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MEDICINAL BIOCHEMISTRY — COMPLETE ANSWERS

Pharm D 1st Year

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UNIT I — Introduction to Biochemistry (Enzymes, Cyclic AMP, Vitamins)

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VERY SHORT ANSWERS (2 marks)

Q1. Structure and Significance of Cyclic AMP (cAMP)

• cAMP (3',5'-cyclic adenosine monophosphate) is formed from ATP by the enzyme adenylate cyclase
• The phosphate group forms a cyclic ester bond between the 3' and 5' carbon of the ribose sugar
• Molecular formula: C₁₀H₁₂N₅O₆P

• Acts as intracellular second messenger for many hormones (glucagon, epinephrine, TSH, LH, FSH)
• Activates Protein Kinase A (PKA), which phosphorylates target proteins
• Regulates glycogenolysis, lipolysis, gene expression
• Broken down by phosphodiesterase to 5'-AMP (inactive)

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Q2. Structure and Significance of ATP

• ATP = Adenosine + 3 phosphate groups (α, β, γ)
• Has two high-energy phosphoanhydride bonds (~P)
• Adenosine = Adenine + Ribose

• Universal energy currency of the cell
• Provides energy for biosynthesis, muscle contraction, active transport, signal transduction
• Produced by: glycolysis (substrate level), oxidative phosphorylation (ETC), and TCA cycle
• Hydrolysis: ATP → ADP + Pi + 7.3 kcal/mol (standard free energy)

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Q3. Allosteric Enzymes — Definition + 2 Examples

1. Phosphofructokinase-1 (PFK-1) — key regulatory enzyme of glycolysis; inhibited by ATP, activated by AMP and ADP
2. Aspartate transcarbamylase (ATCase) — pyrimidine synthesis; inhibited by CTP (feedback inhibition)

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Q4. Biochemical Organisation of the Cell

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Q5. Different Processes Across Cell Membrane

1. Simple diffusion — small nonpolar molecules (O₂, CO₂) down concentration gradient; no energy
2. Facilitated diffusion — polar/charged molecules via protein channels/carriers; no energy (e.g., glucose via GLUT transporters)
3. Active transport — against gradient; requires ATP (e.g., Na⁺/K⁺-ATPase pump)
4. Endocytosis/Exocytosis — bulk transport of macromolecules into/out of cell
5. Osmosis — movement of water through semipermeable membrane from low to high solute concentration

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Q6. Two Factors Affecting Enzyme Activity

1. Temperature: Activity increases with temperature up to an optimum (~37°C in humans); beyond this, denaturation of enzyme occurs and activity falls sharply.
2. pH: Each enzyme has an optimal pH (e.g., pepsin: pH 2, trypsin: pH 8, salivary amylase: pH 6.8). Deviations alter ionization of active site residues.

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Q7. Enzyme Nomenclature

• Each enzyme has a systematic name, a working name, and an EC number
• EC number has 4 digits: EC [class].[subclass].[sub-subclass].[serial number]
• 6 major classes:
  1. Oxidoreductases (oxidation-reduction)
  2. Transferases (group transfer)
  3. Hydrolases (hydrolysis)
  4. Lyases (addition/removal without hydrolysis)
  5. Isomerases (isomerization)
  6. Ligases/Synthetases (bond formation + ATP)

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Q8. Two Models to Explain Active Sites of Enzymes

1. Lock and Key Model (Emil Fischer, 1894): The enzyme active site is rigid and has a fixed shape complementary to the substrate (like a lock and key). Explains specificity but not flexibility.
2. Induced Fit Model (Koshland, 1958): The active site is flexible. Upon substrate binding, the enzyme undergoes conformational change that brings catalytic groups into proper alignment. Better explains broad substrate specificity and allosteric regulation.

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Q9. Enzyme Specificity — Definition + Example

• Absolute specificity — acts on only one substrate (e.g., urease acts only on urea)
• Group specificity — acts on a specific chemical group (e.g., hexokinase phosphorylates hexoses)
• Stereospecificity — acts on only one stereoisomer (e.g., L-amino acid oxidase acts only on L-amino acids)
• Bond specificity — acts on a specific bond type (e.g., lipase on ester bonds)

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Q10. Holoenzyme and Apoenzyme

• Apoenzyme: The protein part of an enzyme without its cofactor; catalytically inactive alone
• Cofactor: Non-protein component needed for activity (metal ions or coenzymes)
• Coenzyme: Organic cofactor, often vitamin-derived (e.g., NAD⁺, FAD, CoA)
• Holoenzyme: Complete, catalytically active enzyme = Apoenzyme + Cofactor

Holoenzyme = Apoenzyme + Cofactor (Coenzyme/Metal ion)

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LONG ANSWERS (10/15 marks)

Q1. IUB Classification of Enzymes + Mechanism of Action + Factors Affecting Activity + Importance

1. Enzyme (E) + Substrate (S) → Enzyme-Substrate complex (ES)
2. ES → Enzyme-Product complex (EP)
3. EP → E + P (product released, enzyme recycled)

1. Temperature — Q₁₀ effect; optimum ~37°C; denaturation above
2. pH — alters ionization of active site residues
3. Substrate concentration — follows Michaelis-Menten kinetics; Vmax and Km
4. Enzyme concentration — activity proportional to [E] at saturating [S]
5. Inhibitors — competitive (increase apparent Km), non-competitive (decrease Vmax), uncompetitive
6. Activators/Cofactors — Mg²⁺ for kinases, Zn²⁺ for carbonic anhydrase
7. Product concentration — accumulation inhibits reaction (product inhibition)

• Diagnostic markers (AST/ALT in liver disease, CK-MB in MI, amylase in pancreatitis)
• Drug targets (ACE inhibitors, statins inhibit HMG-CoA reductase)
• Industrial use (restriction enzymes in biotechnology)

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Q2. Define Enzyme Inhibition — Types

