POWERPOINT

CHAPTER 9
ERYTHROCYTE METABOLISM
Younger RBCs

Have mitochondria
Energy is obtained through

Krebs cycle

Citric acid cycle

Also known as tricarboxylic acid cycle (TCA
cycle), or Szent-GyrgyiKrebs cycle
Series of chemical reactions used by
all aerobicorganisms to generate energy
through the oxidization of acetate derived
from carbohydrates, fats, and proteins into
carbon dioxide

Oxidative phosphorylation

Metabolic
pathway that
uses energy
released by the oxidation of nutrients to
produce adenosine triphosphate

Embden-Meyerhof Pathway

Reticulocytes and Mature RBCs

No nuclei
Source of energy is through
Anaerobic glycolysis
Aerobic glycolysis
Glycolysis
Breakdown of glucose to lactate and
pyruvate

Necessary for generation of ATP

Necessary for RBC to maintain
Hemoglobin function
Membrane
integrity
deformability
RBC volume

RBC METABOLIC PATHWAYS

Embden-Meyerhof pathway
Rapaport-Luebering pathway
Methemoglobin Reductase pathway
Hexose Monophosphate pathway

RBC METABOLISM

90 to 95% of ATP
Aerobic and anaerobic pathway
When 1 mol of glucose breaks down to
lactic acid
Needs 2 ATPs provides 4 ATPs
Net gain of 2 ATPs
When deficiencies of the E-M path exist,
RBC survival is reduced, leading to
hemolysis
Examples of deficiencies
Pyruvate kinase deficiency
Glucose
phosphate
isomerase
deficiency
Both lead to chronic hemolytic
anemia
Also generates NADH which is used in
other metabolic pathways
Blood bank stored blood has decreased
cellular ATP, leading to shorter survival in
transfused patients

1st stage
and

Phosphoryalation isomerization
diphosphorylation
Product is glyceraldehyde-3-phosphate

2nd stage

Product is 2,3-BPG or 3-PG

When 2,3-BPG is produced, there
are less ATPs

3rd stage

Methemoglobin Reductase Pathway

Product is pyruvate

*The next 3 are diversion pathways

Hexose Monophosphate Pathway

Provides 10% of the total energy of RBCs

Also known as Pentose Phosphate Shunt
Diverts G6PD to Pentose phosphate
Produces (reduced) NADPH and reduced
glutathione
G6PD
Provides only means to generate
NADPH and reduced Glutathione
Produces pyridine nucleotides
One of the main lines of defense for
RBC against oxidative injury which
may be caused by infections or
oxidant drugs
Deficiency in this pathway results in
deficiency of glutathione which results in
globin denaturation and precipitation as
aggregates inside the RBC (Heinz bodies)
Ultimately combats the effects of oxygen
stresses (environmental, medications)
If the shunt is defective (as is the case in
patients with G6PD deficiency) oxidative
insults lead to oxidation of globin chains and
denaturation of Hgb leading to precipitates
(Heinz bodies) in the RBC, membrane
damage and, ultimately, cell death

Prevents iron of Hgb from being oxidized

(maintains ferrous state)
Makes NADH which has reducing power
MHb or Hi : 2% (normal condition)
40% is detrimental to patient
Important in maintaining heme iron in the
reduced or ferrous functional state
Hgb iron must be in reduced state
(Fe2+) in order to transport O2
Environment is constantly generating
oxidant stress and therefore a tendency to
oxidize iron to Fe3+
Methemoglobin
reductase
pathway
counteracts this by reducing iron to ferrous
state
Methemoglobin reductase reduces iron as
NADH is converted to NAD
Patients with methemoglobin reductase
deficiency have a substantial quantity of
methemoglobin
Methemoglobin
Hgb with iron in the oxidated state
Associated with reduced O2 carrying
capacity
Non-functional, having lost ability to
transport oxygen
Dependent on the hexose monophosphate
pathway for production of pyridine
nucleotides
In absence of enzyme methemoglobin
reductase, methemoglobin is accumulated

Rapaport-Luebering pathway

Generates 2,3-DPG
Controls affinity of Hgb to O2
2,3-DPG is an important regulator of HgbO2 release
Increased 2,3-DPG, increased O2
release
An increased rate of glycolysis leads to an
increase
in
intracellular
2,3-DPG
concentration
When venous blood is increasingly
deoxygenated, the rate of glycolysis
increases leading to increased 2,3-DPG

Appropriate response
adequate O2 delivery

to

ensure

RBC MEMBRANE

Maintain cell shape and deformability

RBCs stretched 2.5 times so they
can enter capillaries
Maintain osmotic balance b/w plasma and
cytoplasm
Acts as skeletal support for Ag and
receptors
Transports ions and gases
Composition
Lipids: 40%
Proteins: 52%
CHO:
8%
responsible
for
metabolism

