BIOCHEMISTRY OF RED BLOOD

CELLS(ERYTHROCYTES)- RAJESH.P. NARAYANAN

Rajesh.P. Narayanan

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 General details
• Blood is a fluid connective tissue.
• Blood is involved in a variety of functions.
Such as transport of various metabolites, hormones,
metabolic waste products etc. and also involved in
maintenance of physiological temperature.
Blood also play a significant role in providing
immunity.
• The total volume of blood in an adult human being is
4.5- 5 Ltrs.
• Depending on the oxygen concentration, it can be
differentiated in to two- arterial and venous blood.

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 Composition of blood
• On centrifugation, the coagulated blood (without any
anticoagulants) can be separated in to two
compartments.
-Serum & corpuscles ( blood cells)or formed elements.
• Blood containing anticoagulants/un-coagulated blood
(anticoagulants- citrate /heparin /EDTA/ oxalate-
fluoride etc) will be separated in to plasma and
corpuscles.
• The difference between plasma and serum is serum
will not contain clotting factors like fibrinogen as it is
already used up for coagulation( serum can be called
as defibrinated plasma).
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 Composition of blood
• Composition of plasma
– contains mainly water(92%).
- Organic substances ( sugars/fatty acids /
other lipids/ hormones / various plasma
proteins/ waste products vitamins etc).
-Inorganic substances ( Na , K , Ca, Cl. Zn, Mn,
I etc. Etc.)

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 Blood cells (Formed elements)
Blood cells are of three categories:-
1.Red Blood Cells ( RBC) or Erythrocytes-involved
in the transport of molecular oxygen and also
contain various blood group antigens/glyco-
proteins that determines ABO blood types.
2.White blood cells ( leukocytes)
are of 5 different types.
-Neutrophils, basophils and eosinophils are
known as granulocytes.
-Lymphocytes and monocytes are known as
agranulocytes.

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 General functions of blood cells
 Lymphocytes are involved in cell mediated and humoral
(Ab- mediated ) immunity.
 Lymphocytes are of two categories-B lymphocytes (
Plasma cells) and T lymphocytes or helper T cells .
 B Lymphocytes are specifically involved in Ab synthesis
and secretion ,where as helper T cells are helping the B
lymhocytes in its functions.
 Monocytes and neutrophils are involved in macrophage
activity (phagocytosis) and provides cell-mediated
immunity.
 Platelets-involved in blood coagulation and also perform
some specific functions (e.g.. the source of platelet
derived growth factors).

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 Biochemistry of RBC
• RBC are biconcave , enucleated cells without any
cell organelles.
• The space enclosed by the RBC membrane is
called matrix and is filled with a protein called
haemoglobin.
Functions:-
1.RBC are involved mainly in the transport of
oxygen from lungs to tissues.
2.RBC contains certain membrane proteins
(glycoproteins) on its surface that determines
ABO blood groups- called blood group antigens.
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 Biochemistry of RBC
• There are two different types of blood group antigens
on the surface of RBC- antigen A and antigen B. These
antigens determines ABO blood types.
• If antigen A is present alone –that blood group type is
called A group.
• If antigen B is present alone –that blood group is
called B group.
• If A antigen and B antigen are present together –that
blood group is called A B group.
• If both A and B antigens are absent–that blood group
is called O group.

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 Biochemistry of RBC
• Since there is no nucleus in mature RBCs, they do not
posses any genome ( chromosome /genes/DNA)or
genetic information stored in it & it cannot synthesize
any proteins.
• Therefore RBC is not divisible(will not undergo cell
division).
• However, RBC contain certain proteins in its matrix
which is incorporated during erythropesis ( formation
of RBC) in the bone marrow.
• These proteins include – mainly hemoglobin(Hb) and
the enzymes required for two metabolic pathways
• –Glycolysis(anaerobic/Rapaport – Lubering cycle) and
HMP shunt.

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 Metabolic pathways in RBC
1.GLYCOLYSIS
A.ANAEROBIC GLYCOLYSIS- FOR ENERGY
PRODUCTION
B.RAPAPORT LUBERING CYCLE- FOR THE
FORMATION OF 2,3- BIS PHOSPHOGLYCERATE
2.HMP SHUNT
FOR THE FORMATION OF PENTOSE SUGAR
(mainly, Dextro-Ribose)AND NADPH.

