Bilirubin estimation


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Bilirubin estimation

  1. 1. Biochemistry Diploma Students Dr.Ehab Mansoura University Faculty of Science-Damietta Chemistry Department Branch-Biochemistry 2009 For
  2. 2. A. Introduction <ul><li>1. The average life of the red blood cells is 120 days . </li></ul><ul><li>2. At the end of that time, they are removed from circulation by the cells of reticuloendothelial (RE) system present in liver, spleen and bone marrow. </li></ul><ul><li>They are hemolysed and hemoglobin comes out, giving globin and heme molecules. </li></ul><ul><li>a) Globin molecule hydrolyzes into free amino acids . </li></ul><ul><li>b) Heme gives iron and bilirubin </li></ul>
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  5. 5. B. Formation of bilirubin: <ul><li>1. The heme ring is catabolized by </li></ul><ul><li>the microsomal heme oxygenase enzymes of the </li></ul><ul><li>RE cells . </li></ul><ul><li>2. In this reaction (which needs, heme oxygenase enzyme , O 2 and NADPH), iron (Fe++) is removed for re-use. The remaining of heme ring is cleaved between pyrrole rings number I and II to form biliverdin (green pigment) and carbon monoxide (CO) </li></ul><ul><li>3. Biliverdin is then reduced into bilirubin (golden yellow) in a reaction requires biliverdin reductase enzyme . </li></ul>
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  10. 10. C. Transport of bilirubin in the plasma <ul><li>Bilirubin is nonpolar , and is insoluble in plasma . Therefore it binds by noncovalent bonds to plasma albumin . This form is called : unconjugated or indirect bilirubin . </li></ul>
  11. 11. <ul><li>1. Bilirubin dissociates from the carrier albumin molecule and enters hepatocytes. </li></ul><ul><li>2. Bilirubin is conjugated with one or two molecules of glucuronic acid (the acid form of glucose) to form bilirubin mono glucuronide and bilirubin di glucuronide . This reaction needs </li></ul><ul><li>UDP-glucuronyl transferase enzyme : </li></ul>D. Uptake of bilirubin by the liver
  12. 12. E. Secretion of bilirubin into bile <ul><li>Bilirubin diglucuronide is actively transported against concentration gradient into the bile </li></ul>
  13. 13. <ul><li>1. Intestinal bacteria acts on bilirubin diglucuronide leading to: </li></ul><ul><li>a) Removal of glucuronides (by  -glucuronidases enzymes). </li></ul><ul><li>b) Reduction of bilirubin to colorless compounds called: </li></ul><ul><li>Urobilinogens </li></ul><ul><li>2. A small fraction of urobilinogens are reabsorbed from intestine to the liver again and re-excreted in the bile, forming the enterohepatic urobilinogens cycle . </li></ul>F. Formation of urobilin in the intestine
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  15. 15. <ul><li>1. Most of the colorless urobilinogens are oxidized to the colored urobilin .which excreted in the stool giving its brown color . </li></ul><ul><li>2. Part of urobilinogens are reabsorbed to the liver, then to the blood to be excreted by the kidney in urine and converted into urobilin. </li></ul><ul><li>3.Urobilin -together with urochrome-give the characteristic yellow color of urine. </li></ul>G. Excretion of urobilinogens in stool and urine
  16. 16. Van den Bergh reaction <ul><li>1-This is a reaction between bilirubin and Ehrlich diazo reagent giving a reddish purple compound. </li></ul><ul><li>2- Conjugated bilirubin reacts directly with the reagent. Thus it is called: direct bilirubin </li></ul><ul><li>3 - Unconjugated bilirubin does not react with the reagent directly except after addition of methyl alcohol . Thus it may be called: indirect bilirubin </li></ul>
  17. 17. Differences between unconjugated bilirubin and conjugated bilirubin
  18. 18. Heme catabolism is classified into several stages <ul><li>Stage 1: </li></ul><ul><li>Formation of bilirubin in reticuloendothelial system. In reticuloendothelial system (RES), hemoglobin is degraded into heme and globin . Globin is either degraded to amino acids or reused for synthesis of hemoglohin . Heme is converted into biliverdin (green) then to bilirubin (yellow). </li></ul>