Integral/Transmembranous

Lipids

Esterified cholesterol (for tensile strength)

Reduces fluidity of the RBC
membrane
Phospholipids
Phosphatidylcholine,
Phosphatidylethanolamine,
Phosphatidylserine, Sphingomyelin
Maintains
microviscosity
of
membrane
Fatty acids and glycolipids
Gives blood Antigenic properties
(ABH and Lewis Ags)

*NOTES
Cholesterol

Acanthocytes
Enzyme that maintains cholesterol
level is deficient
RBC membrane loses
tensile
strength)
Target cells
More elastic cells due to decrease
cholesterol
Seen in liver disease

Proteins

Shape and deformability of RBC membrane

Pumps and channels for ions and other

substances
To differentiate
Rupture the membrane
PAGE

Polyacrylamide
Gel
Electrophoresis
Stain with Coomasie blue and PAS
(glycophorins)

Penetrates the bilipid layer

Glycophorins M,N, Ss
Glycophorins (A,B,C,D) has sialic
acid residues which is responsible
for zeta potential or net negative
charge
Protein 3 (Band 3) ABO and Ii
Anion exchange channel, attaches
CHON to bilipid layer

Peripheral/Skeletal/Cytoskeletal

Spectrin
Actin
Protein 4.1
Ankyrin
G-3-PD
Adducin
Tropomyosin
Tropomodulin

NOTES
*Spectrin and actin

Structural CHONS, forms the skeletal

framework of the membrane, forms the
biconcave shape

*Ankyrin, G3PD, Protein 4.1

Attaches the structural CHONS (spectrin

and actin ) to the lipid layer

*Cytoskeleton

Network of proteins that contributes shape,

strength, and function to a cell membrane
Elaborate cytoskeleton of erythrocytes gives
them their characteristic biconcave shape

A biconcave, or "donut" shape is an

effective compromise between the need for
gas exchange and the need to pack great
quantities of the oxygen-binding protein
hemoglobin into the cells
Also gives a red cell flexibility, strengthens
the cell membrane, and facilitates its
efficient travel through capillaries
A defect in any of several cytoskeletal
proteins can lead to a disease known as
spherocytosis, in which red cells assume a
spherical shape instead of the normal
biconcave or doughnut shape

*Spectrin, actin, and other associated proteins

Form a meshwork on the cytoplasmic side

of the membrane
To attach the meshwork to the membrane
itself, spectrin is attached to an intrinsic
protein called band 3, also known as the
anion exchanger, via a protein appropriately
called ankyrin and another called protein
4.2, or paladin
Protein, 4.1, helps with the binding of
spectrin to actin and may be responsible for
attaching the meshwork to the intrinsic
protein glycophorin C
Four abnormalities in erythrocyte membrane
proteins have been identified as causing
spherocytosis
Spectrin alone
Spectrin and ankyrin combined
Anion exchanger
Palidin
CHAPTER 10
PART 1: HEMOGLOBIN METABOLISM

HEMOGLOBIN

Conjugated globular protein

Constitutes about 95% of RBC's dry weight
About 65% of Hgb synthesis occurs during
nucleated stages of RBC maturation, and
35% occurs during the reticulocyte stage
Vehicle of transport of O2 in the body
Concentration within the RBC: 34 g/dL
Molecular weight : 64000 D

Approximately 1.34 mL of O2 is bound by 1g

of Hgb
1g Hgb = 3.47 mg Fe
Normal values
Females around 12-15 g/dL
Males around 14-18 g/dL

Hemoglobin consists of
*Globin

A tetramer
Two pairs of unlike globin polypeptide
chains
A protein constituent of hemoglobin
There are 4 globin chains
in the
hemoglobin (Hgb) molecule
Comprises 141 to 146 amino acids
Alpha and zeta chains 141 aa long
Beta, gamma, delta 146 aa long
Epsilon unknown

*Heme

Four heme groups

Each
of
which
contains
a
protoporphyrin ring plus iron
The iron-containing protoporphyrin portion
of the Hgb wherein the iron is in the ferrous
(Fe2+) state

HEMOGLOBIN STRUCTURE
Conformation

Primary type, number, and sequence of

amino acids
Secondary chains arrangements
Tertiary 3-D arrangement of 2 structure
Quaternary complete structure

HEMOGLOBIN SYNTHESIS
Depends on three processes

Adequate supply and delivery of iron

Adequate synthesis of protoporphyrins
Adequate globin synthesis

Iron delivery and supply

Iron delivered to membrane of RBC

precursor by protein carrier transferrin
Most of the iron that crosses membrane and
enters cytoplasm of cell is committed to
hemoglobin synthesis
Proceeds to mitochondria for insertion into
protoporphyrin ring to form heme
Excess iron in cytoplasm aggregates as
ferritin
Amount of ferritin stored depends on
ratio between level of plasma iron
and amount of iron required by
erythrocyte for hemoglobin synthesis
Two-thirds of total iron supply is bound to
heme in hemoglobin
Heme
Protoporphyrin IX ring of C, H, N
Ferrous iron

