Heme Degradation & Bilirubin Metabolism Dr. Shumaila Asim Lecture # 7

FATE OF RED BLOOD CELLS  Life span in blood stream is 60-120 days  RBCs are phagocytosed and/or lysed Normally, lysis occurs extravascularly in the reticuloendothelial system subsequent to RBC phagocytosis Lysis can also occur intravascularly (in blood stream)

Extravascular Pathway for RBC Destruction (Liver, Bone marrow, & Spleen) Phagocytosis & Lysis Hemoglobin Globin Heme Bilirubin Amino acids Fe2+ Amino acid pool Excreted

Handling of Free (Intravascular) Hemoglobin Purposes: 1. Scavenge iron 2. Prevent major iron losses 3. Complex free heme (very toxic) Haptoglobin: hemoglobin-haptoglobin complex is readily metabolized in the liver and spleen forming an iron-globin complex and bilirubin. Prevents loss of iron in urine. Hemopexin: binds free heme. The heme-hemopexin complex is taken up by the liver and the iron is stored bound to ferritin. Methemalbumin: complex of oxidized heme and albumin.

Bilirubin Metabolism Bilirubin formation Transport of bilirubin in plasma Hepatic bilirubin transport Hepatic uptake Conjugation Biliary excretion Excrete through intestine system

Bilirubin is the terminal product of heme metabolism Bilirubin is the terminal product of heme metabolism. Heme is present in hemoglobin and in other oxidative compounds such as hepatic mitochondrial and microsomal cytochromes (P-450). Thus plasma bilirubin is part erythropoietic and part non-erythropoietic. Approximately, 85 % erythropoietic and 15% non-erythropoietic.

The erythropoietic fraction originates from two sources: The circulating normal aging red cells and The immature defective red cells of the bone marrow. The daily production of bilirubin is 250 to 350 mg.

Shunt bilirubin is called that portion that does not originate from circulating red cells but originates from immature and defective red cells (7%) and from non- hemoglobin heme compounds, particularly from hepatic cytochromes and from myoglobin

Bilirubin from erythropoietic heme is produced by monocytic macrophages, reticulo-endothelium, in every organ but especially in the spleen, liver and bone marrow in order of importance. The bilirubin from non-erythropoietic hepatic heme is produced in the hepatocytes.

Bilirubin formation microsomal cytosol The iron-free porphyrin portion of heme is also degraded, mainly in the reticuloendothelial cells of the liver, spleen, and bone marrow.

Heme oxygenase The first step Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. This produces biliverdin, iron, and carbon monoxide.

The tetrapyrrolic ring of heme is broken by an oxygenase at the alpha bridge, the bond between the two carbons opposite to the gamma bridge which is between the two carbons carrying the two propionic acids. The tetrapyrrolic molecule from a ring is transformed into a tetrapyrrolic chain without iron

In mammalian cells Heme oxygenase (HO1) has two basic functions: 1. it recycles iron supplies within the cell to maintain homeostasis. 2. biliverdin and biliruben (its reduced form), are powerful antioxidants believed to aid in the prevention of oxidative cell damage.

M：-CH2 V：-CH=CH2 CH2 HOOC N H CH O M V COOH

The building of intermolecular hydrogen bonds by the NH and COOH groups is spatially hided. Bilirubin is lipophilic and therefore insoluble in aqueous solution.the solubility in water is less.

HEME + Heme oxygenase = OXY- HEME ( closed tetrapyrrolic ring with iron) OXY- HEME + heme reductase = BILIVERDIN (open tetrapyrrolic ring without iron) BILIVERDIN + biliverdin reductase = BILIRUBIN (unconjugated)

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

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

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.