Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st year (First Year) Biochemistry ppt lectures Topic 50 Hemoglobin Metabolism Notes. - biochemistry notes pdf, biochemistry mbbs 1st year notes pdf, biochemistry mbbs notes pdf, biochemistry lecture notes, paramedical biochemistry notes, medical biochemistry pdf, biochemistry lecture notes 2022 ppt, biochemistry pdf.
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.
THANK YOU
This post was last modified on 05 April 2022