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Biochemistry & Cell Biology - Complete Examination Notes

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1. GLYCOLYSIS

• Preparatory (investment) phase: 2 ATP consumed; glucose → glucose-6-phosphate → fructose-6-phosphate → fructose-1,6-bisphosphate → 2 triose phosphates
• ATP-generating phase: 4 ATP + 2 NADH produced per glucose

1. Hexokinase (Step 1) - glucose → G-6-P; inhibited by G-6-P
2. Phosphofructokinase-1 (PFK-1) (Step 3) - rate-limiting enzyme; activated by AMP, ADP, fructose-2,6-bisphosphate; inhibited by ATP, citrate
3. Pyruvate kinase (Step 10) - PEP → pyruvate

• Aerobic: enters mitochondria → Acetyl-CoA (via pyruvate dehydrogenase)
• Anaerobic: → Lactate (by LDH); regenerates NAD+

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2. MITOCHONDRIA

• Double membrane - outer membrane (contains porins/channel proteins) and inner membrane (highly folded into cristae)
• Matrix - contains TCA cycle enzymes, mtDNA, ribosomes
• Cristae - site of oxidative phosphorylation enzymes (ETC + ATP synthase)

1. ATP production via oxidative phosphorylation
2. TCA cycle (Krebs cycle)
3. β-oxidation of fatty acids
4. Urea cycle (partially)
5. Apoptosis regulation (release of cytochrome c)
6. Calcium homeostasis
7. Steroid hormone synthesis (cholesterol side-chain cleavage)

• Self-replicating (contain their own DNA - 37 genes)
• Maternal inheritance of mtDNA
• Number varies: few in adipocytes, many in cardiomyocytes
• Size: 0.2-1 µm diameter; 1-10 µm length
• The inner membrane is impermeable to most ions; uses specific transporters

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3. TCA (KREBS/CITRIC ACID) CYCLE

• Citrate synthase
• Isocitrate dehydrogenase
• α-Ketoglutarate dehydrogenase

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4. CARBOHYDRATES - CLASSIFICATION & PROPERTIES

Classification:

• Trioses: Glyceraldehyde, DHAP (3C)
• Pentoses: Ribose, Deoxyribose (5C) - components of nucleic acids
• Hexoses: Glucose, Fructose, Galactose (6C)

• Sucrose = Glucose + Fructose (α1→β2)
• Lactose = Galactose + Glucose (β1→4); milk sugar
• Maltose = Glucose + Glucose (α1→4); from starch hydrolysis

• Homopolysaccharides: Starch (amylose + amylopectin), Glycogen, Cellulose
• Heteropolysaccharides: Heparin, Hyaluronic acid, Chondroitin sulfate

Properties:

• Reducing sugars: have free anomeric carbon; reduce Benedict's/Fehling's (glucose, fructose, maltose, lactose)
• Non-reducing: sucrose (no free anomeric -OH)
• Optical activity: rotate polarized light (D and L isomers)
• Mutarotation: change in optical rotation due to interconversion of α/β forms

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5. ENZYMES

• Highly specific (substrate and reaction specificity)
• Reusable, not consumed
• Active at physiological pH and temperature
• Regulated

1. Oxidoreductases (oxidation-reduction)
2. Transferases (transfer functional groups)
3. Hydrolases (hydrolysis)
4. Lyases (addition/removal to double bonds)
5. Isomerases (isomerization)
6. Ligases (bond formation with ATP)

Coenzymes:

• NAD+/NADH - derived from Niacin (B3); electron carrier
• FAD/FADH₂ - derived from Riboflavin (B2); electron carrier
• Coenzyme A - derived from Pantothenic acid (B5); acyl group carrier
• TPP (Thiamine pyrophosphate) - from B1; decarboxylation reactions
• Pyridoxal phosphate (PLP) - from B6; transamination, decarboxylation of amino acids
• Biotin - CO₂ carrier (carboxylation reactions, e.g., pyruvate carboxylase)
• Tetrahydrofolate (THF) - one-carbon transfer
• Vitamin B12 - isomerization reactions

Cofactors:

• Zn²+ - carbonic anhydrase, carboxypeptidase
• Mg²+ - kinases (stabilizes ATP), enolase
• Fe²+/Fe³+ - cytochromes, catalase, peroxidase
• Cu²+ - cytochrome oxidase, ceruloplasmin
• Mn²+ - arginase, pyruvate carboxylase
• K+ - pyruvate kinase
• Se - glutathione peroxidase

Factors Affecting Enzyme Activity:

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6. LIPOLYSIS AND β-OXIDATION

Lipolysis:

