C H A P T E R 42
RED BLOOD CELL ENZYMOPATHIES
Elizabeth A. Price, Stavroula Otis, and Stanley L. Schrier
When evaluating the patient with hemolytic anemia, it is good prac-
tice to determine whether the hemolysis is due to an intracorpuscular
or extracorpuscular defect. With the exception of paroxysmal noctur-
nal hemoglobinuria, intracorpuscular causes of hemolysis are all
hereditary and involve the three major compartments of the red
blood cell (RBC): hemoglobin, the plasma membrane, and the RBC’s
metabolic machinery. Abnormalities of hemoglobin and the plasma
membrane are covered elsewhere. This section is devoted to an analy-
sis of the abnormalities of the RBC’s enzymatic-metabolic machinery.
The study of human RBCs has afforded numerous insights into cel-
lular enzymatic pathways. Defects in the enzymes of these pathways
can be categorized according to which essential functions of RBC
metabolism are crippled by their deficiency. The following functions,
when impaired, are deleterious to RBC survival:
1. Maintenance of RBC reducing power: The RBC’s reducing
power is of paramount importance to its survival. Red cells are
loaded with the oxygen they carry to the tissues and are constantly
bombarded with reactive oxygen species. The erythrocyte has a
remarkable ability to absorb extreme oxidative stress by virtue of
the enzymes involved in glutathione metabolism and generation
of the reduced form of nicotinamide adenine dinucleotide phos-
phate (NADPH). These enzymes provide the red cell with tremen-
dous reducing power by serving as a large reservoir of electrons
that can be donated to hemoglobin, lipoproteins, and other intra-
cellular proteins damaged by oxidative stress.
2. Generation of adenosine triphosphate (ATP): The RBC uses
energy in the form of ATP to energize the pumps and membrane
transport mechanisms that allow the red cell to maintain its stable
internal environment. The mature red cell is incapable of oxidative
phosphorylation because it has lost its mitochondria and mito-
chondrial enzymes. As such, the red cell must rely on the less
efficient, classic Embden-Meyerhof pathway of glycolysis for ATP
generation. The glycolytic enzymes catalyzing this evolutionarily
conserved pathway are critical for the production of the ATP
requisite for the pumps and transport mechanisms, as well as the
production of the reduced form of nicotinamide adenine dinucle-
otide (NADH) needed for cytochrome-b5 reductase (Cb5R), the
major methemoglobin reductase.
3. Maintenance of hemoglobin in a functional state: The enzymes
that catalyze the formation of NADH and NADPH play a critical
role in maintaining hemoglobin in its reduced state by supplying
Cb5R (the major methemoglobin reductase) and the alternative
methemoglobin reductase with its cofactors. The enzymes that
catalyze the formation of 2,3-bisphosphoglycerate (2,3-BPG) via
the Rapoport-Luebering shunt also play an important role in
hemoglobin function because 2,3-BGP binds to the end of the
β-globin chain and facilitates the off-loading of oxygen from
hemoglobin to the tissues.
4. Degradation of ribosomal proteins: The RBC is unique in that
it transitions from a typical nucleated cell capable of protein
synthesis and oxidative and anaerobic metabolism to a cell devoid
of organelles, incapable of protein synthesis, and dependent
on glycolysis for energy production. The RBC must modify
its membrane and volume accordingly and expel ribosomal
proteins, which could otherwise prove deleterious. Pyrimidine
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5′-nucleotidase is effective in degrading the ribosomal contents
into smaller components that can pass through the RBC
membrane.
For the most part, these enzyme deficiencies are exceedingly rare,
with a few notable exceptions. The more prevalent enzyme deficien-
cies presumably offer an evolutionary advantage, whereas the rare
deficiencies likely represent spontaneous mutations that offer no
selective advantage.
ENZYMOPATHIES OF GLUTATHIONE METABOLISM
Glutathione Metabolism
Glutathione is a key player in numerous cellular functions, including
free radical scavenging, redox reactions, and biosynthesis of deoxyri-
bonucleic acid (DNA), proteins, and leukotrienes. GSH also plays a
role in neurotransmission and neuromodulation. In the RBC, gluta-
thione is present in extremely high concentrations, higher than any
other cell in the body, and plays a critical role in protecting the RBC
from oxidant injury. The ratio of reduced glutathione (GSH) to
oxidized glutathione (GSSG) is maintained at high levels in the red
cell by two mechanisms: (1) GSSG is converted to GSH by glutathi-
one reductase, and (2) GSSG is actively transported out of the
erythrocyte.
GSH is a tripeptide composed of glutamate, cysteine, and glycine.
γ-glutamylcysteine synthetase catalyzes the first and rate-limiting step
in the synthesis of GSH (Fig. 42-1). The product of this reaction,
γ-glutamylcysteine, is then converted to GSH in the presence of
glycine via glutathione synthetase, the enzyme that catalyzes the
second and final step in GSH synthesis (see Fig. 42-1). Oxidation of
GSH by glutathione peroxidase or other free radicals leads to the
formation of glutathione disulfide (GSSG) and other mixed disulfides
of proteins that contain free –SH groups (like hemoglobin). GSSG
is rapidly restored to reduced glutathione via glutathione reductase
and its cofactor NADPH. NADPH is maintained at high levels by
glucose-6-phosphate dehydrogenase (G6PD). Defects in each of
these enzymes have been described and are extremely rare, with the
notable exception of G6PD deficiency.
G6PD Deficiency
History
G6PD deficiency was first recognized in the early 1950s during the
Korean War when approximately 10% of African American soldiers
given the antimalarial drug primaquine developed a self-limited
hemolytic anemia. This phenomenon led to further investigations on
healthy volunteers who were given 30 mg of primaquine daily. Most
subjects tolerated the drug well, but a minority developed a
self-limited hemolytic anemia. Pursuant studies showed that
51
Cr-labeled RBCs from primaquine-sensitive subjects transfused into
nonsensitive subjects were rapidly destroyed, whereas RBCs from
nonsensitive subjects transfused into primaquine-sensitive subjects
581
 582 Part V  Red Blood Cells
Cysteine Glutamic acid
ATP
γ-Glutamylcysteine
synthetase ADP
γ-Glutamylcysteine
5-Oxoproline
Glycine
RBC membrane
Glutathione ATP
synthetase
ADP
H2O2 H2O
ADP
ATP
Glutathione peroxidase
GSH GSSG
Glutathione reductase
NADP+ NADPH
G6PD
G6P F6P
Pentose
shunt
Figure 42-1  GLUTATHIONE PATHWAY. ADP, Adenosine diphosphate;
ATP, adenosine triphosphate; F6P, fructose 6-phosphate; G6P, glucose
6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; GSH, reduced glu-
tathione; GSSG, oxidized glutathione; NADP+, oxidized form of nicotin-
amide adenine dinucleotide phosphate; NADPH, reduced form of
nicotinamide adenine dinucleotide phosphate; RBC, red blood cell.
survived normally even when the host’s RBCs were rapidly destroyed.1,2
These findings established clearly that primaquine sensitivity was due
to an intrinsic defect of the erythrocyte. Subsequent studies showed
that younger, more metabolically active RBCs were resistant to
destruction, whereas older RBCs were being destroyed, suggestive of
an underlying metabolic defect.3 By 1956, researchers had systemati-
cally deduced that the primary defect was in the G6PD enzyme.4-8 In
1961 the G6PD gene was discovered to be X-linked, due to its linkage
to red-green color blindness.
