Hematology I

ERYTHROCYTE METABOLISM AND ATP

MEMBRANE STRUCTURE AND FUNCTIONS
RBC •
Mechanism that slow the oxidation of
proteins and iron by environment peroxides
• 5 million erythrocyte per microliter of and superoxide anions, maintaining
circulating blood making the RBC the hemoglobin’s function and membrane
primary cell in blood integrity
o Lack nucleus Oxidation
o Biconcave shape • Limiting the RBC circulating life span of 120
o Supports deformation days
o Enabling the circulating cell to pass
smoothly through capillaries Disassembled into its reusable components:
o Exchanges oxygen (o2) and carbon • Globin chains
dioxide (co2) contacting the vessel • Iron from hemoglobin
wall • Phospholipids
o transport carbon dioxide and • Proteins from the cell membrane
bicarbonate from the tissue back to Protoporphyrin
the lungs • Not reusable and excreted as bilirubin
o Average volume of 90 fl
o Cytoplasm of the RBC ENERGY PRODUCTION - ANAEROBIC
▪ Transport oxygen from the GLYCOLYSIS
lungs to the tissue • lacking mitochondria
▪ Abundant hemoglobin o RBC relies on anaerobic glycolysis
▪ A complex f globin, for its energy
protoporphyrin and iron • Exchange of oxygen and carbon dioxide
▪ Produce through o A passive function from high partial
normoblastic proliferation pressure to low partial pressure
and mature in the bone • Energy production slows
marrow o RBC grows senescent and it
removed from the circulation
Hemoglobin • Hereditary glycolytic deficiencies of
glycolytic enzymes
• 4 globin chains: containing a heme
o Shortened RBC survival
molecule with iron in the ferrous state
o Known: collectively as hereditary
• Allows hemoglobin molecule to carry 4 O2
nonspherocytic hemolytic anemia
molecules
• Anaerobic glycolysis or EMP
• Nucleus, present in maturing normoblast
o Requires glucose to generate ATP
• Extruded as part of RBC maturation
• RBC
circulation
o Lack internal energy stores
• Cytoplasmic ribosome & mitochondria
o Rely on plasma glucose to enter the
• Disappear 24 to 28 hours after bone
cell to generate ATP
marrow release, eliminating the cells ability
• Glucose
to produce proteins r support oxidative
o GLUCOSE —enter—> RBC through
metabolism
facilitated diffusion —via
• Without mitochondria (aerobic respiration)
transmembrane protein Glut 1
• Via oxidative phosphorylation
o Catabolized to pyruvate (pyruvic
• ATP adenosine triphosphate: produced
acid) in the EMP generating 4
within the cytoplasm through aerobic
molecules at ATP per molecule of
glycolysis (Embden-Meyrhof pathway EMP)
glucose, for a net gain of 2
for lifetime.
molecules of ATP

1|P age
 Hematology I

• Glycolysis ▪ When the F1,6-BP is

o First phase of glycolysis plentiful, increased activity
o Employs: glucose phosphorylation, of PK favors pyruvate
isomerization, phosphorylation production
—yields—.> fructose 1,6 ▪ Pyruvate
bisphosphate (F1,6-BP) ▪ May diffuse from the
o Intermediate stage employs: erythrocyte or my become a
hexokinase, glucose-6-phosphate substrate for lactate
isomerase and 6- dehydrogenase (LD or LDH)
phosphofructokinase —-> steps with regeneration of
consume a total of 2 ATP molecules oxidized form of
and limit the rate of glycolysis nicotinamide adenine
o Fructose-bisphosphate aldolase— dinucleotide (NAD+)
cleaves—> F1,6-BP —TO ▪ Activity of LD
PRODUCE—> glyceraldehyde-3- ▪ The ratio of NAD+ to
phosphate (G3P) reduced form (NADH)
o Second phase
Glucose catabolism first phase
▪ Glucose catabolism —
Substrates Enzyme Products
converts—> Glyceraldehyde
Glucose, Hexokinase G6P, ADP
3-phosphate (G3P) TO 3-
ATP
phosphoglycerate (3-PG)
o G3P —> oxidized to 1,3 G6P Glucose-6- F6P
bisphosphoglycerate (1,3-BPG) phosphate
through the action of isomerase
Glyceraldehyde3-phosphate
dehydrogenase (G3PD)—-> wit F6P, ATP 6- F1,6-BP,
reduction of NAD to NADH Phosphofructokinase ADP
▪ 1,3 BPG
▪ Dephosphorylated by F1,6-BP Fructose-
phosphoglycerate kinase —- bisphosphate
> Generates 2 ATP aldolase
molecules and 3-pg
o Third phase *ADP, Adenosine diphosphate; ATP, adenosine
triphosphate; DHAP, dihydroxyacetone phosphate;
▪ 3-PG is isomerized by
F1,6-BP, fructose-1,6-bisphosphate; F6P, fructose-
phosphoglycerate mutase
6-phosphate; G3P, glyceraldehyde-3-phosphate;
—-> to 2-phosphoglycerate G6P, glucose-6-phosphate.
(2-PG)
▪ Enolase (phosphohydrolase Glucose catabolism Second phase
hydratase) converts 2-PG — Substrates Enzyme Products
> to phosphoenolpyruvate G3P, NAD Glyceraldehyde-3- 1,3-BPG,
(PEP) phosphate NADH
▪ Pyruvate kinase dehydrogenase
▪ Splits off the phosphates,
forming 2 ATP molecules 1,3-BPG, Phosphoglycerate 3PG, ATP
and pyruvate ADP kinase
▪ Allosterically modulated by 1,3-BPG Bisphosphoglycerate 2,3-BPG
increased concentration of mutase
F1,6-BP which enhances the
affinity of PK for PEP 2,3-BPG Bisphosphoglycerate 3-PG
phosphatase
2|P age
 Hematology I

