Hematology: Hemoglobin Metabolism

Jessica I. Pontanares | ♡♡♡ | Page 1 of 11

OUTLINE

i. Hemoglobin Structure
a. Heme Structure
b. Globin Structure
c. Complete Hemoglobin Molecule
ii. Hemoglobin Biosynthesis
a. Heme Biosynthesis
b. Globin Biosynthesis
c. Hemoglobin Assembly
iii. Hemoglobin Ontogeny
iv. Regulation of Hemoglobin Production Hemoglobin structure
a. Heme Regulation
b. Globin Regulation • Hemoglobin
c. Systemic Regulation of Erythropoiesis o first protein whose structure was
v. Hemoglobin Function described using X-RAY
a. Oxygen Transport CRYSTALLOGRAPHY
b. Carbon Dioxide Transport o globular protein consisting of:
c. Nitric Oxide Transport ▪ 2 different pairs of
vi. Dyshemoglobins polypeptide chains
a. Methemoglobin ▪ 4 heme groups
b. Sulfemoglobin
Heme structure
c. Carboxyhemoglobin
vii. Hemoglobin Measurement • Consist of a RING OF C, H, & N –
PROTOPORPHYRIN IX w/ CENTRAL ATOM OF
Main Topic
DIVALENT FERROUS IRON (Fe2+)
Subtopic • Each of the four heme groups is positioned in a
pocket of the polypeptide chain near the surface
Sub-subtopic
of the hemoglobin molecule.
• The ferrous iron in each heme molecule
reversibly combines with one oxygen molecule
Hemoglobin → When the ferrous irons are oxidized to the
• One of the most studied proteins in the body ferric state (Fe3+), they no longer can bind
• It comprises approx. 95% of the cytoplasmic oxygen → Oxidized hemoglobin is also called
content of RBCs methemoglobin
• Free hemoglobin – generated from RBCs Globin structure
through HEMOLYSIS; has a short half-life
outside of RBCs
• Concentration within RBCs – approx. 34 g/dl
• MW – approx. 64,000 Daltons
• Main function:
o TRANSPORT O2 FROM THE LUNGS →
TISSUES;
o TRANSPORT CO2 FROM TISSUES →
THE LUNGS FOR EXHALATION
• Contributes to the acid-base balance by binding
& releasing hydrogen ions (H+) & transporting • Four globin chains comprising each
nitic oxide (NO) hemoglobin molecule consist of two identical
pairs of unlike polypeptide chains - 141 to 146
amino acids each
Hematology: Hemoglobin Metabolism
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Complete hemoglobin molecule

Primary structure

• the amino acid sequence of the polypeptide

chains

Secondary structure

• chain arrangements in helices and nonhelices

Tertiary structure

• arrangement of the helices into a pretzel-like

configuration

Quaternary structure

• also called a tetramer

• the complete hemoglobin molecule
• spherical, has four heme groups attached to
four polypeptide chains, and may carry up to
four molecules of oxygen

Globin chains

• loop to form a cleft pocket for heme

• Each chain contains a heme group that is
Figure 8.3 Structure of the β-Globin Chain of suspended between the E and F helices of the
Hemoglobin. polypeptide chain
The β-globin chain consists of helical (labeled A through
H) and nonhelical segments. Heme (protoporphyrin IX
with a central iron atom) is suspended in a pocket
between the E and F helices. The iron atom of heme is
linked to the F8 proximal histidine on one side of the
heme plane (solid line). Oxygen binds to the iron atom
on the other side of the plane and is close (but not
linked) to the E7 distal histidine (dotted line)

• Each globin chain is divided into eight helices

separated by seven nonhelical segments
• The helices, designated A to H, contain
subgroup numberings for the sequence of the
amino acids in each helix and are relatively rigid
and linear.
• Flexible nonhelical segments connect the
helices, as reflected by their designations:
o NA for the sequence between the N-
terminus and the A helix Figure 8.4 Hemoglobin Molecule Illustrating Tertiary
o AB for the sequence between the A and Folding of the Four Polypeptide Chains.
B helices, and so forth, with BC, CD, Heme is suspended between the E and F helices of each
DE, EF, FG, GH polypeptide chain. Pink represents α1 (left) and α2
o HC between the H helix and the C- (right); yellow represents non-α2 (left) and non-α1
terminus (right). The polypeptide chains first form α1 -non-α1 and
Hematology: Hemoglobin Metabolism
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α2 -non-α2 dimers and then assemble into a tetramer Hemoglobin biosynthesis
(quaternary structure), with α1 -non-α2 and α2 -non-α1
Heme Biosynthesis
bonds.