Synthesis of Protoporphyrins

Begins in mitochondria with formation of

delta-aminolevulinic from glycine and
succinyl coenzyme A
Enzyme delta aminolevulinic acid
synthetase mediates this reaction
Amount is influenced by EPO and
Vitamin B6 (pyridoxal phosphate)

Zeta Alpha
Epsilon Gamma Delta Beta

*Structure of Protoporphyrins

Porphyrinogens are intermediate products

in heme synthesis
Porphyria
If any one of normal enzyme steps in
heme synthesis blocked, excessive
formation of porphyrins can occur
Protoporphyrinogen IX is last porphyrinogen
formed and becomes Protoporphyrin IX

The various globins that combine with heme

to form Hb are all single chain polypeptides
The synthesis of these globins is under
genetic control
Synthesized in ribosomes
The -like cluster (, , , and globin
genes) on the short arm of chromosome 11

Each globin chain links with heme molecule

(protoporphyrin ring with iron) to form
hemoglobin
Entire hemoglobin molecule has two alpha
chains, two beta chains, and four heme
groups
Precise order of amino acids in globin
chains is critical to hemoglobin molecule's
structure and function
Disorders
Thalassemia

Quantitative
disorders
Hemoglobinopathies Qualitative
disorders

*Ontogeny of Globin Synthesis

Globin Synthesis

The -like cluster ( and globin genes) on

the short arm of chromosome 16
Various globin chains of Hgb differ in the
charge per molecule
Hgb electrophoresis under the influence of
electric fields, Hgbs exhibit different
mobilities, allowing differentiation of subtype
It may be necessary to use different assay
procedures (support media, buffer, pH) for
definitive identification
Throughout
embryonic
and
fetal
development, activation of globin genes
progresses from zeta to alpha, and from
epsilon to gamma to delta to beta

Globin synthesis is first detected in the

primitive erythroid precursors of the yolk sac
at about 3 weeks gestation
Embryonic
Hemoglobin Gower I zeta2,
epsilon2
Hemoglobin Portland zeta2,
gamma2
Hemoglobin Gower II alpha2,
epsilon2
Fetal
HbF alpha2, gamma2
HbA alpha2, beta2
Adult

HbA
HbA2 alpha2, delta2
HbF

ABNORMAL HEMOGLOBIN VARIANTS

HbS

Alpha 2, beta 2 (6th Glu to Val)

HbC

Alpha 2, beta 2 (6th Glu to Lys)

Hb Bart

Tetramer of gamma
Abnormal variant of HbF

HbH

Tetramer of beta

HbM

PART 2: HEMOGLOBIN FUNCTION

HEMOGLOBIN FUNCTION

Alpha 2 (58th His to Tyr), beta 2

2,3-DIPHOSPHOGLYCERATE

Formation of hemoglobin requires iron,

globin chains, protoporphyrin IX, and 2,3DPG
To assemble molecule, ferric iron (Fe3+)
must be obtained from ferritin
Iron is chemically reduced (Fe2+), and then
inserted as ferrous iron into center of
protoporphyrin IX molecule
When globin chain completed on ribosome,
it is released to cytoplasm
Individual alpha and beta chains quickly and
spontaneously form alpha-beta dimers
Two heme molecules bind to each alphabeta dimer
Two dimers quickly form a tetramer and
assume final three dimensional shape
Last step is insertion of 2,3-DPG molecule
into center cavity of hemoglobin molecule.

Organic
phosphate
responsible
for
hemoglobin's affinity for oxygen
Derived from Luebering-Rapaport shunt.
Located in the central cavity of hemoglobin
molecule.
Bound to beta chains

Relaxed form
B chains are
closer
together

Tense form
B chains are
farther

Tense form

ASSEMBLY OF HEMOGLOBIN MOLECULE

Primary function is delivery and release of

oxygen to tissues and the facilitation of
carbon dioxide excretion
One of most important controls of
hemoglobin affinity for oxygen is RBC
organic phosphate: 2,3-DPG
Hemoglobin-oxygen affinity is expressed by
P50 values
P50 is point at which hemoglobin is
50% saturated with oxygen
Increase in P50 values represents a
decrease in hemoglobin-oxygen
affinity (shift to right)
Decrease in P50 values represents
increase
in
hemoglobin-oxygen
affinity (shift to left)
In lungs, where pO2 (oxygen tension) is
very high, hemoglobin is almost 100%
saturated with oxygen
As RBCs travel to tissues where oxygen
tension drops, hemoglobin saturation drops
to about 75%, releasing oxygen to tissues

