1. Porphyrins (Structure of Porphyrins) Objective: In addition to serving as building blocks for proteins, amino acids are precursor of many nitrogen-containing compounds that have important physiologic functions, these molecules include porphyrin.

2. Porphyrins (Structure of Porphyrins) Porphyrins are cyclic compounds that readily bind metal ions, usually Fe 2+, Fe 3+. The most prevalent metallopophyrin in human is heme, which consists of one Fe 2+ coordinated in the center of tetrapyrrol ring protoporphyrin lX through methenyl bridges. Heme is the prothetic group for hemoglopin, myoglopin, the cytochromes, catalase and tryptophane pyrrolase

3. Porphyrins (Structure of Porphyrins)

4. Structure of Porphyrins (1) Side chains :Ex., uroporphyrin contains acetate (-ch 2 -coo - ), and propionate (-ch 2 -ch 2 - coo - ). while coproporphyrin is substituted with methyle (-ch 3 ) and propionate group. Distribution of side chains: The side chains of porphyrins can be ordered around the tetrapyrrol nucleus in four different ways designated by Roman numerals I & IV. Only type III Porphyrins which contains asymmetric substitution on ring D are physiologically important in humans.

5. Biosynthesis of heme Liver is the major site of heme biosynthesis. Liver synthesizes a number of heme proteins, cytochrome P 450, and the erythrocyte-producing cells of the bone marrow which are active in hemoglobin synthesis.

6. Biosynthesis of heme. In the liver: The rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for heme proteins. In contrast, heme synthesis in erythroid cells is relatively constant, and is matched to globin synthesis The initial reaction and the last three steps occur in the mitochondria, whereas the intermediate steps occur in the cytosol.

7. Biosynthesis of heme (1) Formation of δ-aminolevulinic acid (ALA). All the carbon of the porphyrin molecule are provided by by two simple blocks: (glycine and succinyl CoA ). glycine and succinyl CoA condense to form ALA in a reaction catalyzed by ALA synthase. This reaction requires pyridoxal phosphate as a coenzyme. It is a rate limiting steps.

8. Biosynthesis of heme (2) Formation of porphobilinogen. In this reaction: The dehydration of two molecules of ALA to form porphobilinogen by ALA dehydratase ( this enzyme is extremely sensitive to inhibition by heavy metal ions).

9. (1) Formation of δ-aminolevulinic acid (ALA).

10. Biosynthesis of heme (3) Formation of uroporphybilinogen The condensation of four molecules of porphobilinogen results in the formation of uroporphybilinogen lll This reaction requires hydroxymethybilane synthase and uroporphybilinogen lll synthase.

11. Biosynthesis of heme (4) Formation of heme. uroporphybilinogen lll is converted to heme by a series of decarboxylation and oxidation reactions The introducing of Fe +2 into protoporphyrin spontaneously, but the rate is enhanced by ferrochelatase ( an enzyme inhibited by lead.

13. Regulation of heme synthesis (1) The rate controlling step is the formation of ALA in which, when porphyrin production exceeds the availability of globin or other apoproteins. Heme accumulates and is converted to hemin by oxidation of Fe +2 to Fe +3. Hemin decreases the activity of hepatic ALA synthase by causing decreased the synthesis of the enzyme Note: in erythroid cells, heme synthesis is under the control of erythropoietin and the availability of intracellular iron.

14. Regulation of heme synthesis (2) Effect of drugs on ALA Synthase activity: Ex, phenobarbital, griseofulvin or hydantions. These drugs are metabolized microsomal cytochrome P450 monooxygenase system (a heme- protein oxidase system found in the liver) In response to this drugs, the synthesis of cytochrome P450 increases, leading to an enhanced consumption heme.(heme a component of cytochrome P450 ) This in turn, cause a decrease concentration of heme in the liver cells. The lower intracellular heme concentration leads to an increase in the synthesis of ALA Synthase, and prompts a corresponding increase in ALA synthesis.

15. Degradation of heme. After approximately 120 days in the circulation, red blood cells are taken up and degraded by the reticulo- endothelial (RE) system, particularly in the liver and spleen.

16. Degradation of heme. (1) Formation of bilirubin: Microsomal heme oxygenase (HO) system of the RE cells responsible for the first step of heme degradation. (a) In presence of NADH & O 2, the enzyme adds hydroxyl group to the methenyl bridge between two pyrrole rings with a concomitant oxidation of Fe +2 to Fe +3 (b) by the same enzyme system a second oxidation results in cleavage of porphyrin ring, Fe +3 & Co is released in addition to production of green pigment Biliverdin.( c) Biliverdin is reduced by biliverdin reductase forming bilirubin ( red-orange pigment, bile pigment)

18. Degradation of heme. (2) Uptake of bilirubin by the liver. Bilirubin is slightly soluble in plasma, so it transported to the liver by binding non-covalently to albumin. Bilirubin dissociated from albumin and enter hepatocyte, where it binds to intracellular proteins (ligandin)

19. Degradation of heme. (3)Formation of bilirubin diglucuronide: In hepatocytes bilirubin is conjugated to 2 molecules of glucuronic acid in presence of bilirubin glucuronyltransferas using UDP- glucuroic acid (as a glucuronate donor)

20. Degradation of heme (4) Excretion of bilirubin into bile: Bilirubin diglucuronide is actively transported against concentration gradient into the bile canaliculi and then into the bile. This energy –dependant, rate limiting step is susceptible to impairment in the liver disease Unconjugated bilirubin is normally not excreted.

