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Prof. Sasvári Mária
Metabolism of porphyrins
and bile pigments
Gergely Keszler
2009.
1
The biological role of porphyrins
Protoporphyrin IX + Fe2+/Fe3+ → heme →
a prosthetic group of several proteins, such as:
• hemoglobin, myoglobin, neuroglobin
• cytochromes (a, b, c, P450)
• catalase and other enzymes
(• chlorophyll, xanthophyll (Mg2+))
2
vinyl
methyl
methenyl
propionyl
= circular tetrapyrrol – iron chelate complex
3
Myoglobin - monomer
proximal His
distal His
4
Hemoglobin - tetramer
 α2β2, 4 O2
 2,3-BPG
 positive
cooperativity
 sigmoidal
saturation curve
5
Overview of heme biosynthesis
6
The ALA synthase reaction
COO CH2
Succinyl-CoA CH2
(4 C)
C O
COO CoA-SH
S ~ CoA
H
Glycine
(2C + N)
H C
Pyr-P
NH3+
COO -
CH2
COO CO2
CH2
CH2
CH2
C O
C O
H C
NH3+
COO -
a-amino-b-keto-adipate
(6C + N)
H C
NH3+
H
d-aminolevulinate (ALA)
(5C + N)
ALA: irreversible, rate limiting step (mitochondrial)
Regulation of ALA synthase: Repression of transcription by heme
(heme is also an allosteric inhibitor)
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Induction by certain drugs
Formation of porphobilinogen
Acetyl
COO - (A)
COO
CH2
CH2
CH2
C O
H C H
NH3+
ALA
Propionyl
- (P)
A
P
CH2
C
C
C O
C
C
H C H
NH3+
ALA
2 H2O
ALA
dehydratase
CH2
N
H
NH3+
Porphobilinogen
ALA dehydratase: contains Zn2+
inhibition by Pb2+ (lead poisoning) → EDTA!!!
8
Lead poisoning
microcytic anemia
9
Formation of the linear tetrapyrrole
A
CH2
P
N
H
NH3+
4 porphobilinogen
Porphobilinogen deaminase
4 NH3
A
P
A
CH2
N
H
CH2
A
P
N
H
CH2
A
P
N
H
Unstable intermediate
CH2
P
N
H
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Spontaneous or enzyme-assisted ring formation
Uroporphyrinogen III
cosynthase
spontaneous
A
A
P
I.
P
I.
IV.
II.
A
A
A
P
P
III.
P
P
II.
P
III.
P
A
A
uroporphyrinogen I
IV.
A
urophorphyrinogen III
Accumulation in case of porphyrias
II and IV do not exist in nature
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Enzymes converting both type I and type III
A
P
4 CO2
M
I.
A
P
IV.
P
I.
II.
A
P
III.
P
A
M
Uroporphyrinogen
decarboxylase
uroporphyrinogen III
P
IV.
II.
M
P
III.
P
M
coproporphyrinogen III
Acetate group:
- CH2 - COOH
Methyl group:
- CH3
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Enzymes specific for type III
M
2 CO2
4H
P
M
I.
M
P
IV.
II.
I.
M
P
M
P
coproporphyrinogen
oxidase (O2)
III.
P
M
V
coproporphyrinogen III
propionyl group:
IV.
II.
M
V
III.
P
M
protoporphyrinogen IX
- CH2 - CH2 -COOH
ethyl group:
- CH2 - CH3
vinyl group:
- CH - CH2
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Porphyrins are colored compounds
(conjugated double bond system)
V
M
V
M
6H
CH2
CH2
N
H
M
N
H
N
H
V
CH2
CH2
P
P
Protoporphyrinogen
oxidase
M
Protoporphyrinogen III (IX)
M
N
H
P
N
M
N
H
N
H
M
CH
CH
V
N
CH
CH
P
M
Protoporphyrin III (IX)
Conjugated double bond system
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Heme contains iron
Fe2+
Protoporphyrin III (IX)
heme
Ferrochelatase (inhibited by lead)
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Iron metabolism in man
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Porphyrias genetic defects in
porphyrin biosynthesis
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Major symptoms
Photosensitivity
neuropsychiatric problems
fluorescent teeth
red urine
Diagnosis: assay of enzyme activities in RBC
analysis of urine
Erythropoietic porphyrias: Bone marrow (hemoglobin)
Hepatic porphyrias: Liver (cytochrome P450)
Mixed types
Drugs  induction of cytochrome P450  elevated usage of
heme  ALA synthase is released from repression  elevated
level of harmful heme-like products
Treatment: avoid drugs, alcohol
administration of hematin (represses ALA)
sunscreens, carotinoids
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The legend of vampires and werevolves–
victims of porphyria?
 pale skin (anemia)
 thirst for blood (anemia)
 strange behaviour (neurotoxicity)
 night activity (photosensitivity)
 fluorescent teeth (porphyrins)
 fear of garlic (no CYP450 in liver)
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Catabolism of heme
Heme oxygenase: 1. converts hemin (Fe3+) to heme (Fe2+)
2. Circular form  linear form (oxidation)
Coenzyme: NADPH
M
V
M
I.
