Download MBBS Biochemistry PPT 50 Hemoglobin Metabolism Lecture Notes

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Hemoglobin Metabolism

Synopsis
Hemoglobin Biosynthesis

vHeme Synthesis

qPorphyrias (Disorders Of Heme Synthesis)

vGlobin Synthesis

qAbnormal Hb variants/
Hemoglobinopathies(Disorders of Globin Synthesis)

Hemoglobin Breakdown

qFormation and Fate of Bilirubin
qHyperbilirubinemia

Jaundice : Causes Types and Diagnosis

Hemoglobin Biosynthesis
Hemoglobin biosynthesis

includes biosynthesis of:

vHeme
vGlobin Polypeptide chains

Amount of Hb biosynthesized=

6.25 gm/day
Site For Hemoglobin

Biosynthesis

Organs Involved In

Hb Biosynthesis

Bone Marrow-
Immature Erythrocytes ? 85%
Liver ? 15 %
Requirements For

Hemoglobin Biosynthesis

Normal biosynthesis of

Hemoglobin depends upon an

Quality and Quantity of :

qAmino acids
qMinerals
qVitamins
Heme Biosynthesis

OR

Porphyrin Pathway

Biosynthesis

Of

Ferroprotoporphyrin


Site For Heme Biosynthesis

Organs

Bone Marrow-
Immature Erythrocytes ? 85%
Liver ? 15 %

Cellular Site

Mitochondrial Matrix
Cytosol
Requirements For

Heme Biosynthesis

Metabolic Precursors for Heme Biosynthesis:

? Glycine and Succinyl-CoA

Vitamins (5 Hematopoietic Vitamins):

vPantothenic acid (Vitamin B5)
vPyridoxine (Vitamin B6)
vFolate (Vitamin B10)
vCyanocobalamin (Vitamin B12)
vVitamin- C (Ascorbic acid)

Minerals for Heme Biosynthesis:

?Iron ( Fe++)
?Copper (Cu++)
?Zinc ( Zn ++)
Stages and Steps

Of

Heme Biosynthesis

3 Stages Of Heme Biosynthesis
1. Synthesis of -Amino Levulinic Acid

( ALA)

(In Mitochondrial Matrix)

2. Synthesis of CoproPorphyrinogen?III

( CPG-III)

(In Cytoplasm)

3. Synthesis of ProtoPorphyrin IX and

Incorporation of Fe++ to Form Heme

(In Mitochondrial Matrix)

7 steps in Heme Biosynthesis

Step 1 in Mitochondrial Matrix
Steps 2,3,4 in Cytoplasm
Steps 5,6 and 7 in

Mitochondrial matrix



?Important Intermediates of Heme

Synthesis Pathway:

-Aminolevulinic acid

(ALA)
Porphobilinogen

(PBG = Pyrrole derivative)
Uroporphyrinogen III

( UPG? Heme precursor)
Protoporphyrin IX

(Direct Heme precursor)

-Aminolevulinic Acid (ALA)

? Synthesis of Heme starts in Mitochondrial matrix
? Succinyl-CoA and Glycine undergo a condensation ALA
? Reaction is catalyzed by enzyme ALA Synthase
? ALA Synthase requires

?Vitamin B6 (PLP)
? Copper ions

PLP used in first step of

Heme biosynthesis

activates Glycine.
Presence of free Heme inhibits

the synthesis of an enzyme

- ALA Synthase.

This represents a Feedback

mechanism for Heme synthesis.

This first step is a Rate limiting

step of Heme synthesis:

vStimulated by the presence of

Globin chains.

vInhibited by the presence of free

Heme groups.


Rate of Heme

biosynthesis has good

coordination with

Globin synthesis.

Porphobilinogen (PBG)

? ALA leaves the Mitochondria Reach Cytoplasm
? 2x ALA condense together to form Porphobilinogen (PBG).
? Reaction is catalyzed by Porphobilinogen Synthase
/(ALA dehydratase)
Mitochondrial -Aminolevulinic acid

(ALA) is transported to the cytoplasm,

where

ALA Dehydratase /Porphobilinogen

Synthase- Zinc containing enzyme.

Dimerizes 2 molecules of ALA to produce
The Pyrrole ring compound is

Porphobilinogen (PBG).

PBG then biosynthesizes Porphyrin

ring.

The reactions involved for its

synthesis are extremely complex

but can be summarized as follows:
The condensation of four PBG

molecules

Form an asymmetric cyclic

Uroporphyrinogen III(UPG III).

Synthesis of UPG III requires the

presence of two enzymes:

v Uroporphyrinogen I Synthase
v Uroporphyrinogen III Cosynthase
During UPG synthesis there

involves the formation of

short-lived intermediate

Hydroxy Methyl Bilane

(HMB).

UPG I Synthase/PBG

Deaminase /HMB Synthase

Transforms 4 molecules of PBG

to linear Tetrapyrrole

Hydroxy Methyl Bilane

(HMB)
HMB spontaneously

cyclizes to form UPG III

by UPG III Cosynthase

Conversion of

Uroporphyrinogen II Coproporphyrinogen II

4 Acetate residues of

Uroporphyrinogen III are

Decarboxylated into 4 Methyl

groups Coproporphyrinogen III

Coproporphyrinogen III returns to

the Mitochondria again.
UPG III is converted to Coproporphyrinogen III

(CPGIII) by Decarboxylation of the Acetate side

chains

To Methyl groups under the influence of the

enzyme Uroporphyrinogen Decarboxylase.
CPG III enters the Mitochondria where it is

converted to Protoporphyrinogen IX (PPG IX) by an

unknown mechanism.

This reaction is catalyzed by the enzyme

Coproporphyrinogen Oxidase.

Coproporphyrinogen III Protoporphyrinogen IX

In Mitochondria CPG III is oxidized to

PPG IX.

Two Propionyl residues transformed

to Two Vinyl residues.

Removal of two CO2 molecules.
Reaction catalyzed by CPG Oxidase.
Protoporphyrinogen is a

colorless compound

It contains Methylene

bridges in tetrapyrrole

ring structure.

Methylene bridges of

Protoporphyrinogen

are oxidized to

Methenyl bridges to

form ProtoPorphyrin
Protoporphyrinogen IX Protoporphyrin IX

? Oxidation of protoporphyrinogen IX produces the

conjugated Methenyl bonds of Protoporphyrin IX

Final Formation of Heme

? Fe2+ is incorporated into Protoporphyrin IX

? Reaction is catalyzed by enzyme Ferrochelatase/Heme

Synthase to Form Heme.


Iron is chelated within Porphyrin

ring to form Heme by catalytic

activity of Ferrochelatase.

Heme is incorporated into

Proteins to become biologically

functional Hemoproteins.


Heme

MetalloPorphyrin /Ferroprotoporphyrin

Heme forms various: Hemoproteins

Hemoglobin
Myoglobin
Cytochromes
Catalase and Peroxidase
Tryptophan Pyrrolase


Chlorophyll is a Magnesium

containing Porphyrin present

in plants.

Chlorophyll is involved in

photosynthesis of plants.
Regulation Of Heme Biosynthesis

ALA Synthase is an

Allosteric regulatory

Enzyme of Heme

biosynthetic pathway.
ALAS 1 occurs in Hepatocytes
ALAS 2 is found in Erythroid tissue

Rate of Heme biosynthesis is

flexible.

Heme biosynthesis changes

rapidly in response to a wide

variety of external stimuli.
Mechanisms and Factors

Regulating Heme Biosynthesis

Feed Back Inhibition
Repression of ALA Synthase
Inhibition of transport of ALA

Synthase from Cytosol to

Mitochondrial matrix.

Erythropoietin levels
Iron levels
ALA Synthase is a key regulatory

enzyme of Heme biosynthesis.

It is an allosteric enzyme that is

inhibited by an end product-Heme

(Feedback inhibition)

Requires Pyridoxal Phosphate as

a coenzyme

Erythropoietin Stimulates

Heme Biosynthesis
Erythropoietin is a Protein

produced by Kidneys .

Erythropoietin stimulates

the ALA Synthase activity.

Erythropoietin Synthesis

increased in high altitude

dwellers.

Erythropoietin decreased

in chronic renal failure.

(Associated with Anemia)
Iron Levels Affect Heme Synthesis

The amount of cellular

Iron determines

The affinity for Iron

Responsive Element-

Binding Protein(IRE-BP).
When Iron levels are low
There is a high binding

affinity of IRE-BP with

mRNA of ALA.

Which serves to inhibit the

translation of ALA mRNA

Results in decrease of Heme

biosynthesis.

When Iron levels are

sufficient.

There is a low binding affinity

of IRE-BP with mRNA of ALA.

Thus allowing translation of

ALA mRNA

Stimulation of Heme synthesis.


How Iron Levels Affect

Heme Synthesis

Iron and Hemoglobin

Iron deficient red

blood cells

Low number of red

blood cells

Note the hollow and

blanched appearance

of the red blood cells.
If either Heme or Globin

synthesis is impaired

Iron is not utilized and

accumulates in the RBC.



