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LONG ANSWER QUESTIONS (15 Marks Each)

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1. Urea Cycle — Detailed Note with Reactions + Major Metabolic Disorders

Introduction

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Sources of Nitrogen Entering the Urea Cycle

1. NH₄⁺ — derived from:
   • Oxidative deamination of glutamate (via glutamate dehydrogenase)
   • Dehydratase reactions (serine → pyruvate + NH₄⁺; threonine)
   • Bacteria in the gut
2. Aspartate — supplies the second nitrogen of urea directly

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Reactions of the Urea Cycle (5 Steps)

Step 1: Synthesis of Carbamoyl Phosphate (Mitochondria)

NH₄⁺ + HCO₃⁻ + 2 ATP → Carbamoyl phosphate + 2 ADP + Pᵢ

• Enzyme: Carbamoyl phosphate synthetase I (CPS-I) — mitochondrial
• Requires N-acetylglutamate (NAG) as an obligate allosteric activator
• 2 molecules of ATP are consumed
• CPS-II (cytosolic) uses glutamine-N for pyrimidine biosynthesis (different enzyme)

Step 2: Formation of Citrulline (Mitochondria)

Carbamoyl phosphate + Ornithine → Citrulline + Pᵢ

• Enzyme: Ornithine transcarbamoylase (OTC)
• Occurs in the inner mitochondrial matrix
• Citrulline is transported to the cytosol in exchange for ornithine

Step 3: Formation of Argininosuccinate (Cytosol)

Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPᵢ

• Enzyme: Argininosuccinate synthetase
• ATP is hydrolyzed to AMP + pyrophosphate (effectively consumes 2 ATP equivalents)
• Aspartate is the second nitrogen donor (produced by transamination of oxaloacetate)

Step 4: Cleavage of Argininosuccinate (Cytosol)

Argininosuccinate → Arginine + Fumarate

• Enzyme: Argininosuccinate lyase
• Fumarate enters the TCA cycle; can be recycled to oxaloacetate → aspartate (the "Krebs bi-cycle" linking TCA and urea cycles)

Step 5: Hydrolysis of Arginine — Release of Urea (Cytosol)

Arginine + H₂O → Urea + Ornithine

• Enzyme: Arginase (liver-specific)
• Urea is released and excreted by the kidneys
• Ornithine is transported back into the mitochondria to begin the next cycle

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Overall Equation

2 NH₃ + CO₂ + 3 ATP + H₂O → Urea + 2 ADP + AMP + 4 Pᵢ

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Regulation of the Urea Cycle

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Major Metabolic Disorders of the Urea Cycle

• Low-protein diet
• Alternative nitrogen excretion pathways: sodium benzoate conjugates glycine → hippurate; phenylbutyrate → phenylacetylglutamine (both excreted in urine)
• Hemodialysis in acute crisis
• Liver transplantation (curative)
• Gene therapy (experimental)

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2. Transamination Reaction in Catabolism of Amino Acids

Definition

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General Reaction

Amino acid₁ + α-Keto acid₁ ⇌ α-Keto acid₂ + Amino acid₂

Amino acid + α-Ketoglutarate ⇌ α-Keto acid + Glutamate

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Key Example Reactions

Aspartate + α-Ketoglutarate ⇌ Oxaloacetate + Glutamate

Alanine + α-Ketoglutarate ⇌ Pyruvate + Glutamate

R–CH(NH₂)–COOH + HOOC–CO–R' ⇌ R–CO–COOH + R'–CH(NH₂)–COOH

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Enzymes (Transaminases / Aminotransferases)

• All amino acids except lysine and threonine (Harper's: also proline and hydroxyproline) undergo transamination
• Each transaminase is specific for one substrate pair but not the other
• α-Ketoglutarate/glutamate serve as the universal acceptor/donor pair — because glutamate is the only amino acid that undergoes rapid oxidative deamination (via glutamate dehydrogenase), this funnels all amino-group nitrogen toward the urea cycle

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Coenzyme: Pyridoxal Phosphate (PLP)

• PLP is derived from vitamin B₆ (pyridoxine)
• Acts as a carrier of amino groups — forms a Schiff base with the α-amino group of the substrate
• Mechanism: "Ping-pong" (double displacement)
  1. Amino acid₁ reacts with enzyme–PLP → forms Schiff base → releases α-keto acid₁ → enzyme becomes enzyme–PMP (pyridoxamine phosphate)
  2. α-Keto acid₂ reacts with enzyme–PMP → forms new amino acid₂ → enzyme–PLP regenerated
• Vitamin B₆ deficiency impairs all transamination reactions

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Role in Amino Acid Catabolism

1. The amino group is transferred to α-ketoglutarate → glutamate
2. Glutamate undergoes oxidative deamination (glutamate dehydrogenase):

   Glutamate + NAD⁺ → α-Ketoglutarate + NH₄⁺ + NADH

3. The resulting NH₄⁺ enters the urea cycle
4. The carbon skeleton (α-keto acid) is further degraded to acetyl-CoA, TCA cycle intermediates, or glucose — depending on whether the amino acid is glucogenic, ketogenic, or both

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Significance

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3. Bile Pigments — Diseases Associated with Their Metabolism

What Are Bile Pigments?