• The iron atom at the center of the

protoporphyrin IX ring of heme is positioned
between two histidine radicals, forming a
proximal histidine bond within F8 and, through
the linked oxygen, a close association with the
distal histidine residue in E7.
• Globin chain amino acids in the cleft are
hydrophobic, whereas amino acids on the
outside are hydrophilic - renders the molecule
water soluble.
o This arrangement also helps iron
remain in its divalent ferrous form
regardless of whether it is oxygenated
(carrying an oxygen molecule) or
deoxygenated (not carrying an oxygen
molecule).

Hb A (Adult hemoglobin) Figure 8.5 Heme Biosynthesis and Hemoglobin

Assembly.
• predominant adult hemoglobin
• composed of two α-globin chains and two β- Heme biosynthesis begins with the condensation of
globin chains glycine and succinyl coenzyme A (CoA) → forming
• Strong α1- β1 and α2 - β2 bonds hold the aminolevulinic acid (ALA) in a reaction catalyzed by
dimers in a stable form. aminolevulinate synthase.
• α1 - β2 and α2 - β1 bonds are important for the
In the cytoplasm, ALA undergoes several
stability of the quaternary structure in the
transformations from porphobilinogen (PBG) to
oxygenated and deoxygenated forms
coproporphyrinogen III which, catalyzed by
• small percentage of Hb A is glycated.
coproporphyrinogen oxidase, → becomes
• Glycation - a post-translational modification
protoporphyrinogen IX.
formed by the nonenzymatic binding of various
sugars to globin chain amino groups over the In the mitochondria, protoporphyrinogen IX is
life span of the RBC converted to protoporphyrin IX by protoporphyrinogen
oxidase. → Ferrous (Fe2+) ion is added, catalyzed by
Hb A1c
ferrochelatase (heme synthase) to form heme.
• most characterized of the glycated hemoglobin
In the cytoplasm, heme assembles with an α chain and
• glucose attaches to the N-terminal valine of the
non-α chain, → forming a dimer, and ultimately, two
β chain
dimers join to form the hemoglobin tetramer
• Normally, about 4% to 6% of Hb A circulates in
the A1c form. • In the cytoplasm, aminolevulinic acid
• In uncontrolled diabetes mellitus dehydratase (also known as porphobilinogen
o The amount of A1c is increased synthase) converts ALA to porphobilinogen
proportionally to the mean blood (PBG) →
glucose level over the preceding 2 to 3 • PBG undergoes several transformations in the
months. cytoplasm from hydroxymethylbilane to
coproporphyrinogen III →
• This pathway then continues in the
mitochondria until, in the final step of
Hematology: Hemoglobin Metabolism
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production of heme, Fe2+ combines with o After translation is complete, chains are
protoporphyrin IX in the presence of released from the ribosomes in the
ferrochelatase (heme synthase) to make heme cytoplasm.

