Bilirubin formation in detail from basica

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Bilirubin Formation - From Basics

Bilirubin is the major end product of heme catabolism. Its metabolism is a tightly organized, multi-organ process spanning the mononuclear phagocyte system (MPS), blood, liver, and intestine.

1. Sources of Heme (What Gets Degraded)

About 80-85% of daily bilirubin (~4 mg/kg/day) comes from the breakdown of hemoglobin in senescent red blood cells (RBC lifespan ~120 days). The remaining 15-20% comes from:

Premature destruction of erythroid precursors in the bone marrow (ineffective erythropoiesis)
Turnover of non-hemoglobin hemoproteins: myoglobin, cytochrome P-450, catalase, peroxidase

These sources are collectively called the "early-labeled" bilirubin fraction.

Harrison's Principles of Internal Medicine 22E, p. 364

2. Step 1 - RBC Destruction and Heme Liberation

Senescent RBCs are engulfed by macrophages of the MPS - primarily in the spleen, liver (Kupffer cells), and bone marrow. Inside the macrophage:

Hemoglobin is split into globin (recycled as amino acids) and heme (ferroprotoporphyrin IX)
Heme = an iron-containing, cyclic tetrapyrrole (porphyrin ring with Fe²⁺ at the center)

3. Step 2 - Heme → Biliverdin (via Heme Oxygenase)

This is the rate-limiting step in bilirubin formation.

Enzyme: Microsomal heme oxygenase (located in the ER of macrophages)

Reaction: Three successive oxygenations using NADPH + O₂:

The α-methene bridge carbon of the porphyrin ring is oxidatively cleaved
The cyclic heme ring is opened to form a linear tetrapyrrole
Products released: Biliverdin (green pigment) + CO (carbon monoxide) + Fe²⁺

The equation per molecule of heme:

Heme + 3O₂ + 3NADPH → Biliverdin + CO + Fe²⁺ + 3NADP⁺

By-products have functions:

CO acts as a signaling molecule and anti-inflammatory mediator
Fe²⁺ is recycled - bound to transferrin and transported back to bone marrow for re-use in new hemoglobin

In birds, reptiles, and amphibians, biliverdin is the final excretory product. In mammals, it is reduced further to bilirubin - this is evolutionarily important because bilirubin (unlike biliverdin) can cross the placenta, allowing fetal bilirubin to be excreted via the maternal liver.

Heme → Biliverdin → Bilirubin in macrophage, showing heme oxygenase and biliverdin reductase steps with NADPH

4. Step 3 - Biliverdin → Bilirubin (via Biliverdin Reductase)

Enzyme: Cytosolic biliverdin reductase

Reaction: Reduces the central methylene bridge of biliverdin using NADPH:

Biliverdin + NADPH + H⁺ → Bilirubin + NADP⁺

This converts the green biliverdin to the characteristic red-orange bilirubin.

Why bilirubin is insoluble: Bilirubin has tight internal hydrogen bonds between its propionic acid carboxyl groups and the imino/lactam groups of the opposite dipyrrolic half. This "ridge-tile" configuration:

Places hydrophobic residues outward
Buries polar residues inward
Makes bilirubin virtually insoluble in water

Note: Bilirubin in mammals also has antioxidant function at low concentrations. It is oxidized back to biliverdin, which is then reduced again by biliverdin reductase - creating a redox cycle.

Lippincott's Illustrated Reviews: Biochemistry 8e, p. 795

5. Step 4 - Blood Transport (Unconjugated Bilirubin)

Because bilirubin is water-insoluble, it cannot travel freely in plasma. It is:

Bound noncovalently to albumin (2 binding sites per albumin molecule)
This form is called unconjugated (indirect) bilirubin or free bilirubin
It is NOT filtered by the kidney in normal conditions (too large as albumin complex)

Clinical note: Certain drugs (salicylates, sulfonamides, furosemide, radiographic contrast agents) competitively displace bilirubin from albumin. In neonates, this raises free bilirubin which can cross the blood-brain barrier and cause kernicterus.