When Heme

biosynthesis is impaired

Iron is underutilized
Mitochondria or Nucleus

of Erythroblasts become

encrusted with Iron.


A Sideroblast is

a Nucleated Erythrocyte

Containing Iron granules in
its cytoplasm in the bone

marrow.

Sideroblast

Sideroblast is

an Erythroblast with Iron granules

(Pappenheimer bodies) seen in

bone marrow stained by
Prussian blue or Perl stains.
A precursor Red blood

cell (Immature RBC)

with a ring of Iron

around the nucleus is

called a ringed

Sideroblast.

Siderocyte

Siderocyte is a non-

nucleated red cell with

Iron granules
(Pappenheimer bodies)
A mature RBC with an

accumulated Iron is

termed as Siderocyte .

Siderocytes

are abnormally increased in :

Sideroblastic Anemia
Hemosiderosis
Hemoglobinopathies


The Iron within

Sideroblasts and

Siderocytes can be

visualized by staining with

Prussian blue stain.



Ringed Sideroblast


RINGED SIDEROBLASTS AND

SIDEROCYTE

Effect Of Drugs and Other

Substances On Heme Biosynthesis
Certain Drugs and

Steroid Hormones

increases Heme

biosynthesis.

Ingestion of drugs like

Phenobarbitals ,Insecticides,

certain chemical carcinogen,

Markedly increases ALA

Synthase (ALAS1 of Hepatocytes)

activity.

This increases production of Heme.
The biosynthesized Heme in

response to drug administration

Is used for production of

Cytochrome P450

Cyt P450 ?A Hemoprotein is

responsible for drug detoxification.

Lead Poisoning Affects

Heme Biosynthesis

and

Causes Anemia
Following Enzymes of Heme biosynthetic

pathway are inhibited by Lead ions

(Pb2+) in cases of Lead poisoning.

ALA Dehydratase / Porphobilinogen

Synthase

Ferrochelatase / Heme Synthase

Thus Lead

poisoning

Inhibit Heme

biosynthesis and

Leads to Anemia.
Porphyrins

Porphyrins are

chemically cyclic tetra

Pyrrole ring structures

with substituted

groups.
Porphyrins has

conjugated ring system

Alternate single and

double bonds (Methenyl

bonds).

Porphyrins are colored and

Fluorescent compounds with

Methenyl bridges/ Methyne

bonds in it.
The double bonds in Porphyrins

absorb visible light and appear

colored compounds.

The Conjugated bonds of

Porphyrins in UV light shows

fluorescence intense reddish

pink color.

Types Of Porphyrins

Based on arrangements of

Substituted groups on

Tetrapyrrole Rings
Types Of Porphyrins

Type I Porphyrins
Type III Porphyrins
Type I Porphyrins

has symmetric

arrangements of

substituted groups

in tetra pyrrole ring

structure.

Type III Porphyrins

have asymmetrical

distribution of the

substituted groups in

tetra pyrrole rings.
Type III Porphyrins

are most predominant

in biological system.

ProtoPorphyrin IX is of

type III Porphyrins

Fischer placed

ProtoPorphyrin in 9th

series of 15 possible isomers.
In Disorders of Porphyrias

Porphyrins are

abnormally elevated

In blood and excreted in

urine.
Porphyrias

Gk- Porphyria= Purple
Porphyrias

Disorders Due To

Defective

Heme Biosynthesis

Porphyrinurias
What Are Porphyrias?

The Porphyrias are

group of disorders

Associated to

defective Heme

biosynthesis.
Basic Cause Of Porphyrias

Metabolic block in

Heme biosynthesis

leads to Porphyrias.
Defect in any one

Enzyme of Heme

biosynthesis

Defect in Enzyme of Heme

Biosynthesis may be:

?Inherited

?Acquired
Most of the Porphyrias

are of Autosomal

Dominant inheritance .

Types and Classification

Of Porphyrias
6 Common Types Of Porphyrias

S.No Type Of Porphyrias

Enzyme Defect

1

Acute Intermittent

UPG I Synthase/

Porphyria (AIP)

PBG Deaminase

2

Erythropoietic

UPG III Cosynthase

Porphyria

3

Cutanea Tarda

UPG Decarboxylase

4

Coproporphyria

CPG Oxidase

5

Variegate Porphyria

PPG Oxidase

6

Protoporphyria

Ferrochelatase
Pneumonic To Remember

6 Type Of Porphyrias

All Elephants Can

Catch Ved Pathak.

Mitochondria

PORPHYRIAS

GLYCINE + SuccinylCoA

Agent Orange

ALA Synthase

d Aminolevulinic acid(ALA)

3p21/Xp11.21

ALA-Dehydratase

ALA Dehydratase

Deficiency

9q34

Porphobilinogen (PBG)

Porphyria
Acute intermittent

PBG Deaminase

Porphyria

Hydroxymethylbilane

11q23

Uroporphyrinogen III

Congenital Erythropoietic

Cosynthase

Porphyria

Uroporphyrinogen III

10q26

Uroporphyrinogen

Porphyria

Decarboxylase

Cutanea tarda

Coprophyrinogen III

1q34

Coproporphyrinogen

Herediatary

oxidase

coproporphyria

Protoporphyrinogen IX

9

Protoporphyrinogen

Variegate

Protoporphyrin IX

Oxidase

Porphyria

1q14

Ferrochelatase

Erythropoietic

Heme

18q21.3

protoporphyria
Different Basis For

Classification Of Various

Types Of Porphyrias
On Basis Of Cause

Primary/Congenital Porphyrias
Secondary/Acquired Porphyrias

On Basis Of Organ

Hepatic Porphyria
Erythropoietic Porphyria
Inherited Porphyria

Erythropoietic Porphyria - results from

excessive production of Porphyrins in the

bone marrow.

Hepatic Porphyria - results from

excessive production of Porphyrins in the

Liver.

Acquired Porphyria

Lead Intoxication - interferes with

Protoporphyrin synthesis

?Chronic Alcoholic Liver disease

On Basis Of Symptoms

Neurological Porphyrias/Acute Porphyrias

Acute Intermittent Porphyria

Coproporphyria

Variegate Porphyria

?Autonomic Dysfunction
?Abdominal pain
?Chest pain
?Confused Thoughts
?Depression and Psychosis
Photosensitive Porphyria/Chronic

Porphyria

q Erythropoietic Porphyria

q Cutanea Tarda

Porphyrins below skin exposed to

sunlight shows

vRedness
vSwelling
vItching
vBurning Sensation

Biochemical Alterations

And

Consequences Of Porphyrias
Enzyme Defect Of Heme

Pathway

Blocks the Reaction Of Heme Pathway

Accumulates

Porphyrins

Intermediates of

Heme Pathways

High levels of Porphyrin in blood

,Tissues and Urine (Porphyrias)
Porphyrinogens are oxidized

to Porphyrins.

Porphyrins are coloured

pigments.

Porphyrins are more stable

products.

Accumulate in blood, tissues

and get excreted out through

urine

Effects of Accumulated

Porphyrins and their Precursors
?Porphyria Sufferers Shows

qSevere Anemia
qNeurological Disturbances
qExtreme sensitivity to sunlight

Porphyria sufferers has no

normal Heme biosynthesis.

No normal Hb to transport

Oxygen to cells.

Hence suffer from Anemia.
Accumulation of

Porphyrinogens

in Brain and Skin can lead to:

qNeurological symptoms

q Photosensitivity

Enzyme block early in

Porphyrin pathway prior to

formation of Porphyrinogens.

Accumulates ALA and PBG

Exhibits abdominal pain and

neuropsychiatric symptoms.
Enzyme block occur later in

the Porphyrin pathway

Accumulates Porphyrinogens-

CPG/PPG beneath skin.

Causes Photosensitivity

when Porphyrins exposed to

light about 400 nm.

The Porphyrins have

no useful function

Act as highly reactive

oxidants, damaging to

tissues.
Porphyrins get excited at

400 nm.

Shows sharp absorption band

near 400nm (Soret band)

Porphyrins reacts with

molecular Oxygen

To form highly reactive

oxygen free radicals.

Injure Lysosomes and other

cellular organelles.
Damaged Lysosomes release

their degradative enzymes .

Causing variable degrees of

skin damage including

scarring.

Porphyrias are characterized by

?Extreme sensitivity to light

(exposure to sunlight causes

vesicular erythema),

? Excretes reddish-brown urine
? Possess reddish-brown teeth,

and ulcers
Destruction of cartilage and

bone

Causing the deformation of the

nose, ears, and fingers.

Mental aberrations, such as

hysteria, manic-depressive

psychosis, and delirium.

Porphyrias are cruelly referred to

as a Vampire's disease.

Thought to be a cause of the

madness of King George III.

Can be caused by lead poisoning:

The fall of the Roman Empire!




Porphyria
Diagnosis Of Porphyrias

Porphyrias are rare, but

frightening conditions:

Hard to diagnose and there is

no cure for Porphyrias.
Porphyrins Excreted

In Urine and Feces

Uroporphyrin excreted in

urine.

