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3rd Internal Assessment - Biochemistry Paper-2 (2025-26)

Krishnanagar Institute of Medical Science - Complete Answers

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QUESTION 1 (Main Question - 15 Marks)

A. Types of Mutations

Classification of Mutations

• Transition: Purine replaced by another purine (A↔G) or pyrimidine by pyrimidine (C↔T)
• Transversion: Purine replaced by pyrimidine or vice versa (A/G ↔ C/T)

• Missense mutation: One amino acid substituted by another (e.g., Sickle cell - Glu→Val)
• Nonsense mutation: Codon changed to stop codon - truncated protein
• Silent mutation: Base change but same amino acid coded (degeneracy of codons)
• Frameshift mutation: Insertion/deletion of non-multiple of 3 bases - completely altered downstream protein

NORMAL:   ATG - AAA - GGG - TTT - stop
MISSENSE: ATG - AAA - CGG - TTT - stop   (one AA changed)
NONSENSE: ATG - AAA - TAG - TTT          (premature stop)
FRAMESHIFT (deletion of A):
          ATG - AAG - GGT - TT...        (completely altered)

• Forward mutation: Wild type → mutant
• Back (reverse) mutation: Mutant → wild type
• Suppressor mutation: Second mutation at another site corrects the effect

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B. Mutagens (Agents Causing Mutations)

• Ultraviolet (UV) radiation - causes thymine dimers (cyclobutane rings between adjacent thymine bases)
• X-rays and gamma rays - cause DNA strand breaks, base modifications
• Cosmic radiation

• Transposons (jumping genes)
• Certain viruses (retroviruses inserting DNA)

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C. DNA Repair Mechanisms

Common Steps (applicable to most repair mechanisms):

Step 1: RECOGNITION of DNA distortion/damage
          ↓
Step 2: REMOVAL of the damaged region (excision)
          ↓
Step 3: RESYNTHESIS - DNA polymerase fills the gap (5'→3')
          ↓
Step 4: LIGATION - DNA ligase seals the nick
          ↓
     REPAIRED DNA

1. Nucleotide Excision Repair (NER)

1. Endonucleases recognize the helix distortion
2. An oligonucleotide of ~12-29 bases is removed (including the lesion)
3. DNA polymerase I/delta fills the gap using intact strand as template
4. DNA ligase seals the nick

2. Base Excision Repair (BER)

1. DNA glycosylase recognizes the damaged base and cleaves the N-glycosidic bond, releasing the damaged base
2. This creates an AP site (apurinic/apyrimidinic site)
3. AP endonuclease cleaves the backbone at the AP site
4. DNA polymerase fills the gap
5. DNA ligase seals the nick

3. Mismatch Repair (MMR)

• Mismatch recognition proteins (MutS in bacteria, MSH2/MSH6 in humans) bind the mismatch
• MutL (MLH1) nicks the newly synthesized strand
• Exonuclease removes the region containing the mismatch
• DNA polymerase + ligase complete repair
• Clinical: Defective MMR → Hereditary Non-Polyposis Colorectal Cancer (HNPCC/Lynch Syndrome)

4. Direct Repair (Photoreactivation)

• Photolyase enzyme directly cleaves the cyclobutane ring of thymine dimers using energy from visible light (300-500 nm)
• Found in bacteria, plants, some lower eukaryotes; NOT in humans
• No nucleotides are removed

5. Double-Strand Break Repair

• Homologous Recombination (HR): Uses the sister chromatid as template; error-free; active in S/G2 phase. Involves BRCA1, BRCA2, RAD51
• Non-Homologous End Joining (NHEJ): Directly joins broken ends; may be error-prone; active in G1 phase

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D. Initiation of Transcription in Prokaryotes

• RNA polymerase (RNAP): Core enzyme = α2ββ'ω; Holoenzyme = core + sigma (σ) factor
• Sigma (σ) factor: Confers promoter recognition ability (most common: σ70 in E. coli)
• Promoter: DNA sequence upstream of the transcription start site

• -35 element: Consensus sequence TTGACA (17 bp upstream of -10)
• -10 element (Pribnow box): Consensus sequence TATAAT (closest to start site)

