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HAEMATOPOIETIC BIOCHEMISTRY - BDS 1st Year Board Exam Answers

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Q1. Define and classify porphyria. Explain the regulation of heme biosynthesis. [1+2+2=5]

Definition of Porphyria [1 mark]

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Classification of Porphyria [2 marks]

1. Acute porphyrias - present with neurovisceral attacks (acute abdominal pain, neuropsychiatric features, autonomic dysfunction). Examples: AIP, HCP, VP
2. Cutaneous porphyrias - present with photosensitive skin lesions. Example: PCT
3. Mixed (neurocutaneous) - both features. Example: VP, HCP

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Regulation of Heme Biosynthesis [2 marks]

Mitochondria:
1. Glycine + Succinyl-CoA → ALA (enzyme: ALA synthase - rate-limiting, PLP-dependent)

Cytosol:
2. 2 ALA → Porphobilinogen (PBG) (enzyme: ALA dehydratase / PBG synthase)
3. 4 PBG → Hydroxymethylbilane (HMB) (enzyme: PBG deaminase / HMBS)
4. HMB → Uroporphyrinogen III (enzyme: Uroporphyrinogen III synthase)
5. Uroporphyrinogen III → Coproporphyrinogen III (enzyme: Uroporphyrinogen decarboxylase)

Mitochondria:
6. Coproporphyrinogen III → Protoporphyrinogen IX (enzyme: Coproporphyrinogen oxidase)
7. Protoporphyrinogen IX → Protoporphyrin IX (enzyme: Protoporphyrinogen oxidase)
8. Protoporphyrin IX + Fe²⁺ → HEME (enzyme: Ferrochelatase)

1. Feedback inhibition by heme: Heme (the final product) inhibits ALAS-1 (liver isoform) at three levels:
   • Inhibits ALAS transcription
   • Inhibits import of ALAS precursor into mitochondria
   • Directly inhibits ALAS enzyme activity
2. Two isoforms of ALAS:
   • ALAS-1 (liver): regulated by heme feedback; induced by drugs, starvation, hormones
   • ALAS-2 (erythroid cells): regulated by iron - erythropoietin increases its expression; its mRNA has an iron-responsive element (IRE) that responds to intracellular iron levels
3. Drug-induced regulation: Drugs metabolized by cytochrome P450 (e.g., barbiturates, griseofulvin) deplete heme, removing the feedback inhibition and causing massive ALAS-1 induction - this can precipitate acute porphyria attacks
4. Ferrochelatase inhibition: Lead inhibits both ALA dehydratase (step 2) and ferrochelatase (step 8), causing porphyrin accumulation in lead poisoning

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Q2. Write Short Notes

a) Structure and Function of Hemoglobin

• Hemoglobin (Hb) is a tetrameric globular protein - 4 polypeptide subunits, each carrying one heme group
• HbA (adult): 2alpha + 2beta chains (most abundant, ~97%)
• HbA2: 2alpha + 2delta chains (~2.5%)
• HbF (fetal): 2alpha + 2gamma chains (higher O2 affinity - beneficial in fetus)
• Each subunit has a heme prosthetic group: protoporphyrin IX ring + Fe²⁺ (ferrous iron)
• Fe²⁺ can bind one O2 molecule, so each Hb can carry 4 molecules of O2
• The protein exists in two states:
  • T (tense/deoxy) state: low O2 affinity; stabilized by 2,3-BPG, H⁺, CO2
  • R (relaxed/oxy) state: high O2 affinity

1. O2 transport: Picks up O2 in lungs (high pO2) and releases in tissues (low pO2)
2. CO2 transport: ~23% of CO2 transported as carbaminohemoglobin (Hb-NHCOO⁻)
3. Buffering: Histidine residues act as buffers, helping maintain blood pH
4. Bohr Effect: H⁺ and CO2 decrease O2 affinity of Hb, facilitating O2 release in tissues
5. 2,3-BPG effect: Binds beta chains in T state, stabilizing deoxy form and reducing O2 affinity (important in adaptation to altitude/anemia)

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b) G6PD Deficiency

• G6PD catalyzes the first step of the HMP (Hexose Monophosphate) shunt / Pentose Phosphate Pathway:
  • Glucose-6-phosphate + NADP⁺ → 6-Phosphogluconolactone + NADPH
• NADPH is essential for regenerating glutathione (GSH) via glutathione reductase
• GSH neutralizes oxidative radicals (H2O2) via glutathione peroxidase
• RBCs lack mitochondria - they depend entirely on the HMP shunt for NADPH

