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C H A P T E R 42


Elizabeth A. Price, Stavroula Otis, and Stanley L. Schrier

When evaluating the patient with hemolytic anemia, it is good prac- 5′-nucleotidase is effective in degrading the ribosomal contents
tice to determine whether the hemolysis is due to an intracorpuscular into smaller components that can pass through the RBC
or extracorpuscular defect. With the exception of paroxysmal noctur- membrane.
nal hemoglobinuria, intracorpuscular causes of hemolysis are all
hereditary and involve the three major compartments of the red For the most part, these enzyme deficiencies are exceedingly rare,
blood cell (RBC): hemoglobin, the plasma membrane, and the RBC’s with a few notable exceptions. The more prevalent enzyme deficien-
metabolic machinery. Abnormalities of hemoglobin and the plasma cies presumably offer an evolutionary advantage, whereas the rare
membrane are covered elsewhere. This section is devoted to an analy- deficiencies likely represent spontaneous mutations that offer no
sis of the abnormalities of the RBC’s enzymatic-metabolic machinery. selective advantage.
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 ENZYMOPATHIES OF GLUTATHIONE METABOLISM
metabolism are crippled by their deficiency. The following functions,
when impaired, are deleterious to RBC survival: Glutathione Metabolism
1. Maintenance of RBC reducing power: The RBC’s reducing Glutathione is a key player in numerous cellular functions, including
power is of paramount importance to its survival. Red cells are free radical scavenging, redox reactions, and biosynthesis of deoxyri-
loaded with the oxygen they carry to the tissues and are constantly bonucleic acid (DNA), proteins, and leukotrienes. GSH also plays a
bombarded with reactive oxygen species. The erythrocyte has a role in neurotransmission and neuromodulation. In the RBC, gluta-
remarkable ability to absorb extreme oxidative stress by virtue of thione is present in extremely high concentrations, higher than any
the enzymes involved in glutathione metabolism and generation other cell in the body, and plays a critical role in protecting the RBC
of the reduced form of nicotinamide adenine dinucleotide phos- from oxidant injury. The ratio of reduced glutathione (GSH) to
phate (NADPH). These enzymes provide the red cell with tremen- oxidized glutathione (GSSG) is maintained at high levels in the red
dous reducing power by serving as a large reservoir of electrons cell by two mechanisms: (1) GSSG is converted to GSH by glutathi-
that can be donated to hemoglobin, lipoproteins, and other intra- one reductase, and (2) GSSG is actively transported out of the
cellular proteins damaged by oxidative stress. erythrocyte.
2. Generation of adenosine triphosphate (ATP): The RBC uses GSH is a tripeptide composed of glutamate, cysteine, and glycine.
energy in the form of ATP to energize the pumps and membrane γ-glutamylcysteine synthetase catalyzes the first and rate-limiting step
transport mechanisms that allow the red cell to maintain its stable in the synthesis of GSH (Fig. 42-1). The product of this reaction,
internal environment. The mature red cell is incapable of oxidative γ-glutamylcysteine, is then converted to GSH in the presence of
phosphorylation because it has lost its mitochondria and mito- glycine via glutathione synthetase, the enzyme that catalyzes the
chondrial enzymes. As such, the red cell must rely on the less second and final step in GSH synthesis (see Fig. 42-1). Oxidation of
efficient, classic Embden-Meyerhof pathway of glycolysis for ATP GSH by glutathione peroxidase or other free radicals leads to the
generation. The glycolytic enzymes catalyzing this evolutionarily formation of glutathione disulfide (GSSG) and other mixed disulfides
conserved pathway are critical for the production of the ATP of proteins that contain free –SH groups (like hemoglobin). GSSG
requisite for the pumps and transport mechanisms, as well as the is rapidly restored to reduced glutathione via glutathione reductase
production of the reduced form of nicotinamide adenine dinucle- and its cofactor NADPH. NADPH is maintained at high levels by
otide (NADH) needed for cytochrome-b5 reductase (Cb5R), the glucose-6-phosphate dehydrogenase (G6PD). Defects in each of
major methemoglobin reductase. these enzymes have been described and are extremely rare, with the
3. Maintenance of hemoglobin in a functional state: The enzymes notable exception of G6PD deficiency.
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 G6PD Deficiency
methemoglobin reductase with its cofactors. The enzymes that
catalyze the formation of 2,3-bisphosphoglycerate (2,3-BPG) via History
the Rapoport-Luebering shunt also play an important role in
hemoglobin function because 2,3-BGP binds to the end of the G6PD deficiency was first recognized in the early 1950s during the
β-globin chain and facilitates the off-loading of oxygen from Korean War when approximately 10% of African American soldiers
hemoglobin to the tissues. given the antimalarial drug primaquine developed a self-limited
4. Degradation of ribosomal proteins: The RBC is unique in that hemolytic anemia. This phenomenon led to further investigations on
it transitions from a typical nucleated cell capable of protein healthy volunteers who were given 30 mg of primaquine daily. Most
synthesis and oxidative and anaerobic metabolism to a cell devoid subjects tolerated the drug well, but a minority developed a
of organelles, incapable of protein synthesis, and dependent self-limited hemolytic anemia. Pursuant studies showed that
on glycolysis for energy production. The RBC must modify 51
Cr-labeled RBCs from primaquine-sensitive subjects transfused into
its membrane and volume accordingly and expel ribosomal nonsensitive subjects were rapidly destroyed, whereas RBCs from
proteins, which could otherwise prove deleterious. Pyrimidine nonsensitive subjects transfused into primaquine-sensitive subjects
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582 Part V  Red Blood Cells

Cysteine Glutamic acid random X-chromosome inactivation, when compared to parasite

growth in RBCs with normal G6PD activity from the same
ATP individuals.25
γ-Glutamylcysteine Oxidant injury to the parasite itself appears to be one mechanism
synthetase ADP 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
Glycine to the reactive intermediates they generate (particularly oxidized iron)
when they break down hemoglobin.26,27 Other studies have shown

RBC membrane
Glutathione ATP that malaria-infected, G6PD-deficient RBCs undergo phagocytosis
ADP by macrophages at an earlier stage of parasite maturation than do
H2O2 H2O normal malaria-infected RBCs, which could be another means by