ProtoPorphyrin excreted in

feces.

Coproporphyrin excreted

either in urine /feces.

Porphyrins are Colored

and Fluorescent.
Porphyrias are diagnosed by analysis of

Porphyrins in the laboratory.

Spectrophotometry
Fluorometry

Woods lamp- Fluorescence in aqueous

layered viewed.

Based on quantitative Ehrlich's reagent

Watson Schwartz
Hoesch Test

Defective enzymes of

Porphyrias can be assayed

by various methods.

Enzyme Assay- HPLC.
Acute Intermittent

Porphyria

(AIP)

Acute Intermittent Porphyria

vThe most common type of

Porphyria.

vAutosomal Dominant trait.
vSymptoms more common in

females than males.
Acute Intermittent Porphyria

Type Of Porphyria-

Acute/Hepatic/
Neurological Porphyria

Enzyme Defect Of AIP

UPG I Synthase/PBG Deaminase

Biochemical Alteration In AIP

No conversion of PBG to

UPG III.

PBG and -ALA are

abnormally elevated in

blood, tissues and urine.

Manifestations Of AIP
ALA and PBG

accumulates in CNS.

This causes excitation of

visceral pain fibers

Leads to acute pain crises.

ALA blocks the

action of GABA.

Possess neurological

symptoms.
Symptoms of AIP are Acute

and Intermittent.

Symptoms does not occur

before puberty and shown at

Adolescence.

(Due to Steroidal Hormone action)

Person with AIP has

Affected GIT, Heart and Brain.

Abdominal colic pain

No abdominal tenderness

Vomiting, Constipation

Tachycardia, Hypertension

Neuro toxicity

Behavioral changes, seizures
AIP symptoms gets aggravated

during:

vInfections
vFasting
vIntake of drugs

Diagnostic Test For AIP

Watson and Schwartz Test

using Woods lamp ( UV lamp)

Detects urine Porphobilin.
Treatment of AIP

Infusion of Hematin
Represses ALA Synthase

synthesis.

Administration Of

Glucose.

High cellular Glucose

prevents induction of ALA

Synthase.


Use of Sunscreens that

filter out visible light,

Can be used in

management of

Photosensitive Porphyrias

The Madness of Inbreeding

King George III : Severe abdominal pain, mental confusion,

dark urine.
Enzyme Defects Responsible for The Porphyrias

Type

Enzyme Involved

Major Symptoms

Laboratory tests

Acute intermittent Uroporphyrinogen

Abdominal pain

urinary Porphobilinogen

Porphyria

synthase

Neuropsychiatric

Congenital

Uroporphyrinogen

Photosensitivity

urinary uroporphyrin

Erythropoietic

III Cosynthase

Porphyria

Porphobilinogen

Porphyria cutanea

UPG

Photosensitivity

urinary uroporphyrin

tarda

Decarboxylase

Porphobilinogen

Variegate Porphyria

PPG Oxidase

Photosensitivity

urinary uroporphyrin

Abdominal pain

Neuropsychiatric

fecal coproporphyrin
fecal Protoporphyrin

Erythropoietic

Ferrochelatase

Photosensitivity

fecal Protoporphyrin

protoporphyria

red cell Protoporphyrin
Globin Biosynthesis

The biosynthesis of Hemoglobin

Globin Polypeptide chains is

under genetic control.

Using Protein synthetic machinery.

Globin chain biosynthesis occurs

in cytosol on Polyribosomes.
Number And Types Of Globin

Chains Biosynthesized

In Human Life

6 different types of Globin chains are

associated with Normal Hb variants :

Globin

Globin

Globin

Globin

Globin

Globin
To biosynthesize these 6 types of

Globin chains

Human being normally carry

8 functional Globin genes


Arranged in two duplicate gene

clusters.

Globin Gene Clusters
The -like cluster located on the

short arm of chromosome 11

The -like cluster is located on

the short arm of chromosome 16

Globin polypeptide

chain biosynthesis

begins in the yolk sac

At about 3 weeks' of

gestation.
Ontogeny of Globin Synthesis

Ontogeny of Globin Synthesis

Time

Region

Type of Globin

Normal Hb Variant

Gene

Type of Hb

3 weeks of

Yolk Sac

&

(Hb Gower I ( )2

Gestation

5 weeks of

Yolk Sac

&

Hb Portland ( )2

Gestation

Hb G

ower II ( )

2

6-30 weeks of

Liver & spleen

& &

Hb F ( )2

Gestation

30 weeks of

Liver

Hb A2 ( )2

Gestation

At Birth

Bone Marrow

& &

Hb A ( )2

Hb F ( )2
Synthesis of Globin

Primary Structure Of

Globin

The primary structure of globin

refers to the amino acid

sequence of the various chain

types.

Numbering from the N-terminal

end identifies the position of

individual amino acids.
The specific number

,and sequence of amino

acids in Globin chains

Is very important for the

normal structure and

function of Hemoglobin.

Secondary Structure of Globin

The secondary structure of all Globin

chain types comprised of:

9 Non-helical sections joined by 8

Helical sections.

The Helical sections of Globin Chains

are identified by the letters A-H
While the non helical are

identified by a pair of letters

corresponding to the adjacent

helices

e.g. NA (N-terminal end to the

start of A helix), AB (joins the A

helix to the B helix) etc.

Tertiary Structure of Globin

The secondary structure is

further folded and bended on

its own to form 3 dimensional

subunit.

To form a Tertiary structure of

Globin.
Heme gets incorporated in

the Heme pocket formed

inside each Globin subunit.

Quaternary Structure Of Hb

It is native conformational

state of Hb/ Functional

form of Hb.


Four subunits of tertiary

structure ,non covalently

linked

To form quaternary level

of organization of Hb.

Assembly Of Hemoglobin
Although Heme and Globin

synthesis occur separately within

developing red cell precursors,


Their rates of synthesis are

carefully coordinated to ensure

optimal efficiency of Hb

assembly.

Synthesis of Hemoglobin


HEMOGLOBIN SYNTHESIS

Normal structure of Hb

includes the structure and

proportion of Globin chains


Which are necessary for the

normal function of

Hemoglobin
Decreased Concentration of

Hemoglobin in the red blood

cells

Caused due to any abnormality
Results into a clinical

situation called Anemia

Mechanisms Regulating

Hemoglobin Synthesis
Formation of Hemoglobin is

regulated by several

mechanisms:

The rate of Globin

biosynthesis is

directly related to the

rate of Heme

biosynthesis.
Heme stimulates

Globin biosynthesis by


Inactivating an

inhibitor of Globin

translation.

Negative feedback of

Heme.

High concentrations of

Heme

Prevent the mitochondrial

import of the first enzyme in

Heme synthesis, ALA

Synthase ( ALAS).
The Concentration Of Iron

An Iron Responsive Element-

binding protein (IRE-BP)

binds to mRNA

Iron Response Elements (IRE-

BP) affects the translation of the

mRNA for ALAS, Ferritin and

Transferrin receptors.

Low Iron Levels =
Low Heme Synthesis

Sufficient Iron Levels=

Adequate Heme Synthesis
Disorders Associated

To

Globin Chain Synthesis

Of Hemoglobin

Hemoglobinopathies

Caused By

Abnormal Hb Variants
What are Abnormal Hb variants ?

Abnormal Hb variants are

Hemoglobin's with:

Normal Heme and Altered Globin Chain

Abnormal Hb variants are

structurally abnormal

The Abnormal Hb variants may

be abnormal functionally
Approximately 400

abnormal Hb variants

are detected out.

But not all abnormal Hb

variants affect the

normal function of Hb.

Basic Cause For Formation Of

Abnormal Hb Variants
Mutations In Globin Genes

Altered /Mutated

Globin Genes leads to

form Abnormal Hb

variants.

Abnormal Hb Variants

Occurs due to Mutation

in Globin Genes.

Leads to defective

Globin chain synthesis.
Formation Of

Abnormal Hb Variants

Leads To

Hemoglobinopathies

What are Hemoglobinopathies?
Hemoglobinopathies are

genetic disorders

associated to


Structurally and

Functionally Abnormal

Hemoglobin variants.



Structurally and

Functionally Abnormal

Hb variants in human

body leads to

Hemoglobinopathies
Types Of

Abnormal Hemoglobin

Variants

and

Hemoglobinopathies

Broadly two types

of Hb abnormalities
Qualitative

Hb Abnormalities:

Mutations in Structural

Globin genes

e.g. HbS-Sickle cell anemia.

Quantitative

Hb Abnormalities:

Mutations in Regulatory

Globin Genes

e.g. a Thalassemia

b Thalassemia
Qualitative

Abnormal Hb variants:

Caused due to mutations in structural

Globin gene.

Has altered amino acid sequence in

Globin polypeptide chain.

Has altered Globin subunits in Hb

structure

But Has Normal Heme Structure.