A. Reversible Inhibition (inhibitor binds non-covalently)

• Inhibitor resembles substrate; competes for active site
• Km increases; Vmax unchanged
• Overcome by increasing [S]
• Example: Malonate inhibits succinate dehydrogenase; sulfonamides inhibit dihydropteroate synthase

• Inhibitor binds at allosteric site (different from active site)
• Binds to both E and ES complex
• Km unchanged; Vmax decreases
• Cannot be overcome by increasing [S]
• Example: Heavy metals (Pb²⁺, Hg²⁺) inhibiting enzymes with SH groups

• Inhibitor binds only to ES complex, not free enzyme
• Both Km and Vmax decrease proportionally
• Rare; example: certain pesticides (organophosphates)

• Inhibitor binds both E and ES but with different affinities
• Both Km and Vmax are altered

B. Irreversible Inhibition

• Inhibitor forms covalent bond with enzyme
• Permanently inactivates enzyme
• Examples:
  • Organophosphates (e.g., nerve agents, DFP) — irreversibly inhibit acetylcholinesterase
  • Aspirin — irreversibly acetylates cyclooxygenase (COX-1/COX-2)
  • Penicillin — irreversibly inhibits transpeptidase

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Q3. Short Answers on Vitamins (Niacin/B3, B2, B6, B12, Folic Acid)

Vitamin B3 (Niacin) — Biochemical Role and Deficiency

• Coenzyme in over 200 oxidation-reduction reactions
• NAD⁺ — major electron carrier in catabolism (glycolysis, TCA cycle, β-oxidation)
• NADP⁺ — anabolic reactions (fatty acid synthesis, HMP shunt)

• 4 D's: Dermatitis (sun-exposed skin), Diarrhea, Dementia (mental confusion), Death (if untreated)
• Casal's necklace — hyperpigmented rash around neck
• Seen in corn-eating populations (corn lacks tryptophan + niacin)

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Vitamin B2 (Riboflavin) — Biochemical Role and Deficiency

• Coenzyme in flavoproteins (oxidoreductases)
• FAD: electron carrier in ETC (Complex II), β-oxidation, TCA cycle (succinate dehydrogenase)
• FMN: component of Complex I (NADH dehydrogenase)

• Stomatitis (cheilosis — cracks at corners of mouth)
• Glossitis (magenta tongue)
• Corneal vascularization
• Scrotal/vulval dermatitis
• Normocytic anemia

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Vitamin B6 (Pyridoxine) — Biochemical Role and Deficiency

• Coenzyme for transaminases (aminotransferases — AST, ALT)
• Essential for transamination, deamination, decarboxylation of amino acids
• Synthesis of neurotransmitters: serotonin (from tryptophan), dopamine, GABA, norepinephrine
• Synthesis of heme (δ-aminolevulinic acid synthase)
• Glycogen phosphorylase

• Peripheral neuropathy (most common)
• Microcytic hypochromic anemia (impaired heme synthesis)
• Convulsions in infants
• Stomatitis, glossitis, seborrheic dermatitis
• Isoniazid (INH) is a B6 antagonist → gives B6 supplement with INH

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Vitamin B12 (Cobalamin) — Biochemical Role and Deficiency

1. Methylcobalamin — cofactor for methionine synthase
   • Transfers methyl group from methylTHF → homocysteine → methionine
   • "Folate trap" — B12 deficiency traps folate as methylTHF (functional folate deficiency)
2. Adenosylcobalamin — cofactor for methylmalonyl-CoA mutase
   • Converts methylmalonyl-CoA → succinyl-CoA (important in odd-chain FA catabolism, branched AA catabolism)

• Megaloblastic macrocytic anemia (due to folate trap — impaired DNA synthesis)
• Subacute combined degeneration of spinal cord (unique to B12; neurological damage: posterior and lateral columns)
• Hyperhomocysteinemia (risk factor for cardiovascular disease)
• Glossitis, glossy tongue

• Methylcobalamin (cytoplasm)
• Adenosylcobalamin (mitochondria)
• Hydroxycobalamin (therapeutic form)

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Folic Acid — Biochemical Role and Deficiency

• One-carbon carrier (methyl, methylene, formyl, methenyl groups)
• Required for: purine nucleotide synthesis (C2 and C8 of purine ring), thymidylate synthesis (dUMP → dTMP)
• Methionine synthesis (with B12)
• Interconversion of amino acids (serine ↔ glycine)

• Megaloblastic macrocytic anemia (impaired DNA synthesis — no neurological damage, unlike B12)
• Neural tube defects in fetus (spina bifida, anencephaly) — folate supplementation in pregnancy is critical
• Glossitis, angular stomatitis
• Elevated homocysteine

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UNIT II — Carbohydrate Metabolism

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VERY SHORT ANSWERS (2 marks)

Q1. What is Diabetes Mellitus? Mention its types.

• Type 1 DM: Autoimmune destruction of β-cells → absolute insulin deficiency; requires insulin injection; HLA-DR3/DR4 associated; ketosis-prone
• Type 2 DM: Insulin resistance + relative insulin deficiency; associated with obesity; managed with oral hypoglycemics
• Gestational DM: Occurs during pregnancy; risk of fetal macrosomia
• Other specific types: MODY (maturity onset diabetes of youth), secondary DM (pancreatitis, Cushing's, drugs)

• Normal: < 100 mg/dL
• Prediabetes: 100–125 mg/dL
• DM: ≥ 126 mg/dL on two occasions

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Q2. Use of Malate-Aspartate Shuttle

• Transfers NADH reducing equivalents from the cytoplasm into mitochondria (across inner mitochondrial membrane, which is impermeable to NADH directly)
• Operates in heart, liver, kidney (tissues with high energy demand)
• Involves: oxaloacetate → malate (cytoplasm) → enters mitochondria → malate → oxaloacetate (mitochondria), releasing NADH
• Net result: each cytoplasmic NADH yields 2.5 ATP (via Complex I)
• Compare: Glycerol-3-phosphate shuttle (muscle/brain) yields only 1.5 ATP per cytoplasmic NADH

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Q3. Amphibolic Role of TCA Cycle