which G6PD-deficient cells offer a selective advantage.20
Glutathione peroxidase The prevalence of G6PD deficiency in the United States ranges
GSH GSSG from 0.5% to 7% and is most common among African American
Glutathione reductase
males, affecting approximately 10%.10,28 Screening for G6PD defi-
ciency is generally reserved for individuals at increased risk, such as
NADP+ NADPH 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
Pentose approximately 6.8%.29 Because HIV patients are more likely to be
shunt exposed to oxidant drugs like dapsone, primaquine, and sulfon-
Figure 42-1  GLUTATHIONE PATHWAY. ADP, Adenosine diphosphate; amides, primary care guidelines for management of HIV recommend
ATP, adenosine triphosphate; F6P, fructose 6-phosphate; G6P, glucose screening patients from higher prevalence ethnic or racial groups
6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; GSH, reduced glu- (e.g., African and Mediterranean descent) for G6PD deficiency either
tathione; GSSG, oxidized glutathione; NADP+, oxidized form of nicotin- at baseline or before initiating therapy with an oxidant drug.30
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 G6PD is a cellular housekeeping enzyme that catalyzes the first step
These findings established clearly that primaquine sensitivity was due in the hexose monophosphate shunt, wherein glucose 6-phosphate
to an intrinsic defect of the erythrocyte. Subsequent studies showed (G6P) is converted to ribose 5-phosphate (Fig. 42-2), a precursor of
that younger, more metabolically active RBCs were resistant to many important molecules like ribonucleic acid (RNA), DNA, ATP,
destruction, whereas older RBCs were being destroyed, suggestive of coenzyme A, nicotinamide adenine dinucleotide (NAD), flavin
an underlying metabolic defect.3 By 1956, researchers had systemati- adenine dinucleotide (FAD). Another major role of the hexose mono-
cally deduced that the primary defect was in the G6PD enzyme.4-8 In phosphate shunt is to maintain high levels of NADPH, which acts
1961 the G6PD gene was discovered to be X-linked, due to its linkage as a cofactor for GSH. Most cells have alternate enzymatic pathways
to red-green color blindness. 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-
Epidemiology phosphate shunt for maintaining high levels of NADPH and GSH
to protect against oxidative stress.
G6PD deficiency is the most prevalent human enzyme deficiency in G6PD is encoded by a gene located on the telomeric region of
the world, affecting an estimated 350 to 400 million people. The the long arm of the X chromosome (Xq28) that spans 18 kb, and
geographic distribution of G6PD deficiency coincides with the geo- consists of 13 exons and 12 introns. The G6PD gene product com-
graphic distribution of endemic malaria, implicating a survival prises 515 amino acids with a molecular weight of 59 kDa.31 The
benefit. The highest prevalence of G6PD deficiency is in sub-Saharan enzyme is active as a tetramer or dimer. Stability of the active qua-
Africa, followed by the Middle East, Mediterranean Europe, and ternary structure is crucial for normal G6PD activity.
Southeast Asia.9,10 Within the RBC, oxidant injury leads to the oxidation of sulfhy-
The most common allelic variants of G6PD are G6PD A- and dryl groups on the hemoglobin molecule, resulting in the formation
G6PD Mediterranean. In fact, these two variants coexist at polymor- of disulfide bridges, which in turn leads to decreased hemoglobin
phic frequencies in several populations, most notably in countries solubility and ultimately the irreversible precipitation of oxidized
surrounding the Persian Gulf.11 G6PD A- accounts for approximately hemoglobin.32,33 Under normal conditions oxidized hemoglobin is
90% of G6PD in Africa but is also prevalent in North and South reduced by GSH, which itself is oxidized in the process but restored
America, the West Indies, Italy, the Canary Islands, Spain, Portugal, to its reduced form by intracellular NADPH, whose levels are main-
and the Middle East.12-16 G6PD Mediterranean is prevalent in all tained by G6PD. In the G6PD-deficient RBC, GSH is not restored
countries surrounding the Mediterranean Sea, as well as the Middle to adequate levels under oxidative stress, leading to a buildup of free
East, India, and Indonesia.14,17 radicals and insoluble hemoglobin within the cell, which can be
In addition to geographic overlap, numerous studies support the visualized under the microscope as Heinz bodies. Precipitated hemo-
premise that polymorphic variants of G6PD confer a survival benefit globin is disruptive to the structure and function of the RBC mem-
in malaria-endemic regions. Ruwende et al18 showed that the G6PD brane and leads to increased membrane permeability, osmotic fragility,
A- allele is associated with a substantial reduction in the risk for severe and cell rigidity. The compromised integrity of the cell membrane
Plasmodium falciparum malaria for female heterozygotes (46% risk results in both intravascular hemolysis and rapid removal of these
reduction) and male hemizygotes (58% risk reduction). In addition, cells within the splenic pulp.34
several in vitro studies comparing the growth of malarial parasites in Over 400 variants of the G6PD enzyme have been identified by
G6PD-B (wild type) erythrocytes to growth in G6PDA- and G6PD biochemical methods, 100 of which reach polymorphic levels. Most
Mediterranean erythrocytes have shown protracted growth in the variants are due to point mutations and, to a lesser extent, small
G6PD-deficient cells.19-24 Growth is also blunted in G6PD-deficient in-frame deletions that result in missense mutations. Gross deletions,
RBCs taken from female heterozygotes, whose cells have undergone nonsense mutations, frame-shift mutations, and splicing defects are

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Chapter 42  Red Blood Cell Enzymopathies 583

