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8/4/2019 Porphyrins and Bile Pigments
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PORPHYRINS AND
BILE PIGMENTS
.
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WHAT ARE PORPHYRINS?
Porphyrins are cyclic
compounds formed by
the linkage of 4 pyrrole
rings throughHC=methenyl bridges
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A characteristic property of the porphyrins is
the formation of complexes with metal ions
bound to the nitrogen atom of the pyrrole
rings
Examples are the iron porphyrins such as
heme of hemoglobin and the magnesium-
containing porphyrin chlorophyll, thephotosynthetic pigment of plants.
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WHAT IS HEME?
Heme is a prosthetic group that consists of
an iron atom contained in the center of the
porphyrin ring
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A hemoprotein or heme protein, is a
metalloprotein containing a heme prosthetic
group, either covalently or noncovalently
bound to the protein itself
The iron in the heme is capable of undergoing
oxidation and reduction (usually to +2 and +3)
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Examples of Some Important Human and
Animal Hemoproteins
Protein Function
Hemoglobin Transport of oxygen in blood
Myoglobin Storage of oxygen in muscle
Cytochrome c Involvement in electron transport
chain
Cytochrome P450 Hydroxylation of xenobiotics
Catalase Degradation of hydrogen peroxide
Tryptophan pyrrolase Oxidation of tryptophan
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HEME NOMENCLATURE
The porphyrins found in nature are
compounds in which various side chains are
substituted for the eight hydrogen atoms
numbered in the porphyrin nucleus
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As a simple means of showing these
substitutions, Fischer proposed a shorthand
formula in which the methlene bridges are
omitted and each pyrrole ring is shown with
the eight substituent positions
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UROPORPHYRIN III
A (acetate) =
CH2COOHP (propionate) =CH2CH2COOH
Note the asymmetry of substituents in ring IV
A porphyrin with this type ofasymmetric substitution is
classified as a type III porphyrin
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TYPES OF PORPHYRINS
A porphyrin with a completely symmetric arrangement of
the substituents is classified as a type I porphyrin.
Only types I and III are found in nature, and the type III
series is far more abundant
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PROTOPORPHYRIN IX AND HEME
Heme and its immediate precursor, protoporphyrin IX, areboth type III porphyrins
However, they are sometimes identified as belonging to
series IX, because they were designated ninth in a series of
isomers postulated by Hans Fischer
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HEME SYNTHESIS
Starting materials: Succinyl-CoA and Glycine
Condensation with the help of pyridoxal phosphate produces
-amino--ketoadipate
cytosol
mitochondria
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HEME SYNTHESIS
Decarboxylation produces ALA, catalyzed by the rate controlling enzyme in
porphyrin biosynthesis, ALA synthase
2 ALA molecules condense to form porphobilinogen (PBG), catalyzed by
ALA dehydratase
ALA dehydratase is a zinc-containing enzyme and is inhibited by lead
cytosol
mitochondria
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HEME SYNTHESIS
4 PBG molecules
condense to form
hydroxymethylbilane
(HMB), catalyzed byuroporphyrinogen I
synthase (PBG
deaminase/HMB
synthase)
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HEME SYNTHESIS
HMB cyclizes
spontaneously to form
uroporphyrinogen I or
is converted touroporphyrinogen III
by the action of
uroporphyrinogen III
synthase
Also called PBG deaminase
or HMB synthase.
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HEME SYNTHESIS
Uroporphyrinogens Iand III have the pyrrole
rings connected by
methylene bridges
(CH2), which do
not form a conjugated
ring system.
Thus, these compoundsare colorless
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HEME SYNTHESIS
The porphyrinogensare readily auto-
oxidized to their
respective colored
porphyrins.