Epidemiology
G6PD deficiency is the most prevalent human enzyme deficiency in
the world, affecting an estimated 350 to 400 million people. The
geographic distribution of G6PD deficiency coincides with the geo-
graphic distribution of endemic malaria, implicating a survival
benefit. The highest prevalence of G6PD deficiency is in sub-Saharan
Africa, followed by the Middle East, Mediterranean Europe, and
Southeast Asia.9,10
The most common allelic variants of G6PD are G6PD A- and
G6PD Mediterranean. In fact, these two variants coexist at polymor-
phic frequencies in several populations, most notably in countries
surrounding the Persian Gulf.11 G6PD A- accounts for approximately
90% of G6PD in Africa but is also prevalent in North and South
America, the West Indies, Italy, the Canary Islands, Spain, Portugal,
and the Middle East.12-16 G6PD Mediterranean is prevalent in all
countries surrounding the Mediterranean Sea, as well as the Middle
East, India, and Indonesia.14,17
In addition to geographic overlap, numerous studies support the
premise that polymorphic variants of G6PD confer a survival benefit
in malaria-endemic regions. Ruwende et al18 showed that the G6PD
A- allele is associated with a substantial reduction in the risk for severe
Plasmodium falciparum malaria for female heterozygotes (46% risk
reduction) and male hemizygotes (58% risk reduction). In addition,
several in vitro studies comparing the growth of malarial parasites in
G6PD-B (wild type) erythrocytes to growth in G6PDA- and G6PD
Mediterranean erythrocytes have shown protracted growth in the
G6PD-deficient cells.19-24 Growth is also blunted in G6PD-deficient
RBCs taken from female heterozygotes, whose cells have undergone
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random X-chromosome inactivation, when compared to parasite
growth in RBCs with normal G6PD activity from the same
individuals.25
Oxidant injury to the parasite itself appears to be one mechanism
by which G6PD deficiency confers its protective effects. Due to the
host cell’s impaired ability to restore intracellular NADPH and GSH,
malarial parasites in G6PD-deficient RBCs may be more vulnerable
to the reactive intermediates they generate (particularly oxidized iron)
when they break down hemoglobin.26,27 Other studies have shown
that malaria-infected, G6PD-deficient RBCs undergo phagocytosis
by macrophages at an earlier stage of parasite maturation than do
normal malaria-infected RBCs, which could be another means by
which G6PD-deficient cells offer a selective advantage.20
The prevalence of G6PD deficiency in the United States ranges
from 0.5% to 7% and is most common among African American
males, affecting approximately 10%.10,28 Screening for G6PD defi-
ciency is generally reserved for individuals at increased risk, such as
human immunodeficiency virus (HIV)–infected individuals from
susceptible racial and ethnic groups. The overall prevalence rate of
G6PD deficiency among HIV-infected patients is estimated to be
approximately 6.8%.29 Because HIV patients are more likely to be
exposed to oxidant drugs like dapsone, primaquine, and sulfon-
amides, primary care guidelines for management of HIV recommend
screening patients from higher prevalence ethnic or racial groups
(e.g., African and Mediterranean descent) for G6PD deficiency either
at baseline or before initiating therapy with an oxidant drug.30
Pathobiology
G6PD is a cellular housekeeping enzyme that catalyzes the first step
in the hexose monophosphate shunt, wherein glucose 6-phosphate
(G6P) is converted to ribose 5-phosphate (Fig. 42-2), a precursor of
many important molecules like ribonucleic acid (RNA), DNA, ATP,
coenzyme A, nicotinamide adenine dinucleotide (NAD), flavin
adenine dinucleotide (FAD). Another major role of the hexose mono-
phosphate shunt is to maintain high levels of NADPH, which acts
as a cofactor for GSH. Most cells have alternate enzymatic pathways
that can generate NADPH, but RBCs lack a nucleus, mitochondria,
and other organelles necessary to produce these enzymes and are
therefore particularly dependent on G6PD and the hexose mono-
phosphate shunt for maintaining high levels of NADPH and GSH
to protect against oxidative stress.
G6PD is encoded by a gene located on the telomeric region of
the long arm of the X chromosome (Xq28) that spans 18 kb, and
consists of 13 exons and 12 introns. The G6PD gene product com-
prises 515 amino acids with a molecular weight of 59 kDa.31 The
enzyme is active as a tetramer or dimer. Stability of the active qua-
ternary structure is crucial for normal G6PD activity.
Within the RBC, oxidant injury leads to the oxidation of sulfhy-
dryl groups on the hemoglobin molecule, resulting in the formation
of disulfide bridges, which in turn leads to decreased hemoglobin
solubility and ultimately the irreversible precipitation of oxidized
hemoglobin.32,33 Under normal conditions oxidized hemoglobin is
reduced by GSH, which itself is oxidized in the process but restored
to its reduced form by intracellular NADPH, whose levels are main-
tained by G6PD. In the G6PD-deficient RBC, GSH is not restored
to adequate levels under oxidative stress, leading to a buildup of free
radicals and insoluble hemoglobin within the cell, which can be
visualized under the microscope as Heinz bodies. Precipitated hemo-
globin is disruptive to the structure and function of the RBC mem-
brane and leads to increased membrane permeability, osmotic fragility,
and cell rigidity. The compromised integrity of the cell membrane
results in both intravascular hemolysis and rapid removal of these
cells within the splenic pulp.34
Over 400 variants of the G6PD enzyme have been identified by
biochemical methods, 100 of which reach polymorphic levels. Most
variants are due to point mutations and, to a lesser extent, small
in-frame deletions that result in missense mutations. Gross deletions,
nonsense mutations, frame-shift mutations, and splicing defects are
 Chapter 42  Red Blood Cell Enzymopathies 583

Glutathione production Table 42-1  World Health Organization Classification

GSH GSSG of G6PD Variants
Glucose
ATP NADP+ NADPH Class Enzymatic Activity Degree of Associated Hemolysis
Glycolysis ADP I Severely deficient Chronic hemolysis
Glucose 6-P G6PD 6PG
II Severely deficient (<10% Acute, episodic
Hexose
residual activity)
monophosphate
Fructose 6-P shunt III Moderately to mildly deficient Acute, episodic
ATP Phosphofructokinase (10%-60% residual activity)
ADP
Fructose 1,6-biP IV Mildly deficient to normal Absent
Aldolase (60%-150%)
DHAP V Increased (>150%) Absent
Hemoglobin NAD+ TPI
GAPD From WHO Working Group: Glucose-6-phosphate dehydrogenase deficiency.
Methemoglobin NADH Bull World Health Organ 67:601, 1989.
G6PD, Glucose-6-phosphate dehydrogenase.
1,3-BPG Rapoport-
ADP Luebering 2,3-BPG
PGK
ATP
3-P-Glycerate shunt
Phosphoglyceromutase agents like drugs, fava beans, chemicals (e.g., naphthalene, antifungal
2-P-Glycerate sprays), and herbs (e.g., Coptis sinensis, calculus bovis), or the result
of a systemic process like infection, liver injury, and diabetic
Enolase
ketoacidosis.35
Phosphoenolpyruvate
ADP Pyruvate
ATP kinase Clinical Manifestations
NADH Lactate Most people with G6PD deficiency have no clinical symptoms and
NAD+ dehydrogenase
are not anemic. In fact, the majority of affected individuals live out
Lactate their lives unaware of their status. Diagnosis typically occurs when
Figure 42-2  GLYCOLYSIS (EMBDEN-MEYERHOF PATHWAY). ADP, an episode of acute hemolysis is triggered by exposure to oxidant
Adenosine diphosphate; ATP, adenosine triphosphate; 1,3-BPG, 1,3- bisphos- drugs, infection, or ingestion of fava beans. Unusual presentations
phoglycerate; 2,3-BPG, 2,3-bisphosphoglycerate; DHAP, dihydroxyacetone include hemolysis precipitated by complications of diabetes, myocar-
phosphate; GAPD, glyceraldehyde phosphate dehydrogenase; G6PD, glucose- dial infarction, and strenuous physical exercise.42-44
6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glu- The symptoms are characteristic of acute hemolysis and include
tathione; NAD+, nicotinamide adenine dinucleotide; NADP+, oxidized form fatigue, jaundice, pallor, dark urine, and abdominal and low back
of nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of pain. In the case of drug-induced hemolysis, symptoms occur 2 to 4
nicotinamide adenine dinucleotide phosphate; 6PG, 6-phosphogluconate; days after drug ingestion and are associated with a 3 to 4 g/dL drop
PGK, phosphoglycerate kinase; TPI, triose-phosphate isomerase. in hemoglobin level. The bone marrow responds appropriately by
generating a reticulocytosis that peaks approximately 7 to 10 days
after the onset of hemolysis.1
not reported for this gene, because mutants showing 100% deficiency Depending on the G6PD variant, hemolysis can be self-limited
of the G6PD enzyme would presumably be incompatible with despite continuation of the offending drug, or it can be protracted
