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NARAYANA MEDICAL COLLEGE — Biochemistry 2nd Internal Assessment

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ESSAY QUESTIONS (15 marks each)

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Q1. Vitamin D: Synthesis, Biochemical Functions, Daily Requirement, Sources, and Deficiency Manifestations

Structure

• Vitamin D₂ (Ergocalciferol) — from plants/fungi
• Vitamin D₃ (Cholecalciferol) — from animal sources and skin synthesis

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Synthesis of Vitamin D (Step-by-step)

7-Dehydrocholesterol (in skin)
        ↓  [UV light / sunlight, 290–315 nm]
Previtamin D₃  →  Vitamin D₃ (Cholecalciferol)
        ↓  [Liver: 25-hydroxylase / CYP27A1]
25-Hydroxycholecalciferol [25(OH)D₃] — major storage form
        ↓  [Kidney: 1α-hydroxylase / CYP27B1]  ← PTH stimulates; Ca²⁺ inhibits
1,25-Dihydroxycholecalciferol [1,25(OH)₂D₃]
       = CALCITRIOL — the active form

• PTH ↑ → activates renal 1α-hydroxylase → more calcitriol
• Low serum Ca²⁺/PO₄³⁻ → stimulates calcitriol production
• 24,25-dihydroxycholecalciferol is the inactive/storage metabolite (formed by 24-hydroxylase)

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Daily Requirement

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Dietary Sources

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Biochemical Functions of Vitamin D (Calcitriol)

1. Intestinal Ca²⁺ absorption:
   • Calcitriol binds to nuclear VDR (Vitamin D Receptor)
   • Induces synthesis of Calbindin-D (calcium-binding protein)
   • Increases transcellular absorption of Ca²⁺ and phosphate from gut
2. Bone mineralization:
   • Promotes osteoblast activity and calcium deposition
   • Maintains adequate serum Ca²⁺ × PO₄³⁻ product for hydroxyapatite formation
   • At high doses: promotes osteoclast activity → bone resorption
3. Renal reabsorption:
   • Enhances reabsorption of Ca²⁺ and phosphate in distal tubules
4. Parathyroid gland:
   • Suppresses PTH synthesis (negative feedback)
5. Immunomodulation:
   • Activates macrophages; anti-inflammatory; reduces risk of autoimmune diseases
6. Cell differentiation:
   • Promotes differentiation and inhibits proliferation (anticancer role)
7. Muscle function:
   • Required for normal muscle contraction; deficiency causes myopathy

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Deficiency Manifestations

In Children — RICKETS

• Defective mineralization of growing bones
• Craniotabes (softening of skull)
• Frontal bossing (prominent forehead)
• Rachitic rosary (beading at costochondral junctions)
• Harrison's sulcus (horizontal groove at diaphragm)
• Bow legs (genu varum) or knock knees (genu valgum)
• Widened epiphyses (wrist widening — "rickety rosary of wrist")
• Delayed dentition; dental caries
• Hypocalcemia → tetany, convulsions

In Adults — OSTEOMALACIA

• Softening of bones due to inadequate mineralization
• Bone pain, muscle weakness, fractures
• Looser zones (pseudo-fractures) on X-ray
• Waddling gait

Biochemical findings in rickets/osteomalacia:

• ↓ Serum Ca²⁺, ↓ Serum PO₄³⁻
• ↑ Serum Alkaline Phosphatase (ALP)
• ↑ PTH

Other deficiency effects:

• Osteoporosis (elderly)
• Increased susceptibility to infections
• Muscle weakness (proximal myopathy)

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Q2. Metabolism of Phenylalanine and Tyrosine; Biologically Important Products; Inborn Errors

Overview

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Metabolism Pathway

Phenylalanine
    ↓  [Phenylalanine hydroxylase + BH4 (tetrahydrobiopterin)]
Tyrosine
    ├──→ Catecholamines (DOPA → Dopamine → Norepinephrine → Epinephrine)
    ├──→ Thyroid hormones (T3, T4)
    ├──→ Melanin (via DOPA → Dopaquinone → Melanin)
    ├──→ Fumarate + Acetoacetate (energy catabolism)
    └──→ Homogentisate pathway

