Hemoglobin function &

Thalassemia

Dr. Vinayak Bhat MBBS, MD, DGM

Assistant professor
Dept. of Biochemistry
IMS & SUM Hospital, BBSR
 Normal Hemoglobin
• Normal level of Hemoglobin (Hb) in blood in
males is 12-16 g/dl and in females, 11-15 g/dl.
• Hb is globular in shape.
• The adult Hb (HbA) has 2 alpha chains and 2
beta chains.
• Molecular weight of HbA is 67,000 Daltons.
• Hb F (Fetal Hb) is made up of 2 alpha and 2
gamma chains.
• Hb A2 has 2 alpha and 2 delta chains.
• Normal adult blood contains 97% HbA, about 2%
HbA2 and about 1% HbF.
• Each alpha chain has 141 amino acids.
• The beta, gamma and delta chains have 146
amino acids.
 Fetal Hemoglobin (HbF)
• HbF has 2 alpha chains and 2 gamma chains.
Gamma
chain has 146 amino acids.
• The differences in physicochemical properties
when compared with HbA are:
o a. Increased solubility of deoxy HbF
o b. Slower electrophoretic mobility for HbF
o c. Increased resistance of HbF to alkali denaturation
o d. HbF has decreased interaction with 2,3-BPG.
• The synthesis of HbF starts by 7th week of
gestation; it becomes the predominant Hb by 28th
week.
• At birth, 80% of Hb is HbF.
• During the first 6 months of life, it decreases to
about 5% of total.
• HbF level may remain elevated in children with
anaemia and beta-thalassemia as a compensatory
measure.
 Hemoglobin A2
• It is a normal adult Hb; it is about 2% of total Hb.
• It has 2 alpha chains and 2 delta chains.
• The delta chain has sequence homology with beta
chain.
• In beta thalassemia, as a compensation, HbA2 is
increased.
• The iso-electric pH of HbA2 is 7.4, while HbA has
the pI value of 6.85.
• HbA2 is slower moving on electrophoresis.
Hemoglobin molecule is made up of two alpha and two beta chains.
• The alpha and beta subunits are connected by
relatively weak non-covalent bonds like van der
Waals forces, hydrogen bonds and electrostatic
forces.
• There are 4 heme residues per Hb molecule, one
for each subunit in Hb.
• The 4 heme groups account for about 4% of the
whole mass of Hb.
• The heme is located in a hydrophobic cleft of
globin chain.
 N
H
pyrrole

pyrrole rings
hem
e
• The iron atom of heme occupies the central
position of the porphyrin ring.
• The reduced state is called ferrous (Fe++) and the
oxidized state is ferric (Fe+++).
• The ferrous iron has 6 valencies and ferric has 5
valencies.
• In hemoglobin, iron remains in the ferrous state.
 Iron Carries Oxygen

• The iron is linked to the pyrrole nitrogen by 4 co-

ordinate valency bonds and a fifth one to the
imidazole nitrogen of the proximal histidine.
• In oxy-Hb, the 6th valency of iron binds the O2.
• The oxygen atom directly binds to Fe, and forms
a hydrogen bond with an imidazole nitrogen of
the distal histidine.
 Transport of Oxygen by Hemoglobin
• Hemoglobin is an ideal oxygen carrying pigment
because –
o a. It can transport large quantities of oxygen
o b. It has great solubility
o c. It can take up and release oxygen at appropriate
partial pressures
o d. It is a powerful buffer.
 Subunit Interaction in Hemoglobin

Pink rectangles represent hemoglobin (Hb) monomers. Black

connection lines represent salt bridges. As oxygen is added, salt
bridges are successively broken and finally 2,3-bisphosphoglycerate
(BPG) is expelled. Simultaneously the T (taught) confirmation of
deoxy-Hb is changed into R (relaxed) confirmation of oxy-Hb. Blue
circle represents 2,3- BPG.
Oxygen dissociation curve (ODC). A = Theoretical curve as per mass
action. B = Sigmoid curve, due to heme-heme interaction (Hill effect). C =
Further shift to right due to carbon dioxide (Bohr effect) and PG. This
curve represents the pattern under normal conditions. D = Further shift to
right when temperature is increased to 42oC.
In Tissues, Oxy Hb Releases O2
Affinity of Oxygen with Oxygen