1. Hormone-sensitive lipase (HSL) - rate-limiting; activated by glucagon/epinephrine (via cAMP-PKA); inhibited by insulin
2. Monoacylglycerol lipase

• Fasting/stress → glucagon/catecholamines → ↑cAMP → PKA activates HSL → lipolysis ↑
• Fed state → insulin inhibits HSL → lipolysis ↓

β-Oxidation (Fatty Acid Oxidation):

• Carnitine palmitoyltransferase I (CPT-I) on outer membrane - rate-limiting step; inhibited by malonyl-CoA
• Carnitine palmitoyltransferase II (CPT-II) on inner membrane

1. Oxidation (FAD) → FADH₂ + trans-enoyl-CoA
2. Hydration → L-hydroxyacyl-CoA
3. Oxidation (NAD+) → NADH + β-ketoacyl-CoA
4. Thiolysis → Acetyl-CoA + shortened fatty acyl-CoA

• 7 cycles → 7 FADH₂ + 7 NADH + 8 Acetyl-CoA
• Total ATP ≈ 106 (net ~129 ATP) per palmitate molecule

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7. LIPOPROTEINS

• ApoB-48: intestinal chylomicrons
• ApoB-100: VLDL, IDL, LDL; ligand for LDL receptor
• ApoA-I: HDL; activates LCAT
• ApoC-II: activates lipoprotein lipase (LPL)
• ApoE: hepatic clearance (binds to LDL-R)

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8. ACID-BASE BALANCE AND BLOOD BUFFERS

• Acidosis: pH < 7.35
• Alkalosis: pH > 7.45

Buffer Systems in Blood:

• pH = pKa + log [HCO₃⁻]/[H₂CO₃] (Henderson-Hasselbalch)
• pH = 6.1 + log [HCO₃⁻]/[0.03 × pCO₂]
• Normal: HCO₃⁻ = 24 mEq/L; pCO₂ = 40 mmHg; ratio = 20:1
• Regulated by: kidneys (HCO₃⁻) and lungs (CO₂)

• H₂PO₄⁻ ⇌ H+ + HPO₄²⁻; pKa = 6.8

• Imidazole groups of histidine; N-terminal amino groups

• Deoxyhemoglobin is a better buffer than oxyhemoglobin
• Bohr effect: CO₂ + H₂O → H₂CO₃ → H+ + HCO₃⁻; H+ binds Hb

Acid-Base Disorders:

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9. ESSENTIAL AMINO ACIDS

• Arginine (children, pregnancy)
• Tyrosine (when phenylalanine is deficient or in PKU)
• Cysteine (when methionine is deficient)
• Glutamine (critically ill patients)

• Positive: growth, pregnancy (synthesis > degradation)
• Negative: starvation, illness, burn injuries (degradation > synthesis)
• Equilibrium: healthy adult

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10. VITAMINS - FAT-SOLUBLE VITAMINS

Vitamin A (Retinol)

• Forms: Retinol, Retinal, Retinoic acid; provitamin = β-carotene
• Functions: Vision (retinal + opsin = rhodopsin), epithelial differentiation, reproduction, immune function
• Deficiency: Night blindness, Bitot's spots, xerophthalmia, keratomalacia
• Toxicity: Pseudotumor cerebri, hepatotoxicity, teratogenic

Vitamin D (Calciferol)

• Forms: D2 (ergocalciferol, plants), D3 (cholecalciferol, skin/sunlight)
• Activation: Liver (25-OH-D3) → Kidney (1,25-(OH)₂-D3 = calcitriol; active form); enzyme = 1α-hydroxylase
• Functions: Intestinal absorption of Ca²+ and PO₄³⁻, bone mineralization, regulates PTH
• Deficiency: Rickets (children), Osteomalacia (adults), hypocalcemia
• Toxicity: Hypercalcemia, nephrocalcinosis

Vitamin E (Tocopherol)

• Function: Antioxidant - protects polyunsaturated fatty acids (PUFAs) from peroxidation; maintains RBC integrity; prevents oxidative hemolysis
• Deficiency: Rare; hemolytic anemia (neonates), peripheral neuropathy, spinocerebellar ataxia
• Toxicity: Rare; interferes with Vitamin K function (anticoagulant effect)

Vitamin K (Phylloquinone, Menaquinone)

• Forms: K1 (phylloquinone, plants), K2 (menaquinone, bacteria), K3 (menadione, synthetic)
• Function: Cofactor for γ-carboxylase; activates clotting factors II, VII, IX, X and anticoagulants Protein C and S; bone protein osteocalcin
• Deficiency: Bleeding tendency, prolonged PT, hemorrhagic disease of newborn
• Antagonist: Warfarin (inhibits VKOR - Vitamin K epoxide reductase)

