1. METABOLISM OF BILE 22.11.2012 V.Sridevi

2. LEARNING OBJECTIVES Biosynthesis of bilirubin and bile acids/salts Causes of hyperbilirubinemia

3. FATE OF RED BLOOD CELLS  Life span in blood stream is 60-120 days  Senescent 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)

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

5. Metabolism of bilirubin Bilirubin is potentially toxic catabolic product of heme metabolism. When Hb degraded- polypetides are degraded to aminoacids while the heme group are freed of their iron- salvaged and converted to bilirubin.

6. Metabolism of bilirubin… Approximately 300-400 mg of heme are degraded in the human body/day

7. Metabolism of bilirubin… Heme oxygenase is the only known CO-forming enzyme in the human body. Bilirubin is transported into the liver in tight, non covalent binding to serum albumin. Taken up into hepatocyte by a facilitated diffusion. Conjugation with glucouronic acid in ER to make it water soluble by the help of UDP glucouronic acid and UDP- glucouronyl transferase which forms UDP-diglucuronide. Water soluble bilirubin diglucuronide is actively secreted into the bile canaculi.

8. Metabolism of bilirubin… In the intestine, bilirubin diglucouronide is deconjugated by bacterial ß- glucouronidases and reduced to uncoloured urobilinogen. Urobilinogens Urobilins Stercobilinogen

9. DEGRADATION OF HEME TO BILIRUBIN P 450 cytochrome  75% is derived from RBCs  In normal adults this results in a daily load of 250-300 mg of bilirubin  Normal plasma concentrations are less then 1 mg/dL  Hydrophobic – transported by albumin to the liver for further metabolism prior to its excretion “unconjugated” bilirubin

10. NORMAL BILIRUBIN METABOLISM  Uptake of bilirubin by the liver is mediated by a carrier protein (receptor)  Uptake may be competitively inhibited by other organic anions  On the smooth ER, bilirubin is conjugated with glucoronic acid.  Glucoronic acid is the major conjugate - catalyzed by UDP glucuronyl tranferase  “Conjugated” bilirubin is water soluble and is secreted by the hepatocytes into the biliary canaliculi  Converted to stercobilinogen (urobilinogen) (colorless) by bacteria in the gut  Oxidized to stercobilin which is colored  Excreted in feces  Some stercobilin may be re-adsorbed by the gut and re-excreted by either the liver or kidney

12. Elevation of serum bilirubin cause jaundice Hyperbilirubinemia: Increased plasma concentrations of bilirubin (> 3 mg/dL) occurs when….? There is an imbalance between its production and excretion. Either unconjugated bilirubin or conjugated bilirubin or both are elevated All types of hyperbilirubinemia lead to the deposition of bilirubin in the tissues, skin and sclera of the eye which it imparts yellow colour. This condition is called jaundice or icterus

13. Jaundice… The sclera of the eye is affected early- because of its high content of elastin for which bilirubin has high affinity. Types of jaundice: Prehepatic Intrahepatic Post hepatic (obstructive)

15. Prehepatic (hemolytic) jaundice Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis Excess RBC lysis is commonly the result of autoimmune disease; hemolytic disease of the newborn (Rh- or ABO- incompatibility); structurally abnormal RBCs (Sickle cell disease); or breakdown of extravasated blood High plasma concentrations of unconjugated bilirubin (normal concentration ~0.5 mg/dL)

16. Intrahepatic jaundice Impaired uptake, conjugation, or excretion of bilirubin Reflects a generalized liver (hepatocyte) dysfunction – hepatitis, Cirrhosis, Leptospirosis etc In this case, hyperbilirubinemia is usually accompanied by other abnormalities in biochemical markers of liver function- AST and ALT

17. Posthepatic jaundice Caused by obstruction- gallstones (cholelithiasis), carcinoma of the head of pancreas, hepatitis etc Plasma bilirubin is conjugated, and other biliary metabolites, such as bile acids accumulate in the plasma. Characterized by pale colored, whitish stools (absence of stercobilin), and dark urine (increased conjugated bilirubin) In a complete obstruction, urobilin is absent from the urine The enzyme alkaline phosphatase (ALP) is typically elevated much more than the AST or ALT in obstructive jaundice

18. Neonatal Jaundice Common, particularly in premature infants. Transient (resolves in the first 10 days) Due to immaturity of the enzymes (UDP- glucuronyl transferase) involved in bilirubin conjugation If bilirubin levels are judged to be too high, then phototherapy with UV light is used to convert it to a water soluble, non-toxic form If necessary, exchange blood transfusion is used to remove excess bilirubin Phenobarbital is oftentimes administered to Mom prior to an induced labor of a premature infant – crosses the placenta and induces the synthesis of UDP glucuronyl transferase High levels of unconjugated bilirubin (lipid soluble) can enter the brain especially in infants. Bilirubin deposition in the basal ganglia can cause irreversible brain damage, a condition known as “kernicterus”