Affinity Affinity Affinity Affinity

1 time 2 times 4 times 18 times

Hb + O2 → HbO2 HbO2 + O2 → HbO4 + O2 → HbO6 + O2 →

HbO4 HbO6 HbO8
Chloride Shift in Tissues
Chloride Shift, in Lungs
Embryonal, Fetal and Adult Hemoglobins

Name of hemoglobin Globin chains present Period of life

Hb Gower I 2 Zeta, 2 epsilon Embryonal
Hb Gower2 2 alpha, 2 epsilon hemoglobins.
Hb Portland 2 gamma, 2 delta Upto the end of first
trimester of pregnancy

Fetal Hb or Hb F 2 alpha 2 gamma Second trimester to

early post natal life,
upto 2 years

Hb A (adult Hb) 2 alpha ,2 Beta Immediately after birth

to end of life span

Hb A2 (adult) 2 alpha ,2 delta Adult life

 Clinical Applications
• In all hypoxic states, the O2 affinity is decreased
with a shift in ODC to the right and an increase in
2,3-BPG inside RBC.
• The adaptation to the high altitude where pO2
is low, includes increased pulmonary ventilation,
polycythemia and increase in 2,3-BPG level with
a shift in ODC to right.
2. In anemia, where the total concentration of Hb is
reduced, increased oxygen unloading alone will
ensure proper oxygenation of tissues.
3. In many cases 2,3-BPG level varies inversely as the
hemoglobin concentration.
4. In chronic pulmonary diseases and cyanotic
cardiac diseases also the 2,3-BPG level is increased
ensuring maximum unloading of O2 to tissues.
 Hemoglobin Derivatives
• Hemoglobin derivatives are formed by the
combination of different ligands with the heme
part, or change in the oxidation state of iron.
• Oxy-Hb is dark red, deoxy-Hb is purple, met-Hb is
dark brown, COHb is cherry red and sulph-Hb is
green in color.
• Normally concentration of deoxy-Hb is less than
5% of the total Hb.
• If the level increases cyanosis occurs.
 Carboxy-Hemoglobin
(Carbon Monoxy Hb) (CO-Hb)

• Hemoglobin binds with carbon monoxide (CO) to

form carboxy-Hb.
• The affinity of CO to Hb is 200 times more than
that of oxygen.
• It is then unsuitable for oxygen transport.
• When one molecule of CO binds to one
monomer of the hemoglobin molecule, it
increases the affinity of others to O2; so that the
O2 bound to these monomers are not released.
• This would further decrease the availability of
oxygen to the tissues.
 Carbon Monoxide Poisoning

• CO is a colorless, odorless, tasteless gas

generated by incomplete combustion.
• CO poisoning is a major occupational hazard for
workers in mines.
• Breathing the automobile exhaust in closed
space is the commonest cause for CO poisoning.
• Clinical symptoms manifest when carboxy-Hb
levels exceed 20%.
• Symptoms are breathlessness, headache,
nausea, vomiting, and pain in chest.
• At 40-60% saturation, death can result.
• Administration of O2 is the treatment.
• In severe cases, oxygen under high pressure
(hyperbaric oxygen) is helpful.
 Met-hemoglobin (Met-Hb)
• When the ferrous (Fe++) iron is oxidized to ferric
(Fe+++) state, met-Hb is formed.
• Small quantities of met-Hb formed in the RBCs
are readily reduced back to the ferrous state by
met-Hb reductase enzyme systems.
• About 75% of the reducing activity is due to
enzyme system using NADH and cytochrome b5.
 Methemoglobin Reductase System

Another 20% of the reducing activity is due to NADPH dependent

system.
Glutathione dependent Met-Hb-reductase accounts for the rest
5% activity.
 Met-hemoglobinemias
• Normal blood has only less than 1% of met-
hemoglobin.
• It has markedly decreased capacity for oxygen
binding and transport.
• An increase in methemoglobin in blood, (met-
hemoglobinemia) is manifested as cyanosis.
• Causes may be congenital or acquired.
Congenital Met-hemoglobinemia
• Cytochrome b5 reductase deficiency is
characterized bycyanosis from birth.
• 10-15% of hemoglobin may exist as met-
hemoglobin.
• Oral administration of methylene blue, 100-300
mg/day or ascorbic acid 200-500 mg/day
decreases met-Hb level to 5-10% and reverses
the cyanosis.
 Acquired or Toxic Met-hemoglobinemia
• Met-hemoglobinemia may develop by intake of
water containing nitrates or due to absorption of
aniline dyes.
• Aniline dye workers have been known to
develop met-hemoglobinemia.
• Drugs which produce met-hemoglobinemia are:
acetaminophen, phenacetin, sulphanilamide,
amyl nitrite, and sodium nitroprusside.
 Glucose-6-phosphate Dehydrogenase
Deficiency