Glutathione production Table 42-1  World Health Organization Classification

GSH GSSG of G6PD Variants
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
residual activity)
Fructose 6-P shunt III Moderately to mildly deficient Acute, episodic
ATP Phosphofructokinase (10%-60% residual activity)
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
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
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 enzyme activity, whereas absence of fluorescence indicates enzyme
hemolysis by further impairing the reductive capacity of the G6PD- deficiency. Other semiquantitative tests have been used but require
deficient RBC.53 Favism develops within 5 to 24 hours of bean inges- definitive testing to confirm an abnormal result.64,65 False negatives
tion and is characterized by the typical symptoms of acute intravascular are a concern when diagnostic tests are performed during or right
hemolysis: headache, nausea, back pain, chills, fever, hemoglobinuria, after an acute hemolytic episode in the midst of a reticulocytosis.
and jaundice. Favism has also been reported in breastfed babies whose Younger RBCs have higher levels of G6PD than older RBCs, so when
mothers consumed fava beans.54 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
Neonatal Jaundice X-chromosome enzymes. Heterozygotes with extremely skewed
Among the clinical manifestations associated with G6PD deficiency, X-inactivation can have enzymatic activity ranging anywhere from
neonatal jaundice now carries the highest risk for potentially devastat- hemizygote to normal. Molecular diagnostic methods are more reli-
ing clinical consequences, namely, kernicterus.55 Contrary to what is able for making the diagnosis of females suspected of being hetero-
commonly believed, jaundice in neonates with G6PD deficiency is zygous for G6PD deficiency.
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- Therapy
ondary to the newborn liver’s inability to adequately conjugate bili-
rubin.57 This problem is compounded when the newborn also inherits Treatment of the clinical sequelae of G6PD deficiency is straightfor-
a mutation of the uridine diphosphate-glucuronosyltransferase 1 ward. In the case of acute hemolysis, removal of the inciting agent
(UGT1A1) gene promoter that is associated with Gilbert syndrome, (e.g., drug, fava beans) is recommended. This maneuver is of particu-
increasing the risk for neonatal jaundice.58 Over 30% of kernicterus lar importance in patients with Class I and II variants, in whom even
cases are associated with G6PD deficiency,59 raising the question of the young RBCs lack adequate G6PD activity to resist ongoing
whether G6PD deficiency should be included in routine newborn exposure to oxidative agents. Class III variants, on the other hand,
screening programs. 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
Congenital Nonspherocytic Hemolytic Anemia with a blunted erythropoietic response (e.g., HIV, infection, drug-
As mentioned earlier, class I G6PD variants have very low enzymatic related myelosuppression). Very rarely, congenital nonspherocytic
activity and/or marked instability, resulting in lifelong hemolysis due hemolytic anemia is severe enough to mandate ongoing transfusions,
to the everyday oxidative stresses encountered by the circulating which may lead to iron overload in the absence of appropriate iron
erythrocyte. These variants are sporadic, and almost all arise from chelation. Folic acid supplementation is also recommended for
independent mutations clustering in exons 10 and 11, which are close patients with congenital nonspherocytic hemolysis. Antioxidants like
to the substrate binding domain in the folded protein.60 The disorder vitamin E and selenium may also be beneficial in these patients, but
is first suspected when affected infants develop severe neonatal jaun- there are no consistent data to support their use.66
dice, or it is discovered later when the typically mild anemia is exac- In the event of neonatal jaundice, the treatment is the same as
erbated by oxidant drug exposure, infection, or parvovirus-induced that recommended for neonatal jaundice arising from other causes.
aplastic crisis. 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
Laboratory Manifestations of its clinical sequelae, which requires awareness of the disorder on
the part of both the physician and the patient. The disease should
Under normal conditions, most G6PD-deficient individuals (class II always be suspected in patients with a nonimmune hemolytic anemia
and III variants) are not anemic and have no laboratory evidence of and patients with an otherwise unexplained personal or family history
hemolysis. In the setting of oxidative stress, the laboratory findings of recurrent jaundice, splenomegaly, or cholelithiasis. G6PD defi-
are those of any acute hemolytic process and include anemia, reticu- ciency should be considered in any neonate with hyperbilirubinemia,
locytosis, hyperbilirubinemia, increased lactate dehydrogenase especially those of high-risk ethnic descent. There has been consider-
(LDH), decreased haptoglobin, and hemoglobinuria. The peripheral able debate in recent years as to whether testing for G6PD deficiency
blood smear is notable for Heinz bodies (precipitated sulfhemoglo- should be a routine part of neonatal screening tests. Interestingly, in
bin), and on occasion for “bite cells” (also known as hemiblister cells, a study done in Cleveland, Ohio, investigators found that the routine
eccentrocytes, and cross-bonded cells), which occur when denatured cord blood screening protocol for isoimmune hemolytic disease had
hemoglobin binds to the cell membrane, creating a puddling of a lower yield and higher cost than their pilot protocol for cord blood
hemoglobin to one side of the cell with an adjacent membrane-bound G6PD deficiency screening,67 both of which are considered major risk
clear zone.61,62 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
Diagnosis classified as “normal” due to the limitations of the fluorescent spot
test,68,69 instilling a false sense of security and subverting the preven-
The enzymatic activity of G6PD can be quantitated by measuring tion of complications.
the rate of NADPH production in red cell hemolysates that contain Once the diagnosis of G6PD deficiency is established, patients
G6P and NADP+ (the enzyme’s substrates); this is done using a must be counseled to avoid drugs, chemicals, and foods known to
spectrophotometer, which can measure the rate of NADPH forma- precipitate hemolysis. Although there are some medications classically
tion at wavelength 340 nm. This method is used to make a biochemi- connected to hemolysis in G6PD patients, there are a number of
cal definitive diagnosis. For more rapid screening of at-risk medications for which there is considerable confusion as to whether
populations, there are several semiquantitative methods available. they are safe in G6PD-deficient patients. In an attempt to better
The most simple, sensitive, and inexpensive screening test is the fluo- clarify which are truly high-risk drugs, investigators in Israel per-
rescent spot test.63 In this procedure, NADPH production is detected formed a comprehensive literature search and categorized drugs
by virtue of its fluorescence under ultraviolet light. As with the quan- according to how much evidence there is in the literature to contra-
titative test, G6P and NADP+ are added to a hemolysate of the indicate their use.70 They found only seven medications for which
patient’s red cells, and fluorescence under ultraviolet light indicates there is solid evidence to prohibit their use in G6PD-deficient

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Chapter 42  Red Blood Cell Enzymopathies 585

patients: dapsone, methylthionine chloride (methylene blue), nitro- content. The diagnosis is confirmed by demonstrating low levels of
furantoin, phenazopyridine, primaquine, rasburicase, and tolonium glutathione synthetase in cultured skin fibroblasts and/or RBCs or
chloride (toluidine blue). Their review found no substantial evidence by demonstrating mutations in the glutathione synthetase gene.74
to absolutely contravene the use of other medications in normal Treatment involves correcting the metabolic acidosis with bicarbon-
therapeutic doses. 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
Prognosis longer recommended because studies have shown that cysteine, which
is known to be neurotoxic at high concentrations, accumulates in the
Except for rare cases of severe congenital nonimmune hemolytic tissues of patients with glutathione synthetase deficiency.80,81 Patients
anemia and severe neonatal jaundice, the prognosis of patients with with glutathione synthetase deficiency should avoid the same agents
G6PD deficiency is excellent with no apparent impact on life known to precipitate acute hemolysis in patients with G6PD
expectancy. deficiency.

Future Directions Glutathione Reductase Deficiency

Current screening tests for G6PD deficiency do not reliably identify Glutathione reductase restores intracellular GSH by reducing GSSG
female heterozygotes or male hemizygotes with greater than 20% in the presence of NADPH and FAD, a derivative of the water-
residual enzyme activity. This is problematic because these groups soluble vitamin riboflavin. Consequently, malnourished patients, or
remain at risk for hemolytic complications. It would be useful to patients who are otherwise deficient in riboflavin, develop an acquired
devise a rapid and inexpensive screening test that could detect patients partial deficiency of glutathione reductase. Riboflavin deficiency can
with moderately reduced enzymatic activity (20% to 60%), so they be demonstrated by testing the in vitro activity of glutathione reduc-
too may be counseled on avoiding exogenous agents that could pre- tase with and without exogenously added FAD. Acquired glutathione
cipitate a hemolytic crisis. It would also be useful to devise a simple reductase deficiency has no documented hematologic phenotype and
in vitro method to determine whether new drugs will cause hemoly- is easily corrected by administration of physiologic quantities of
sis. Simply incubating RBCs with the drug in question is unreliable, riboflavin.
most likely because drug metabolites are often the culprits, rather Congenital deficiency of glutathione reductase has been reported
than the original drug. And finally, the value of G6PD screening of in two Dutch families. In the first family, the eldest of three siblings
all newborns must be flushed out, because this is a major cause of from a consanguineous marriage developed a hemolytic crisis after
neonatal kernicterus, even in first world countries.71-73 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
γ-Glutamylcysteine Synthetase Deficiency the enzyme.83 In the second family, an infant was born with severe
neonatal jaundice and found to be a compound heterozygote for a
As described earlier, γ-glutamylcysteine synthetase catalyzes the first nonsense and missense mutation in the glutathione reductase gene.83
step in glutathione synthesis. Deficiency of this enzyme is exceedingly There was no evidence of enhanced hemolysis in this infant, which
rare. Only nine patients in seven families have been reported world- is consistent with the neonatal jaundice associated with G6PD
wide.74 All nine patients developed a relatively mild hemolytic anemia, deficiency.
and four of the nine manifested neurologic problems, including
spinocerebellar degeneration, peripheral neuropathy, and mental
retardation.75-78 Glutathione Peroxidase Deficiency
Glutathione peroxidase is a selenium-containing enzyme that cata-
Glutathione Synthetase Deficiency lyzes the oxidation of GSH by hydrogen peroxide, producing GSSG
and water. Although rare cases of glutathione peroxidase deficiency
Glutathione synthetase deficiency is the most common of the inborn have been described in association with hemolysis,84-86 a causative
errors of GSH metabolism, with more than 70 patients in over 50 relationship between the two has not been clearly established. There
families reported worldwide.74 Patients are homozygous or compound are several reported cases of normal individuals with reduced gluta-
heterozygous for mutations in the glutathione synthetase gene. The thione peroxidase activity and no evidence of hemolysis.87,88
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 ENZYMOPATHIES OF THE GLYCOLYTIC PATHWAY
the buildup of 5-oxoproline, a metabolite of γ-glutamylcysteine (the
substrate of glutathione synthetase). Under normal circumstances Pyruvate Kinase Deficiency
GSH negatively feeds back on γ-glutamylcysteine synthetase, but in
patients with moderate to severe glutathione synthetase deficiency Epidemiology
there is loss of negative feedback, leading to the accumulation of
5-oxoproline in body fluids, resulting in metabolic acidosis and Pyruvate kinase (PK) deficiency is the most common red cell enzy-
massive 5-oxoprolinuria. Severely affected patients may also develop mopathy leading to congenital nonspherocytic hemolytic anemia.
recurrent bacterial infections and progressive dysfunction of the The prevalence has been estimated by gene frequency studies to be
central nervous system (CNS). The mechanism behind CNS involve- 51 cases per million in the general white population.89 However,
ment remains unclear. Autopsy of the first patient described with recognized clinical cases in the Northern United Kingdom based on
glutathione synthetase deficiency revealed selective atrophy of the a registry study initiated in 1974 had a much lower prevalence,90
granule cell layer of the cerebellum, focal lesions in the frontoparietal suggesting premature disease-related death, clinically mild disease, or
cortex, and bilateral focal lesions in the thalamus and visual cortex. diagnostic error.91 Conversely, the prevalence of homozygous PK defi-
The diagnosis of glutathione synthetase deficiency is suspected in ciency was found to be quite high (1 in 830) in a small, remote town
patients with a nonimmune hemolytic anemia, elevated levels of in the Western United States,92 likely due to consanguinity within the
5-oxoproline in the urine, and markedly reduced RBC glutathione community. Common mutations have well-defined geographic