*1,3-BPG, 1,3-Bisphosphoglycerate; 2,3-BPG, 2,3- o Enzyme, helps prevent damage in

bisphosphoglycerate; 3-PG, 3-phosphoglycerate; the RBC
ADP, adenosine diphosphate; ATP, adenos- ine o G6PD deficient: yield
triphosphate; G3P, glyceraldehyde-3-phosphate;
denaturation and precipitation of
NAD, nicotinamide adenine dinucleotide (oxidized
form); NADH, nicotinamide adenine dinucleotide the hemoglobin
(reduced form). o Heinz bodies- kind of RBC
abnormality
Glucose catabolism Third Phase ▪ One of the screened
Substrates Enzyme Products disorder for infants: G6PD
3-PG Phosphoglycerate 2-PG deficiency
mutase • Oxidative glycolysis
o occurs through a diversion of
glucose catabolism into the HMP,
2-PG Enolase PEP
also known as the pentose
(phosphopyruvate
hydratase phosphate shunt
PEP, ADP Pyruvate kinase Pyruvate, • HMP detoxifies peroxide (H2O2)
ATP o arises from O2 reduction in the
Pyruvate, Lactate Lactate, cell’s aqueous environment.
NADH dehydrogenase NAD o H2O2 oxidizes heme iron to the
non-functional ferric state and
oxidizes and denatures proteins
*The activity of lactate dehydrogenase to convert and lipids
pyruvate to lactate is modulated by the ratio of • Extends the functional life span of the RBC
NAD to NADH. by maintaining membrane
2-PG, 2-Phosphoglycerate; 3-PG, 3- o proteins and lipids
phosphoglycerate; ADP, adenos- ine diphosphate; o enzymes
ATP, adenosine triphosphate; NAD, nicotinamide o Hemoglobin iron in the functional
adenine dinucleotide (oxidized form); NADH,
• ferrous state
nicotinamide adenine dinucleotide (reduced form);
• Diverts glucose-6-phosphate (G6P) to
PEP, phosphoenolpyruvate.
6- phosphogluconate (6-PG)
GLYCOLYSIS DIVERSION PATHWAYS o by the action of glucose-6-
(SHUNTS) phosphate dehydrogenase (G6PD)
• Shunts, branch from the glycolytic pathway o In the process, oxidized
o Three alternate pathways nicotinamide adenine dinucleotide
1. hexose monophosphate phosphate (NADP) is converted to
pathway (HMP) its reduced form (NADPH).
2. methemoglobin reductase • NADPH is then available to reduce oxidized
pathway glutathione (GSSG) to reduced glutathione
3. Rapoport-Luebering (GSH) in the presence of glutathione
pathway reductase
• Glutathione
HEXOSE MONOPHOSPHATE PATHWAY o cysteine
• 5 to 10 % RBC energy o containing tripeptide, and the
• Provides: reduced glutathione designation GSH highlights the
o Detoxifying material sulfur in the cysteine moiety.
o Prevents denaturation of the o Reduced glutathione becomes
hemoglobin oxidized as it reduces peroxide to
• G6PD water and oxygen via glutathione
peroxidase
3|P age
 Hematology I

• During steady-state glycolysis electron carrier, returning the oxidized

o 5% to 10% of G6P ferric iron to its ferrous, oxygen-carrying
▪ diverted to the HMP state
o After oxidative challenge o Using H+ from NADH
▪ increase up to thirtyfold • More than 65% of the methemoglobin-
the HMP activity reducing capacity within the RBC
o HMP further catabolizes 6-PG to o enzyme accounts
ribulose 5-phosphate (R5P), carbon
RAPOPORT-LUEBERING PATHWAY
dioxide, and NADPH by the action
• Generation of the 2,3 DPG
of 6-phosphogluconate
• 2,3 DPG- regulates hemoglobin affinity in
dehydrogenase
the oxygen
• G6PD
o provides the only means of • Increase 2,3- DPG- decreased 02 affinity
generating NADPH for (right shift)
glutathione reduction, and in its o Right shift- cells that are normally
absence erythrocytes are in the bloodstream are increased
particularly vulnerable to oxidative number, larger than normal cells
damage ▪ Associated with increased
*NADP-oxidaized form Glucose Catabolism: Hexose
**NADPH-reduced form Monophosphate Pathway
Substrate Enzyme Product
METHEMOGLOBIN REDUCTASE PATHWAY s
• Focused in maintaining iron present in your (G6P), Glucose-6- (6-PG), 6-
hemoglobin in a functional state (iron glucose-6- phosphate Phosphogluco
content of the body which is ferrous phosphate dehydrogenase nate
and 6-
fe2+(more absorb by the body)
(NADP), phosphoglucono (NADPH)nico
▪ Important enzyme: methemoglobin nicotinamid lactonase tinamide
cytochrome b5 reductase e adenine adenine
o Maintaining iron in a reduced state dinucleotid dinucleotide
o Responsible for the reduction of e phosphate
methemoglobin phosphate (reduced
• Heme iron (oxidized form)
form)
o constantly exposed to oxygen and
(6-PG),6- 6- (R5P),
peroxide
Phosphoglu Phosphoglucona ribulose 5-
• Peroxide oxidizes heme iron from the
conate te phosphate
ferrous (2+) to the ferric (3+) state dehydrogenase
• methemoglobin (NADP), (NADPH)nico
o iron state is ferric, the affected nicotinamid tinamide
hemoglobin molecule e adenine adenine
• HMP prevents hemoglobin oxidation by dinucleotid dinucleotide
reducing peroxide, it is not able to reduce e phosphate
methemoglobin once it forms phosphate (reduced
o Reduction of methemoglobin by (oxidized form)
NADPH is rendered more efficient in form)
the presence of methemoglobin CO2
mature neutrophils
reductase, also called cytochrome
b5 reductase. (polymorphonuclear cells)-
• G3P is converted to 1,3-BPG, cytochrome increased number of
b5 reductase acts as an intermediate nuclear lobes
4|P age
 Hematology I

