature atherosclerosis seen in these patients.

QUESTION 8: Methotrexate is given for Lymphoblastic Leukaemia treatment

• ALL (Acute Lymphoblastic Leukaemia) cells are rapidly dividing lymphoblasts with very high demand for nucleotide synthesis (for DNA replication).
• MTX, as a DHFR inhibitor, blocks reduction of DHF → THF.
• Without THF:
  • No 5,10-methylene-THF → thymidylate synthase cannot synthesize dTMP → DNA replication stops (thymineless death).
  • No 10-formyl-THF → de novo purine synthesis blocked at two steps → purine nucleotide pools depleted → DNA and RNA synthesis halted.
• Why ALL specifically?
  • Lymphoblasts have high DHFR expression and rapid proliferation.
  • They also have high expression of RFC (reduced folate carrier) - good drug uptake.
  • They have high FPGS activity → efficient polyglutamation of MTX → prolonged intracellular drug retention.
  • Leukaemia cells lack the normal mechanisms to reduce folate dependency.
• MTX is used in ALL as part of induction, consolidation, and CNS prophylaxis (intrathecal MTX crosses the blood-CSF barrier poorly with systemic dosing, so direct intrathecal administration is used).
• Leucovorin (folinic acid) rescue is given after high-dose MTX to rescue normal cells - it bypasses DHFR and provides reduced folate directly.

QUESTION 9: Human beings and higher primates cannot synthesize Ascorbic Acid

• Most mammals synthesize ascorbic acid (Vitamin C) from glucose via the glucuronate pathway:
  • Glucose → UDP-glucose → UDP-glucuronate → L-gulonate → L-gulonolactone → 2-oxogulonolactone → L-ascorbic acid (last step by L-gulonolactone oxidase (GULO)).
• Humans, apes, monkeys, guinea pigs, and some fruit bats carry a non-functional pseudogene for GULO (GULOP) on chromosome 8.
• During evolution (~40-61 million years ago), a series of mutations inactivated the GULO gene in a common primate ancestor.
• This was not lethal because the ancestral diet was rich in fresh fruits and vegetables containing ascorbic acid.
• The GULOP pseudogene in humans retains significant homology to the functional GULO gene in other mammals but contains multiple exon deletions and point mutations rendering it non-functional.
• Consequence: humans must obtain all Vitamin C from diet. Deficiency causes scurvy because:
  • Vitamin C is required as a cofactor for prolyl hydroxylase and lysyl hydroxylase (both Cu-containing enzymes that require ascorbic acid to maintain Fe²⁺ in the active site).
  • These enzymes hydroxylate proline and lysine residues in procollagen → hydroxylation is necessary for cross-linking of collagen fibres.
  • Without Vitamin C → defective collagen → weakened blood vessels, poor wound healing, perifollicular haemorrhages, bleeding gums.

QUESTION 10: Glycine residues in proteins are important in metabolic pathways

• Glycine is the only amino acid that fits in the tight interior of a collagen triple helix. Every third residue in collagen is glycine (Gly-X-Y repeat). The H side chain allows the tight winding. Substitution of any Gly → mutations cause Osteogenesis Imperfecta.

1. Purine synthesis - Entire glycine molecule (C2, N1 of the purine ring) is incorporated in step 2 of de novo purine synthesis.
2. Haem synthesis - Glycine + Succinyl-CoA → delta-ALA (by ALA synthase, rate-limiting step), the first committed step in porphyrin synthesis.
3. Creatine synthesis - Glycine + Arginine → guanidinoacetate → creatine (by creatine synthase with methyl group from SAM).
4. Glutathione synthesis - Gly is the third residue of the tripeptide Glu-Cys-Gly (glutathione), essential for antioxidant defence.
5. Hippurate formation - Glycine conjugates with benzoate (a xenobiotic) in the liver to form hippuric acid (urine excretion of benzoate).
6. Bile acid conjugation - Primary bile acids (cholate, chenodeoxycholate) are conjugated with glycine or taurine in the liver → glycocholate, glycochenodeoxycholate → more soluble bile salts.
7. Serine and one-carbon metabolism - Glycine ↔ Serine (by serine hydroxymethyltransferase, SHMT), interconverting with THF/methylene-THF, linking glycine to one-carbon metabolism.
8. Inhibitory neurotransmitter - Glycine is the main inhibitory neurotransmitter in the spinal cord and brainstem (glycine receptor is a Cl⁻ channel). Strychnine poisons by blocking glycine receptors.