DNA:  5'--TTGACA--(17 bp)--TATAAT--(~6 bp)--[+1 start site]--3'
              ↑                   ↑
           -35 box           -10 box (Pribnow box)

1. SIGMA FACTOR binds to RNA polymerase (holoenzyme formation)
          ↓
2. HOLOENZYME binds loosely to DNA (closed complex / recognition)
          ↓
3. HOLOENZYME scans DNA and binds to PROMOTER (-35 and -10 elements)
          ↓
4. DNA UNWINDING (~17 bp) → forms OPEN COMPLEX (melting)
          ↓
5. First phosphodiester bond formed (start with ATP or GTP)
          ↓
6. SIGMA FACTOR RELEASED (elongation begins)
          ↓
7. RNA chain elongation in 5'→3' direction

• No primer needed (unlike DNA polymerase)
• Transcription starts at +1 site
• The template strand is read 3'→5'; RNA is synthesized 5'→3'

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OR: BMI and Nutrition (Alternative for Q1)

• Reduce constipation and colon cancer risk (bulk, decreased transit time)
• Lower serum cholesterol (bind bile acids - soluble fibres like pectin, oat bran)
• Reduce glycaemic index of meals (slow glucose absorption)
• Decrease risk of type 2 diabetes
• Reduce cardiovascular disease risk (by lowering LDL cholesterol)
• Prebiotic effect - feed beneficial gut bacteria

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QUESTION 2: Explain Why (Any Five) - 5 × 3 = 15 Marks

i) Free Radicals Play a Beneficial Role by Killing Bacteria in Our Body

• Phagocytes (neutrophils and macrophages) deliberately generate free radicals to kill engulfed bacteria through a process called the Respiratory Burst (Oxidative Burst)
• When a phagocyte engulfs a bacterium:

RESPIRATORY BURST CASCADE:
O2 + NADPH → O2•- + NADP+   [NADPH oxidase enzyme]
         ↓
2 O2•- + 2H+ → H2O2 + O2    [Superoxide dismutase - SOD]
         ↓
H2O2 + Cl- → HOCl + OH-     [Myeloperoxidase - MPO]
         ↓
HOCl (hypochlorous acid) → DESTROYS bacterial cell walls/proteins

• HOCl (similar to bleach) is the most potent bactericidal agent produced
• Hydroxyl radicals (via Fenton reaction) also directly damage bacterial DNA, lipids, and proteins
• Clinical: Chronic Granulomatous Disease (CGD) - NADPH oxidase deficiency → recurrent bacterial infections because free radical killing is defective

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ii) Haemorrhagic Disease of the Newborn is More in Breast-Fed Babies

1. Low Vitamin K in breast milk: Human breast milk contains very low levels of Vitamin K (1-4 µg/L), while formula milk is supplemented with Vitamin K (50-100 µg/L)
2. Sterile gut: Newborns are born with a sterile gut; intestinal bacteria (that synthesize Vitamin K2/menaquinones) are not yet colonized at birth
3. Immature liver: The newborn liver is immature and has limited stores of Vitamin K
4. Vitamin K does not cross the placenta well

• Vitamin K is a cofactor for γ-carboxylation of glutamate residues in clotting factors II (prothrombin), VII, IX, X (also Protein C and S)
• Without carboxylation, these clotting factors cannot bind Ca2+ and phospholipid surfaces - rendering them inactive
• Result: defective clotting cascade → bleeding tendency

Without Vitamin K:
Factors II, VII, IX, X (PIVKA = Protein Induced by Vitamin K Absence)
         ↓
Cannot bind calcium → inactive
         ↓
Prolonged PT and aPTT
         ↓
BLEEDING (haemorrhagic disease of the newborn)

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iii) P53 is Termed the "Guardian of the Genome"

DNA DAMAGE (radiation, mutagens, hypoxia)
          ↓
P53 PROTEIN STABILIZED (normally rapidly degraded by MDM2)
          ↓
         / \
        /   \
       ↓     ↓
CELL CYCLE    APOPTOSIS
  ARREST       (if irreparable)
   (G1/S)
       ↓
 DNA REPAIR
       ↓
Cell survives with corrected DNA