• Acetyl-CoA oxidized → 3 NADH + 1 FADH₂ + 1 GTP per turn
• Complete glucose oxidation via TCA + ETC yields ~30–32 ATP

• α-Ketoglutarate → glutamate → other amino acids (transamination)
• Oxaloacetate → aspartate (transamination) → pyrimidines
• Succinyl-CoA → porphyrin/heme synthesis
• Citrate (exported) → Acetyl-CoA for fatty acid synthesis
• Malate → glucose (gluconeogenesis via PEP carboxykinase)

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Q4. Anaplerotic Reactions of TCA Cycle

1. Pyruvate carboxylase: Pyruvate + CO₂ + ATP → Oxaloacetate (liver, kidney; biotin coenzyme)
2. PEP carboxykinase: Replenishes OAA from PEP during gluconeogenesis
3. Glutamate dehydrogenase: Glutamate → α-ketoglutarate (replenishes α-KG)
4. Propionyl-CoA → Succinyl-CoA (replenishes succinyl-CoA; odd-chain FA catabolism)

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Q5. ATP Yield in Glycolysis

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Q6. Glucose Tolerance Test (GTT) and Significance

• Patient fasts overnight, fasting blood glucose measured
• 75 g oral glucose load given
• Blood glucose measured at 30 min, 1 hr, 2 hr intervals

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Q7. Enzymes and Coenzymes of Pyruvate Dehydrogenase Complex

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Q8. Significance of HMP Shunt (Hexose Monophosphate Shunt / Pentose Phosphate Pathway)

1. NADPH generation — for reductive biosynthesis (fatty acid synthesis, cholesterol synthesis), and maintaining glutathione in reduced form (protects RBCs from oxidative damage)
2. Ribose-5-phosphate — precursor for nucleotide/nucleic acid synthesis
3. Operates in liver, adrenal cortex, mammary gland, RBCs
4. G6PD deficiency — impaired HMP shunt → RBC oxidative damage → hemolytic anemia (especially with primaquine, fava beans)

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Q9. Glycogen Storage Diseases

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Q10. Gluconeogenesis (Definition)

1. Pyruvate carboxylase (pyruvate → OAA)
2. PEPCK (OAA → PEP)
3. Fructose-1,6-bisphosphatase (F-1,6-BP → F-6-P)
4. Glucose-6-phosphatase (G-6-P → glucose)

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LONG ANSWERS

Q1. Glycolysis — Biochemical Pathway, Enzymes, Energetics

1. Glucose + ATP → Glucose-6-phosphate (G-6-P) — Hexokinase (liver: glucokinase)
2. G-6-P → Fructose-6-phosphate — Phosphoglucose isomerase
3. F-6-P + ATP → Fructose-1,6-bisphosphate — Phosphofructokinase-1 (PFK-1) — rate-limiting step; most regulated
4. F-1,6-BP → DHAP + Glyceraldehyde-3-phosphate (G3P) — Aldolase
5. DHAP ↔ G3P — Triosephosphate isomerase

• 2 ATP consumed, 4 ATP produced → Net: 2 ATP
• 2 NADH produced in cytoplasm (= 5 ATP via malate-aspartate shuttle or 3 ATP via G3P shuttle)
• Total from glycolysis alone: 2 ATP + 2 NADH

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Q2. TCA Cycle — Reaction Sequence, Significance, Enzymes

• Citrate synthase: inhibited by ATP, NADH, succinyl-CoA
• Isocitrate dehydrogenase: activated by ADP; inhibited by ATP, NADH
• α-KG dehydrogenase: inhibited by NADH, succinyl-CoA

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Q3. Gluconeogenesis — Reactions, Significance

1. Maintains blood glucose during fasting/starvation
2. Utilizes lactate from anaerobic glycolysis (Cori cycle: muscle lactate → liver glucose)
3. Prevents lactic acidosis
4. Glucose-alanine cycle: muscle alanine → liver → glucose (amino acid carbon skeleton utilized)
5. Regulated by glucagon (stimulates) and insulin (inhibits)

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Q4. Glycogenesis and Glycogenolysis

• Glucose → G-6-P (hexokinase) → G-1-P (phosphoglucomutase) → UDP-glucose (UDP-glucose pyrophosphorylase) → added to glycogen chain by glycogen synthase (α-1,4 bonds)
• Branching: Branching enzyme transfers 6-7 glucose units to form α-1,6 branches

• Glycogen phosphorylase (rate-limiting): cleaves α-1,4 bonds → G-1-P
• Debranching enzyme: removes α-1,6 branches
• G-1-P → G-6-P (phosphoglucomutase) → glucose (glucose-6-phosphatase in liver, not muscle)
• Activated by glucagon (liver), epinephrine (liver + muscle) via cAMP → PKA → phosphorylase kinase → glycogen phosphorylase

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Q5. HMP Shunt — Biochemical Reactions and Significance

1. G-6-P + NADP⁺ → 6-Phosphogluconate + NADPH — G6PD (rate-limiting)
2. 6-Phosphogluconate + NADP⁺ → Ribulose-5-phosphate + CO₂ + NADPH

• Involves transketolase (TPP-dependent) and transaldolase
• Converts pentose phosphates back to F-6-P and G3P (re-enters glycolysis)

• Insulin (from β-cells): ↓ blood glucose → activates glycolysis, glycogenesis, lipogenesis; inhibits gluconeogenesis
• Glucagon (from α-cells): ↑ blood glucose → activates glycogenolysis, gluconeogenesis, lipolysis
• Epinephrine: emergency hyperglycemia (glycogenolysis)
• Cortisol: promotes gluconeogenesis
• Growth hormone: anti-insulin effect

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UNIT III — Lipid Metabolism

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VERY SHORT ANSWERS (2 marks)