Transferrin Hemoglobin assembly

• a plasma protein, carries iron in the ferric (Fe3+) • After their release from ribosomes, each globin
form to developing erythroid cells chain binds to a heme molecule and then forms
• binds to transferrin receptors on erythroid a heterodimer
precursor cell membranes, and the receptors • The non-α chains have a charge difference that
and transferrin (with bound iron) are brought determines their affinity to bind to α chains.
into the cell in an endosome • The α chain has a positive charge and has the
• Acidification of the endosome releases iron highest affinity for a β chain because of its
from transferrin → Iron is transported out of the negative charge
endosome and into the mitochondria, where it • The γ-globin chain has the next highest
is reduced to the ferrous state and is united with affinity, followed by the δ-globin chain.
protoporphyrin IX to make heme → Heme • Two heterodimers then combine to form a
leaves the mitochondria and is joined to the tetramer.
globin chains in the cytoplasm. • Two α and two β chains form Hb A, the major
hemoglobin present from 6 months of age
Globin Biosynthesis
through adulthood.
• Six structural genes code for six globin chains • Hb A2 contains two α and two δ chains.
• α- and ζ globin genes • Owing to a mutation in the promoter region of
o are on the short arm of chromosome 16 the δ-globin gene, production of the δ chain
• ∈-, γ-, δ-, and β-globin gene cluster polypeptide is very low
o is on the short arm of chromosome 11 • Consequently, Hb A2 comprises less than 3.5%
• In the human genome, there is one copy of each of total hemoglobin in adults.
globin gene per chromatid for a total of two • Fetal hemoglobin (Hb F) contains two α and
genes per diploid cell, with the exception of α two γ chains.
and γ • In healthy adults, Hb F comprises 1% to 2% of
• There are two copies of the α- and γ-globin total hemoglobin, and it is present only in a
genes per chromatid for a total of four genes small proportion of the RBCs (uneven
per diploid cell. distribution).
• Production of globin chains takes place in • These RBCs with Hb F are called F or A/F cells
erythroid precursors from the pronormoblast • The various amino acids that comprise the
through the circulating polychromatic globin chains affect the net charge of the
erythrocyte but not in mature erythrocytes hemoglobin tetramer.
• Transcription of the globin genes to messenger • Electrophoresis and high-performance liquid
ribonucleic acid (mRNA) occurs in the nucleus, chromatography (HPLC) are used for
and translation of mRNA to the globin fractionation, presumptive identification, and
polypeptide chain occurs on ribosomes in the quantification of normal hemoglobins and
cytoplasm. hemoglobin variants
o Although transcription of α-globin • Molecular genetic testing of globin gene DNA
genes produces more mRNA than the provides definitive identification of variant
β-globin gene, there is less effcient hemoglobins.
translation of the α-globin mRNA.
o Therefore the α and β chains are
produced in approximately equal
amounts.
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Hemoglobin ontogeny Regulation of Hemoglobin production

