Bilirubin metabolism

Degradation of Hemoglobin, Jaundice and case studies

1. Haemoglobin Degradation Akanksha Dubey

2. Erythrocytes have a life span of 120 days in adults(60-90 days in neonates , 35-50 days in pre-mature neonates). At the end of this period, they are removed from the circulation. Erythrocytes are taken up and degraded by the macrophages of the reticulo -endothelial (RE) system in the spleen and liver.

3. The haemoglobin is cleaved to the protein part globin and non-protein heme. About 6 g of haemoglobin per day is broken down, and resynthesized in an adult man.

4. Fate of globin : The globin may be reutilized as such for the formation of haemoglobin or degraded to the individual amino acids. The Iatter undergo their own metabolism, including participation in fresh globin synthesis.

5. Sources of heme : 80% of the heme is derived from the erythrocytes and the rest (2O%) comes from immature RBC, myoglobin and cytochromes .

6. Heme oxygenase is present in microsomes of spleen, liver and bone marrow Heme oxygenase cleaves methylene bridges b/w I and II pyrrole rings. Iron is used for recycling into Iron pool

7. Biliverdin is green colored bile pigment which is excretory product in birds and amphibians but in humans it is further degraded. Biliverdin is linear tetrapyrrole compound An enzyme Biliverdin Reductase cleaves methylene bridges of III and Iv pyrrole rings to form Yellow colored pigment Bilirubin. 1 gm of Hb is converted to 35 mg of Bilirubin, 250-350 mg of Bilirubin is produced everyday

8. Bilirubin formed is insoluble in water hence it is bound to albumin 100 ml of plasma can carry 25 mg of bilirubin-albumin complex beyond this bilirubin is loosely bound to albumin and hence can be easily released into circulation. Certain drugs like aspirin, sulfa -antibiotics salicylates compete with bilirubin to bind to albumin , which makes free bilirubin to cross blood brain barrier . As the albumin-bilirubin complex enters the liver, bilirubin dissociates and is taken up by sinusoidal surface of the hepatocytes by a carrier mediated active transport. The transport system has a very high capacity and therefore is not a limitation for further metabolism of bilirubin. Inside the hepatocytes, bilirubin binds to a specific intracellular protein namely Ligandin .

9. Conjugation in Liver Inside the liver cell, the bilirubin is conjugated with Glucuronic Acid , to make it water soluble(conjugation disturbs internal hydrogen bonding that limits solubility of bilirubin) The first carbon of glucuronic acid is combined with the carboxyl group of the propionic acid side chains of the bilirubin molecule. About 80% molecules are in the diglucuronide form, while 20% are monoglucuronides .

10. iii. Drugs like primaquine , novobiocin , chloramphenicol , and androgens may interfere in this conjugation process and may cause jaundice.

11. The water soluble conjugated bilirubin is excreted into the bile by an active process and this occurs against a concentration gradient. This is the rate limiting step in the catabolism of heme . It is induced by phenobarbitone . Excretion of conjugated bilirubin into bile is mediated by an ATP binding cassette protein which is called Multispecific organic anion transporter (MOAT), located in the plasma membrane of the biliary canaliculi . .The conjugated bilirubin reaches the intestine through the bile. Intestinal bacteria deconjugate the conjugated bilirubin. This free bilirubin is further reduced to a Colorless Tetrapyrrole Urobilinogen (UBG) Further reduction of the vinyl substituent groups of UBG leads to formation of stercobilinogen (SBG) The SBG is mostly excreted through feces (250-300 mg/day)

12. Enterohepatic Circulation 20% of the UBG is reabsorbed from the intestine and returned to the liver by portal blood. The UBG is again re-excreted ( enterohepatic circulation). Since the UBG is passed through blood, a small fraction is excreted in urine (less than 4 mg/day)

13. Fate of Bilirubin: Bilirubin Glucuronides are hydrolysed in the intestine by specific bacterial enzymes namely Beta- glucuronidases to liberate bilirubin. The latter is then converted to urobilinogen (colourless compound) , a small part of which may be reabsorbed into the circulation. Urobilinogen be converted to urobilin (an yellow color compound) in the kidney and excreted. The characteristic colour of urine is due to urobilin

14. A major part of urobilinogen is converted by intestinal bacteria to stercobilin which is excreted along with feces . The characteristic brown colour of feces is due to stercobilin .