▪ Associated: Severe liver • Acidic pH and low concentrations of 3-PG

disease and advanced and 2-PG
anemia o inhibit the activity of
o Left shift - lesser oxygen in the bisphosphoglycerate mutase, thus
blood steam inhibiting the shunt and retaining
▪ Associate with increased 1,3-BPG in the EMP.
number of immature WBC, o These conditions decreased ATP
activate bisphosphoglycerate
indicates infection or
phosphatase, which returns
inflammation
2,3-BPG to the glycolysis
• Decrease 2,3 DPG- increase 02 affinity (left
mainstream
shift) • These conditions favor generation of ATP
• third metabolic shunt by causing the conversion of 1,3- BPG
• shunt generates 2,3-bisphosphoglycerate directly to 3-PG and returning 2,3-BPG to
(2,3- BPG0 3-PG for ATP generation downstream by
o Also called 2,3- PK.
diphosphoglycerate or (2,3-
DPG) EMBDEN-MEYERHOF
• diverted by bisphosphoglycerate mutase to PATHWAY
form 2,3-BPG ● primary source of energy, primary way of
o 2,3-BPG binds between the globin how red blood cells obtain energy
chains in the interior cavity of the ● Anaerobic glycolysis
hemoglobin tetramer to stabilize it ● 90-95% how the red blood cells gain it’s
in the deoxygenated state (tense or required energy
low oxygen affinity state). ● Glucose——>2 ATP’s (lactic acid) waste
o binding shifts the hemoglobin- product ———> (yielded) controls Na & K
oxygen dissociation curve to the
- prevents oxidation of the membrane lipids
right, which enhances delivery of
RBC MEMBRANE
oxygen to the tissues
• RBCs are biconcave
• 2,3-BPG forms 3-PG
• 7 to 8 µ in diameter
o by the action of
• Volume range of 80 to 100 fL and a mean
bisphosphoglycerate phosphatase.
volume of 90 fL
o diversion of 1,3-BPG to form 2,3-
• Their average surface area is 140 µm ,
BPG sacrifices the production of
which is a 40% excess of surface area
two ATP molecules. There is
compared with a sphere of 7 to 8 µ in
further loss of two ATP molecules
diameter
at the level of PK, because fewer
• This excess surface area-to-volume ratio
molecules of PEP are formed.
enables RBCs to stretch undamaged up to
o two ATP molecules were used to
2.5 times their resting diameter as they
generate 1,3-BPG
pass through narrow capillaries and
o production of 2,3-BPG eliminates
through splenic pores 2 µm in diameter.
the production of four ATP
This property is called RBC deformability.
molecules, the cell is put into ATP
• RBC Plasma Membrane
deficit by this diversion.
o 100 times more elastic than a
• Delicate balance between ATP generation
comparable latex membrane, yet it
o to support the energy requirements
has tensile (lateral) strength
of cell metabolism and the need to
greater than that of steel.
the maintenance
• The deformable RBC membrane provides
o oxygenation and deoxygenation
the broad surface area and close tissue
status of hemoglobin.
5|P age
 Hematology I

contact necessary to support the delivery o Resides parallel to the acyl tails of
of O2 from the lungs to body tissues and to the phospholipids
transport CO2 from body tissues to the o It is equally distributed between the
lungs. outer and inner layer
• RBC deformability depends not only on RBC o Evenly dispersed within each layer
geometry but also on relative cytoplasmic o Approximately one cholesterol
(hemoglobin) viscosity. molecule per phospholipid molecule
• The normal mean cell hemoglobin • β-hydroxyl
concentration (MCHC) ranges from 32% to o A group of cholesterol that anchors
36% and as MCHC rises, internal viscosity within the phospholipid polar head
rises. • As cholesterol rises, the membrane gains
• MCHCs greater than 36% compromise strength but loses elasticity
deformability and shorten the RBC life span • Membrane enzymes maintain the
because viscous cells become damaged as cholesterol concentration by regularly
they stretch to pass through narrow exchanging membrane and plasma
capillaries or splenic pores. cholesterol.
• As the MCHC rises, the RBC, unable to pass • Deficiencies in these enzymes are
through the splenic pores, is phagocytized associated with RBC membrane
and destroyed by splenic macrophages abnormalities such as acanthocytes and
target cells (codocytes)
RBC MEMBRANE LIPIDS
• Phospholipids are asymmetrically
• Consists of approximately 8%
distributed
carbohydrates, 52% proteins, and 40%
• Phosphatidylcholine and sphingomyelin
lipids.
predominate in the outer layer
• The lipid portion, equal parts of cholesterol
• Phosphatidylserine and
and phospholipids, forms a bilayer
phosphatidylethanolamine form most of
universal to all animal cells
the inner layer
• Hydrophilic Polar Head Group
o Arrayed on the membrane’s
surfaces
o oriented toward both the aqueous
plasma and the cytoplasm
o Depicted in the fluid mosaic
membrane model (FMMM)
• Hydrophobic Nonpolar Acyl Tails
o Arranged themselves a form a
central layer sequestered from the • When phospholipid distribution is
aqueous plasma and cytoplasm disrupted, as in sickle cell anemia and
• The phospholipids provide a dynamic thalassemia or in aging RBCs, PS, the only
fluidity to the membrane; if a portion of the negatively charged phospholipid,
lipid bilayer is lost, the membrane can self- redistributes to the outer layer.
seal to retain the cytoplasmic contents • Splenic macrophages possess receptors
• The membrane also maintains extreme that bind to the PS displayed on senescent
differences in osmotic pressure, cation and damaged RBCs and remove them from
concentration between external plasma circulation.
and the cytoplasm through the interaction • C-reactive protein and inflammatory
of the lipids and proteins conditions increase the PS distribution in
• Cholesterol the outer layer of the RBC membrane
o Esterified and largely hydrophobic leading to increased RBC death (also called
eryptosis).
6|P age
 Hematology I