Q1. Role of Citrate in Fatty Acid Synthesis

• When TCA cycle intermediates accumulate (high energy state), citrate is exported from mitochondria to cytoplasm via the citrate shuttle (tricarboxylate transporter)
• In cytoplasm, ATP-citrate lyase cleaves citrate → Acetyl-CoA (substrate for fatty acid synthesis) + OAA
• Citrate also allosterically activates acetyl-CoA carboxylase (ACC) — the rate-limiting enzyme of fatty acid synthesis (ACC converts Acetyl-CoA → Malonyl-CoA)
• Therefore, citrate signals "high energy" → promotes fat storage

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Q2. Ketolysis — Definition and Conditions

1. Prolonged fasting/starvation
2. Uncontrolled Type 1 DM (with diabetic ketoacidosis — high ketone body production due to unregulated lipolysis + fatty acid oxidation)
3. Low-carbohydrate (ketogenic) diet
4. Prolonged exercise

• Acetoacetate + Succinyl-CoA → Acetoacetyl-CoA (by succinyl-CoA transferase/thiophorase) → 2 Acetyl-CoA → TCA cycle
• Note: Liver cannot use ketone bodies (lacks thiophorase) — only produces them

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Q3. Ketosis — Definition and Significance

• Mild ketosis = normal adaptive response to starvation (glucose-sparing)
• Severe ketosis = Ketoacidosis (DKA in Type 1 DM or alcoholic ketoacidosis)
• DKA features: Kussmaul breathing (deep, rapid), fruity breath (acetone), nausea, vomiting, dehydration, metabolic acidosis

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Q4. Functional Significance of Fatty Acid Synthetase (FAS) Complex

• FAS is a multienzyme complex in the cytoplasm (especially liver, mammary gland, adipose)
• Catalyzes synthesis of palmitate (C16:0) from malonyl-CoA and acetyl-CoA
• Has 7 enzymatic activities on a single polypeptide chain
• Uses NADPH (from HMP shunt) as reducing equivalents
• Each cycle adds 2 carbons to growing chain
• 7 cycles: Acetyl-CoA (2C) + 7 Malonyl-CoA → Palmitate (16C) + 7 CO₂ + 8 CoA + 14 NADPH
• ACP (Acyl Carrier Protein) holds the growing chain (pantothenic acid component)

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Q5. Three Unsaturated Fatty Acids

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Q6. Structure of Cholesterol and its Role in the Body

• Steroid nucleus: cyclopentanoperhydrophenanthrene (4 fused rings — 3 cyclohexane + 1 cyclopentane)
• 27 carbons; hydroxyl group at C3; double bond at C5-C6; methyl groups at C10 and C13; side chain at C17
• Amphipathic molecule (OH group is hydrophilic; steroid rings + side chain are hydrophobic)

1. Cell membrane — structural component; maintains fluidity and permeability
2. Precursor of bile acids (cholate, chenodeoxycholate) — for fat digestion/absorption
3. Precursor of steroid hormones (cortisol, aldosterone, estrogen, testosterone, progesterone)
4. Precursor of Vitamin D (7-dehydrocholesterol → UV light → cholecalciferol)
5. Myelin formation in nervous system

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Q7. β-Oxidation in Fatty Acids

• Site: Mitochondrial matrix
• Activation: Fatty acid → Acyl-CoA (in cytoplasm, uses 2 ATP equivalents)
• Transport into mitochondria: Via carnitine shuttle (carnitine acyltransferase I — rate-limiting step)
• Steps (per cycle): Oxidation (FAD → FADH₂) → Hydration → Oxidation (NAD⁺ → NADH) → Thiolysis → Acetyl-CoA released
• Products per cycle: 1 FADH₂ + 1 NADH + 1 Acetyl-CoA

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Q8. Biologically Important Compounds from Cholesterol Catabolism

1. Bile acids (cholic acid, chenodeoxycholic acid) — conjugated with glycine/taurine → bile salts
2. Steroid hormones — glucocorticoids (cortisol), mineralocorticoids (aldosterone), sex hormones (estrogen, testosterone, progesterone)
3. Vitamin D3 (cholecalciferol)
4. Oxysterols — regulatory molecules

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Q9. Hypercholesterolemia — Definition and 2 Disorders

1. Familial Hypercholesterolemia (FH): Autosomal dominant; LDL receptor mutation → severely elevated LDL; xanthomas, premature coronary artery disease (MI in 3rd–4th decade)
2. Familial Combined Hyperlipidemia: Elevated LDL + VLDL; associated with Type 2 DM, obesity, metabolic syndrome; atherosclerosis risk

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Q10. Atherosclerosis

1. LDL enters intima → oxidized → taken up by macrophages → foam cells
2. Foam cells accumulate → fatty streaks → fibrous plaque → complicated plaque (calcification, ulceration, thrombosis)

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LONG ANSWERS

Q1. Biosynthesis of Cholesterol (15 marks)

1. 2 Acetyl-CoA → Acetoacetyl-CoA (thiolase)
2. Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA (HMG-CoA synthase)
3. HMG-CoA + 2 NADPH → Mevalonate — catalyzed by HMG-CoA reductase (rate-limiting step)
   • Target of statins (lovastatin, atorvastatin — competitive inhibitors of HMG-CoA reductase)

• HMG-CoA reductase: inhibited by cholesterol (feedback), oxysterols; activated by insulin; inhibited by glucagon
• Statins are the primary pharmacological intervention

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Q2. Ketone Bodies — Formation and Importance

1. Excess Acetyl-CoA (from β-oxidation) + Acetyl-CoA → Acetoacetyl-CoA (thiolase)
2. Acetoacetyl-CoA + Acetyl-CoA → HMG-CoA (HMG-CoA synthase — mitochondrial)
3. HMG-CoA → Acetoacetate + Acetyl-CoA (HMG-CoA lyase)
4. Acetoacetate + NADH → β-Hydroxybutyrate (β-hydroxybutyrate dehydrogenase)
5. Acetoacetate → Acetone + CO₂ (spontaneous decarboxylation)

• High OAA diverted to gluconeogenesis → TCA cycle cannot accept Acetyl-CoA → Acetyl-CoA overflow → ketone body synthesis
• High NADH (from β-oxidation) shifts OAA → malate (depleting OAA)