15. Summary of bilirubin metabolism in liver showing its transfer from blood to bile involving three processes - uptake, conjugation and secretion. ( = represents blockage at the respective steps showing the diseases that occur due to the blockage). SUMMARY OF BILIRUBIN METABOLISM IN LIVER

16. Plasma Bilirubin Normal plasma bilirubin level ranges from 0.2– 0.8 mg/dl. The Unconjugated bilirubin is about 0.2–0.6 mg/dl The Conjugated bilirubin is only 0–0.2 mg/dl. If the plasma bilirubin level exceeds 1 mg/dl, the condition is called Hyperbilirubinemia . Levels between 1- 2 mg/dl are indicative of latent jaundice(without any visible symptoms)

17. When the bilirubin level exceeds 2 mg/dl, it diffuses into tissues producing yellowish discoloration of sclera, conjunctiva, skin and mucous membrane resulting in JAUNDICE. Icterus is the Greek term for jaundice

18. Van den Bergh reaction This is a specific reaction to identify the increase in serum bilirubin. Normal serum gives a negative van den Bergh reaction. Mechanism of the reaction : van den Bergh reagent is a mixture of equal volumes of sulfanilic acid (in dilute HCI) and sodium nitrite. The principle of the reaction is that diazotised sulfanilic acid reacts with bilirubin to form a purple coloured azobilirubin .

19. Direct and indirect reactions : Bilirubin as such is insoluble in water while the conjugated bilirubin is soluble. van den Bergh reagent reacts with conjugated bilirubin and gives a purple colour immediately (normally within 30 seconds). This is referred to as a direct positive van den Bergh reaction . Addition of methanol (or alcohol) dissolves the unconjugated bilirubin which then gives the van den Bergh reaction (normally within 30 minutes) positive and this is referred to as indirect positive .

20. lf the serum contains both unconjugated and conjugated bilirubin in high concentration, the purple colour is produced immediately (direct positive) which is further intensified by the addition of alcohol (indirect positive). This type of reaction is known as Biphasic .

21. van den Bergh reaction and jaundice : This reaction is highly useful in understanding the nature of jaundice. This is due to the fact that the type of jaundice is characterized by increased serum concentration of unconjugated bilirubin ( hemolytic ), conjugated bilirubin (obstructive) or both of them (hepatic).

22. Therefore, the response of van den Bergh reaction can differentiate the jaundice as follows Indirect positive - Hemolytic jaundice Direct positive - Obstructive jaundice Biphasic - Hepatic jaundice.

23. HYPERBILIRUBINEMIAS Depending on the nature of the bilirubin elevated, the condition may be grouped into conjugated or unconjugated hyperbilirubinemia . Based on the cause it may also be classified into congenital and acquired.

24. Congenital Hyperbilirubinemias

25. Crigler-Najjar Syndrome Here the defect is in conjugation. In Type 1 (Congenital non- hemolytic jaundice), there is severe deficiency of UDP Glucuronyl Transferase . The disease is often fatal and the children die before the age of 2. Jaundice usually appears within the first 24 hours of life. Unconjugated bilirubin level increases to more than 20 mg/dl, and hence Kernicterus results.

26. Type 2 disease is a milder form; the process of conjugation is partly impaired(addition of second glucoronide is defective). When barbiturates are given, some response is seen and jaundice improves. Bilirubin level in blood exceeds 20 mg/dl in Crigler-Najjar syndrome Type 1 and does not exceed 20 mg/ dl in Crigler-Najjar syndrome Type 2.

27. The gene encoding Bilirubin-UDP Glucuronyl Transferase is a part of large gene complex present in chromosome number 2. This contains 13 substrate specific exons , each with its own promoter. Exon A1 is involved with conjugation of bilirubin.

28. Gilbert's Disease It is inherited as an autosomal dominant trait affecting mostly males The defect is in the uptake of bilirubin by the liver and activity of UDP- Glucoronyl Transferase is partly defective hence conjugation is affected. Even bilirubin secretion in bile is also affected. Bilirubin level is usually around 3 mg/dl, and patient is asymptomatic, except for the presence of mild jaundice( Unconjugated )

29. Dubin -Johnson Syndrome It is an autosomal recessive trait leading to defective excretion of conjugated bilirubin; so conjugated bilirubin in blood is increased. The disease results from the defective ATP dependent organic anion transport in bile canaliculi . There is a mutation in the MRP-2 (Multidrug Resistance Associated Protein) protein which is responsible for transport of conjugated bilirubin into bile. The bilirubin gets deposited in the liver and the liver appears black.

30. The condition is referred to as Black liver jaundice . In normal persons, when 250 mg BSP ( Bromosulphthalein ) is given intravenously, blood contains only 5% of original value at 45 minutes, and only 2% at 2 hr. BSP is taken up by hepatocytes, conjugated and then excreted through liver. In Dubin -Johnson's syndrome, the BSP level in serum at 2 hours is more than the value at 45 min. This is because by 45 min, BSP is taken by hepatocytes, and hence blood level is reduced. But excretory defect for conjugated BSP leads to regurgitation into plasma. Hence higher value at 2 hr.

31. Rotor Syndrome It is a similar condition, but the exact defect is not identified. Bilirubin excretion is defective, but there is no staining of the liver. It is an autosomal recessive condition.