Examples of Common

Abnormal Hb variants

And Corresponding

Hemoglobinopathy

Due to Structural Globin Gene

Mutations

OR

Symptomatic Abnormal

Hb Variants
Abnormal Hb

Globin

Amino acid Altered

Variants

Gene/Chain

In Globin Chain

Altered

Hb S

6 GLU VAL

Sickle cell Hb

Hb C

6 GLU LYS

Cooley's Hb

Hb D

121 GLU GLN

Punjab Hb

Hb E

26 GLU LYS

Hb M

87 HIS TYR

Hb has Fe+3

Proximal

If noted most common abnormal Hb

variants has:

Altered Globin genes and Globin

chains.

Substitution of Polar amino acid

"GLUTAMATE" with another amino

acid.
Non Symptomatic

Abnormal Hb Variants

Abnormal Hb Variants

?Hb P
?Hb Q
?Hb N
?Hb J
Consequences Of

Abnormal Hb Variants

Presence of

Symptomatic Abnormal Hb Variants

In RBCs

Alters normal structure and

function of Hb

Alters morphology of RBC's
Make RBC's fragile.
Causes Hemolysis, reduces Hb

content and affects its function.

Leads to Hemolytic Anemia
Increases Unconjugated serum

Bilirubin

Causes Hemolytic Jaundice
Possess Splenomegaly -

Increased function of Spleen to

remove defective Erythrocytes

from the blood circulation.

Detection Of

Abnormal Hb Variants
CBC and Blood Film Evaluation
Solubility Test
Electrophoresis
(Cellulose Acetate and Citrate Agar)
DNA Technology- PCR based

techniques:

DNA Finger Print Technique
Hybridization Technique

Hemoglobinopathy-Antenatal

Diagnosis

Check the Test partners of

heterozygous or affected

individuals

Antenatal diagnosis from DNA

is obtained by chorionic villus

sampling, or by Amniocentesis
Common

Abnormal Hb Variant

Causing

Hemoglobinopathy

Sickle Cel Hemoglobin (HbS)



Hb S is most

commonly occurring

abnormal Hb variant.
Hb S leads to Hemoglobinopathy

Sickle Cell Anemia

Biochemical Defect TO Form HbS
Formation of

Sickle Cell Hemoglobin (HbS)



Is a classic example of

point mutation

(Transversion)

Point mutation is a

substitution of

Nitrogen base in a

normal Globin gene

sequence.
Substitution Of Nitrogen

Base Which Forms Hb S

Altered Nitrogen base

sequence in Beta Globin gene.

There is substitution of

Nitrogen base Thymine to

Adenine (T to A).

On transcription of mRNA it

has altered codon, GAG to

GUG

Altered amino acid

substitution in the beta Globin

chain.

Glutamate substituted by

Valine
On translation at 6th position

of Globin chain polar amino

acid GLU is substituted by

non polar amino acid VAL .

This transforms HbA1 to HbS.

Deoxystate of HbS

Affects Solubility
HbS ? Is sickle cell Hb.
Altered HbS affects

the solubility of Hb

at Deoxystate in RBCs.

HbS at low oxygen tension /

deoxy state forms Deoxy HbS.

DeoxyHb S looses its polarity

/solubility

Deoxy HbS forms protrusion on

the globin chain.

Sticky patch appears on HbS at

deoxy state.


Each Hb S fits into this

complementary site of another

globin chain.

Many Hb S polymerizes inside

the RBC's

Forming a network of fibrous

polymers.


HbS aggregates into long, rigid

polymers are called Tactoids.

This makes HbS relatively

insoluble and non functional.

EM of Red

Blood Cell

showing

`Tactoids'
Tactoids stiffen and distort

the red blood cells.

RBC's changes morphology

and appear sickled/crescent

shaped.

Thus HbS Leads To

Sickling of Erythrocytes and

hemolysis.
Sickled Erythrocytes

may return to their

original shape when

oxygenated.

Effect of Sickled RBC's

And its

Associated Complications
HbS Causes

Reduction of RBC life span to

just 20 days.

Sickling of RBC's

Distortion and lysis of RBC's

Hemolysis

Sickling distorts and make

RBC's fragile.

After several sickling

episodes of RBC's

There is irreversible damage

to RBC membrane.
Thus Sickling Of RBC's

causes Sickle Cell

Anemia/Hemolytic

Anemia.

Sickled cells are phagocytized

by macrophages.

In the spleen, Liver or bone

marrow.
Sickling of RBC's

makes blood viscous

Lowers the rate of

blood circulation.

Sickled cells has

increased tendency to

adhere to blood vessels.


Rigid sickled cells unable to

squeeze out through small

capillaries

Sickled cells get trapped in

small capillaries and block

them.


Sickling produces localized

Anoxia/Tissue Hypoxia

Oxygen deprivation in the

tissues.

Lowers ATP production in

cells.

Anoxia in turn leads to

increased sickling process.
Sickling causes pain

and infarction (death)

of cells in tissues.

Sickling causes

Spleen Dysfunction

Making the spleen

non functional
Sickling Increases

susceptibility towards tissue

infection

Premature death of

individuals before 20 years

due to infections.

Factors Increasing

Sickling Of RBC's
Extent of RBC's sickling

is related to

Amount of Hb S

present in Erythrocytes.

Conditions Creating Hb S in Deoxy

state:

Decreased pO2

Increased pCO2

Decreased pH

Increased 2,3 BPG

Dehydration


Sickle Cel Anemia

Sickle Cell Anemia is a genetic disorder

due to presence of abnormal Hb variant

HbS

It is a Commonest type of a

Hemoglobinopathy

It is a type of Hemolytic Anemia due to

Sickling of Erythrocytes
Inheritance of Sickle Cell Anemia

Sickle Cell Disease is an

Autosomal recessive

disorder

Prevalence and Incidence

Prevalence of HbS

Tropical areas
Africa
South America

Incidence of HbS

1:5000 births.
Biochemical Defect

To Cause

Sickle Cell Anaemia

Sickle Cell Anemia is caused by

a point mutation in structural

beta Globin gene

Characterized by the presence

of abnormal HbS in

Erythrocytes.
Hb S in Deoxy state promotes

formation of hard, sticky,

sickled-shaped red blood

cells ? Sickling of RBCs.

Types of Sickle Cell Anemia




HbSS

HbSS is sickle cell disease
Homozygous state
Full blown disease
100% HbS concentration.
Both Globin genes of 2

chromosomes are

mutated.

Globin chain has

alteration at 6 Glu to Val

HbAS

HbAS is sickle cell trait
Heterozygous state.
50% HbA1 and 50 % HbS
Symptoms are mild and less

severe.

Fatality can be delayed.
Sickle cell trait offers

protection from

Malarial parasites-

Plasmodium falciparum.

Hb SC Disease

Another red cell sickling disease
Individual has mutant genes for

both Hb S and Hb C.

Has significant clinical variability
Less severe anemia
Less painful crises.


Effects And Complications

Of

Sickle Cell Anemia
Sickle Cell Anemia

Leads To

Hemolytic Anemia
Hemolytic Jaundice

Sickle Cell Anemia

Main Clinical Features

?Hemolysis
?Occlusion of blood

vessels by sickled red

cells



Hemolysis /Lysis of Sickled RBC
Low Oxygen transport to tissues

(Hemolytic Anemia)

Hemolytic Jaundice
Tissue Hypoxia
Tissue Infarction
Tissue Infection
Painful Crisis
Fatality in severe cases

Site of

Clinical Features Management

Sickling
Bone

Painful crises

Pain relief and hydration

Hydroxyurea

Lung

Acute chest

Transfusion regimen, pain

syndrome

relief and hydration

Brain

Stroke

Transfusion regimen.

Heart

Myocardial

Transfusion regimen, pain

infarction

relief and hydration

Spleen

Acute splenic

Transfusion, pain relief

sequestration

and hydration

Spleen

Hyposplenism

Pneumovax

Retina

Proliferative

Retinal surveil ance.

retinopathy

Laser
`Tactoids' form at low oxygen

tension

Stiff Sickled red cells occlude
small blood vessel
Tissue Hypoxia and Infarction
Tissue Infections
Symptoms are more severe
Fatality confirmed

Sudden death during

intensive training

Hematuria, Isosthenuria
(Renal Papillary Necrosis)
Infected RBC has incomplete life

cycle of parasites.

Sickled erythrocytes efficiently

phagocytized and destroyed.

Low K+ ion concentration in Hb S

is unfavorable for malarial

parasites to develop.

Diagnosis of Sickle Cell Anemia

Sickling Test-

Using Sodium Dithionite

reducing agent on blood

smear

Watch microscopically for

sickled RBC's.
Sickle Cel Anemia ? Blood film

Sickle

Cel s

Erythroblasts

Howel -

Jol y Body

Electrophoresis

Hb S is confirmed with

Cellulose Acetate

Electrophoresis



At Alkaline pH during

Electrophoresis.

Hb S is less negatively

charged than Hb A1.

Hb S moves in a

position between Hb

A1 and Hb A2.
Hb S migrates more slowly

towards anode than Hb A1.

Altered mobility of HbS is due

to absence of negatively

charged Glutamate residue.