Goldman-Cecil Medicine, Chapter 133

6. Step 5 - Hepatic Uptake

When albumin-bilirubin complex reaches the liver sinusoids:

Bilirubin dissociates from albumin (albumin stays in blood)
Unconjugated bilirubin enters hepatocytes by facilitated diffusion (specific transporters, including OATP1B1/OATP1B3)
Inside the hepatocyte cytosol, bilirubin binds to ligandin (glutathione-S-transferase Y) and protein Z - these reduce back-diffusion and shuttle bilirubin to the ER

7. Step 6 - Conjugation in the Liver (Making Bilirubin Water-Soluble)

Location: Endoplasmic reticulum of hepatocytes

Enzyme: Bilirubin UDP-glucuronosyltransferase (UGT1A1), encoded by the UGT1 gene complex

Reaction: Sequential addition of two glucuronic acid molecules from UDP-glucuronic acid:

Bilirubin + UDP-glucuronic acid → Bilirubin monoglucuronide (BMG)
BMG + UDP-glucuronic acid → Bilirubin diglucuronide (BDG) ← predominant form

The conjugation disrupts the internal hydrogen bonds, making bilirubin water-soluble (conjugated/direct bilirubin). It can now be excreted.

According to Guyton and Hall: ~80% is conjugated as bilirubin glucuronide, ~10% as bilirubin sulfate, and ~10% with other substances.

Bilirubin conjugation in the liver - UDP-glucuronosyltransferase adds 2 glucuronic acid molecules to form bilirubin diglucuronide, excreted into bile

Clinical relevance of UGT1A1:

Gilbert syndrome: Mild UGT1A1 reduction → mild unconjugated hyperbilirubinemia
Crigler-Najjar syndrome type I: Complete absence of UGT1A1 → severe, life-threatening unconjugated hyperbilirubinemia
Crigler-Najjar syndrome type II (Arias): Markedly reduced UGT1A1

8. Step 7 - Biliary Secretion

Conjugated bilirubin is actively transported across the canalicular membrane into bile by:

MRP2 (Multidrug Resistance-associated Protein 2), also called cMOAT/ABCC2 - an ATP-dependent transporter
This is the rate-limiting step for bilirubin excretion (not conjugation)
MRP3 transports some conjugated bilirubin back into sinusoidal blood when MRP2 is overloaded (regurgitation pathway)

Clinical note: Deficiency of MRP2 (canalicular transport) = Dubin-Johnson syndrome (conjugated hyperbilirubinemia, benign).

Conjugated bilirubin flows into bile, is stored in the gallbladder, and enters the duodenum.

9. Step 8 - Intestinal Fate and Enterohepatic Circulation

In the small intestine, conjugated bilirubin is NOT absorbed (too hydrophilic and large). In the terminal ileum and colon, intestinal bacteria:

Hydrolyze the glucuronide conjugate (beta-glucuronidase) → free unconjugated bilirubin
Reduce bilirubin → urobilinogen (colorless compound), a series of compounds (mesobilirubinogen, stercobilinogen)

Fate of urobilinogen:

Most (~80%) is further oxidized by bacteria to stercobilin (brown pigment) → excreted in feces (gives stool its brown color)
~20% is reabsorbed from the gut into portal blood:
- Most is re-extracted by the liver and re-secreted into bile = enterohepatic circulation of urobilinogen
- A small fraction escapes into systemic blood → kidneys → oxidized to urobilin (yellow) → excreted in urine (gives urine its yellow color)

Complete bilirubin catabolism pathway showing 10-step process from senescent RBCs through macrophage breakdown, liver conjugation, biliary excretion, intestinal conversion to urobilinogen/stercobilin, and renal excretion of urobilin

Summary Table

Stage	Location	Key Enzyme/Protein	Product
RBC destruction	Spleen/liver/BM macrophages	-	Heme liberated
Heme → Biliverdin	Macrophage ER	Heme oxygenase	Biliverdin + CO + Fe²⁺
Biliverdin → Bilirubin	Macrophage cytosol	Biliverdin reductase	Unconjugated bilirubin
Blood transport	Plasma	Albumin	Bilirubin-albumin complex
Hepatic uptake	Hepatocyte cytosol	Ligandin/OATP	Unconjugated bilirubin (intrahepatic)
Conjugation	Hepatocyte ER	UGT1A1	Bilirubin diglucuronide (conjugated)
Biliary secretion	Bile canaliculus	MRP2 (ATP-dependent)	Conjugated bilirubin in bile
Intestinal reduction	Colon bacteria	Bacterial reductases	Urobilinogen
Fecal excretion	Intestine	Bacterial oxidases	Stercobilin (brown)
Urinary excretion	Kidney	-	Urobilin (yellow)

Normal Reference Values

Total serum bilirubin: ≤1 mg/dL
Conjugated (direct): ≤0.3 mg/dL
Jaundice clinically visible: ≥2-3 mg/dL

Sources: Lippincott's Illustrated Reviews: Biochemistry 8e (pp. 794-799) | Harrison's Principles of Internal Medicine 22E (p. 364) | Goldman-Cecil Medicine, Chapter 133 | Guyton and Hall Medical Physiology 14e, Chapter 71

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