QUESTION 11: Biochemical Reasons

a) p53 mutation is dangerous in cancer

• Normal p53 functions:
  1. Activated by DNA damage, oncogenic stress, hypoxia.
  2. Transcriptionally activates p21 (CDKN1A) → inhibits CDK4/6-Cyclin D → G1 arrest → time for DNA repair.
  3. Activates GADD45 (DNA repair genes).
  4. Activates BAX, PUMA, NOXA (pro-apoptotic) → apoptosis if damage is irreparable.
  5. Inhibits MDM2 (which normally ubiquitinates p53 for proteasomal degradation - feedback loop).
• Why p53 mutation is dangerous:
  • p53 mutations (most commonly missense in the DNA-binding domain - hotspots: R175H, G245S, R248W, R248Q, R249S, R273H, R282W) prevent DNA binding → loss of transactivation.
  • Loss of G1 checkpoint → cells with damaged DNA continue replicating → accumulate mutations.
  • Loss of apoptosis → damaged cells survive and proliferate.
  • Dominant negative effect - mutant p53 can form tetramers with wild-type p53 (from the other allele) and inactivate it → one mutant allele can knock out p53 function even before LOH.
  • Gain-of-function mutations - Some p53 mutants actively promote oncogenesis by binding and inhibiting other tumour suppressors (p63, p73) and activating oncogenic transcription programs.
  • p53 is mutated in >50% of all human cancers (the most commonly mutated gene in cancer).
  • Germline p53 mutations → Li-Fraumeni Syndrome - multiple cancers at young age.

b) Cancer cells have increased and persistent activity of telomerase

• Telomeres are repetitive TTAGGG sequences at chromosome ends, protecting them from exonucleolytic degradation and end-to-end fusions.
• With each cell division, telomeres shorten by ~50-200 bp (because DNA polymerase cannot replicate the 3' end of a linear chromosome - the end-replication problem).
• After ~50-70 divisions (Hayflick limit), telomeres reach a critical length → DNA damage response is triggered → replicative senescence (p53/p21 pathway) or apoptosis.
• Telomerase (hTERT - reverse transcriptase + hTR - RNA template) elongates telomeres using RNA template (3'-AAUCCC-5') - but is silenced in most somatic cells.
• In cancer cells:
  • Cells that have overcome senescence (often through p53 and Rb mutation) eventually reach crisis (massive telomere shortening → chromosomal fusions → anaphase bridges → breakage-fusion-bridge cycles → genomic instability).
  • To achieve immortality (hallmark of cancer), cells must reactivate telomerase - occurs in ~90% of cancers (mostly by hTERT promoter mutations C228T and C250T, which create new ETS transcription factor binding sites).
  • Persistent telomerase maintains telomere length → cells bypass senescence and crisis → unlimited proliferative potential.
  • This is why telomerase is an attractive cancer therapeutic target (e.g., imetelstat).
  • ~10% of cancers use the ALT (Alternative Lengthening of Telomeres) mechanism (homologous recombination-based) instead.

c) Overdose of acetaminophen/paracetamol causes liver toxicity

• Paracetamol is glucuronidated (~55%) and sulphated (~30%) in the liver → non-toxic conjugates excreted in urine.
• A small fraction (~5-10%) is oxidised by CYP2E1 (and CYP3A4) via phase I reactions → generates NAPQI (N-acetyl-p-benzoquinone imine), a highly reactive electrophile.
• NAPQI is normally rapidly detoxified by conjugation with glutathione (GSH) → non-toxic cysteine/mercapturic acid conjugates.

