Metabolism of porphyrins and bile pigments. Mária Sasvári 2017

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1 Metabolism of porphyrins and bile pigments Mária Sasvári

2 The biological role of porphyrins rotoporphyrin IX + Fe 2+ heme the prosthetic group of several proteins, such as: hemoglobin, myoglobin cytochromes (a, b, c, 450) catalase, peroxidase nitric oxide synthase 2

3 vinyl Structure of heme B (cytochrome b, b, Mb) methyl Order of substituents: MVMVMM pyrrol ring methinyl bridge propionate (not propionyl!) protoporphyrin IX Fe 2+ chelate complex 3

4 Myoglobin: monomer proximal is distal is globin: 8 α-helical domains and linker sequences 4

5 emoglobin: tetramer α 2 β 2, 4 O 2 2,3-BG positive cooperativity sigmoidal saturation curve 5

6 succinyl-coa δ-amino-levulinate (ALA) porphobilinogen glycine heme B Overview of heme biosynthesis hydroxymethylbilane protoporphyrin IX mitochondrium cytoplasm uroporphyrinogen III protoporphyrinogen IX coproporphyrinogen III 6

7 The ALA synthase reaction COO - C 2 Succinyl-CoA (4 C) C 2 C O S ~ CoA CoA-S COO - C 2 C 2 CO 2 COO C 2 C 2 - Glycine (2C + ) C 3 + COO - L C O C 3 + COO - C O C 3 + a-amino-b-keto-adipate (6C + ) d-aminolevulinate (ALA) (5C + ) ALA synthase: irreversible, rate-limiting step (mitochondrial) Regulation of ALA synthase: repression of transcription by heme (heme is also an allosteric inhibitor) 7 induction by certain drugs

8 2 ALA molecules yield porphobilinogen acetate (A) COO - propionate () COO - C 2 C 2 C O C 3 + ALA C 2 C 2 C O C 3 + ALA 2 2 O ALA dehydratase (porphobilinogen synthase) C C 2 A C 3 + C C porphobilinogen ALA dehydratase: contains Zn 2+ inhibition by b 2+ (lead poisoning) EDTA!!! 8

9 Lead poisoning microcytic anemia 9

10 Formation of linear terapyrrole A A C A 4 porphobilinogen porphobilinogen deaminase (hydroxymethylbilane synthase) A A O C 2 C 2 C 2 unstable intermediate (hydroxymethylbilane) C 2 10

11 Spontaneous or enzyme-catalysed ring closure spontaneous uroporphyrinogen III synthase A A I. IV. III. A II. A A inversion of ligands A I. IV. III. A II. A uroporphyrinogen I slow synthesis, false endproduct, excreted in urine urophorphyrinogen III rapid synthesis 11

12 Modification of substituents begins with decarboxylation A A I. IV. III. A II. A 4 CO 2 uroporphyrinogen decarboxylase M M I. IV. II. III. M M uroporphyrinogen III coproporphyrinogen III Acetate group: - C 2 - COO Methyl group: - C 3 12

13 and gets completed by an oxidase M I. M M IV. II. III. M coproporphyrinogen III 2 CO 2 4 coproporphyrinogen III oxidase (O 2 ) M IV. M V I. III. M II. M V protoporphyrinogen IX propionate group: - C 2 - C 2 -COO ethyl group: - C 2 - C 3 vinyl group: - C - C 2 13

14 The ring system is completed M V M V M C 2 C 2 M 6 M C C M C 2 C 2 V protoporphyrinogen oxidase C C V M M protoporphyrinogen IX colourless protoporphyrin IX conjugated double bond system, 14 coloured

15 Formation of the iron (II) chelate complex Fe 2+ rotoporphyrin IX heme ferrochelatase (inhibited by lead) 15

16 Iron metabolism in man Fe 3+ 16

17 Iron uptake and storage Fe 2+ ferritin ferritin reductase ferritin Fe 3+ 17

18 The iron-ferritin complex 18

19 Regulation of ALA synthase isoenzymes Liver-specific isoform: ALAS-L (ALAS-1) heme inhibits the transcription, mra export, mitochondrial uptake and enzymatic activity of this isoenzyme Erythroid (bone marrow) isoform: ALAS-E (ALAS-2) - deficiency: sideroblastic anemia (hypochromic) - not inhibited by heme - heme inhibits iron release from ferritin and stimulates globin production - transcription is stimulated by erythropoetin 19

20 Mutations in heme biosynthesizing enzymes result in porphyria most frequent types: porphobilinogen acute intermittent porphyria hydroxymethylbilane congenital erythropoetic porphyria uroporphyrinogen III porphyria cutanea tarda coproporphyrinogen III 20

21 Major symptoms photosensitivity neuropsychiatric symptoms fluorescent teeth red urine Diagnosis: assay of enzyme activities in RBC analysis of urine Erythropoietic porphyrias: bone marrow (hemoglobin) epatic porphyrias: liver (cytochrome 450) Treatment: administration of hemin (represses ALAS) sunscreens, carotinoids no drugs and alcohol! 21