• Without sufficient NADPH, GSH cannot be maintained
• Oxidative stress (drugs, infections, fava beans) causes:
  • Oxidation of Hb → Heinz bodies (denatured Hb precipitates)
  • RBC membrane damage → Hemolytic anemia
• Triggers: Primaquine, dapsone, nitrofurantoin, aspirin, infection, fava beans
• Labs: Heinz body stain positive, decreased G6PD enzyme assay, normocytic hemolytic anemia

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c) Sickle Cell Haemoglobin and Its Laboratory Diagnosis

• Autosomal recessive point mutation in the beta-globin gene (chromosome 11)
• Glutamate (Glu) → Valine (Val) at position 6 of the beta chain
• This creates HbS (alpha2 beta2S) instead of normal HbA

• Under deoxygenated conditions, HbS polymerizes (forms long, rod-like fibers)
• These fibers distort RBCs into a sickle (crescent) shape
• Sickled cells:
  • Are rigid and non-deformable - obstruct small vessels (vaso-occlusive crisis)
  • Have shortened lifespan (~20 days vs normal 120 days) - hemolytic anemia
  • Cause ischemia, pain crises, acute chest syndrome, stroke

• Reduced O2 affinity compared to HbA
• HbS is insoluble when deoxygenated - this is the basis of the sickling test

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d) Glycolytic Products in RBCs

1. ATP (2 mol per mol glucose):
   • Powers Na⁺/K⁺-ATPase - maintains RBC shape and volume
   • Powers Ca²⁺-ATPase
   • Maintains membrane flexibility
2. NADH:
   • Reduces methemoglobin (Fe³⁺) back to functional hemoglobin (Fe²⁺) via methemoglobin reductase
3. Pyruvate → Lactate:
   • In RBCs, pyruvate is converted to lactate (anaerobic end product)
   • Regenerates NAD⁺ to keep glycolysis running
4. 2,3-Bisphosphoglycerate (2,3-BPG):
   • Unique to RBCs via the Rapoport-Luebering shunt (bypasses the ATP-generating step)
   • Produced from 1,3-BPG by bisphosphoglycerate mutase
   • Key allosteric regulator of Hb-O2 affinity: binds to deoxyhemoglobin between beta chains, stabilizes T-state, reduces O2 affinity, facilitating O2 delivery to tissues
   • Increased in altitude, anemia, chronic lung disease
5. NADPH (via HMP shunt):
   • Maintains glutathione in reduced form (GSH) - protects against oxidative damage

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e) Regulation of Heme Synthesis

• Main regulator: ALA synthase (ALAS) - rate-limiting enzyme
• ALAS-1 (hepatic): Inhibited by heme (end-product feedback); induced by drugs (P450 inducers)
• ALAS-2 (erythroid): Regulated by iron via IRE in mRNA; induced by erythropoietin
• Lead poisoning: Inhibits ALA dehydratase and ferrochelatase - causes accumulation of ALA and Zn-protoporphyrin

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f) Porphyria

• Enzyme defect: PBG deaminase (HMBS) deficiency (step 3)
• Autosomal dominant
• Hepatic type; NO skin manifestations
• Clinical triad:
  1. Acute abdominal pain (colicky)
  2. Neuropsychiatric symptoms (anxiety, hallucinations, seizures)
  3. Peripheral neuropathy (ascending motor neuropathy)
• Urine turns dark reddish-brown on standing (oxidation of PBG to porphyrins)
• Diagnosis: Elevated urine ALA + PBG during acute attacks
• Precipitating factors: Drugs (barbiturates, sulfonamides, OCP), fasting, alcohol, infection, stress, hormones
• Treatment: IV glucose (10-20 g/hr), IV hematin; Givosiran (siRNA) for recurrent attacks

• Most common porphyria
• Enzyme defect: Uroporphyrinogen decarboxylase (step 5)
• Photosensitive skin blisters on sun-exposed areas
• Associated with alcohol, estrogen, hepatitis C, HIV, iron overload
• Treatment: Phlebotomy, low-dose hydroxychloroquine

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Q3. Discuss diagrammatically the absorption of non-heme iron, its transport and storage. How does hepcidin regulate iron absorption? [4+2=6]

Non-Heme Iron Absorption [Diagrammatic]

