BDS 1st Year Biochemistry - Haematopoietic Questions (KU)

Q1. Define and Classify Porphyria. Explain the Regulation of Heme Biosynthesis. [1+2+2=5]

Definition of Porphyria

Classification of Porphyrias

• Acute (neuropsychiatric) - colicky abdominal pain, peripheral neuropathy, psychiatric features (e.g., AIP)
• Cutaneous (photosensitive) - blistering, scarring of sun-exposed skin (e.g., PCT)
• Mixed - both features (e.g., Variegate Porphyria)

Regulation of Heme Synthesis (Biosynthesis Pathway)

1. Glycine + Succinyl CoA → δ-aminolevulinic acid (δ-ALA) - enzyme: ALA synthase (requires pyridoxal phosphate/B6; this is the rate-limiting step, located in mitochondria)
2. 2 δ-ALA → Porphobilinogen (PBG) - enzyme: ALA dehydratase (cytoplasm)
3. 4 PBG → Hydroxymethylbilane - enzyme: PBG deaminase
4. Hydroxymethylbilane → Uroporphyrinogen III - enzyme: Uroporphyrinogen III synthase
5. Uroporphyrinogen III → Coproporphyrinogen III - enzyme: Uroporphyrinogen decarboxylase
6. Coproporphyrinogen III → Protoporphyrinogen IX - enzyme: Coproporphyrinogen oxidase (returns to mitochondria)
7. Protoporphyrinogen IX → Protoporphyrin IX - enzyme: Protoporphyrinogen oxidase
8. Protoporphyrin IX + Fe2+ → Heme - enzyme: Ferrochelatase

• The rate-limiting enzyme is ALA synthase (also called ALAS)
• Heme regulates its own synthesis by:
  • Feedback inhibition - directly inhibiting ALA synthase activity
  • Repression - suppressing transcription of the ALA synthase gene
• Inducers of cytochrome P450 (e.g., drugs like barbiturates, alcohol, steroids) increase consumption of heme, lower free heme levels, and thus upregulate ALA synthase - this can precipitate attacks of acute porphyria
• In erythroid cells, a separate isoform, ALAS2, is regulated by iron availability through an iron-responsive element (IRE) in its mRNA

Q2. Write Short Notes On:

a) Structure and Function of Hemoglobin

• Hemoglobin A (HbA), the major adult hemoglobin, is a tetramer: 2 alpha (α) chains + 2 beta (β) chains
• Each subunit contains a heme prosthetic group (protoporphyrin IX + Fe2+)
• The heme group sits in a hydrophobic pocket; the iron is bound to a proximal histidine (His F8) and O2 is stabilized by the distal histidine (His E7)
• The four subunits are held by noncovalent interactions; they exist as two dimers (αβ1 and αβ2) with flexible contacts between dimers

• T (tense) form: deoxy conformation, low O2 affinity
• R (relaxed) form: oxy conformation, high O2 affinity
• Cooperative binding means each successive O2 molecule binds with greater affinity - giving the sigmoidal O2-dissociation curve (P50 ~26 mm Hg)

1. Transport O2 from lungs to tissues (main function)
2. Transport CO2 from tissues to lungs (as carbaminohemoglobin)
3. Transport H+ (buffer) - Bohr effect
4. Bind 2,3-BPG (allosteric effector), H+, and CO2 to modulate O2 delivery

• Decreased pH (H+)
• Increased CO2 (Bohr effect)
• Increased 2,3-BPG (important in RBCs)
• Increased temperature

b) G6PD Deficiency

• G6PD (Glucose-6-phosphate dehydrogenase) is the first enzyme of the HMP shunt (pentose phosphate pathway) in RBCs
• It generates NADPH, which maintains glutathione in its reduced form (GSH) to protect RBCs against oxidative damage
• G6PD deficiency is X-linked (gene on X chromosome), so males are predominantly affected
• It is the most common enzyme deficiency in humans - heterozygous females have partial protection against malaria (balanced polymorphism)
• All G6PD variant genes have small in-frame deletions or missense mutations; complete absence is lethal (embryonic lethality in mice)

