STRUCTURE AND FUNCTION OF
ERYTHROPOIETIC TISSUE
The RBCs
ERYTHROPOIESIS (RBC PRODUCTION)
 Mature
erythrocytes are derived
from committed erythroid proginator
cells through a series of mitotic divisions
and maturation phases.
 Erythropoietin, a humoral agent produced
mainly by the kidneys stimulates
erythropoiesis by acting on committed
stem cells to induce proliferation and
differentiation of erythrocytes in the bone
marrow.
ERYTHROPOIESIS
Tissue hypoxia (lack of oxygen) is the
main stimulus for erythropoietin
production.
 Nucleated red cell precursors in the
bone marrow are collectively called
normoblasts or erythroblasts.
 RBCs that have matured to the nonnucleated stage gain entry to the
peripheral blood.
 Once the cells have lost their nuclei,
they are called erythrocytes.

ERYTHROPOIESIS
Young erythrocytes that contain
residual RNA are called reticulocytes.
 Bone marrow normoblast proliferation
and maturation occurs in an orderly
and well defined sequence.


The process involves a gradual decrease
in cell size, condensation and eventual
expulsion of the nucleus, and an increase in
hemoglobin production.
BASIC BLOOD CELL MATURATION

Nearly all hematopoietic cells mature in the
manner shown below. For RBCs the nucleus is
eventually extruded and the cytoplasm increase
correlates with hemoglobin increase.
ERYTHROPOIESIS

For red cell production to be efficient , 85%
or more of the erythroid activity must have a
balanced incorporation of heme and globin to
form hemoglobin.
The immature, nucleated RBC must have an
adequate supply of iron‚ as well as normal production
of porphyrin and globin polypeptide chains‚ for
adequate synthesis of hemoglobin.
 Folic acid and vitamin B12‚ are also needed in
adequate amounts to maintain proliferation
and differentiation.
 Defects may occur at any stage of development and
these defects will lead to the death of the cell.

ERYTHROPOIESIS
Normally 1-15% of the RBCs die
during maturation.
 Ineffective erythropoiesis occurs when
there is a failure to deliver the
appropriate number of erythrocytes to
the peripheral blood.

Normoblasts normally spend 4-7 days
proliferating and maturing in the bone
marrow.
 The stages of maturation from the most
immature to the most mature are:

PRONORMOBLAST OR RUBRIBLAST
Pronormoblast (A)
BASOPHILIC NORMOBLAST OR
PRORUBICYTE
Basophilic normoblast
POLYCHROMATOPHILIC NORMOBLAST OR
RUBICYTE
Polychromatophilic normoblasts
ORTHOCHROMIC NORMOBLAST OR
METARUBICYTE
Orthochromic normoblast
RETICULOCYTE OR POLYCHROMATOPHILIC
ERYTHROCYTE
Reticulocyte
MATURE ERYTHROCYTE
ERYTHROPOIESIS
ERYTHROPOIESIS
Reticulocytes are released from the bone marrow into the
peripheral blood where they mature into erythrocytes ,
usually within 24 hours.
 It is rare to see more than 1% reticulocytes in the
peripheral smear from an adult, but common in healthy
newborns.
 They can be visualized more easily by staining with new
methylene blue which allows for visualization of the
remnants of the ribosomes on the endoplasmic reticulum.

ERYTHROPOIESIS
Mature RBCs have a lifespan of 100-120 days and
senescent RBCs are removed by the spleen.
 3 areas of RBC structure/metabolism are crucial for
normal erythrocyte maturation, survival and
function:

The RBC membrane
 Hemoglobin structure and function
 Cellular energetics

ERYTHROPOIESIS
Defects or problems associated with any of these will
result in impaired RBC survival.
 The RBC must be flexible in order to squeeze through the
capillaries of the spleen.



Flexibility is a property of the membrane and the fluidity of
the cell’s content.
Any decrease in flexibility results in a decrease in RBC
deformability and a decrease in RBC survival in passage
through the spleen.
THE RBC MEMBRANE
 The
RBC membrane is a semi-permeable
lipid bilayer supported by a protein
cytoskeleton (contains both integral and
peripheral proteins).
 Since the mature cells lack enzymes and
cellular organelles necessary to synthesize
new lipid or protein, extensive damage
cannot be repaired and the cell will be
culled in the spleen.
THE RBC MEMBRANE
 The
constituents of the RBC membrane
include:

Phospholipids- exchange between
phospholipids in the membrane and the
plasma may occur.