• In persons with this enzyme deficiency, the

condition may be manifested even with small
doses of drugs.
• In such persons, NADPH is not available in the
RBC.
• In such individuals, disease is manifested easily.
• In such patients, intravenous leuko methylene
blue 2 mg/kg is effective, which will substitute for
the NADPH.
 Sulf-hemoglobinemia
• When hydrogen sulfide acts on oxy-hemoglobin,
sulf-hemoglobin is produced.
• It can occur in people taking drugs like
sulphonamides, phenacetin, acetanilide,
dapsone, etc.
• It cannot be converted back to oxy-hemoglobin.
• It is seen as basophilic stippling of RBC,
throughout its lifespan.
Hemoglobinopathies and Thalassemias
Abnormalities in the primary sequence of globin chains
lead to hemoglobinopathies, e.g. hemoglobin S (HbS).

Abnormalities in the rate of synthesis would result in

thalassemias. In other words, normal globin chains in
abnormal concentrations result in thalassemias, e.g. beta
thalassemia.
 Hemoglobinopathies
• Hundreds of hemoglobin variants
• Alpha chain variants or beta chain variants.
• Gamma and delta chain variants - Rare
• The hemoglobin variants may be classified into 5
major types, based on their clinical
manifestations.
1. Sickle syndromes
o A. Sickle-cell trait (AS)
o B. Sickle-cell disease with SS, SC, SD, SO varieties
and S beta thalassemia
2. Unstable hemoglobins
3. Hemoglobins with abnormal O2 affinity
4. Structural variations leading to thalassemia
5. Non symptomatic Hb variants – HbP, Q, N, J
 CASE
• One year old child was brought to the hospital by her
father with complaints of failure to thrive for 5 – 6
months and jaundice for 1 month. Child also had
severe pain and swelling in hands and feet. On asking
her mother, she told history of sickle cell in their family.
On examination icterus was present. Peripheral smear
showed sickle-shaped RBCs. Lab reports showed,
• Hb – 5.5 g% RBC count- 2.5 million/mm3, total bilirubin
– 5 mg/dl, Indirect bilirubin – 3.7 mg/dl urine routine –
within normal limits.
Hemoglobin S (HbS) (Sickle Cell Hemoglobin)
• HbS constitutes the most common variety
worldwide.
• Hemoglobin with abnormal electrophoretic
mobility is responsible for the sickling disease
(Pauling, 1949).
 Sickle Cell Disease
• The glutamic acid in the 6th position of beta
chain ofHbA is changed to valine in HbS.
• Leads to polymerization of hemoglobin
molecules inside RBCs.
• Causes a distortion of cell into sickle shape.
• The substitution of hydrophilic glutamic acid by
hydrophobic valine causes a localized stickiness
on the surface of the molecule.
• The deoxygenated HbS depicted with a
protrusion on one side and a cavity on the other
side, so that many molecules can adhere and
polymerize.
• HbA and HbF will prevent sickling, because they
do not co-polymerize with HbS.
• HbS can bind and transport oxygen.
• The sickling occurs under deoxygenated state.
Sticky Patches on HbS Molecule
• The sickled cells form small plugs in capillaries.
• Occlusion of major vessels can lead to infarction in
organs
like spleen.
• Death usually occurs in the second decade of life.
• Heterozygous state is very common in Central and
West Africa, East and Central India.
• Tribals all over India show an increased incidence
of SS and AS.
• The slave trade has played an important role in
spreading the gene from Africa to different parts
of America.
Mechanism of vascular
occlusion in sickle cell
anemia.
 Sickle Cell Trait
• Heterozygotes (AS) - 50% of Hb normal.
• Sickle cell trait as such does not produce clinical
symptoms.
• Such persons can have a normal lifespan.
• At higher altitudes, hypoxia may cause
manifestation of the disease.
• Chronic lung disorders may also produce
hypoxia-induced sickling in HbS trait.
 HbS gives Protection Against Malaria
• The high incidence of the sickle cell gene in
population
coincides with the area endemic for malaria.
• HbS affords protection against Plasmodium
falciparum infection.
• Hence the abnormal gene was found to offer a
biologic advantage.
Sickle Cell Trait Protects from Malaria
 Electrophoresis
• Electrophoresis at alkaline pH shows a slower
moving band than HbA.
• At pH 8.6, carboxyl group of glutamic acid is
negatively charged.
• Lack of this charge on HbS makes it less negatively
charged, and decreases the electrophoretic
mobility towards positive pole.
• At acidic pH, HbS moves faster than HbA. In sickle
cell trait, HbA and HbS.
 Electrophoresis at pH 8.6