• Redistribute laterally so that the RBC • Any change affecting adhesion proteins
membrane may respond to stresses and permits RBCs to adhere to one another and
deform within 100 milliseconds of being to the vessel walls, promoting
challenged by the presence of a narrow fragmentation (vesiculation), reducing
passage, such as a capillary. membrane flexibility, and shortening the
• In liver disease, membrane cholesterol RBC life span
concentration becomes increased because • Signaling receptors
of an increased plasma bile salt o Binds plasma ligands
concentration. o Trigger activation of intracellular
• Glycolipids signaling proteins, which initiate
o (sugar-bearing lipids) make up 5% various energy-dependent cellular
of the external half of the RBC activities called signal
membrane transduction
o They associate in clumps or rafts • Glycosylation
and support carbohydrate side o Through this, transmembrane
chains that extend into the aqueous proteins also support surface
plasma to anchor the glycocalyx carbohydrates, which join with
• Glycocalyx glycolipids to make up the
o layer of carbohydrates whose net protective glycocalyx
negative charge prevents microbial • Most transmembrane proteins assemble
attack and mechanical damage into one of the two major macromolecular
caused by adhesion to neighbor complexed named by their respective
RBCs or to the endothelium. cytoskeletal anchorages: the ankyrin
complex and the actin junctional complex,
RBC MEMBRANE PROTEINS
also called protein 4.1 complex
• Transmembrane (integral) and cytoskeletal
• The anchoring of these transmembrane
(skeletal, peripheral)
complexes to cytoskeletal proteins
o Make up 52% of the membrane
(adjacent to the inner or cytoplasmic side
structure by mass
of the membrane) prevents loss of the lipid
• Proteomic study
bilayer
o Revealed that there are at least 300
• The linking of cytoskeletal proteins by the
RBC membrane proteins, including
actin junctional complex provides
105 transmembrane proteins
membrane structural integrity
o Some proteins have a few hundred
because the cell relies on an intact
copies per cell, and others have
cytoskeleton to maintain its biconcave
more than a million copies per cell
shape despite deformability
• Of the reported 300 membrane proteins,
• Provide vertical membrane structure
about 5- have been characterized and
named
Blood group antigens
Transmembrane Proteins • Located in membrane macromolecules
complexes that serve as:
• Serve many functions:
o Transporters
o Transport sites
o Structural components
o Adhesion sites
o Enzymes
o Signaling receptors
o Receptors
• Any disruption in transport protein function
o Adhesion molecules
changes the osmotic tension of the
• Transmembrane proteins
cytoplasm, which leads to a rise in
viscosity and loss of deformability

7|P age
 Hematology I

o Supports carbohydrate-defined o Few copies of this reside in the

blood group antigens in the RBC outer, plasma-side layer of the
membrane membrane
o Approximately 25 (half) are o Serves as a base on which a glycan
involved in the macromolecular core of sugar molecules is
complexes that define blood synthesized, forming the
antigen groups glycosylphosphatidylinositol
• Support the majority of ABH system (GPI) anchor
carbohydrate determinants: • More than 30 proteins
o Band 3 (anion transport) o Bind to the GPI anchor and appear
o Glut-1 (glucose transport) to float on the surface of the
• Several transmembrane proteins provide membrane
peptide epitopes o 2 of these protect the RBC
o Glycophorin A membrane from lysis by
• Carries the peptide-defined complement
M • Decay-accelerating factor
o N determinants and glycophorin B (DAF, or CD55)
• Carries the Ss determinants, • Membrane inhibitor of
which together comprise reactive lysis
the MNSs system (MIRL, or CD59)
o Minor glycophorins C and D • Phosphatidylinositol glycan anchor
• Carry the Gerbich system biosynthesis class A
antigens (PIGA)
o Rh system o Gene codes for a
• 2 transmembrane glycosyltransferase required to add
lipoproteins N-acetylglucosamine to the PI base
• 1 multipass glycoprotein early in the biosynthesis of the GPI
• Crosses the membrane 12 anchor on the membrane
times • Paroxysmal nocturnal hemoglobinuria
• The 2 lipoproteins present o An acquired mutation in the PIGA
the D and CcEe epitopes gene affects the cells’ ability to
o Requires the synthesize the GPI anchor
separately inherited • Without the GPI anchor, the cell membrane
glycoprotein RhAG, becomes deficient in CD55 and CD59, and
which localizes near the cells are susceptible to complement
the Rh lipoproteins mediated hemolysis
in the ankyrin
complex Nomenclature
• Loss of the RhAG glycoprotein prevents • Preproteomics
expression of both the D and CcEe antigens o Protein identification techniques
(Rh-null) and is associated with RBC that distinguished bilayer
morphologic abnormalities membrane to the spectrin
• Additional blood group antigens localize to cytoskeleton through ankyrin
the acting junctional (4.1) complex or • Major components of the actin junctional
specialized proteins (4.1) complex
o Band 3
The GPI anchor and paroxysmal nocturnal o Protein 4.3
hemoglobinuria o Adducin
• Phosphatidylinositol (PI) o Actin