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586 Part V  Red Blood Cells

associations. Mutation 1529A is frequently seen in the United States93 jaundice that persists in childhood.92 The anemia is relatively stable
and central Europe,94 1456T is commonly found in Southern in adults,121 although transient worsening may occur with infection,122
Europe,95-97 and 1468T is the most common mutation found in pregnancy, or the use of certain medications, including oral contra-
Asia.89,98 PK deficiency is an autosomal recessive disease, and affected ceptives.123 Gallstones may be present. Extramedullary hematopoiesis
patients are typically double heterozygotes, or, less commonly, homo- can occur even in patients who are transfusion independent124 and
zygous for the same mutation. Homozygous mutations are usually may lead to characteristic bone deformities.125 Extradural extramedul-
seen in groups with marked consanguinity, and homozygous PK lary hematopoiesis leading to spinal cord compression and neurologic
deficiency has been well described in the Amish populations of Penn- deficits has been reported,126 with good response to surgical excision
sylvania and Ohio.99,100 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-
Pathobiology nary arterial hypertension was reported in another patient who had
undergone splenectomy at age 5.128
PK catalyzes the irreversible transfer of phosphate from phosphoenol- Iron overload has been reported in nontransfused129,130 and
pyruvate to adenosine diphosphate (ADP), forming ATP and pyru- transfusion-dependent patients and may be severe, leading to cir-
vate in the second ATP-generating step of the glycolytic pathway. rhosis and cardiac and endocrine dysfunction.131 Splenectomy and the
There are four distinct PK isoforms, M1, M2, L, and R types, encoded presence of hemochromatosis-related genes may increase the risk for
by two separate genes (PKM and PKLR).101 PKM1 and PKM2 are iron overload.132 Serum levels of the iron regulatory protein hepcidin
produced from the PKM gene by alternative RNA splicing.102 The were lower in PK-deficient patients compared to controls, and growth
M2 isoform is expressed in early fetal and proliferating tissues and differentiation factor 15 (GDF15) levels were higher, suggesting sup-
remains the dominant form in adults in leukocytes and platelets. pression of hepcidin as a contributing mechanism of iron overload
PKM1 is found in skeletal muscle, heart, and brain.103 PKL, found in in some PK-deficient patients.133
the liver, and PKR, in red cells, are both encoded by the PKLR gene Although PK deficiency does not localize to geographic areas of
on chromosome 1q21 through the use of alternate promoters.104 malarial endemicity, PK deficiency may be protective against
PKM2 is expressed in normal erythroid precursors105 and is gradually malaria.134 In a mouse model of infection with Plasmodium chabaudi,
replaced during maturation by the PK-R isoform. The functional mice carrying a loss-of-function mutation in the PKLR gene had
enzyme is a tetramer, and each isoform has characteristic reduced blood-stage parasite replication and improved survival com-
kinetic properties,103 although all isoforms except M1 are allosterically pared to control mice.135 In vitro, erythrocytes from homozygous
activated by fructose 1,6-diphosphate (FDP).106,107 More than PK-deficient patients have decreased invasion by P. falciparum com-
190 mutations in the PKLR gene encoding the red cell PK have pared to normal or heterozygous PK-deficient control erythrocytes.136
been identified, most of which are missense mutations (www. In addition, phagocytosis of ring-stage infected erythrocytes from
pklrmutatinodatabase.com). Mutations may alter the stability of the patients with homozygous and heterozygous mutations was increased
enzyme or the enzyme’s affinity for its substrate, phosphoenolpyru- compared to control subjects.
vate (PEP), or its allosteric activator, FDP.106,108 There appears to be Laboratory findings are those typical for hemolytic anemia,
some correlation with the location of the mutation and the severity including low hemoglobin, increased reticulocyte count, high LDH,
of the hemolytic anemia, with more severe disease being associated low haptoglobin, and elevated indirect bilirubin. Reticulocytosis may
with disruptive mutations and with missense mutations directly be suppressed in the context of the normal hypoplastic phase of
involving the active site or protein stability.92,101 erythropoiesis following birth, making the diagnosis more challeng-
The mechanism of hemolysis in PK deficiency is not fully under- ing.137 The bilirubin is usually less than 6 mg/dL: if higher, the patient
stood. Reticulocytes are preferentially destroyed in the spleen and the may have coexisting Gilbert syndrome.101,138 Red cells are commonly
liver,109 and following splenectomy a striking reticulocytosis may be without characteristic morphologic abnormalities; however, following
seen.110,111 It has been suggested that the higher metabolic requirement splenectomy they may become markedly abnormal, with numerous
of reticulocytes renders them more vulnerable to PK deficiency than crenated cells, lobulated cells, and target cells.125 Establishing the
mature erythrocytes, leading to increased destruction.109 The defect diagnosis requires the demonstration of low enzyme activity.112 Care
in ATP generation may contribute to the anemia, although it is likely must be taken in interpreting in vitro test results, because contamina-
not the sole cause, because other disorders with more severe ATP tion with transfused red cells or leukocytes can increase measured
deficiency do not have significant hemolytic anemia.112 The metabolic enzyme activity.101 Specialized laboratories can assess for kinetically
derangements found in PK deficiency may also lead to increased abnormal mutant PKs.112 Molecular diagnostic methods can be used
apoptosis and ineffective erythropoiesis, as seen in the spleen of a if the likely mutation is known, as in the case of prenatal diagnosis.
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 Therapy
2,3-BPG may lead to further impairment in glycolysis through inhi-
bition of hexokinase,101 the resultant shift in the oxyhemoglobin Treatment for PK deficiency remains supportive. Exchange transfu-
dissociation curve to the right115 leads to improved tolerance of the sion and/or intense phototherapy may be required in the neonatal
anemia.116 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-
Clinical and Laboratory Manifestations fusion may be needed during pregnancy both for maternal supportive
care and to prevent miscarriage.100 Splenectomy typically leads to an
Clinical severity in patients with PK deficiency is widely variable, increase in hemoglobin by 1 to 3 g/dL121 and can lead to transfusion
ranging from fully compensated hemolysis to a transfusion-dependent independence. In one reported case, partial splenectomy failed to
anemia. Newborns may present with severe hemolytic anemia and ameliorate transfusion needs secondary to rapid regeneration of the
pronounced jaundice, requiring exchange transfusion.117 In the worst spleen.139 General recommendations include, where possible, delaying
cases, hydrops fetalis118 with intrauterine or neonatal death may rarely splenectomy until the patient is 3 years of age or older to reduce the
occur.119 Severe liver dysfunction has also been reported as an unusual risk for postsplenectomy sepsis.112 If iron overload occurs, both phar-
cause of death in infants with PK deficiency.120 Early onset of symp- macologic iron chelation therapy140 and, if the anemia is not too
toms is typically associated with a more severe clinical course.121 severe, phlebotomy,141 can successfully reduce excess iron. Hemato-
Heterozygotes for PK deficiency who are heterozygotes or homozy- poietic stem cell transplant was reportedly successful in one 5-year-
gotes for the Gilbert syndrome polymorphism may have neonatal old boy with PK deficiency and hemoglobin E trait.142