Since the fatty acid content of the diet and the plasma
are correlated, changes in the diet may have an effect
on the fatty acid composition of the phospholipids in
the RBC membrane which can adversely effect the
flexibility of the RBC and may result in an RBC with
a decreased survival time.
THE RBC MEMBRANE

Cholesterol- membrane cholesterol exists in
free equilibrium with plasma cholesterol.

Therefore, an increase in free plasma cholesterol
results in an accumulation of cholesterol in the RBC
membrane.
RBCs with increased cholesterol appear distorted and the
increased cholesterol results in the formation of target
cells, and acanthocytes.
 An increase in the cholesterol to phospholipid ratio
results in a cell membrane that is less deformable and
therefore, the RBC has a decreased survival time.

ACANTHOCYTES
TARGET CELLS
THE RBC MEMBRANE

RBC membrane proteins- 10 major and over
200 minor proteins are asymmetrically
organized in the RBC membrane.

Integral proteins- many carry RBC antigens and act
as receptors or are transport proteins. Glycophorins
are the major integral membrane proteins in the
RBC.

Located in the membrane are proteins that function as
cationic pumps.
 The RBC maintains its volume and water homeostasis
by controlling the intracellular concentrations of Na+
and K+ via these cationic pumps which require ATP.
 ATP is also required in the Ca++ pump system that
prevents excessive intracellular build-up of Ca++.
 In ATP depleted cells there is an intracellular build-up
of Na+ and Ca++ and a loss of K+ and water. This
leads to dehydrated, rigid cells that are culled by the
spleen.
THE RBC MEMBRANE


Any abnormality that increases membrane permeability
or alters cationic transport may lead to decreased RBC
survival.
The major peripheral protein is spectrin and it binds
with other peripheral proteins such as actin to form a
skeleton of microfilaments on the inner surface of the
membrane. This strengthens the membrane and gives
it its elastic properties.
For spectrin to participate in this interaction, it must be
phosphorylated by a protein kinase that requires ATP.
 Thus, a decrease in ATP leads to decreased
phosphorylation of spectrin.
 Unphosphorylated spectrin can no longer bind to actin to
give the membrane its elastic properties.
 This then leads to a loss in membrane deformability and
a decreased RBC survival time.

RBC MEMBRANE STRUCTURE
HEMOGLOBIN STRUCTURE AND FUNCTION

Hemoglobin occupies 33% of the RBC volume and
90-95% of the dry weight.
65% of the hemoglobin synthesis occurs in the
nucleated stages of RBC maturation and 35% during
the reticulocyte stage.
 Normal hemoglobin consists of 4 heme groups, which
contain a protoporphyrin ring plus iron, and globin,
which is a tetramer of 2 pairs of polypeptide chains.

STRUCTURE OF HEMOGLOBIN
HEMOGLOBIN STRUCTURE AND FUNCTION

Normal hemoglobin production is dependent
upon 3 processes: Adequate iron delivery and
supply, adequate synthesis of protoporphyrins
and adequate globin synthesis.

Iron delivery and supply:


Iron is delivered to the RBC precursor by transferrin. It
goes to the mitochondria where it is inserted into
protoporphyrin to form heme.
Synthesis of protoporphyrin:
Begins in the mitochondria where glycine + succinyl CoA
 delta aminolevulenic acid ( ALA). This is the rate
limiting step.
 In the cytoplasm 2  ALA prophobilinogen (PBG)

HEMOGLOBIN STRUCTURE AND FUNCTION
4 prophobilinogen (PBG) uroporphyrinogen I and III
(UPG I and III). Only type III is used. Type I represents
a dead-end pathway. PBG deaminase and UPG
cosynthase are both required for UPG III synthesis.
UPG I synthesis requires only PBG deaminase. In the
absence of UPG cosynthase large amounts of UPG I
accumulate in the RBCs , bone marrow, and urine
causing a condition called congenital erythropoietic
porphyria (more on this later).
 Decarboxylation of UPG III  coproporphyrinogen III
(CPG III). This moves to the mitochondria.
 In the mitochondria CPG III  protoporphyrin IX
 Fe is added to form ferroprotoporphyrin IX= HEME