• Glucuronidation and sulphation pathways are saturated.
• Proportionally more is shunted through CYP2E1 → massive NAPQI generation.
• Hepatic GSH is depleted (liver GSH stores are limited ~5-10 mM).
• NAPQI, no longer neutralised, covalently binds to protein sulfhydryl groups in hepatocytes (arylation) → mitochondrial dysfunction, oxidative stress, lipid peroxidation.
• Mitochondrial damage: NAPQI binds mitochondrial proteins → inhibits electron transport → ATP depletion → necrosis (Zone 3/centrilobular - where CYP2E1 is most concentrated).
• Triggers MPTP (mitochondrial permeability transition pore) opening → cytochrome c release → apoptosis + necrosis.

d) Glucuronic acid has an important role in the metabolism of xenobiotics

• UDP-glucuronic acid (UDPGA) is the activated form, synthesized from UDP-glucose by UDP-glucose dehydrogenase.
• UDP-glucuronosyltransferases (UGTs) catalyse the transfer of glucuronic acid from UDPGA to a nucleophilic substrate (Phase II conjugation reaction - occurs mainly in hepatic ER).
• Glucuronidation increases water solubility (glucuronic acid is highly polar, carboxylic acid group ionised at physiological pH) → facilitates renal or biliary excretion.
• Substrates include:
  • Drugs: paracetamol, morphine, lorazepam, bilirubin, NSAIDs.
  • Endogenous compounds: bilirubin (glucuronidation is essential for bile excretion - failure causes jaundice as in Crigler-Najjar syndrome and Gilbert's syndrome where UGT1A1 is deficient/reduced).
  • Steroid hormones, thyroid hormones (for excretion and enterohepatic cycling regulation).
  • Environmental toxins, carcinogens.
• Special example: Morphine-6-glucuronide is actually more potent than morphine itself (active metabolite) - an exception where glucuronidation doesn't inactivate.
• Also important in GAG synthesis (glucuronic acid is a component of hyaluronate, heparan sulphate, dermatan sulphate, chondroitin sulphate) and ascorbic acid synthesis (in animals that can make it).

e) Taq Polymerase is preferred in PCR over other DNA polymerases

• Each PCR cycle involves denaturation at ~95°C to separate the double-stranded DNA template.
• Ordinary mesophilic DNA polymerases (like E. coli DNA Pol I, T4 DNA polymerase) are irreversibly denatured at these temperatures (proteins unfold, active site is destroyed).
• If regular polymerase were used, new enzyme would have to be added after each cycle - impractical and extremely expensive (PCR runs 30-40 cycles).

• Derived from Thermus aquaticus, a thermophilic bacterium found in Yellowstone hot springs.
• Optimally active at 72-75°C (extension temperature) and stable at 95°C for extended periods.
• Its thermostability is due to: higher content of ionic interactions and hydrophobic core packing; reduced surface exposure of hydrophobic residues; rigid protein structure (less conformational flexibility at high temperature).
• Has 5'→3' polymerase and 5'→3' exonuclease activity but lacks 3'→5' proofreading exonuclease → error rate ~1/1000 bases (higher than Pfu polymerase, which has proofreading).
• This allows PCR to be performed using a thermal cycler without adding fresh enzyme each cycle - the key innovation that made PCR practical (Kary Mullis, 1983, Nobel Prize 1993).
• When proofreading is needed (e.g., cloning), Pfu polymerase (from Pyrococcus furiosus) is preferred - it has 3'→5' exonuclease and lower error rate.

QUESTION 12

a) Hyperkalaemia is seen in metabolic acidosis

1. H⁺/K⁺ exchange across cell membranes: In acidosis, excess H⁺ enters cells (buffering intracellular proteins). To maintain electrochemical neutrality, K⁺ exits cells into the extracellular fluid → hyperkalaemia. Approximately for every 0.1 unit fall in pH, serum K⁺ rises by ~0.5-0.6 mEq/L.
2. Insulin resistance in acidosis: Insulin normally drives K⁺ into cells (via Na⁺-K⁺-ATPase). Acidosis impairs insulin signalling → less cellular K⁺ uptake.
3. Reduced Na⁺-K⁺-ATPase activity: Acidosis directly inhibits the Na⁺-K⁺-ATPase → less K⁺ pumped into cells.
4. In DKA specifically: Also volume depletion → reduced GFR → less renal K⁺ excretion. Despite total body K⁺ depletion, serum K⁺ is often normal or high at presentation.