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Chapter 42  Red Blood Cell Enzymopathies 587

Glucose Phosphate Isomerase Deficiency Triose-Phosphate Isomerase Deficiency

Glucose phosphate isomerase (GPI) catalyzes the interconversion of Triose-phosphate isomerase (TPI) catalyzes the reversible interconver-
G6P and fructose 6-phosphate in the second step of glycolysis. GPI sion of the triose phosphate isomers, dihydroxyacetone phosphate,
deficiency is an autosomal recessive disorder and is one of the four and glyceraldehyde 3-phosphate. TPI deficiency is a rare, autosomal
most common erythrocyte enzymopathies, including deficiencies of recessive, multisystemic disease, characterized by chronic nonsphero-
G6PD, PK, and pyrimidine 5′-nucleotidase.143 The hemolytic anemia cytic hemolytic anemia, frequent bacterial infections, and progressive
is of variable severity and may require chronic transfusions.144 Hydrops neuromuscular disease.157-159 Cardiomyopathy may also be present.
fetalis has been reported.143 Splenectomy can improve the anemia, Hemolytic anemia manifests before 14 months of age157 and may be
eliminating transfusion requirements.143,145 GPI deficiency can also be severe enough to require ongoing red cell transfusions.160 In cases with
associated with neurologic impairment, including myopathy and a milder degree of anemia, intermittent transfusions may be required
mental retardation.146 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
Hexokinase Deficiency adult cases with less severe involvement are reported.162 A high preva-
lence of heterozygous TPI deficiency has been found in African
Hexokinase catalyzes the initial step of glycolysis, the phosphoryla- American newborns.163 No specific therapy for this disorder is
tion of glucose to G6P (see Fig. 42-2) and is one of the rate-limiting available.
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 Pyrimidine 5′-Nucleotidase Deficiency
that can include other cytopenias, diabetes mellitus, skeletal
malformations, hypogonadism, and abnormal pigmentation.147 Pathobiology
Severe iron overload can occur.148 Most patients are of European
descent, although an affected Chinese kindred has also been Red cell pyrimidine 5′-nucleotidase type 1 (P5′NT-1) deficiency,
described.147 A mouse model of generalized hexokinase deficiency initially described in 1974,164 is the third most common red cell
has been described, with manifestations of severe hemolytic anemia, enzymopathy causing hemolytic anemia, after G6PD deficiency and
marked reticulocytosis, and extensive tissue iron deposition.149 PK deficiency.91 It is transmitted as an autosomal recessive trait.
Splenectomy may provide benefit.150 In contrast to PK deficiency, Maturation of the reticulocyte results in degradation of ribosomal
the defect occurs proximal to the Rapoport-Leubering shunt, and RNA into pyrimidine 5′ nucleoside monophosphates, which must be
thus the concentration of 2,3-BPG is reduced, shifting the oxyhemo- dephosphorylated in order to freely diffuse across the cell membrane.
globin dissociation curve to the left115 and decreasing patient tolerance P5′NT-1 is a member of a family of enzymes that catalyze this
to the anemia.116 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-
Phosphofructokinase Deficiency 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
Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose are highest in the youngest cells.169 Mutations in P5′NT-1 lead to the
6-phosphate to fructose-1,6-bisphosphate (see Fig. 42-2).151,152 Two of accumulation of pyrimidine nucleotides, which impede RNA break-
the three identified isoenzymes (muscle type [PFKM] and liver type down. The resultant ribosomal aggregates are visible as the character-
[PFKL]) are expressed in erythrocytes. Mutations in the muscle-type istic coarse basophilic stippling seen on the peripheral smear.164
PFKM isoenzyme cause type VII glycogen storage disease (Tarui Characterized mutations in P5′NT-1 lead to decreased thermal stabil-
disease), a rare autosomal recessive disorder. Such mutations lead to ity and/or impaired catalytic ability of the enzyme,170 although resid-
an almost complete loss of PFK activity in muscle, but only partial ual measured enzyme activity in some cases suggests that enzyme
loss of activity in erythrocytes. Clinical manifestations of type VII deficiency is at least in part compensated by other nucleotidases.
glycogen storage disease may present in infancy or older adulthood Acquired P5′NT deficiency occurs in the instance of lead intoxica-
and include hypotonia, exercise intolerance, and weakness.152 The tion, manifesting as hemolytic anemia, reticulocytosis, and striking
severity of the hemolytic anemia is highly variable, ranging from a basophilic stippling.167
mild, compensated hemolysis153 to severe chronic nonspherocytic
hemolytic anemia.152
Clinical and Laboratory Manifestations
ALDOLASE DEFICIENCY P5′NT-1 deficiency is inherited in an autosomal recessive manner,
and patients are usually homozygotes, or, less commonly, compound
Aldolase catalyzes the interconversion of fructose 1,6-diphosphate to heterozygotes.166 The disease is characterized by mild to moderate
glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.107 One hemolytic anemia, reticulocytosis, indirect hyperbilirubinemia, and
of the three distinct isoenzymes, aldolase A, is present in erythrocytes, variable hepatosplenomegaly.171,172 The anemia is occasionally severe.
muscle, and brain. Aldolase deficiency is a very rare cause of nons- Gallstones may be present, and ulcers are seen rarely.171 Learning dis-
pherocytic hemolytic anemia, and clinical manifestations may also abilities have been described in a few cases.171 Iron overload occurs in
include myopathy and mental retardation.154 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-
Phosphoglycerokinase Deficiency tion with homozygous hemoglobin E in one described case led to a
particularly severe hemolytic anemia, which responded well to
Phosphoglycerate kinase catalyzes the reversible conversion of splenectomy.175
1,3-bisphosphoglycerate to 3-phosphoglycerate. Deficiency of this X The laboratory hallmark of the disorder is the pronounced baso-
chromosome–encoded enzyme is associated with an array of findings philic stippling seen on the peripheral smear.164 Thus in contrast to
that can variably include hemolytic anemia, myopathy, rhabdomyoly- most cases of congenital nonspherocytic hemolytic anemia, the
sis, mental retardation, and other neurologic symptoms.155 Splenec- peripheral smear provides a rapid, inexpensive diagnostic clue to the
tomy may improve the anemia.156 underlying disorder. This is a sensitive but not specific finding,

