Module 2:

2.1) Haemoglobin and

2.2) Biological Pathways of Red cell

By: Edwig Hauwanga

For: Haematology A
HAM611S
2.1 Haemoglobin

Learning Objectives

• HB function
• HB Synthesis
• How to test for HB
• Different kinds of HB (fetal, adult….)
Haemoglobin- Intro

Haemoglobin is
It is synthesized during
responsible for the
About 640 million HB nucleated (65%) and
delivery of O2 to
molecules per red cell reticulocyte (35%)
tissues and CO2 to
stage.
lungs for excretion.

Enzyme δ-
Synthesized mainly in aminolaevulinic acid VitaminB6 (Porydoxal
the mitochondria & (ALA) synthase key phosphate) is a co-
Ribosome enzyme for HB enzyme.
synthesis
Types of
Haemoglobin
Normal adult RBCs contain the following type of
HB:
• 95% to 97% of HbA consisting of α2β2
• 2 to 3% of HB in HbA2 with α2δ2 chains
• 1% to 2% of HbF with α2γ2 chains
HB Into cont.….
• Adult haemoglobin (HbA) Consists of globin (tetramer of
two pairs of globin peptide pairs) 2alpha & 2beta(β)
• And 4 heme groups (protopophyrin rings plus Fe2+)
• Small quantities of fetal haemoglobin (HbF) and adult
haemoglobin 2 (HbA2) are also present in normal adult
blood. Have γ and ε chains instead of β .
• Major switch between fetal and adult HB takes place 3-6
months after birth.
Structure of HB molecule

https://sciencing.com/hemoglobin-show-four-
levels-protein-structure-8806.html
Structures of Hb

• In the cytoplasm, globin chains link with

heme (ferroproporohyrin IX) to form HB.
• Structure of HB molecule described as;
1. Primary structure- number and
sequence of amino acid constituting
each chain. Alpha chains(141 amino
acids) and beta (146)
2. Secondary structure- twisting of the
amino acid chain around an axis in
helical conformation.
3. Tertiary structure- bending twisted
chain into 3 dimensional “pretzel”
shape.
4. Quaternary structure- 4 dimensional
chains with their respected heme
groups.
HB Molecular
Structure

The HB molecule is comprised of

the following:
• Two alpha chains
• Two Beta Chains
• Four iron containing heme
groups
HB
structure
• Haem group with an iron (Fe) atom as
central atom.
• Fe reduced to ferrous Fe2+ state to
bind oxygen.
• Oxidation of HB can result in
methaemoglobin which cannot bind
oxygen.
• Results in conditions with abnormal HB
that cannot maintain reduced iron
(ferrous)
• E.g. Certain drugs, infections and other
conditions that result in oxidation of
HB
Methaemoglob
in

• Haemoglobin iron is oxidized

(Fe3+) instead of reduced (Fe 2+)
• May be hereditary or due to
drugs or toxic substance that
oxidize HB
• Patients will show cyanosis
Hypoxia Vs Anaemia

• Hypoxia- insufficient oxygen to meet metabolic demands.

• Anaemia- when hypoxia is due erythrocyte or HB defect
HB synthesis
HB production is dependent on the following:
1. Iron delivery and supply
2. Synthesis of protorphyrins (precursor of heme)
3. Adequate globin synthesis

• Iron is delivered by transferrin through the membrane to the cytoplasm and

then proceeds to mitochondria.
• In the mitochondria it is reduced to the ferrous state for insertion into
protoporphyrin ring.
Heme Synthesis

• Protoporphyrin synthesis begins in mitochondria and is mediated by the

enzyme delta-aminolevulinic (δALA) synthase.
• δALA is formed from glycine and sccinyl coenzyme A (CoA) which is a major
rate limiting step in the biosynthesis of heme
• Influenced by erythropoietin and requires cofactor pyridoxal phosphate
(vitamin B6) .
• Porphyrinogens are intermediates of heme synthesis not porphyrins.
• Porphyrinogens are oxidized to form porphyrins.
• Porphyrins found in small quantities in urine as a result of RBC breakdown.
Heme synthesis: Enzymes and their
products
• Eight enzymes accomplish this process, four of which work in the mitochondria and
four in the cytosol.
• The process starts in the mitochondria, where ALA (aminolevulinic acid) synthase
links glycine and succinyl coenzyme A to form ALA. Steps 2 through 5 occur in the
cytosol. Next, ALA dehydratase takes two molecules of ALA and produces
porphobilinogen (PBG). In the third step, porphobilinogen deaminase takes four
molecules of PBG and produces hydroxymethylbilane. Next, uroporphyrinogen III
cosynthase takes hydroxymethylbilane and produces uroporphyrinogen III. In the
fifth step, uroporphyrinogen decarboxylase takes uroporphyrinogen III and
produces coproporphyrinogen III. The final three steps of heme synthesis occur in
the mitochondria. Coproporphyrinogen III is then transformed to
protoporphyrinogen IX by coproporphyrinogen oxidase. The seventh step occurs
when protoporphyrinogen oxidase converts protoporphyrinogen IX to
protoporphyrin IX. The eighth and final step of heme synthesis is the addition of Fe
to protoporphyrin IX by ferrochelatase, producing a heme molecule

