Metabolism of and bile pigments

Mária Sasvári 2017

1 The biological role of porphyrins

Protoporphyrin IX + Fe2+ → → the of several proteins, such as:

• , • cytochromes (a, b, c, P450) • catalase, peroxidase •

2 Structure of (cytochrome b, Hb, Mb) vinyl

methyl Order of substituents: MVMVMPPM methinyl bridge pyrrol ring

propionate (not propionyl!)

protoporphyrin IX – Fe2+ chelate complex 3 Myoglobin: monomer

proximal His

distal His

globin: 8 α-helical domains and linker sequences 4 Hemoglobin: tetramer

 α2β2, 4 O2  2,3-BPG

 positive cooperativity

 sigmoidal saturation curve

5 δ-amino-levulinate (ALA) succinyl-CoA glycine

heme B Overview of heme

proto- mito- cytoplasm III IX chondrium

proto- copro- IX porphyrinogen III 6 The ALA synthase reaction

COO -

CH2 CH Succinyl-CoA 2 COO - COO - (4 C) C O CH2 CH CoA-SH CO2 2 S ~ CoA CH2 CH2 C O C O H PLP Glycine + + + H C NH3 H C NH3 H C NH3 (2C + N) - COO - COO H a-amino-b-keto-adipate d-aminolevulinate (ALA) (6C + N) (5C + N)

ALA synthase: irreversible, rate-limiting step (mitochondrial) Regulation of ALA synthase: repression of transcription by heme

(heme is also an allosteric inhibitor7 ) induction by certain drugs 2 ALA molecules yield porphobilinogen

acetate propionate - (A) (P) COO COO - CH 2 CH2 2 H2O A P CH 2 CH2 C C

C O C O C C N H C H H C H ALA dehydratase CH2 (porphobilinogen H + + + NH NH NH3 3 3 synthase) ALA ALA porphobilinogen

ALA dehydratase: contains Zn2+ inhibition by Pb2+ (lead poisoning) → EDTA!!! 8 Lead poisoning

microcytic anemia 9 Formation of linear terapyrrole A P

CH2 N + H NH3 4 porphobilinogen porphobilinogen deaminase (hydroxymethylbilane synthase) 4 NH3 A P A P A P A P

HO

CH2 N CH2 N CH2 N CH2 N H H H H

unstable intermediate (hydroxymethylbilane) 10 Spontaneous or -catalysed ring closure

uroporphyrinogen III spontaneous synthase

A P A P I. I. P A A A IV. II. IV. II. A P P P III. inversion III. P A of ligands P A

urophorphyrinogen III slow synthesis, false endproduct, rapid synthesis excreted in urine

11 Modification of substituents begins with decarboxylation…

A P 4 CO2 M P I. I. A A M M IV. II. IV. II. P P uroporphyrinogen P P III. III. decarboxylase P A P M

uroporphyrinogen III coproporphyrinogen III

Acetate group: - CH2 - COOH

Methyl group: - CH3

12 … and gets completed by an oxidase

2 CO2 M P 4 H M V I. M M I. IV. II. M M P P IV. II. P V III. coproporphyrinogen III oxidase (O ) III. P M 2 P M coproporphyrinogen III protoporphyrinogen IX

propionate group: - CH2 - CH2 -COOH

ethyl group: - CH2 - CH3

vinyl group: - CH - CH2 13 The ring system is completed

M V M V

6H

CH2 CH2 CH CH M N M N

H M M

H N

H N

N

H

N H H

P N V P N V CH2 CH2 protoporphyrinogen CH CH oxidase

P M P M

protoporphyrinogen IX protoporphyrin IX colourless conjugated double bond system, coloured 14 Formation of the iron (II) chelate complex

Fe2+

Protoporphyrin IX heme ferrochelatase (inhibited by lead)

15 Iron metabolism in man

Fe3+

16 Iron uptake and storage

2+ Fe 3+ ferritin ferritin reductase ferritin Fe

17 The iron-ferritin complex

18 Regulation of ALA synthase isoenzymes

Liver-specific isoform: ALAS-L (ALAS-1) heme inhibits the transcription, mRNA 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 Mutations in heme biosynthesizing result in porphyria

most frequent types: porphobilinogen acute intermittent porphyria hydroxymethylbilane congenital erythropoetic porphyria uroporphyrinogen III porphyria cutanea tarda coproporphyrinogen III