• Without NADPH, glutathione cannot be maintained in reduced form
• Oxidative stress (from antimalarial drugs like primaquine, infection, fava beans, aspirin) leads to Heinz body formation (denatured hemoglobin)
• Hemolytic anemia (episodic) - the RBCs lyse prematurely

c) Sickle Cell Haemoglobin and Its Laboratory Diagnosis

• Point mutation: Glutamic acid (Glu) → Valine (Val) at position 6 of the beta-globin chain (β6 Glu→Val)
• This substitution is caused by a single nucleotide change (A→T in the sixth codon of the β-globin gene)

• Deoxygenated HbS molecules polymerize into long, rigid fibers that distort RBCs into a sickle shape
• Sickle cells obstruct microcirculation (vaso-occlusion) causing pain crises, organ damage
• Repeated sickling causes membrane damage and hemolytic anemia
• Heterozygotes (HbAS - sickle cell trait) are largely asymptomatic and have some protection against malaria

1. Peripheral blood smear: sickle-shaped RBCs, target cells
2. Sickle solubility test (Sickling test): sodium metabisulfite reduces pO2, causing sickling of HbS
3. Hemoglobin electrophoresis (definitive): HbS migrates differently from HbA; in sickle disease: ~95% HbS, 2-3% HbF, trace HbA2
4. HPLC (High Performance Liquid Chromatography): gold standard for screening
5. DNA analysis: confirms β-globin gene mutation

d) Glycolytic Products in RBCs

1. ATP: Used for Na+/K+ ATPase (maintains cell shape and membrane), cation pumps, and phosphorylation reactions
2. NADH: Reduces methemoglobin (Fe3+) back to functional hemoglobin (Fe2+) via methemoglobin reductase
3. 2,3-Bisphosphoglycerate (2,3-BPG): Formed via the Rapoport-Luebering shunt (a detour from standard glycolysis); 2,3-BPG binds to the central cavity of deoxy-hemoglobin and decreases its O2 affinity, facilitating O2 unloading to tissues
4. Lactate: End-product of anaerobic glycolysis (pyruvate → lactate via LDH to regenerate NAD+)
5. Pyruvate: Minor product; mostly converted to lactate in RBCs

e) Regulation of Heme Synthesis

f) Porphyria

Q3. Diagrammatically Discuss the Absorption of Non-Heme Iron, Its Transport and Storage. How Does Hepcidin Regulate Iron Absorption? [1+2=6]

Absorption of Non-Heme Iron

• Non-heme iron (from plant sources) is in the Fe3+ (ferric) form, which must be reduced to Fe2+ (ferrous) before absorption
• Reduction is accomplished by duodenal cytochrome B (DcytB) on the brush border of enterocytes, using vitamin C as a cofactor
• Fe2+ is taken up into the enterocyte via DMT-1 (Divalent Metal Transporter 1) - a proton-coupled transporter on the apical membrane
• Inside the enterocyte, iron can be: (a) stored as ferritin (if excess), or (b) exported across the basolateral membrane
• Export is via ferroportin (basolateral transporter); once in plasma, Fe2+ is oxidized to Fe3+ by hephaestin (a copper-containing ferroxidase) and then bound to apotransferrin to form transferrin

• Vitamin C (ascorbic acid) - reduces Fe3+ to Fe2+
• Alcohol, fructose
• Acidic gastric pH
• Hypoxia, anemia, hemorrhage (physiological upregulation)

• Phytates (cereals), oxalates, tannins
• Calcium (milk inhibits iron absorption)
• Alkaline pH
• High iron stores