In the electrophoresis, the abnormal HbS can be detected along

with normal Hb in persons with HbS trait.
 Sickling Test
• A blood smear is prepared.
• A reducing agent such as sodium dithionite is
added.
• Blood smear examined under the microscope
shows sickled RBCs.
Management of Sickle Cell Disease
• Repeated blood transfusions may be required in
severeanemia.
• But this can lead to iron overload and cirrhosis.
• Treatment with anti-sickling agents like urea,
cyanate and aspirin, that interfere with
polymerization are tried.
• Sodium butyrate will induce HbF production with
clinical improvement.
 Hemoglobin E
• It is the second most prevalent variant.
• It is due to the replacement of beta 26 glutamic
acid by Lysine.
• It is primarily seen in orientals of South-East Asia
(Thailand, Myanmar, Bangladesh, etc).
• The variant is very prevalent in West Bengal.
• Heterozygotes are completely asymptomatic.
• Similar mobility as of A2 on electrophoresis.
 Other Hemoglobin Variants
1. Hb C- 6th amino acid beta chain glutamic acid is
replaced by lysine.
• Double heterozygotes (HbSC) have moderate
disease.
• Homozygotes (CC) have hemolytic anemia.
2. Hb D – Beta 121 glutamic acid to glutamine (HbD
Punjab). HbSD is severe disease.
3. Hb M – Proximal or distal hisitidine substitutions
(HbM Boston, HbM Hyde Park).
Important Hemoglobinopathies

Hemo- Point mutation position Amino acid Codon and base

globin substitution substitution

HbS Beta 6 Glu → Val GAG→ GUG

HbC Beta 6 Glu → Lys GAG → AAG

HbE Beta 26 Glu → Lys GAG → AAG

HbD Beta 121 Glu → Gln GAG → CAG

(Punjab)
HbM Proximal or distal His → Tyr CAC → UAC
histidine in α or β chains
 Inheritance of HbC Trait
and Generation of HbS-C Disease
 CASE
• 9 year old child was brought to the hospital with chief
complaints of breathlessness, weakness, failure to thrive.
Mother also complained of her poor appetite and frequent
infections. On examination pallor and icterus was present.
Abdominal ecxamination showed massive
hepatosplenomegaly. There were signs of slowed growth and
tachycardia. There was history of multiple blood transfusions.
Lab findings showed,

• Hb – 5.5 g% MCV- 60 fl (80 – 100) ,MCH- 15 pg (27 – 32)

• LFT – raised serum bilirubin and liver enzymes AST, ALT
• Hb electrophoresis showed the presence of increased
ammounts of HbF.
 Thalassemias
• The name is derived from the Greek word,
"thalassa", which means "sea".
• Greeks identified this disease present around
Mediterranean sea.
• Thalassemia may be defined as the normal
hemoglobins in abnormal proportions.
• The gene function is abnormal, but there is no
abnormality in the polypeptide chains.
• Reduction in alpha chain synthesis is called
alpha thalassemia
deficient beta chain synthesis is the beta-
thalassemia