  19. 19. <ul><li>One gram of hemoglobin yields 35 mg of bilirubin . The daily bilirubin formation in adult human is about 250 mg . Bilirubin is transported to the liver in conjunction with plasma albumin (termed indirect or unconjugated bilirubin ). Each molecule of albumin has one high affinity site and one low affinity site for bilirubin. </li></ul>
  20. 20. <ul><li>This stage occurs in the liver and can be divided into three processes : </li></ul><ul><li>1-Uptake of bilirubin by liver parenchymal cells. </li></ul><ul><li>This is followed by removal of albumin and release of free bilirubin. </li></ul><ul><li>2-Synthesis of bilirubin-diglucuronide. </li></ul><ul><li>This is catalyzed by bilirubin-glucuronyl-transterase to form bilirubin diglucuronide . UDP-glucuronate (UDP-GlcUA) acts as donor or the glucuronate group to form bilirubin-monoglucuronide then bilirubin-diglucuronide (termed chole bilirubin. direct or conjugated bilirubin ) and it is water soluble . </li></ul><ul><li>3-Bilirubin-diglucuronide is actively secreted into bile. </li></ul>Stage II
  21. 21. Stage III : This is produced by the action of intestinal bacteria. <ul><li>Bilirubin is deconjugated from glucuronate , and then reduced to urobilinogens (colorless) . Most of urobilinogens are excreted in feces and oxidized to the colored pigments urobilins by oxygen. This explains darkening of feces upon standing in air. Normally small amounts of urobilinogens are re absorbed by portal blood and excreted by liver cells into bile (enterohepatic circulation) and traces escape and excreted in urine. </li></ul>
  22. 22. Plasma Bilirubin or Bile Pigments <ul><li>Plasma Bilirubin or Bile Pigments </li></ul><ul><li>The nonnal level of bilirubin in plasma (or serum) is less than 1.2 mg/dL and it is present in two forms , indirect and direct . </li></ul><ul><li>1-Indirect reacting (bilirubin-albumin complex or unconjugated bilirubin). </li></ul><ul><li>It requires the addition of methanol to react with the diazo reagent of Van den Bergh (used for its estimation). It is the main fonn present nonnally in plasma and it ranges from 0.3 to 0.8 mg/dL . It increases in all cases of hemolytic anemia and when liver fails to take up or conjugate bilirubin . </li></ul><ul><li>  </li></ul>
  23. 23. <ul><li>2-Direct reacting (bilirubin-diglucuronide or conjugated bilirubin). </li></ul><ul><li>It is water-soluble and reacts directly with the diazo reagent of van den Bergh. It represents the bilirubin-diglucuronide which escapes from liver to systemic blood. It increases when liver fails to excrete bilirubin after conjugation or when there is obstruction in the biliary system that prevents its passage to the intestine. Normally its level in plasma is less than 0.3 mg/ldL. </li></ul>
  24. 24. <ul><li>Hyper bilirubin emia exists when bilirubin levels exceeds 1.2 mg/dL . Jaundice (yellow discoloration of skin and sclera of eyes) becomes manifest usually at levels above 2 mg/dL . </li></ul><ul><li>Depending on the type of bilirubin present in plasma, hyper bilirubin emias include two main types , unconjugated and conjugated hyperbilirubinemias. </li></ul> HYPERBILIRUBINEMIA
  25. 25. A-Unconjugated Hyperbilirubinemia <ul><li>It is due to overproduction of bilirubin by reticuloendothelial system over the capacity of the liver to remove and clear from blood. It is characterized by high level of indirect or unconjugated bilirubin. This type of bilirubin can cross the blood-brain barrier into the central nervous system and cause kernicterus (encephalopathy). Unconjugated bilirubin is water insoluble and not excreted in urine ( acholuric jaundice ). </li></ul>
  26. 26. Unconjugated hyperbilirubinemia occurs in the following conditions <ul><li>I-Neonatal or Physiologic Jaundice : This is the most common cause of jaundice in neonatal age. It results from accelerated hemolysis and immature hepatic system for uptake, conjugation and secretion of bilirubin. It is characterized by high plasma level of unconjugated bilirubin, if it exceeds 20-25 mg/dL </li></ul>
  27. 27. <ul><li>These cases are treated by: </li></ul><ul><li>a-Phenobarbital which acts as inducer for UDP-glucuronyl transferase enzyme. </li></ul><ul><li>b-Phototherapy (exposure to visible light) helps hepatic excretion of bilirubin by converting bilirubin to other derivatives that are more soluble and easily excreted in bile. </li></ul>