LUMEN (duodenum/upper jejunum)
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   Non-heme iron (Fe³⁺)
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    Duodenal cytochrome b (DCYTB)  ← reduces Fe³⁺ → Fe²⁺
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   Fe²⁺ enters enterocyte via
   DMT-1 (Divalent Metal Transporter-1)
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   Inside enterocyte:
   - Small pool stored as FERRITIN
   - Rest transferred across basolateral membrane
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   FERROPORTIN (FPN-1) exports Fe²⁺ from enterocyte → portal blood
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   HEPHAESTIN (copper-containing ferroxidase) oxidizes Fe²⁺ → Fe³⁺
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   Fe³⁺ binds TRANSFERRIN (each molecule binds 2 Fe³⁺)
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   Transported via BLOOD to tissues

• Vitamin C (ascorbic acid) - reduces Fe³⁺ to Fe²⁺
• Acidic pH (gastric HCl)
• Animal protein (MFP factor)
• Iron deficiency state
• Pregnancy, erythropoiesis

• Phytates (cereals), oxalates (spinach), polyphenols (tea, coffee)
• Alkaline pH, antacids
• Calcium, zinc (compete with DMT-1)
• Iron overload (hepcidin upregulation)

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Iron Transport and Storage

• Transferrin: Major iron transport protein; a beta-1 globulin glycoprotein made in liver; carries Fe³⁺; normally ~30% saturated (Transferrin Saturation)
• Transferrin receptor (TfR1): On all cells, especially erythroid precursors and liver; binds transferrin-Fe complex; endocytosed; iron released in endosome (acidification)

• Ferritin: Cytosolic storage protein; spherical shell of 24 apoferritin subunits; stores up to 4,500 iron atoms as Fe³⁺-phosphate complex; major storage in liver, spleen, bone marrow, muscle
• Hemosiderin: Aggregated, partially degraded ferritin; insoluble; mobilized slowly; seen in iron overload (Golden-brown granules with Prussian Blue staining)

• Macrophages phagocytose old RBCs, break down heme via heme oxygenase, release iron via ferroportin back into circulation

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Hepcidin Regulation of Iron Absorption [2 marks]

• A 25 amino acid antimicrobial peptide hormone produced by the liver
• The master regulator of systemic iron homeostasis

• Hepcidin binds to Ferroportin (FPN-1) on enterocytes, macrophages, and hepatocytes
• This binding causes internalization and degradation of ferroportin
• Without ferroportin, iron is trapped inside enterocytes and macrophages
• Result: reduced iron absorption from gut and reduced iron release from stores

• Iron overload (high transferrin saturation, high ferritin)
• Inflammation/infection (IL-6 induces hepcidin) - this causes anemia of chronic disease
• BMP-SMAD signaling pathway activated by BMP6 (liver) when iron stores are high

• Iron deficiency
• Hypoxia (via HIF-2alpha signaling)
• Increased erythropoiesis (erythropoietin, growth differentiation factor 15/ERFE)
• Anemia

• Hemochromatosis (type 1): HFE gene mutation → hepcidin not produced → unregulated iron absorption → iron overload (liver, heart, pancreas, joints)
• Anemia of chronic disease: High hepcidin (from inflammation) → iron trapped → functional iron deficiency despite adequate stores
• Hepcidin mimetics are being developed to treat iron overload disorders

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Q4. Write down the absorption, transport, storage and release of iron in our body [5]

• Total body iron: ~3-4 g in men, ~2-3 g in women
• Daily dietary requirement: 8-10 mg/day (men), 18 mg/day (women)
• Daily absorption: ~1-2 mg (equal to daily loss)
• Most iron (~25 mg/day) is recycled from senescent RBCs by macrophages

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Q5. Importance of HMP Shunt Pathway

1. Oxidative (irreversible) phase: Glucose-6-P → Ribulose-5-P
   • G6PD catalyzes: G-6-P + NADP⁺ → 6-Phosphogluconolactone + NADPH + H⁺ (rate-limiting)
   • 6-Phosphogluconate dehydrogenase: 6-PG + NADP⁺ → Ribulose-5-P + NADPH + CO2
   • Yield: 2 NADPH per glucose
2. Non-oxidative (reversible) phase: Interconverts pentose phosphates → fructose-6-P and glyceraldehyde-3-P (enters glycolysis)

