THE RED BLOOD CELL
Three aspects of red cell metabolism are
crucial for normal erythrocyte survival and
function:
•
the red blood cell membrane
•
haemoglobin structure and function
•
active red cell metabolic pathways
A tissue section
showing red cells in a
small blood vessel.
The red cell must be able to
change shape and squeeze
through small capillaries.
THE RED CELL MEMBRANE
The red cell membrane consists of a
bipolar lipid layer supported by
structural proteins.
50% of the membrane is protein
40% is lipid
10% is carbohydrate.
Structure of the red cell membrane.
Actin
Spectrin
HAEMOGLOBIN
Ankyrin
INSIDE CELL
Band 4.1
Lipid
bilayer
Cholesterol
Fatty
acid
chain
Phospholipid
Glycophorin
PLASMA
Band 3
OUTSIDE CELL
Lipids consist of
60% phospholipid
30% neutral lipids (mainly cholesterol)
10% glycolipids.
The phospho- and glycolipids are structural
with polar groups (hydrophilic) on the external
and internal surfaces of the cell.
Non-polar groups (hydrophobic)
form a
barrier at the centre of the membrane.
The lipid bilayer forms the wall of the red
cell and separates the contents from the
external environment.
Oxygen can pass through the lipid barrier
and bind to the Hb inside.
Alteration in
lipid
composition
can produce
target cells
Carbohydrates are mostly found on the external
surface of the red cell membrane.
Monosaccharides are associated with specific blood
group antigens, e.g. ABH and Lewis.
Donor nucleotide
(UDP-GalNAc)
A gene product
(A transferase)
Gal
GalNAc
1
4
3
2
Specific 13
linkage
Acceptor sugar
(Galactose)
GlcNAc
Gal
1
4
3
2
Glc
band 3
Type 2 precursor
Note: 14 linkage
Red cell membrane
Fuc
Producing blood group A antigen on the red cell of a group A
individual.
Proteins are either peripheral or integral.
Integral proteins, e.g. glycophorin, are
important for the active transport of solutes
across the membrane.
Spectrin, actin and ankyrin are peripheral
proteins on the inner surface and maintain
the biconcave shape of the red cell.
Subnormal spectrin produces
spherocytes or elliptocytes.
THE RED CELL "SKELETON"
The lipid bilayer is stabilised by a protein
framework on the inside of the cell.
The "skeleton" is made of spectrin, an
asymmetric two-chained molecule which is
attached to the inside of the cell wall by other
proteins including actin and ankyrin.
The ankyrin binds to an integral protein (band 3)
and the actin to a peripheral protein (band 4.1).
Spectrin gives the cell membrane its
flexibility and strength.
The red cell distorts as it passes through
tiny capillaries, but once through the
capillary, it immediately returns to its
biconcave shape.
If spectrin is denatured, e.g. by heat, the
red cell assumes a spherical shape and
loses its flexibility (spherocytosis).
PROTEIN
IDENTIFICATION
Proteins in the red cell
membrane can be
solubilised by a
detergent called
sodium dodecyl
sulphate (SDS) and
then be separated
according to their size
using polyacrylamide
gel-electrophoresis
(SDS-PAGE).
RED CELL ANTIGENS
Blood group antigens are associated with the red cell
membrane and are either integral to its structure or are
adsorbed onto it from the plasma.
They are made of proteins or carbohydrates.
An individual has a particular antigen if the genes
controlling its production are inherited. Protein
antigens are under direct genetic control, whereas
genetically controlled transferase enzymes assemble
carbohydrate antigens.
HAEMOGLOBIN STRUCTURE
Haemoglobin (MW 68,000) constitutes 95%
of the red blood cell’s dry weight.
65% of haemoglobin synthesis occurs during
the nucleated stages of RBC maturation and
35% occurs during the reticulocyte stage.
Normal Hb consists of globin (a tetramer of two pairs
of polypeptide chains) and four haem groups.
Each haem group contains a protoporphyrin ring plus
ferrous iron (Fe++).