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11. PHOSPHOLIPIDS

Classification:

• Phosphatidylcholine (lecithin) - most abundant; lung surfactant
• Phosphatidylethanolamine (cephalin) - brain, nervous tissue
• Phosphatidylserine - intracellular; flips to outer leaflet during apoptosis
• Phosphatidylinositol - signal transduction (PIP₂ → IP₃ + DAG)
• Phosphatidylglycerol - mitochondrial membrane
• Cardiolipin (diphosphatidylglycerol) - inner mitochondrial membrane; antigen in syphilis (VDRL test)

• Abundant in nerve myelin sheath
• Cleaved by sphingomyelinase → ceramide (deficiency causes Niemann-Pick disease)

Functions:

1. Major structural components of cell membranes (bilayer)
2. Signal transduction (phosphatidylinositol)
3. Pulmonary surfactant (dipalmitoyl-phosphatidylcholine) - prevents alveolar collapse
4. Emulsification (lecithin in bile)
5. Blood coagulation (phosphatidylserine)
6. Acetylcholine precursor

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12. IMMUNOGLOBULINS

Basic Structure:

• 2 heavy chains (H) + 2 light chains (L) linked by disulfide bonds
• Heavy chain types: γ(IgG), α(IgA), µ(IgM), ε(IgE), δ(IgD)
• Light chain types: κ or λ
• Variable (V) region: antigen-binding site (Fab fragment)
• Constant (C) region: effector functions (Fc fragment)
• Fab: 2 fragments antigen binding
• Fc: 1 fragment crystallizable - binds complement, Fc receptors

Classes (Isotypes):

Functions:

1. Neutralization (IgG, IgA)
2. Opsonization (IgG promotes phagocytosis)
3. Complement activation (IgM > IgG via classical pathway)
4. ADCC - antibody-dependent cellular cytotoxicity (IgG)
5. Mast cell/basophil activation (IgE - Type I hypersensitivity)
6. Neonatal immunity (IgG via placenta; IgA via breast milk)

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13. MICROSCOPY

Types:

• Uses visible light + glass lenses
• Resolution: ~0.2 µm
• Used for routine histology, blood films, urine analysis
• Subtypes: Brightfield, Darkfield, Phase contrast, Fluorescence

• Uses electron beams; much higher resolution (~0.1 nm)
• Transmission EM (TEM): Internal ultrastructure (organelles, viruses)
• Scanning EM (SEM): 3D surface images
• Requires: fixation, dehydration, embedding, sectioning

• Uses fluorescent dyes/antibodies (immunofluorescence)
• Direct: primary antibody labeled with fluorochrome
• Indirect: secondary antibody labeled; more sensitive
• Applications: ANA, ANCA, glomerulonephritis diagnosis

• Converts phase differences in light into amplitude differences
• Visualizes living, unstained cells

• Background dark; object appears bright
• Used to visualize Treponema pallidum (syphilis)

• Laser scanning; 3D reconstruction from serial optical sections

Common Stains:

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14. GLUCONEOGENESIS

1. Lactate (Cori cycle - from muscle and RBCs)
2. Glycerol (from lipolysis)
3. Glucogenic amino acids (most; especially alanine - glucose-alanine cycle)
4. Propionate (odd-chain fatty acids)

• Activated by: glucagon, cortisol, epinephrine, fasting, high acetyl-CoA
• Inhibited by: insulin, AMP, ADP, fructose-2,6-bisphosphate

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15. DIGESTION OF PROTEINS

Digestion Steps:

• HCl (pH ~2): denatures proteins, converts pepsinogen → pepsin
• Pepsin (endopeptidase): cleaves aromatic/large hydrophobic AA peptide bonds; pH optimum 1.5-2

• Aminopeptidases (N-terminal)
• Dipeptidases
• Final absorption as single amino acids + di/tripeptides

• Enterokinase deficiency → all pancreatic proteases remain inactive → malnutrition
• Hartnup disease: defect in neutral AA transport
• Cystinuria: defect in cationic AA transport

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16. GLYCOGENESIS

1. Glucose → Glucose-6-phosphate (hexokinase/glucokinase)
2. G-6-P → Glucose-1-phosphate (phosphoglucomutase)
3. G-1-P + UTP → UDP-glucose + PPi (UDP-glucose pyrophosphorylase) - activated glucose
4. UDP-glucose added to existing glycogen primer (glycogenin - self-glucosylating protein) at α1→4 bonds by glycogen synthase (rate-limiting)
5. Branching: Branching enzyme (amylo-α1,4→1,6-transglycosylase) moves terminal 6-7 glucosyl units to form α1→6 branch points