Unloading of oxygen by hemoglobin is

accompanied by widening of space between

beta chains and binding of 2,3-DPG to beta

chains
Has lower oxygen affinity
Also known as deoxyhemoglobin

Relaxed form

When hemoglobin binds oxygen, 2,3-DPG

and beta chain bonds break, Beta chains
close up and 2,3-DPG expelled
Has high affinity for oxygen
Also called oxyhemoglobin

THE BOHR EFFECT

*NOTE

Relationship between hemoglobin and

oxygen has a sigmoid curve
Shape of curve means lots of oxygen can
be delivered to tissues with small drop in
oxygen tension

Shift to the right

In hypoxia, a compensatory "shift to right" of

hemoglobin dissociation curve occurs to
relieve tissue oxygen deficit
Right shift of curve, mediated by increase in
2,3-DPG,
results
in
decrease
in
hemoglobin's affinity for oxygen and an
increase in oxygen delivery to tissues
Shifts to right commonly occur
Hypoxia
Anemia
Acidosis
Rise in body temperature

Shift to the left

The effect pH has on Hgb-oxygen affinity

Increase pH shift to left (increased Hgb
affinity for O2)
Hyperventilation
Decrease pH shift to right (decreased
Hgb affinity for O2)
Exercise
Renal failure

Conversion between tense form and relaxed

form referred to as respiratory movement

OXYGEN DISSOCIATION CURVE

In "shift to left" of hemoglobin dissociation

curve, there is an increase in hemoglobinoxygen affinity and decrease in amount of
oxygen being delivered to tissues
Conditions causing
Alkalosis
Increase in the amount of abnormal
hemoglobins (methemoglobin or
carboxyhemoglobin)

Increased amount of Hemoglobin F

Multiple transfusions of 2,3-DPG
depleted stored blood

PART 3: CHEMICALLY MODIFIED

HEMOGLOBINS
Carboxyhemoglobin

Oxygen bound to hemoglobin replaced by

carbon monoxide (CO)
Replacement process relatively slow and
dependent upon blood concentration of
carbon monoxide
Heme binds to carbon monoxide about 200
times tighter than it binds to oxygen
Reversible condition (oxygen inhalation)

*Values

Reference interval: 0.2-0.8%

Smokers: 4%-20%
Toxic levels: 10-15%
Coma and convulsions: more than 50%
Cherry red color
Measured at 541 nm

Methemoglobin

Formed when iron of hemoglobin molecule

oxidized to ferric state (Fe3+)
Methemoglobinemia
Acquired
Hereditary Hgb M
Congenital reduction of Mhb
reductase enzyme

Acquired

Due to strong oxidizing drug

Reversible condition

Administration of strong reducing

substances such as ascorbic acid
and methylene blue

*Values

Normal concentration: 1% of the total Hgb

Greater than 30%: hypoxia and cyanosis
Brownish to bluish
Measured at 620 to 640 nm at pH 7.1

Sulfhemoglobin

*Acid Hematin

Occurs when sulfur content in blood builds

up (ingestion of sulfur-containing drug or
chronic constipation)
Irreversible condition (RBCs must be
removed from circulation)
Ineffective for O2 transport (100x less
affinity)
Measured at 620 nm
Green in color but when mixed in blood
becomes mauve/lavander

Dares

Uses 0.1 N HCl

Add HCl to blood
Add distilled water until same color with
standard
HgbF resists acid elution therefore it is not
recommended for measuring newborns
Hgb
Elution/Kleuhauer betke

*Alkaline Hematin

Uses 0.1 N NaOH

Denaturation

Photometric

Uses spectrophotometry

*Oxyhemoglobin

Uses 0.007 N NH4OH read at 540 nm

HEMOGLOBIN MEASUREMENT
*Cyanmethemoglobin
Copper sulfate or Specific Gravity method

Copper sulfate sp. g. is 1.053

Blood sinks at once not qualified
Floats not qualified
Stationary then gradually sinks qualified

Iron Content (Kennedy/Wongs) method

=

(/)
3.47 /

O2 combining (Van Slyke/Gasometric) method

=

2 /
1.34 /

Colorimetric Method

Convert 1st Hgb to its derivatives

Visual
*Direct matching

Blot and match with color

Tallquist

Uses Drabkins reagent

Most reliable
Measures all Hgb except sulfhemoglobin
With K ferricyanide ferrous to ferric
KCN + ferric Hgb = cyanmethemoglobin
Read at 540nm
Compare with standard
Calculate using Beers law
Automation
Uses sodium lauryl sulfate to convert
Hb to SLS-methemoglobin
[GAPANG MEDTECH]