1. Cell cycle arrest: Activates transcription of p21 (CIP1), which inhibits CDK4/6-cyclin D complex → arrests cell at G1/S checkpoint
2. DNA repair: Upregulates DNA repair genes (GADD45)
3. Apoptosis induction: Activates pro-apoptotic genes (BAX, PUMA, NOXA) if DNA damage is irreparable
4. Senescence: Permanently halts division of cells with damaged DNA
5. Antiangiogenesis: Inhibits tumour blood vessel formation

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iv) Genomic DNA Library and cDNA Library are NOT the Same

Genomic Library:
Genomic DNA → Restriction Enzymes → Fragments (with introns) → Cloned into vector

cDNA Library:
mRNA → Reverse Transcriptase → cDNA → Cloned into vector
(introns already removed by splicing)

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v) Defective Caspases in the Apoptotic Pathway May Lead to Cancer

EXTRINSIC PATHWAY              INTRINSIC PATHWAY
(Death receptors:               (Mitochondrial):
FasL, TNF-α)                   DNA damage, stress
      ↓                               ↓
FADD recruitment               BAX/BAK activation
      ↓                               ↓
Procaspase-8 →                 Cytochrome C release
Caspase-8                            ↓
      ↓                        APOPTOSOME formation
      ↓                        (Cytochrome C + Apaf-1 + Caspase-9)
      ↓←─────────────────────────────↓
           CASPASE-3 (Executioner)
                   ↓
      APOPTOSIS: protein/DNA degradation
                   ↓
           Phagocytosis of debris

1. When caspase-3, -8, or -9 are defective or absent, cells cannot execute apoptosis even when they have accumulated DNA damage
2. Normally, cells with irreparable DNA damage are eliminated by apoptosis (a surveillance mechanism)
3. Without apoptosis:
   • Genetically damaged/mutated cells survive
   • Continue to proliferate
   • Accumulate further mutations
   • Eventually transform into cancer cells
4. Many tumours show downregulation of caspases (e.g., reduced caspase-8 in neuroblastoma)
5. Also, Bcl-2 overexpression (which inhibits apoptosis) is found in follicular lymphoma

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vi) In Scurvy, Collagen Loses its Tensile Strength

1. Hydroxylation of proline → hydroxyproline (by prolyl hydroxylase)
2. Hydroxylation of lysine → hydroxylysine (by lysyl hydroxylase)

NORMAL COLLAGEN SYNTHESIS:
Proline/Lysine in pro-collagen
          ↓ (Vitamin C needed)
Hydroxylation → Hydroxyproline/Hydroxylysine
          ↓
Triple helix formation (stabilized by H-bonds with hydroxyproline)
          ↓
Secreted → Lysyl oxidase cross-links → STRONG COLLAGEN FIBERS

IN SCURVY (no Vitamin C):
Proline/Lysine NOT hydroxylated
          ↓
Unstable triple helix
          ↓
Cross-links CANNOT form
          ↓
WEAK, DISORGANIZED collagen
          ↓
Loss of tensile strength

• Bleeding gums, perifollicular haemorrhages
• Poor wound healing
• Corkscrew hairs
• Painful joints (collagen in tendons/cartilage weak)
• "Scorbutic rosary" in children (rib cartilage)
• Subperiosteal haemorrhages (collagen in periosteum weak)

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QUESTION 3: Short Notes (4 × 5 = 20 Marks)

i) Mucosal Block Theory of Iron Absorption

INTESTINAL LUMEN
Fe3+ (dietary) → Reduced to Fe2+ by:
                 - Stomach acid (HCl)
                 - Ferrireductase (DcytB) on brush border
                 - Vitamin C
          ↓
Fe2+ enters mucosal cell via DMT-1 (Divalent Metal Transporter-1)
          ↓
         INSIDE MUCOSAL CELL:
              ┌──────────────────────────────────┐
              │  Iron STORED as FERRITIN          │
              │  OR                               │
              │  Transported via FERROPORTIN-1    │
              │  to blood                         │
              └──────────────────────────────────┘
          ↓                           ↓
    MUCOSAL BLOCK:            WHEN BODY NEEDS IRON:
 Iron stored in ferritin     Ferritin stores empty
 in mucosal cells            → More ferroportin
 (when body stores full)     → More transfer to blood