  28. 28. II-Hemolytic jaundice (Anemia) <ul><li>Hemolytic anemia is caused by one of the following causes: </li></ul><ul><li>1-The presence of abnormal hemoglobins e.g. sickle cell anemia and thalathemia. </li></ul><ul><li>2- Erythro blastosis fetalis : when Rh negative mother has Rh positive fetus (due to Rh positive father ). </li></ul><ul><li>3- Congenital spherocytosis where the red cells are abnormally fragile. </li></ul><ul><li>4- Favism , due to deficiency of G 6 PD. </li></ul><ul><li>5-Deficiency of Pyruvate kinase or other glycolytic enzymes </li></ul><ul><li>6-Incompatibl blood transfusion. </li></ul><ul><li>7-Some diseases e.g. malaria and black water fever </li></ul>
  29. 29. <ul><li>III-Congenital Syndromes related to uptake and conjugation of bilirubin as follow : </li></ul><ul><li>Crigler-Najjar Syndrome </li></ul><ul><li>Type 1: Due to severe decrease in the activity of UDP- </li></ul><ul><li>glucuronyl transferase. </li></ul><ul><li>Type II: Due to decreased activity of UDP-glucuronyl transferase that adds the second glucuronide group . </li></ul><ul><li>Gilbert Disease </li></ul><ul><li>It is mainly due to hepatic defect in the uptake of bilirubin by liver cells. </li></ul><ul><li>IV-Toxic Hyperbilirubinemia </li></ul><ul><li>This is due to toxin-induced liver dysfunctions e.g. chloroform, carbon tetrachloride and mushroom poisoning. </li></ul>
  30. 30. B-Conjugated Hyperbilirubinemia <ul><li>Conjugated hyperbilirubinemia is due to reflux of direct or conjugated bilirubin into blood due to biliary obstruction , conjugated bilirubin is water soluble, so it is excreted in urine and darken its colour (Choluric jaundice) . </li></ul><ul><li>It occurs in the following conditions: </li></ul><ul><li>1-Obstructive Jaundice (Cholestatic Jaundice) </li></ul><ul><li>Conjugated hyperbilirubinemia results from blockage of hepatic or common bile duct (stones and tumors), bilirubin diglucuronide is not excreted and returns to hepatic veins and lymphatics and appears in blood and urine. </li></ul>
  31. 31. <ul><li>It may be produced by micro-obstruction of intrahepatic biliary ductules by swollen damaged hepatocytes e.g. viral hepatitis and liver cirrhosis . Both cases are associated with marked increase of conjugated bilirubin and slight to moderate increase of unconjugated bilirubin (mixed hyperbilirubinemia). </li></ul><ul><li>2-Chronic Idiopathic Jaundice (Dubin-Johnson Syndrome) </li></ul><ul><li>This is a genetic disease characterized by conjugated hyperbilirubinemia and defect in secretion of other conjugated compounds e.g. estrogens and sulfobromophethalein. </li></ul><ul><li>3-Rotor's Syndrome </li></ul><ul><li>It is characterized by conjugated hyperbilirubinemia with normal liver </li></ul>
  32. 32. Hyperbilirubinemia <ul><li>Hyperbilirubinemia or jaundice is classified clinically into three main types as follows : </li></ul><ul><li>1-Prehepatic (or hemolytic) jaundice </li></ul><ul><li>It is characterized by the following: </li></ul><ul><li>1. Anemia </li></ul><ul><li>2. High levels of indirect bilirubin in plasma, urobilinogen in urine and stool </li></ul><ul><li>3. Dark stool (contains excessive amounts of bilirubin and urobilinogen) </li></ul><ul><li>4. Dark urine on standing (contains excessive amounts of urobilinogen, which gives urobilin by oxidation, so the color of urine becomes dark on standing). </li></ul>
  33. 33. <ul><li>II-Hepatic jaundice </li></ul><ul><li>It is characterized by the following: </li></ul><ul><li>1. Elevated transaminases (ALT & AST) in plasma </li></ul><ul><li>2. Elevated levels of both direct and indirect bilirubin in plasma. </li></ul><ul><li>3. Dark urine and faint stool (due to decreased excretion of bile) </li></ul><ul><li>III-Obstructive jaundice </li></ul><ul><li>It is characterized by the following: </li></ul><ul><li>1. Marked elevation of direct bilirubin and alkaline phosphatase in plasma. </li></ul><ul><li>2. Dark urine (excess bilirubin) and very faint stool (white clay appearance). </li></ul>