Catabolism (convergent pathway):

Tyrosine
  ↓ [Tyrosine aminotransferase]
p-Hydroxyphenylpyruvate
  ↓ [p-hydroxyphenylpyruvate dioxygenase]
Homogentisate
  ↓ [Homogentisate oxidase]
Maleylacetoacetate
  ↓
Fumarylacetoacetate
  ↓
Fumarate + Acetoacetate  (→ TCA cycle + ketone bodies)

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Biologically Important Products

Tyrosine → [Tyrosine hydroxylase, BH4] → DOPA
  → [DOPA decarboxylase] → Dopamine
  → [Dopamine β-hydroxylase] → Norepinephrine
  → [PNMT, SAM] → Epinephrine

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Inborn Errors of Metabolism

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SHORT ESSAY QUESTIONS (10 marks each)

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Q3. Products Obtained from Glycine

1. Heme (Porphyrins)

• Glycine + Succinyl-CoA → δ-Aminolevulinic acid (ALA) [by ALA synthase, rate-limiting; requires pyridoxal phosphate]
• 8 ALA → Protoporphyrin IX → Heme (with Fe²⁺)

2. Purines

• Glycine contributes C4, C5, N7 of the purine ring
• Essential for de novo purine synthesis

3. Glutathione (GSH)

• Tripeptide: Glu–Cys–Gly
• Glycine is the third amino acid
• Key antioxidant

4. Creatine

• Glycine + Arginine → Guanidinoacetate [in kidney]
• Guanidinoacetate + methyl group (from SAM) → Creatine [in liver]
• Creatine → Creatine phosphate (energy storage in muscle)

5. Conjugated Bile Acids

• Cholic acid + Glycine → Glycocholic acid (bile salt)
• Facilitates fat digestion/absorption

6. Hippuric Acid

• Benzoic acid + Glycine → Hippuric acid
• Detoxification product; excreted in urine

7. Serine and One-Carbon Units

• Glycine ↔ Serine (reversible; serine hydroxymethyltransferase + THF)
• Provides 1-carbon units for methylation reactions

8. Glycine as Neurotransmitter

• Inhibitory neurotransmitter in spinal cord and brainstem

9. Glyoxylate and Oxalate

• Glycine → Glyoxylate → Oxalate (risk for kidney stones if excess)

10. Collagen

• Glycine is every third residue in collagen (Gly-X-Y repeat) — essential for triple helix structure

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Q4. Purine Catabolism

Pathway

AMP                          GMP
 ↓ [5'-nucleotidase]         ↓ [5'-nucleotidase]
Adenosine                   Guanosine
 ↓ [Adenosine deaminase]     ↓ [Purine nucleoside phosphorylase]
Inosine                     Guanine
 ↓ [Purine nucleoside         ↓ [Guanase/Guanine deaminase]
    phosphorylase]
Hypoxanthine                Xanthine
      ↘                      ↗
       [Xanthine oxidase]
           Xanthine
              ↓ [Xanthine oxidase]
           Uric Acid (end product)

Uric Acid

• End product in humans, great apes, Dalmatian dogs
• Other mammals have uricase → allantoin → urea
• Normal serum uric acid: Men: 3.5–7.2 mg/dL; Women: 2.6–6.0 mg/dL
• Excreted mainly by kidneys (~70%) and gut (~30%)

Clinical Significance

Salvage Pathway (recap)

• HGPRT: Hypoxanthine + PRPP → IMP; Guanine + PRPP → GMP
• APRT: Adenine + PRPP → AMP
• Saves energy vs. de novo synthesis

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Q5. Iron Absorption and Mucosal Block Theory

Forms of dietary iron

• Haem iron (Fe²⁺ in heme) — from meat; 20–30% absorbed; direct uptake via HCP1
• Non-haem iron (Fe³⁺) — from plants; 1–5% absorbed; must be reduced to Fe²⁺ first