These oxidations are
catalyzed by light
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HEME SYNTHESIS
Uroporphyrinogen III is converted to coproporphyrinogen
III by decarboxylation of all the acetate groups
Coproporphyrinogen III enters the mitochondria where it is
converted to protoporphyrinogen III and then to
protoporphyrin III, the parent porphyrin of heme
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HEME SYNTHESIS
85% of heme synthesis occurs in the bone marrow and
majority of the remainder is made in hepatocytes
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ALA SYNTHASE
The key regulatory enzyme in the biosynthesis
of heme
ALA synthase occurs in both hepatic (ALAS 1)
and erythroid (ALAS 2) forms
Heme acts as a negative regulator of the
synthesis of ALAS I
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ALA SYNTHASE
Many drugs can result in an increase in ALAS 1
due to heme utilization by cytochrome p450
for their metabolism e.g. Morphine,
Phenobarbital
ALAS 2 does not undergo feedback regulation
by heme
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PORPHYRIAS
A group of disorders due to abnormalities in
the pathway of biosynthesis of heme
Genetic or acquired
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PORPHYRIAS
If the enzyme lesion
occurs early in the
pathway prior to
formation of
porphyrinogens, ALA andPBG accumulates in body
tissues causing abdominal
pain and neuropsychiatric
symptoms
Later blocks cause
photosensitivity
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Biochemical causes of the major
signs and symptoms of the
porphyrias
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HEME CATABOLISM
Under physiologic conditions, 1-2 x 108erythrocytes are destroyed per hour
When hemoglobin is destroyed, globin isdegraded into its constituent amino acids,which are then reused
Iron is also reused
The iron-free porphyrin is also degraded inthe reticuloendothelial cells of the liver,spleen, and bone marrow
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HEME CATABOLISM
In birds and amphibians the final product is thegreen biliverdin IX
In mammals, biliverdin reductase reduces themethenyl bridge between pyrrole III and IV to a
methylene group to produce a yellow pigment,bilirubin
The chemical conversion of heme to bilirubin byreticuloendothelial cells can be observed in vivo
as the purple color of the heme in a hematoma isslowly converted to the yellow pigment ofbilirubin.
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BILIRUBIN CATABOLISM
Divided into three processes:
1. Uptake of bilirubin by liver parenchymal cells
2. Conjugation of bilirubin with glucuronate in
the endoplasmic reticulum
3. Secretion of conjugated bilirubin into the
bile.
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BILIRUBIN CATABOLISM
Bilirubin is nonpolar, so hepatocytes conjugate it
to make it water-soluble by adding glucuronic
acid; then, it is excreted in the bile
The conjugation of bilirubin is catalyzed by aglucuronosyltransferase
The enzyme is mainly located in the endoplasmic
reticulum, uses UDP-glucuronic acid as theglucuronosyl donor, and is referred to as bilirubin-
UGT
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Structure of bilirubin diglucuronide
(conjugated, "direct-reacting"
bilirubin)
Glucuronic acid is attached via ester linkage to the two
propionic acid groups of bilirubin to form an
acylglucuronide
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Conjugation of bilirubin with
glucuronic acid
The glucuronate donor, UDP-glucuronic acid, is formedfrom UDP-glucose
The UDP-glucuronosyltransferase is also called bilirubin-
UGT.
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BILIRUBIN CATABOLISM
Most bilirubin is excreted in the form of
bilirubin diglucuronide
Phenobarbital induces bilirubin-UGTactivity, which makes it effective in the
treatment of unconjugated
hyperbilirubinemia in infants
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BILIRUBIN SECRETION INTO BILE
Occurs by active transport
Protein involved is MRP-2 (Multidrug-
resistance-like protein 2) or MOAT
(Multispecific organic ion transporter) located
in the plasma membrane of the bile
canalicular membrane
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BILIRUBIN SECRETION INTO BILE
As the conjugated bilirubin reaches the
terminal ileum and large intestine, the
glucuronides are removed by -
glucuronidases, which are bacterial enzymes
The pigment is subsequently reduced to
urobilinogen, a colorless compound
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BILIRUBIN SECRETION INTO BILE
Most of the colorless urobilinogens are
oxidized to colored urobilins in the colon and
are excreted in feces
A small fraction of urobilinogens is reabsorbed
and reexcreted through the liver
(enterohepatic urobilinogen cycle )
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In the lower small intestine and colon,
bacteria remove glucuronic acid residues and
reduce bilirubin to the colorless urobilinogen
and stercobilinogen.
Exposure to air oxidizes these to urobilin and
stercobilin, respectively, (red-orange pigments
that contribute to the normal color of stooland urine).
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Urobilinogen is excreted mostly in the feces,
but a small fraction is absorbed from the
colon, enters the portal circulation, is removed
by the liver, and is secreted into bile.