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Metabolism of Bilirubin (Step-by-Step)

Step 1: Heme Catabolism (Reticuloendothelial System)

• Aged RBCs are phagocytized by macrophages in spleen, liver (Kupffer cells), and bone marrow
• Hemoglobin is broken down: globin → amino acids; heme → iron + protoporphyrin
• Heme oxygenase converts heme → biliverdin (green) + CO + Fe²⁺
• Biliverdin reductase converts biliverdin → bilirubin (yellow-orange)

Step 2: Transport in Blood (Pre-hepatic)

• Bilirubin is unconjugated (indirect bilirubin) — water-insoluble
• Transported bound to albumin in plasma (not filtered by kidneys; urine is normal colored)
• Normal serum total bilirubin: 0.2–1.0 mg/dL (or up to 1.2 mg/dL)

Step 3: Hepatic Uptake and Conjugation

• Hepatocytes take up bilirubin via carrier proteins (ligandin/Y protein)
• In the endoplasmic reticulum, UDP-glucuronosyltransferase (UGT1A1) conjugates bilirubin with glucuronic acid:

  Bilirubin + 2 UDP-glucuronate → Bilirubin diglucuronide (conjugated/direct bilirubin)

• Conjugated bilirubin is water-soluble

Step 4: Excretion into Bile

• Conjugated bilirubin is excreted into bile canaliculi via the MRP2 (ABCC2) transporter
• Passes into the intestine

Step 5: Intestinal Conversion

• Intestinal bacteria reduce bilirubin → urobilinogen (colorless)
• Most urobilinogen is oxidized in the colon → stercobilin (brown color of feces)
• A small amount (~20%) is reabsorbed (enterohepatic circulation):
  • Most is re-excreted by the liver
  • A small amount reaches the kidney → oxidized to urobilin → gives urine its yellow color

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Summary Table of Bile Pigments

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Diseases Associated with Bile Pigment Metabolism

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4. Transaminases — Definition and Diagnostic Importance

Definition

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Normal Serum Values

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Diagnostic Importance of Transaminases

1. Liver Disease

• ALT is the most liver-specific transaminase
• AST/ALT De Ritis ratio > 2 strongly suggests alcoholic liver disease

2. Myocardial Infarction (MI)

• AST rises within 6–8 hours, peaks at 24–48 hours, normalizes by 4–6 days after MI
• Less specific than troponin/CK-MB; now rarely used as primary MI marker
• Historically called GOT (Glutamate-Oxaloacetate Transaminase)

3. Skeletal Muscle Diseases

• Both AST and ALT rise in muscular dystrophy, polymyositis, rhabdomyolysis
• Distinguish from liver disease by checking CK (creatine kinase)

4. Monitoring Disease Progression

• Serial ALT/AST values track the course of hepatitis, efficacy of antiviral therapy, and liver recovery

5. Drug Monitoring

• Statin-induced hepatotoxicity is monitored by periodic ALT/AST
• If ALT > 3× upper limit of normal, drug is discontinued

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Summary: Diagnostic Uses at a Glance

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5. Porphyrins — Types and Clinical Importance of Porphyria

What Are Porphyrins?

• Protoporphyrin IX — the precursor of heme
• Heme — protoporphyrin IX + Fe²⁺ (found in hemoglobin, myoglobin, cytochromes, catalase)

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Heme Biosynthesis Pathway (Overview)

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What Is Porphyria?

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Classification of Porphyrias

A. By Site of Overproduction

B. Major Clinical Types

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Clinical Features of Acute Porphyrias (AIP, HCP, VP)

1. Abdominal pain (colicky, severe) — most common presenting feature
2. Neuropsychiatric symptoms — anxiety, hallucinations, psychosis, seizures
3. Autonomic dysfunction — tachycardia, hypertension, constipation, urinary retention

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Treatment of Porphyria

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SHORT ANSWER QUESTIONS (5 Marks Each)

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S1. Transamination Reaction in Catabolism of Amino Acids

Amino acid + α-Ketoglutarate ⇌ α-Keto acid + Glutamate

• Removes the amino group from amino acids as the first step in degradation
• All amino acids (except Lys and Thr) can undergo transamination
• The amino group concentrates in glutamate, which then undergoes oxidative deamination by glutamate dehydrogenase → releasing NH₄⁺ for the urea cycle
• The resulting α-keto acid enters central metabolic pathways (TCA cycle, gluconeogenesis)

• AST: Aspartate + α-KG ⇌ Oxaloacetate + Glutamate
• ALT: Alanine + α-KG ⇌ Pyruvate + Glutamate