• Insulin → activates glycogen synthase (dephosphorylation via PP1)
• Glucagon/Epinephrine → activate glycogen synthase kinase → inactivate glycogen synthase
• Allosteric: Glucose-6-phosphate activates glycogen synthase

• Core protein: glycogenin
• Linear: α1→4 linkages
• Branch points: α1→6 linkages every 8-12 glucose units
• More branches = more non-reducing ends = faster mobilization

• Von Gierke disease (Type I): G-6-Pase deficiency → hypoglycemia
• Pompe disease (Type II): Lysosomal acid maltase deficiency → cardiomegaly
• McArdle disease (Type V): Muscle phosphorylase deficiency → exercise intolerance

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17. DIGESTION OF LIPIDS

Steps:

• Lingual lipase (minor) and gastric lipase: begin TG hydrolysis (short-chain FA)

1. Bile salts (from liver/gallbladder): emulsify fats → micelles (↑ surface area for lipase)
2. Pancreatic lipase (main enzyme): hydrolyzes TG at sn-1 and sn-3 positions → 2-monoacylglycerol + 2 FFAs; requires colipase as cofactor
3. Cholesterol esterase: hydrolyzes cholesterol esters → free cholesterol
4. Phospholipase A2: hydrolyzes phospholipids at sn-2 position → lysophospholipid + FA

• Free fatty acids, monoglycerides, cholesterol, fat-soluble vitamins are incorporated into mixed micelles
• Absorbed into enterocytes (brush border)
• Short/medium-chain FA: directly into portal blood (albumin-bound)
• Long-chain FA + monoglycerides: re-esterified in smooth ER → TG → chylomicrons (with ApoB-48) → lymphatics (lacteals) → thoracic duct → blood

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18. BILIRUBIN METABOLISM

Steps:

• Hemoglobin → Heme + Globin
• Heme → Biliverdin (by heme oxygenase; CO released)
• Biliverdin → Unconjugated (indirect) bilirubin (by biliverdin reductase)
• Unconjugated bilirubin is lipid-soluble, bound to albumin in blood; cannot be excreted in urine

• Bilirubin dissociates from albumin; taken up by hepatocytes (carrier: ligandin/Y protein)
• Conjugated with glucuronic acid by UDP-glucuronyl transferase → Conjugated (direct) bilirubin (water-soluble, non-toxic)

• Conjugated bilirubin secreted into bile (active transport by MRP2)
• In gut: bacterial action (deconjugation + reduction) → urobilinogen
  • Some urobilinogen → absorbed → portal blood → kidney → excreted as urobilin (urine, yellow)
  • Most urobilinogen → oxidized → stercobilin (stool, brown)

Clinical Jaundice (Normal serum bilirubin: 0.2-1.0 mg/dL):

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19. CHOLESTEROL

• Occurs in liver (major), intestine, adrenal cortex
• Substrate: Acetyl-CoA
• Rate-limiting enzyme: HMG-CoA reductase (converts HMG-CoA → Mevalonate)
• Inhibited by statins, cholesterol (feedback)
• Activated by insulin; inhibited by glucagon

1. 3 Acetyl-CoA → HMG-CoA
2. HMG-CoA → Mevalonate (HMG-CoA reductase) ← STATIN target
3. Mevalonate → Isoprene units → Squalene → Lanosterol → Cholesterol

1. Cell membrane component (regulates fluidity)
2. Precursor of steroid hormones (cortisol, aldosterone, estrogen, testosterone)
3. Precursor of bile acids/salts (digestion of fat)
4. Precursor of Vitamin D3
5. Component of myelin sheath

• Endogenous path: Liver synthesizes → VLDL → IDL → LDL → tissues (via LDL receptor/ApoB-100)
• Reverse cholesterol transport: Peripheral tissues → HDL (via ABCA1) → liver (via SR-B1)

• High intracellular cholesterol: ↓ LDL receptor expression, ↑ PCSK9 (degrades LDL-R), ↑ ACAT (stores as CE)
• Low cholesterol: ↑ SREPB-2 → ↑ LDL-R and HMG-CoA reductase

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20. ELISA (Enzyme-Linked Immunosorbent Assay)

Types:

• Antigen coated on plate → enzyme-labeled primary antibody added → substrate → color change
• Fast, less sensitive; eliminates secondary Ab variability

• Antigen coated → unlabeled primary antibody → enzyme-labeled secondary antibody → substrate
• More sensitive (signal amplification); most common format
• Used to detect antibodies in patient serum (e.g., HIV, H. pylori)