Sickle Cell Anemia - Treatment

Adequate hydration
Analgesics to relive pain
Aggressive Antibiotic therapy

to arrest the infection.
Ingestion of 0.01 M of

Potassium or Sodium

Cyanate.

Prevents sickling of RBC's and

its complications.

Opiates and hydration

for painful crises

Pneumococcal

vaccination

Retinal surveillance
Hydroxyurea an antitumor drug
Used in therapy of Sickle cell

anemia.

Increases circulating levels of Hb F
Decreases Sickling
Decreases painful crises
Reduces mortality

Blood Transfusion

for serious

manifestations

Support with Folate,

Iron chelation.

Stem cell transplant
Thalassemias

Thalassemia's are

Hemoglobinopathies

Caused due to

defect/mutations in

Regulatory Globin

genes of Globin chain

synthesis.
Individual suffering from

Thalassemia's has

Structurally and

functionally unfavorable

abnormal Hb variants.



Thalassemias are

Autosomal recessive

blood disorders.
Thalassemias are

Characterized with Anemia

Thalassemias mostly

occur in regions of

Mediterranean sea.

Also termed as

Mediterranean Anemia.
Thalassemias are also

prevalent

In Arab,Americans, and

Asians

In populations where

Malaria is endemic

Causes Of Thalassemias
Thalassemias due to

Regulatory Gene mutations

is a quantitative abnormality

of Hemoglobin.

Mutations in Regulatory

Genes of Globin chain

synthesis.

Suppression of Globin

chain synthesis.
Reduced/Absent of one or

more of Globin polypeptide

chain synthesis of Hb.

Globin chains has normal amino

acid sequence.

Alpha Thalassemia ?

reduced alpha chain synthesis

Beta Thalassemia ?
reduced beta chain synthesis


+ Reduced production of chains

0 complete absence of chains

Compensatory

Globin chain synthesis

occurs in Thalassemias.


Unbalanced production of

Globin chains in

Thalassemias causes


Erythrocytes to be small,

hypochromic and sometimes

deformed.

Blood Picture Of Thalassemia
There occurs intracellular

accumulation of unmatched

Globin chains in the developing

Erythrocytes

Precipitation of the Proteins,

which leads to cell destruction in

the bone marrow.

Infective Erythropoiesis.
Mature functional RBC's

do not reach the

peripheral blood to carry

oxygen.
Types of Thalassemia

Thalassemia Minor

Heterozygous State
Asymptomatic

Thalassemia Major

Homozygous Type
Lethal at birth or in childhood.
Has many complications
Early and Continuous treatment

of Thalassemia allows survival to

young adulthood .
-Thalassemia

Suppression of Globin

genes

No/reduced globin chains

synthesis.

Compensatory more /

globin chains synthesized.

Abnormal Hb in -Thalassemia

vHbH ? 4
vHb Bart - 4

?Affect normal function of Hb
?Anemia
?Fetal death
Types Of Alpha Thalassemias

/ Normal

/-

Mild microcytosis

/- -

Mild microcytosis

-/-
-/- -

Hemoglobin H disease

- -/- -

Hemoglobin Barts Disease ? Hydrops Fetalis

Silent Carriers of

Thalassemia

Out of 4 Gene there is missing

of only 1 Gene.

Remaining 3 genes produces

sufficient chains for normal

Hb production.

1-2 % of Hb Bart in cord blood.
Thalassemia Trait

2 Genes are deleted
Shows mild microcytic

hypochromic anemia

Occasionally Hb H inclusions
Cord blood contains 2-10% of Hb Bart.

Hb H inclusions can be seen

in RBC's after supra vital

stain.

Cord blood contains 10-20%

Hb Bart.
Hb H Disease

Type of Thalassemia where 3

genes absent

Hb H present-Tetramer of chains.
Alters shape of RBC's
Shorten RBC life span.
Moderate hemolytic anemia.

Hb Bart Disease

Most clinically severe form
Thalassemia.
Where all 4 genes deleted
Total absence of chain

biosynthesis
Hb Bart major Hb found-

tetramer of chains.

Hydrops Fetalis
(Fetal Anemia causes Edema)

Hb Bart has extremely high

Oxygen affinity

Allows Oxygen transport but no

release at tissues.

Hypoxia
Still born infants/ die shortly

after birth.
Beta Thalassemia

Beta Thalassemia

Suppression of Globin gene.
Reduced/ no production of

beta globin chains.

Compensatory / Globin

chains biosynthesized.
Abnormal Hb in beta Thalassemia:

HbF (22)

HbA2(22)

Types Of Beta Thalassemia

Beta Thalassemia minor

? heterozygous (or trait)

Beta Thalassemia major

- homozygous
Beta Thalassemia Trait

No symptoms
Mild microcytic anemia

Beta Thalassemia Major

No beta chain produced

(no HbA)

Cooley's Anemia
Homozygous disease
Beta Thalassemia Major

Crippling disease of

childhood

Persistent HbF in age above 1

year

Reduced unloading of oxygen at

tissues.

Premature destruction of RBC's.
Severe hypochromic

microcytic anemia occurs

gradually in the first year of life

Bone Marrow expansion
Hypersplenism
Hepatosplenomegaly

MCV low
Severe Anemia
Reticulocytosis
Extreme Poikilocytosis

(Different Shape ) and

Anisocytosis (Different Size).
HPFH

Hereditary Persistence of

Fetal Hb (HPFH)

It is a Genetic heterogeneous

disorder

Caused due to deletions of

Genes in chromosome 11.

Exhibits total absence of

and Globin chain synthesis.

Hb F(2 2) is the

predominant Hb present.
HPFH patients are

asymptomatic


If they are sedentary

and slow workers.

Diagnosis Of Thalassemia's

PCR based methods.

Gene Mapping
For families that carry

a Thalassemia trait.

Genetic counseling

and genetic testing is

recommended

Treatment Of Thalassemias

Repeated / frequent

blood transfusions.

(After every 3- 4

months)
Due to repeated blood transfusions

in patients of Beta Thalassemia.

There exhibits Iron overload
Iron toxicity is noted since Iron is

one way element

Iron once entered in blood do not get

excreted out.

Iron gets accumulated in functional

tissues.

Tissue dysfunctions,

Growth failure and death

occurs before puberty due to

Iron toxicity.

However Iron chelation-

Reduces Iron toxicity.
Folate supplementation

for promotion of

Erythropoiesis

Azacytidine ? Drug used

with limited success

Bone marrow

transplantation

Stem cell transplant
Gene therapy


Abnormal Hb Variants

With Increased Oxygen Affinity

Hb Bart
Hb H
Hb Chesapeake
Hb Rainier


Catabolism/Breakdown Of Hb

OR

Formation and Fate Of Bilirubin

OR

How Bilirubin is Formed and

Excreted ?
Catabolism of Hemoglobin

begin after destruction of

RBC's.

RBC destruction is normally

the result of senescence

(Old/Aged).

Red cell destruction

usually occurs after a

mean life span of

120 days.
The old red blood

cells are removed

Extravascularly by

Macrophages of R.E

System.

Reticuloendothelial

system (RES), specially of

Spleen, Bone marrow and

Liver are involved in RBC

destruction.
Essentials for Erythrocyte

Membrane Integrity

Continuous supply of Glucose to

Erythrocytes

Continuous and uninterrupted

Glycolysis in RBCs

Continuous minimal ATP

production in RBCs

RBC aging is characterized by:
Decreased Glycolytic enzyme

activity

Which leads to decreased

Glycolysis and ATP production

Subsequent loss of deformability

and membrane integrity of RBCs.
Each day ~ 1% of the

RBCs are removed

and replaced.

Approximately 2- 3 million old

RBCs removed and same amt

of new red blood cells enter

the circulation per second.

This maintains constant RBC

count in blood.
80-90% of aged RBC destruction

is Extravascular

Occurs mainly in the

Macrophages, Mononuclear

phagocytic cells of Spleen

Spleen is grave yard for RBC's

Small amount occurring in the

RE system of Liver and Bone

marrow.

10-20 % of RBC destruction

is Intravascular, occurring

in the lumen of the blood

vessels.
Senescent /old RBC's

in RES are lysed to

release its contents-

Hemoglobin (Hb)

Hb is degraded to:

Globins Amino Acids

Recycled Metabolism
Heme Bilirubin

excreted out of the body.

Fe2+ Transported as

Transferrin

Iron stored as Ferritin

and reused in the next

Heme biosynthesis

Not only Hb but

other Hemoproteins

containing Heme

groups are degraded

by the same pathway.


Extravascular Erythrocyte

Destruction

Extravascular Erythrocyte Destruction

is a normal pathway.

80-90% Erythrocytes destructed in

this manner.

Outside the circulatory system.
Inside the phagocytic cells of Spleen,

Liver and Bone marrow.


Extravascular destruction of

RBCs
Intravascular Erythrocyte

Destruction

Erythrocytes destructed in

circulatory system.

Normally 10 -20% erythrocytes

destructed in this manner.

Hb is directly released into blood

stream.

Hb in blood is bound to

Haptoglobin.