SUMMARY OF HEME SYNTHESIS
HEME SYNTHESIS
STRUCTURE OF HEME
HEMOGLOBIN SYNTHESIS
HEMOGLOBIN STRUCTURE AND FUNCTION

Since porphyrinogens are readily oxidized to form
porphyrins, excess formation of porphyrins can occur if
any of the normal enzymatic steps in heme synthesis is
blocked. Metabolic disorders in which this occurs are
called porphyrias. There are 2 categories of porphyrias:
inherited and acquired
 Inherited
Erythropoietic porphyria - results from
excessive production of porphyrins in the
bone marrow.
Hepatic porphyria - results from excessive
production of porphyrins in the liver.
 Acquired
Lead intoxication - interferes with
protoporphyrin synthesis
Chronic alcoholic liver disease
HEMOGLOBIN STRUCTURE AND
FUNCTION

Globin Synthesis
In the yolk sac, the embryonic hemoglobins epsilon and
zeta are produced.
 In the fetus and the adult, 4 types of hemoglobin chains
may be formed: alpha ( α), beta (β ), gamma ( γ), and
delta ( δ).
 Normal hemoglobin's contain 4 globin chains.
 Hemoglobin (hgb) F= α2 γ2 and is the predominant hgb
formed during liver and bone marrow erythropoiesis in
the fetus. A normal, full term baby has 50-85% hgbF.
 Near the end of the first year of life, normal adult hgb
levels are reached. All adult normal hgbs are formed as
tetramers containing 2 α chains + 2 non-α chains.
Normal adult RBCs contain:

HEMOGLOBIN STRUCTURE AND FUNCTION




92-95% hgb A=α2β2
3-5% hgb Ac= glycosylated α2β2
2-3% hgb A2= α2δ2
1-2% hgb F (fetal hgb)= α2γ2
Each globin chain links with heme to form hgb= 4 globin
+ 4 heme.
 The precise order of the amino acids is critical for hgb
structure and function.
 An adequate amount of globin synthesis is also
important. A decreased production in 1 chain results in
thalassemia (discussed later).

GLOBIN SYNTHESIS
ASSEMBLY OF HEMOGLOBIN
HEMOGLOBIN STRUCTURE AND FUNCTION

Hemoglobin formation is regulated by several
mechanisms:
The regulation of globin chain synthesis. The rate of
globin synthesis is directly related to the rate of heme
synthesis because heme stimulates globin synthesis
by inactivating an inhibitor of globin translation.
 Negative feedback of heme. High concentrations of
heme prevent the mitochondrial import of the first
enzyme in heme synthesis,  ALA synthase ( ALAS).
 The concentration of iron. An iron responsive
element-binding protein (IRE-BP) binds to mRNA
iron response elements (IRE) to affect the translation
of the mRNA for  ALAS, ferritin (discussed later),
and transferrin receptors (discussed later).

HEMOGLOBIN STRUCTURE AND FUNCTION

The affinity of IRE-BP for IRE is determined by the amount
of cellular iron.
 When iron levels are low, there is a high binding affinity
which acts to inhibit the translation of  ALAS mRNA
resulting in a decrease in heme synthesis.
 When iron levels are sufficient, the binding affinity is
low, thus allowing translation of  ALAS mRNA and
stimulation of heme synthesis.
HOW IRON LEVELS AFFECT HEME
SYNTHESIS
HEMOGLOBIN STRUCTURE AND FUNCTION

If either heme or globin synthesis is impaired,
iron accumulates in the RBC.


An RBC with accumlated iron is then called a
siderocyte and the iron can be visualized using a
Prussian blue stain.
When protoporphyrin synthesis is impaired,
mitochondria become encrusted with iron.
This is visible as a ring around the nucleus of
the RBC precursor when stained with
Prussian blue.