life.35-37 despite discontinuation of the offending drug. Class III G6PD vari-
The World Health Organization classifies the G6PD variants ants have a moderately shortened half-life (e.g., the half-life of G6PD
based on their enzymatic activity and the degree of associated hemo- A- is approximately 13 days) when compared to the half-life of wild-
lysis (Table 42-1).38,39 type G6PD (approximately 62 days).45,46 Consequently, hemolysis is
Wild-type G6PD is referred to as G6PD B (Western). It is the restricted to older RBCs that are more deficient in G6PD. As the
most common worldwide normal isoenzyme and is the ancestral older RBCs are eradicated, they are replaced by reticulocytes with
human sequence, as demonstrated by showing that G6PD B matches higher concentrations of G6PD. The concentration of G6PD in these
the sequence of G6PD in the chimpanzee, our nearest relative, and younger cells is sufficient to overcome the oxidative stress induced by
by analysis of linkage disequilibrium.12,40 The numerous G6PD vari- the offending agent, and the hemolytic process is thereby self-limited,
ants differ in molecular stability and enzymatic activity. The degree even when the offending agent is continued. In contrast, individuals
of hemolysis associated with each isoenzyme is dependent on the type with a Class II variant of G6PD (such as G6PD Mediterranean)
of defect and severity of oxidant injury. Class I variants are severe, experience a more severe drug-induced hemolysis because the half-life
occur sporadically, and lead to chronic hemolytic anemia. Class II of these variants is on the order of hours,45 making all erythrocytes,
and III variants are found at much higher frequencies than class I young and old, more vulnerable to oxidant injury. In these individu-
variants, and are implicated in providing protection against malaria. als, hemolysis continues well after discontinuation of the culprit
Class IV and V variants are of no clinical significance. drug.47,48
In normal erythrocytes the G6PD enzyme operates at 1% to 2%
of its maximum potential, leaving a large reserve of reductive poten-
tial for times of severe oxidative stress.41 This potential is variably Favism
decreased in G6PD-deficient erythrocytes. Under normal conditions For centuries fava beans have been associated with the clinical sequelae
a mild to moderate deficiency in the activity of G6PD is not deleteri- of acute hemolysis.49 The beans are a staple food in the Mediterra-
ous, and there are no clinical or laboratory signs of hemolysis in these nean, Middle East, and Far East, and their ingestion results in acute
individuals. Under conditions of oxidative stress, however, the reduc- hemolysis in susceptible individuals. Only a minority of G6PD-
tive potential of G6PD-deficient erythrocytes is overwhelmed, result- deficient individuals develop hemolytic anemia after ingestion of the
ing in acute hemolysis. Oxidative stress may be due to exogenous bean, suggesting other genetic factors at play.50-52 Two components of

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 584 Part V  Red Blood Cells
the fava bean, divicine and isouramil, may play a role in driving
hemolysis by further impairing the reductive capacity of the G6PD-
deficient RBC.53 Favism develops within 5 to 24 hours of bean inges-
tion and is characterized by the typical symptoms of acute intravascular
hemolysis: headache, nausea, back pain, chills, fever, hemoglobinuria,
and jaundice. Favism has also been reported in breastfed babies whose
mothers consumed fava beans.54
Neonatal Jaundice
Among the clinical manifestations associated with G6PD deficiency,
neonatal jaundice now carries the highest risk for potentially devastat-
ing clinical consequences, namely, kernicterus.55 Contrary to what is
commonly believed, jaundice in neonates with G6PD deficiency is
not the result of hemolysis. In fact, most neonates have normal
hemoglobin and reticulocyte levels, and the RBC life span is only
modestly shortened, if at all.56 Rather, the hyperbilirubinemia is sec-
ondary to the newborn liver’s inability to adequately conjugate bili-
rubin.57 This problem is compounded when the newborn also inherits
a mutation of the uridine diphosphate-glucuronosyltransferase 1
(UGT1A1) gene promoter that is associated with Gilbert syndrome,
increasing the risk for neonatal jaundice.58 Over 30% of kernicterus
cases are associated with G6PD deficiency,59 raising the question of
whether G6PD deficiency should be included in routine newborn
screening programs.
Congenital Nonspherocytic Hemolytic Anemia
As mentioned earlier, class I G6PD variants have very low enzymatic
activity and/or marked instability, resulting in lifelong hemolysis due
to the everyday oxidative stresses encountered by the circulating
erythrocyte. These variants are sporadic, and almost all arise from
independent mutations clustering in exons 10 and 11, which are close
to the substrate binding domain in the folded protein.60 The disorder
is first suspected when affected infants develop severe neonatal jaun-
dice, or it is discovered later when the typically mild anemia is exac-
erbated by oxidant drug exposure, infection, or parvovirus-induced
aplastic crisis.
Laboratory Manifestations
Under normal conditions, most G6PD-deficient individuals (class II
and III variants) are not anemic and have no laboratory evidence of
hemolysis. In the setting of oxidative stress, the laboratory findings
are those of any acute hemolytic process and include anemia, reticu-
locytosis, hyperbilirubinemia, increased lactate dehydrogenase
(LDH), decreased haptoglobin, and hemoglobinuria. The peripheral
blood smear is notable for Heinz bodies (precipitated sulfhemoglo-
bin), and on occasion for “bite cells” (also known as hemiblister cells,
eccentrocytes, and cross-bonded cells), which occur when denatured
hemoglobin binds to the cell membrane, creating a puddling of
hemoglobin to one side of the cell with an adjacent membrane-bound
clear zone.61,62
Diagnosis
The enzymatic activity of G6PD can be quantitated by measuring
the rate of NADPH production in red cell hemolysates that contain
G6P and NADP+ (the enzyme’s substrates); this is done using a
spectrophotometer, which can measure the rate of NADPH forma-
tion at wavelength 340 nm. This method is used to make a biochemi-
cal definitive diagnosis. For more rapid screening of at-risk
populations, there are several semiquantitative methods available.
The most simple, sensitive, and inexpensive screening test is the fluo-
rescent spot test.63 In this procedure, NADPH production is detected
by virtue of its fluorescence under ultraviolet light. As with the quan-
titative test, G6P and NADP+ are added to a hemolysate of the
patient’s red cells, and fluorescence under ultraviolet light indicates
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enzyme activity, whereas absence of fluorescence indicates enzyme
deficiency. Other semiquantitative tests have been used but require
definitive testing to confirm an abnormal result.64,65 False negatives
are a concern when diagnostic tests are performed during or right
after an acute hemolytic episode in the midst of a reticulocytosis.
Younger RBCs have higher levels of G6PD than older RBCs, so when
the patient’s red cell population is weighted toward younger cells, the
deficiency may go undetected. Females heterozygous for G6PD are
particularly difficult to diagnose due to the natural mosaicism for
X-chromosome enzymes. Heterozygotes with extremely skewed
X-inactivation can have enzymatic activity ranging anywhere from
hemizygote to normal. Molecular diagnostic methods are more reli-
able for making the diagnosis of females suspected of being hetero-
zygous for G6PD deficiency.
Therapy
Treatment of the clinical sequelae of G6PD deficiency is straightfor-
ward. In the case of acute hemolysis, removal of the inciting agent
(e.g., drug, fava beans) is recommended. This maneuver is of particu-
lar importance in patients with Class I and II variants, in whom even
the young RBCs lack adequate G6PD activity to resist ongoing
exposure to oxidative agents. Class III variants, on the other hand,
usually undergo a self-limited hemolysis that resolves despite ongoing
exposure to the inciting agent. In cases of severe, symptomatic
anemia, blood transfusion may be necessary, especially in patients
with a blunted erythropoietic response (e.g., HIV, infection, drug-
related myelosuppression). Very rarely, congenital nonspherocytic
hemolytic anemia is severe enough to mandate ongoing transfusions,
which may lead to iron overload in the absence of appropriate iron
chelation. Folic acid supplementation is also recommended for
patients with congenital nonspherocytic hemolysis. Antioxidants like
vitamin E and selenium may also be beneficial in these patients, but
there are no consistent data to support their use.66
In the event of neonatal jaundice, the treatment is the same as
that recommended for neonatal jaundice arising from other causes.