• Capture antibody coated on plate → antigen added → detection antibody (enzyme-labeled) → substrate
• Most sensitive; used to quantify antigens (e.g., ELISA for cytokines, hepatitis B surface Ag)

• Sample antigen competes with labeled antigen for limited antibody binding
• Inverse relationship: more antigen in sample → less color

Components:

• Microplate (96-well)
• Blocking agent (BSA, milk protein)
• Primary and secondary antibodies
• Enzyme conjugate (HRP - Horseradish Peroxidase, or ALP - Alkaline Phosphatase)
• Substrate (TMB for HRP → blue/yellow; pNPP for ALP → yellow)
• Stop solution (H₂SO₄)
• Read at specific wavelength (450 nm for HRP/TMB)

Applications:

• Diagnosis of infections (HIV, hepatitis B/C, dengue, COVID-19)
• Drug/hormone quantification (pregnancy tests for hCG)
• Food safety testing
• Research (cytokine measurement)

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21. CELL ORGANELLES

• Pompe: acid maltase deficiency
• Gaucher: glucocerebrosidase deficiency
• Tay-Sachs: hexosaminidase A deficiency
• Niemann-Pick: sphingomyelinase deficiency

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22. CELL-MEDIATED IMMUNITY

CD4+ T Helper Cells (Th):

• Th1 cells: IL-12 driven; produce IFN-γ, TNF-α, IL-2; activate macrophages and CTLs; defend against intracellular bacteria/viruses
• Th2 cells: IL-4 driven; produce IL-4, IL-5, IL-13; activate B cells and eosinophils; defend against parasites, mediate allergy
• Th17 cells: IL-6+TGF-β driven; produce IL-17; defend against extracellular bacteria and fungi; involved in autoimmunity
• Treg cells: IL-10, TGF-β; suppress immune responses; prevent autoimmunity

CD8+ Cytotoxic T Cells (CTL):

• Kill via: Perforin-granzyme pathway (granzyme B → apoptosis), Fas-FasL interaction
• Clear virus-infected cells, tumor cells

Process of Cell-Mediated Immunity:

1. Antigen presenting cell (DC) ingests antigen, processes it
2. Presents peptide on MHC II to CD4+ T cells (via TCR + CD4)
3. Co-stimulation required: B7 (APC) + CD28 (T cell)
4. T cell activation → proliferation (IL-2 autocrine) → effector and memory T cells
5. Effector functions: macrophage activation, cytotoxicity, delayed-type hypersensitivity (DTH/Type IV)

• Organ transplant rejection (cell-mediated)
• Contact dermatitis, tuberculin skin test (DTH)
• HIV - destroys CD4+ T cells → AIDS
• Immunosuppressants (cyclosporin - inhibits IL-2 production)

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23. UREA CYCLE

1. Carbamoyl phosphate synthetase I (CPS-I) [mitochondria] - NH₃ + CO₂ + 2ATP → Carbamoyl phosphate; rate-limiting; activated by N-acetylglutamate
2. Carbamoyl phosphate + Ornithine → Citrulline (ornithine transcarbamylase; OTC) [mitochondria → cytoplasm]
3. Citrulline + Aspartate → Argininosuccinate (argininosuccinate synthetase) + ATP [cytoplasm]
4. Argininosuccinate → Arginine + Fumarate (argininosuccinate lyase)
5. Arginine → Ornithine + Urea (arginase) - ornithine reenters mitochondria; urea excreted in urine

• N-acetylglutamate (NAG) activates CPS-I; NAG synthetase activated by arginine

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24. DIABETIC INVESTIGATIONS

Diagnosis of Diabetes Mellitus (ADA Criteria - any one):

1. Fasting plasma glucose ≥ 126 mg/dL (fasting ≥ 8 hours)
2. 2-hour plasma glucose ≥ 200 mg/dL during 75g OGTT
3. HbA1c ≥ 6.5%
4. Random plasma glucose ≥ 200 mg/dL with symptoms (polyuria, polydipsia, weight loss)

• FPG: 100-125 mg/dL (Impaired Fasting Glucose)
• 2h-PG: 140-199 mg/dL (Impaired Glucose Tolerance)
• HbA1c: 5.7-6.4%

Key Investigations:

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25. LIVER FUNCTION TESTS (LFTs)

Tests Grouped by Function:

• Hepatitis: ALT/AST markedly elevated; ALP mildly elevated
• Cholestasis: ALP/GGT/direct bilirubin markedly elevated; minimal transaminase elevation
• Cirrhosis: ↓ albumin, ↑ PT, ↑ bilirubin, transaminases mildly elevated

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QUICK EXAM SUMMARY TABLE

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