22 Vampires and werevolves victims of porphyria? pale skin (anemia) blood-thirst (anemia) psychiatric disorders (neurotoxicity) night activity (photosensitivity) fluorescent teeth (porphyrins) fear of garlic (no CY450 in liver) 22

23 Enzymatic breakdown of heme eme oxygenase: linearizes the ring system by elimination of one of the methinyl bridges M M V I. IV. Fe 2+ biliverdin III. M II. M V 3 AD + + +O 2 CO M 3 AD O Fe 2+ M V I. O M IV. Fe 2+ II. V III. M O 2 heme: red; biliverdin: green; bilirubin: yellow (the colour of hematomas changes) 23

24 Reduction of biliverdin yields bilirubin O M V M M M V I IV III II C C C biliverdin O biliverdin reductase AD + + AD O 2 O 2 important endogenous antioxidant O M V M M M V I IV III II C C 2 bilirubin C O 24

25 Bilirubin contains polar groups, but due to its special conformation - cis double bonds ( ) and intramolecular hydrogen bonds ( ) - its outer surface is rather apolar. Therefore, it is carried in blood plasma by albumin 25

26 Mechanism of bilirubin excretion from the body blood: bound to albumin uptake in hepatocytes (ligandin) formation of bilirubin in the monocyte-macrophage system liver: conjugation with glucuronic acid in ER bilirubin diglucuronide excretion into bile canaliculi (MR2 efflux pump) colon: bacterial deconjugation and reduction to urobilinogen (Ubg) 90 % faeces: urobilin and stercobilin (brown pigment) diarrhea: fast passage, no time to yield stercobilin: yellow feces 10 % reabsorption excreted in urine 26

27 Conjugation of bilirubin in the ER of hepatocytes 1. UD-glucuronic acid formation from UD-glucose C 2 O UD-glucose dehydrogenase COO - UD-glucose O-UD 2 O + 2 AD + 2AD O-UD UD-glucoronic acid 2. UD-glucuronidation of bilirubin UD-glucuronosyl transferase Bilirubin bilirubin monoglucuronide bilirubin diglucuronide UD-glucuronate UD UD-glucuronate UD 27

28 Bilirubin diglucuronide (conjugated bilirubin) COO - COO - O O O C O C O M V M C 2 C 2 M M V I IV III II C C 2 C 2 C 2 C O 28

29 Conformation of conjugated bilirubin polar, water-soluble molecule 29

30 yperbilirubinemia leads to jaundice I. indirect hyperbilirubinemia indirect-reacting bilirubin: methanol soluble (OT conjugated) prehepatic or hepatic jaundice (e.g. hemolysis, hepatitis) retention of water-insoluble bilirubin II. direct hyperbilirubinemia direct-reacting bilirubin: water soluble (conjugated) posthepatic jaundice: reflux of conjugated bilirubin into the blood appearance in the urine 30

31 rehepatic jaundice: elevated levels of indirect bilirubin in plasma emolytic anemias: the liver has a large capacity, so unconjugated bilirubin will elevate only if other defects exist too eonatal jaundice: accelerated hemolysis, immature hepatic system low activity of UD-glucuronyl transferase low rate of UD-glucuronic acid production unconjugated bilirubin can pass the blood-brain barrier hyperbilirubinemic toxic encephalopathy (kernicterus) (mental retardation) Treatment: phenobarbital administration: induction of the conjugating system phototherapy: photoisomerization of bilirubin water-soluble products excretion via kidneys 31

32 hotoisomerisation of bilirubin blue light 32

33 33

34 epatic jaundice: elevated levels of indirect bilirubin in plasma Crigler-ajjar syndrome, type I full deficiency of bilirubin UD-glucuronosyl transferase fatal within the first 15 month of life phenobarbital treatment does not help phototherapy Crigler-ajjar syndrome, type II partial enzyme defect, milder symptoms patient might respond to phenobarbital Gilbert s Disease 5% of mankind affected unconjugated bilirubinemia, promoter mutations slightly reduced UD-glucuronosyl transferase activity Toxic hyperbilirubinemia Toxin-induced liver dysfunction (e.g. hepatitis, liver chirrhosis, mushroom poisoning) 34

35 osthepatic jaundice: direct hyperbilirubinemia Obstruction of the biliary tree Blockade of the bile duct Bilirubin conjugates cannot be excreted Regurgitates into hepatic veins and lymphatics Conjugated bilirubin appears in the serum and in the urine Dubin-Johnson and Rotor-syndromes Defect in the hepatic secretion of conjugated bilirubin due to congenital MR2 transporter mutations 35

36 Microscopic pigmentation of the liver in Dubin-Johnson syndrome 36

37 Urinanalysis helps discriminate between pre- and posthepatic forms of jaundice osthepatic jaundice no urobilinogen in urine (as it is produced in the intestine) high conjugated bilirubin levels in the blood and urine dark urine, pale feces emolytic (posthepatic) jaundice: increased production of bilirubin and urobilinogen large amounts of urobilinogen in the urine 37