1. HEME DEGRADATION AND JAUNDICE xiaoli Molecular Biochemistry II

2. RBCs last 120 days, degraded by reticuloendothelial (RE) system most heme from RBCs (85%) - rest from turnover of cytochromes, p450s, immature erythrocytes  Under physiologic conditions in the human adult, 1~2×10 8 erythrocytes are destroyed per hour. Thus, in 1 day, a 70kg human turns over approximately 6g of hemoglobin.  When hemoglobin is destroyed in the body, globin is degraded to its constituent amino acids, which are reused, and the iron of heme enters the iron pool, also for reuse. globin Heme Amino acids iron + Bile pigments

3. iron Bile pigments

4. Bile pigments Any of several coloured compounds derived from porphyrin that are found in bile; principally bilirubin and biliverdin.

5. Bilirubin Metabolism Bilirubin formation Transport of bilirubin in plasma Hepatic bilirubin transport  Hepatic uptake  Conjugation  Biliary excretion Excrect through intestine system

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

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

8. Heme oxygenase There are three known isoforms of heme oxygenase.  Heme oxygenase 1 (HO-1) is an inducible isoform in response to stress such as oxidative stress, hypoxia, heavy metals, cytokines, etc. Its activity is induced by its substrate heme and by various nonheme substances.  Heme oxygenase 2 (HO-2) is a constitutive isoform which is expressed under homeostatic conditions. Both HO-1 and HO-2 are ubiquitously expressed and catalytically active.  A third heme oxygenase (HO-3) is not catalytically active, but is thought to work in oxygen sensing.

9. 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.

10. CH 2 HOOC N H CH O N H CH 2 N H CH N H O M M M M V V CH 2 COOH M ： -CH 2 V ： -CH=CH 2

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

12. Albumin + free Bilirubn Bilirubin ~ Albumin Complex Bilirubin on release from macrophages circulates as unconjugated bilirubin in plasma tightly bound to albumin. Transport of Bilirubin in Plasma Why bound to albumin? Significance: ★ Increase the solubility of whole molecule ★ Prevent unconjugated bilirubin freely come into other tissue, cause damage. unconjugated bilirubin

13. Transport of Bilirubin in Plasma can be replaced by Other organic anions PH  UB  Molar Ratio Bilirubin H affinity binding sites 2:1 Bilirubin Plasma protein Albumin L affinity binding sites >2:1 Albumin has two binding sites for bilirubin---a high affinity site and a low affinity site. Albumin per 1L of plasma can bind about 342 ～ 427.5μmol bilirubin, but 1.7 ～ 17.1μ bilirubin per 1L.

14. blood [bilirubin-albumin] Other antibiotics [albumin]↑ [bilirubin] membrancecytosol [bilirubin-lipids] [bilirubin] Certain drugs as sulfonamides and salicylates compete with bilirubin for albumin binding and displace bilirubin to enter into the brain in neonates and increase the risk of kernicterus (a type of brain damage that can result from high levels of bilirubin in a baby’s blood. It can cause athetoid cerebral palsy and hearing loss).

15. Hepatic phase On coming in contact with the hepatocyte surface, unconjugated bilirubin is preferentially metabolized which involved 3 steps: Hepatic uptake Conjuation Secretion in bile

16. Hepatic uptake of Bilirubin Bilirubin ~ Albumin Complex Albumin Bilirubin (lipid soluble) carrier proteinBilirubin transfer ER ligation (Y protein, Z protein) (be) taken up by membrane of the liver (be) bound to

17. Conjugation of bilirubin Conjugation occur in endoplasmic reticulum UDP- glucuronosyl transferase, Product: mono or diglucuronides. The process of conjugation can be induced by drugs phenobarbital. Bilirubin +UDP-gluconic acid Bilirubin monoglucuronides + UDP UDPGT Bilirubin diglucuronide + UDP UDP-gluconic acid

18. CH 2 HOOC N H CH O N H CH 2 N H CH N H O M M M M V V CH 2 COOH H H H CH 2 COCO N CH O N CH 2 N H CH N O M M M M V V CH 2 CO O O OH HO COOH OH O HOOC OH O bilirubin diglucuronides conjugated bilirubin ★ Increase the solubility of whole molecule ★ delete the toxicity of bilirubin.

19. Unconjugated bilirubin: Bilirubin that are not conjugated with gluconic acid, also called hemobilirubin, indirected bilirubin. conjugated bilirubin: Bilirubin that are conjugated with gluconic acid, also called hepatic bilirubin, directed bilirubin.

20. Major differences between unconjugated and conjugated bilirubin FEATURE Unconjugated bilirubin CONJUGATED BILIRUBIN Normal serum levelMoreLess (less than 0.25mg/dl) Water solubilityAbsentPresent Affinity to lipids (alcohol solubilty) PresentAbsent Serum albumin bindingHighLow Van den Bergh reactionIndirect (Total minus direct) Direct Reanal excretionAbsentPresent Affinity to brain tissuePresent (kernicterus)Absent

21. 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.