• When body iron is sufficient: mucosal cells are pre-loaded with ferritin (apoferritin + iron)
• New dietary iron entering the mucosal cell quickly binds to this apoferritin - it is sequestered inside the cell
• The mucosal cells are shed every 2-3 days, taking their iron stores with them back into the intestinal lumen (excreted)
• Hepcidin (liver peptide) is the master regulator: high iron → high hepcidin → hepcidin degrades ferroportin → blocks iron export to blood

• Low hepcidin levels
• Ferroportin increases on basolateral surface
• More iron transferred to blood via transferrin

• Iron poisoning: Large doses overwhelm the mucosal block
• Haemochromatosis: Defective HFE gene → hepcidin is not induced → ferroportin not degraded → excessive iron absorption

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ii) Post-Translational Modifications (PTMs)

• Attachment of sugar chains (oligosaccharides) to protein
• N-linked (to asparagine-Asn): in ER and Golgi; e.g., antibodies, hormones
• O-linked (to serine/threonine): mainly in Golgi
• Function: protein folding, stability, cell recognition, receptor binding

• Addition of phosphate group (-PO4) to Ser, Thr, or Tyr residues by kinases (removed by phosphatases)
• Most common regulatory PTM
• Activates or inhibits enzyme/receptor activity
• Examples: Insulin receptor tyrosine kinase, glycogen phosphorylase

• Hydroxylation of proline (→ hydroxyproline) in collagen (requires Vit C)
• Adds -OH group, enables cross-linking

• Addition of -COOH to glutamate (→ γ-carboxyglutamate) in clotting factors II, VII, IX, X
• Requires Vitamin K

• Addition of acetyl group to N-terminal amino acids or Lys residues
• Important in histone modification (gene regulation)

• Attachment of ubiquitin peptide to Lys residues
• Tags proteins for proteasomal degradation
• Key in cell cycle regulation and protein quality control

• Addition of methyl groups (histones, DNA regulation)

• Removal of signal peptides, propeptides
• Activation of zymogens (e.g., trypsinogen → trypsin; proinsulin → insulin)

• S-S bonds between cysteine residues stabilize 3D structure
• Occurs in ER (oxidizing environment)

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iii) Type-1 Xenobiotic Reactions (Phase I / Dehydrogenase Reactions)

• Microsomal oxidation (CYP450 system): Located in smooth ER of liver; uses Cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP3A4, etc.)

RH + O2 + NADPH + H+ → ROH + H2O + NADP+
(xenobiotic)           (hydroxylated product)

• Types: Aromatic hydroxylation, aliphatic hydroxylation, N-dealkylation, O-dealkylation, S-oxidation, Deamination, Epoxidation

• Azo-reduction: Breaks azo linkage (-N=N-) → aromatic amines
• Nitro-reduction: -NO2 → -NH2
• Carbonyl reduction: Ketones/aldehydes → alcohols

• Ester hydrolysis (by esterases/pseudocholinesterases): Esters → acids + alcohols
• Amide hydrolysis: Amides → acids + amines
• Epoxide hydration

• Alcohol dehydrogenase (ADH): Ethanol → Acetaldehyde
• Aldehyde dehydrogenase (ALDH): Acetaldehyde → Acetate
• These are important Phase I steps in alcohol metabolism

CH3CH2OH → [ADH, NAD+] → CH3CHO → [ALDH, NAD+] → CH3COO-
 Ethanol                Acetaldehyde              Acetate

• May activate pro-drugs (codeine → morphine)
• May generate toxic metabolites (e.g., paracetamol → NAPQI by CYP2E1)
• Products may then undergo Phase II conjugation reactions

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iv) Inhibitors of Protein Synthesis

PROKARYOTIC TRANSLATION:

                    AMINOGLYCOSIDES (streptomycin, gentamicin)
                    - Cause misreading of mRNA (30S subunit)
                              ↓
         30S ────────────────────────────────────────
INITIATION:                                             ← LINEZOLID (30S initiation)
        mRNA + tRNA(fmet) + 30S + 50S
                    ↓
ELONGATION:
  A-site: aminoacyl-tRNA binding  ← TETRACYCLINES (block A-site, 30S)
  Peptide bond formation          ← CHLORAMPHENICOL (inhibits peptidyl transferase, 50S)
  Translocation                   ← FUSIDIC ACID (blocks EF-G)
                    ↓
TERMINATION/TRANSLOCATION         ← ERYTHROMYCIN/MACROLIDES (block translocation, 50S, tunnel)
                    ↓
         50S ─────────────────────────────────────────

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QUESTION 4: Short Notes (3 × 6 = 18 Marks)

i) Mechanism of Action of Glucagon, Norepinephrine, and Epinephrine

Mechanism of Action via cAMP Pathway:

HORMONE (Glucagon/Epinephrine/Norepinephrine)
          ↓  (binds to GPCR on cell membrane)
G-PROTEIN COUPLED RECEPTOR (GPCR)
          ↓  (Gs protein activated - GDP→GTP)
ADENYLYL CYCLASE (activated)
          ↓
ATP → cAMP (cyclic AMP)
          ↓
PROTEIN KINASE A (PKA) activated
[Regulatory subunits dissociate from catalytic subunits upon cAMP binding]
          ↓
PHOSPHORYLATION OF TARGET ENZYMES

Effects in Different Tissues:

• In liver: Same as glucagon (via β2 receptors / cAMP) - glycogenolysis
• In muscle: Glycogenolysis (via β2 receptors) - glucose for immediate energy
• In adipose tissue: Activates HSL → fatty acid release (energy source)
• In heart: (+) chronotropic and inotropic effects via β1 receptors
• Also acts via α1 receptors (IP3/DAG pathway) in some tissues

• Primarily acts via α1 and α2 receptors
• α1: vasoconstriction (IP3/DAG pathway) → increases blood pressure
• α2: presynaptic inhibition (decreases cAMP)
• Less effect on heart rate than epinephrine
• Metabolic effects similar to epinephrine but less pronounced

SUMMARY OF cAMP PATHWAY:

Hormone → Receptor → Gs → Adenylyl cyclase → ATP → cAMP
                                                       ↓
                                                    PKA activated
                                                       ↓
                                          Phosphorylates enzymes
                                         (cascade amplification)
                                                       ↓
                              GLYCOGENOLYSIS + LIPOLYSIS + GLUCONEOGENESIS
                              (Metabolic effects)
cAMP degraded by Phosphodiesterase (PDE) → 5'-AMP (terminates signal)

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ii) Proto-Oncogenes, Oncogenes, and Tumour Suppressor Genes

Proto-Oncogenes

• Definition: Normal cellular genes that promote cell growth, proliferation, and differentiation
• They encode proteins like growth factors, growth factor receptors, signal transducers, and transcription factors
• Examples: RAS (G-protein signal transducer), MYC (transcription factor), ERBB2/HER2 (growth factor receptor), SRC (tyrosine kinase)
• Under tight regulatory control in normal cells

Oncogenes

• Definition: Mutated/overexpressed versions of proto-oncogenes that cause uncontrolled cell proliferation
• Act as "gain-of-function" mutations - dominant (one mutant allele is sufficient)
• A proto-oncogene is converted to an oncogene by:
  1. Point mutation (e.g., RAS → KRAS mutation in codons 12, 13 - constitutively active)
  2. Chromosomal translocation (e.g., BCR-ABL in CML - Philadelphia chromosome t(9;22))
  3. Gene amplification (e.g., HER2/neu amplification in breast cancer)
  4. Retroviral insertion (provirus inserted near proto-oncogene → its overexpression)

PROTO-ONCOGENE (normal):
Growth signal → Receptor → Signal → Response → STOPS when signal stops

ONCOGENE (mutated):
NO growth signal needed → CONSTITUTIVELY ACTIVE SIGNAL → continuous proliferation
(like a car accelerator stuck down)