• https://www.youtube.com/watch?v=DoGPUKsuxV4
Synthesis of Globins
• Occurs in ribosomes
• Initiated from inheritance of certain structural genes.
• Genes result in specific polypeptide chains
• Different chains occur at different stages in life (Embryonic-ε and ζ plus α
and γ; )
• Αalpha (α) chains are always present
• Production of β chain rise gradually after birth and reaches adult percentage
at 3 to 6 months
Globin Synthesis
cont….
• Occurs in RBC-specific cytoplasmic ribosomes initiated by inheritance of
structural genes
• Genes directs formation of structural genes of a specific polypeptide gene
• Genes in each diploid: four alpha (α), two beta(β), four gamma (γ), two delta
(δ), two epsilon (ε) and two zeta (ζ), which are located on chromosome 11
(alpha and zeta) and the rest on chromosome 16
• The gene products are chains called by the name of the gene
The Hb Function
• Red cells carry O2 from the lungs to the tissues and
• Return venous blood with CO2 from tissues to lungs.
• HB loads oxygenated blood and unloads deoxygenated blood.
• HB binds oxygen molecules using co-operativity.
• When first O2 molecule binds, it attracts other O2 molecules to bind by
widening the spaces between the chains. (relaxed state)
• It the unloads the O2 in the tissues relaxing the spaces between the chains.
(Tense state)
How Hb performs its
functions
• 2,3-diphosphoglycerate (2,3-DPG) controls HB affinity for oxygen.
• O2 is loaded onto the HB molecule in a relaxed state, beta chains are pulled
together and 2.3 DPG is expelled. The relax form HB has high affinity for O2
• The unloading of oxygen to the tissues, spaces between beta chains widen
and 2.3 DPG binds to the HB molecule this is known as a tense stage and has
lower affinity for oxygen.
• Deoxyhaemoglobin-not saturated with oxygen
• Oxyhaemoglobin-saturated with oxygen
• This flexibility of HB molecules allows higher affinity for oxygen.
HB
Functio
n
HB Oxygen Dissociation Curve

• The function of HB is responsible for the sigmoid form of the HB O 2

curve
• P50 is the partial pressure of O2 which haemoglobin is half saturated
with oxygen
• P50 of normal blood is 26.6mmHg
• As affinity for O2 increases, the curve shifts to the left (in cases where
P50 falls) while in decreased affinity the curve shifts to the right
• In normal circumstances in vitro oxygen exchange operates at 95%
saturation (arterial blood) with 95mmHg O2 tension
• 70% saturation in venous blood which has 40mmHg O2
• The concentration of 2.3 DPG, H+ ions and CO2 determines normal
position of the curve
HB Oxygen Dissociation
Curve
• High concentrations of 2.3 DPG, H+ ions and
CO2 and in cases of sickle haemoglobin shift
curve to the right
• While in cases of low 2.3 DPG such as
haemoglobin F (unable to bind 2.3 DPG) shifts
curve to the left as they give up O2 less readily
than normal
• His is the case with polycythaemia vera where
there is an abnormal high numbers of red cells

• Assessment: O2 saturation in covid

How do we test for HB?

1. Quantification of concentration of HB- Haemoglobin cynide method

using spectrophotometry recording absorbance at 540nm and the
calculating the concentration. HB concentration usually between 11-14
g/dl
2. Electrophoresis- Different amino acids in globin chains, carry different
electrical charges in buffered solutions.Globins migrate at different rates
and are separated and identified.
-Pertinent in identifying abnormal haemoglobins
HB electrophoresis
Other tests of HB
• Visual inspection of smear with light microscope
• HB solubility-screens for certain HB types
• Spectra analysis- detects HB variants combined or modified forms (carboxy-
haemoglobin, sulfhaemoglobin and porphyrin precursor)
Disorders associated with defective HB

• Thalassaemia- defective globin

chains
• Iron deficiency anaemia- Lack
of iron
• Sideroblastic anaemia- Lack of
haem
2.2 Cell Metabolic Pathways

Learning Objectives

• How the red cell metabolism takes place

• Different metabolic pathway red cell uses to metabolize
• Overview of red cell membrane structure
Red cell metabolism
Introduction
• Like any cell, red cells need energy to carry out their functions effectively
• RBC uses energy in the form of ATP (adenosine triphosphate) and must have
a viable or workable membrane - any abnormality will decrease cell survival
Red cell
metabolism
Remember!!!
• Red cells unlike normal cells do not have organelles.
Advantages:
-Biconcave shape that enables flexibility to pass
through vessels and have optimum oxygen exchange
Disadvantage
-No mitochondria to utilize oxygen to metabolize and
produce energy