Steps of Iron Absorption

LUMEN                           ENTEROCYTE               BLOOD
Fe³⁺ [dietary]
  ↓ [Duodenal cytochrome b (DcytB), vitamin C helps]
Fe²⁺
  ↓ [DMT-1 (Divalent Metal Transporter 1)]
                       Fe²⁺ in enterocyte
                       ↙              ↘
              [stored as Ferritin]    [Ferroportin → exports Fe²⁺]
                                           ↓ [Hephaestin, copper-enzyme]
                                         Fe³⁺
                                           ↓ [Transferrin in plasma]
                                    Transferrin-Fe³⁺ complex

Mucosal Block Theory (Hahn, 1943)

• Proposed that the intestinal mucosal cell acts as a "block" to prevent iron overload
• When iron stores are replete:
  • Intracellular ferritin levels rise in enterocytes
  • Incoming Fe²⁺ is stored as ferritin rather than exported
  • When the enterocyte is shed (every 3–5 days), stored iron is lost
  • This limits further absorption → "mucosal block"
• When body needs more iron:
  • Ferritin synthesis decreases
  • More iron is exported via ferroportin
• Hepcidin (from liver) is the master regulator:
  • ↑ Hepcidin → degrades ferroportin → blocks iron export → reduces absorption
  • ↑ Iron stores, infection, inflammation → ↑ Hepcidin
  • ↓ Iron stores, hypoxia, erythropoiesis → ↓ Hepcidin

Factors enhancing absorption

• Ascorbic acid (Vitamin C) — reduces Fe³⁺ → Fe²⁺
• HCl (gastric acid) — keeps iron soluble
• Meat factor
• Low body iron stores

Factors reducing absorption

• Phytates (cereals), oxalates (spinach)
• Phosphates, tannins (tea/coffee)
• Antacids (↑ pH)
• ↑ Body iron stores (via hepcidin)

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Q6. Functions of Glutathione

• The sulfhydryl (–SH) group of cysteine is the active site
• Exists in reduced (GSH) and oxidized (GSSG) forms
• GSH:GSSG ratio normally >10:1 in cells
• Regenerated by Glutathione reductase using NADPH

Functions

1. Antioxidant (primary role)
   • 2 GSH + H₂O₂ → GSSG + 2H₂O [by Glutathione peroxidase]
   • Neutralizes free radicals and reactive oxygen species (ROS)
2. Protection of RBCs
   • Reduces oxidized hemoglobin (MetHb → Hb)
   • Maintains membrane protein thiols
   • Deficiency of G6PD → ↓ NADPH → ↓ GSH → RBC hemolysis (G6PD deficiency)
3. Detoxification / Conjugation
   • GSH conjugates with xenobiotics (drugs, carcinogens) in Phase II liver detoxification
   • GSH + Paracetamol toxic metabolite (NAPQI) → safe conjugate
   • Mercapturic acid pathway
4. Amino acid transport (γ-glutamyl cycle)
   • γ-Glutamyl transpeptidase transfers Glu from GSH to amino acids on cell surface
   • Transports amino acids across cell membranes (especially kidney, intestine)
   • Meister cycle — active transport of amino acids
5. Reduction of ribonucleotides
   • Ribonucleotide reductase uses glutaredoxin (reduced by GSH) to make deoxyribonucleotides
6. Maintenance of protein –SH groups
   • Keeps enzyme active-site cysteines in reduced (active) state
7. Leukotriene synthesis
   • GSH is substrate for leukotriene (eicosanoid) synthesis
8. Sperm function
   • High GSH in sperm; protects from oxidative damage

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Q7. Van Den Berg Reaction

Definition

Principle

• Bilirubin + Diazo reagent → Azobilirubin (purple-pink color)
• The reaction gives a pink/violet color measured at 540 nm

Types of Bilirubin

Procedure

• Direct bilirubin (conjugated): Sample + diazo reagent → pink color in aqueous medium within 1 min
• Total bilirubin: Sample + methanol (alcohol) + diazo reagent → all bilirubin reacts
• Indirect bilirubin (unconjugated): Total − Direct