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 Metabolic pathways in RBC
GLYCOLYSIS
(Embedden – Mayerhoff- Paranas Pathway)
• Glycolysis is a biochemical pathway in which the 6-
carbon glucose is enzymatically degraded in to 2
molecules of 3- carbon pyruvate.
• It is the chief oxidative (degradative) pathway of D-
Glucose.
• Only D. Glucose undergo degradation within the RBC and
not other hexoses.
• It takes place in the cytosol ( soluble fraction of the
cytoplasm ) of the cell.
• The pathway consist of 10 steps, each steps being
catalyzed by separate enzymes ie. There are 10 enzymes.

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 Metabolic pathways in RBC
• Glycolysis is the only source of energy for the
RBC s.
• Glycolysis is the pathway by which other
hexoses such as mannose, Galactose and
Fructose are also oxidized, but with some
modifications.
• In RBC only glucose is catabolized.
• It is occurring in all the living organisms .
& takes place in all the cells and all the tissues
( universal Pathway).

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 Metabolic pathways in RBC
• Glycolytic pathway is having two phases
• Phase - I : Praparatory phase–consists of the
first five reactions.
• Phase - II : Pay off phase or energy conserving
phase –consists of the last five reactions.

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 Metabolic pathways in RBC
• There are three irreversible steps in glycolysis
which are called rate limiting steps or key
enzyme steps.
• The steps are step I (HK) step III (PFK) and
step X( PK).
• Glycolysis is regulated mainly by controlling
the activity of these key enzymes .

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 Metabolic pathways in RBC
Regulation of Glycolysis
 When the blood Glucose level increases
1. Insulin- will be secreted which induces the
synthesis of enzymes like HK and PFK .
 This in turn stimulate glycolysis and helps to
decrease the blood sugar level.
2. Feed back control-HK is inhibited by Glucose- 6
P in a feedback manner.
3. ATP -inhibits PFK(step-3).
4. Citrate- inhibits PFK.

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Steps Enzyme ATP
/NADH
(produced
or utilized)
Step I HK/GK -1 ATP

StepII PFK -1ATP

StepVI Gly.3-P-DH +2
NADH+H+(
=+6ATP)
StepVII PGK +2 ATP

Step X PK + 2ATP

Total Yield of ATP = 10

Total no. of ATP utilized =2
Net yield of ATP= 8/ Glucose molecule

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 Metabolic pathways in RBC
• When the blood Glucose level decreases
1. Glucagon and Epinephrine are released
which activate protein kinases (enzymes).
2. Protein kinases phosphorylates pyruvate
kinase leading to its inactivation. Thus the
rate of Glycolysis decreases.
3. AMP is an inhibitor for PFK.

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 Regulation of Glycolysis
2 Ethyl alcohol (in yeast)

Glucose 2,pyruvate 2-AcetylCOA (under aerobic.condition)

2 Lactate (in muscles & RBC under

anaerobic condition)

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 Metabolic pathways in RBC
Anaerobic glycolysis ( under anaerobic condition)
• The conversion of Pyruvate in to lactate is called
anaerobic glycolysis.
• It takes place in RBC and also in the cytosol of skeletal
muscle cells (In the absence of sufficient oxygen).
• The reaction is catalyzed by an enzyme called Lactate
dehydrogenase (LDH) which requires NADH+H+ as a
co-enzyme.
• The net energy production in anaerobic glycolysis is
only 2 molecules of ATP per molecule of Glucose.

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steps Enzyme ATP /NADH (produced or
utilized)
Step I HK/GK -1 ATP
StepII PFK -1ATP
StepVI Gly.3-P-DH +2 NADH+2H
StepVII PGK +2 ATP