Mild cases do not require treatment, intermediate cases require pho-
totherapy, and severe cases require exchange transfusion (total biliru-
bin >20 mg/dL). The true key to management of G6PD is prevention
of its clinical sequelae, which requires awareness of the disorder on
the part of both the physician and the patient. The disease should
always be suspected in patients with a nonimmune hemolytic anemia
and patients with an otherwise unexplained personal or family history
of recurrent jaundice, splenomegaly, or cholelithiasis. G6PD defi-
ciency should be considered in any neonate with hyperbilirubinemia,
especially those of high-risk ethnic descent. There has been consider-
able debate in recent years as to whether testing for G6PD deficiency
should be a routine part of neonatal screening tests. Interestingly, in
a study done in Cleveland, Ohio, investigators found that the routine
cord blood screening protocol for isoimmune hemolytic disease had
a lower yield and higher cost than their pilot protocol for cord blood
G6PD deficiency screening,67 both of which are considered major risk
factors for neonatal jaundice. One caveat to routine screening of all
newborns is that heterozygous females and hemizygous males with
residual enzyme activity greater than 20% are likely to be erroneously
classified as “normal” due to the limitations of the fluorescent spot
test,68,69 instilling a false sense of security and subverting the preven-
tion of complications.
Once the diagnosis of G6PD deficiency is established, patients
must be counseled to avoid drugs, chemicals, and foods known to
precipitate hemolysis. Although there are some medications classically
connected to hemolysis in G6PD patients, there are a number of
medications for which there is considerable confusion as to whether
they are safe in G6PD-deficient patients. In an attempt to better
clarify which are truly high-risk drugs, investigators in Israel per-
formed a comprehensive literature search and categorized drugs
according to how much evidence there is in the literature to contra-
indicate their use.70 They found only seven medications for which
there is solid evidence to prohibit their use in G6PD-deficient

patients: dapsone, methylthionine chloride (methylene blue), nitro-
furantoin, phenazopyridine, primaquine, rasburicase, and tolonium
chloride (toluidine blue). Their review found no substantial evidence
to absolutely contravene the use of other medications in normal
therapeutic doses.
Prognosis
Except for rare cases of severe congenital nonimmune hemolytic
anemia and severe neonatal jaundice, the prognosis of patients with
G6PD deficiency is excellent with no apparent impact on life
expectancy.
Future Directions
Current screening tests for G6PD deficiency do not reliably identify
female heterozygotes or male hemizygotes with greater than 20%
residual enzyme activity. This is problematic because these groups
remain at risk for hemolytic complications. It would be useful to
devise a rapid and inexpensive screening test that could detect patients
with moderately reduced enzymatic activity (20% to 60%), so they
too may be counseled on avoiding exogenous agents that could pre-
cipitate a hemolytic crisis. It would also be useful to devise a simple
in vitro method to determine whether new drugs will cause hemoly-
sis. Simply incubating RBCs with the drug in question is unreliable,
most likely because drug metabolites are often the culprits, rather
than the original drug. And finally, the value of G6PD screening of
all newborns must be flushed out, because this is a major cause of
neonatal kernicterus, even in first world countries.71-73
γ-Glutamylcysteine Synthetase Deficiency
As described earlier, γ-glutamylcysteine synthetase catalyzes the first
step in glutathione synthesis. Deficiency of this enzyme is exceedingly
rare. Only nine patients in seven families have been reported world-
wide.74 All nine patients developed a relatively mild hemolytic anemia,
and four of the nine manifested neurologic problems, including
spinocerebellar degeneration, peripheral neuropathy, and mental
retardation.75-78
Glutathione Synthetase Deficiency
Glutathione synthetase deficiency is the most common of the inborn
errors of GSH metabolism, with more than 70 patients in over 50
families reported worldwide.74 Patients are homozygous or compound
heterozygous for mutations in the glutathione synthetase gene. The
clinical presentation is variable, and patients are categorized as mildly,
moderately, or severely affected.79 Mildly affected patients have an
isolated hemolytic anemia, whereas moderately affected patients
develop a metabolic acidosis within the first few days of life due to
the buildup of 5-oxoproline, a metabolite of γ-glutamylcysteine (the
substrate of glutathione synthetase). Under normal circumstances
GSH negatively feeds back on γ-glutamylcysteine synthetase, but in
patients with moderate to severe glutathione synthetase deficiency
there is loss of negative feedback, leading to the accumulation of
5-oxoproline in body fluids, resulting in metabolic acidosis and
massive 5-oxoprolinuria. Severely affected patients may also develop
recurrent bacterial infections and progressive dysfunction of the
central nervous system (CNS). The mechanism behind CNS involve-
ment remains unclear. Autopsy of the first patient described with
glutathione synthetase deficiency revealed selective atrophy of the
granule cell layer of the cerebellum, focal lesions in the frontoparietal
cortex, and bilateral focal lesions in the thalamus and visual cortex.
The diagnosis of glutathione synthetase deficiency is suspected in
patients with a nonimmune hemolytic anemia, elevated levels of
5-oxoproline in the urine, and markedly reduced RBC glutathione
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Chapter 42  Red Blood Cell Enzymopathies 585
content. The diagnosis is confirmed by demonstrating low levels of
glutathione synthetase in cultured skin fibroblasts and/or RBCs or
by demonstrating mutations in the glutathione synthetase gene.74
Treatment involves correcting the metabolic acidosis with bicarbon-
ate and protecting the cells against further oxidant injury by admin-
istering antioxidants like vitamin C and vitamin E. N-acetylcysteine
was used in the past to protect against oxidant damage but is no
longer recommended because studies have shown that cysteine, which
is known to be neurotoxic at high concentrations, accumulates in the
tissues of patients with glutathione synthetase deficiency.80,81 Patients
with glutathione synthetase deficiency should avoid the same agents
known to precipitate acute hemolysis in patients with G6PD
deficiency.
Glutathione Reductase Deficiency
Glutathione reductase restores intracellular GSH by reducing GSSG
in the presence of NADPH and FAD, a derivative of the water-
soluble vitamin riboflavin. Consequently, malnourished patients, or
patients who are otherwise deficient in riboflavin, develop an acquired
partial deficiency of glutathione reductase. Riboflavin deficiency can
be demonstrated by testing the in vitro activity of glutathione reduc-
tase with and without exogenously added FAD. Acquired glutathione
reductase deficiency has no documented hematologic phenotype and
is easily corrected by administration of physiologic quantities of
riboflavin.
Congenital deficiency of glutathione reductase has been reported
in two Dutch families. In the first family, the eldest of three siblings
from a consanguineous marriage developed a hemolytic crisis after
ingesting fava beans.82 All three siblings (one male, two female) were
found to be homozygous for a large deletion in the glutathione
reductase gene encoding almost the entire dimerization domain of
the enzyme.83 In the second family, an infant was born with severe
neonatal jaundice and found to be a compound heterozygote for a
nonsense and missense mutation in the glutathione reductase gene.83
There was no evidence of enhanced hemolysis in this infant, which
is consistent with the neonatal jaundice associated with G6PD
deficiency.
Glutathione Peroxidase Deficiency
Glutathione peroxidase is a selenium-containing enzyme that cata-
lyzes the oxidation of GSH by hydrogen peroxide, producing GSSG
and water. Although rare cases of glutathione peroxidase deficiency
have been described in association with hemolysis,84-86 a causative
relationship between the two has not been clearly established. There
are several reported cases of normal individuals with reduced gluta-
thione peroxidase activity and no evidence of hemolysis.87,88
ENZYMOPATHIES OF THE GLYCOLYTIC PATHWAY
Pyruvate Kinase Deficiency
Epidemiology
Pyruvate kinase (PK) deficiency is the most common red cell enzy-
mopathy leading to congenital nonspherocytic hemolytic anemia.
The prevalence has been estimated by gene frequency studies to be
51 cases per million in the general white population.89 However,
recognized clinical cases in the Northern United Kingdom based on
a registry study initiated in 1974 had a much lower prevalence,90
suggesting premature disease-related death, clinically mild disease, or
diagnostic error.91 Conversely, the prevalence of homozygous PK defi-
ciency was found to be quite high (1 in 830) in a small, remote town
in the Western United States,92 likely due to consanguinity within the
community. Common mutations have well-defined geographic
 586 Part V  Red Blood Cells
associations. Mutation 1529A is frequently seen in the United States93
and central Europe,94 1456T is commonly found in Southern
Europe,95-97 and 1468T is the most common mutation found in
Asia.89,98 PK deficiency is an autosomal recessive disease, and affected
patients are typically double heterozygotes, or, less commonly, homo-
zygous for the same mutation. Homozygous mutations are usually
seen in groups with marked consanguinity, and homozygous PK
deficiency has been well described in the Amish populations of Penn-
sylvania and Ohio.99,100
Pathobiology
PK catalyzes the irreversible transfer of phosphate from phosphoenol-
pyruvate to adenosine diphosphate (ADP), forming ATP and pyru-
vate in the second ATP-generating step of the glycolytic pathway.