19. Diagnoses of Jaundice

20.  Autosomal dominant disorder  Characterized by mild, fluctuating increases in unconjugated bilirubin caused by decreased ability of the liver to conjugate bilirubin – often correlated with fasting or illness  Due to decreased UDP-glucuronyltransferase.  Serum bilirubin concentration rarely exceeds 5mg/dl.  Males more frequently affected then females  Can be treated with small doses of phenobarbital to stimulate UDP glucuronyl transferase activity Gilbert’s Syndrome

21. Crigler-Najjar Syndrome type I:  Autosomal recessive  Extremely rare < 200 cases worldwide – gene frequency is < 1:1000  High incidence in the “plain people of Pennsylvania” (Amish and Mennonites)  Characterized by a complete absence or marked reduction in bilirubin conjugating enzyme,UDP-glucuronosyl transferase.  Present with a severe unconjugated hyperbilirubinemia that usually presents at birth  Affected individuals are at a high risk for kernicterus Crigler-Najjar Syndrome

22. Crigler-Najjar Syndrome type II(Arias syndrome) Milder and caused by partial deficiency of bilirubin UDP-glucuronyltransferase. Because of lower serum bilirubin, kernicterus is rare in type II.

23. Dubin-johnson syndrome and Rotors syndrome Associated with conjugated hyperbilirubinemia. Uncommon, rare. Associated with a defect in the ability of hepatocytes to secrete conjugated bilirubin into the bile, and is similar to Rotors syndrome.

24. Table 2- Genetic Disorders of Bilirubin Metabolism ConditionDefectBilirubin Clinical Findings Crigler-Najjar syndrome severely defective UDP-glucuronyl transferase Unconjugated bilirubin  Profound jaundice Gilberts syndrome reduced activity of UDP-glucuronyl transferase Unconjugated bilirubin  Very mild jaundice during illnesses Dubin- Johnson syndrome abnormal transport of conjugated bilirubin into the biliary system Conjugated bilirubin  Moderate jaundice

25. Unconjugated hyperbilirubinemia: Prehepatic jaundice Neonatal jaundice Gilbert’s disease Criggler najjar syndrome Conjugated hyperbilirubinemia: Post hepatic jaundice Dubin-johnson syndrome Rotors syndrome

26. Cholesterol and Bile Acid/Salt Metabolism Synthesis of bile acids is the predominant mechanism by which excess cholesterol is eliminated from the body Major excretory form of cholesterol Occurs in liver Bile acid/salts involved in dietary lipid digestion as emulsifiers

27. Bile Acids and Salts (Biological detergents) Bile acids are synthesized from cholesterol and is major metabolic fate of cholesterol. Bile acids are synthesized and secreted by the liver and conjugated with glycine or taurine to form bile salts before leaving the liver. Bile salts are stored in gallbladder and passed through the bile duct into the intestine.

28. Synthesis of bile acid

29. Types of Bile Acids/Salts Primary bile acids: End product of cholesterol catabolism in liver. chenodeoxycholic acid (45%) and cholic acid (31%).

30. Bile salts The carboxyl group of primary bile acids is conjugated with glycine or taurine to yield glycocholic acid and taurocholic acid which are called as bile salts. Lithocholic acid and deoxycholic acid are the secondary bile acids formed by the action of intestinal bacterial enzyme 7- alpha dehydroxylase. Ursodeoxycholic acid is the tertiary bile acid.

31. Role of Bile Salts in Fat Digestion Bile salt molecule has both hydrophobic and hydrophilic surfaces (amphipathic). Bile salts able to orient at an oil-water interface, with the hydrophobic surface in contact with the a polar phase and the hydrophilic surface in contact with the aqueous phase. Detergent action emulsifies lipids and yields micelles hence allowing attack by water-soluble enzymes. Most of the digestion carried out by pancreatic lipase in water-oil interface.

32. Action of Bile Salts in Emulsifying Fats in the Intestine

33. Learning outcomes At the end of the lecture student should be able to Describe the synthesis and excretion of bilirubin List the primary, secondary and tertiary bile acids Describe the pathway of synthesis and excretion of bile acids/salts Classify hyperbilirubinemia and list the underlying causes Explain the differences between conjugated and unconjugated types Classify heriditary hyperbilirubinemia and state the defects and mode of inheritance [Gilberts syndrome, Criggler Najjar syndrome and Dubin johnson and Rotor syndrome].