8|P age
 Hematology I

• Dematin o Forms short filaments of 14 to 16

o Links with transmembrane protein monomers whose length is regulated
Glut-1 by tropomyosin
• Adducin and tropomodulin
Cytoskeletal Proteins o Cap the ends of actin
• Principal cytoskeletal proteins • Dematin
o α-spectrin o Appears to stabilize the actin junctional
o β-spectrin complex and helps maintain the RBC
o These assemble to form an antiparallel shape
heterodimer held together with a series
of lateral bonds Membrane deformation
• Antiparallel • Spectrin dimer bonds that appear along the
o Means that the carboxyl (COOH) end of length of the molecules disassociate and
one strand associates with the amino reassociate (open and close) during RBC
(NH3) end of the other, and the two deformation
heterodimers self-associate head-to- • The 20 α-spectrin and 16 β-spectrin
head to form a tetramer repeated helices unfold and refold
• The ends of the spectrin tetramers are linked • Key regulators of membrane elasticity and
in the actin junctional complex, forming a mechanical stability
hexagonal cytoskeletal lattice adjacent to the o Flexible interactions plus the
inner (cytoplasmic) lipid bilayer spectrinprotein 4.1 junctions in the
o Provides lateral or horizontal actin junctional complexes between
membrane stability the tetramers
• Peripheral proteins • Abnormalities in any of these proteins
o Because the cytoskeletal proteins do result in deformation-induced
not penetrate the bilayer membrane fragmentation
o • Hereditary elliptocytosis
Spectrin stabilization o Arises from one of several
• The secondary structure of both α- and β- autosomal dominant mutations
spectrin features triple-helical repeats of 106 affecting the spectrin dimer-to-
amino acids each; 20 such repeats make up %- dimer lateral bonds or the
spectrin, and 16 make up β-spectrin spectrinprotein 4.1 junction
• Essential to the cytoskeleton are the previously • Horizontal interaction
mentioned: o Defects inhibit the membrane’s
o Anykrin ability to rebound from deformation
o Protein 4.1 • RBCs progressively elongate to form
o Adducin visible elliptocytes, which causes a mild to
o Dematin severe hemolytic anemia
o Actin • Autosomal dominant mutations that affect
o Tropomysin the integrity of band 3, ankyrin, protein
o Tropomodulin 4.2, or α- or β-spectrin are associated with
• Single helix at the amino terminus of α-spectrin hereditary spherocytosis
o Consistently binds a pair of helices at o In these cases, there are too few
the carboxyl terminus of the β-spectrin vertical anchorages to maintain
chain, forming a stable triple helix that membrane stability
holds together the ends of the • The lipid membrane peels off in small
heterodimers fragments called blebs, or vesicles and
• Actin immediately reseals keeping the
cytoplasmic volume intact

9|P age
 Hematology I

o Results in: reduced surface area- • Defects in the ion channels result in red cell
tovolume ratio and the formation of volume disorders:
spherocytes o Overhydrated stomatocytosis
(hereditary hydrocytosis)
OSMOTIC BALANCE AND PEARMEABILITY o Dehydrated stomatocytosis
• RBC membrane (hereditary xerocytosis)
o Impermeable to cations Na+, K+, • Sickle cell disease
and Ca2+ o Also provides an example of
o Permeable to water and the anions increased cation permeability
bicarbonate (HCO3-) and chloride o When hemoglobin S polymerizes on
(Cl-), which freely exchange deoxygenation, the cell deforms
between plasma and RBC into a sickle shape and the
cytoplasm membrane becomes more
• Aquaporin 1 permeable to Ca 2+

o A transmembrane protein that • This causes a downstream

forms pores or channels whose effect of increased Na+ and
surface charges create inward K+, resulting in hemolysis
water flow in response to internal
osmotic changes
o Decreases in aquaporin 1
expression has been linked with
hereditary spherocytosis
• ATP-dependent cation
o Pumps Na+ -ATPase and K+ -
ATPase
o Regulate the concentrations of Na+
and K+, maintaining intracellular-to-
extracellular ratios of 1:12 and
25:1
• Ca2 -ATPase
+

o Expels calcium from the cell,

maintaining low intracellular levels
of 30 to 60 nm compared with 1.8
mM in the plasma
• Calmodulin
o A cytoplasmic Ca2+ binding protein
o Controls the function of Ca2+ -
ATPase
• These pumps, in addition to aquaporin,
maintain osmotic balance in the RBC.
• Cation pumps
o Consume a significant portion of
RBC ATP production
• Colloid osmotic hemolysis
o ATP loss or pump damage permits
Ca2+ and Na+ influx, with water
following osmotically
o Cell swells, becomes spheroid, and
eventually ruptures

