Heme Catabolism and Fate of Bilirubin - Biochemistry

1. Overview

• 70-80% from hemoglobin in senescent red blood cells (sequestered by reticuloendothelial cells)
• 20-30% from other heme proteins (myoglobin, cytochromes P450, catalase, peroxidase) mainly in hepatocytes
• Minor fraction from ineffective erythropoiesis (premature destruction of newly formed erythrocytes)

2. Step-by-Step Heme Catabolism

Step 1 - Heme to Biliverdin (in Reticuloendothelial Cells)

Fe³⁺-Heme + 3 O₂ + 7 e⁻ → Biliverdin + CO + Fe³⁺

• Heme oxygenase is substrate-inducible (heme both induces and acts as substrate)
• The iron in heme is typically already oxidized to ferric form (hemin) before reaching HO
• 3 moles of O₂ are consumed; electrons are provided by NADPH via cytochrome P450 reductase
• The heme ring is opened at the α-methene bridge, releasing a green pigment - biliverdin IXα
• CO is released (has signaling and anti-inflammatory roles; excreted via lungs)
• Fe³⁺ is released and reutilized (enters the iron pool)
• Two isoforms: HO-1 (inducible, ER, ubiquitous) and HO-2 (constitutive, mitochondria, in selected tissues including hepatocytes)

Step 2 - Biliverdin to Bilirubin

Biliverdin + NADPH + H⁺ → Bilirubin IXα + NADP⁺

• The central methylene bridge of biliverdin is reduced, forming the yellow-orange pigment bilirubin
• This step is unique to mammals (birds and amphibians excrete biliverdin directly)
• Bilirubin also serves as an antioxidant at physiologic concentrations; it cycles bilirubin → biliverdin → bilirubin via BVR, regenerating protective antioxidant capacity

Visual clue: The color changes of a bruise (purple → green → yellow) directly reflect heme → biliverdin → bilirubin conversion in the tissues.

3. Transport to the Liver

• Binds noncovalently to serum albumin for transport in the blood
• Albumin has a high-affinity site (~25 mg bilirubin/100 mL plasma) and a low-affinity site
• Certain drugs (salicylates, sulfonamides, rifampin) can displace bilirubin from albumin, increasing free bilirubin and risk of kernicterus in neonates

4. Hepatic Processing of Bilirubin (3 Stages)

Stage 1: Hepatic Uptake

• Bilirubin is removed from albumin at the sinusoidal surface of hepatocytes via a large-capacity, saturable facilitated transport system
• Internalized bilirubin binds to cytosolic ligandin (glutathione S-transferase), which prevents it from re-entering the bloodstream
• This uptake step is not normally rate-limiting

Stage 2: Conjugation with Glucuronic Acid

• Bilirubin is nonpolar and must be made water-soluble for excretion
• Bilirubin UDP-glucuronosyltransferase (UGT1A1) in the ER catalyzes sequential conjugation:

Bilirubin + UDP-glucuronate → Bilirubin monoglucuronide + UDP

Bilirubin monoglucuronide + UDP-glucuronate → Bilirubin diglucuronide + UDP

• The diglucuronide (conjugated/direct bilirubin) is the predominant form in bile (~80%)
• This transformation makes bilirubin water-soluble, non-toxic, and excretable

Stage 3: Secretion into Bile

• Conjugated bilirubin is actively transported into bile canaliculi by MOAT (Multispecific Organic Anion Transporter), also called MRP2 (Multidrug-Resistant Protein 2) - a member of the ATP-binding cassette (ABC) transporter family
• This is the rate-limiting step for overall hepatic bilirubin metabolism
• Both conjugation and excretion are induced by the same drugs (e.g., phenobarbital)
• Deficiency in MRP2 → Dubin-Johnson syndrome (conjugated hyperbilirubinemia)

5. Fate in the Intestine - The Enterohepatic Circulation

1. Bacterial deconjugation cleaves glucuronate
2. Bacterial reduction produces urobilinogen (colorless)
3. Most urobilinogen is further oxidized by bacteria to stercobilin → excreted in feces (gives stool its characteristic brown color)
4. A small fraction of urobilinogen is reabsorbed from the gut into portal blood:
   • Most is re-extracted by the liver and re-excreted in bile (enterohepatic circulation)
   • A small amount escapes into the systemic circulation and is excreted by the kidneys as urobilin (gives urine its yellow color)

6. δ-Bilirubin

7. Laboratory Parameters in Jaundice

8. Diseases of Bilirubin Metabolism

Classification by Site

A. UNCONJUGATED HYPERBILIRUBINEMIAS

1. Hemolytic (Pre-hepatic) Jaundice

• Excessive RBC destruction overwhelms the liver's conjugation capacity
• ↑ Indirect bilirubin; bilirubin usually modest (<4 mg/dL) because the healthy liver has large reserve capacity
• No bilirubinuria (unconjugated bilirubin cannot pass glomerulus)
• ↑ Urobilinogen in urine and stool
• Examples: sickle cell anemia, thalassemia, G6PD deficiency, autoimmune hemolysis