Heme regulation

• The key rate-limiting step in heme synthesis is

the initial reaction of glycine and succinyl CoA
to form ALA, catalyzed by ALA synthase
• Heme inhibits the transcription of the ALA
synthase gene, which leads to a decrease in
heme production (a negative feedback
mechanism).
• Heme inhibits other enzymes in the
biosynthesis pathway, including ALA
dehydrase and PBG deaminase. A negative
feedback mechanism by heme or substrate
inhibition by protoporphyrin IX is believed to
inhibit the ferrochelatase enzyme.
• Conversely, an increased demand for heme
• Hemoglobin changes reflect the sequential induces an increased synthesis of ALA
activation and inactivation (or switching) of the synthase.
globin genes, progressing from the ζ- to the α-
Globin regulation
globin gene on chromosome 16 and from the
∈- to the γ-, δ-, and β-globin genes on • Globin synthesis is highly regulated so that
chromosome 11. there is a balanced production of globin and
• The ζ- and ∈-globin chains normally appear heme.
only during the first 3 months of embryonic o This is critical because an excess of
development. globin chains, protoporphyrin IX, or
o These two chains, when paired with the iron can accumulate and damage the
α and γ chains, form the embryonic cell, reducing its life span.
hemoglobins • Globin production is mainly controlled at the
• During the second and third trimesters of fetal transcription level by a complex interaction of
life and at birth, Hb F (α2 γ2 ) is the DNA sequences (cis-acting promoters,
predominant hemoglobin. enhancers, and silencers) and soluble
• By 6 months of age and through adulthood, Hb transcription factors (trans-acting factors) that
A (α2β2) is the predominant hemoglobin, with bind to DNA or to one another to promote or
small amounts of Hb A2 (α2δ2 ) and Hb F. suppress transcription.
• Initiation of transcription of a particular globin
gene requires:
(1) the promoter DNA sequences immediately
before the 5′ end or the beginning of the
gene; (2) a key transcription factor called
Krüppel-like factor 1
(2) (KLF1);
(3) a number of other transcription factors
(such as GATA1, Ikaros, TAL1, p45-NF-E2,
and LDB1);
(4) an enhancer region of DNAse 1
hypersensitive nucleic acid sequences
located more than 20 kilobases upstream
(before the 5′ start site of the gene) from
Hematology: Hemoglobin Metabolism
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the globin gene called the locus control defective in transporting oxygen, tissue hypoxia
region (LCR). occurs.
• For example, to activate transcription of the β- • Hypoxia
globin gene in the β-globin gene cluster on o is detected by the peritubular cells of
chromosome 11, the LCR, the promoter for the the kidney, which respond by
β-globin gene, and various transcription factors increasing production of erythropoietin
join together to form a three-dimensional active (EPO).
chromosome hub (ACH), with KLF1 playing a o EPO increases the number of
key role in connecting the complex. erythrocytes produced and released
• Because the LCR is located a distance upstream into the periphery; it also accelerates
from the β-globin gene complex, a loop of DNA the rate of synthesis of erythrocyte
is formed when the LCR and β-globin gene components, including hemoglobin
promoter join together in the chromosome hub.
• The other globin genes in the cluster (∈-, γ-, and Hemoglobin concentration reference intervals:
δ-globin) are maintained in the inactive state in Men: 13.5–18.0 g/dL (135–180 g/L)
the DNA loop, so only the β-globin gene is
transcribed. Women: 12.0–16.0 g/dL (120–160 g/L)
• KLF1 also plays a key regulatory role in the Newborns: 16.5–21.5 g/dL (165–215 g/L)
switch from γ chain to β chain production (γ β
switching) that begins in late fetal life and • Reference intervals for infants and children vary
continues through adulthood. according to age group.
• KLF1 is an exact match for binding to the DNA o Individuals living at high altitudes have
promoter sequences of the β globin gene, slightly higher levels of hemoglobin as
whereas the γ-globin gene promoter has a a compensatory mechanism to provide
slightly di erent sequence.6 is results in a more oxygen to the tissues in the
preferential binding to and subsequent oxygen-thin air.
activation of transcription of the β-globin gene.
Hemoglobin function
• KLF1 also regulates the expression of
repressors of γ-globin gene transcription, such Oxygen transport
as BCL11A and MYB.
• Globin synthesis is also regulated during
translation when mRNA coding for the globin
chains associates with ribosomes to produce
the polypeptide.
• Many protein factors are required to control
initiation, elongation, and termination steps of
translation.
• Heme is an important regulator of globin mRNA
translation at the initiation step by promoting
activation of a translation initiation factor and
inactivating its repressor.
• Conversely, when the heme level is low, the
repressor accumulates and inactivates the
initiation factor, thus blocking translation of the
globin mRNA. Figure 8.7 Hemoglobin-Oxygen Dissociation Curves.

Systemic Regulation of Erythropoiesis Normal hemoglobin-oxygen dissociation curve (A). P50