Normal Values

Clinical Significance

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Q8. Products of Transmethylation

Definition

Formation of SAM

Methionine + ATP → S-Adenosylmethionine (SAM)   [by Methionine adenosyltransferase]

Products of Transmethylation via SAM

After Methylation: SAM → SAH

SAM + Acceptor → SAH (S-Adenosylhomocysteine) + Methylated product
SAH → Adenosine + Homocysteine [by SAH hydrolase]
Homocysteine → Methionine [by methionine synthase, needs B₁₂ + folate]
         or → Cystathionine → Cysteine [transsulfuration, needs B₆]

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Q9. Calcium Homeostasis

Normal values

• Total serum calcium: 8.5–10.5 mg/dL (2.1–2.6 mmol/L)
• Ionized (free) Ca²⁺: 4.5–5.5 mg/dL (~50%)
• Protein-bound: ~40% (mainly albumin)
• Complexed: ~10%

Body distribution

• 99% in bone (hydroxyapatite)
• 1% in blood and soft tissues

Three Hormones Regulating Calcium

1. Parathyroid Hormone (PTH)

• Released when serum Ca²⁺ ↓
• Bone: ↑ Osteoclast activity → ↑ Ca²⁺ and PO₄³⁻ release
• Kidney: ↑ Ca²⁺ reabsorption (distal tubule); ↓ PO₄³⁻ reabsorption; activates 1α-hydroxylase → ↑ calcitriol
• Gut: Indirect (via calcitriol) → ↑ Ca²⁺ absorption
• Net: ↑ serum Ca²⁺, ↓ serum PO₄³⁻

2. Vitamin D (Calcitriol — 1,25(OH)₂D₃)

• Activated by PTH in kidney
• Gut: ↑ Ca²⁺ and PO₄³⁻ absorption (via calbindin-D synthesis)
• Bone: At physiological levels → promotes mineralization; at high levels → resorption
• Kidney: ↑ Ca²⁺ reabsorption
• Net: ↑ serum Ca²⁺ and PO₄³⁻

3. Calcitonin

• Released from parafollicular (C) cells of thyroid when Ca²⁺ ↑
• Bone: ↓ Osteoclast activity → ↓ Ca²⁺ release
• Kidney: ↑ Ca²⁺ and PO₄³⁻ excretion
• Net: ↓ serum Ca²⁺ (opposes PTH)

Summary diagram:

Low Ca²⁺ → ↑PTH → ↑ Bone resorption, ↑ Renal Ca²⁺ reabsorption, ↑ Calcitriol
                 ↓
           ↑ Gut absorption (calcitriol)
                 ↓
           ↑ Serum Ca²⁺ → negative feedback → ↓ PTH; ↑ Calcitonin

Clinical Disorders

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Q10. Transmethylation and Deamination

A. TRANSMETHYLATION

• Transfer of methyl group (–CH₃) from SAM to various acceptors
• SAM = "active methionine" (methionine + ATP)
• Products: creatine, epinephrine, phosphatidylcholine, melatonin, methylated DNA/RNA

Methionine + ATP → SAM → [Methylation] → SAH → Homocysteine → (back to Methionine via B₁₂/folate)

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B. DEAMINATION

Types of Deamination:

Glutamate + NAD⁺ → α-Ketoglutarate + NH₃ + NADH
[Enzyme: Glutamate dehydrogenase, in liver mitochondria; requires NAD⁺ or NADP⁺]

• Allosteric inhibited by GTP, NADH (energy surplus)
• Activated by ADP, NAD⁺ (energy deficit)
• This is the major source of free ammonia for urea synthesis

Amino acid + α-Ketoglutarate → Keto acid + Glutamate [by aminotransferases, PLP]
Glutamate → deaminated by GDH → releases NH₃

• Serine → Pyruvate + NH₃ [serine dehydratase, B₆]
• Threonine → α-ketobutyrate + NH₃ [threonine dehydratase]
• Cysteine → Pyruvate + NH₃ + H₂S