Prevents renal excretion of

plasma Hb

Circulating Hb is removed

by Liver.


Intravascular destruction of RBCs


Globin chains are broken

down to amino acids


Which are reutilized for

general protein synthesis

in the body.
Heme part is

catabolized to

Bilirubin and

excreted out of the

body.

Microsomal enzyme Heme

Oxygenase of RE cells acts on

Heme

Requires NADPH+H+ as a coenzyme
Cleaves Methenyl bridge of

cyclic tetrapyrrole ring of Porphyrin

.

Forms Biliverdin ? A linear tetra

Pyrrole ring structure.
Iron is released in Ferrous is

oxidized to Ferric and

transported by Transferrin.

CO released is expired out.

Enzyme Biliverdin Reductase

Reduces Methenyl bridges of Biliverdin

to Methylene bridges.

Reduces Biliverdin (Green bile pigment )

to Bilirubin ( Yellow bile pigment).

NADPH+H+ is used as reducing equivalent

for this reduction reaction by Biliverdin

Reductase.
Albumin Transports Unconjugated

Bilirubin Through Blood.

1 gram of Hb yields 35 mg

of Bilirubin.

Daily 250-300 mg of

Bilirubin is produced by an

adult.
Bilirubin formed in RE cells of a

Spleen after Heme catabolism and

released in the blood circulation is:

Non polar
Insoluble
Free or Unconjugated Bilirubin

Albumin a polar moiety

helps in transporting this

non polar Bilirubin


Through aqueous phase of

blood circulation up to

Liver.
Albumin has two binding

sites for Bilirubin.

?High affinity binding site
?Low affinity binding site

Bilirubin first tightly binds

to high affinity binding site

of Albumin.

25 mg of Bilirubin tightly

binds with Albumin in 100 ml

blood.
Bilirubin bound

with Albumin

Prevents urinary

excretion of

Bilirubin in urine.

Drugs like Sulfonamides,

Penicillin, Salicylates

Compete with Bilirubin

for its binding to

Albumin.
Hypoalbuminemia

affects transport

and excretion of

Bilirubin.

Hypoalbuminemia may

lead to retention of

Unconjugated Bilirubin

in blood circulation.

May cause Bilirubin

Encephalopathy.
Facilitated transport

system helps in uptake of

Bilirubin in sinusoidal

surface of Hepatocytes.

Ligandin and Protein

Y of Hepatocytes

Prevent efflux of

Bilirubin back into

blood stream.
Conjugation Of Bilirubin

In Liver

Non polar Bilirubin

entered in Liver

Undergoes conjugation

reaction

Conjugating agent is two

molecules of UDP-

Glucuronic acid
In presence of conjugating

enzyme UDP Glucuronyl

Transferase

Forms Conjugated Bilirubin-

Bilirubin Diglucuronide.

Conjugated Bilirubin is:

Polar
Soluble form
Readily excretable form.
Conjugated Bilirubin is carried

through Bile via common bile duct

and excreted in the intestine


Secretion of Bilirubin into the

bile occurs by an active

transport mechanism.

Conjugated Bilirubin reaches terminal

ileum and large intestine.

Glucuronides are removed by specific

bacterial enzymes- Glucuronidase.

Bilirubin is reduced to colorless

compound Stercobilinogen in

intestine.
Small amount of Stercobilinogen is

reabsorbed and re excreted through

the Liver ? Enterohepatic

circulation.

Stercobilinogen is partly reabsorbed

enters in blood circulation is excreted

in urine as Urobilinogen and Urobilin.

Most of the

Stercobilinogen of

intestine is oxidized to

Stercobilin a orange

yellow colored

compound.
Stercobilin is a

major excretable

form of Bilirubin in

feces.

Futher Fate of Bilirubin

Bilirubin (Bil) is released from RES into the blood.
BUT! Bil is only poorly soluble in plasma, and therefore

during transport it is bound to albumin (Unconjugated

Bilirubin").



LIVER
In the hepatocytes, Bil is conjugated by 2 molecules of

glucuronic acid bilirubin diglucuronide (soluble in

water, ,,conjugated Bil")



BILE



INTESTINE
Bilirubin is reduced to urobilinogen and stercobilinogen


BLOOD

CELLS

Stercobilin

excreted in

Urobilin

Hemoglobin

feces

excreted in

urine

Globin

Urobilinogen

Heme

formed by bacteria

KIDNEY

O2

reabsorbed

Heme

INTESTINE

into blood

oxCyO

genase

Biliverdin IX

via bile duct to intestines

NADPH

Biliverdin

Bilirubin diglucuronide

reductase

(water-soluble)

NADP+

2 UDP-glucuronic acid

Bilirubin

Bilirubin

LIVER

(water-

(water-insoluble)

via blood

insoluble)

to the

liver

Catabolism of Hemoglobin


Normal Red Cell Breakdown

Hemoglobin

Heme

Globin

Iron

Protoporphyrin

Amino acids

CO

Bilirubin

Transferrin

Expired air

(free)

Liver

conjugation

Erythroblast

Bilirubin Glucuronides

Urobilin(ogen)

Stercobilin(ogen)

Urine

Feces


Red Blood Cell Turnover
What Is Bilirubin ?

Bilirubin is

Metabolic waste, an end product of Heme

catabolism

Formed in cells of RE system mainly in Spleen

Richly present in Bile

Yellow colored Bile pigment

Carried through Bile for its excretion through

feces.
Recently a research

study has depicted


Bilirubin has an

Antioxidant capacity.

Bilirubin is majorly

excreted out through

feces in the form of

Stercobilin.

(Yellow Orange pigment)
Types Of Bilirubin

Free/ Unconjugated /Indirect

Bilirubin

Non polar / insoluble form

Formed in RES- Spleen ,

Liver

Present in blood circulation

before entry into Liver cells.


Bilirubin Diglucuronide /Conjugated/

Direct Bilirubin

Polar/ Soluble form of

Bilirubin

Unconjugated Bilirubin is

transformed to conjugated

Bilirubin in Liver.

Bilirubin-diglucuronide = Conjugated Bilirubin

is soluble in water "Direct Bilirubin"
Conjugated Bilirubin is formed

after conjugation reaction,

Conjugated with Glucuronate
Conjugated Bilirubin is

readily mixed with bile and

excreted out through feces.

Normal Levels

Serum Bilirubin

S.

Type Of Bilirubin

Normal

No

Ranges

1

Total Bilirubin

0.2- 1 mg %

Direct+ Indirect

2 Unconjugated/Indirect 0.2-0.8 mg%

Bilirubin

3

Conjugated / Direct 0- 0.2 mg%

Bilirubin
In normal healthy

conditions there is

No conjugated

Bilirubin in

circulating blood.

When Is Conjugated Bilirubin

is present in blood?

During obstruction in common

bile duct (CBD)

(Obstructive Jaundice)
Forms of Bilirubin

Bilirubin - In Bile
Urobilin ? In Urine
Stercobilin - In Stool

Hyperbilirubinemia
Hyperbilirubinemia

Increased levels of serum

Total Bilirubin

Above 1 mg% is termed

as Hyperbilirubinemia.

Types Of Hyperbilirubinemia

Unconjugated

Hyperbilirubinemia

Conjugated

Hyperbilirubinemia

Biphasic Hyperbilirubinemia.
Causes of Hyperbilirubinemia

Over production Of Bilirubin

Less excretion of Bilirubin

Leads to retention of Bilirubin in

blood causing

Hyperbilirubinemia.
Causes of Hyperbilirubinemia

Hemolytic anemia

Hepatitis

Biliary duct stone

excess

hemolysis

Unconjugated Bilirubin

Unconjugated

conjugated Bilirubin

(in blood)

Bilirubin

(in blood)

conjugated Bilirubin

(in blood)

(released to bile duct)

conjugated Bilirubin

(in blood)

Causes of Hyperbilirubinemia

1.Conditions which

form excess Bilirubin

than the excreting

capacity of body.
Abnormal excessive intravascular

hemolysis

Overproduction of Bilirubin from Heme

catabolism.

More than the capacity of Liver to

conjugate and excrete

Leads to Unconjugated

Hyperbilirubinemia.

2.Conditions which

block excretion of

Bilirubin out of the

body.
Failure of diseased Liver to

conjugate and excrete

Bilirubin through bile.

In viral hepatitis
Leads to biphasic

Hyperbilirubinemia.

Obstruction to the flow of bile
Due to obstruction in bile duct.
Regurgitation of bile in systemic

blood circulation.

Leads to conjugated

Hyperbilirubinemia.
3.Congenital / Inherited

defects in uptake and

excretion of Bilirubin by

Liver
Leads to congenital

Hyperbilirubinemia.