is the oxygen (O2) partial pressure needed for 50% O2
• When there is an insufficient quantity of saturation of hemoglobin. Leftshifted curve with reduced
hemoglobin or if the hemoglobin molecule is P50 can be caused by decreases in 2,3-
bisphosphoglycerate (2,3-BPG), H+ ions (raised pH),
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partial pressure of carbon dioxide (PCO2), and/or • Conversely, with relatively low oxygen tension
temperature (B). A left-shifted curve is also seen with in the tissues, the affinity of hemoglobin for
hemoglobin F and hemoglobin variants that have oxygen is low, and hemoglobin rapidly releases
increased oxygen affinity. A right-shifted curve with oxygen.
increased P50 can be caused by elevations in 2,3-BPG; • Normally, a PO2 of approximately 27 mm Hg
H+ ions (lowered pH); PCO2; and/ or temperature (C). A results in 50% oxygen saturation of the
right-shifted curve is also seen with hemoglobin variants hemoglobin molecule.
that have decreased oxygen affinity. Myoglobin, a • If there is a shift of the curve to the left, 50%
muscle protein, produces a markedly left-shifted curve, oxygen saturation of hemoglobin occurs at a
indicating a very high oxygen affinity. It is not effective PO2 of less than 27 mm Hg.
in releasing oxygen at physiologic oxygen tensions. • If there is a shift of the curve to the right, 50%
oxygen saturation of hemoglobin occurs at a
• The function of hemoglobin is to readily bind
PO2 higher than 27 mm Hg.
oxygen molecules in the lung, which requires
• Reference interval for arterial oxygen saturation
high oxygen affinity; to transport oxygen; and to
is 96% to 100%.
efficiently unload oxygen to the tissues, which
• If the oxygen dissociation curve shifts to the left,
requires low oxygen affinity.
a patient with arterial and venous PO2 levels in
• During oxygenation, each of the four heme iron
the reference intervals (80 to 100 mm Hg
atoms in a hemoglobin molecule can reversibly
arterial and 30 to 50 mm Hg venous) will have
bind one oxygen molecule.
a higher percent oxygen saturation and a higher
• Approximately 1.34 mL of oxygen is bound by
affinity for oxygen than a patient for whom the
each gram of hemoglobin.
curve is normal.
• Affinity of hemoglobin for oxygen relates to the
• With a shift in the curve to the right, a lower
partial pressure of oxygen (PO2 ), often defined
oxygen affinity is seen.
in terms of the amount of oxygen needed to
• In addition to the PO2 , shifts of the curve to the
saturate 50% of hemoglobin, called P50 value.
left or right occur if there are changes in the pH
• The relationship is described by the oxygen
of the blood.
dissociation curve of hemoglobin, which plots
• In the tissues, a lower pH shifts the curve to the
the percent oxygen saturation of hemoglobin
right and reduces the affinity of hemoglobin for
versus the PO2
oxygen, and the hemoglobin more readily
• The curve is sigmoidal, which indicates low
releases oxygen.
hemoglobin affinity for oxygen at low oxygen
• A shift in the curve because of a change in pH
tension and high affinity for oxygen at high
(or hydrogen ion concentration) is termed the
oxygen tension.
Bohr effect.
• Cooperation among hemoglobin subunits
o It facilitates the ability of hemoglobin to
contributes to the shape of the curve.
exchange oxygen and carbon dioxide
Hemoglobin that is completely deoxygenated (CO2).
has little affinity for oxygen.
• The concentration of 2,3-bisphosphoglycerate
• However, with each oxygen molecule that is
(2,3-BPG, formerly 2,3-diphosphoglycerate)
bound, there is a change in the conformation of
also has an effect on oxygen affinity.
the tetramer that progressively increases the
• In the deoxygenated state, the hemoglobin
oxygen affinity of the other heme subunits.
tetramer assumes a tense or T conformation
• Once one oxygen molecule binds, the remainder
that is stabilized by the binding of 2,3-BPG
of the hemoglobin molecule quickly becomes
between the β-globin chains
fully oxygenated
• The formation of salt bridges between the
• Therefore with high oxygen tension in the lungs,
phosphates of 2,3-BPG and positively charged
the affinity of hemoglobin for oxygen is high,
groups on the globin chains further stabilizes
and hemoglobin becomes rapidly saturated with
the tetramer in the T conformation.
oxygen.
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• The binding of 2,3-BPG shifts the oxygen effective as hemoglobin in releasing
dissociation curve to the right, favoring the oxygen to the tissues at physiologic
release of oxygen. oxygen tensions.
• In addition, a lower pH and higher partial o Myoglobin is released into the plasma