Transport of Iron

• In plasma, iron circulates bound to transferrin (as Fe3+)
• Normal transferrin saturation: ~33% (transferrin is one-third saturated)
• Total iron binding capacity (TIBC): ~300 μg/dL
• Cells acquire iron via transferrin receptor (TfR1) on the cell surface
• Transferrin-TfR1 complex is internalized by receptor-mediated endocytosis into an endosome
• The acidic pH of the endosome causes iron release; Fe3+ is reduced to Fe2+ by a membrane oxidoreductase and transported into cytoplasm via DMT-1
• The apotransferrin-TfR1 complex is recycled back to the cell surface

Storage of Iron

• Primary storage sites: liver, spleen, bone marrow
• Iron is stored as ferritin (Fe2+ bound to apoferritin shell; soluble, readily mobilizable)
• When iron stores are excessive, it is stored as hemosiderin (ferritin-iron complexes that have aggregated; insoluble, poorly mobilizable)
• Serum ferritin level is the most sensitive indicator of total body iron stores (low = iron deficiency; high = iron overload)

How Hepcidin Regulates Iron Absorption

• Hepcidin is a small antimicrobial peptide hormone secreted by the liver
• It is the master regulator of iron homeostasis
• Hepcidin works by binding to ferroportin on enterocytes, macrophages, and hepatocytes, causing ferroportin's internalization and degradation
• Without ferroportin, iron cannot be exported from enterocytes into the blood - it stays trapped inside mucosal cells and is lost when those cells are shed
• This reduces circulating iron

• High iron stores
• Inflammation (via IL-6 signaling - this is why anemia of chronic disease occurs)

• Hypoxia
• Anemia
• Hemorrhage
• Erythropoietic activity (via erythroferrone from erythroblasts)

Q4. Importance of HMP Shunt Pathway (Hexose Monophosphate Shunt / Pentose Phosphate Pathway)

Products

1. NADPH (reduced nicotinamide adenine dinucleotide phosphate) - 2 molecules per glucose-6-P
2. Ribose-5-phosphate - a 5-carbon sugar

Importance

• NADPH reduces glutathione disulfide (GSSG) → glutathione (GSH) via glutathione reductase
• GSH is used by glutathione peroxidase to neutralize H2O2 and other peroxides
• RBCs have no mitochondria and cannot regenerate NADPH any other way - the HMP shunt is their only source of NADPH
• G6PD deficiency impairs this pathway, leaving RBCs vulnerable to oxidative hemolysis

• Required for synthesis of purines and pyrimidines (nucleotides, RNA, DNA)
• Critical for rapidly dividing cells

• Fatty acid synthesis (fatty acid synthase requires NADPH)
• Cholesterol synthesis
• Steroid hormone synthesis (in adrenal cortex)
• Synthesis of deoxyribonucleotides (ribonucleotide reductase)

• NADPH oxidase in neutrophils and macrophages uses NADPH to generate superoxide (O2·-) for killing pathogens (respiratory burst)
• G6PD deficiency impairs this function, increasing susceptibility to infections

• The non-oxidative phase of the pathway interconverts 3-, 4-, 5-, 6-, and 7-carbon sugars, feeding them into glycolysis

Q5. Reactions of Glycolysis with Rate-Limiting Enzymes

Phase 1: Preparative Phase (Energy Investment: 2 ATP consumed)

Phase 2: ATP-Generating Phase (Energy Recovery: 4 ATP produced)

Net Equation

Glucose + 2 NAD+ + 2 Pi + 2 ADP → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O

1. Hexokinase/Glucokinase (Step 1)
2. PFK-1 (Step 3) - the main rate-limiting enzyme of glycolysis
3. Pyruvate kinase (Step 10)

Q6. Account of Acute Intermittent Porphyria (AIP)

Definition

Inheritance

• Autosomal dominant (heterozygous mutation in the HMBS gene on chromosome 11q23)
• Low penetrance - most carriers never have symptoms

Pathogenesis

• Reduced PBG deaminase → PBG and ALA accumulate in liver and plasma
• Attacks are precipitated when demand for heme increases (e.g., for cytochrome P450 induction by drugs)
• ALA and PBG accumulation causes neurovisceral toxicity
• Heme levels fall (end-product deficiency) → loss of feedback inhibition of ALA synthase → even more ALA and PBG accumulate