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 Hematology I

HEMOGLOBIN METABOLISM YG Gamma G 146 (position

• Hemoglobin is one of the most studied 136: glycine)
proteins in the body because of the ability Delta 146
to easily isolate it from red blood cells Epsilon 146
(RBCs). Zeta 141
• 95% of the cytoplasmic content of RBCs. Theta Unknown
• provides protection from denaturation in *Oxidized hemoglobin is also called
the plasma and loss through the kidneys methemoglobin
• Free (non-RBC) hemoglobin HEME STRUCTURE
o generated from RBCs through • consists of a ring of carbon, hydrogen,
hemolysis, has a short half-life and nitrogen atoms called
outside of RBCs protoporphyrin IX
o When released into the plasma, it o with a central atom of divalent
is rapidly salvaged to preserve its ferrous iron (Fe2+)
iron and amino acid components. • Each of the four heme groups is
o When salvage capacity is positioned in a pocket of the polypeptide
exceeded, it is excreted by the chain near the surface of the
kidneys. hemoglobin molecule
• Ferrous iron in each heme molecule
Concentration Molecular weight reversibly combines with one oxygen
• approximately • approximately molecule.
34 g/dL 64,000 Daltons o When the ferrous irons are
oxidized to the ferric state (Fe3+),
they no longer can bind oxygen.
GLOBIN STRUCTURE
• Hemoglobin’s main function? • four globin chains comprising each
o transport oxygen from the lungs to hemoglobin molecule consist of two
tissues and transport carbon identical pairs of unlike polypeptide chains
dioxide from the tissues to the o 141 to 146 amino acids each
lungs for exhalation • Each globin chain is divided into eight
• Hemoglobin also contributes to acid-base helices separated by seven nonhelical
balance segments
o by binding and releasing hydrogen • Helices
ions and transports nitric oxide, a o designated to H, contain subgroup
numberings for the sequence of
regulator of vascular tone
the
HEMOGLOBIN STRUCTURE
amino acids in each helix and are
• first protein whose structure was
relatively rigid and linear.
described using x-ray crystallography • Flexible nonhelical segments
• molecule is a globular protein consisting of o connect the helices
two different pairs of polypeptide o reflected by their designations:
chains and four heme groups, with 1. NA for the sequence between
one heme group imbedded in each of the N-terminus and the A helix
the four polypeptide chains. 2. AB between the A and B
helices, and so forth, with BC,
Globin Chains CD, DE, EF, FG, GH
Symbol Name Number of 3. HC between the H helix and
Amino Acids the C-terminus
a Alpha 141
B Beta 146 COMPLETE HEMOGLOBIN MOLECULE
YA Gamma A 146 (position • Hemoglobin molecule can be described by
136: alanine) o primary structure

11 | P a g e
 Hematology I

o secondary structure o Oxygen binds to the iron atom on

o tertiary structure the other side of the plane and is
o quaternary structure close (but not linked) to the E7
primary structure refers to the amino distal histidine (dotted line)
acid sequence of the • Globin chains loop to form a cleft pocket
polypeptide for heme
chains. Each chain contains a heme group that is
secondary refers to chain suspended between the E and F helices of
structure arrangements the polypeptide chain
in helices and • The iron atom at the center of the
nonhelical. protoporphyrin IX ring of heme
tertiary structure refers to the o positioned between two histidine
arrangement of the radicals, forming a proximal
helices into a pretzel- histidine bond within F8 and,
like configuration. through the linked oxygen, a close
quaternary also called a association with the distal histidine
structure tetramer, residue in E7.
describes the o Amino acids on the outside are
complete hydrophilic, which renders the
hemoglobin molecule molecule water soluble.
• Arrangement also helps iron remain in its
STRUCTURE OF THE B-GLOBIN CHAIN divalent ferrous form regardless of
OF HEMOGLOBIN whether it is oxygenated (carrying an
oxygen molecule) or deoxygenated (not
carrying an oxygen molecule).
• Complete hemoglobin molecule
1. Spherical
2. four heme groups attached to four
polypeptide chains
3. carry up to four molecules of
oxygen
• Predominant adult hemoglobin
o Hb A
o composed of two a-globin chains
and two b-globin chains
▪ hold the dimers in a stable
form
▪ important for the stability
of the quaternary structure
in the oxygenated and
deoxygenated form
• Glycated
o consists of helical (labeled A o small percentage of Hb A
through H) and nonhelical o post translational modification
segments formed by the nonenzymatic
o Heme (protoporphyrin IX with a binding of various sugars to globin
central iron atom) is suspended in chain amino groups over the life
a pocket between the E and F span of the RBC.
helices. • Most characterized of the glycated
o The iron atom of heme is linked to hemoglobin is Hb A
the F8 proximal histidine on one o glucose attaches to the N-terminal
side of the heme plane (solid line) valine of the b-chain
• 4% to 6% of Hb A circulates at A1c

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• Uncontrolled diabetes mellitus • The relationship is described by the

o the amount of A1c is increased oxygen dissociation curve of hemoglobin,
proportionally to the mean which plots the percent oxygen saturation
blood glucose level over the of hemoglobin versus the PO2
preceding 2 to 3 months. • Sigmoidal
o The curve that indicates low
hemoglobin affinity for oxygen at
low oxygen tension and high
affinity for oxygen at high oxygen
tension
• Hemoglobin that is completely
deoxygenated has little affinity for oxygen.
• with each oxygen molecule that is bound,
there is a change in the conformation of
the tetramer that progressively increases
the oxygen affinity of the other heme
subunits.
• a PO2 of approximately 27 mm Hg results
in 50% oxygen saturation of the
hemoglobin molecule.
o Heme is suspended between the E and F
• The reference interval for arterial oxygen
helices of each polypeptide chain. Pink saturation is 96% to 100%.
represents "1 (left) and "2 (right); • If the oxygen dissociation curve shifts to
o yellow represents non-"2 (left) and non-"1 the left, a patient with arterial and venous
(right). PO2 levels in the reference intervals (80 to
o The polypeptide chains first form "1-non- 100 mm Hg arterial and 30 to 50 mm Hg
"1 and "2-non-"2 dimers, and then venous) will have a higher percent oxygen
assemble into a tetramer (quaternary saturation and a higher affinity for oxygen
structure) with "1-non-"2 and "2-non-"1 than a patient for whom the curve is
bonds. normal.
• addition to the PO2, shifts of the curve to
Oxygen Transport the left or right occur if there are changes
in the pH of the blood.
• Functions: • In the tissues, a lower pH shifts the curve
o Readily bind oxygen molecules in to the right and reduces the affinity of
the lungs hemoglobin for oxygen, and the
o Requires high oxygen affinity hemoglobin more readily releases oxygen.
o To transport oxygen • Bohr effect
o Efficiency unload oxygen to the o It facilitates the ability of
tissues hemoglobin to exchange oxygen
o Requires low oxygen affinity and carbon dioxide (CO2).
• During oxygenation, each of the four • The concentration of 2,3
heme iron atoms in a hemoglobin bisphosphoglycerate (2,3-BPG, formerly
molecule can reversibly bind one oxygen 2,3-diphosphoglycerate) also has an effect
molecule on oxygen affinity.
• Approximately 1.34 mL of oxygen is • In the deoxygenated state, the
bound by each gram of hemoglobin hemoglobin tetramer assumes a tense or
• The affinity of hemoglobin for oxygen T conformation that is stabilized by the
relates to the partial pressure of oxygen binding of 2,3-BPG between the β-globin
(PO2) chains
• often defined in terms of the amount of • The binding of 2,3-BPG shifts the oxygen
oxygen needed to saturate 50% of dissociation curve to the right, favoring
hemoglobin, called P50 value. the release of oxygen. In addition, a

