Porphyrins and Bile Pigments

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 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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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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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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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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Porphyrins and Bile Pigments

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