• Drugs: barbiturates, anticonvulsants (phenytoin), sulfonamides, rifampicin, alcohol
• Hormones: estrogens, progesterone (explains female predominance after puberty)
• Fasting/low carbohydrate intake
• Infection, stress

Clinical Features ("Rule of 5 P's")

1. Pain - severe colicky abdominal pain (most common), without peritoneal signs
2. Polyneuropathy - peripheral motor neuropathy (ascending weakness, can mimic Guillain-Barre)
3. Psychiatric/Psychological - anxiety, psychosis, confusion, seizures
4. Paralysis - respiratory failure in severe cases
5. Pigment (Urine) - port-wine colored urine (ALA + PBG oxidize to porphobilin on standing)

Laboratory Diagnosis

• Urine ALA and PBG: markedly elevated during attacks (Watson-Schwartz test - urine turns pink/red with Ehrlich's aldehyde reagent)
• Urine porphyrins: mildly elevated
• Normal urine porphyrins (no skin manifestations - distinguishes from PCT)
• Fecal porphyrins: normal (distinguishes from Variegate Porphyria and Hereditary Coproporphyria)
• RBC PBG deaminase activity: reduced (~50%)

Treatment

1. Remove precipitants (stop offending drugs, treat infection)
2. Carbohydrate loading (glucose 300-500 g/day IV) - represses ALAS1, reduces ALA/PBG
3. IV Heme (Hematin/Hemin) - directly inhibits ALAS1, most effective treatment for severe attacks
4. Symptomatic: analgesics, beta-blockers for tachycardia, safe anticonvulsants (gabapentin, levetiracetam)
5. Avoid fasting; maintain adequate caloric intake

Q7. Glycolysis in RBC (Red Blood Cells)

Why RBCs Depend Exclusively on Glycolysis

• Mature RBCs have no mitochondria, no nucleus, no ribosomes
• Cannot perform oxidative phosphorylation, TCA cycle, or fatty acid oxidation
• Anaerobic glycolysis is the sole source of ATP in RBCs

Pathway in RBCs - Key Features

• At step 7, instead of transferring phosphate from 1,3-BPG to ADP (via phosphoglycerate kinase), the enzyme bisphosphoglycerate mutase converts 1,3-BPG → 2,3-bisphosphoglycerate (2,3-BPG)
• 2,3-BPG is then hydrolyzed to 3-phosphoglycerate by 2,3-bisphosphoglycerate phosphatase (enters main glycolysis)
• This shunt sacrifices ATP production (bypasses one substrate-level phosphorylation) in exchange for 2,3-BPG formation
• Importance of 2,3-BPG: Binds to the central cavity between the beta chains of deoxy-hemoglobin → stabilizes the T (deoxy) form → decreases O2 affinity of hemoglobin → promotes O2 delivery to tissues

• Via standard glycolysis: 2 ATP net per glucose
• Via Rapoport-Luebering shunt: 1 ATP net per glucose (if shunt is used instead of PGK step)

• Na+/K+ ATPase pump: maintains cell volume and membrane integrity
• Ca2+ ATPase: prevents premature hardening/sickling
• Protein kinases: membrane cytoskeleton phosphorylation

• Regenerated by lactate dehydrogenase (pyruvate → lactate), which oxidizes NADH → NAD+
• NADH also reduces methemoglobin (Fe3+) back to hemoglobin (Fe2+) via cytochrome b5 reductase (methemoglobin reductase)

• Pyruvate is reduced to lactate (regenerating NAD+ for continued glycolysis)
• Lactate is released into blood and taken up by liver (Cori cycle)

Clinical Relevance

• Pyruvate kinase (PK) deficiency: The most common glycolytic enzyme deficiency in RBCs; causes chronic hemolytic anemia due to ATP depletion; diagnosis by RBC PK enzyme assay
• G6PD deficiency: Impairs the HMP shunt (not glycolysis per se) but causes oxidative hemolysis