The mitochondria are the main site of protoporphyrin
synthesis.
Iron is supplied from circulating transferrin and globin
chains are synthesised on ribosomes.
HAEMOGLOBIN
SYNTHESIS
The role of red cells is to carry oxygen to the
tissues and to return carbon dioxide from the
tissues to the lungs.
Haemoglobin is essential for normal red cell
function.
Six Hb variants are normally formed.
Embryonic haemoglobins include Gower 1,
Gower 2 and Hb Portland.
HbF is the predominant haemoglobin of
fetal life (65-95%). Adults have only trace
amounts of HbF (<1%).
HbA (>95%) and HbA2 (2.5-3.5%) are the
main adult haemoglobins.
Globin chains found in adult haemoglobins are
designated
alpha (α)
beta (β)
gamma (γ)
delta (δ).
Hb A has two alpha and two beta chains (α2β2),
Hb F has two alpha and two gamma chains (α2γ2)
Hb A2 has two alpha and two delta chains (α2δ2).
The alpha chain is thus common to all three types
of adult Hb.
Alpha chain synthesis is directed by two α
genes, α1 and α2, on chromosome 16.
Beta and delta chains result from single genes
on chromosome 11.
The gamma chain is directed by two genes, G
and A, on chromosome 11.
Alpha chains have 141 amino acids and nonalpha chains have 146; the exact sequence of
the amino acids has been determined.
HAEM consists of four pyrrole rings with a
central iron atom linked to the four nitrogen
atoms.
The iron atom has two further binding sites,
one of which is bound to a globin histidine
residue and the other binds reversibly to
oxygen.
Haem is synthesised mainly in mitochondria of
erythroblasts, some steps occur in the cytoplasm.
The initial and rate-limiting step is the fusion of
succinyl-Co A with glycine mediated by ALA
synthetase to form δ-aminolaevulinic acid (ALA).
This occurs in the mitochondrion and depends on
the presence of vitamin B6 (pyridoxal phosphate).
The reaction is stimulated by erythropoietin and
inhibited by haem.
The next step occurs in the cytoplasm.
Two molecules of ALA fuse to form
porphobilinogen. A double enzyme step forms
uroporphyrinogen that is decarboxylated to
coproporphyrinogen.
At this point, the pathway re-enters the
mitochondrium where protoporphyrin is formed.
Finally, ferrous iron is inserted to form haem
by haem synthetase.
The ferrous iron atom in each haem molecule is
attached to the proximal histidine residue of a
globin chain, but not to the distal histidine
residue.
Amino acids lying in the loop between
proximal and distal histidine residues form the
haem pocket, essential for the O2 carrying
capacity.
HAEMOGLOBIN FUNCTION
Red cells carry oxygen from the lungs to the tissues and
return in venous blood with carbon dioxide.
As the haemoglobin molecule loads and unloads O2, the
individual globin chains in the haemoglobin molecule
move in relation to each other.
When O2 is unloaded, the β chains are pulled apart,
permitting entry of the metabolite 2,3 diphosphoglycerate (2,3-DPG) resulting in a lower affinity for O2.
During oxygenation the two β chains move
together to give a species more avid for O2. Hence
as the initial oxygen is taken up by Hb it increases
its affinity for oxygen to bind to the remaining
haem groups in the molecule.
The relationship between oxygen concentration in
the blood (the partial pressure of oxygen) and the
proportion of oxygen bound to haemoglobin (the
percentage oxygen saturation) forms a sigmoid
curve called the Hb oxygen-dissociation curve.
The P50O2 (the partial pressure of O2 at which Hb is
half saturated) of normal blood is 27 mmHg.
With increased affinity for O2, the curve shifts to the
left (the P50 falls).
With decreased affinity for O2, the curve shifts to the
right (the P50 rises).
Normally, O2 exchange operates between 95%
saturation (arterial blood) with a mean arterial O2
tension of 95 mmHg and 70% saturation (venous
blood) with a mean venous O2 tension of 40 mmHg.