pressure of carbon dioxide (PCO2) in the when there is damage to the muscle in
tissues further shifts the curve to the right, myocardial infarction, trauma, or severe
favoring the release of oxygen. muscle injury, called rhabdomyolysis.
• As hemoglobin binds oxygen molecules, a o Myoglobin is normally excreted by the
change in conformation of the hemoglobin kidney, but levels may become elevated
tetramer occurs, with a change in hydrophobic in renal failure.
interactions at the α1β2 contact point, a o Serum myoglobin levels aid in
disruption of the salt bridges, and release of diagnosis of myocardial infarction in
2,3-BPG patients who have no underlying
• A 15-degree rotation of the α1β1dimer relative trauma, rhabdomyolysis, or renal
to the α2β2 dimer occurs along the α1β2 failure.
contact point. o Myoglobin in the urine produces a
• When the hemoglobin tetramer is fully positive result on urine biochemical
oxygenated, it assumes a relaxed or R state analysis (by reagent test strip) for
• Clinical conditions that produce a shift of the blood; this must be differientiated from
oxygen dissociation curve to the left include: a positive result caused by hemoglobin.
o lowered body temperature as a result • Hb F (the primary hemoglobin in newborns) has
of external causes; a P50 of 19 to 21 mm Hg, which results in a left
o multiple transfusions of stored blood shift of the oxygen dissociation curve and
with depleted 2,3-BPG; increased affinity for oxygen relative to that of
o alkalosis; and Hb A.
o presence of hemoglobin variants with o This increased affinity for oxygen is due
a high affinity for oxygen. to its weakened ability to bind 2,3-BPG.
• Conditions producing a shift of the curve to the o There is only one amino acid difference
right include: in a critical 2,3-BPG binding site
o increased body temperature; between the γ chain and the β chain that
o acidosis; presence of hemoglobin accounts for this difference in binding.
variants with a low affinity for oxygen; • In fetal life, the high oxygen affinity of Hb F
and provides an advantage by allowing more
o increased 2,3-BPG concentration in effective oxygen withdrawal from the maternal
response to hypoxic conditions such as circulation.
high altitude, pulmonary insufficiency, o At the same time, Hb F has a
congestive heart failure, and severe disadvantage in that it delivers oxygen
anemia less readily to tissues.
• Sigmoidal oxygen dissociation curve generated o bone marrow in the fetus and newborn
by normal hemoglobin contrasts with compensates by producing more RBCs
myoglobin’s hyperbolic curve to ensure adequate oxygenation of the
• Myoglobin tissues.
o present in cardiac and skeletal muscle, ▪ This response is mediated by
is a 17,000-Dalton, monomeric, EPO
oxygen-binding heme protein. It binds • Consequently, the RBC count, hemoglobin
oxygen with greater affinity than concentration, and hematocrit of a newborn are
hemoglobin. higher than adult values, but they gradually
o Its hyperbolic curve indicates that it decrease to normal physiologic levels by 6
releases oxygen only at very low partial months of age as the γ β switching is completed
pressures, which means it is not as and most of the Hb F is replaced by Hb A.
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Carbon Dioxide Transport o secreted by vascular endothelial cells
and causes relaxation of vascular wall
• A second crucial function of hemoglobin is the
smooth muscle and vasodilation.
transport of carbon dioxide.
o When released, free nitric oxide has a
• In venous blood, the carbon dioxide diffuses
very short half-life, but some enters
into the RBCs and combines with water to form
RBCs and can bind to cysteine in the β
carbonic acid (H2CO3).
chain of hemoglobin, forming S-
o This reaction is facilitated by the RBC
nitrosohemoglobin.
enzyme carbonic anhydrase.
o Some investigators propose that
• Carbonic acid then dissociates to release H+ and
hemoglobin preserves and transports
bicarbonate (HCO3) nitric oxide to hypoxic microvascular
• H+ from the second reaction binds oxygenated areas, which stimulates vasodilation
hemoglobin (HbO2 ), and the oxygen is released and increases blood low (hypoxic
from the hemoglobin because of the Bohr vasodilation).
effect. o In this way, hemoglobin may work with
• oxygen then diffuses out of the cell into the other systems in regulating local blood
tissues. ow to microvascular areas by binding
• As the concentration of the negatively charged and inactivating nitric oxide (causing
bicarbonate increases, it diffuses across the vasoconstriction and decreased blood
RBC membrane into the plasma. low) when oxygen tension is high and