32. CONGENITAL HYPERBILIRUBINEMIAS

33. Acquired Hyperbilirubinemias or Jaundice

34. Jaundice

35. Physiological Jaundice It is also called as Neonatal Hyperbilirubinemia . In all newborn infants after the 2nd day of life, mild jaundice appears. This transient hyperbilirubinemia is due to an accelerated rate of destruction of RBCs and also because of the immature hepatic system of conjugation of bilirubin(activity of enzyme UDP Glucoronyl Transferase is low).

36. In such cases, bilirubin does not increase above 5 mg/dl. It disappears by the second week of life.

37. However in some cases Bilirubin level increases above 25 mg/dl , which crosses blood brain barrier and causes Hyperbilirubinemic Toxic Encephalopathy or Kernicterus which leads to mental retardation. Drug phenobarbital is used for treatment becoz it can induce conjugation Above 5 mg/dl bilirubin is treated by Phototherapy

38. Phototherapy : Bilirubin can absorb blue light (420-470 nm) maximally. Phototherapy deals with the exposure of the jaundiced neonates to blue light. By a process called photoisomerization , the toxic native unconjugated bilirubin gets converted into a non-toxic isomer namely lumirubin .

39. Lumirubin can be easily excreted by the kidneys in the unconjugated form (in contrast to bilirubin which cannot be excreted). Serum bilirubin is monitored every 12-24 hours, and phototherapy is continuously carried out till the serum bilirubin becomes normal (< 1 mg/dl).

40. Breast milk jaundice In some breast-fed infants, unconjugated hyperbilirubinemia occurs. This is because of the presence of enzyme Beta Glucoronidase in breast milk which de-conjugates bilirubin in the intestine.

41. Jaundice is a yellow discoloration of the skin, mucous membranes, and sclera caused by increased amounts of bilirubin in the blood. Jaundice is a sign of an underlying disease process. Jaundice results from the accumulation of bilirubin. Hyperbilirubinemia may be due to abnormalities in the formation, transport, metabolism, and excretion of bilirubin.

42. Jaundice results from one or more of the following mechanisms: (1) excessive production of bilirubin, (2) reduced hepatic uptake, (3) impaired conjugation, (4) decreased hepatocellular excretion, and (5) impaired bile flow (both intra hepatic and extra hepatic).

43. Hemolytic Jaundice This condition results from increased hemolysis of erythrocytes(Malaria, hemolytic anaemia, Sickle cell anaemia, incompatible blood transfusion, G6PD Deficiency, Toxins like carbon tetrachloride)

44. This results in production of bilirubin beyond the capability of liver to conjugate(however liver can conjugate ten times more bilirubin than normally 0.3mg/day) More bilirubin in bile, more formation of stercobilinogen and urobilinogen

45. However bilirubin is more but can’t appear in urine ( unconjugated is non-polar) In hemolytic jaundice: Increased serum uncojugated Bilirubin Increased urine urobilinogen Increased feces stercobilinogen (dark color stool)

46. Hepatocellular Jaundice This kind of Jaundice is caused by perenchymal liver disease The most common cause is viral hepatitis, chronic alcohol abuse leading to cirrhosis , cystic fibrosis As a result of damaged hepatocytes bilirubin secretion in Bile is impaired (this can be anywhere from uptake of uncojugated bilirubin to transport of conjugated bilirubin into bile duct)

47. Hepatic jaundice is characterized by: Increased levels of conjugated and unconjugated bilirubin in the serum. Dark coloured urine due to the excessive excretion of bilirubin and urobilinogen lncreased activities of alanine transaminase (SGPT) and aspartate transaminase (SGOT) released into circulation due to damage to hepatocytes.

48. The patients pass pale, clay coloured stools due to the absence of stercobilinogen . The affected individuals experience nausea and anorexia (loss of appetite)

49. Obstructive Jaundice This occurs d/t obstruction of bile duct that prevents the flow of bile from liver to the intestine. The common causes of obstructive jaundice may be gallstones or tumours Due to blockage in the bile duct conjugated bilirubin enters into the circulation and slowly diffuses into tissues producing jaundice.

50. Obstructive Jaundice is characterized by Increased concentration of conjugated bilirubin in serum. Serum alkaline phosphatase is elevated as it is released from the cells of the damaged bile duct. Dark coloured urine due to elevated excretion of bilirubin and clay coloured feces due to absence of stercobilinogen .

51. Feces contain excess fat indicating impairment in fat digestion and absorption in the absence of bile (specifically bile salts) The patients experience nausea and gastrointestinal pain.

52. van den Bergh reaction and jaundice : This reaction is highly useful in understanding the nature of jaundice. This is due to the fact that the type of jaundice is characterized by increased serum concentration of unconjugated bilirubin ( hemolytic ), conjugated bilirubin (obstructive) or both of them (hepatic).