• Other types like delta-beta thalassemia, Hb

Lepore, hereditary persistence of HbF (HPF) are
related conditions.
 Beta Thalassemia
• Beta thalassemia is more common than alpha
variety.
• Beta type is characterized by a decrease or
absence of synthesis of beta chains.
• As a compensation, gamma or delta chain
synthesis is increased.
 Inheritance
• Beta thalassemias are phenotypically described
as beta (+) or beta (o) depending on whether
there is beta chain synthesis or not.
• Beta (o) thalassemia may result from base
substitutions.
• Beta (+) thalassemias are produced from defects
in posttranscriptional processing of mRNA.
 Alpha Thalassemias
• They may result from different types of gene
deletions.
• Since there are 2 pairs of alpha genes per cell, a
single gene deletion in one chromosome or a
pair of genes in the chromosomes does not have
much effect on a chain production.
• Alpha thalassemia is rarer because alpha chain
deficiency is incompatible with life.
 Thalassemia Syndromes
• These syndromes are mainly seen in people of
Asian, African and Mediterranean origin.
• All cases of thalassemias are characterized by
deficit of HbA synthesis.
• Hypochromic microcytic anemia is seen
• In homozygous state, clinical manifestations are
severe, and hence called Thalassemia major.
• There will be nucleated RBCs in peripheral
circulation.
• In heterozygous conditions, the clinical signs and
symptoms are minimal; they are called Thalassemia
minor.
• The synthesis of unaffected chains occurs at the
normal rate.
• Since they do not have complementary chains to bind,
they form aggregates and precipitate within the cell.
• These precipitates or inclusion bodies lead to
membrane damage and destruction of red cells.
• The co-existence of HbS and beta thalassemia
trait is fairly common.
• Homozygous beta thalassemia is characterised
by severe anemia, hypersplenism and hepato
splenomegaly.
• The marrow in the skull bones expand producing
the “hair-on-end appearance” described in X-ray.
Expansion of marrow of skull bones lead to Hair-
on-end appearance in beta Thalassemia
• It is the radiologic pattern seen as calcified spicules perpendicular
to bone surface
• Repeated transfusion is the only available
treatment.
• This may lead to iron overload.
• Splenectomy may also lessen the anemia.
• Marrow transplantation has been successfully
tried in a few cases.
 MYOGLOBIN (Mb)
• One molecule of Mb can combine with 1 molecule
of oxygen.
• In the muscles, the oxygen is taken up by Mb for
the sake of tissue respiration.
• Mb has higher affinity for oxygen than that of Hb.
• The pO2 in tissue is about 30 mm of Hg, when Mb
is 90% saturated.
• At this pO2, Hb saturation will be only 50%.
• In severe physical exercise, pO2 in muscles
lowers to 5 mm Hg, when myoglobin releases all
the bound oxygen.
• Mb has a high oxygen affinity while Bohr effect,
co-operative effect and 2,3-BPG effect are
absent.
• Severe crush injury causes release of myoglobin
from the damaged muscles.
Myoglobin Chain.
Causes of Anemias
1. Hemolysis due to impaired production of RBCs
a. Defect in heme synthesis: Nutritional deficiency of iron,
copper, pyridoxal phosphate, folic acid, vitamin B12 or
vitamin C. Lead will inhibit heme synthesis.
b. Defect in regulators: Erythropoietin synthesis is reduced in
chronic renal failure.
c. Defect in stem cells: Aplastic anemia due to drugs (e.g.
Chloramphenicol), infections or malignant infiltrations.
2. Hemolysis due to intracorpuscular defect
a. Hemoglobinopathies such as HbS, HbC:
b. Thalassemias—major and minor
c. Abnormal shape: Spherocytosis and elliptocytosis.
d.Enzyme deficiencies: Deficiency of glucose-6- phosphate
dehydrogenase.
Causes of Anemias, Continued
3. Hemolysis due to extracorpuscular causes
a. Infections: Malarial parasites
b. Autoimmune hemolysis: Antibodies are seen against RBC
membrane components.
c. Isoimmune hemolysis: Rh incompatibility.
d. Hemolysis due to drug sensitisation: Many drugs (e.g. alpha-
methyl dopa, quinine) may fix on RBC membrane, and
produce antibodies against the altered membrane.
4. Hemorrhage
Hematuria, hematemesis, hemoptysis, peptic ulcer metrorrhagia and
hemorrhoides are the usual causes for hemorrhage. Hemophilia
(absence of AHG) and thrombocytopenia are other major causes for
bleeding tendencies.
Common symptoms of anemia.