• Chloride (Cl ), also negatively charged, diffuses releasing nitric oxide (causing
from the plasma into the cell to maintain vasodilation and increased blood low)
electroneutrality across the membrane; this is when oxygen tension is low.
called the chloride shift
• In the lungs, oxygen diffuses into the cell and Dyshemoglobins
binds to deoxygenated hemoglobin (HHb)
• (dysfunctional hemoglobins that are unable to
because of the high oxygen tension. H+ is
transport oxygen) include methemoglobin,
released from hemoglobin and combines with
sulfhemoglobin, and carboxyhemoglobin.
bicarbonate to form carbonic acid. Carbonic acid
• Dyshemoglobins form and may accumulate to
is converted to water and CO2; the latter
toxic levels after exposure to certain drugs or
diffuses out of the cells and is expelled by the
environmental chemicals or gases.
lungs.
• The offending agent modifies the structure of
• As more bicarbonate diffuses into the cell to
the hemoglobin molecule, preventing it from
produce carbonic acid, chloride diffuses back
binding oxygen. Most cases of
out into the plasma.
dyshemoglobinemia are acquired; a small
• Approximately 85% of the CO2 produced in the
fraction of methemoglobinemia cases are
tissues is transported by hemoglobin as H+ 1 In
hereditary
this capacity, hemoglobin provides a buffering
effect by binding and releasing H+ 1 A small Methemoglobin
percentage of CO2 remains in the cytoplasm,
• Methemoglobin (MetHb) is formed by the
and the remainder binds to the globin chains as
reversible oxidation of heme iron to the ferric
a carbamino group.
state (Fe3+). Normally, a small amount of
Nitric Oxide Transport methemoglobin is continuously formed by
oxidation of iron during normal oxygenation and
• A third function of hemoglobin involves the
deoxygenationofhemoglobin.
binding, inactivation, and transport of nitric
• However, methemoglobin reduction systems,
oxide.
predominantly the NADH-cytochrome b5
• Nitric oxide
reductase 3 (NADH-methemoglobin reductase)
pathway, normally limit its accumulation to only
1% of total hemoglobin
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• Methemoglobin cannot carry oxygen because • Hb M is inherited in an autosomal dominant
oxidized ferric iron cannot bind it. pattern, with methemoglobin comprising 30% to
• An increase in methemoglobin level results in 50% of total hemoglobin.
decreased delivery of oxygen to the tissues. • There is no effective treatment for this form of
• Individuals with methemoglobin levels less than methemoglobinemia.
25% are generally asymptomatic. • Cytochrome b5 reductase defficiency is an
• If the methemoglobin level increases to more autosomal recessive disorder, and
than 30% of total hemoglobin, cyanosis (bluish methemoglobin elevations occur in individuals
discoloration of skin and mucous membranes) who are homozygous or compound
and symptoms of hypoxia (dyspnea, headache, heterozygous for a CYB5R3 mutation.
vertigo, change in mental status) occur. • Most individuals with Hb M or homozygous
• Levels of methemoglobin greater than 50% can cytochrome b5 reductase defficiency maintain
lead to coma and death. methemoglobin levels less than 50%; they have
• An increase in methemoglobin, called cyanosis but only mild symptoms of hypoxia
methemoglobinemia, can be acquired or that do not require treatment.
hereditary. • Individuals heterozygous for the CYB5R3
• Acquired form, also called toxic mutation have normal levels of methemoglobin
methemoglobinemia, occurs in individuals after but develop methemoglobinemia, cyanosis, and
exposure to an exogenous oxidant, such as hypoxia when exposed to an oxidant drug or
nitrites, primaquine, dapsone, or benzocaine. chemical.
• As the oxidant overwhelms the hemoglobin • Methemoglobin is assayed by spectral
reduction systems, the level of methemoglobin absorption analysis instruments such as the CO-
increases, and the patient may exhibit cyanosis oximeter. Methemoglobin shows an absorption
and symptoms of hypoxia. peak at 630 nm.
• In many cases, withdrawal of the offending • With high levels of methemoglobin, the blood
oxidant is sufficient for a recovery, but if the takes on a chocolate brown color and does not
level of methemoglobin increases to 30% or revert back to normal red color after oxygen
more of total hemoglobin, intravenous exposure.
methylene blue is administered. • The methemoglobin in Hb M disease has
• Methylene blue reduces methemoglobin ferric different absorption peaks, depending on the
iron to the ferrous state through NADPH- variant.
methemoglobin reductase and NADPH • Hemoglobin electrophoresis, HPLC, and DNA