53. Therefore, the response of van den Bergh reaction can differentiate the jaundice as follows Indirect positive - Hemolytic jaundice Direct positive - Obstructive jaundice Biphasic - Hepatic jaundice.

54. A 24 year old male suffering from Malaria was put on Primaquine . He developed malaise, fatigue and yellow discolouration of sclera and skin. On examination – There was pallor- ++, icterus ++, Pulse – 100/min. Liver and spleen were palpable. The investigation report was as follows Hb-5gm% Serum bilirubin- 6 mg% Van den Bergh- Indirect positive. Urine-  Hemoglobin + and   Urobilinogen + The color of the urine was brownish black What is the probable diagnosis? What is the relationship of primaquine intake and the present manifestations?

55. Case Details- This is a case of primaquine induced Hemolytic anemia , progressing to jaundice. Glucose-6-P dehydrogenase deficiency seems to be the underlying defect. High fever is due to malaria, while pallor and icterus are due to hemolytic anemia and underlying jaundice as apparent from low Hb and high bilirubin levels. Primaquine being an oxidant drug precipitates the underlying defect to induce hemolysis . However massive lysis of red blood cells, as in Glucose-6 –phosphate dehydrogenase deficiency, may produce bilirubin faster than it can be conjugated.

56. A 50 –year-old woman had 8 day history of loss of appetite, nausea and flu-like symptoms. She had noticed that her urine had been dark in color over the past two days. On examination she had tenderness in the right upper quadrant.  Laboratory investigations showed; Serum Total bilirubin 4.5 mg% Direct bilirubin 2.5 mg% Indirect bilirubin 2.0 mg% Serum AST- 40 IU/L Serum ALT-115 IU/L Serum ALP- 20 Units (KA) What is the probable diagnosis? What will be the observation regarding bile pigments in urine?

57. Case details   Flu like symptoms are indicative of viral hepatitis. Damage to liver cells can cause unconjugated bilirubin to increase in the blood as a result of decreased conjugation. The bilirubin that is conjugated is not efficiently secreted in to the bile, but instead diffuses in to the blood. Urobilinogen is increased in urine because hepatic damage decreases the enterohepatic circulation of this compound allowing more to enter blood, from which it is filtered in to the urine. The urine thus becomes dark in color , whereas stools are pale colored . Plasma levels of AST and ALT are elevated. This is a case of hepatic jaundice .

58. Based on the following clinical laboratory data, give the most probable diagnosis Serum bilirubin 4 mg% (N- 0.2-0.8 mg%) Direct bilirubin 0.2 mg% (N-0.1-0.4 mg%) Serum Alkaline phosphatase 6 units( KA) (N-3-13 KAU/100 ml) SGOT- 30 IU/L (N-5-35 IU/L) SGPT- 26 IU/L (N- 7-21 IU/L) Urine Bilirubin- Negative Urine urobilinogen -Positive Urine Bile Salts- Negative

59. Case details  Normal enzyme profile, Hyperbilirubinemia , absence of urinary bilirubin and positive urobilinogen are indicative of Hemolytic jaundice .

60. A 40 –year- old, fat female, presents with intolerance to fatty foods, pain in the right side of abdomen, yellowing of eyes and passage of clay colored stools. Laboratory Investigations revealed Serum Total Bilirubin – 20 mg% Direct Bilirubin- 16 mg% ALP- 800 U(KA) SGPT- 90 IU/L Urine Color - deep yellow Bilirubin- ++ Urobilinogen - absent Stools Clay colored Stercobilnogen - absent What is the likely diagnosis?

61. Case details This is a case of Obstructive Jaundice Due To Gall Stones . This patient fits the “classic” criteria of gallbladder disease: female, middle-aged, overweight. Gallstones may cause pain. Pain develops when the stones pass from the gallbladder into common bile duct and block the duct..The pain is felt in the upper abdomen, usually on the right side.  In this instance jaundice is not due caused due to overproduction of bilirubin, but instead results from obstruction of  the bile duct  from the gall stones. The liver regurgitates conjugated bilirubin in to the blood ( Hyperbilirubinemia ) High direct bilirubin (Conjugated hyperbilirubinemia ), high alkaline phosphatase (marker of cholestasis ),slightly increased SGPT level are suggestive of post hepatic or obstructive jaundice. Furthermore the diagnosis is supported by the presence of bilirubin (since it is conjugated) and absence of urobilinogen (Since there is obstruction to the out flow of bile) in urine. Due to the same reason of obstruction stool is clay colored as stercobilnogen is absent. Prolonged obstruction of the bile duct can lead to liver damage and a subsequent rise in unconjugated hyperbilirubinemia and a rise in SGPT levels.