1. Alternative fuel during starvation — brain uses β-hydroxybutyrate (after 3 days of starvation, provides 60–70% brain energy)
2. Glucose-sparing — reduces protein catabolism
3. Heart and renal cortex prefer ketone bodies over glucose
4. Diagnostic value — ketonuria/ketonemia in DM, starvation

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Q3. β-Oxidation of Palmitic Acid — ATP Calculation

• 7 FADH₂ × 1.5 ATP = 10.5 ATP
• 7 NADH × 2.5 ATP = 17.5 ATP
• 8 Acetyl-CoA × 10 ATP (from TCA + ETC) = 80 ATP

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Q4. Bile Acids/Bile Salts Synthesis and Enterohepatic Circulation

1. Bile salts secreted from liver → bile → duodenum → emulsify fats
2. ~95% reabsorbed in terminal ileum → portal blood → liver → re-secreted
3. Only ~5% lost in feces (replaced by new synthesis)
4. Circulate 6–10 times per day

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UNIT IV — Biological Oxidation

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VERY SHORT ANSWERS (2 marks)

Q1. Uncouplers of Oxidative Phosphorylation

1. 2,4-Dinitrophenol (DNP) — carries H⁺ across membrane; was used as a weight-loss drug (dangerous)
2. Thermogenin (UCP-1) — natural uncoupler protein in brown adipose tissue (BAT); generates heat (non-shivering thermogenesis in neonates, hibernating animals)
3. CCCP (carbonyl cyanide m-chlorophenylhydrazone) — ionophore uncoupler
4. Aspirin (salicylate) — at high doses uncouples
5. Long-chain fatty acids — natural uncouplers
6. Bilirubin — at high concentrations (kernicterus)

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Q2. Two Inhibitors of ETC and Their Site of Action

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Q3. Substrate-level vs. Oxidative Phosphorylation

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Q4. Components of Electron Transport Chain (ETC)

1. Complex I — NADH dehydrogenase (NADH-CoQ reductase); contains FMN + Fe-S centers; pumps 4H⁺
2. Coenzyme Q (Ubiquinone) — mobile lipid-soluble carrier; accepts electrons from Complex I and II
3. Complex II — Succinate dehydrogenase (Succinate-CoQ reductase); contains FAD + Fe-S; does NOT pump H⁺
4. Complex III — Cytochrome bc1 complex (CoQ-Cyt c reductase); contains Cyt b, Fe-S, Cyt c₁; pumps 4H⁺
5. Cytochrome c — mobile protein carrier (water-soluble) in intermembrane space
6. Complex IV — Cytochrome c oxidase (Cyt aa₃); contains Cu centers (CuA, CuB) and Cyt a, a₃; pumps 2H⁺; reduces O₂ → H₂O
7. Complex V — ATP synthase (F₀F₁-ATPase); F₀ in membrane (H⁺ channel), F₁ in matrix (ATP synthesis)

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Q5. Chemiosmotic Theory of Oxidative Phosphorylation (Mitchell's Theory)

1. ETC pumps H⁺ from matrix to intermembrane space → creates electrochemical proton gradient (proton motive force)
2. Proton motive force has two components: pH gradient (ΔpH) + membrane potential (ΔΨ)
3. H⁺ flow back into matrix only through F₀ subunit of ATP synthase
4. This H⁺ flow drives rotation of ATP synthase's γ-subunit (rotor) → conformational changes in β-subunits → synthesis of ATP from ADP + Pi (binding change mechanism)
5. H⁺ pumped per NADH: Complex I (4H⁺) + Complex III (4H⁺) + Complex IV (2H⁺) = 10 H⁺/NADH
6. ~4 H⁺ required per ATP synthesized → 2.5 ATP per NADH (modern value)
7. FADH₂ enters at Complex II → only 6H⁺ pumped → 1.5 ATP per FADH₂

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Q6. Free Energy — Definition and Types

• ΔG < 0: spontaneous (exergonic)
• ΔG > 0: non-spontaneous (endergonic)
• Standard free energy (ΔG°') measured at pH 7, 25°C, 1 M concentrations

1. Chemical energy — stored in chemical bonds (ATP, NADH, glucose)
2. Kinetic energy — energy of motion (muscle contraction)
3. Electrical energy — membrane potential, nerve impulses
4. Thermal energy — heat (from uncoupled oxidation in BAT)
5. Osmotic energy — concentration gradients

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Q7. P:O Ratio

• For NADH: P:O ≈ 2.5 (modern values)
• For FADH₂: P:O ≈ 1.5

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Q8. Enzymes in Biological Oxidation

1. Oxidases — use O₂ directly (e.g., cytochrome c oxidase, amino acid oxidases)
2. Dehydrogenases — remove H from substrate (e.g., LDH, succinate DH, isocitrate DH)
3. Oxygenases — incorporate O₂ into substrate (monooxygenases/hydroxylases; dioxygenases)
4. Dismutases — e.g., Superoxide dismutase (SOD: 2O₂⁻ + 2H⁺ → H₂O₂ + O₂)
5. Peroxidases — reduce H₂O₂ (e.g., glutathione peroxidase)

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LONG ANSWERS

Q1. ETC — Steps and Mechanism (15 marks)

NADH → Complex I → CoQ → Complex III → Cyt c → Complex IV → O₂ (→H₂O)
FADH₂ → Complex II → CoQ → Complex III → Cyt c → Complex IV → O₂

• Accepts 2e⁻ from NADH → FMN → Fe-S clusters → CoQ
• Pumps 4H⁺ into intermembrane space
• Inhibited by rotenone, amytal, piericidin A

• Accepts 2e⁻ from FADH₂ (succinate oxidation) → Fe-S → CoQ
• Does NOT pump H⁺ — that's why FADH₂ yields less ATP
• Also part of TCA cycle (dual role)

• Accepts electrons from CoQH₂ → Cyt b → Fe-S → Cyt c₁ → Cyt c
• Q cycle doubles H⁺ pumping: pumps 4H⁺ per pair of electrons
• Inhibited by antimycin A