Tumour Suppressor Genes (Anti-Oncogenes)

• Definition: Genes that normally inhibit cell division, promote apoptosis, or maintain genomic stability
• Act as "loss-of-function" mutations - recessive (BOTH alleles must be inactivated - "Two-Hit Hypothesis" by Knudson)
• Examples:

TWO-HIT HYPOTHESIS (Knudson):

Sporadic cancer:          Familial cancer:
Hit 1: Somatic mutation  Hit 1: Germline mutation (inherited)
Hit 2: Somatic mutation  Hit 2: Somatic mutation in target tissue
Both alleles lost → TSG inactivated → Cancer

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iii) Changes in Serum LFT (Liver Function Tests) in Different Types of Jaundice

Haemoglobin (RBC breakdown)
          ↓ (in reticuloendothelial system)
Unconjugated (indirect) bilirubin [insoluble, bound to albumin]
          ↓  (uptake by liver hepatocytes)
Conjugated (direct) bilirubin [water-soluble, glucuronide]
          ↓
Secreted into bile → intestine → urobilinogen/stercobilin (stool)
                    ↑
              Part reabsorbed (enterohepatic circulation)
              → urobilinogen in urine

Comparison Table of LFT in Different Types of Jaundice:

• In obstructive jaundice: ALP is very high (produced by bile duct epithelium under bile pressure), direct bilirubin predominates, acholic stools, no urobilinogen in urine
• In hepatocellular: ALT/AST dramatically elevated (hepatocyte necrosis releases enzymes), mixed bilirubinemia
• In pre-hepatic: Only unconjugated bilirubin rises, all liver enzymes normal, dark urine (urobilinogen), dark stools

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QUESTION 5: Short Notes (4 × 5 = 20 Marks)

i) Renal Defence in Acid-Base Imbalance

1. Bicarbonate Reabsorption (Proximal Tubule - 85%)

In tubular lumen:
HCO3- + H+ → H2CO3 → CO2 + H2O
(CO2 diffuses into cell)

Inside tubular cell:
CO2 + H2O → [Carbonic anhydrase] → H2CO3 → H+ + HCO3-

H+ secreted back into lumen (via Na+/H+ antiporter)
HCO3- enters blood via Na+/HCO3- cotransporter

2. Titratable Acid Excretion (Distal Tubule)

• H+ secreted by H+-ATPase combines with HPO4²- in tubule:
• HPO4²- + H+ → H2PO4- (excreted in urine)
• This allows excretion of H+ while regenerating HCO3-

3. Ammonium Excretion (Distal Tubule/Collecting Duct)

Glutamine → [glutaminase in renal cells] → NH3 + Glutamate
NH3 + H+ → NH4+ (trapped in tubular lumen, excreted in urine)

Response to Acid-Base Disorders:

• ↑ H+ secretion
• ↑ NH4+ excretion (NH3 production from glutamine increases)
• ↑ Titratable acid excretion
• ↑ HCO3- reabsorption → Net result: Urine becomes acidic (pH < 5.5), new HCO3- generated

• ↓ H+ secretion
• ↑ HCO3- excretion in urine → Urine becomes alkaline (pH > 7)

• Same as metabolic acidosis response (kidneys compensate over 3-5 days)

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ii) Lac Operon

Structure:

REGULATORY GENE    OPERATOR    STRUCTURAL GENES
      |               |         |
    lacI ── P ── O ── lacZ ── lacY ── lacA
              ↑
           Promoter

lacZ = β-galactosidase (breaks lactose → glucose + galactose)
lacY = β-galactoside permease (transports lactose into cell)
lacA = β-galactoside transacetylase (minor role)

Regulation:

lacI gene → constitutively expressed → REPRESSOR PROTEIN synthesized
                                              ↓
                                       Binds to OPERATOR
                                              ↓
                            RNA polymerase CANNOT transcribe lacZ, Y, A
                                              ↓
                                       Operon is SWITCHED OFF