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 Hematology I

lower pH and higher PCO2 in the tissues • Chloride Shift

further shifts the curve to the right, o The negative charge that diffuses
favoring the release of oxygen. from the plasma into the cell to
• The sigmoidal oxygen dissociation curve maintain electroneutrality across
generated by normal hemoglobin the membrane
contrasts with myoglobin’s hyperbolic • In the lungs, oxygen diffuses into the cell
curve and binds to deoxygenated hemoglobin
• Myoglobin, present in cardiac and skeletal (HHb) because of the high oxygen
muscle, is a 17,000-Dalton, monomeric, tension. H+ is released from hemoglobin
oxygen-binding heme protein. and combines with bicarbonate to form
• Its hyperbolic curve indicates that it carbonic acid.
releases oxygen only at very low partial • Carbonic Acid acid is converted to water
pressures, which means it is not as and Co2 ; the latter diffuses out of the
effective as hemoglobin in releasing cellls and is expelled by the lungs
oxygen to the tissues at physiologic • As more bicarbonate diffuses into the cell
oxygen tensions. to produce carbonic acid, chloride diffuses
• Rhabdomyolysis back out into the plasma.
oMyoglobin is released into the • Approximately 85% of the CO2 produced
plasma when there is damage to in the tissues is transported byhemoglobin
the muscle in myocardial as H+
infarction, trauma, or severe • In this capacity, hemoglobin provides a
muscle injury buffering effect by binding and releasing
• In fetal life the high oxygen affifinity of Hb H+
F provides an advantage by allowing more • A small percentage of CO2 remains in the
effective oxygen withdrawal from the cytoplasm and the remainder binds to the
maternal circulation. globin chains as a carbamino group
• Hb F has a disadvantage in that it delivers
oxygen less readily to tissues. The bone
marrow in the fetus and newborn Nitric Oxide Transport
compensates by producing more RBCs to • Third function of hemoglobin involves:
ensure adequate oxygenation of the o Binding
tissues. o Inactivation
o Transport of nitric oxide
• Nitric oxide
Carbon Dioxide Transport
o Secreted by vascular endothelial
cells
• The second crucial function of hemoglobin o Causes relaxation of vascular wall
• In venous blood, the carbon dioxide smooth muscle and vasodilation
diffuses into the RBCs and combines with o When released, free nitric oxide
water to form carbonic acid (H2CO3) has a very short half-life, but some
• This reaction is facilitated by the RBC enters RBCs and can bind to
enzyme carbonic anhydrase. cysteine in the β chain of
• Carbonic acid then dissociates to release hemoglobin, forming S-
H+ and bicarbonate (HCO3,)
nitrosohemoglobin
• The H+ from the second reaction binds
• Hemoglobin preserves and transports
oxygenated hemoglobin (HbO2), and the
oxygen is released from the hemoglobin nitric oxide to hypoxic microvascular
because of the Bohr effect. areas, which stimulates vasodilation and
• The oxygen then diffuses out of the cell increases blood flow (hypoxic
into the tissues. vasodilation)
• As the concentration of the negatively o Hemoglobin may work with other
charged bicarbonate increases, it diffuses systems in regulating local blood
across the RBC membrane into the plasma flow to microvascular areas by
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 Hematology I

binding and inactivating nitric • Dyspnea

oxide (causing vasoconstriction • Headache
and decreased blood flow) • Vertigo
when oxygen tension is high and • Change in mental status
releasing nitric oxide (causing • Levels of methemoglobin greater than
vasodilation and increased 50%
blood flow) when oxygen tension o Coma
is low o Death
• Methemoglobinemia
DYSHEMOGLOBINS
o Increase in hemoglobin
• Dysfunctional hemoglobins that are unable
o Can be acquired or hereditary
to transport oxygen
o Toxic methemoglobinemia
• Include:
• Acquired form
o Methemoglobin
• Occurs in normal
o Sulfhemoglobin
individuals after exposure
o Carboxyhemoglobin
to an exogenous oxidant
• Form and may accumulate to toxic levels,
o Nitrites
after exposure to certain drugs or
o Primaquine
environmental chemicals or gasses
o Dapsone
• Offending agent
o Benzocaine
o Modifies the structure of the
• As the oxidant overwhelms the
hemoglobin molecule, preventing it
hemoglobin reduction systems, the level
from binding oxygen
of methemoglobin increases, and the
• Most cases of dyshemoglobinemia are
patient may exhibit cyanosis and
• acquired; a small fraction of
symptoms of hypoxia
methemoglobinemia cases are hereditary.
• If the level of methemoglobin increases to
Methemoglobin 30% or more of total hemoglobin,
• MetHb intravenous methylene blue is
• Formed by the reversible oxidation of administered
heme iron to the ferric state (Fe3+) • Methylene blue
• NADH-cytochrome b5 reductase 3 (NADH- o Reduces methemoglobin ferric iron
methemoglobin reductase) pathway to the ferrous state through
o Methemoglobin reduction system NADPH-methemoglobin
o Normally limit its accumulation to reductase and NADPH produced
only 1% of total hemoglobin by glucose-6-phosphate
• Cannot carry oxygen because oxidized dehydrogenase in the hexose
ferric iron cannot bind it monophosphate shunt
• An increase in methemoglobin level • In life-threatening cases, exchange
results in decreased delivery of oxygen transfusion may be required
to the tissues • Hereditary causes are rare
• Individuals with methemoglobin levels less o Include mutations in the gene for
than 25% NADH-ctochrome b6 reductase 3
o Asymptomatic (CYB5R3)
• If the methemoglobin level increases to • Resulting in a diminished
more than 30% of total hemoglobin capacity to reduce
o Cyanosis methemoglobin
• Bluish discoloration of skin o Mutations in the α-, β-, or y- globin
and mucous membranes gene
o Symptoms of hypoxia