• Accepts 4e⁻ from 4 Cyt c → CuA → Cyt a → Cyt a₃-CuB → O₂
• Reduces O₂ + 4H⁺ → 2H₂O
• Pumps 2H⁺
• Inhibited by CN⁻, CO, azide, H₂S

• F₁ (matrix): α₃β₃γδε — β-subunits do catalysis (Boyer's binding change mechanism)
• F₀ (membrane): a, b₂, c₁₀₋₁₄ ring — H⁺ channel (oligomycin-sensitive)
• H⁺ flow rotates c-ring → rotates γ-subunit → β-subunit conformational changes: Open (O) → Loose (L) → Tight (T) → ATP released

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Q2. Oxidative Phosphorylation vs. Substrate-level Phosphorylation (see VSA Q3 above, expanded)

1. NAD⁺/NADH — niacin-derived; accepts 2e⁻ + H⁺ from dehydrogenation reactions; major electron carrier
2. FAD/FADH₂ — riboflavin-derived; in flavoproteins; accepts 2H
3. CoQ (Ubiquinone) — lipid-soluble; mobile carrier
4. Cytochromes — iron-containing; transfer single electrons
5. Lipoic acid — in PDH complex; covalently bound
6. CoA (pantothenic acid) — thioester bond; carries acyl groups

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UNIT V — Protein and Amino Acid Metabolism

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VERY SHORT ANSWERS (2 marks)

Q1. Transaminases (Aminotransferases)

• ALT (Alanine Aminotransferase = SGPT): Alanine + α-KG ↔ Pyruvate + Glutamate (liver-specific)
• AST (Aspartate Aminotransferase = SGOT): Aspartate + α-KG ↔ OAA + Glutamate (liver + heart + muscle)

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Q2. Diagnostic Importance of Transaminases

• ALT: 7–56 U/L
• AST: 10–40 U/L

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Q3. Normal Blood Urea Level and Conditions of Elevation

• Pre-renal: dehydration, heart failure, shock (↓ GFR)
• Renal: glomerulonephritis, renal failure (↓ GFR)
• Post-renal: urinary obstruction
• High protein diet; GI bleeding (protein absorption); increased catabolism (fever, infections)

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Q4. Metabolic Disorders of Urea Cycle (with enzyme defects)

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Q5. Maple Syrup Urine Disease (MSUD)

• Maple syrup odor of urine (due to sotolon)
• Feeding difficulties, vomiting in neonates
• Progressive neurological deterioration, seizures, mental retardation
• Ketoacidosis
• Diagnosis: Newborn screening (tandem mass spectrometry)
• Treatment: Dietary restriction of BCAAs + BCKD cofactor thiamine (B1) in some variants

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Q6. Albinism

• Absent pigmentation: white hair, pink/white skin, pink eyes (lack of melanin in iris)
• Photophobia, nystagmus, reduced visual acuity
• Increased risk of skin cancer (no UV protection from melanin)
• Autosomal recessive inheritance

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Q7. Jaundice (Icterus)

1. Prehepatic (Hemolytic): Excessive RBC breakdown → excess unconjugated bilirubin; dark stool; urobilinogen in urine; no bilirubin in urine (unconjugated is water-insoluble)
2. Hepatic (Hepatocellular): Liver disease (hepatitis, cirrhosis) → both conjugated and unconjugated ↑; mixed picture
3. Posthepatic (Obstructive/Cholestatic): Bile duct obstruction → conjugated bilirubin regurgitates into blood → pale stool; dark urine (bilirubinuria); no urobilinogen in urine

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Q8. Bile Pigments

• Hemoglobin (RBCs) → Heme → Biliverdin (green; by heme oxygenase; CO + Fe²⁺ also released) → Bilirubin (orange-yellow; by biliverdin reductase)
• Bilirubin (unconjugated = indirect) + albumin in blood → liver → conjugated with glucuronic acid → Bilirubin diglucuronide (conjugated = direct = water-soluble)
• Excreted in bile → gut → urobilinogen (by intestinal bacteria) → stercobilin (brown stool color) or reabsorbed → urobilin (yellow urine color)

• Neonatal jaundice (physiological; excess unconjugated bilirubin due to immature conjugation)
• Crigler-Najjar syndrome (severe UGT1A1 deficiency)
• Gilbert's syndrome (mild UGT1A1 deficiency)
• Dubin-Johnson, Rotor syndrome (conjugated hyperbilirubinemia)

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Q9. Porphyria

• Acute intermittent porphyria (AIP): PBG deaminase deficiency; abdominal pain, neuropsychiatric symptoms, NO skin photosensitivity; dark (port-wine) urine; induced by drugs (barbiturates, alcohol)
• Porphyria cutanea tarda (PCT): Uroporphyrinogen decarboxylase deficiency; skin photosensitivity, blistering; most common porphyria
• Erythropoietic porphyria (Gunther's disease): uroporphyrinogen III cosynthase deficiency; severe photosensitivity from birth; red urine; teeth fluoresce pink

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Q10. Nitrogen Balance

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LONG ANSWER

Q1. Urea Cycle — Detailed Note with Major Metabolic Disorders

• N-acetylglutamate (NAG) — obligate allosteric activator of CPS-I
• NAG synthase activated by arginine
• High protein diet → high ammonia → stimulates cycle

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Q2. Transamination Reactions in Amino Acid Catabolism

• Links amino acid catabolism to glycolysis/gluconeogenesis
• Pyruvate can enter gluconeogenesis → glucose (glucose-alanine cycle)

• Links amino acid catabolism to TCA cycle
• OAA enters TCA or gluconeogenesis

• Oxidative deamination: Glutamate + NAD⁺ → α-KG + NH₃ (by glutamate dehydrogenase) — main source of free NH₃ for urea cycle
• Transamination to other amino acids

• ALT/SGPT elevated in liver damage (hepatitis, drug toxicity)
• AST/SGOT elevated in liver disease + MI + muscle disease
• AST:ALT ratio > 2 = alcoholic liver disease