1. Production of NADPH:
   • Maintains glutathione (GSH) in reduced form - protects RBCs and other cells from oxidative damage
   • Required for fatty acid synthesis (liver, mammary gland, adipose)
   • Required for steroid/cholesterol synthesis (adrenal cortex, gonads)
   • Required for cytochrome P450 reactions (liver detoxification)
   • Phagocytic killing in neutrophils (NADPH oxidase generates superoxide)
2. Production of Ribose-5-phosphate:
   • Precursor for nucleotide and nucleic acid synthesis (ATP, GTP, NAD, FAD, RNA, DNA)
   • Essential for proliferating cells
3. Critical for RBCs:
   • Only source of NADPH in RBCs (no mitochondria)
   • Protects hemoglobin from oxidation (met-Hb formation)
   • G6PD deficiency disrupts this - causes hemolytic anemia with oxidative drugs
4. Provides erythrose-4-phosphate:
   • For synthesis of aromatic amino acids (via shikimate pathway in bacteria - basis of antibiotic targets)
5. Reductive biosynthesis in tissues:
   • NADPH used in synthesis of fatty acids, bile acids, neurotransmitters, and nitric oxide (by NO synthase)

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Q6. Reactions of Glycolysis with Rate-Limiting Enzymes

The 10 Steps of Glycolysis:

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Three Rate-Limiting (Irreversible) Enzymes:

1. Hexokinase / Glucokinase (Step 1)
   • Inhibited by: Glucose-6-P (product inhibition) - hexokinase
   • Glucokinase (liver/pancreas): NOT inhibited by G6P; induced by insulin; low affinity (high Km) - acts as glucose sensor
2. Phosphofructokinase-1 - PFK-1 (Step 3) - THE most important
   • Inhibited by: High ATP, citrate, H⁺ (acidosis)
   • Activated by: AMP, ADP, Fructose-2,6-bisphosphate (most potent activator), Pi
   • Fructose-2,6-bisP is made by PFK-2 (activated by insulin, inhibited by glucagon)
3. Pyruvate Kinase (Step 10)
   • Inhibited by: ATP, alanine, glucagon (via phosphorylation)
   • Activated by: Fructose-1,6-bisP (feedforward activation), AMP, insulin
   • Pyruvate kinase deficiency causes hemolytic anemia (important in RBC)

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Glycolysis in RBCs - Special Features:

• Anaerobic only (no mitochondria)
• ATP produced: 2 mol per mol glucose (net)
• Pyruvate is converted to lactate (by lactate dehydrogenase) to regenerate NAD⁺
• Rapoport-Luebering shunt: 1,3-BPG → 2,3-BPG (bypasses ATP production at step 7)
  • 2,3-BPG produced is crucial for O2 delivery regulation
• Pyruvate kinase deficiency in RBCs: most common glycolytic enzyme deficiency causing hemolytic anemia; buildup of 2,3-BPG; right-shifted O2 dissociation curve (cells tolerate anemia better)

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Q7. Give an Account of Acute Intermittent Porphyria (AIP)

• Autosomal dominant, chromosome 11q23
• Penetrance is low - most carriers never develop attacks
• More common in women (female hormones trigger ALAS-1 induction)

1. When demand for heme increases (drugs, fasting, hormones) → ALAS-1 is induced
2. Due to PBGD deficiency, ALA and PBG accumulate
3. ALA is neurotoxic - interferes with GABA receptors and inhibitory neurons
4. This causes the neurovisceral symptoms

1. Pain - severe, acute abdominal pain (most common presenting symptom)
2. Polyneuropathy - peripheral and autonomic neuropathy
3. Psychiatric - anxiety, confusion, hallucinations, psychosis
4. Port-wine urine - reddish-brown urine on standing (PBG oxidizes to porphyrins)

• Fasting / low carbohydrate diet
• Alcohol
• Chemicals/drugs (barbiturates, sulfonamides, rifampicin, OCP, griseofulvin, anticonvulsants)
• Toxins
• Stress / infection / surgery / menstrual cycle (luteal phase)

• Urine: elevated ALA and PBG (Watson-Schwartz test - urine turns pink-red with Ehrlich reagent for PBG)
• Red fluorescence of urine under UV light
• Erythrocyte PBGD activity reduced (~50%)
• No photosensitivity (unlike other porphyrias)
• Normal during remission - diagnosis missed unless tested during attack

1. Remove precipitating factors (stop offending drug)
2. IV Glucose (300-400 g/day) - reduces ALAS-1 induction (glucose effect)
3. IV Hematin (3-4 mg/kg/day x 4 days) - provides exogenous heme, inhibits ALAS-1
4. Supportive: IV fluids, pain management (opioids), beta-blockers for tachycardia/hypertension, benzodiazepines for seizures (NOT barbiturates or phenytoin - these are UNSAFE)
5. Givosiran (siRNA) - monthly subcutaneous injection for recurrent attacks; neutralizes ALA mRNA in hepatocytes (approved 2019)

• Chronic renal failure
• Hepatocellular carcinoma (liver)
• Chronic neuropathy

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