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S2. Phenylketonuria (PKU) and Alkaptonuria

Phenylketonuria (PKU)

• Deficient enzyme: Phenylalanine hydroxylase (PAH) — EC 1.14.16.1
• Inheritance: Autosomal recessive
• Frequency: ~1 in 10,000 births
• Biochemistry: Phenylalanine cannot be converted to tyrosine → accumulates → shunted to phenylpyruvate, phenylacetate, phenyllactate (phenylketones), which are excreted in urine
• Clinical features:
  • Mental retardation (if untreated) — phenylpyruvate is toxic to the developing brain
  • Fair skin, blue eyes, blonde hair (tyrosine deficiency → reduced melanin)
  • Musty/"mousy" odor of urine (phenylacetate)
  • Eczema, seizures
• Screening: Guthrie test (newborn heel-prick); now tandem mass spectrometry
• Treatment: Low-phenylalanine diet commenced in first weeks of life; sapropterin (BH₄ cofactor) for BH₄-responsive forms
• Maternal PKU: Untreated PKU in pregnant women causes fetal brain damage even if the fetus is heterozygous

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Alkaptonuria

• Deficient enzyme: Homogentisate oxidase (homogentisate 1,2-dioxygenase) — a step in tyrosine catabolism
• Inheritance: Autosomal recessive
• Biochemistry: Homogentisic acid (homogentisate) cannot be cleaved → accumulates → excreted in urine; darkens on exposure to air (oxidizes to benzoquinone polymers → black pigment)
• Clinical features:
  • Dark urine (first sign; may be noticed from infancy)
  • Ochronosis — dark blue-black pigmentation of connective tissue (sclera, ears, cartilage) in adults
  • Arthritis (ochronotic arthropathy) — large joints, spine; due to deposition of benzoquinone-acetic acid polymers in cartilage
• Historical significance: First described in the 16th century; Archibald Garrod used it to formulate the concept of inborn errors of metabolism (1908)
• Treatment: Nitisinone (NTBC) — inhibits p-hydroxyphenylpyruvate dioxygenase, reducing homogentisate production; low-protein diet

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S3. What Is Porphyria?

1. Hepatic porphyrias — enzyme defect in the liver; accumulate precursors (ALA, PBG) → neurovisceral attacks: acute abdominal pain, psychosis, peripheral neuropathy, autonomic dysfunction
   • Examples: Acute Intermittent Porphyria (AIP), Hereditary Coproporphyria, Variegate Porphyria
2. Erythropoietic porphyrias — defect in bone marrow; accumulate porphyrins → cutaneous photosensitivity: blistering, scarring, disfigurement on sun-exposed areas
   • Examples: Congenital Erythropoietic Porphyria (Günther disease), Erythropoietic Protoporphyria, Porphyria Cutanea Tarda

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S4. Jaundice — Definition and Types

Definition

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Types of Jaundice

• Dark urine: conjugated bilirubin is water-soluble → filtered by kidneys
• Pale stools: no bilirubin reaches intestine → no stercobilin
• Pruritus: accumulation of bile salts in skin
• Malabsorption/steatorrhea: absence of bile salts impairs fat digestion

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S5. Normal Blood Urea Level and Conditions with Elevated Blood Urea

Normal Blood Urea Level

• Blood Urea: 15–40 mg/dL (2.5–6.7 mmol/L)
• Blood Urea Nitrogen (BUN): 7–20 mg/dL (2.5–7.1 mmol/L)

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Conditions with Elevated Blood Urea (Azotemia/Uremia)

Pre-renal Causes (↑ production or ↓ renal perfusion)

1. High-protein diet — increased amino acid catabolism → more urea synthesis
2. Dehydration / hypovolemia — reduced renal blood flow → reduced urea excretion
3. Congestive heart failure — reduced renal perfusion
4. GI bleeding — blood proteins digested and absorbed → large nitrogen load
5. Prolonged fasting/starvation — muscle protein catabolism

Renal Causes (Impaired excretion)

1. Acute kidney injury (AKI) — tubular necrosis, nephrotoxins, ischemia; BUN rises rapidly
2. Chronic kidney disease (CKD) — progressive loss of nephrons; BUN rises as GFR falls; uremia develops (BUN > 100 mg/dL with symptoms)
3. Glomerulonephritis — impaired filtration
4. Nephrotic syndrome — proteinuria + reduced GFR

Post-renal Causes (Obstruction)

1. Ureteral obstruction (stones, tumors) — urine backpressure → impaired filtration
2. Prostatic hypertrophy — urinary retention → hydronephrosis

• > 20:1 → pre-renal (or GI bleed, high protein intake)
• 10–20:1 → intrinsic renal disease
• < 10:1 → post-renal or starvation/liver disease

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Quick Reference Guide

Long Answer Questions (15 Marks)

Short Answer Questions (5 Marks)