M
P
IV.
Fe3+
III.
P
M
V
M
I.
II.
M
M
V
P
NADPH
+H+
NADP+
IV.
Fe2+
III.
P
M
V
I.
II.
M
M
V
P
NADPH
+H+
+O2
biliverdin:bilirubin = 1:1
NADP+
IV.
Fe2+
OH
II.
III.
P
M
O2
Fe2+
CO
Linearization: production of biliverdin and bilirubin in equimolar ratio
heme: purple
bilirubin: yellow
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(see: the colour of hematomas changes)
M
V
Biliverdin → bilirubin
V
M
I
O
M
CH
P
IV
N
P
M
III
CH
N
H
M
II
CH
N
O
N
H
biliverdin
V
H
NADPH + H+
biliverdin reductase
V
M
I
O
N
H
M
CH
NADP+
P
IV
P
CH2
M
III
N
N
H
H
M
CH
V
II
N
O
H
bilirubin
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Excretion of bilirubin
BLOOD: transport (bound to albumin)
uptake in hepatocytes (ligandin)
LIVER: conjugation in ER with glucuronate
Bilirubin diglucuronide
Active excretion into bile canaliculi (MRP2)
Reabsorbtion, reexcretion
intrahepatic urobilingen cycle
LARGE INTESTINE, gut bacteria
Deconjugation, colourless urobilinogen formation
FECES
Coloured uro- and stercobilins
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Conjugation of bilirubin
1. UDP-glucuronic acid formation from UDP glucose
CH2OH
UDP-gl-dehydrogenase
COO -
O-UDP
UDP-glucose
H2O + 2 NAD+
O-UDP
2NADH + 2H+
UDP-glucoronic acid
2. UDP-glucuronidation of bilirubin
UDP-glucuronyl transferase
Bilirubin  bilirubin monoglucuronide  bilirubin diglucuronide
UDP-gl UDP
UDP-gl UDP
dismutase
2 bilirubin monoglucuronide  bilirubin diglucoronide + bilirubin
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Bilirubin diglucuronide
COO COO -
V
M
I
O
N
H
O
O
O C
O C
M
CH
IV
N
H
CH2
CH2
CH2
CH2
CH2
M
III
N
H
M
CH
V
II
N
O
H
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Hyperbilirubinemia:
jaundice
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 (choluric jaundice)
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Elevated unconjugated bilirubin in the blood PREHEPATIC JAUNDICE
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!
Albumin binding capacity: 20-25 mg/dL
Over this value:
hyperbilirubinemic toxic encephalopathy (kernicterus)
(mental retardation)
Treatment:  phenobarbital administration:
induction of the conjugating system
 phototherapy: photoisomerization of bilirubin →
water-soluble products →
excretion via kidneys
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Elevated unconjugated bilirubin in the blood –
HEPATIC JAUNDICE
Congenital nonhemolytic jaundice (Crigler-Najjar Syndrome) Type I
Enzyme defect of bilirubin UDP-glucosyl transferase
fatal within the first 15 month of life
phenobarbiturate treatment does not help → phototherapy
(Crigler-Najjar Syndrome) Type II
Partial enzyme defect, milder symptomes
Patient might respond to phenobarbital
Gilbert’s Disease
Problem with the uptake of bilirubin into the liver
Unconjugated bilirubinemia
UDP-glucuronyl transferase activity is normal
Toxic hyperbilirubinemia
Toxin-induced liver dysfunction
(e.g. Hepatitis, liver chirrhosis, mushroom poisoning)
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Conjugated Hyperbilirubinemia
Obstruction of the biliary tree
Blockage 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’s syndromes
Defect in the hepatic secretion of conjugated bilirubin
due to MRP2 transporter mutations
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Micro- and macroscopic pigmentation of the liver
in Dubin-Johnson syndrome
Binding of conjugated bilirubin to albumin (abnormal)
Covalent bound
Remains elevated
after the recovery phase of obstructive jaundice
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The clinical importance of
urine analysis in jaundice
Complete obstruction of bile duct:
Urine: No urobilinogen (this is produced in the intestine)
High conjugated bilirubin
Hemolytic jaundice:
Increased production of bilirubin  increased urobilinogen
Large amount of urobilinogen in the urine
Absence of bilirubin in the urine
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