There are four distinct PK isoforms, M1, M2, L, and R types, encoded
by two separate genes (PKM and PKLR).101 PKM1 and PKM2 are
produced from the PKM gene by alternative RNA splicing.102 The
M2 isoform is expressed in early fetal and proliferating tissues and
remains the dominant form in adults in leukocytes and platelets.
PKM1 is found in skeletal muscle, heart, and brain.103 PKL, found in
the liver, and PKR, in red cells, are both encoded by the PKLR gene
on chromosome 1q21 through the use of alternate promoters.104
PKM2 is expressed in normal erythroid precursors105 and is gradually
replaced during maturation by the PK-R isoform. The functional
enzyme is a tetramer, and each isoform has characteristic
kinetic properties,103 although all isoforms except M1 are allosterically
activated by fructose 1,6-diphosphate (FDP).106,107 More than
190 mutations in the PKLR gene encoding the red cell PK have
been identified, most of which are missense mutations (www.
pklrmutatinodatabase.com). Mutations may alter the stability of the
enzyme or the enzyme’s affinity for its substrate, phosphoenolpyru-
vate (PEP), or its allosteric activator, FDP.106,108 There appears to be
some correlation with the location of the mutation and the severity
of the hemolytic anemia, with more severe disease being associated
with disruptive mutations and with missense mutations directly
involving the active site or protein stability.92,101
The mechanism of hemolysis in PK deficiency is not fully under-
stood. Reticulocytes are preferentially destroyed in the spleen and the
liver,109 and following splenectomy a striking reticulocytosis may be
seen.110,111 It has been suggested that the higher metabolic requirement
of reticulocytes renders them more vulnerable to PK deficiency than
mature erythrocytes, leading to increased destruction.109 The defect
in ATP generation may contribute to the anemia, although it is likely
not the sole cause, because other disorders with more severe ATP
deficiency do not have significant hemolytic anemia.112 The metabolic
derangements found in PK deficiency may also lead to increased
apoptosis and ineffective erythropoiesis, as seen in the spleen of a
patient with PK deficiency113 and in a mouse model.114 The metabolic
defect is distal to the Rapoport-Leubering shunt, and thus the con-
centration of 2,3-BPG is increased. Although accumulation of
2,3-BPG may lead to further impairment in glycolysis through inhi-
bition of hexokinase,101 the resultant shift in the oxyhemoglobin
dissociation curve to the right115 leads to improved tolerance of the
anemia.116
Clinical and Laboratory Manifestations
Clinical severity in patients with PK deficiency is widely variable,
ranging from fully compensated hemolysis to a transfusion-dependent
anemia. Newborns may present with severe hemolytic anemia and
pronounced jaundice, requiring exchange transfusion.117 In the worst
cases, hydrops fetalis118 with intrauterine or neonatal death may rarely
occur.119 Severe liver dysfunction has also been reported as an unusual
cause of death in infants with PK deficiency.120 Early onset of symp-
toms is typically associated with a more severe clinical course.121
Heterozygotes for PK deficiency who are heterozygotes or homozy-
gotes for the Gilbert syndrome polymorphism may have neonatal
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jaundice that persists in childhood.92 The anemia is relatively stable
in adults,121 although transient worsening may occur with infection,122
pregnancy, or the use of certain medications, including oral contra-
ceptives.123 Gallstones may be present. Extramedullary hematopoiesis
can occur even in patients who are transfusion independent124 and
may lead to characteristic bone deformities.125 Extradural extramedul-
lary hematopoiesis leading to spinal cord compression and neurologic
deficits has been reported,126 with good response to surgical excision
and radiation therapy.126 Leg ulcers are seen rarely.127 Recurrent venous
thromboembolic disease was reported in one adult patient who
underwent splenectomy as a young child,110 and idiopathic pulmo-
nary arterial hypertension was reported in another patient who had
undergone splenectomy at age 5.128
Iron overload has been reported in nontransfused129,130 and
transfusion-dependent patients and may be severe, leading to cir-
rhosis and cardiac and endocrine dysfunction.131 Splenectomy and the
presence of hemochromatosis-related genes may increase the risk for
iron overload.132 Serum levels of the iron regulatory protein hepcidin
were lower in PK-deficient patients compared to controls, and growth
differentiation factor 15 (GDF15) levels were higher, suggesting sup-
pression of hepcidin as a contributing mechanism of iron overload
in some PK-deficient patients.133
Although PK deficiency does not localize to geographic areas of
malarial endemicity, PK deficiency may be protective against
malaria.134 In a mouse model of infection with Plasmodium chabaudi,
mice carrying a loss-of-function mutation in the PKLR gene had
reduced blood-stage parasite replication and improved survival com-
pared to control mice.135 In vitro, erythrocytes from homozygous
PK-deficient patients have decreased invasion by P. falciparum com-
pared to normal or heterozygous PK-deficient control erythrocytes.136
In addition, phagocytosis of ring-stage infected erythrocytes from
patients with homozygous and heterozygous mutations was increased
compared to control subjects.
Laboratory findings are those typical for hemolytic anemia,
including low hemoglobin, increased reticulocyte count, high LDH,
low haptoglobin, and elevated indirect bilirubin. Reticulocytosis may
be suppressed in the context of the normal hypoplastic phase of
erythropoiesis following birth, making the diagnosis more challeng-
ing.137 The bilirubin is usually less than 6 mg/dL: if higher, the patient
may have coexisting Gilbert syndrome.101,138 Red cells are commonly
without characteristic morphologic abnormalities; however, following
splenectomy they may become markedly abnormal, with numerous
crenated cells, lobulated cells, and target cells.125 Establishing the
diagnosis requires the demonstration of low enzyme activity.112 Care
must be taken in interpreting in vitro test results, because contamina-
tion with transfused red cells or leukocytes can increase measured
enzyme activity.101 Specialized laboratories can assess for kinetically
abnormal mutant PKs.112 Molecular diagnostic methods can be used
if the likely mutation is known, as in the case of prenatal diagnosis.