Step X PK +2ATP
Step XI LDH - 2 NADH+2H+

Energy yield of anaerobic

glycolysis

Total Yield of ATP = 4

Total no. of ATP utilized =2

Net yield of ATP= 2 ATP/

Glucose molecule 39
 Metabolic pathways in RBC
Rapaport – Lubering cycle
This is a modified pathway of glycolysis taking
place in R BC.
-Usually, the 7th step of normal glycolytic
pathway is the conversion of 1,3 bis-
phosphoglycerate in to 3- phosphoglycerate –
catalysed by an enzyme called phospho
glycerate kinase (PGK).
This is an ATP forming step(by Substrate level
phosphorylation).
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 Metabolic pathways in RBC
• But, in the RBCs 1,3- bisphospho glycerate is
converted to 2,3 bis-phosphoglycerate ( 2,3-
BPG) by an enzyme called 1,3- bis-
phosphoglycerate mutase .
• 2,3-BPG is then converted to 3 –
phosphoglycerate by an enzyme called 2,3 bis-
phosphoglycerate phosphatase.
• In this way Rapaport- Lubering cycle in the RBC
bypasses(called shunting) an ATP forming step in
normal glycolysis (VII step).
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 Metabolic pathways in RBC
Significance of 2,3 -BPG( Rapaport – Lubering cycle)
• 2,3 BPG binds to oxy-hemoglobin in the tissues and reduces its
affinity towards oxygen. It will help in fast oxygen unloading.
• Under hypoxic condition like in high altitude, 2,3 BPG concentration in
the RBC increases. This favours the release of oxygen in the tissues-
acclimatization.
• No, ATP is generated in this pathway(wasting of ATP).
• Relatively high oxygen affinity of Foetal blood ( Hb F) is due to the
inability of Hb-gamma chains to bind 2,3 BPG. This helps the foetus
tissue to receive more oxygen from the mothers circulation across the
placenta.
• When the blood is stored in the blood bank for long duration the 2,3 BPG
concentration will be decreased. Such old blood is not ideal for
transfusion particularly during complicated surgical procedures like
neuro and cardiac surgery as these tissues require more amount of
oxygen after the surgery.

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 Metabolic pathways in RBC
• 2,3- BPG level in normal blood approximately
is 15 mg/g Hb.
• 2,3- BPG level is higher in children compared
to elderly.
• 2,3-BPG binds to the Globin part of Hb at the
junction between two beta chains by ionic
interaction.

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 Metabolic pathways in RBC
• HEXOSE MONOPHOSPHATE PATHWAY(HMP)
• Also called,
-Hexose monophosphate shunt/Pentose phosphate
pathway /Phosphogluconate pathway)
• alternate pathway of glucose oxidation other than
glycolysis.
• Active mainly in RBC.
• Also, active in liver, adipose tissue, lactating
mammary glands, gonads, adrenal cortex and thyroid
tissue.
• Operated in the cytosol.
• Glucose 6-P is the starting substrate of this pathway.

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 Metabolic pathways in RBC
• Consists of two different phases- oxidative
phase and non-oxidative phase.
1.Oxidative phase – This phase consists of the
first 5- reactions by which Glucose-6-P is
enzymatically converted to Ribulose -5-P.
2.Non-oxidative phase – glucose -6-P is
regenerated from Ribulose-5-P by the
involvement of trans-aldolase and trans-
ketolase enzymes.

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 Metabolic pathways in RBC
Significance of HMP shunt
1. HMP shunt is the source of NADPH which
is essential for -
– Required for the reductive biosynthesis of fatty
acids, cholesterol, other steroids, and certain
amino acids.
– NADPH provides reducing power for the
antioxidant enzymes present in the RBCs thereby
helps to maintain the stability of RBC membrane.

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 Metabolic pathways in RBC
Glutathione reductase
G-S-S-G 2-G-SH
(oxidized glutathione) (reduced glutathione)

Glutathione peroxidase
2-G-SH + H2O2 G-S-S-G + 2H2O
• This reaction is important to prevent the
accumulation of H2O2 and helps to increase
the life span of the erythrocyte.
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 Metabolic pathways in RBC
• HMP shunt produces Ribose -5-P important
in the synthesis of Nucleotides (DNA and
RNA).
• HMP shunt is an alternate pathway of
glucose oxidation other than glycolysis.
• Only 10 % of D. Glucose is oxidized via HMP
shunt in all tissues except RBCs.
• In RBCs it is 30%.

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 Metabolic pathways in RBC
Regulation of HMP shunt.
The regulatory enzyme is Glucose -6-P
dehydrogenase.
• NADP+, High carbohydrate diet, insulin,
Thyroid hormones etc. activates this enzyme.
• Whereas, NADPH inhibit this enzyme.

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 BIOCHEMISTRY HEMOGLOBIN
• Haemoglobin is a conjugated protein.
• Haemoglobin consists of heme(non –protein)
and globin(protein)components.
• Adult Human Hemoglobin ( Hb A) is a hetero-
tetrameric protein ( alpha-2, beta -2, hetero-
tetramer)-consisting of 2 alpha and 2 beta
chains( subunits).
• MW of Hb is 67,000 Daltons.
• In males , normal concentration of Hb is 14-16 g
/L of blood.
• In females, it is 13-15 g/ L.