A precursor cell with a ring of iron around the nucleus
is called a ringed sideroblast.
RINGED SIDEROBLASTS AND
SIDEROCYTE
RINGED SIDEROBLAST
HEMOGLOBIN STRUCTURE AND FUNCTION
 Hemoglobin

function
The primary function of hgb is gas transport.
The hgb molecule is capable of a considerable
amount of allosteric movement as it loads and
unloads O2. This is due to the multichain
structure of the molecule.
In unloading of O2, the space between the chains
widens and 2,3 diphosphoglycerate (DPG) binds. This
is the T (tense) form of hgb and it is called deoxyhgb.
It has a lower affinity for O2, so O2 unloads from the
hbg.
 When hgb loads O2 and becomes oxyhgb, the chains
are pulled together, expelling 2,3 DPG. This is the R
(relaxed) form of hgb. It has a higher affinity for O2,
so O2 binds to or loads onto the hgb.

OXY VERSUS DEOXY HEMOGLOBIN
HEMOGLOBIN STRUCTURE AND FUNCTION

Binding and dissociation of O2 are not
directly proportional to the O2 concentration.
Note the hgb-O2 dissociation curve below:
HEMOGLOBIN STRUCTURE AND FUNCTION
The sigmoid shape of the curve shows that a
significant amount of O2 delivery will occur
with a small drop in O2 tension.
 O2 affinity of hgb is expressed as the partial
(P) O2 pressure (in mm Hg) at which hgb is
50% saturated with O2.

Increased O2 affinity means that hgb does not
readily give up its O2.
 Decreased O2 affinity means that hgb releases the
O2 more readily.


Normally the partial O2 pressure in the
lungs is 100 mm. and the hgb is 100
saturated with O2. In tissues the partial
pressure is 40mm. and the hgb is 75%
saturated with O2. Therefore 25% of the O2
is delivered to the tissues
HEMOGLOBIN STRUCTURE AND FUNCTION
In hypoxia there is a compensatory
shift to the right in the dissociation
curve. This is mediated by an
increase in 2,3 DPG and results in
decreased hgb affinity for O2 and
increased O2 delivery to the tissues.
Therefore the RBCs are more efficient
in O2 delivery.

A patient suffering from anemia due to
blood loss may compensate by shifting the
O2 dissociation curve to the right.
 A shift to the right also occurs in acidosis
and when the body temperature is
increased.

RIGHT SHIFT IN O2 DISSOCIATION
CURVE
HEMOGLOBIN STRUCTURE AND FUNCTION
A shift to the left in the O2
dissociation curve results in decreased
O2 delivery to the tissues. This occurs:

In alkalosis
 When there are increased quantities of
abnormal hemoglobins such as methgb and
carboxyhgb
 When there is an increase in hgb F which
has a higher affinity for O2 than does hgb
A or
 When a patient has received multiple
transfusions with 2,3 DPG depleted blood.

LEFT SHIFT IN O2 DISSOCIATION
CURVE
COMPARISON OF AN O2 DISSOCIATION
CURVE AT NORMAL PH AND WITH
ACIDOSIS OR ALKALOSIS
HEMOGLOBIN STRUCTURE AND FUNCTION

Inherited abnormalities in hgb may result in either
type of shift and can have profound effects on the
RBCs ability to provide the tissues with O2. Acquired
abnormal hgbs of clinical importance are those that
have been altered post- translationally to produce
hgbs that are unable to transport or deliver O2 and
they include:
Carboxyhgb - CO replaces O2 and binds 200X tighter
than O2.
 This may be seen with heavy smokers
 This may be reversed with high concentrations of O2
 Methgb - occurs when iron is oxidized to the +3 (ferric)
state. In order for hgb to carry O2 the iron must be in the
+2 (ferrous) state. In the body, normally~ 2% is formed
and reducing systems prevent an increase beyond 2%.
 Increases above 2% can occur with the ingestion of
strong oxidant drugs or
 As a result of enzyme deficiency.

HEMOGLOBIN STRUCTURE AND FUNCTION


Methgb can be reduced by treatment with methylene blue or
ascorbic acid.
Sulfhgb - occurs when the sulfur content of the blood
increases due to ingestion of sulfur containing drugs or to
chronic constipation.

