Bilirubin Metabolism and the Biochemical Basis of Jaundice.pptx

This presentation, “Bilirubin and Jaundice,” provides a detailed overview of the biochemistry, metabolism, and clinical aspects of bilirubin and its role in different types of jaundice. It explains the formation of bilirubin from the breakdown of red blood cells and heme metabolism, its transport, conjugation, and excretion by the liver, and the conversion of bilirubin to urobilinogen and stercobilin in the intestine. The slides discuss normal bilirubin values, the classification of jaundice into prehepatic, hepatic, and posthepatic types, and their biochemical patterns. Important hereditary conditions like Gilbert’s syndrome, Crigler–Najjar syndrome, Dubin–Johnson syndrome, Rotor syndrome, and neonatal jaundice are described in detail, along with their causes, enzyme defects, and clinical features. The presentation also highlights the laboratory methods used for bilirubin estimation (Van den Bergh, Malloy & Evelyn, Jendrassik & Grof), bilirubinometry for neonates, and interpretation of urinary urobilinogen findings. It serves as a complete educational resource for medical, biochemistry, and pathology students studying liver function, bilirubin metabolism, and the diagnosis of jaundice.

1. Bilirubin And Jaundice Presented by: Kainaat Mahzaib John

2. Bilirubin Formation Liver function: Processing and excretion of endogenous and exogenous substances into the bile or urine such as the major heme waste product, bilirubin. The liver is the only organ that has the capacity to rid the body of heme waste products.

3. + O2 in presence of heme oxygenase In presence of biliverdin reductase Bilirubin Formation Breakdown into amino acids to make amino acid stores of the body Binds to transferrin to move into liver or bone marrow to be used for Hb or other iron containing substances like cytochrome synthesis

4. RBC Breakdown & Hemoglobin Metabolism RBC lifespan: ~126 days → then phagocytized by reticuloendothelial system (RES). Hemoglobin breakdown : Heme → bilirubin (in 2–3 hours). Iron → bound to transferrin, returned to stores (liver/bone marrow). Globin → broken to amino acids, reused. 85% bilirubin → from hemoglobin breakdown (RBC destruction). 15% bilirubin → from non- hemoglobin hemoproteins (myoglobin, cytochromes, catalase, peroxidase, etc.). Transport & Conjugation of Bilirubin Intestinal Conversion & Excretion Unconjugated bilirubin (indirect): Insoluble in water. Bound to albumin → transported to liver. In hepatocyte: Albumin releases bilirubin → picked by ligandin (carrier protein). Sent to endoplasmic reticulum. Conjugated by enzyme UDPGT (uridine diphosphate glucuronyl transferase) → forms bilirubin diglucuronide (conjugated bilirubin). Conjugated bilirubin: Water soluble. Secreted into bile canaliculi → bile ducts → intestines. Intestinal bacteria ( glucuronidase ) convert conjugated bilirubin: Mesobilirubin → mesobilirubinogen → urobilinogen ( colorless ). Fate of urobilinogen: 80% → oxidized to urobilin ( stercobilin ) → gives stool brown color . 20% → reabsorbed into circulation: Majority recycled to liver (enterohepatic circulation). Small amount → filtered by kidney → excreted in urine.

5. Summary of bilirubin metabolism

6. Bile Bile composition: Contains bile acids/salts, bile pigments, cholesterol, and other blood-derived substances.
Bile volume: ~3 L produced daily; ~1 L excreted.
Bilirubin source: Major pigment in bile, derived from breakdown of RBCs

7. Jaundice Jaundice : Yellow discoloration of skin, sclera, mucous membranes Caused by retention of bilirubin (or other pigments) Derived from French jaune = yellow Described since Hippocratic times

8. Bilirubin and Normal Values Normal total bilirubin : 1.0–1.5 mg/ dL Clinical jaundice visible at ~3 mg/ dL Fractions of bilirubin: Unconjugated (indirect) – water insoluble, bound to albumin Conjugated (direct) – water soluble Delta bilirubin – conjugated bilirubin bound to albumin (seen in obstruction ). Conjugated bilirubin has a reactive glucuronic acid group . When it circulates in high amounts for a long time, it forms covalent bonds with albumin in plasma . This albumin-bound bilirubin is delta bilirubin.

9. Classification of Jaundice Based on site of disorder: Prehepatic – abnormality before liver. Hepatic – intrinsic liver defect/disease. Posthepatic – abnormality after liver (obstruction). Helps plan appropriate treatment/management.

10. Prehepatic Jaundice An increased rate of bilirubin production exceeds normal excretory capacity of liver Cause: Excess RBC breakdown → bilirubin overload Common in: Hemolytic anemias (acute/chronic) Features: ↑ RBC destruction → ↑ bilirubin presented to liver. Mostly unconjugated hyperbilirubinemia Liver works at max capacity; Bilirubin rarely > 5 mg/ dL No bilirubinuria (not water-soluble) ↑ Urobilinogen in urine/stool

11. Hepatic Jaundice Normal load of bilirubin cannot be conjugated/excreted by damaged liver cells Cause: Intrinsic liver disease 1. Inherited metabolic/transport defects: Gilbert syndrome – mild, benign, ↓ UGT1A1 activity, unconjugated hyperbilirubinemia Crigler-Najjar syndrome – rare, severe, absent or deficient UGT enzyme → unconjugated hyperbilirubinemia Dubin -Johnson syndrome – impaired excretion, conjugated hyperbilirubinemia, dark granules in liver biopsy Rotor syndrome – benign, similar to Dubin -Johnson but no pigmentation 2 . Acquired causes: Hepatitis ( viral or drug induced ), cirrhosis, Drugs ( rifampicin that interfere with bilirubin uptake prematurity, Methyltestosterone ), lymphoma, amyloid, tumors , sepsis.