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 BIOCHEMISTRY HEMOGLOBIN
• In foetus, the Hb is specifically called HbF( foetal
haemoglobin)- consisting of 2 alpha and 2
gamma chains and is different from adults.
• In adults , there are 3 different type of Hb ,
1) Hb A ( 97 %)- major.
2) Hb A 2( 2%)- consisting of 2 alpha and 2 delta
chains and
3) Hb F( 1 %)- consisting of 2 alpha and 2 gamma
chains.
• Alpha chain has 141 amino acids and beta and
delta chains have 146 amino acids each.

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 BIOCHEMISTRY HEMOGLOBIN
• Heme is the prosthetic group of haemoglobin
molecule and each subunit has one heme
unit.
• Each heme unit binds one molecular oxygen.
There fore 4 molecules of oxygen can bind to
one hemoglobin molecule at a time.
• The heme is located in the hydrophobic cleft
of the globin subunits.

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 BIOCHEMISTRY HEMOGLOBIN
Hemoglobin derivatives
1. Carboxy haemoglobin ( carbon monoxy Hb/ CO Hb)
- Hb combines with CO to form CO Hb. The affinity of CO to
Hb is 200 times more than that of Oxygen.
• Carbon monoxide poisoning:-Co poisoning is a major
occupational hazard for workers in mines. Breathing the
automobile exhaust in closed space is a very common
cause of CO poisoning.
Cigarette smoking is another common cause. The CO- Hb
level of a normal person is 0.16 %. An average cigarette
smoker has an additional 4 % of CO Hb in their blood.
One cigarette releases 10-20 ml CO to the lung.
• CO poisoning can even lead to death.

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 BIOCHEMISTRY HEMOGLOBIN
2. Met – Hemoglobin ( Met Hb):-
• In a normal Hb Fe is in the Fe ++ state ( Ferrous ). Maintenance of
the ferrous state is essential for oxygen binding .
• If Hb contain Fe in the Fe +++ state ,it is called Met Hb. It cannot
bind oxygen.
• In the normal condition little amount of Met Hb will be formed in
the RBC ,but it will be reduced to normal Hb by the action of Met
Hb reductase enzyme ( 75 % NADH and cyt. B 5 dependent, 20 %
NADPH dependent and 5 % Glutathione dependent).
• A normal person contain 1 % Met Hb. If it is more( a condition
called met hemoglobinemia) can lead to cyanosis.
• The causes of Met hemoglobinemia are both
acquired (e.g. Aniline dye workers) and congenital (Glucose 6- P
deficiency).

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 BIOCHEMISTRY HEMOGLOBIN
3.Hemin crystal (Fe+++ & Cl2):-When Fe+++ of
Hb combines with chloride Hb undergo
crystallization to form hemin crystals (hematin
chloride).
It is having application in forensic analysis
and medico-legal cases.

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 BIOCHEMISTRY HEMOGLOBIN
4.Sulfhemoglobinemia:-When hydrogen
sulphide reacts with Hb sulfhemoglobin will
be formed.
• It is usually seen in people who take drugs
like sulphonamides , phenacetin, acetanilide
etc.
• Sulfhemoglobin cannot be converted back to
normal Hb. It will remain throughout the life
span of RBC.
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 BIOCHEMISTRY HEMOGLOBIN
5.Glycosylated Hemoglobin:-
• Glycosylated Hb is a characteristic feature of
long term untreated diabetes mellitus.
• Glucose molecule become attached to the
globin subunits of the Hb to form a conjugant
called glycosylated Hb.
• The measurement of Glycosylated Hb has
significant value in understanding the
background/duration of untreated DM.

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 BIOCHEMISTRY HEMOGLOBIN
Hemoglobin – chain variants
1.Hemoglobin S ( sickle cell Hb)
• Glutamic acid in the 6th position of beta chain is replaced with
valine.
• This lead to polymerisation of Hb inside the RBC leading to the
distortion of RBC in to sickle shape.
• HbS can bind and transport oxygen. But, the oxygen carrying
capacity of the polymerised HbS is less than that of normal Hb-
leading to anemia – called sickle cell anemia.
• The sickle cells from small plugs in the capillaries leading to
infarction of organs like spleen.
• Death usually occurs in the second decade of life.
• Hb S provide protection against malaria- and is a biological
advantage.

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 BIOCHEMISTRY HEMOGLOBIN
• Management of sickling:-
1. repeated blood transfusions.
2. treatment with anti-sickling agents like urea
and aspirin.
3. Sodium butyrate will induce HBF production
with clinical improvement( Hb F will not
polymerise and there fore will not produce
sickling).