The normal position of the curve depends on the
concentration of 2,3-DPG, H+ ions and CO2 in the red
cell and on the structure of the haemoglobin molecule.
High concentrations of 2,3-DPG, H+ or CO2, and the
presence of certain haemoglobins, e.g. HbS, shifts the
curve to the right.
This facilitates the release of oxygen from the red cells.
HbF (unable to bind 2,3-DPG) and some rare abnormal
haemoglobins associated with polycythaemia shift the
curve to the left and so give up O2 less readily.
Mechanisms to Compensate for Anaemia
A patient suffering from an anaemia caused
by a loss of red cells may be able to
compensate by shifting the oxygen
dissociation curve to the right, making
available red cells, though fewer in number,
more efficient.
A shift to the right may also occur in response
to acidosis or a rise in body temperature.
A right shift of the oxygen dissociation
curve is only one way in which patients
may compensate for various types of
hypoxia; other ways include increase in
total cardiac output and in
erythropoiesis.
RED BLOOD CELL METABOLISM
Red cells generate energy almost exclusively
through the anaerobic breakdown of glucose.
The metabolism of the anucleated erythrocyte is
more limited than that of other body cells.
Mature red cells possesses little ability to
metabolise fatty acids or amino acids and have no
mitochondrial apparatus for oxidative metabolism.
Red cells deliver oxygen, not consume it.
THE EMBDEN-MEYERHOF PATHWAY
(EMP)
In the Embden-Meyerhof Pathway (anaerobic)
glucose is metabolised to lactate.
For each molecule of glucose, two molecules of
ATP are generated.
ATP provides energy for maintenance of red cell
volume, shape and flexibility.
The red cell has an osmotic pressure five times
that of plasma. A membrane ATP-ase sodium
pump ensures the correct concentrations of Na+
and K+ within the cell to protect the membrane
from lysis.
One molecule of ATP moves three sodium ions
out and two potassium ions into the cell.
The Embden-Meyerhof pathway also generates
NADH that is used to reduce non-functional
methaemoglobin, containing ferric (Fe+++) iron,
to functional reduced Hb, containing ferrous
(Fe++) iron.
2,3-diphosphoglycerate
(2,3-DPG)
is
generated by the Luebering-Rapoport shunt
and forms a 1:1 complex with Hb to regulate
haemoglobin's oxygen affinity.
THE PENTOSE PHOSPHATE PATHWAY
(hexose monophosphate shunt)
5 to 10% of glycolysis occurs through the Pentose
Phosphate Pathway (aerobic). Glucose 6-phosphate
is converted to 6-phospho-gluconate and so to
ribulose 5-phosphate.
NADPH is generated and linked with glutathione to
protect against oxidative stress e.g. hydrogen
peroxide produced by oxidative drugs or phagocytes.
NADPH is also used to maintain Hb in the active
ferrous (Fe++) state.
One of the commonest inherited abnormalities of
red cells is glucose-6-phosphate dehydrogenase
(G6PD) deficiency in which the red cells are
susceptible to oxidant stress.
Heinz Body Haemolytic Anaemia
G6PD deficiency is the most
common enzymopathy causing
hereditary haemolytic anaemia.
Heinz bodies
Bite cells and helmet cells
Blister cell
Extravascular
Haemolysis
Architecture of the Spleen
LABORATORY FINDINGS
Features of increased erythrocyte breakdown:
•
•
•
•
Unconjugated bilirubinaemia.
Urobilinogenuria.
Haptoglobins decreased.
Radioisotope red cell
survival studies
can
quantitate rate and site of
destruction.
Features of increased erythrocyte production:
•
•
•
•
Reticulocytosis
Polychromasia and nucleated red cells in
peripheral blood film.
Erythroid hyperplasia in bone marrow
aspirate.
Radiological changes, e.g. "hair on end"
appearance of cranial X-ray.
Features specific to intravascular haemolysis:
•
•
•
•
Haemoglobinaemia
(haptoglobin and haemopexin exhausted).
Methaemoglobinaemia.
Haemoglobinuria.
Haemosiderinuria.