12. Gilbert Syndrome Benign hereditary disorder, affects ~5% of population. Most common cause of jaundice. Features: Intermittent unconjugated hyperbilirubinemia. No hemolysis or liver disease. Appears in adolescence/early adulthood. Bilirubin usually 20–50 μ mol /L, rarely > 85 μ mol /L. Due to UGT1A1 gene promoter mutation (extra TA insertion). Conjugation system at only 30% of normal. No morbidity, mortality, or clinical consequences.

13. Crigler-Najjar Syndrome First described in 1952. Inherited defect in bilirubin conjugation enzyme (UGT). Type I: complete absence of conjugation → severe, fatal. Type II: severe deficiency → less serious. Both cause unconjugated hyperbilirubinemia. Rare but serious compared to Gilbert.

14. Dubin -Johnson Syndrome Rare inherited disorder. Deficiency of canalicular transporter cMOAT /MDR2. Features: Conjugation intact, but excretion into bile defective. ↑ conjugated bilirubin (+ delta bilirubin). Bilirubinuria present. Dark pigmented granules in liver biopsy. Bilirubin ~2–5 mg/ dL . Prognosis: mild, normal life expectancy, no treatment required.

15. Rotor Syndrome Similar to Dubin -Johnson. Exact defect not known – thought due to ↓ ligandin binding. No pigmented granules in liver biopsy. Benign, excellent prognosis. Important to distinguish from more serious liver diseases.

16. Neonatal Jaundice Cause: deficiency of glucuronyl transferase (late-activating enzyme). Common in premature infants. Leads to high unconjugated bilirubin → kernicterus. Kernicterus = bilirubin deposition in brain nuclei → cell damage/death. Management: Careful monitoring of bilirubin (~20 mg/ dL dangerous). Phototherapy (UV light destroys bilirubin). Exchange transfusion in severe cases. Normal babies show jaundice btw days 2 and 8 of life. Jaundice on the first day of life is invariably pathological.

17. Posthepatic Jaundice Biliary flow is obstructed so that conjugated bilirubin cannot be excreted into intestine and is regurgitated into systemic circulation Cause: Biliary obstruction (gallstones, tumors , biliary stricture, carcinoma of head of pancreas or biliary tree, primary biliary cirrhosis , Cholangiocarcinoma, Parasitic infections ) Pathophysiology: Liver conjugates bilirubin normally But excretion into bile blocked Clinical: Clay- colored stools (lack of stercobilin ) Dark urine ( bilirubinuria ) Pruritus Lab: Predominantly conjugated hyperbilirubinemia

18. Bilirubin Fractions Unconjugated (indirect): Nonpolar, water-insoluble. Albumin-bound, not in urine. Conjugated (direct): Polar, water-soluble, free in plasma. Excreted in urine. Delta bilirubin: Conjugated bilirubin bound to albumin. Appears in obstruction, not filtered in urine. Total bilirubin = unconjugated + conjugated + delta.

19. Specimen Protect from light (bilirubin degrades 30–50%/hr). Stable 2 days RT, 1 week at 4°C, indefinitely at –20°C . Fasting sample preferred → lipemia falsely increases bilirubin levels. Avoid hemolyzed samples → free hemoglobin decreases bilirubin reactivity with diazo reagent.

20. Laboratory Testing 1883 (Ehrlich ): First described bilirubin reaction with diazotized sulfanilic acid using urine samples . Known as the classic diazo reaction . 1913 (van den Bergh ): Applied diazo reaction to serum . Required an accelerator ( solubilizer ). Methodology had errors . 1937 (Malloy & Evelyn ): Developed the first clinically useful method for serum bilirubin quantitation . Used diazo reaction with 50% methanol as accelerator . 1938 ( Jendrassik & Grof ): Introduced diazo reaction with caffeine-benzoate-acetate as accelerator . Modern Methods : All current bilirubin methods are modifications of Malloy & Evelyn’s technique . Measure total bilirubin and conjugated bilirubin . Unconjugated bilirubin = Total – Conjugated.

21. Bilirubinometry (Neonatal Measurement) Use : Quantifies bilirubin in neonates. Limitation: Not useful in adults due to carotenoid interference. Principle: Measures reflected light from skin at two wavelengths. Provides bilirubin index based on spectral reflectance. Newer bilirubinometers : Use microspectrophotometers . Measure optical densities of bilirubin, hemoglobin , and melanin in infant’s skin . Apply mathematical correction to isolate bilirubin.

22. Differentials

23. Reference ranges

24. Urobilinogen in Urine & Feces Colorless bilirubin end-product, converted to urobilin (brown) by gut bacteria. Excretion: Feces (main route). Reabsorbed → liver. Some excreted in urine. Clinical findings: ↑ urine urobilinogen: hemolysis , hepatitis. Absent: complete biliary obstruction. Detection: Ehrlich’s reagent test (red color ).

25. Summary Prehepatic : unconjugated ↑, no bilirubin in urine. Hepatic: mixed pattern (depends on disorder). Posthepatic : conjugated ↑, bilirubinuria , clay- colored stools.

26. Questions?