Therapy
Treatment for PK deficiency remains supportive. Exchange transfu-
sion and/or intense phototherapy may be required in the neonatal
period to prevent kernicterus and its resultant sequelae, including
permanent hearing loss.92 Patients with more severe disease may
require periodic or even regular red cell transfusions. Increased trans-
fusion may be needed during pregnancy both for maternal supportive
care and to prevent miscarriage.100 Splenectomy typically leads to an
increase in hemoglobin by 1 to 3 g/dL121 and can lead to transfusion
independence. In one reported case, partial splenectomy failed to
ameliorate transfusion needs secondary to rapid regeneration of the
spleen.139 General recommendations include, where possible, delaying
splenectomy until the patient is 3 years of age or older to reduce the
risk for postsplenectomy sepsis.112 If iron overload occurs, both phar-
macologic iron chelation therapy140 and, if the anemia is not too
severe, phlebotomy,141 can successfully reduce excess iron. Hemato-
poietic stem cell transplant was reportedly successful in one 5-year-
old boy with PK deficiency and hemoglobin E trait.142

Glucose Phosphate Isomerase Deficiency
Glucose phosphate isomerase (GPI) catalyzes the interconversion of
G6P and fructose 6-phosphate in the second step of glycolysis. GPI
deficiency is an autosomal recessive disorder and is one of the four
most common erythrocyte enzymopathies, including deficiencies of
G6PD, PK, and pyrimidine 5′-nucleotidase.143 The hemolytic anemia
is of variable severity and may require chronic transfusions.144 Hydrops
fetalis has been reported.143 Splenectomy can improve the anemia,
eliminating transfusion requirements.143,145 GPI deficiency can also be
associated with neurologic impairment, including myopathy and
mental retardation.146
Hexokinase Deficiency
Hexokinase catalyzes the initial step of glycolysis, the phosphoryla-
tion of glucose to G6P (see Fig. 42-2) and is one of the rate-limiting
steps of this pathway.107 Hexokinase deficiency is a rare cause of
congenital nonspherocytic anemia. Hemolytic anemia may occur
as a sole manifestation or as part of a constellation of abnormalities
that can include other cytopenias, diabetes mellitus, skeletal
malformations, hypogonadism, and abnormal pigmentation.147
Severe iron overload can occur.148 Most patients are of European
descent, although an affected Chinese kindred has also been
described.147 A mouse model of generalized hexokinase deficiency
has been described, with manifestations of severe hemolytic anemia,
marked reticulocytosis, and extensive tissue iron deposition.149
Splenectomy may provide benefit.150 In contrast to PK deficiency,
the defect occurs proximal to the Rapoport-Leubering shunt, and
thus the concentration of 2,3-BPG is reduced, shifting the oxyhemo-
globin dissociation curve to the left115 and decreasing patient tolerance
to the anemia.116
Phosphofructokinase Deficiency
Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose
6-phosphate to fructose-1,6-bisphosphate (see Fig. 42-2).151,152 Two of
the three identified isoenzymes (muscle type [PFKM] and liver type
[PFKL]) are expressed in erythrocytes. Mutations in the muscle-type
PFKM isoenzyme cause type VII glycogen storage disease (Tarui
disease), a rare autosomal recessive disorder. Such mutations lead to
an almost complete loss of PFK activity in muscle, but only partial
loss of activity in erythrocytes. Clinical manifestations of type VII
glycogen storage disease may present in infancy or older adulthood
and include hypotonia, exercise intolerance, and weakness.152 The
severity of the hemolytic anemia is highly variable, ranging from a
mild, compensated hemolysis153 to severe chronic nonspherocytic
hemolytic anemia.152
ALDOLASE DEFICIENCY
Aldolase catalyzes the interconversion of fructose 1,6-diphosphate to
glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.107 One
of the three distinct isoenzymes, aldolase A, is present in erythrocytes,
muscle, and brain. Aldolase deficiency is a very rare cause of nons-
pherocytic hemolytic anemia, and clinical manifestations may also
include myopathy and mental retardation.154
Phosphoglycerokinase Deficiency
Phosphoglycerate kinase catalyzes the reversible conversion of
1,3-bisphosphoglycerate to 3-phosphoglycerate. Deficiency of this X
chromosome–encoded enzyme is associated with an array of findings
that can variably include hemolytic anemia, myopathy, rhabdomyoly-
sis, mental retardation, and other neurologic symptoms.155 Splenec-
tomy may improve the anemia.156
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Chapter 42  Red Blood Cell Enzymopathies 587
Triose-Phosphate Isomerase Deficiency
Triose-phosphate isomerase (TPI) catalyzes the reversible interconver-
sion of the triose phosphate isomers, dihydroxyacetone phosphate,
and glyceraldehyde 3-phosphate. TPI deficiency is a rare, autosomal
recessive, multisystemic disease, characterized by chronic nonsphero-
cytic hemolytic anemia, frequent bacterial infections, and progressive
neuromuscular disease.157-159 Cardiomyopathy may also be present.
Hemolytic anemia manifests before 14 months of age157 and may be
severe enough to require ongoing red cell transfusions.160 In cases with
a milder degree of anemia, intermittent transfusions may be required
during acute exacerbations triggered by bacterial infection.161 Hydrops
fetalis can rarely occur.143 The typical course involves progressive
neurologic degeneration with frequent early death; however, rare
adult cases with less severe involvement are reported.162 A high preva-
lence of heterozygous TPI deficiency has been found in African
American newborns.163 No specific therapy for this disorder is
available.
Pyrimidine 5′-Nucleotidase Deficiency
Pathobiology
Red cell pyrimidine 5′-nucleotidase type 1 (P5′NT-1) deficiency,
initially described in 1974,164 is the third most common red cell
enzymopathy causing hemolytic anemia, after G6PD deficiency and
PK deficiency.91 It is transmitted as an autosomal recessive trait.
Maturation of the reticulocyte results in degradation of ribosomal
RNA into pyrimidine 5′ nucleoside monophosphates, which must be
dephosphorylated in order to freely diffuse across the cell membrane.
P5′NT-1 is a member of a family of enzymes that catalyze this
dephosphorylation.165 Two main types of 5′NT, P5′NT-1 and P5′NT-
2, have been isolated from RBCs.166 Only deficiency of P5′NT-1,
encoded by a gene found on chromosome 7, is associated with hemo-
lytic anemia. P5′NT-1 is dependent on Mg2+ for activity and is readily
inhibited by lead167 and other heavy metals.168 P5′NT-1 activity levels
are highest in the youngest cells.169 Mutations in P5′NT-1 lead to the
accumulation of pyrimidine nucleotides, which impede RNA break-
down. The resultant ribosomal aggregates are visible as the character-
istic coarse basophilic stippling seen on the peripheral smear.164
Characterized mutations in P5′NT-1 lead to decreased thermal stabil-
ity and/or impaired catalytic ability of the enzyme,170 although resid-
ual measured enzyme activity in some cases suggests that enzyme
deficiency is at least in part compensated by other nucleotidases.
Acquired P5′NT deficiency occurs in the instance of lead intoxica-
tion, manifesting as hemolytic anemia, reticulocytosis, and striking
basophilic stippling.167
Clinical and Laboratory Manifestations
P5′NT-1 deficiency is inherited in an autosomal recessive manner,
and patients are usually homozygotes, or, less commonly, compound
heterozygotes.166 The disease is characterized by mild to moderate
hemolytic anemia, reticulocytosis, indirect hyperbilirubinemia, and
variable hepatosplenomegaly.171,172 The anemia is occasionally severe.
Gallstones may be present, and ulcers are seen rarely.171 Learning dis-
abilities have been described in a few cases.171 Iron overload occurs in
both severe transfusion-dependent cases and mild cases.173 Conversely,
iron deficiency secondary to intravascular hemolysis was described in
one case.174 Homozygous P5′NT-1 deficiency inherited in combina-
tion with homozygous hemoglobin E in one described case led to a
particularly severe hemolytic anemia, which responded well to
splenectomy.175
The laboratory hallmark of the disorder is the pronounced baso-
philic stippling seen on the peripheral smear.164 Thus in contrast to
most cases of congenital nonspherocytic hemolytic anemia, the
peripheral smear provides a rapid, inexpensive diagnostic clue to the
underlying disorder. This is a sensitive but not specific finding,
 588 Part V  Red Blood Cells
because basophilic stippling can also be seen in other causes of
anemia, including lead poisoning, some hemoglobin variants,176 and
sideroblastic anemia. Confirmation of the diagnosis requires demon-
stration of decreased P5′NT-1 activity and high concentrations of
pyrimidine nucleotides in red cells.166
Therapy
There is no targeted therapy for this disorder. In the atypical case
with severe anemia, patients may be red cell transfusion dependent.177
In other cases, transfusions may be needed sporadically, such as
during pregnancy or acute infections.178 Splenectomy in a few cases
has provided benefit,179 but in other cases has not led to any significant
improvement in the anemia.177 Patients should be monitored for iron
overload, and iron chelation may be required.
Red Cell Enzymopathies Causing Methemoglobinemia
Pathobiology
Normal hemoglobin A is composed of two α-chains and two β-chains,
and each one of the globin chains carries a heme group in the center
of which is a molecule of ferrous iron (Fe2+). In oxyhemoglobin, each
ferrous atom links reversibly to a molecule of oxygen. As is shown in
the sigmoidal oxyhemoglobin association curve, the binding of
oxygen is cooperative, such that the binding of the first molecule of
oxygen facilitates the binding of the second molecule of oxygen.
Methemoglobin occurs when the ferrous molecule is oxidized to
ferric (Fe3+), either in the usual course of events (Fig. 42-3) or due to
inadequately controlled oxidant attack. Methemoglobin causes two
sorts of problems: methemoglobin cannot bind oxygen, and, further,
if a methemoglobin becomes part of the hemoglobin tetramer, the
oxygen affinity is increased with a left shift in the oxyhemoglobin
dissociation curve and a biologically important fall in the partial
pressure of oxygen at which hemoglobin is 50% saturated with
oxygen (P50).180 The combined effect of decreased oxygen-carrying
capacity and decreased oxygen release may lead to a profound func-
tional impairment,181 particularly in the patient with underlying car-
diopulmonary disease.