b) High anion gap is observed in Diabetic Ketoacidosis (DKA)

• Absolute insulin deficiency → unrestrained lipolysis (HSL active) → massive release of FFA from adipose tissue.
• FFA flood the liver → hepatic fatty acid oxidation overwhelms TCA cycle capacity → acetyl-CoA accumulates → ketogenesis via HMG-CoA pathway:
  • Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA → Acetoacetate + Acetyl-CoA
  • Acetoacetate → Beta-hydroxybutyrate (by BHBD, using NADH) and Acetone
• Acetoacetate and beta-hydroxybutyrate are organic acids - they dissociate at physiological pH, donating H⁺ → metabolic acidosis.
• These ketoacid anions (acetoacetate⁻, beta-hydroxybutyrate⁻) are "unmeasured" anions - not included in the routine measurement of Cl⁻.
• HCO₃⁻ falls (consumed buffering H⁺), but Cl⁻ does NOT rise proportionally (ketoanions replace bicarbonate, not chloride) → the "gap" between Na⁺ and (Cl⁻ + HCO₃⁻) widens.
• Therefore: high anion gap metabolic acidosis in DKA.
• Contrast with hyperchloraemic (normal AG) acidosis where Cl⁻ rises as HCO₃⁻ falls (e.g., renal tubular acidosis, diarrhoea).

QUESTION 13: G6PD deficiency is protective against malaria

• G6PD (Glucose-6-phosphate Dehydrogenase) catalyses the first step of the Pentose Phosphate Pathway: Glucose-6-phosphate + NADP⁺ → 6-Phosphogluconate + NADPH.
• NADPH is essential for regenerating reduced glutathione (GSH) via glutathione reductase: GSSG + NADPH + H⁺ → 2GSH
• GSH protects red blood cells from oxidative damage (neutralizes H₂O₂ via glutathione peroxidase).

• Plasmodium falciparum parasites multiply inside RBCs and generate significant oxidative stress (H₂O₂, free radicals).
• Normally, these are neutralized by GSH (RBCs have no mitochondria, so PPP/G6PD is the ONLY source of NADPH for antioxidant defense).
• In G6PD-deficient RBCs (G6PD-A⁻, G6PD Mediterranean etc.):
  • Basal parasite survival is impaired (parasite itself needs some G6PD activity and the oxidative environment is hostile).
  • Plasmodium-infected G6PD-deficient RBCs are preferentially phagocytosed by macrophages in the spleen (they are more rigid and oxidatively stressed).
  • Immune clearance is enhanced - the host's immune system more readily recognizes and removes parasitized G6PD-deficient cells.
  • Additionally, the haemolysis of parasitized cells is faster, reducing parasite burden.
• This explains why G6PD deficiency alleles are maintained at high frequency (Hardy-Weinberg selection) in malaria-endemic regions (Africa, Mediterranean, Middle East, Southeast Asia) - balanced polymorphism, similar to sickle cell trait protecting against malaria.

QUESTION 14: Glycogenolysis in muscle does not contribute to maintaining blood glucose

• During exercise or fasting, glycogenolysis in muscle breaks down glycogen → Glucose-1-phosphate → Glucose-6-phosphate.
• Muscle lacks glucose-6-phosphatase (the enzyme that cleaves the phosphate from G6P to release free glucose into the blood).
• Therefore, G6P produced in muscle cannot be dephosphorylated → cannot exit the muscle cell as free glucose (the glucose transporter GLUT4 can only transport free glucose, not G6P).
• Instead, muscle G6P is committed to:
  • Glycolysis → pyruvate → lactate or acetyl-CoA (energy for the muscle itself).
  • The lactate produced can enter the blood and travel to the liver, where it is used as a gluconeogenic substrate (Cori cycle) - but this is the liver making glucose, not the muscle.
• Contrast with liver - hepatocytes have abundant glucose-6-phosphatase in the ER → can release free glucose into the blood → maintain blood glucose during fasting.
• This is also why, in Von Gierke's disease (G6Pase deficiency in the liver), severe hypoglycaemia occurs, but muscle glycogen is unaffected functionally.

