HEME DEGRADATION AND JAUNDICE

1. Molecular Biochemistry II HEME DEGRADATION AND JAUNDICE xiaoli

2. RBCs last 120 days, degraded by reticuloendothelial (RE) system • Under physiologic conditions in the human adult, 1~2×108 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. iron + Bile pigments Heme most heme from RBCs (85%) - rest from turnover of cytochromes, p450s, immature erythrocytes globin Amino acids

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

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

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. M：-CH2 V：-CH=CH2 HOOC COOH CH2 CH2 M V M V M M CH2 CH2 O CH CH O CH2 N N N N H H H H

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

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

13. H affinity binding sites 2:1 Bilirubin Plasma protein Albumin Molar Ratio L affinity binding sites >2:1 Bilirubin Other organic anions PH can be replaced by UB Transport of Bilirubin in Plasma 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. membrance [bilirubin-lipids] Other antibiotics [albumin]↑ [bilirubin] [bilirubin] blood cytosol [bilirubin-albumin] • Certain drugs assulfonamides and salicylatescompete with bilirubin for albumin binding and displace bilirubin to enter into the brain in neonatesand 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. Bilirubin carrier protein Hepatic uptake of Bilirubin Bilirubin ~ Albumin Complex Albumin Bilirubin (lipid soluble) (be) taken up by membrane of the liver ligation (Y protein, Z protein) (be) bound to transfer ER

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

18. HOOC COOH CH2 CH2 M V M V M M CH2 CH2 O CH CH O CH2 N N N N H H H H OH COOH O O O OH OH HOOC CO CO O OH HO CH2 CH2 OH M M V M V M CH2 CH2 O CH CH O CH2 N N N N H H H H 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

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. urobilin Bilirubin diglucuronide urobilinogen bilin stercobilin 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)

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

24. Stercobilin excreted in feces Urobilin excreted in urine Hemoglobin Globin Urobilinogen formed by bacteria Heme O2 Heme oxygenase CO Biliverdin IX NADPH Bilirubin diglucuronide (water-soluble) Biliverdin reductase NADP+ 2 UDP-glucuronic acid Bilirubin (water-insoluble) Bilirubin (water-insoluble) via blood to the liver BLOOD CELLS KIDNEY reabsorbed into blood INTESTINE via bile duct to intestines LIVER unconjugated Catabolism of hemoglobin

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. Hyperbilirubinemia: the concentration of blood bilirubin are more than 1mg/dl. 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.

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: • Pre-hepatic (hemolytic jaundice) • Hepatic jaundice • 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

40. Diagnoses of Jaundice