• D-amino acid oxidase: D-amino acids + O₂ → keto acid + NH₃ + H₂O₂

Fate of NH₃:

• In liver → Urea cycle (detoxification)
• In brain/peripheral tissues → Glutamine synthesis (Glu + NH₃ → Glutamine, by glutamine synthetase)
• In kidney → Excreted as NH₄⁺ (acid-base regulation)

Clinical: Hyperammonemia

• Urea cycle enzyme defects → toxic accumulation of NH₃
• Symptoms: encephalopathy, vomiting, cerebral edema, coma
• NH₃ depletes α-ketoglutarate → TCA cycle impaired

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Q11. Lesch-Nyhan Syndrome and Orotic Aciduria

A. LESCH-NYHAN SYNDROME

• HGPRT normally salvages hypoxanthine and guanine back to IMP and GMP
• In its absence, hypoxanthine and guanine cannot be recycled
• PRPP accumulates → drives de novo purine synthesis → excess purine catabolism → ↑↑ Uric acid
• Loss of IMP/GMP feedback → unregulated PRPP amidotransferase

• Allopurinol — reduces uric acid (controls gout/stones but NOT neurological symptoms)
• No cure; neurological damage is irreversible

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B. OROTIC ACIDURIA

• Orotic acid is an intermediate in pyrimidine synthesis pathway
• CAD → Orotic acid → [OPRT] → Orotidine-5'-phosphate → [ODC] → UMP
• Enzyme defect → orotic acid cannot proceed → accumulates → excreted in urine

• OTC (ornithine transcarbamylase) deficiency — accumulated carbamoyl phosphate is shunted into pyrimidine pathway → orotic acid overflow
• Allopurinol therapy (minor orotic aciduria)

• Megaloblastic anemia (not responsive to B₁₂ or folate)
• Failure to thrive, growth retardation
• Crystalluria (orotic acid crystals in urine)
• Developmental delay (if untreated)
• No hyperammonemia (distinguishes from OTC deficiency)

• Elevated orotic acid in urine
• Megaloblastic anemia unresponsive to B₁₂/folate

• Uridine supplementation (bypasses the block)
  • Uridine → UMP → UTP → CTP (pyrimidines replenished)
  • UTP feedback-inhibits carbamoyl phosphate synthetase → reduces orotic acid accumulation

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Q12. Urea Cycle — Illustrative Diagram

Enzymes, substrates, and products:

                    MITOCHONDRIA
                    ┌────────────────────────────────┐
                    │                                │
  NH₃ + CO₂        │                                │
  (from GDH)       │                                │
       ↓            │                                │
  Carbamoyl phosphate                               │
  [CPS-I: Carbamoyl  ← 2 ATP                        │
   Phosphate         ← H₂O                          │
   Synthetase I]     ← N-acetylglutamate (activator) │
       ↓            │                                │
  Carbamoyl phosphate + Ornithine                   │
       ↓ [OTC: Ornithine Transcarbamylase]           │
    CITRULLINE                                       │
       │             └────────────────────────────────┘
       │  (exits mitochondria via transporter)
       ↓
                    CYTOPLASM
    CITRULLINE + Aspartate  (ATP → AMP + PPᵢ)
       ↓ [ASS: Argininosuccinate Synthetase]
    ARGININOSUCCINATE
       ↓ [ASL: Argininosuccinate Lyase]
    ARGININE  +  Fumarate
       |                 ↓ (→ TCA cycle → regenerates aspartate)
       ↓ [Arginase + H₂O]
    ORNITHINE  +  UREA
       |
       ↓ (Ornithine re-enters mitochondria)

Summary Table of Urea Cycle Reactions

Urea — key points:

• 2 nitrogen atoms in urea: one from NH₃ (glutamate deamination), one from aspartate
• 1 carbon from CO₂
• Net cost: 3 ATP (4 high-energy bonds) per urea molecule
• Urea is water-soluble; excreted by kidneys
• Blood urea nitrogen (BUN): normal 7–20 mg/dL

Inborn errors of the Urea Cycle:

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Quick Reference — All Questions Answered