produced by glucose-6-phosphate mutation testing are used for identification of Hb
dehydrogenase in the hexose monophosphate M variants.
shunt • Cytochrome b5 reductase 3 deficiency is
• In life-threatening cases, exchange transfusion diagnosed by enzyme assays and DNA mutation
may be required testing.
• Hereditary causes of methemoglobinemia are
Sulfhemoglobin
rare and include mutations in the gene for
NADH-cytochrome b5 reductase 3 (CYB5R3), • formed by irreversible oxidation of hemoglobin
resulting in a diminished capacity to reduce by drugs (such as sulfanilamides, phenacetin,
methemoglobin, and mutations in the α-, β-, or nitrites, and phenylhydrazine) or exposure to
γ-globin gene, resulting in a structurally sulfur chemicals in industrial or environmental
abnormal polypeptide chain that favors the settings
oxidized ferric form of iron and prevents its • It is formed by the addition of a sulfur atom to
reduction. the pyrrole ring of heme and has a greenish
• Methemoglobin produced by the latter group is pigment.
called hemoglobin M (Hb M) • ineffective for oxygen transport, and patients
with elevated levels present with cyanosis.
Hematology: Hemoglobin Metabolism
Jessica I. Pontanares | ♡♡♡ | Page 11 of 11
• cannot be converted to normal Hb A; it persists • Carboxyhemoglobin may be detected by
for the life of the cell. spectral absorption instruments at 540 nm.
• Treatment consists of prevention by avoidance • It gives blood a cherry red color, which is
of the offending agent. sometimes imparted to the skin of victims.
• has a similar peak to methemoglobin on a • A diagnosis of carbon monoxide poisoning is
spectral absorption instrument. made if the COHb level is greater than 3% in
• Sulfhemoglobin spectral curve, however, does nonsmokers and greater than 10% in smokers.
not shift when cyanide is added, a feature that • Treatment involves removing the carbon
distinguishes it from methemoglobin. monoxide source and administration of 100%
oxygen.
Carboxyhemoglobin
• Use of hyperbaric oxygen therapy is
• Carboxyhemoglobin (COHb) results from the controversial; it is primarily used to prevent
combination of carbon monoxide (CO) with neurologic and cognitive impairment after acute
heme iron. carbon monoxide exposure in patients whose
• affnity of carbon monoxide for hemoglobin is COHb level exceeds 25%.
240 times that of oxygen.
Hemoglobin measurement
• Once one molecule of carbon monoxide binds
to hemoglobin, it shifts the hemoglobin-oxygen Cyanmethemoglobin method
dissociation curve to the left, further increasing
• reference method for hemoglobin assay.
its affinity and severely impairing release of
• A lysing agent present in the
oxygen to the tissues.
cyanmethemoglobin reagent frees hemoglobin
• Carbon monoxide has been termed the silent
from RBCs.
killer because it is an odorless and colorless
• Free hemoglobin combines with potassium
gas, and victims may quickly become hypoxic.
ferricyanide contained in the
• Some carboxyhemoglobin is produced
cyanmethemoglobin reagent, which converts
endogenously, but it normally comprises less
hemoglobin iron from the ferrous to the ferric
than 2% of total hemoglobin.
state to form methemoglobin.
• Exogenous carbon monoxide is derived from
• Methemoglobin combines with potassium
the exhaust of automobiles; tobacco smoke;
cyanide to form the stable pigment
and industrial pollutants such as coal, gas, and
cyanmethemoglobin.
charcoal burning]
• The cyanmethemoglobin color intensity, which
• In smokers, COHb levels may be as high as
is proportional to hemoglobin concentration, is
15%.
measured at 540 nm spectrophotometrically
• As a result, smokers may have a higher
and compared with a standard
hematocrit and polycythemia to compensate for
• performed manually but has been adapted for
the hypoxia.
use in automated blood cell analyzers.
• Exposure to carbon monoxide may be
• Many instruments now use sodium lauryl
coincidental, accidental, or intentional (suicidal).
sulfate (SLS) to convert hemoglobin to SLS-
Many deaths from house fires are the result of
methemoglobin – this method does not
inhaling smoke, fumes, or carbon monoxide.
generate toxic wastes
• Even when heating systems in homes are
• Hemoglobin electrophoresis and HPLC are used
properly maintained, accidental poisoning with
to separate the different types of hemoglobins
carbon monoxide may occur.
such as Hb A, A2 , and F
• Toxic effects, such as headache, dizziness, and
disorientation, begin to appear at blood levels of
20% to 30% COHb
• Levels of more than 40% of total hemoglobin
may cause coma, seizure, hypotension, cardiac
arrhythmias, pulmonary edema, and death