Lactose enters cell → converted to ALLOLACTOSE
                              ↓
                    Allolactose binds to REPRESSOR
                              ↓
                   Repressor INACTIVATED (allosteric change)
                              ↓
               Operator FREE → RNA polymerase binds promoter
                              ↓
            lacZ, lacY, lacA genes TRANSCRIBED
                              ↓
          Lactose metabolised (β-galactosidase cleaves it)

• When glucose is available, cAMP levels are LOW → CAP (Catabolite Activator Protein) is inactive → weak transcription even if repressor removed
• When glucose is absent, cAMP levels RISE → cAMP binds CAP → CAP-cAMP binds to DNA upstream of promoter → ENHANCES RNA polymerase binding → strong transcription

BOTH required for full expression:
1. Lactose present (removes repressor) AND
2. Glucose absent (high cAMP → CAP activated)

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iii) Different Renal Clearance Tests in Chronic Kidney Disease (CKD)

• U = urinary concentration of substance
• V = urine volume per minute
• P = plasma concentration of substance

Tests Used in CKD:

• Inulin is freely filtered, not reabsorbed, not secreted, not metabolized
• Inulin clearance = True GFR = 125 mL/min (normal)
• Not used routinely (requires IV infusion, complex)

• Endogenous marker, 24-hour urine collection
• Slightly overestimates GFR (some tubular secretion of creatinine)
• Normal: ~90-120 mL/min
• Formula: (Urine Cr × Urine Vol/min) / Plasma Cr
• In CKD: decreases proportionally to GFR loss

• Uses serum creatinine + age + sex + race
• CKD staging based on eGFR:

• PAH is filtered + actively secreted → completely cleared in one pass
• PAH clearance = Effective Renal Plasma Flow = ~625 mL/min
• Renal Blood Flow = RPF / (1 - Hct) = ~1200 mL/min

• Glucose Tm: Threshold = ~180 mg/dL plasma glucose; Tm glucose = ~375 mg/min (tubular reabsorption capacity)
• Tm PAH: Measures maximal tubular secretion capacity = ~80 mg/min

• Older test, rarely used now
• Partially reabsorbed in tubules; underestimates GFR
• Normal: 70 mL/min

• Creatinine and urea rise in serum (azotemia)
• GFR progressively falls
• Phosphate clearance falls → hyperphosphatemia
• Bicarbonate clearance affected → metabolic acidosis

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iv) Types of Hypersensitivity Reactions

Type I - Immediate (IgE-Mediated / Anaphylactic)

First exposure:
Antigen (allergen) → IgE antibodies produced → IgE binds to mast cells/basophils

Second exposure:
Allergen crosslinks IgE on mast cells
          ↓
DEGRANULATION of mast cells
          ↓
Release of: Histamine, Leukotrienes (LTC4, LTD4), PGD2, Tryptase
          ↓
Vasodilation, bronchoconstriction, ↑ vascular permeability, ↑ mucus

• Time: Within minutes (15-30 min)
• Examples: Allergic rhinitis, asthma, urticaria, anaphylaxis, food allergy
• Mediators: Histamine, leukotrienes, prostaglandins
• Test: Skin prick test, serum IgE, RAST

Type II - Cytotoxic (IgG or IgM antibody against cell-surface antigens)

Antigen on cell surface → IgG/IgM antibodies
          ↓
Complement activation (MAC - membrane attack complex)
OR
ADCC (Antibody-Dependent Cellular Cytotoxicity)
OR
Opsonization → phagocytosis
          ↓
Cell destruction

• Time: Hours
• Examples: Haemolytic anaemia (drug-induced), transfusion reactions, Rh incompatibility, Goodpasture's syndrome, Myasthenia gravis (receptor blockade)

Type III - Immune Complex-Mediated

Antigen + Antibody → IMMUNE COMPLEXES
          ↓
Deposited in blood vessel walls, glomeruli, joints, skin
          ↓
Complement activation → neutrophil recruitment → lysosomal enzymes
          ↓
TISSUE DAMAGE (vasculitis, nephritis)

• Time: 4-8 hours (Arthus reaction); 10-21 days (serum sickness)
• Examples: SLE, post-streptococcal glomerulonephritis, serum sickness, farmer's lung
• Lab: Low complement (C3, C4), immune complexes in blood/tissue