Methemoglobin is being formed continuously, but there are
mechanisms in place that normally keep the methemoglobin level at
about 1% of the total hemoglobin.182 The most important of these is
the NADH-dependent Cb5R (also known as methemoglobin reduc-
tase).183,184 The electrons from NADH are transferred first to FAD,
reducing it to FADH2, which is a prosthetic group of the enzyme.
FADH2 then transfers these electrons to the enzyme Cb5R, which
completes the transfer of electrons to Fe3+, completing the reduction
of methemoglobin to Fe2+ hemoglobin (Fig. 42-4).
There is a backup flavin-dependent methemoglobin reductase that
uses the electrons catalyzed by G6PD when it reduces NADP+ to
NADPH. This flavin NADPH methemoglobin reductase, lacking a
physiologic electron acceptor, is of minimal physiologic importance,
but becomes important when methylene blue is used in the treatment
of toxic causes of methemoglobinemia, where the normal Cb5R
system is inadequate. In that case the flavin NADPH-dependent
O2– O2–
Fe2+ Fe3+ - O2– Fe3+ - OH–
(deoxyHb) (deoxyHb) (deoxyHb)
Figure 42-3  AUTO-OXIDATION OF HEMOGLOBIN. Iron is in the
ferrous state (Fe2+) in deoxyhemoglobin (deoxyHb). When oxygen is bound,
an electron is partially transferred from the iron moiety to the bound oxygen,
forming a ferric-superoxide anion complex (Fe3+-O2−). During deoxygenation,
some of the oxygen leaves as a superoxide (O2−) radical. The partially trans-
ferred electron is not returned to the iron moiety, leaving the iron in the ferric
state (Fe3+) and forming methemoglobin (metHb).
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methemoglobin reductase reduces methylene blue as an electron
acceptor to leukomethylene blue, which then directly reduces met-
hemoglobin (Fig. 42-5).185
Acquired or Acute Toxic Methemoglobinemia
Most cases of methemoglobinemia are acquired, resulting from either
exposure to oxidizing agents or from pathologic underlying condi-
tions. Oxidizing agents may accelerate the rate of formation of met-
hemoglobin up to 1000-fold182 and eventually overwhelm the capacity
of the methemoglobin reduction pathways. Numerous toxins and
drugs or their metabolites have been implicated in causing methemo-
globinemia (see box on Substances Associated With Methemoglobin-
emia), including the more common culprits, dapsone, local anesthetics
(benzocaine, lidocaine, prilocaine), and derivatives of the anesthetic
phenacetin. In a retrospective study of pediatric oncology patients
who were treated with dapsone for the prevention of Pneumocystis
carinii pneumonia, methemoglobinemia was documented in 32 of
167 (19.2%) patients.186 In this study, higher dapsone dosing was
associated with increased risk. In another retrospective study of 138
cases of acquired methemoglobinemia, 42% of cases were due to
dapsone.181 In this series, methemoglobinemia was mild (<8% of total
hemoglobin) in the majority of cases. Five of the most severe cases
were caused by topical 20% benzocaine spray. In 2006 the U.S. Food
and Drug Administration (FDA) issued a public health advisory
regarding the risk for methemoglobinemia with the use of benzocaine
sprays.187 In a follow-up safety announcement in 2011, the FDA
reported a total of 319 cases of methemoglobinemia due to topical
benzocaine sprays, including 32 life-threatening and 7 fatal cases.188
A single spray of benzocaine, and doses well below the maximum
standard allowable dose of prilocaine, particularly in the presence of
predisposing factors, have been reported to lead to methemoglobin-
emia.189 The FDA has recommended that benzocaine products not
G3P NAD+ FADH2 Fe3+ - b5 Fe2+ - Hb
Cytochrome-b5
G3 PD
reductase
1,3-BPG NADH FAD Fe2+ - b5 Fe3+ - Hb
Figure 42-4  NADH-DEPENDENT METHEMOGLOBIN REDUC-
TION. The reduced form of nicotinamide adenine dinucleotide (NADH) is
generated during glycolysis in the reaction mediated by glucose-3-phosphate
dehydrogenase (G3PD). A pair of electrons from NADH is transferred to the
flavin adenine dinucleotide (FAD) prosthetic group of cytochrome-b5 reduc-
tase, reducing it to FADH2. Two molecules of ferric (Fe3+) cytochrome b5 are
then sequentially bound and reduced, forming ferrous (Fe2+) cytochrome b5.
An ionic complex between ferrous (Fe2+) cytochrome b5 and a ferric (Fe3+)
subunit of a hemoglobin (methemoglobin) tetramer is formed and an electron
transferred between the two hemes, creating ferrous (Fe2+) hemoglobin. 1,3-
BPG, 1,3- Bisphosphoglycerate; G3P, glyceraldehyde 3-phosphate; Hb, hemo-
globin; NAD+, nicotinamide adenine dinucleotide.
G6P NADP+ Leukomethylene Fe2+ - Hb
blue
G6 PD
6PG NADPH Methylene blue Fe3+ - Hb
Figure 42-5  NADPH-DEPENDENT METHEMOGLOBIN REDUC-
TION. NADPH methemoglobin reduction can be activated by exogenously
administered methylene blue. G6P, Glucose 6-phosphate; G6PD, glucose-6-
phosphate dehydrogenase; 6PG, 6-phosphogluconate; NADP+, oxidized
form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced
form of nicotinamide adenine dinucleotide phosphate.

Substances Associated With Methemoglobinemia
Acetaminophen (nitrobenzene derivative)
Acetanilide
Local anesthetics
Benzocaine
Lidocaine
Prilocaine
Aniline dyes
Celecoxib
Dapsone
Flutamide
Ifosfamide
Metoclopramide
Nitric oxide
Nitrites
Amyl nitrite
Isobutyl nitrite
Sodium nitrite
Nitrates (bacterial conversion to nitrites)
Nitrobenzenes/nitrobenzoates
Nitroethane (nail polish remover)
Nitrofurans
Nitroglycerin
Paraquat/monolinuron
Phenacetin
Phenazopyridine (Pyridium)
Primaquine
Rasburicase
Sulfamethoxazole
be used on children less than 2 years of age, such as in use of over-the
counter teething medicine,190 except under the advice and supervision
of a health care professional.188 The offending agent may be hidden,
as is seen when local anesthetics are used in the preparation of
cocaine.191,192 Inhalation and/or ingestion of volatile nitrites is another
recreational drug practice that can cause methemoglobinemia.192
Exposure to nitrates and nitrites, widely used as food preservatives
and found in well water,193 can also cause methemoglobinemia.194
Infants less than 6 months of age may have increased susceptibility
to methemoglobinemia due to low gastric pH, which allows prolifera-
tion of intestinal flora that reduces ingested nitrates to nitrites. Neo-
nates are at particularly increased risk due to decreased Cb5R activity
(50% to 60% of adult activity).195 Homemade baby food purees of
high-nitrate-containing foods such as carrots, spinach and silver beets
may cause methemoglobinemia, as can acquired illnesses, including
diarrheal disease in infants and sepsis.
Clinical Manifestations
Clinical manifestations develop secondary to impaired tissue oxygen-
ation. Typical “cyanotic” slate-blue coloring of the skin and mucous
membranes will be visible when 5% to 15% of the total hemoglobin
is methemoglobin.189,196 Although methemoglobin levels up to 20%
or even higher may be well tolerated in some individuals, others may
experience tachypnea, shakiness, altered consciousness, and signs of
myocardial ischemia at methemoglobin levels of 10% to 20%.189,190
As methemoglobin levels rise above 20% to 30%, patients can experi-
ence progressive respiratory compromise, myocardial ischemia, sei-
zures, and coma.189 Death typically ensues at methemoglobin levels
above 70%197 but can occur at lower levels.189 Signs and symptoms
may be potentiated by factors such as concomitant use of other oxi-
dizing agents, age less than 6 months, anemia, or significant underly-
ing comorbid disease. The onset of disease may be abrupt, and the
clinician must maintain a high index of suspicion in at-risk situations
(i.e., procedures in which topical anesthetics are used).
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Chapter 42  Red Blood Cell Enzymopathies 589
Laboratory Manifestations and Diagnosis
Methemoglobinemia should be suspected when the patient appears
cyanotic but has a normal PaO2 as measured by arterial blood gas
assessment. The blood will typically be a dark purple to chocolate
color, and the blood will not become more red on exposure to oxygen.