Therefore:
-RBC use anaerobic means to produce energy
-They ferment lactic acid to produce adenosine
triphosphate (ATP)
-This happens in biochemical pathways
Energy for
the Red Cell
• ATP is main source of energy for
the cells
• When ATP becomes depleted,
the cell will become a sphere
(becomes rigid and stiff)
• These spherical cells are
removed by the spleen.
Energy requirements
Why red cells need energy
• To carry its the function
• Movement of electrolytes sodium, calcium and potassium.
• Oxidation
• Membrane integrity and preservation of shape. (so its able to bend through
vessels)
• Maintain iron in a Ferrous (FE++) state
Biochemical Pathways

• RBC do not use the aerobic Krebs cycle to

produce energy.
• Utilise anaerobic glycolysis
• Pathways make use of enzymes to produce
energy
• Enzymes are synthesized before nucleus is
expelled in a immature red cell
• The enzymes produce ATP in different pathways
• Cell can longer produce energy when enzymes
are depleted and therefore the cell is removed
from circulation.
The Metabolic
Pathways of RBC

1. Embden-Meyerhof Pathway
2. Leubering-Rapoport Pathway
3. Hexose Monophosphate
(pentose phosphate) Pathway
4. Methaemoglobin Pathway
 • Non-oxidative or anaerobic Pathway
1.Embd • Produces 90% of ATP
• Glucose that enters the cell by diffusion is
en- metabolized to lactate.

Meyerh
• For every 1 glucose molecule, 2 ATP high
energy phosphate bonds are generated.

of • ATP provides energy for maintenance of red

cell volume, shape & flexibility

Pathwa
• Energy is also utilized regulate electrolyte
movement in and out of the cell.
*ATPase Na pump
y • Also generates NADH needed by
methaemoglobin reductase to reduce
dead methaemoglobin to activate it.
2. Leubering-
Rapaport or
Shunt Pathway
• Part (side arm) of the Embden-Meyerhof
pathway
-One of the ATP used by the cell is directed to
this pathway.
• Produces 2,3-PDG (diphosphoglycerate)
-2,3 DPG affects oxygen affinity of HB
-When haemoglobin binds 2,3-DPG, oxygen
is released.
-Regulates oxygen delivery to the tissues
 Its an oxidative pathway

in which glucose-6-phosphate is converted to 6-

phosphogluconate and so to ribulose-5-phosphate.

Uses the enzyme G6PD (glucose-6-phosphate

dehydrogenase)
3. Hexose Produces NADPH (Nicotinamide adenine
Monophosp dinucleotide phosphate)
hate Shunt
-NADPH protects cell from oxidative stress

-Biosynthetic reactions such as fatty acid synthesis,

cholesterol synthesis, drug reduction

-Used by another methaemoglobin reductase to

maintain iron in the active Fe++
 • A defective pathway leads to the
denaturing of HB chains and precipitates
as aggregates.
• These aggregates are called Heinz Bodies
Hexose • Heinz bodies damages the red cell
Monophosp membrane leading to destruction of the cell
hate Shunt • They can be seen supravital stain
(methylene blue), crystal blue, brilliant
cont…. cresyl blue
• Present in congenital haemolytic anaemia,
G6PD deficiency
 • An off shoot Embden-Meyerhof pathway
• Maintains iron in the reduced ferrous
(Fe+2) state
-Ferrous carries oxygen and is functional
• Although methaemoglobin is produced, it is
4.Methaemo reduced back to the ferrous state.
globin • Methaemoglobin reductase is the enzyme
that is responsible for the reduction.
Reductase
• Oxidant drugs can decrease
Pathway methaemoglobin reductase therefore
increasing methaemoglobin.
• Methaemoglobin cannot carry and combine
oxygen.
• Leads to cyanosis in the patient, which
leads bluish discoloration
2.3 Red cell
Membrane

• A normal intact membrane is essential for

normal erythrocyte function and survival -
cells must be able to flexibly move through
the vessels
• Normal erythrocyte is 6-8 um
• Must be flexible to squeeze through 3 um
capillaries in the spleen - Becomes a
microcyte
Red cell membrane

• RBC membrane has 3 layers

• Outer hydrophilic composed if glycolipid, glycoprotein and protein
• A central hydrophobic layer consisting protein, cholesterol and phospholipid
• An inner hydrophilic layer containing protein