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588 Part V  Red Blood Cells

because basophilic stippling can also be seen in other causes of methemoglobin reductase reduces methylene blue as an electron
anemia, including lead poisoning, some hemoglobin variants,176 and acceptor to leukomethylene blue, which then directly reduces met-
sideroblastic anemia. Confirmation of the diagnosis requires demon- hemoglobin (Fig. 42-5).185
stration of decreased P5′NT-1 activity and high concentrations of
pyrimidine nucleotides in red cells.166
Acquired or Acute Toxic Methemoglobinemia
Therapy Most cases of methemoglobinemia are acquired, resulting from either
exposure to oxidizing agents or from pathologic underlying condi-
There is no targeted therapy for this disorder. In the atypical case tions. Oxidizing agents may accelerate the rate of formation of met-
with severe anemia, patients may be red cell transfusion dependent.177 hemoglobin up to 1000-fold182 and eventually overwhelm the capacity
In other cases, transfusions may be needed sporadically, such as of the methemoglobin reduction pathways. Numerous toxins and
during pregnancy or acute infections.178 Splenectomy in a few cases drugs or their metabolites have been implicated in causing methemo-
has provided benefit,179 but in other cases has not led to any significant globinemia (see box on Substances Associated With Methemoglobin-
improvement in the anemia.177 Patients should be monitored for iron emia), including the more common culprits, dapsone, local anesthetics
overload, and iron chelation may be required. (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
Red Cell Enzymopathies Causing Methemoglobinemia carinii pneumonia, methemoglobinemia was documented in 32 of
167 (19.2%) patients.186 In this study, higher dapsone dosing was
Pathobiology associated with increased risk. In another retrospective study of 138
cases of acquired methemoglobinemia, 42% of cases were due to
Normal hemoglobin A is composed of two α-chains and two β-chains, dapsone.181 In this series, methemoglobinemia was mild (<8% of total
and each one of the globin chains carries a heme group in the center hemoglobin) in the majority of cases. Five of the most severe cases
of which is a molecule of ferrous iron (Fe2+). In oxyhemoglobin, each were caused by topical 20% benzocaine spray. In 2006 the U.S. Food
ferrous atom links reversibly to a molecule of oxygen. As is shown in and Drug Administration (FDA) issued a public health advisory
the sigmoidal oxyhemoglobin association curve, the binding of regarding the risk for methemoglobinemia with the use of benzocaine
oxygen is cooperative, such that the binding of the first molecule of sprays.187 In a follow-up safety announcement in 2011, the FDA
oxygen facilitates the binding of the second molecule of oxygen. reported a total of 319 cases of methemoglobinemia due to topical
Methemoglobin occurs when the ferrous molecule is oxidized to benzocaine sprays, including 32 life-threatening and 7 fatal cases.188
ferric (Fe3+), either in the usual course of events (Fig. 42-3) or due to A single spray of benzocaine, and doses well below the maximum
inadequately controlled oxidant attack. Methemoglobin causes two standard allowable dose of prilocaine, particularly in the presence of
sorts of problems: methemoglobin cannot bind oxygen, and, further, predisposing factors, have been reported to lead to methemoglobin-
if a methemoglobin becomes part of the hemoglobin tetramer, the emia.189 The FDA has recommended that benzocaine products not
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 G3P NAD+ FADH2 Fe3+ - b5 Fe2+ - Hb
oxygen (P50).180 The combined effect of decreased oxygen-carrying
capacity and decreased oxygen release may lead to a profound func- Cytochrome-b5
tional impairment,181 particularly in the patient with underlying car- G3 PD
diopulmonary disease.
Methemoglobin is being formed continuously, but there are 1,3-BPG NADH FAD Fe2+ - b5 Fe3+ - Hb
mechanisms in place that normally keep the methemoglobin level at
about 1% of the total hemoglobin.182 The most important of these is Figure 42-4  NADH-DEPENDENT METHEMOGLOBIN REDUC-
the NADH-dependent Cb5R (also known as methemoglobin reduc- TION. The reduced form of nicotinamide adenine dinucleotide (NADH) is
tase).183,184 The electrons from NADH are transferred first to FAD, generated during glycolysis in the reaction mediated by glucose-3-phosphate
reducing it to FADH2, which is a prosthetic group of the enzyme. dehydrogenase (G3PD). A pair of electrons from NADH is transferred to the
FADH2 then transfers these electrons to the enzyme Cb5R, which flavin adenine dinucleotide (FAD) prosthetic group of cytochrome-b5 reduc-
completes the transfer of electrons to Fe3+, completing the reduction tase, reducing it to FADH2. Two molecules of ferric (Fe3+) cytochrome b5 are
of methemoglobin to Fe2+ hemoglobin (Fig. 42-4). then sequentially bound and reduced, forming ferrous (Fe2+) cytochrome b5.
There is a backup flavin-dependent methemoglobin reductase that An ionic complex between ferrous (Fe2+) cytochrome b5 and a ferric (Fe3+)
uses the electrons catalyzed by G6PD when it reduces NADP+ to subunit of a hemoglobin (methemoglobin) tetramer is formed and an electron
NADPH. This flavin NADPH methemoglobin reductase, lacking a transferred between the two hemes, creating ferrous (Fe2+) hemoglobin. 1,3-
physiologic electron acceptor, is of minimal physiologic importance, BPG, 1,3- Bisphosphoglycerate; G3P, glyceraldehyde 3-phosphate; Hb, hemo-
but becomes important when methylene blue is used in the treatment globin; NAD+, nicotinamide adenine dinucleotide.
of toxic causes of methemoglobinemia, where the normal Cb5R
system is inadequate. In that case the flavin NADPH-dependent
G6P NADP+ Leukomethylene Fe2+ - Hb
O2– O2– G6 PD
Fe2+ Fe3+ - O2– Fe3+ - OH–
(deoxyHb) (deoxyHb) (deoxyHb)
Figure 42-3  AUTO-OXIDATION OF HEMOGLOBIN. Iron is in the 6PG NADPH Methylene blue Fe3+ - Hb
ferrous state (Fe2+) in deoxyhemoglobin (deoxyHb). When oxygen is bound, Figure 42-5  NADPH-DEPENDENT METHEMOGLOBIN REDUC-
an electron is partially transferred from the iron moiety to the bound oxygen, TION. NADPH methemoglobin reduction can be activated by exogenously
forming a ferric-superoxide anion complex (Fe3+-O2−). During deoxygenation, administered methylene blue. G6P, Glucose 6-phosphate; G6PD, glucose-6-
some of the oxygen leaves as a superoxide (O2−) radical. The partially trans- phosphate dehydrogenase; 6PG, 6-phosphogluconate; NADP+, oxidized
ferred electron is not returned to the iron moiety, leaving the iron in the ferric form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced
state (Fe3+) and forming methemoglobin (metHb). form of nicotinamide adenine dinucleotide phosphate.