QUESTION 15: Methylmalonic aciduria occurs in Vitamin B12 deficiency

• Odd-chain fatty acids and some amino acids (Ile, Val, Thr, Met) are catabolised to propionyl-CoA.
• Propionyl-CoA carboxylase (requires biotin) converts propionyl-CoA → D-methylmalonyl-CoA.
• Methylmalonyl-CoA racemase converts D to L-methylmalonyl-CoA.
• Methylmalonyl-CoA mutase converts L-methylmalonyl-CoA → succinyl-CoA (enters TCA cycle).
• Methylmalonyl-CoA mutase requires adenosylcobalamin (AdoCbl, a coenzyme form of Vitamin B12) for its activity.

• Adenosylcobalamin is depleted → methylmalonyl-CoA mutase activity is impaired.
• Methylmalonyl-CoA accumulates → hydrolysed to methylmalonic acid → excreted in urine → methylmalonic aciduria.
• Methylmalonate is toxic: it inhibits succinate dehydrogenase (Complex II), disrupts the TCA cycle and myelin formation.
• This explains the neurological manifestations of B12 deficiency (subacute combined degeneration of the spinal cord) - myelin synthesis requires succinyl-CoA (for ALA synthesis → haem in neurons) and is disrupted by methylmalonate accumulation.
• Diagnostic utility: Serum methylmalonic acid is a sensitive and specific marker of functional B12 deficiency (elevated even before macrocytic anaemia appears or serum B12 is low).
• Note: B12 deficiency ALSO causes elevated homocysteine (because methylcobalamin is needed for methionine synthase to convert homocysteine → methionine).

QUESTION 16: Viper snake bite results in massive haemolysis

• PLA2 hydrolyses the sn-2 ester bond of phospholipids (phosphatidylcholine → lysophosphatidylcholine + arachidonic acid).
• Acts on the RBC membrane phospholipid bilayer → destroys membrane integrity → direct lysis of RBCs → intravascular haemolysis.
• Also generates lysophosphatidylcholine, which is itself a powerful detergent-like lytic molecule (lyso-PC inserts into membranes and disrupts them).

• Directly damage RBC membranes and vascular endothelium.
• Activate or consume coagulation factors → Disseminated Intravascular Coagulation (DIC) → microangiopathic haemolytic anaemia (RBCs sheared by fibrin strands in microvasculature).

• Degrades hyaluronic acid in connective tissue, allowing venom to spread.

• Directly lyse RBC membranes.

• Thrombin-like enzymes (e.g., Ancrod in Agkistrodon) cleave fibrinogen → consumptive coagulopathy → haemolysis from DIC.

QUESTION 17: Big toe is the first and common site affected in gout

• Purines are catabolised to hypoxanthine → xanthine → uric acid (by xanthine oxidase).
• Uric acid (pKa 5.75) is mostly ionised as urate at physiological pH (pH 7.4) but is at the limit of its solubility (~6.8 mg/dL in plasma).
• In hyperuricaemia (>7.0 mg/dL in men, >6.0 in women), urate supersaturates and precipitates as monosodium urate (MSU) crystals.

1. Temperature - The first MTP joint is the most distal and coolest joint in the body (~29-32°C vs 37°C core temperature). Uric acid solubility decreases with temperature - at lower temperatures, urate is less soluble and precipitates more readily.
2. Hydrostatic pressure and trauma - The first MTP joint bears the greatest mechanical load during walking. Repetitive trauma and pressure promote crystal nucleation and deposition.
3. pH - Slightly lower local pH in cartilage and synovial fluid reduces solubility further.
4. Poor circulation - Extremities have slower blood flow, especially at night → local urate concentration increases.
5. Avascular cartilage - Articular cartilage is avascular; urate that enters is not efficiently washed out.
6. Nocturnal attacks - During sleep, dehydration concentrates urate; cooler temperature; lower cortisol (anti-inflammatory) → crystals form and trigger acute inflammation.

• MSU crystals are phagocytosed by macrophages and neutrophils → activate the NLRP3 inflammasome → caspase-1 activation → cleavage and release of IL-1β → acute inflammatory response (pain, redness, swelling, heat).