2. Neonatal "Physiologic" Jaundice

• Results from accelerated hemolysis + immature UGT1A1 activity (low enzyme + low UDP-glucuronate synthesis)
• UGT1A1 levels are low at birth; do not reach adult levels until 3-4 months
• Breast milk jaundice: bilirubin-deconjugating enzymes in breast milk may worsen hyperbilirubinemia
• Risk: unconjugated bilirubin >20-25 mg/dL can cross the blood-brain barrier → kernicterus (toxic encephalopathy causing mental retardation)
• Treatment: phototherapy with blue light (converts bilirubin to water-soluble photoisomers excreted in urine/bile without needing conjugation); phenobarbital (induces UGT1A1)

3. Gilbert Syndrome

• Most common hereditary hyperbilirubinemia
• Autosomal recessive - homozygous polymorphism in the UGT1A1 promoter (UGT1A1*28: extra TA repeat in the TATA box → ↓ UGT1A1 transcription → ~30% residual UGT1A1 activity)
• Allele frequencies: 42% Black, 30% White, 10% Asian populations
• Mild unconjugated hyperbilirubinemia (<3 mg/dL); increases 2-3 fold with fasting, stress, illness
• Clinically benign - no liver damage
• Clinical relevance: Irinotecan (CPT-11) toxicity - active metabolite SN-38 is glucuronidated by UGT1A1; patients with Gilbert's get severe diarrhea and myelosuppression. Also affects raloxifene metabolism.
• Phenobarbital normalizes bilirubin

4. Crigler-Najjar Syndrome Type I

• Complete absence of UGT1A1 activity (autosomal recessive; nonsense mutations in UGT1A1 gene)
• Severe congenital jaundice: bilirubin >20 mg/dL
• Fatal within the first 15 months if untreated (kernicterus)
• Phenobarbital has no effect (no enzyme to induce)
• Treatment: prolonged phototherapy (12-16 hrs/day) + ultimately liver transplantation

5. Crigler-Najjar Syndrome Type II (Arias Syndrome)

• Reduced (but not absent) UGT1A1 activity
• Serum bilirubin <20 mg/dL
• More benign; responds to phenobarbital (which induces residual enzyme)

6. Toxic Hyperbilirubinemia

• Hepatocyte damage → impaired conjugation
• Causes: chloroform, CCl₄, acetaminophen overdose, hepatitis viruses, cirrhosis, Amanita mushroom poisoning, certain drugs (isoniazid, diclofenac)
• Mostly unconjugated initially; mixed (conjugated + unconjugated) with cell necrosis

B. CONJUGATED (DIRECT) HYPERBILIRUBINEMIAS

7. Dubin-Johnson Syndrome

• Autosomal recessive - mutation in MRP2 gene (canalicular transporter for organic anions including conjugated bilirubin)
• Impaired secretion of conjugated bilirubin into bile canaliculi
• Conjugated hyperbilirubinemia (mild, <7 mg/dL); usually presents in childhood/adulthood
• Striking feature: deposition of brown-black melanin-like pigment in hepatocytes → can blacken the liver grossly
• Clinically benign; normal life expectancy
• Total urinary coproporphyrin normal but ratio of isomers I/III is reversed (>80% isomer I vs normally <25%)

8. Rotor Syndrome

• Autosomal recessive - mutation in SLCO1B1/SLCO1B3 genes (organic anion transporting polypeptides OATP1B1/1B3) - defective reuptake of bilirubin conjugates secreted into the space of Disse back into hepatocytes
• Similar to Dubin-Johnson but no liver pigment deposition
• Total urinary coproporphyrin elevated (isomers I + III both high)
• Clinically benign

9. Obstructive (Post-hepatic/Cholestatic) Jaundice

• Blockage of hepatic or common bile duct prevents excretion of conjugated bilirubin
• Most common causes: gallstone in CBD or carcinoma of the head of the pancreas
• Conjugated bilirubin regurgitates into hepatic veins and lymphatics → enters blood and urine (choluria/bilirubinuria)
• Pale/clay-colored stools (acholic - no stercobilin reaches gut)
• No urobilinogen in urine (no bilirubin reaches the intestine)
• ↑ Alkaline phosphatase, ↑ GGT (cholestatic markers)
• Persistent obstruction → biliary cirrhosis, ascending cholangitis

10. Hepatocellular Disease (Hepatitis/Cirrhosis)

• Mixed conjugated + unconjugated hyperbilirubinemia
• Impaired uptake, conjugation, AND secretion
• Both types of bilirubin elevated; ↑ ALT, ↑ AST (hepatocellular injury markers)
• Urobilinogen may be decreased (if micro-obstruction of bile ductules occurs)

9. Summary Table: Hereditary Hyperbilirubinemias

10. Key Biochemical Points for Exams

1. Heme oxygenase is microsomal, substrate-inducible; uses 3 O₂ + 7 electrons (NADPH)
2. CO is a byproduct (excreted via lungs) - also a signaling molecule
3. Biliverdin reductase uses NADPH; mammals unique in this reduction step
4. 1 g hemoglobin → ~35 mg bilirubin
5. Ligandin (glutathione S-transferase) - intrahepatic bilirubin binding protein
6. UGT1A1 - key conjugating enzyme; inducible by phenobarbital
7. MRP2 - canalicular ABC transporter; rate-limiting step for bilirubin excretion
8. Conjugated bilirubin is water-soluble, appears in urine; unconjugated does not (albumin-bound)
9. Jaundice clinically detectable when bilirubin >3-4 mg/dL
10. Kernicterus risk when unconjugated bilirubin >20-25 mg/dL (crosses BBB)