The diagnosis of methemoglobinemia relies on analysis of its absor-
bance spectrum. Pulse oximetry is unreliable in the presence of met-
hemoglobinemia, due to its light absorbance properties198; however,
cooximetry can determine the methemoglobin fraction.199 The pres-
ence and percentage of methemoglobin can be confirmed by use of
the Evelyn-Molloy method.200
Therapy
Treatment is guided by the level of methemoglobinemia and patient
symptoms. In the case of acute onset due to oxidizing agents or
disease states, any potential offending agents should be immediately
discontinued. The asymptomatic patient with methemoglobin levels
of less than 20% may only need observation. If the patient is symp-
tomatic or if methemoglobin levels are greater than 20%, interven-
tion with methylene blue is indicated. Methylene blue is given in a
dose of 1 to 2 mg/kg intravenously over 5 minutes, and the dose may
be repeated after 60 minutes if necessary. Cumulative doses greater
than 4 to 7 mg/kg (or even lower in infants) may cause cyanosis,
dyspnea, and acute hemolysis.201,202 Methemoglobinemia generally
resolves promptly with treatment, and within 20 hours in those who
receive no treatment.189 Rebound methemoglobinemia189 has been
reported to occur after methylene blue administration, in one case
several days following intentional massive nitrobenzene ingestion.203
Therefore treated patients should be carefully monitored for recur-
rence of methemoglobinemia. The therapeutic effect of methylene
blue is dependent on the NADPH generated by G6PD. As such,
methylene blue will probably not be effective in treating toxic met-
hemoglobinemia in patients who are also G6PD deficient and may
even cause hemolysis due to its oxidant effects.204 Patients with G6PD
deficiency who require therapy can be treated with exchange transfu-
sion.205 Hyperbaric oxygen has also been used successfully in severe
cases of methemoglobinemia.206
Hereditary Methemoglobinemia
Hereditary causes of methemoglobinemia are secondary to deficiency
of Cb5R, very rarely described deficiency of cytochrome b5,207 or
inheritance of an abnormal hemoglobin in hemoglobin M disease
(see Chapter 41).
Cytochrome-b5 Reductase Deficiency
Cb5R deficiency is the most common cause of congenital methemo-
globinemia and is inherited in an autosomal recessive manner.208
Cb5R is present in two forms, a soluble form present mainly in red
cells183 and a membrane-bound form found in the endoplasmic retic-
ulum and outer mitochondrial membrane.209,210 Type I Cb5R defi-
ciency is characterized by a deficiency of the soluble, red cell form of
Cb5R and manifests as cyanosis, fatigue, and dyspnea. This is the
more common form of hereditary methemoglobinemia and is
endemic in certain populations, including Navajo211 and Athabasca212
Native Americans and natives of Yakutsk, Siberia.213 Compensatory
polycythemia is seen only rarely. Methemoglobinemia, even up to
levels of 40%, may be well tolerated.208 The less common type II
Cb5R deficiency, characterized by deficiency of the enzyme in all
tissues, has a devastating clinical course, characterized, in addition to
cyanosis, by progressive microcephaly, severe mental retardation, dys-
tonia, and early death.208,214 The types can be differentiated by the
clinical phenotype, as well as by assaying enzyme activity in erythroid
and nonerythroid tissues. The cyanosis can be treated with oral doses
 590 Part V  Red Blood Cells
of methylene blue (100 to 300 mg/day) or ascorbic acid (300 to
1000 mg/day in divided doses). There is currently no treatment for
the neurologic manifestations of type II Cb5R deficiency.
POLYCYTHEMIA DUE TO CONGENITAL RED BLOOD CELL
ENZYME DEFICIENCY
Polycythemia is defined as an absolute increase in RBC mass. Primary
polycythemia refers to an inherent defect (acquired or inherited)
within the RBCs that results in increased proliferation, whereas sec-
ondary polycythemia refers to a reactive process that is usually driven
by elevated levels of serum erythropoietin (EPO) as a consequence
of chronic tissue hypoxia or EPO-secreting tumors. The vast majority
of primary and secondary polycythemias are acquired. The congenital
secondary polycythemias are extremely rare and occur when germline
mutations alter hemoglobin oxygen affinity or decrease the concen-
tration of 2,3-BPG, resulting in impaired oxygen delivery to the
tissues. The latter condition is a result of a congenital deficiency of
the erythrocyte enzyme bisphosphoglycerate mutase (BPGM).
Epidemiology and Clinical Manifestations
Only two families with complete BPGM deficiency have been com-
prehensively studied because of the rarity of the condition. The first
case of complete BPGM deficiency was described in 1978 in a family
in France.215 The propositus was a 42-year-old male with a hemoglo-
bin level of 19 g/dL. He had a ruddy complexion but was otherwise
clinically well. His 2,3-BPG levels were 3% of normal, and BPGM
activity was undetectable.215 Both the patient and three of his sisters
were found to be compound heterozygotes for mutations in the
BPGM gene.216 A second family with complete BPGM deficiency was
described in 2004 and included the first and only reported instance
(to date) of a patient homozygous for a mutation in the BPGM gene.
The mutation was determined to be a missense mutation near the
active binding site of the enzyme.217
Pathobiology
2,3-BPG is an important modifier of oxygen delivery by RBCs. It
binds to a central cavity of the hemoglobin tetramer and allosterically
converts hemoglobin to a low oxygen affinity state, resulting in a
rightward shift of the oxygen dissociation curve. Patients with
chronic hypoxia have elevated levels of 2,3-BPG to facilitate oxygen
unloading and increase oxygen delivery to the tissues (Fig. 42-6).
BPGM is the enzyme responsible for the synthesis of 2,3-BPG via
the Rapoport-Luebering shunt. BPGM is a homodimer with both
synthase and phosphatase activity; its synthetic function converts
1,3-bisphosphoglycerate to 2,3-BPG, and its phosphatase activity
metabolizes 2,3-BPG to 3-phosphoglycerate, an intermediate of the
glycolytic pathway. A deficiency of BPGM leads to a decrease in 2,3-
BPG. The ensuing increase in hemoglobin oxygen affinity results in
a decrease in oxygen delivery to the tissues, which in turn leads to a
compensatory polycythemia.
Diagnosis and Therapy
The diagnosis of BPGM deficiency is first suspected when a patient
with an otherwise unexplained polycythemia is noted to have a
decreased P50 on the oxygen dissociation curve and a normal hemo-
globin electrophoretic profile. These studies should be followed by
an assay measuring 2,3-BPG activity and, if decreased, an assay mea-
suring BPGM activity. The condition is confirmed by demonstrating
a mutation in the BPGM gene.
Due to the rarity of the condition, there is limited experience to
guide treatment. Presumably the reactive polycythemia compensates
for the diminished oxygen delivery to the tissues and thereby
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G3P
1,3-BPG
BPG synthase
ADP
Phosphoglycerate 2,3-BPG
kinase
ATP
BPG phosphatase
3PG Pi
Phosphoenolypyruvate
Figure 42-6  RAPOPORT-LUEBERING SHUNT.  2,3-BPG metabolism
is regulated by a multifunctional enzyme, bisphosphoglycerate mutase,
with both synthase (BPG synthase) and phosphatase (BPG phosphatase)
activity. This reaction bypasses the formation of adenosine triphosphate
(ATP) in the glycolytic pathway. ADP, Adenosine diphosphate; 1,3-BPG,
1,3-bisphosphoglycerate; 2,3-BPG, 2,3-bisphosphoglycerate; G3P, glyceralde-
hyde 3-phosphate; 3PG, 3-phosphoglycerate; Pi, inorganic phosphate.
circumvents frank tissue hypoxia. These patients may, however, be
more sensitive to decreases in hemoglobin and become symptomatic
from what would otherwise be deemed a mild anemia.
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Chapter 42  Red Blood Cell Enzymopathies 591
Serpa JA, Villarreal-Williams E, Giordano TP: Prevalence of G6PD
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For complete list of references log on to www.expertconsult.com.
 Chapter 42  Red Blood Cell Enzymopathies 591.e1

Key Words
Anemia
Congenital nonspherocytic hemolytic anemia
Glucose-6-phosphate dehydrogenase deficiency
Methemoglobinemia
Polycythemia
Pyrimidine 5′-nucleotidase
Pyruvate kinase
Red cell enzymes

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591.e2 Part V  Red Blood Cells
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