22. 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 diglucuronideurobilinogen urobilin stercobilin bilin

23. Portal vein 10 ～ 20% reabsorbed bilinogen Sterobilin excrection 80% ～ 90% bilinogen prototype Conjugated bilirubin bilinogen enterohepatic circulation 0.5 ～ 4mg/day blood urobilin

24. BLOOD CELLS LIVER Bilirubin diglucuronide (water-soluble) 2 UDP-glucuronic acid via bile duct to intestines Stercobilin excreted in feces Urobilinogen formed by bacteria KIDNEY Urobilin excreted in urine CO Biliverdin IX  Heme oxygenase O2O2 Bilirubin (water-insoluble) NADP + NADPH Biliverdin reductase Heme Globin Hemoglobin reabsorbed into blood Bilirubin (water-insoluble) via blood to the liver INTESTINE Catabolism of hemoglobin unconjugated

25. Summary of bilirubin metabolism Senescent 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.

27. ＊ normal range of bilirubin: 1 ～ 16  mol/l (0.1 ～ 1mg/dl) 4/5 are unconjugated bilirubin, others are conjugated bilirubin. <1mg/dl normal 1-2mg/dl occult >2mg/ dl jaundice Hyperbilirubinemia

28. Occult: the concentration of blood bilirubin are increased, but have no clinic sympotom, normally 1-2mg/dl. Jaundice : ( also called icterus) refers to the yellow color of the skin and scleare caused by deposition of bilirubin, secondry to increased bilirubin levels in the blood. Although not a disease itself, jaundice is usually a symptom of an underlying disorder. Hyperbilirubinemia: the concentration of blood bilirubin are more than 1mg/dl.

31. Based on pathophysiology, jaundice may result from one or more of the following mechanism: 1. Increased bilirubin production ( excessive red cell destruction) 2. Decreased hepatic uptake ( ligandin, drug, prolonged starvation, and sepsis) Decreased hepatic conjugation (enzyme,drugs, cirrhossis) 3. Decreeased excretion of bilirubin into bile ( gallstone, tumour)

32. Simple Classification of jaundice Accordingly, a simple classification of jaundice is to divided into 3 predominant type: ① Pre-hepatic (hemolytic jaundice) ② Hepatic jaundice ③ Post – hepatic cholestatic (obstructive jaundice)

33. Hemolytic jaundice massive lysis of red blood cells (for example, in patients with sickel cell anemia or malaria) may produce bilirubin faster than the liver can conjuagte it. More bilirubin is excreted into the bile, the amount of the urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased. Unconjugated bilirubin is elevated in blood.

34. Causes of hemolytic jaundice  Malaria  Side effects of certain drugs :antibiotic and anti-tuberculosis medicines, levodopa,  Certain drugs in combination with a hereditary enzyme deficiency known as glucose-6-phosphate dehydrogenase (G6PD)  Poisons Snake and spider venom, certain bacterial toxins, copper, and some organic industrial chemicals directly attack the membranes of red blood cells  Artificial heart valves  Hereditary RBC disorders sickle cell disease  Enlargement of the spleen  Diseases of the small blood vessels  Immune reactions to RBCs cancer  Transfusions  Kidney failure and other serious diseases  Erythroblastosis fetalis

35. Hepatocellular jaundice Damage to liver cells( for example in patient with cirrhosis or hepatitis) causes a decrease in both bilirubin uptake and production of conjuagted bilirubin. Unconjugated bilirubin occur in the blood and increased urobilinogen in the urine. The urine is dark in color and stool are pale, clay color. Plasma level of AST and ALT are elevated and the patient experience nausea and anorexia.

36. Obstructive jaundice In this instance jaundice is results from obstruction of the bile duct. For example, the presence of a hepatic tumor or bile stone may block the bile ducts, preventing passage of bilirubin into the intestine, patients with obstructive jaundice experience GI pain, nausea and produce stools that are a pale, clay color.

37. 65 year-old lady presenting with obstructive jaundice Biliary stones

38. SampleIndicesNormalObstructive Jaundice Hemolytic Jaundice Hepatic Jaundice Serum Total Bil ＜ 1mg/dl ＞ 1mg/dl Direct Bil 0 ～ 0.8mg/dl ↑↑↑ Indirect Bil ＜ 1mg/dl ↑↑ Urine Colornormaldeepdeeperdeep Bilirubin — ＋＋ — Urobilinogen a little ↓↑uncertain Urobilin a little ↓↑uncertain Stool ColornormalArgilous (complete obstruction) deeperlighter or normal

40. Diagnoses of Jaundice