4.Hypoalbuminemia / high

drug concentration in blood
Affects Bilirubin transportation

by Albumin

Retains Bilirubin in blood
Leads to Unconjugated

Hyperbilirubinemia.
Diagnosis of Hyperbilirubinemia

Diagnosis of Hyperbilirubinemia

Vanden Bergh's Reaction
Quantitative estimation of

serum:

Total Bilirubin
Unconjugated Bilirubin
Conjugated Bilirubin
Types of Vanden Bergh's Reaction

Direct Vanden Bergh's Reaction

Estimates serum Conjugated

Bilirubin (soluble form)

Serum Conjugated Bilirubin +

Diazo Reagent = Pink Azobilirubin
In the Direct Vanden Bergh

Reaction soluble form of a Bilirubin

is directly and immediately

reacted with the Diazo Reagent.

Conjugated Bilirubin requires no

solubilizing agent.

Hence Conjugated Bilirubin is

also termed as Direct Bilirubin

Indirect Vanden Bergh's Reaction

Estimates Serum Unconjugated

Bilirubin (Insoluble form)

Serum Unconjugated Bilirubin +

Methanol/Surfactant (solubilizing

agent)+ Diazo Reagent = Pink

Azobilirubin
In an Indirect Vanden Bergh

Reaction

Insoluble form of Unconjugated

Bilirubin is first solubilized with a

solubilizing agent (Methanol)

Then the solubilized form reacts with

Diazo reagent to form a pink

Azobilirubin complex

Unconjugated Bilirubin indirectly

reacts with the Diazo Reagent.

Since Unconjugated Bilirubin

requires solubilizing agent for

reaction with Diazo reagent.

Hence Unconjugated Bilirubin is

also termed as Indirect Bilirubin
Results and Significance

Of

Vanden Bergh's Reaction

S.No

Results of

Type of Hyperbilirubinemia/

Vanden Bergh

Jaundice

1

Direct Vanden Conjugated Hyperbilirubinemia

Bergh's Reaction

Obstructive Jaundice

Positive

2

Indirect Vanden

Unconjugated

Bergh's Reaction

Hyperbilirubinemia.

Positive

Hemolytic Jaundice

3

Both Direct and Biphasic Hyperbilirubinemia

Indirect Vanden

means

Bergh's Reaction

Both conjugated and

positive

Unconjugated Bilirubin

increased.

Hepatic Jaundice.
Significance Of

Vanden Bergh's Reaction

I. Quantitatively Estimates serum

Total, conjugated and

Unconjugated Bilirubin.

II. From the levels of serum total

Bilirubin- Diagnoses Jaundice

III. From the serum levels of Direct and

Indirect Bilirubin levels-

Differentiate- Type of Jaundice

IV. From the values of serum Bilirubin-

Indicate-Severity of Jaundice
Jaundice/Icterus Condition

Jaundice is a

pathological/Clinical

condition

Characterized by

Hyperbilirubinemia
In Jaundice Total

Serum Bilirubin levels

are more than 2.5 mg %.

High circulating

Bilirubin more than

2.5 mg% in blood

and tissues causes

jaundice.


Jaundice is a yellow

discoloration to:

Skin
Sclera of eyes
Nails
Mucous membrane
Causes and Types Of Jaundice

Basic Cause and Consequence

Of Jaundice

Defect in Heme catabolism

vOverproduction of Bilirubin

Defect in Bile excretion

?Less excretion of Bilirubin

Retains Bilirubin in blood and

body


Jaundice

Types Of Jaundice
Hemolytic/Pre Hepatic /Retention/

Acholuric Jaundice

Hepatic/Infectitious Jaundice

Obstructive/Post Hepatic/
Regurgitation /Choluric/Cholestatic

Jaundice.

Neonatal Physiological Jaundice

Neonatal Pathological Jaundice

Erythroblastosis Foetalis

Breast Feeding Jaundice

Breast Milk Jaundice
Sub Clinical Jaundice/ Latent

Jaundice-

Serum Bilirubin levels between 1-3

mg%

Clinical Jaundice-

Serum Bilirubin levels more than

3 mg%.

Hemolytic/ Prehepatic

/Retention Jaundice

Due to abnormal excessive

intravascular

hemolysis(premature) .

Characterized by Unconjugated

Hyperbilirubinemia.


Healthy Body RBC Destruction

Normal human body brings approx.

200 billion RBC lysis per day

v 160 billion is Extravascular Lysis
v 40 billion is Intravascular lysis

Intravascular destruction of RBCs
Conditions Causing Abnormal

Excessive Intravascular Hemolysis

Sickle Cell Anemia
Thalassemia's
Glucose-6-Phosphate Dehydrogenase

deficiency

Mismatched blood transfusion
Malaria
Burns
Spherocytosis
Drug interactions
Excessive abnormal intravascular

hemolysis

Increased Heme catabolism
Increased Unconjugated Bilirubin

in blood

Levels of the Unconjugated Bilirubin

more than normal capacity of Liver

to conjugate and excrete (3 gm/day).

Unconjugated Bilirubin is in

queue to enter Liver for

conjugation and excretion.

Thus there occurs retention of

Unconjugated Bilirubin in

blood.
Laboratory Findings

In

Hemolytic Jaundice.

Blood Investigations

Results of Vanden Bergh's

Reaction

Total Bilirubin Increased
Indirect Bilirubin Increased
Direct Bilirubin Normal
Urine Investigations

Ehrlich's Test for urine

Urobilinogen ? Positive

Urine Urobilin increased.
Hays sulfur test for Bile salts-

Negative

Fouchet's Test for Bilirubin-

Negative

Stool Appearance

Dark brown color feces in

Hemolytic Jaundice

Due to more Stercobilin

excreted out in feces.
Bone Marrow Examination

Hyperplasia of Bone marrow
Reticulocytosis in Peripheral

blood smear

Immature form of RBC's

increased in blood circulation.

Hepatic/ Infectitious Jaundice

Liver Parenchymal damage
Due to Viral Hepatitis
Biphasic

Hyperbilirubinemia.
Conditions Affecting Liver

Parenchymal Damage

Viral infection of Liver
( Viral Hepatitis)
Liver Cirrhosis- Alcoholism
Drug Effects:

vRifampicin -affect cellular

uptake of Bilirubin by Liver cells

vNovobiocin -affect conjugation

of Bilirubin in Liver.
Causes Of Biphasic

Hyperbilirubinemia in Hepatic

Jaundice

In Hepatitis damage and

inflammation of Liver parenchymal

cells.

This impairs and delays the

conjugation and excretion of Bilirubin

by Liver.

This retains Unconjugated Bilirubin

in blood.
Inflammation of Hepatocytes

and intra hepatic obstruction

in hepatitis

Leaks out conjugated

Bilirubin in blood.

Thus biphasic

Hyperbilirubinemia is noted.

In Hepatic Jaundice

there is a Marginal

increase

In both serum

Unconjugated and

Conjugated Bilirubin
Laboratory Findings

In

Hepatic Jaundice

Blood Investigations

Results of Vanden Bergh's Reaction

Total Bilirubin increased
Indirect Bilirubin increased (Marginal)
Direct Bilirubin increased (Marginal)

Serum SGPT/ALT and SGOT/AST

activity increased.
Urine Investigations

Ehrlich's Test for urine

Urobilinogen ? Normal

Urine Urobilin normal.
Hays sulfur test for Bile salts-

may be positive in severe cases.

Fouchet's Test for Bilirubin-may

be positive in severe cases.

Stool Appearance

Normal/ slightly pale colored

feces in Hepatic Jaundice.
Obstructive/ Post hepatic /

Regurgitative Jaundice

Due to obstruction in bile

flow to reach small intestine.

Characterized with

Conjugated

Hyperbilirubinemia.

Conditions Causing Obstructive

Jaundice
Obstruction of Bile duct due to

Gall stones in Common Bile

Duct(CBD).

Narrowing of bile duct due to

surgery.

Tumor of head of Pancreas.
Enlargement of lymph glands

near Gall bladder /bile duct.

Causes Of Conjugated

Hyperbilirubinemia In Obstructive

Jaundice.
Partial/ Complete Obstruction of

bile duct

Regurgitation of bile into systemic

circulation

Bile contains bile salts and bile

pigment- Bilirubin .

Hydrophilic Conjugated Bilirubin

is now in blood circulation.

In Obstructive Jaundice

Bile get excreted out through urine.
Bile salts and conjugated Bilirubin

present in urine.

Dark yellow colored urine noted

due to presence of Bilirubin occurs in

Obstructive Jaundice patients.
Laboratory Findings

In

Obstructive Jaundice.

Blood Investigations

Results of Vanden Bergh's

Reaction

Total Bilirubin increased
Indirect Bilirubin normal
Direct Bilirubin increased
Serum ALP activity increased.
Urine Investigations

Ehrlich's Test for urine

Urobilinogen ? Negative

Urine Urobilin decreased.
Hays sulfur test for Bile salts-

Significantly Positive

Fouchet's Test for Bilirubin-

Significantly Positive

Stool Appearance

Clay colored stools due to

absence of Stercobilin in feces of

Obstructive Jaundice.

Fatty stools due to excretion of

Lipids in feces

(Absence of Bile salts in intestine).
Neonatal

Physiological Jaundice

Neonatal - Physiological Jaundice

Noted in premature, low

birth weight infants.