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Q3. Bile Pigments — Diseases Associated with Metabolism

1. Hemoglobin degraded in reticuloendothelial system (spleen, liver, bone marrow)
2. Globin → amino acids (recycled)
3. Fe²⁺ → ferritin/hemosiderin (recycled)
4. Heme → Biliverdin (by heme oxygenase; CO released)
5. Biliverdin → Unconjugated bilirubin (lipid-soluble; toxic at high levels → kernicterus)
6. Bilirubin binds albumin → transported to liver
7. Uptake by hepatocytes → conjugated with 2 glucuronic acid (by UDP-glucuronosyltransferase/UGT1A1) → Bilirubin diglucuronide (water-soluble, non-toxic)
8. Secreted into bile → intestine
9. Bacteria → urobilinogen → stercobilin (excreted in feces) or reabsorbed → portal blood → liver (re-secreted) or kidney (urobilin in urine)

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UNIT VI — Nucleic Acid Metabolism

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VERY SHORT ANSWERS (2 marks)

Q1. Okazaki Fragments

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Q2. Gout

• Purine nucleotides → IMP → Xanthine → Uric acid (by xanthine oxidase)
• Normal uric acid: 3.5–7 mg/dL (men), 2.5–6 mg/dL (women)

• Acute: Colchicine, NSAIDs
• Chronic: Allopurinol (inhibits xanthine oxidase; structural analog of hypoxanthine — competitive/mechanism-based inhibitor)

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Q3. Leading and Lagging Strands in DNA Replication

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Q4. Nucleotides — Definition + 4 Nucleotides

1. dAMP — Deoxyadenosine monophosphate (Adenine + deoxyribose)
2. dGMP — Deoxyguanosine monophosphate (Guanine + deoxyribose)
3. dCMP — Deoxycytidine monophosphate (Cytosine + deoxyribose)
4. dTMP — Deoxythymidine monophosphate (Thymine + deoxyribose)

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Q5. Mutation — Types

1. Point Mutations (single nucleotide changes):
   • Transition — purine ↔ purine or pyrimidine ↔ pyrimidine (A↔G, C↔T)
   • Transversion — purine ↔ pyrimidine (A/G ↔ C/T)
2. By consequence:
   • Silent (synonymous) — codon changes but same amino acid (due to degeneracy of genetic code)
   • Missense — codon changes → different amino acid (e.g., sickle cell: Glu→Val at β-globin position 6)
   • Nonsense — codon → stop codon → truncated protein
   • Frameshift — insertion/deletion of nucleotides (not multiple of 3) → completely altered reading frame
3. Chromosomal mutations: Deletions, inversions, translocations, duplications

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Q6. Semiconservative Replication of DNA

1. Initiation at origin of replication (ori)
2. Unwinding: Helicase unwinds double helix; Topoisomerase relieves supercoiling ahead
3. Primase synthesizes RNA primers (5'→3')
4. DNA Pol III (prokaryotes) / DNA Pol δ/ε (eukaryotes) elongates (5'→3')
5. Editing: 3'→5' exonuclease proofreading
6. RNA primers removed, replaced with DNA; DNA Ligase seals nicks

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Q7. Purines and Pyrimidines in DNA and RNA

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Q8. Inhibitors of Protein Synthesis and Site of Action

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Q9. Okazaki Fragments + DNA Repair Mechanisms (short note)

1. Direct reversal: Photolyase repairs pyrimidine dimers (UV damage) using light energy
2. Base Excision Repair (BER): DNA glycosylase removes damaged base → AP site → AP endonuclease → gap filled by Pol β
3. Nucleotide Excision Repair (NER): Removes bulky adducts (UV dimers); 12–13 nucleotides excised; deficient in Xeroderma pigmentosum (extreme skin cancer sensitivity)
4. Mismatch Repair (MMR): Corrects mismatches after replication; deficient in HNPCC (hereditary non-polyposis colorectal cancer)
5. Double-strand break repair: Homologous recombination (BRCA1/BRCA2) or Non-homologous end-joining (NHEJ)

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LONG ANSWERS

Q1. De Novo Synthesis of Purine Nucleotides

• N1 — Aspartate
• C2 — Formate (from THF/N¹⁰-formyl-THF)
• N3 — Glutamine
• C4, C5, N7 — Glycine
• C6 — CO₂
• N9 — Glutamine
• C8 — Formate (from THF)

1. PRPP + Glutamine → 5-Phosphoribosylamine (by PRPP amidotransferase — rate-limiting step; inhibited by AMP, ADP, ATP, GMP in feedback inhibition)
2. Addition of glycine → GAR
3. Formylation, amination, cyclization steps → IMP (the common purine precursor)

• AMP inhibits conversion of IMP → AMP (adenylosuccinate synthetase)
• GMP inhibits conversion of IMP → GMP (IMP dehydrogenase)
• Both inhibit PRPP amidotransferase (cross-regulation)

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Q2. Biosynthesis of Pyrimidine Nucleotides

1. Carbamoyl phosphate synthesized by CPS-II (cytoplasm; different from CPS-I of urea cycle which is mitochondrial)
   • Glutamine + CO₂ + 2ATP → Carbamoyl phosphate (CPS-II uses glutamine as N-donor)
2. Carbamoyl phosphate + Aspartate → Carbamoyl aspartate (Aspartate transcarbamoylase — ATCase)
3. Carbamoyl aspartate → Dihydroorotate (closure of ring)
4. Dihydroorotate → Orotate (by dihydroorotate dehydrogenase in mitochondria — only mitochondrial step)
5. Orotate + PRPP → OMP (orotate phosphoribosyltransferase)
6. OMP → UMP (OMP decarboxylase) ← rate-limiting step regulated

• dUMP + N⁵,N¹⁰-methylene-THF → dTMP + DHF (by thymidylate synthase)
• DHF → THF (by dihydrofolate reductase/DHFR) — target of methotrexate (anticancer)
• 5-Fluorouracil (5-FU) inhibits thymidylate synthase (anticancer)

