Dr. Shumaila Asim Lecture # 8 Bilirubin Metabolism Dr. Shumaila Asim Lecture # 8

Definition of bilirubin Bilirubin is the water insoluble breakdown product of normal heme catabolism It’s a yellow pigment present in bile ( a fluid made by the liver) , urine and feces . Heme is found in hemoglobin, a principal component of RBCs [Heme: iron + organic compound “porphyrin”]. Heme source in body: 80% from hemoglobin 20% other hemo-protein: cytochrome, myoglobin)

Heme and bilirubin Heme four pyrrols rings connected together to form (porphyrin). Bilirubin consists of open chain of four pyrrols-like rings

Hemoglobin degrading and bilirubin formation Spleen Plasma Protein and a.a pool Iron pool TO LIVER Liver globin iron Heme Hemoglobin Bilirubin Binds with albumin Conjugation process

Transport of Bilirubin in Plasma Bilirubin on release from macrophages circulates as unconjugated bilirubin in plasma tightly bound to albumin. Albumin + free Bilirubn Bilirubin ~ Albumin Complex unconjugated bilirubin Why bound to albumin? Significance: ★Increase the solubility of whole molecule ★ Prevent unconjugated bilirubin freely come into other tissue, cause damage.

Transport of Bilirubin in Plasma Molar Ratio Bilirubin H affinity binding sites 2:1 Bilirubin Plasma protein Albumin L affinity binding sites >2:1 can be replaced by Other organic anions PH UB Albumin has two binding sites for bilirubin---a high affinity site and a low affinity site.

Bilirubin formed in peripheral tissues is transported to liver by albumin IN LIVER: 1)Uptake of bilirubin by liver paranchmal cells 2)conjugation of bilirubin with glucuronate in endoplasmic reticulum 3)secretion of conjugated bilirubin into bile

Uptake of bilirubin by liver Bilirubin is only sparingly soluble in water İts solubility in plasma is increased by noncovalent binding to albumin Albumin has one high affinity site and one low affinity site for bilirubin In 100 ml plasma = 25 mg bilirubin can be tightly bound to albumin at high affinity site

Antibiotics and other drugs compete with bilirubin to bind to high_affinity site of albumin. In liver, bilirubin is removed from albumin and taken up at the sinusoidal surface of hepatocytes by a facilated transport system. In the hepatocytes bilirubin binds to cytosolic proteins : ligandin (a family of glutathione s-transferases) and protein γ_to be kept solubilized prior to conjugation.

Conjugation of bilirubin with glucuronic acid Bilirubin is non-polar. Hepatocytes convert bilirubin to a polar form by adding glucuronic acid to it (conjugation) Enzyme: glucuronosyl transferase Location:endoplasmic reticulum Glucuronyl donor:UDP-GLUCURONIC ACID

Bilirubin monoglucuronide is an intermediate, subsequently converted to diglucuronide. Phenobarbital induces UDP-glucoronoyl-transferase activity

Major differences between unconjugated and conjugated bilirubin FEATURE Unconjugated bilirubin CONJUGATED BILIRUBIN Normal serum level More Less (less than 0.25mg/dl) Water solubility Absent Present Affinity to lipids (alcohol solubilty) Serum albumin binding High Low Van den Bergh reaction Indirect (Total minus direct) Direct Reanal excretion Affinity to brain tissue Present (kernicterus)

Excretion of bilirubin into bile Bilirubin diglucuronide is actively transported against a concentration gradient into bile duct. This energy-dependent, rate –limiting step is susceptible to impairment in liver disease. Uncojugated bilirubin is normally not excreated.

Formation of urobilins in the intestine Bilirubin diglucuronide is hydrolysed and reduced by bacteria in the gut to yield urobilinogen, a colorless compound. Most of the urobilinogens of the feces are oxidized by intestinal bacteria to stercobilin, which gives stools their characteristic brown color. Some urobilinogen is reabsorbed from the gut into the portal blood and transported to the kidney, where it is converted to the yellow urobilin and excreted, giving urine its characteristic color. (bilinogen enterohepatic circulation) Bilirubin diglucuronide urobilinogen urobilin stercobilin bilin

Catabolism of hemoglobin BLOOD CELLS Stercobilin excreted in feces Urobilinogen formed by bacteria Urobilin excreted in urine Heme Globin Hemoglobin KIDNEY CO Biliverdin IX Heme oxygenase O2 reabsorbed into blood INTESTINE via bile duct to intestines Bilirubin (water-insoluble) NADP+ NADPH Biliverdin reductase Bilirubin diglucuronide (water-soluble) 2 UDP-glucuronic acid Bilirubin (water-insoluble) via blood to the liver LIVER unconjugated Catabolism of hemoglobin

Summary of bilirubin metabolism red cells are major source of hemeproteins Breakdown of heme to bilirubin occur in macrophage of reticuloendithelial system ( tissue macrophages, spleen and liver). Unconjugated bilirubin is transported through blood ( complex to albumin) to liver. Bilirubin is taken into liver and conjugate with glucuronic acid. Bile is secreted into intestine where glucuronic acid is removed and the resulting bilirubin is converted to urobilinogen. A portion of urobilinogen is reabsorbed into blood, where it is converted to the yellow urobilin and excreted by kidneys. Urobilinogen is oxidized by intestinal bacteria to the brown stercobilin.