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Chapter 42  Red Blood Cell Enzymopathies 589

Substances Associated With Methemoglobinemia

Laboratory Manifestations and Diagnosis

Acetaminophen (nitrobenzene derivative) Methemoglobinemia should be suspected when the patient appears
Acetanilide cyanotic but has a normal PaO2 as measured by arterial blood gas
Local anesthetics assessment. The blood will typically be a dark purple to chocolate
Benzocaine color, and the blood will not become more red on exposure to oxygen.
Lidocaine The diagnosis of methemoglobinemia relies on analysis of its absor-
Prilocaine bance spectrum. Pulse oximetry is unreliable in the presence of met-
Aniline dyes hemoglobinemia, due to its light absorbance properties198; however,
Celecoxib cooximetry can determine the methemoglobin fraction.199 The pres-
Dapsone ence and percentage of methemoglobin can be confirmed by use of
Flutamide the Evelyn-Molloy method.200
Nitric oxide Therapy
Amyl nitrite Treatment is guided by the level of methemoglobinemia and patient
Isobutyl nitrite symptoms. In the case of acute onset due to oxidizing agents or
Sodium nitrite disease states, any potential offending agents should be immediately
Nitrates (bacterial conversion to nitrites) discontinued. The asymptomatic patient with methemoglobin levels
Nitrobenzenes/nitrobenzoates of less than 20% may only need observation. If the patient is symp-
Nitroethane (nail polish remover) tomatic or if methemoglobin levels are greater than 20%, interven-
Nitrofurans tion with methylene blue is indicated. Methylene blue is given in a
Nitroglycerin dose of 1 to 2 mg/kg intravenously over 5 minutes, and the dose may
Paraquat/monolinuron be repeated after 60 minutes if necessary. Cumulative doses greater
Phenacetin than 4 to 7 mg/kg (or even lower in infants) may cause cyanosis,
Phenazopyridine (Pyridium) dyspnea, and acute hemolysis.201,202 Methemoglobinemia generally
Primaquine resolves promptly with treatment, and within 20 hours in those who
Rasburicase receive no treatment.189 Rebound methemoglobinemia189 has been
Sulfamethoxazole 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,
be used on children less than 2 years of age, such as in use of over-the methylene blue will probably not be effective in treating toxic met-
counter teething medicine,190 except under the advice and supervision hemoglobinemia in patients who are also G6PD deficient and may
of a health care professional.188 The offending agent may be hidden, even cause hemolysis due to its oxidant effects.204 Patients with G6PD
as is seen when local anesthetics are used in the preparation of deficiency who require therapy can be treated with exchange transfu-
cocaine.191,192 Inhalation and/or ingestion of volatile nitrites is another sion.205 Hyperbaric oxygen has also been used successfully in severe
recreational drug practice that can cause methemoglobinemia.192 cases of methemoglobinemia.206
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 Hereditary Methemoglobinemia
to methemoglobinemia due to low gastric pH, which allows prolifera-
tion of intestinal flora that reduces ingested nitrates to nitrites. Neo- Hereditary causes of methemoglobinemia are secondary to deficiency
nates are at particularly increased risk due to decreased Cb5R activity of Cb5R, very rarely described deficiency of cytochrome b5,207 or
(50% to 60% of adult activity).195 Homemade baby food purees of inheritance of an abnormal hemoglobin in hemoglobin M disease
high-nitrate-containing foods such as carrots, spinach and silver beets (see Chapter 41).
may cause methemoglobinemia, as can acquired illnesses, including
diarrheal disease in infants and sepsis.
Cytochrome-b5 Reductase Deficiency
Clinical Manifestations Cb5R deficiency is the most common cause of congenital methemo-
globinemia and is inherited in an autosomal recessive manner.208
Clinical manifestations develop secondary to impaired tissue oxygen- Cb5R is present in two forms, a soluble form present mainly in red
ation. Typical “cyanotic” slate-blue coloring of the skin and mucous cells183 and a membrane-bound form found in the endoplasmic retic-
membranes will be visible when 5% to 15% of the total hemoglobin ulum and outer mitochondrial membrane.209,210 Type I Cb5R defi-
is methemoglobin.189,196 Although methemoglobin levels up to 20% ciency is characterized by a deficiency of the soluble, red cell form of
or even higher may be well tolerated in some individuals, others may Cb5R and manifests as cyanosis, fatigue, and dyspnea. This is the
experience tachypnea, shakiness, altered consciousness, and signs of more common form of hereditary methemoglobinemia and is
myocardial ischemia at methemoglobin levels of 10% to 20%.189,190 endemic in certain populations, including Navajo211 and Athabasca212
As methemoglobin levels rise above 20% to 30%, patients can experi- Native Americans and natives of Yakutsk, Siberia.213 Compensatory
ence progressive respiratory compromise, myocardial ischemia, sei- polycythemia is seen only rarely. Methemoglobinemia, even up to
zures, and coma.189 Death typically ensues at methemoglobin levels levels of 40%, may be well tolerated.208 The less common type II
above 70%197 but can occur at lower levels.189 Signs and symptoms Cb5R deficiency, characterized by deficiency of the enzyme in all
may be potentiated by factors such as concomitant use of other oxi- tissues, has a devastating clinical course, characterized, in addition to
dizing agents, age less than 6 months, anemia, or significant underly- cyanosis, by progressive microcephaly, severe mental retardation, dys-
ing comorbid disease. The onset of disease may be abrupt, and the tonia, and early death.208,214 The types can be differentiated by the
clinician must maintain a high index of suspicion in at-risk situations clinical phenotype, as well as by assaying enzyme activity in erythroid
(i.e., procedures in which topical anesthetics are used). and nonerythroid tissues. The cyanosis can be treated with oral doses

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590 Part V  Red Blood Cells

of methylene blue (100 to 300 mg/day) or ascorbic acid (300 to G3P

1000 mg/day in divided doses). There is currently no treatment for
the neurologic manifestations of type II Cb5R deficiency.


Phosphoglycerate 2,3-BPG
Polycythemia is defined as an absolute increase in RBC mass. Primary ATP
polycythemia refers to an inherent defect (acquired or inherited) BPG phosphatase
within the RBCs that results in increased proliferation, whereas sec-
ondary polycythemia refers to a reactive process that is usually driven 3PG Pi
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 Phosphoenolypyruvate
secondary polycythemias are extremely rare and occur when germline
mutations alter hemoglobin oxygen affinity or decrease the concen- Figure 42-6  RAPOPORT-LUEBERING SHUNT.  2,3-BPG metabolism
tration of 2,3-BPG, resulting in impaired oxygen delivery to the is regulated by a multifunctional enzyme, bisphosphoglycerate mutase,
tissues. The latter condition is a result of a congenital deficiency of with both synthase (BPG synthase) and phosphatase (BPG phosphatase)
the erythrocyte enzyme bisphosphoglycerate mutase (BPGM). 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-
Epidemiology and Clinical Manifestations hyde 3-phosphate; 3PG, 3-phosphoglycerate; Pi, inorganic phosphate.