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Q3. DNA Replication (Semiconservative) — Detailed

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Q4. Protein Biosynthesis (Translation)

• Start codon: AUG (methionine in eukaryotes; fMet in prokaryotes)
• mRNA + small subunit (30S/40S) + initiator tRNA → assembled with large subunit
• Requires initiation factors (IF1, IF2, IF3 in prokaryotes; eIFs in eukaryotes)

• Aminoacyl-tRNA enters A-site (codon-anticodon recognition)
• Transpeptidation: Peptide bond formed by peptidyl transferase (23S rRNA — ribozyme activity)
• Translocation: Ribosome moves 3' (one codon); EF-G (prokaryotes) or EF-2 (eukaryotes); GTP required

• Stop codons: UAA, UAG, UGA
• Release factors recognize stop codons → polypeptide released → ribosome dissociates

• Glycosylation, phosphorylation, acetylation, proteolytic cleavage, disulfide bond formation

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Q5. Mutations — Types + Repair (Detailed)

• Spontaneous: Tautomeric shifts, deamination (C→U), depurination, oxidative damage
• Induced: UV light (pyrimidine dimers), ionizing radiation (double-strand breaks), chemical mutagens (alkylating agents, intercalating agents, base analogs like 5-BU/2-aminopurine)

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UNIT VII — Introduction to Clinical Chemistry

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VERY SHORT ANSWERS (2 marks)

Q1. Semiquantitative Urine Analysis

• Physical: Color, clarity, specific gravity, volume
• Chemical: pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrites, leukocyte esterase
• Microscopic: Casts, cells, crystals, bacteria

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Q2. Urine Concentration Test + Significance

• Fishberg concentration test: Patient water-deprived for 16 hours; urine collected in 3 specimens (6-hourly); maximum specific gravity measured
• ADH test: Desmopressin administered; urine concentration measured

• Reduced concentrating ability: chronic renal failure, pyelonephritis, hypokalemia, hypercalcemia, diabetes insipidus
• Tests distal tubule and collecting duct function (ADH response)

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Q3. Creatinine Clearance Test + Significance

Creatinine clearance (mL/min) = (Urine creatinine × Urine volume) / Plasma creatinine

• Monitors progression of CKD (chronic kidney disease)
• Guides drug dosing in renal impairment
• < 60 mL/min = CKD stage 3 or higher
• < 15 mL/min = kidney failure → dialysis needed

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Q4. Urinary Tract Calculi (Kidney Stones)

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Q5. Role of Clinical Laboratory

1. Diagnosis — confirm/exclude diseases (blood tests, urine analysis, cultures, biopsies)
2. Monitoring — track disease progression and treatment response (HbA1c, LFTs, renal function)
3. Screening — identify disease in asymptomatic individuals (newborn screening, cancer markers)
4. Prognosis — predict disease outcome (troponin in MI, INR in liver failure)
5. Drug monitoring — therapeutic drug monitoring (digoxin, vancomycin levels)
6. Research — reference values, new diagnostic tests

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Q6. Normal Urine Constituents (4 examples)

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Q7. NPN (Non-Protein Nitrogen) Constituents

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Q8. Functions of the Kidney

1. Excretion — wastes (urea, creatinine, uric acid, drugs, toxins)
2. Fluid balance — regulates blood volume; tubular reabsorption and secretion
3. Electrolyte regulation — Na⁺, K⁺, Ca²⁺, Cl⁻, HCO₃⁻
4. Acid-base balance — H⁺ excretion, HCO₃⁻ reabsorption
5. Endocrine functions: Erythropoietin (EPO — stimulates RBC production), Renin (RAAS activation — blood pressure), Calcitriol (Vitamin D activation — calcium absorption)
6. Gluconeogenesis (renal cortex during prolonged starvation)

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LONG ANSWERS

Q1. Kidney Function Tests (KFTs) — Complete

1. Blood urea estimation — BUN; elevated in pre-renal, renal, post-renal causes; normal 7–20 mg/dL
2. Serum creatinine — more specific than BUN; normal 0.6–1.2 mg/dL; ↑ in CKD
3. Creatinine clearance (GFR) — best indicator of overall renal function; 90–120 mL/min
4. Inulin clearance — gold standard for GFR (research only)
5. Urea clearance — measures volume of blood cleared per minute; normal 70–75 mL/min

1. Urine concentration test — tests distal tubule/ADH response
2. Urine dilution test — patient drinks water; urine should dilute to sp. gravity < 1.003
3. Phenolsulfonphthalein (PSP) test — tubular secretion test (largely obsolete)
4. Glucose TM (tubular maximum) — threshold ~180 mg/dL; above this → glycosuria

• Serum urea (Diacetyl monoxime method / Urease method)
• Serum creatinine (Jaffe reaction — creatinine + picric acid → orange Janovsky complex)
• Serum uric acid (Uricase/phosphotungstic acid method)

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Q2. Abnormal Urine Constituents

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Q3. NPN Constituents — Tests and Significance

• Diacetyl monoxime method: Urea + diacetyl monoxime + H₂SO₄ + heat → pink/red color → spectrophotometry
• Urease method: Urea → NH₃ + CO₂ (urease) → NH₃ measured by Berthelot reaction (indophenol blue)
• Normal BUN: 7–20 mg/dL; Blood urea: 15–45 mg/dL

• Jaffe reaction (1886): Creatinine + alkaline picrate → orange-red Janovsky complex; measured at 520 nm
• Normal: 0.6–1.2 mg/dL (men); 0.5–1.0 mg/dL (women)
• ↑ in CKD, muscle disease, dehydration

• Uricase method (enzymatic): Uric acid + uricase → allantoin + H₂O₂; H₂O₂ measured (peroxidase + chromogen)
• Phosphotungstic acid method: Uric acid reduces phosphotungstic acid → tungsten blue color
• Normal: 3.5–7.0 mg/dL (men); 2.5–6.0 mg/dL (women)
• ↑ in gout, renal failure, tumor lysis, leukemia