21. Degradation of heme (5)Formation of urobilins in the intestine. In the gut, bilirubin diglucuronide is hydrolyzed by bacteria to yield urobilinogen ( a colourless compound), (a) Most urobilinogen is oxidized to sterbilin( brown color & excreted in feces). (b) Some is reabsorbed from the gut and enter portal blood to participate in the enterohepatic urobilinogen cycle. © The remainder urobilinogen is transported by the blood to the kidney where it is converted to yellow urobilinogen (give urine its characteristic color)

23. Disorders in heme metabolism In heme synthesis: (1) Porphyrias are classified as erythropoietic or hepatic, depending on location of the enzyme deficiency occurs in the erythropoietic cells or in the liver.

25. Porphyrias (1) Porphyria cutanea tarda: A chronic disease caused by a deficiency in uroporphyrinogen decarboxylase. Uroporphyrin accumulates in the urine It is the most common Porphyria Patients are photosenstive, their skin itches and burn (pruritis) when exposed to visible light.

26. (1) Porphyria cutanea tarda:

27. Porphyrias (2) Acute hepatic Porphyrias: they are characterized by acute attacks of gastrointestinal, neurologic/ psychatric and cardiovascular symptoms. (A) Acute intermittent Porphyria: An acute disease caused by a deficiency in hydroxymethylbilane synthase. Porphobilinogen and δ-aminolevulinic acid accumulate in the urine. Urine darkens on expoure to light and air. Patients are not photosenstive

28. Porphyrias : Acute hepatic Porphyrias (2) Hereditary coproPorphyria: An acute disease caused by a deficiency in coproPorphyrinogen oxidase CoproPorphyrinogen lll and other intermediates prior to the block accumulate in the urine. Patients are photosenstive.

29. Porphyrias : Acute hepatic Porphyrias (3) Varigate Porphyria: An acute disease caused by a deficiency in protoporphyrinogen oxidase. ProtoPorphyrinogen lX and other intermediates prior to the block accumulate in the urine. Patients are photo-senstive.

30. Porphyrias : Erythropoietic Porphyrias Erythropoietic Porphyrias are characterized by skin rashes and blisters that appear in the early childhood. The disease are complicated by cholestatic liver cirrhosis and progressive hepatic failure.

31. Porphyrias : Erythropoietic Porphyrias (1) Erythropoietic Porphyrias: The disease caused by a deficiency in ferrochelates. ProtoPorphyrin accumulate in the erythrocytes, bone marrow and plasma Patients are photosenstive.

32. Porphyrias : Erythropoietic Porphyrias (2) Congenital erythropoietic Porphyrias: The disease caused by a deficiency in uroporphyrinogen lll synthase. Uroporphyrinogen l & coproPorphyrinogen l accumulate in urine Patients are photosenstive.

33. Porphyrias Increased in ALA synthase activity: One of the common feature of the Porphyrias is a decreased in heme synthesis. In the liver heme functions as a repressor of ALA synthase.Therefore, the absence of this end product results in an increase in the synthesis of ALA synthase (derepression). This leads to an increased in synthesis of intermediates (they are the major pathophysiology of the Porphyrias

34. Porphyrias Treatment: Treatment of vomiting and pain. I.V injection by hemin, which decrease the synthesis of ALA synthase Avoidance of sunlight and ingestion of β-carotene ( free-radical scavenger) are also helpful.

35. Disorders in heme metabolism In Degradation of Heme Jaundice: it is also called icterus, which refers yellow color of skin, nail beds, and sclerae caused by deposition of bilirubin. Types of Jaundice: (1) Hemolytic Jaundice (2) obstructive Jaundice (3) Hepatocellular Jaundice

36. Jaundice

37. Disorders in heme metabolism Jaundice (1) Hemolytic Jaundice: In massive lysis of RBCs (in patients with sickle cell anemia, PK, G-6-P dehydrogenase deficiency or malaria) Bilirubin produced faster than it can be conjugated. So more bilirubin is excreted in the bile, the amount of urobilinogen enter enterohepatic circulation is increased. Uncojugated bilirubin level is increased.

38. Disorders in heme metabolism Jaundice (2) obstructive Jaundice: Jaundice results from obstruction of the bile duct ( due to hepatic tumor or bile stone may block the bile duct), preventing passage of bilirubin in the intestine. Patients with obstructive jaundice suffer from gastrointestinal pain and nausea and produce stools that are a pale,clay color The liver regurgitates bilirubin into the blood. The compound is excreted in the blood.

39. Disorders in heme metabolism Jaundice (3) Hepatocellular Jaundice. Damage of the liver cells ( Ex. Liver cirrhosis or hepatitis) can cause increased in the unconjugated bilirubin level in the blood The bilirubin that conjugated is not efficiently secreted in the bile but diffuses into the blood. The urine becomes dark ( urobilinogen increased), whereas stools are a pale,clay color. Patients nausea Plasma level of (SGOT), (SGPT) are elevated.

40. Jaundice

41. (4) Jaundice in new born: Premature babies often accumulate bilirubin ( activity of bilirubin glucuronyl transferase is low) Elevated bilirubin, more than albumin capacity, can diffuse into the basal ganglia and cause toxic encephalopathy Treatment: By blue fluorescent light, which converts bilirubin to more polar and water- soluble isomers.

42. Jaundice in new born

43. Determination of bilirubin By Van de Bergh reaction: in which diazotized sulfanilic acid reacts with bilirubin to form red azodipyrroles that measured colorimetry. Direct bilirubin: In aqueous solution conjugated bilirubin reacts rapidly with the reagent Indirect bilirubin: when the reaction carried out in methanol, both conjugated bilirubin and unconjugated bilirubin are soluble and reacted with the reagent and give total bilirubin