Only two families with complete BPGM deficiency have been com-
prehensively studied because of the rarity of the condition. The first circumvents frank tissue hypoxia. These patients may, however, be
case of complete BPGM deficiency was described in 1978 in a family more sensitive to decreases in hemoglobin and become symptomatic
in France.215 The propositus was a 42-year-old male with a hemoglo- from what would otherwise be deemed a mild anemia.
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 Aberg, JA., Kaplan JE, Libman H, et al: Primary care guidelines for the
BPGM gene.216 A second family with complete BPGM deficiency was management of persons infected with human immunodeficiency virus:
described in 2004 and included the first and only reported instance 2009 update by the HIV Medicine Association of the Infectious Diseases
(to date) of a patient homozygous for a mutation in the BPGM gene. Society of America. Clin Infect Dis 49:651, 2009.
The mutation was determined to be a missense mutation near the Ash-Bernal R, Wise R, Wright SM: Acquired methemoglobinemia: A
active binding site of the enzyme.217 retrospective series of 138 cases at 2 teaching hospitals. Medicine 83:265,
Baronciani L, Beutler E: Molecular study of pyruvate kinase deficient patients
Pathobiology with hereditary nonspherocytic hemolytic anemia. J Clin Invest 95:1702,
2,3-BPG is an important modifier of oxygen delivery by RBCs. It Beutler E: G6PD deficiency. Blood 84:3613, 1994.
binds to a central cavity of the hemoglobin tetramer and allosterically Beutler E: PGK deficiency. Br J Haematol 136:3, 2007.
converts hemoglobin to a low oxygen affinity state, resulting in a Beutler E: Glucose-6-phosphate dehydrogenase deficiency: A historical per-
rightward shift of the oxygen dissociation curve. Patients with spective. Blood 111:16, 2008.
chronic hypoxia have elevated levels of 2,3-BPG to facilitate oxygen Cappadoro M, Giribaldi G, O’Brien E, et al: Early phagocytosis of glucose-
unloading and increase oxygen delivery to the tissues (Fig. 42-6). 6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by
BPGM is the enzyme responsible for the synthesis of 2,3-BPG via Plasmodium falciparum may explain malaria protection in G6PD defi-
the Rapoport-Luebering shunt. BPGM is a homodimer with both ciency. Blood 92:2527, 1998.
synthase and phosphatase activity; its synthetic function converts Cappellini MD, Fiorelli G: Glucose-6-phosphate dehydrogenase deficiency.
1,3-bisphosphoglycerate to 2,3-BPG, and its phosphatase activity Lancet 371:64, 2008.
metabolizes 2,3-BPG to 3-phosphoglycerate, an intermediate of the Clark IA, Hunt NH: Evidence for reactive oxygen intermediates causing
glycolytic pathway. A deficiency of BPGM leads to a decrease in 2,3- hemolysis and parasite death in malaria. Infect Immun 39:1, 1983.
BPG. The ensuing increase in hemoglobin oxygen affinity results in Delivoria-Papadopoulos M, Oski FA, Gottlieb AJ: Oxygen-hemoglobulin
a decrease in oxygen delivery to the tissues, which in turn leads to a dissociation curves: Effect of inherited enzyme defects of the red cell.
compensatory polycythemia. Science 165:601, 1969.
Esbenshade AJ, Ho RH, Shintani A, et al: Dapsone-induced methemoglo-
binemia: A dose-related occurrence? Cancer 117:3485, 2011.
Diagnosis and Therapy FDA: FDA Drug Safety Communication: FDA continues to receive reports
of a rare, but serious and potentially fatal adverse effect with the use of
The diagnosis of BPGM deficiency is first suspected when a patient benzocaine sprays for medical procedures. 2011 August 1. Available from:
with an otherwise unexplained polycythemia is noted to have a http://www.fda.gov/Drugs/DrugSafety/ucm250040.htm#data, 2011.
decreased P50 on the oxygen dissociation curve and a normal hemo- Friedman, MJ: Oxidant damage mediates variant red cell resistance to
globin electrophoretic profile. These studies should be followed by malaria. Nature 280:245, 1979.
an assay measuring 2,3-BPG activity and, if decreased, an assay mea- Gaetani, GD, Parker JC, Kirkman HN: Intracellular restraint: A new basis
suring BPGM activity. The condition is confirmed by demonstrating for the limitation in response to oxidative stress in human erythrocytes
a mutation in the BPGM gene. containing low-activity variants of glucose-6-phosphate dehydrogenase.
Due to the rarity of the condition, there is limited experience to Proc Natl Acad Sci U S A 71:3584, 1974.
guide treatment. Presumably the reactive polycythemia compensates Guay J: Methemoglobinemia related to local anesthetics: A summary of 242
for the diminished oxygen delivery to the tissues and thereby episodes. Anesth Analg 108:837, 2009.

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Chapter 42  Red Blood Cell Enzymopathies 591

Kaplan M, Hammerman C: The need for neonatal glucose-6-phosphate Serpa JA, Villarreal-Williams E, Giordano TP: Prevalence of G6PD
dehydrogenase screening: A global perspective. J Perinatol 29:S46, 2009. deficiency in a large cohort of HIV-infected patients. J Infect 61:399,
Kwiatkowski, DP: How malaria has affected the human genome and what 2010.
human genetics can teach us about malaria. Am J Hum Genet 77:171, Tantular IS, Kawamoto F: An improved, simple screening method for detec-
2005. tion of glucose-6-phosphate dehydrogenase deficiency. Trop Med Int Health
Lenzner C, Nürnberg P, Jacobasch G, et al: Molecular analysis of 29 pyruvate 8:569, 2003.
kinase-deficient patients from central Europe with hereditary hemolytic Valentine WN, Fink K, Paglia DE, et al: Hereditary hemolytic anemia with
anemia. Blood 89:1793, 1997. human erythrocyte pyrimidine 5’-nucleotidase deficiency. J Clin Invest
Miller J, Golenser J, Spira DT, et al: Plasmodium falciparum: Thiol status and 54:866, 1974.
growth in normal and glucose-6-phosphate dehydrogenase deficient Youngster I, Arcavi L, Schechmaster R, et al: Medications and glucose-6-
human erythrocytes. Exp Parasitol 57:239, 1984. phosphate dehydrogenase deficiency: An evidence-based review. Drug Saf
Nkhoma ET, Poole C, Vannappagari V, et al: The global prevalence of 33:713, 2010.
glucose-6-phosphate dehydrogenase deficiency: A systematic review and Zaffanello M, Rugolotto S, Zamboni G, et al: Neonatal screening for glucose-
meta-analysis. Blood Cells Mol Dis 42:267, 2009. 6-phosphate dehydrogenase deficiency fails to detect heterozygote females.
Nock ML, Johnson EM, Krugman RR, et al: Implementation and analysis Eur J Epidemiol 19:255, 2004.
of a pilot in-hospital newborn screening program for glucose-6-phosphate Zanella A, Bianchi P, Fermo E, et al: Hereditary pyrimidine 5′-nucleotidase
dehydrogenase deficiency in the United States. J Perinatol 31:112, 2011. deficiency: From genetics to clinical manifestations. Br J Haematol 133:113,
Percy MJ, Lappin TR: Recessive congenital methaemoglobinaemia: Cyto- 2006.
chrome b(5) reductase deficiency. Br J Haematol 141:298, 2008. Zanella A, Fermo E, Bianchi P, et al: Pyruvate kinase deficiency: The genotype-
Ruwende C, Khoo SC, Snow RW, et al: Natural selection of hemi- and phenotype association. Blood Rev 21:217, 2007.
heterozygotes for G6PD deficiency in Africa by resistance to severe malaria.
Nature 376:246, 1995. For complete list of references log on to www.expertconsult.com.

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Chapter 42  Red Blood Cell Enzymopathies 591.e1

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

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591.e2 Part V  Red Blood Cells

24. Roth EF, Jr, Raventos-Suarez C, Rinaldi A, et al: Glucose-6-phosphate

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591.e4 Part V  Red Blood Cells

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Chapter 42  Red Blood Cell Enzymopathies 591.e5

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591.e6 Part V  Red Blood Cells

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