Unlike the formation of carboxyhgb and methgb, the
formation of sulfhgb is an irreversible change of hgb.
CELLULAR ENERGETICS
 Maintenance
of hgb function requires
active RBC metabolic pathways for ATP
production. ATP is required for:




Maintaining hgb in the reduced form
Membrane integrity and deformability
Maintaining the RBC intracellular volume
Producing adequate amounts of NADH,
NADPH, and GSH
 RBCs
generate energy almost exclusively
from the anaerobic breakdown of glucose 4 metabolic pathways are important for
maintaining cellular energetics.
CELLULAR ENERGETICS


Glycolysis- generates 90% of the required ATPthe breakdown of 1 glucose generates 2 ATP
and 2 NADH.
Hexose monophosphate shunt (pentose
phosphate shunt) - 5- 10% of the glucose is
metabolized this way. It produces NADPH
and GSH which protect the RBC from
oxidative injury.

If the concentrations of these are too low, the globin
will denature and precipitate in the cell. This is seen
as Heinz bodies which attach to the membrane
causing membrane damage and RBC destruction.
CELLULAR ENERGETICS

Inherited defects in the pathway result in the
formation of Heinz bodies with subsequent
extravascular hemolysis (more on this later).
Heinz bodies can only be seen with a supravital stain
such as new ethylene blue.
 The most common deficiency is Glucose-6-Phosphate
Dehydrogenase deficiency.

Heinz
bodies
CELLULAR ENERGETICS

Methgb Reductase Pathway- maintains iron in
the reduced functional state.
There are 2 pathways, the NADH and the NADPH
reductase pathways. They are dependent upon NADH and
NADPH respectively.
 In the absence of the enzymes or NADH and NADPH,
methgb, which can't transport O2, is formed.


Leubering-Rapoport shunt - causes the
accumulation of 2,3 DPG which is important in
decreasing the hgb affinity for O2 during O2
unloading.
ERYTHROCYTE KINETICS
 The
normal erythrocyte concentration
varies with age, sex, and geographic
location.



There is a high RBC count at birth which
decreases until the age of 2-3 months where
physiologic anemia is seen due to low levels of
erythropoietin production.
The RBC count will then gradually increase
until adult levels are reached at about 14
years of age.
Males have higher RBC counts because
testosterone stimulates erythropoietin
production.
ERYTHROCYTE KINETICS

A
Individuals living at high altitudes have
increased RBC levels because of the decreased
partial pressure of O2 at high altitudes which
leads to decreased O2 saturation.
decrease in RBC mass and therefore, a
decrease in hemoglobin concentration
results in tissue hypoxia and can lead to
anemia. Anemia is not necessarily a
diagnosis in itself, but is a clinical sign of
many different pathologies.
ERYTHROCYTE KINETICS
 An
increase in RBC mass is called
polycythemia and it may lead to an
increase in blood viscosity.

Polycythemia may be relative or absolute
Relative polycythemia occurs with a decreased
plasma volume. This occurs with dehydration.
 Absolute polycythemia results from an actual
increase in RBC mass. This may occur in disorders
that prevent adequate tissue oxygenation such as:
 High affinity hemoglobins
 Pulmonary disorders
 Occasionally this is due to a primary defect
resulting in an unregulated proliferation of RBCs
(polycythemia vera)

ERYTHROCYTE DESTRUCTION
 RBC
destruction is normally the result of
senescsence.




Each day ~ 1% of the RBCs are removed and
replaced.
RBC aging is characterized by decreased
glycolytic enzyme activity which leads to
decreased energy production and subsequent
loss of deformability and membrane integrity.
90% of aged RBC destruction is extravascular
and occurs mainly in the phagocytic cells in
the spleen, with a small amount occurring in
the liver and bone marrow.
5-10% of RBC destruction is intravascular,
occurring in the lumen of the blood vessels
Extravascular destruction of RBCs
EXTRAVASCULAR DESTRUCTION OF RBCS
Intravascular destruction of RBCs
INTRAVASCULAR
DESTRUCTION OF
RBCS