20 Major symptoms

photosensitivity neuropsychiatric symptoms fluorescent teeth red urine

Diagnosis: assay of enzyme activities in RBC analysis of urine

Erythropoietic porphyrias: bone marrow (hemoglobin) Hepatic porphyrias: liver ()

Treatment: administration of hemin (represses ALAS) sunscreens, carotinoids no drugs and alcohol! 21 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 CYP450 in liver) 22 Enzymatic breakdown of heme

Heme oxygenase: linearizes the ring system by elimination of one of the methinyl bridges

M V M V I. I. OH M M M M IV. Fe2+ II. IV. Fe2+ II. P V P V III. III. P M P M 3 NADPH 3 NADP+ +H+ + H O 2 +O2 O2 Fe2+ CO

heme: red; biliverdin: green; : yellow (the colour of hematomas changes) 23 Reduction of biliverdin yields bilirubin

M V M P P M M V I IV III II CH CH CH O O N N N N H H H biliverdin

important NADPH + H+ 2 H O 2 endogenous biliverdin reductase + NADP H2O2 antioxidant

M V M P P M M V I IV III II CH CH2 CH O O N N N N H H H H bilirubin

24 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 Mechanism of bilirubin excretion from the body

blood: bound to albumin formation of bilirubin uptake in hepatocytes (ligandin) in the monocyte-macrophage system

liver: conjugation with in ER 

excretion into bile canaliculi (MRP2 efflux pump)

colon: bacterial deconjugation and reduction to (Ubg) 10 %

90 % reabsorption excreted in urine faeces: and (brown pigment) diarrhea: fast passage, no time to yield stercobilin: yellow feces 26 Conjugation of bilirubin in the ER of hepatocytes

1. UDP-glucuronic acid formation from UDP-glucose CH2OH COO - UDP-glucose dehydrogenase

O-UDP O-UDP UDP-glucose UDP-glucoronic acid + + H2O + 2 NAD 2NADH + 2H

2. UDP-glucuronidation of bilirubin UDP-glucuronosyl transferase Bilirubin  bilirubin monoglucuronide  bilirubin diglucuronide

UDP-glucuronate UDP UDP-glucuronate UDP 27 Bilirubin diglucuronide (conjugated bilirubin)

COO -

COO - O O O C O C CH 2 CH2 CH M V M 2 CH2 M M V I IV III II CH CH2 CH O O N N N N H H H H

28 Conformation of conjugated bilirubin

polar, water-soluble molecule

29 Hyperbilirubinemia leads to

I. indirect hyperbilirubinemia “indirect-reacting” bilirubin: methanol soluble (NOT 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 Prehepatic jaundice: elevated levels of indirect bilirubin in plasma

Hemolytic anemias: the liver has a large capacity, so unconjugated bilirubin will elevate only if other defects exist too

Neonatal jaundice: accelerated hemolysis, immature hepatic system low activity of UDP-glucuronyl transferase low rate of UDP-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 Photoisomerisation of bilirubin

blue light

32 33 Hepatic jaundice: elevated levels of indirect bilirubin in plasma

Crigler-Najjar syndrome, type I full deficiency of bilirubin UDP-glucuronosyl transferase fatal within the first 15 month of life phenobarbital treatment does not help → phototherapy

Crigler-Najjar 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 UDP-glucuronosyl transferase activity

Toxic hyperbilirubinemia Toxin-induced liver dysfunction (e.g. hepatitis, liver chirrhosis, mushroom poisoning) 34 Posthepatic 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 MRP2 transporter mutations

35 Microscopic pigmentation of the liver in Dubin-Johnson syndrome

36 Urinanalysis helps discriminate between pre- and posthepatic forms of jaundice

Posthepatic 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

Hemolytic (posthepatic) jaundice: increased production of bilirubin and urobilinogen large amounts of urobilinogen in the urine

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