Type IV - Delayed (Cell-Mediated / T-cell mediated)

Antigen presented to T cells (by APC via MHC II)
          ↓
Sensitized CD4+ Th1 cells (memory cells form)

Second exposure:
CD4+ T cells activated → release cytokines (IFN-γ, TNF-α, IL-2)
          ↓
Macrophage activation → granuloma formation
CD8+ CTLs directly kill antigen-bearing cells
          ↓
TISSUE DAMAGE

• Time: 48-72 hours (delayed)
• Examples: Contact dermatitis (nickel, poison ivy), tuberculin test (Mantoux), graft rejection, type 1 diabetes

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QUESTION 6: Mark the Correct Response (12 × 1 = 12 Marks)

i) In Obstructive Jaundice, Which Enzyme is NOT Significantly Elevated?

• ALP (Alkaline Phosphatase): ↑↑↑↑ (markedly elevated) - produced by bile duct epithelium in response to back-pressure of bile; hallmark of cholestasis
• GGT (Gamma-Glutamyl Transferase): ↑↑↑ - also elevated in obstructive jaundice; confirms biliary source of ALP rise
• 5'-NT (5'-Nucleotidase): ↑↑ - liver-specific enzyme elevated in cholestasis; differentiates hepatic from bone source of ALP
• ALT (Alanine Aminotransferase): NOT significantly elevated - ALT is a marker of hepatocellular damage/necrosis. In pure obstructive jaundice (without secondary hepatocellular damage), ALT is normal or only mildly raised. It rises dramatically only when hepatocytes are damaged (hepatitis, acute liver injury).

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ii) A 10-Year-Old Girl with Pellagra-Like Rash and Increased Urinary Indole Acetic Acid and Indole Pyruvic Acid

1. Tryptophan cannot be absorbed from intestine → passes to colon → bacterial metabolism of tryptophan → produces Indole Acetic Acid and Indole Pyruvic Acid → excreted in large amounts in urine (aminoaciduria of neutral amino acids)
2. Tryptophan cannot be reabsorbed in renal tubules → neutral aminoaciduria
3. Tryptophan is precursor to Niacin (Vitamin B3) - without tryptophan, Niacin cannot be synthesized → Pellagra-like symptoms (4 D's: Dermatitis, Diarrhoea, Dementia, Death)
4. Neurological symptoms - tryptophan is also the precursor to serotonin and affects brain function

• Phenylketonuria: Deficiency of phenylalanine hydroxylase; elevated phenylalanine and phenylpyruvic acid in urine, NOT indole compounds; presents with intellectual disability and fair skin
• Alkaptonuria: Deficiency of homogentisic acid oxidase; homogentisic acid in urine → darkens on standing (ochronosis); no neurological symptoms initially
• Homocystinuria: Deficiency of cystathionine synthase; elevated homocysteine; Marfan-like features, lens dislocation, thrombosis; no pellagra

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iii) Hyperventilation, pH 7.3, pCO2 20 mmHg, Serum Bicarbonate 23 mEq/L

• pH = 7.3 → ACIDOSIS (below 7.35)
• pCO2 = 20 mmHg → very LOW (normal 35-45) → points to respiratory ALKALOSIS (excess CO2 blown off by hyperventilation)
• HCO3- = 23 mEq/L → near normal

• The hyperventilation is compensatory for an underlying metabolic acidosis
• HCO3- of 23 is still near normal initially (the question cuts off)
• The pCO2 of 20 with pH 7.3 suggests metabolic acidosis with respiratory compensation (Kussmaul breathing)

• pH ↑ (alkalosis)
• pCO2 ↓ (PRIMARY change due to hyperventilation)
• HCO3- ↓ slightly (renal compensation, takes days)

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Sources: Basic Medical Biochemistry - A Clinical Approach 6e (Lieberman & Peet); Robbins & Kumar Pathologic Basis of Disease; Janeway's Immunobiology 10e; Thompson & Thompson Genetics & Genomics in Medicine 9e; Tietz Textbook of Laboratory Medicine 7e; Harrison's Principles of Internal Medicine 22e.