• The RBC membrane is highly elastic

• Structure consisting of semipermeable lipid bilayer supported by membrane
cytoskeleton structure
• Membrane cytoskeleton is network of proteins responsible fpr maintaining
shape, stability and deformability
 Red cell membrane
Red cell
membrane
composition
Protein
10% Phospholipids
• Membrane composed of lipid bilayer, 20% cholesterol
carbohydrate
50%
membrane proteins and membrane
skeleton. 20%
Red cell
membrane
composition

• Any defect in structure or change in

percentages in compounds will alter
function and lead to premature cell death
Red cell membrane carbohydrates

• They occur only on the

external surface of the red
cell.
• They are composed of
glycoproteins and
glycolipids
• Glycolipids are your
surface antigens (A, B, H-
blood group antigens)
Red Cell
Membrane Lipids

Lipid bilayer composed of cholesterol and

phospholipids in equal portions.
• Lipid is crucial for physical properties
1. Membrane permeability
2. Fluidity
3. Facilitate activity of proteins
Red Cell Membrane Lipids

• Phospholipid molecules are characterized by a polar head group

attached to a non-polar fatty acid tail.
• The polar head group is hydrophilic ( water loving), while the fatty
acid tail is hydrophobic (water fearing).
• Thus the phospholipids in the cell membrane tend to arrange
themselves in a bilayer with their hydrophilic heads pointing towards
the inner and outer aqueous phases ( the cytoplasm and plasma),
while the hydrophobic tails point towards each other.
Red Cell Membrane Lipids

• Cholesterol responsible for passive transport of ions and gases and

active transport of cation
• Cholesterol and lecithin affect surface area
• If blood cholesterol increases, it increases membrane surface area
-leads to formation of target cells/acanthocytes.
-Red cell is destroyed
Red Cell Membrane
Proteins

PERIPHERAL PROTEINS
• The red cell peripheral proteins interact to form a cytoskeleton.
• The cytoskeleton acts as a tough supporting framework for the lipid bilayer.
• Peripheral proteins: Spectrin, ankyrin, protein 4.1 and actin
• Spectrin, is the most abundant protein and consists of two chains alpha and
beta Spectrin.
Red Cell Membrane Proteins

INTEGRAL PROTEINS
• Extend from the outer surface and transverse the entire membrane
to the inner cytoplasmic side of the RBC.
-Band 3 (acts as anion transport channel).
-Glycophorins A, B, and C.
-Na+/K+ ATPase-enzyme catalysing hydrolysis of ATP to ADP
-glucose transport protein.
-surface receptors. (the most important is the transferrin
receptors)
Functions of membrane proteins

• Binding
• Catalytic activities
• Transporters
• Signalling (communication)
• Structural (Shape maintenance ,Deformability, Flexibility, Durability)

Defect leads to:

• abnormal cell shape
• decreased stability
• haemolytic anaemia
Deformability
• Red cell deformability or flexibility is critical to RBC survival and function as it
travels through the microvascular
• Decrease deformability and red cell shape changes are used to distinguish
features of a number of congenital or hereditary haemolytic anaemias
• Loss of ATP lead to decreased spectrin phosphorylation leading to a loss of
deformability
• Increased membrane calcium also leads to increased membrane rigidity and
loss of pliability
• Spherocytes are RBC with reduced surface to volume ration and are an
example of loss of RBC membrane
Permeability
• The RBC membrane is freely permeable to water and anions (Cl- and HCO3-)
• Channels for these anions are formed by membrane proteins
• RBC membrane is impermeable to cations (Na+ and K+) and are controlled by cationic
pumps that are energy (ATP) depended and requires the enzyme sodium-potassium ATPase
• Calcium is also pumped from extracellular to the intracellular through the calcium-ATPase
pump
• The RBC maintains its volume and water homeostasis through the sodium and potassium
intracellular concentration
• K+ is found inside the cell while Na+ is found outside the cel
• Permeability of RBC and cationic transport is crucial in preventing colloid osmotic
haemolysis
• Disorders increasing membrane permeability or altering cationic transport lead to a
decrease in RBC survival
Defects in red cell membrane

While alterations in lipid composition

Defects of the proteins may explain
because of congenital or acquired
some of the abnormalities of the
abnormalities in plasma cholesterol
shape of the red cell membrane, e.g.
or phospholipids may be associated
hereditary spherocytosis and
with other membrane abnormalities,
elliptocytosis.
e.g. target cells and acanthocytes
References

• A.V. Hoffbrand, P.AH. Moss and J.E. Pettit (2006)5th Ed.Essential

Haematology.Massaxhusetts,USA.Blackwell
• A.V Hoffbrand, PAH. Moss.(2016) 7th Edition. Hoffbrand’s Essential
Haematology.West Sussex. Whiley Blackwell
• Denis M.Harmening. (2009).5th Edition. Clinical Haematology and
fundamentals of haemostasis.Philadelphia.USA. F.A.Davis