That which is not removed from the portal
blood by the liver enters the systemic
circulation and is excreted by the kidneys.
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Urobilinogen excretion in urine normally
amounts to 1-4 mg per 24 hours, as opposed
to the 40-280 mg excreted in feces.
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Lack of urobilinogen in the urine and feces
indicates biliary obstruction
Stools are whitish ("clay-colored") owing to
the absence of bile pigment.
Urinary and fecal urobilinogen excretion
increases in hemolytic anemia.
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HYPERBILIRUBINEMIA AND JAUNDICE
Hyperbilirubinemia occurs when bilirubin in theblood exceeds 1 mg/dL (17.1 mol/L)
May be due to excess bilirubin production or liverfailure
Obstruction of the excretory ducts of the liver alsoprevents bilirubin excretion and causeshyperbilirubinemia
When bilirubin reaches 2-2.5 mg/dL in blood, itdiffuses into tissues causing jaundice or icterisia
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TYPES OF HYPERBILIRUBINEMIA
1. Retention hyperbilirubinemia due tooverproduction of bilirubin (Unconjugated
bilirubin)2. Regurgitation hyperbilirubinemia due to the
reflux into the bloodstream secondary tobiliary obstruction (Conjugated bilirubin)
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Because of its hydrophobicity, only
unconjugated bilirubin can cross the blood-
brain barrier into the central nervous system
Thus, encephalopathy due to
hyperbilirubinemia (kernicterus) can occur
only in connection with unconjugated
bilirubin, as found in retentionhyperbilirubinemia.
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On the other hand, because of its water-solubility, only conjugated bilirubin canappear in urine
Choluric jaundice (choluria is the presenceof bile pigments in the urine) occurs only inregurgitation hyperbilirubinemia
Acholuric jaundice occurs only in thepresence of an excess of unconjugatedbilirubin
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EHRLICH TEST FOR BILIRUBIN
Based on the coupling of diazotized sulfanilic
acid and bilirubin to produce a reddish-purple
azo compound
A reaction without methanol being added
means that conjugated bilirubin is present;
with methanol it is unconjugated bilirubin
UNCONJUGATED
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UNCONJUGATED
HYPERBILIRUBINEMIA
1. Hemolytic anemia
2. Neonatal Physiologic jaundice
A transient condition
Most common cause of unconjugated
hyperbilirubinemia
Due to accelerated hemolysis with animmature liver
UNCONJUGATED
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UNCONJUGATED
HYPERBILIRUBINEMIA
3. Crigler-Najjar syndrome type I
Congenital nonhemolytic jaundice
Autosomal recessive
Due to mutations in the gene encoding
bilirubin-UGT activity in hepatic tissues
4. Crigler-Najjar syndrome type II Like type I but more benign
UNCONJUGATED
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UNCONJUGATED
HYPERBILIRUBINEMIA
5. Gilbert syndrome
Also has mutations in genes encodingbilirubin-UGT but retains 30% of enzyme
activity6. Toxic hyperbilirubinemias
Chloroform, carbon tetrachloride,
acetaminophen, viral hepatitis, cirrhosis,Amanita mushroom
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CONJUGATED HYPERBILIRUBINEMIA
1. Biliary Obstruction Regurgitation into hepatic veins and lymphatics
Conjugated bili in blood and urine
2. Dubin-Johnson syndrome
Benign autosomal recessive
Mutation in the gene encoding MRP-2 for thesecretion of conjugated bili into bile
3. Rotor syndrome
Chronic conjugated hyperbilirubinemia and normalliver
L b t R lt i N l
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Laboratory Results in NormalPatients and Patients with ThreeDifferent Causes of Jaundice.
Serum
Bilirubin
Urine
Urobilinogen
Urine
Bilirubin
Fecal
Urobilinogen
Normal Direct: 0.10.4
mg/dL
04 mg/24 h Absent 40280 mg/24 h
Indirect: 0.20.7
mg/dL
Hemolytic
anemia
Indirect Increased Absent Increased
Hepatitis Direct andindirect
Decreased if micro-obstruction is present
Present if micro-obstruction
occurs
Decreased
Obstructive
jaundice1
Direct Absent Present Trace to absent
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