•Haemoglobin

Haemoglobin
 It is a red, oxygen carrying pigment.

 It is found within the cytoplasm of RBC of

vertebrates.

 It consists of protein globin

united with pigment haem.
 Normal values of Hb

• At birth 23 gm%

• After one year: 12 gm%

• Adults-
 Male (14 - 18 gm%)

 Female (12 - 15.5 gm%)

 Haemoglobin (Hb)
Structure of Haemoglobin:-
 Spherical molecule

 Molecular weight of 68,000.

 Each Hb molecule contains -

Globin + Haem

 Globin made up of 4 polypeptide chains

(2 alpha and 2 beta chains in adult Hb)
 Haemoglobin

• Haem pigment (Fe protoporphyrin IX).

• The porphyrin nucleus made up of

• 4 pyrrole rings joined by
• 4 methine bridges.
Structure of Hb molecule
Methine
bridges
Pyrrole ring

I II

Fe2+
(Ferrous)

III IV
 Haemoglobin
• Each haem has 1 Fe2+ combined loosely and
reversibly with 1 molecule of O2 (oxygenation
because iron in haem stays in Fe2+ state).

Thus one molecule of Hb has

• 4 Polypeptide chains

• 4 haem containing 4 Fe2+ atoms

• and can carry 4 molecules of oxygen /

8 atoms of oxygen.
Spherical molecule, Molecular weight of 68,000.
 Synthesis of Hb
It starts from stage of intermediate Normoblast
during erythropoiesis & continues till stage of
reticulocyte.
• (ALA – Aminolevulinic acid)
• 2 succinyl CoA + 2 glycine ALA pyrrole
synthetase
• 4 pyrrole protoporphyrin IX

• protoporphyrin IX + Fe Haem

• Haem + PP Hb chain
• 2α + 2β HbA
 Synthesis of Hb
• Synthesis of Hb requires provision of nutrients
• Proteins - for globin formation

• Vitamins
Vit-C, B12 & FA help in synthesis of nucleic acid.

• Minerals
Fe for haem formation
Cu, Ca2+ for Fe absorption (Vit C )
Co - Cobalt for B12 formation
 O2-Hb dissociation curve
• Shifting affinity of Hb for oxygen results in Sigmoid shape
of O2-Hb dissociation curve. (T to R interconversion)

• Shift to right
• increase in Carbon dioxide
• increase in H+ ions
• increase in Temperature
• increase in 2,3 DPG
• (more oxygen is released by blood to tissue)
O2 - Hb dissociation curve
 Derivatives of Haemoglobin
1. Oxy-Hb :- oxygen reacts with Hb

2. Carbamino-Hb :- CO2 reacts with Hb

3. Reduced-Hb :- Hb from which oxygen has been

removed.
Cyanosis : >5gm of reduced Hb

Causes of cyanosis – hypoxia (hypoxic & stagnant

hypoxia), polycythemia.
 Reduced

Cyanosis is bluish discoloration of mucus membrane & /or

skin due to presence of >5gm of reduced Hb
(Sites: tongue, lips, tip of nose, ear lobes, nail beds )
 Derivatives of Haemoglobin
4. Carboxy-Hb :- CO reacts with Hb

5. Meth-Hb :- when reduced or oxygenated Hb is

exposed to Oxidising agents
Fe2+ Fe3+
and the resulting compound is meth-Hb

(cannot combine reversibly with oxygen)

 Oxygen carrying capacity-

• 1 gm of Hb carries 1.34 ml of oxygen.

• Therefore Hb concentration is index of oxygen

carrying capacity of blood.

• Males - 21 ml /dl (15.5 gm/dl)

• Females - 19 ml /dl (14gm/dl)

 Functions of Hb

1. Transport of oxygen from lungs to tissues.

2. Transport of carbon dioxide from tissues to lungs.

3. Acts as an acid base buffer.

4. Role in regulation of blood flow & blood

pressure.

Has an additional NO binding site on beta chain.

Therefore binds with NO in lungs and releases it
in tissue. (vasodilation)
 Methods of Hb estimation
Direct Methods

1. Van Slykes

O2 content of Hb estimated & from that amount

of Hb

2. Estimation of Fe content

1 gm of Hb contains 3.3mg of Fe So according

to Fe content Hb conc. calculated
 Direct Methods
3. Spectrophotometric method - based on
measurement of absorption of light

4. Talliquist method - drop of blood placed on

filter paper & matched with standard. (mass
screening)

5. Gower's method -
Same as Haldane. Standard solution of
picrocarmine gelatin.
 Methods of Hb estimation
Indirect Methods
1. Haldanes method-
Hb converted to Carboxy Hb, color matched with
standard.

2. Wu´s method
(alkaline haematin method)
 Indirect Methods
3. Oxy Hb method

4. Sahli´s Acid Haematin method

5. Determination of specific gravity of blood by

CuSO4 method.

6. Cyanmethemoglobin method best method for Hb

estimation.
 Fate of Hb
Old RBC are destroyed in tissue macrophage system

Enters
AA Hb
Pool

Globin Haem Reused for

synthesis

Remaining part Fe2+

Haem oxygenase Combines with
Biliverdin Appoferritin
Reduced by biliverdin Ferritin
reductase
(stored in liver)
Bilirubin
(excreted in bile)
 Disadvantages of free Hb
1. Increased viscosity

2. Increased Blood pressure

3. Increased osmotic pressure, interferes with

fluid exchange at tissue level

4. Haemoglobinuria & kidney damage

 Varieties of Hb
In all varieties of Hb, haem moiety is same

 Physical and chemical difference being due

to variation in composition of globin part.
 Adult Haemoglobin (α2β2 )

• Appears in foetus after 5 months of Intrauterine

life, when bone marrow begins to function.