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 Hematology I

• Resulting in a structurally • Cytochrome b5 reductase 3 deficiency

abnormal polypeptide chain o Diagnosed by:
that favors the oxidized • Enzyme assays
ferric form of iron and • DNA mutation testing
prevents its reduction
Sulfhemoglobin
• M hemoglobin or Hb M
• Formed by irreversible oxidation of
o It is inherited in an autosomal
hemoglobin by drugs
dominant pattern, with
o Sulfanilamides
methemoglobin comprising 30% to
o Phenacetin
50% of total hemoglobin
o Nitrites
o No effective treatment for this
o Phenylhydrazine
form of methemoglobinemia
• Or exposure to sulfur chemicals in
• Cytochrome b5 reductase deficiency
industrial or environmental settings
o An autosomal recessive disorder
• It is formed by the addition of a sulfur
• Methemoglobin elevations
atom to the pyrrole ring of heme
o Occur in individuals who are
o Green pigment
homozygous or compound
• Ineffective for oxygen transport
heterozygous for a CYB5R3
• Patients with elevated levels: cyanosis
mutation
• Cannot be converted to normal Hb A; it
• Most individuals with Hb M or homozygous
persists for the life of the cell
cytochrome b5 reductase deficiency
• Treatment: prevention by avoidance of
maintain methemoglobin levels less than
the offending agent
50%
• Has a similar peak to methemoglobin on a
o They have cyanosis but only mild
spectral absorption instrument
symptoms of hypoxia that do not
• Sulfhemoglobin spectral curve
require treatment
o Does not shift when cyanide is
• Individuals heterozygous for the CYB5R3
added
mutation have normal levels of
• A feature that
methemoglobin but develop the following
distinguishesit from
when exposed to an oxidant drug or
methemoglobin
chemical:
o Methemogobinemia Carboxyhemoglobin
o Cyanosis • Fesults from the combination of carbon
o Hypoxia monoxide (CO) with heme iron
• Methemoglobin • Affinity: 240 times of oxygen
o Assayed by spectral absorption • Once one molecule of carbon monoxide
analysis instruments such as the binds to hemoglobin, it shifts the
CO-oximeter hemoglobin-oxygen dissociation curve to
o Absorption peak: 630 nm the left, further increasing its affinity
o High levels of methemoglobin and severely impairing release of oxygen
• Blood takes on a chocolate to the tissues
brown color and does not • Carbon monoxide
revert back to normal red o Silent killer
color after oxygen exposure o Because it is an odorless and
o Used for identification of Hb M colorless gas, and victims may
variants quickly become hypoxic
• Hemoglobin electrophoresis • Some is produced endogenously but it
• HPLC normally comprises less than 2% of total
• DNA mutation testing hemoglobin

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• Exogenous carbon monoxide • Use of hyperbaric oxygen therapy is

o Derived from the exhaust of: controversial
• Autonomous biles o Primarily used to prevent
• Tobacco smoke neurologic and cognitive
• Industrial pollutants impairment after acute carbon
o Coal monoxide exposure in patients
o Gas whose COHb level exceeds 25%
o Charcoal burning
HEMOGLOBIN MEASUREMENT
• In smokers, COHb levels may be as high
• Cyanmethemoglobin method
as 15%
o Reference method for hemoglobin
o Result: smokers may have a
assay
higher hematocrit and
• A lysing agent present in the
polycythemia to compensate for
cyanmethemoglobin reagent frees
the hypoxia
hemoglobin from RBCs
• Exposure to carbon monoxide may be:
o Free hemoglobin combines with
o Coincidental
potassium ferricyanide
o Accidental
contained in the
o Intentional (suicidal)
cyanmethemoglobin reagent,
• Deaths from house fire are result of
which converts hemoglobin iron
inhaling:
from the ferrous to the ferric state
o Smoke
to form methemoglobin
o Fumes
o Methemoglobin combines with
o Carbon monoxide
potassium cyanide to form the
• Toxic effects that begin to appear at blood
stable pigment
levels of 20% to 30% COHb:
cyanmethemoglobin
o Headache
• Cyanmethemoglobin color intensity
o Dizziness
o Proportional to hemoglobin
o Disorientation
concentration
• Levels of more than 40% of total
o Measured at 540 nm
hemoglobin may cause:
spectrophotometricallyand
o Coma
compared with a standard
o Seizure
• Performed manually but has been adapted
o Hypotension
for use in automated blood cell analyzers
o Cardiac arrhythmias
• Sodium lauryl sulfate (SLS)
o Pulmonary edema
o Used to convert hemoglobin to
o Death
SLS-metheoglobin
• May be detected by spectral absorption
o This method does not generate
instruments at 540 nm
toxic wastes
• Gives blood a cherry red color, which is
• Hemoglobin electrophoresis and HPLC
sometimes imparted to the skin of victims
o Used to separate the different
• A diagnosis of carbon monoxide poisoning
types of hemoglobins such as:
is made if the COHb level is greater than
• Hb
3% in nonsmokers and greater than 10%
• A
in smokers
• A2
• Treatment
• F
o Removing the carbon monoxide
source
o Administration of 100% oxygen

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