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 BIOCHEMISTRY HEMOGLOBIN
4.Hb E : In the beta chain Glutamic acid at 26th
position is replaced by Lysine.
It is asymptomatic
2.Hb C: Glutamic acid present at the 6 th
position of beta chain is replaced by Lysine.
Moderate type of hemolytic anemia is seen.
3.Hb D: in the beta chain Glutamic acid present
at 121st position is replaced by Glutamine .
HbD causes sickling of RBC like Hb S.

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 BIOCHEMISTRY HEMOGLOBIN
Thalasemia
Thalasemia is a condition in which the Hb is abnormal
because of the absence of alpha /beta chain.
• There are two types
1. Alpha chain thalasemia- in which alpha chain is absent in
the Hb because of defect in the alpha chain gene.
2. Beta chain thalasemia – in which beta chain is absent
because of a defect in beta chain gene.
 As a compensation gamma or delta chain synthesis is
increased resulting in HbF and Hb A 2 production.
• Beta chain thalasemia is more common as compared to
alpha chain thalasemia.

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 Lymphocytes
• Lymphocytes constitute about 28–42 percent of the white cells of
the blood,
• they are part of the immune response to foreign substances in
the body.
• Most lymphocytes are small, only slightly larger than
erythrocytes, with a nucleus that occupies most of the cell.
• Some are larger and have more abundant cytoplasm that contains
a few granules.
• Lymphocytes are sluggishly motile, and their paths of migration
outside of the bloodstream are different from those of
granulocytes and monocytes.
• Lymphocytes are found in large numbers in the lymph nodes,
spleen, thymus, tonsils, and lymphoid tissue of the
gastrointestinal tract.

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 Lymphocytes
• They enter the circulation through lymphatic
channels that drain principally into the
thoracic lymph duct, which has a connection
with the venous system.
• Unlike other blood cells, some lymphocytes
may leave and reenter the circulation,
surviving for about one year or more.

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 Lymphocytes
• The principal paths of re-circulating
lymphocytes are through the spleen or lymph
nodes.
• Lymphocytes freely leave the blood to enter
lymphoid tissue, passing barriers that
prevent the passage of other blood cells.
• When stimulated by antigen and certain
other agents, some lymphocytes are
activated and become capable of cell division
(mitosis).
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 Lymphocytes
• The lymphocytes regulate or participate in the
acquired immunity to foreign cells and antigens.
• They are responsible for immunologic reactions to
invading organisms, foreign cells such as those of a
transplanted organ, and foreign proteins and other
antigens not necessarily derived from living cells.
• The two classes of lymphocytes are not distinguished
by the usual microscopic examination but rather by
the type of immune response they elicit.
• The B lymphocytes (or B cells) are involved in what is
called humoral immunity.

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 Lymphocytes
• Upon encountering a foreign substance (or antigen),
the B lymphocyte differentiates into a plasma cell,
which secretes immunoglobulin (antibodies).
• The second class of lymphocytes, the T lymphocytes
(or T cells), are involved in regulating the antibody-
forming function of B lymphocytes as well as in
directly attacking foreign antigens.
• T lymphocytes participate in what is called the cell-
mediated immune response.
• T lymphocytes also participate in the rejection of
transplanted tissues and in certain types of allergic
reactions.

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 Lymphocytes
• Two classes of T lymphocytes can be involved in the
response to those cell-associated viral antigens:
1)cytotoxic T lymphocytes, which destroy the infected cells by a
lytic mechanism.
2)Helper T lymphocytes: which assist B cells to produce
antibodies against the microbial antigens.
 It exert their influence on B lymphocytes through several
hormone like peptides termed interleukins (IL).
 Five different T lymphocyte interleukins (IL-2, IL-3, IL-4, IL-5,
and IL-6) have been discovered, each with different (and
sometimes overlapping) effects on B lymphocytes and other
blood cells.
 Interleukin-1, produced by macrophages, is a peptide that
stimulates T lymphocytes and that also acts on the
hypothalamus in the brain to produce fever. 78
 Lymphocytes
• The ability to develop an immune response (i.e.,
the T cell-mediated and humoral immune
responses) to foreign substances is called
immunologic competence (immuno
competence).
• Immunologic competence, which begins to
develop during embryonic life, is incomplete at
the time of birth but is fully established soon
after birth.
• If an antigen is introduced into the body before
immunologic competence has been established,
an immune response will not result.

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THE END
THANK YOU FOR YOUR ATTENTION

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