• In normal adults about 97.5 % of Hb is HbA1

(α2β2 )(141AA and 146AA respectively).

• About 2.5% of total Hb is HbA2

(α₂δ₂, 141AA and 146AA respectively).
 Foetal Haemoglobin (α2γ2 )
• Structure same as adult Hb except β-chains are
replaced by γ-chains
(α2γ2 141AA and 146AA respectively, 37 AA
differ) .

1. More resistant to action of alkalis

2. Greater affinity for oxygen because of poor

binding with 2, 3DPG, therefore carries greater
volume of oxygen at low pressure.
 Foetal Haemoglobin (α2γ2 )

3. Hb F (fetal Hb)
70% saturated at 20mm of Hg of Po2,
Hb A only 30-35%

4. Life span 80 days

5. At birth Hb F predominates, disappears

2-3 months after birth.
O2 - Hb dissociation curve
 Glycosylated Hb
• Glucose attaches itself to terminal valine in each
β-chain & such compounds are known as
glycosylated Hb (HbA1C).

• Normal value <6%.

• It is an index of control of diabetes.

• To identify 3 months average plasma glucose

concentration.
 Pathological varieties
2 major types of inherited disorders of
Hb

1. Thalassaemia (polypeptide chains are

normal )

2. Haemoglobinopathy (Polypeptide chains

abnormal )

Haem synthesis is normal in both

 1. Thalassaemia
• Inherited disorder of Hb – Inheritance autosomal
recessive.

• It is of 2 types α & β thalassaemia ,

Major & Minor thalassaemia
• Haem synthesis is normal

• Polypeptide chains are normal

• But produced in decreased amount or absent

• Cause : defect in globin genes.

 Thalassaemia minor
• Only one of your parents is a carrier,

• Thalassaemia Minor , may not have symptoms, but

can be carrier of disease.

• Most common in ASIA, Middle East, Africa,

Mediterranean countries such as Greece & Turkey.
 a) Major β thalassaemia
• Less common. Also k/a
Cooley’s Anemia/Mediterranean Anemia

• Homozygous transmission : identical, abnormal

genes inherited from both parents.

• Total absence of β chain synthesis

• Fetal Hb (Hb F - (α2γ2 ) markedly increased.

• Requires regular blood transfusions.

 a) Major β thalassaemia
• Free α chains w/c form inclusions, precipitate in
RBC & are haemolysed.

• Have haemolytic anaemia & ineffective

erythropoiesis

• Free Haem released is converted to bilirubin.

Over time Increased bilirubin leads to jaundice
 Major β thalassaemia
Symptoms : develop in first 3-6 months of life
• Moderate to severe anemia, pallor, dyspnoea
• Frequent infections,

• Jaundice, hemochromatosis
• Hepatospleenomegaly

• Growth retardation
• Skeletal deformities
• & cardiovascular deformities
• Life span short 17yrs
 Major β thalassaemia
• Chipmunk facies – frontal bossing,
maxillary marrow hyperplasia

• Long bone fractures,

vertebral fractures due to cortical
invasion by erythroid elements

• Microcytic hypochromic anemia

• Hb, MCV, MCH decreased
• RBC’s show anisocytosis, poikilocytosis,
nucleated RBC’s, target cells
 Blood test

Hb

MCV All
decreased
MCH
 Blood test
• Hb, MCV,MCH - All decreased

• Blood smear – Microcytic , hypochromic

RBC’s, anisocytosis, poikilocytosis, target cells

• Serum Iron
• Serum Ferritin All increased
• Serum Transferritin
 Thalassaemia
Major β Thalassaemia Minor β Thalassaemia
1. Less common more common
2. Homozygous transmission Heterozygous transmission

3 Moderate to severe Mild anaemia

anaemia
4. Total absence of β- chain Partial β- chain synthesis
synthesis
5. Hb-F level markedly Hb-F level normal or
increased slightly increased
6. Life span shorter Life span longer
 Management
• Suppress excess erythropoiesis &
prevent Fe overload (chelation therapy)
• Blood transfusion – when Hb < 7gm%
• Bone marrow transplantation – only definitive
cure (limitation identical sibling donor)

• Prevention – can be diagnosed antenatally by

genetic analysis. Amniocentesis/ chorionic villus
sampling (DNA sample)

• Thalassemia minor – excellent prognosis

 2. Haemoglobinopathy
• Abnormal polypeptide chains are produced.

Sickle Cell Anemia (Hb S)

• Inheritence Mendelian Recessive.

• Defect-
• in each β chain of HbA at position 6 one
glutamic acid is replaced by valine.
 Sickle Cell Anemia (Hb S)
• When HbS is reduced it becomes much less soluble
than HbA and
• Hb precipitates into crystals within RBC‘s & causes
damage of cell membrane with increase in
Osmotic Fragility.

• RBCs become sickle shaped which decreases blood

flow to tissue due to increased viscosity.

• Patient dies of severe anemia & secondary

infection.
Sickle Cell Anemia (Hb S)
 Miscellaneous Hb

• Hb C, E, I, J, M. All these abnormal Hb

leads to hemolytic anemias. (γ ζ ε)

• Fetal Hb - Hb F (α2γ2 )
Hb Bart γ4 minor Hb present in fetal life.
 Miscellaneous Hb
Embryonic Hb

1. Gower Hb 1 - two ζ ( Zeta ) two ε (epsilon) chains.

2. Gower Hb 2 - two α & two ε chains.

3. Hb Portland - two ζ & two γ chains.