After 1- 7 days of birth.
Causes

Immature hepatic system

in premature born infants

Poor uptake and

conjugation of

Unconjugated Bilirubin

from blood by Liver

Low levels of Conjugating Enzyme
UDP- Glucuronyl Transferase
Delays the conjugation and

excretion of Bilirubin
Consequences

Physiological Jaundice exhibits

Unconjugated

Hyperbilirubinemia

Serum Bilirubin may raise up to

20 mg % or more

Unconjugated Bilirubin is

hydrophobic

It can easily cross blood brain

barrier to enter central nervous

system.

Leading to Kernicterus

(Bilirubin Encephalopathy)
Bilirubin accumulates in

Neurons of Basal Ganglia,

Hippocampus Cerebellum
Medulla of Brain.
This causes necrosis of

nerve cells and brain

damage.

Symptoms

Fits/ Convulsions
Mental Retardation
Encephalitis
Spasticity
(Skeletal muscle tightness and

stiffness)
Treatment

Phototherapy at 450 nm.

Exchange transfusions of blood
(When Serum Bilirubin > 20 mg%)

During Phototherapy baby is

exposed to uv light

The Bilirubin is transformed to

solubilized form of Bilirubin


Which is readily excretable out

through urine.
?Blue / white UV light induces

isomerization

?Of non polar , insoluble form of

Unconjugated Bilirubin ?Z isomer

?To water soluble, polar form of

Bilirubin- E isomer.

The phototherapy

should be exposed to

child's skin

Breast feed the child

every 2 to 3 hours

(10 to 12 times a day).
Feeding prevents

dehydration and helps Bilirubin

to excrete out the body.

Phototherapy will continue until

the baby's serum Bilirubin

levels are low enough to be

safe.

Neonatal-Pathological Jaundice
Erythroblastosis Foetalis

Hemolytic condition in

neonates

Extrinsic Cause of Hemolysis

Exhibits Unconjugated Hyper

Bilirubinemia

Cause

Caused due to Rh incompatibility

When Rh ? ve mother

conceives Rh + ve baby.

This causes excessive

hemolysis of Erythrocytes at

the time of birth.
Neonatal

Pathological Jaundice

Non-Hemolytic Cause

Breast Feeding Failure

Jaundice

Jaundice caused in new

born infants.

Due to insufficient/lack

in breast feeding of milk
Infants born by cesarean

section are at higher risk

for this condition.

Due to no normal

Lactation phase.

Inadequate quantities of

milk reached to infants

body.

Decreases body fluids

,lowers bowel movements

,impair to remove

Bilirubin from an infants

body.
Proper feeding

prevents dehydration

Helps Bilirubin to

excrete out of the body

without its retention.

Condition of breast feeding

Jaundice can be ameliorated by

Frequent breast feeding

sessions of sufficient

duration(10-12 /day)

This stimulate adequate milk

production by mothers breast.
Extra fluids are helpful for babies who

have not been getting enough breast

milk.

Nursing more often (up to 12 times a day)
Will increase the baby's fluid levels

Can cause the Bilirubin level to drop.


Breast Milk Jaundice
Breast Milk Jaundice

Non Organic Cause

Breast milk Jaundice is more of a

biochemical problem

Probably caused by

factors/chemicals present in the

Breast milk.

These may block certain Proteins/

Enzymes in the infant Liver that

metabolize Bilirubin.

Breast Milk Jaundice tends to

run in families

It occurs equally often in Males

and Females

Affects 0.5 % to 2.4% of all

newborns.
Hypothesized Mechanisms

Increased levels of Epidermal

Growth Factor (EGF) in Breast

milk.

Increased Bilirubin uptake from

the gut (enterohepatic

circulation) in breast fed babies.

In a new born Liver, activity of

Glucuronyl Transferase is only at 0.1-

1% of adult levels

Conjugation of Bilirubin in infants is

reduced in comparison to adults.

Further inhibition of Bilirubin

conjugation by other agents leads

to increased levels of Bilirubin in the

blood.
Breast-milk of some women contains a

metabolite of Progesterone called

3-Alpha-20-beta Pregnanediol.

This metabolite inhibits the action of

the conjugating enzyme Uridine Di

Phospho (UDPGA) Glucuronyl

Transferase.

This brings poor conjugation and

subsequent excretion of Bilirubin.

An enzyme in breast milk called

Lipoprotein Lipase produces increased

concentration of non esterified free

fatty acids

That inhibit Hepatic UDP Glucuronyl

Transferase

Which again leads to decreased

conjugation and subsequent excretion of

Bilirubin.
Mothers taking drugs like Novobiocin,

Steroidal derivatives or Rifampicin

Drugs secreted through breast milk
Infant fed by this milk has drug inhibitory

effect on Bilirubin metabolism.

Delay in Bilirubin uptake and

conjugation in infants Liver.

Leads to Unconjugated Hyperbilirubinemia.

Management Of

Breast Milk Jaundice

Temporary stoppage of breast

milk feeding

Till the drug is cleared away

from the breast fed milk.
Congenital Hyperbilirubinemia

Congenital Hyperbilirubinemia

Genetic defects in Bilirubin

uptake, conjugation and

excretion of Bilirubin

Leads to elevated levels of

Bilirubin in infants blood and

body tissues.
Congenital Defect

Type Of

Disorders

Hyperbilirubinemia

Gilbert's

Defect in

Unconjugated

Syndrome

uptake

Hyperbilirubinemia

of

Bilirubin

by Liver

cells

Congenital Defect

Type Of

Disorders

Hyperbilirubinemia

Crigler

Complete

Unconjugated

Najjar

absence of

Hyperbilirubinemia

Syndrome-I

enzyme

UDP-

Glucuronyl

Transferase
Congenital Defect

Type Of

Disorders

Hyperbilirubinemia

Crigler

Partial

Unconjugated

Najjar

absence of

Hyperbilirubinemia

Syndrome-

enzyme

II

UDP-

Glucuronyl

Transferase

Congenital Defect

Type Of

Disorders

Hyperbilirubinemia

Dubin

Defect in

Conjugated

Johnson's Hepatic

Hyperbilirubinemia

Syndrome/ excretion

of

Deposition of Bilirubin

Black Liver conjugated

in Liver

Jaundice

Bilirubin
Congenital Defect

Type Of

Disorders

Hyperbilirubinemia

Rotors

Unknown

Conjugated

Syndrome Autosomal

Hyperbilirubinemia

Recessive

Inheritance Deposition of Bilirubin

in Liver

Conditions Causing

Unconjugated Hyperbilirubinemia
Conditions Causing

Unconjugated Hyperbilirubinemia

Hemolytic Jaundice
Hepatic Jaundice
Neonatal/ Physiological Jaundice
Breast Milk Jaundice
Gilbert's Syndrome
Crigler Najjar Syndrome
Hypoalbuminemia
High Drug Concentration lowering Albumin

activity.

Conditions Causing Conjugated

Hyperbilirubinemia

Obstructive Jaundice
Hepatic Jaundice
Dubin Johnson's Syndrome
Rotors Syndrome
Differential Diagnosis Of Jaundice

Parameters

Hemolytic

Hepatic

Obstructive

Jaundice

Jaundice

Jaundice

Serum Bilirubin Indirect Bilirubin/

Biphasic

Direct Bilirubin/

Unconjugated

Both Direct

Conjugated

Bilirubin

and

Bilirubin

increased

Indirect

Increased

Bilirubin

Increased

Urine

Increased

Normal or

Absent or

Urobilinogen

Decreased

Decreased

Urine Bilirubin

Absent

May present in

Present in high

and Bile Salts

small amounts

amounts

Fecal Stercobilin

Increased

Normal or

In traces or

Color Of Feces

Dark color feces

Decreased

absent

Clay colored

feces

Serum ALT

Normal

Significantly

May be slightly

activity

Increased

increased

Serum ALP

Normal

May slightly

Significantly

activity

increased

Increased
Questions

SHORT NOTES

Outline of Heme Biosynthesis
Porphyrias: Types , Causes

and Consequences

Acute Intermittent Porphyria

(AIP).
Catabolism of Hb /Fate &

Formation of Bilirubin.

Jaundice & its types.
Vanden-Berg's Reaction

and its significance.

Name the enzymes of Heme

Biosynthesis inhibited during

lead poisoning. What is its

consequence?

Comparison between abnormal

Hb derivatives & abnormal Hb

variants.
Abnormal Hb variants
Hemoglobinopathies
Sickle Cell Anemia
Thalassemias
Thalassemia
Thalassemia

Name the Bile pigments &

give the significance of their

presence in blood & urine.

Laboratory reports of

Obstructive Jaundice.

When and Why Bilirubin

comes in Urine Explain?
Neonatal Jaundice
Breast Milk and Breast

Feeding Jaundice

Congenital

Hyperbilirubinemias

Write the defect & type of

Hyperbilirubinemia in

Crigler's Najjar Syndrome-I
Crigler's Najjar Syndrome-II
Gilbert's Disease.
Dubin Johnson Syndrome.


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This post was last modified on 05 April 2022