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Journal of
of Nepal
PATHOLOGY
www.acpnepal.com
Journal of Pathology of Nepal (2016) Vol. 6, 1034 -
Review Article
Glusoce-6-phosphate dehydrogenase- History
and diagnosis
Correspondence:
Dr. Keyoor Gautam, MBBS, MD
Consultant Pathologist
Department of Pathology, Samyak Diagnostic Pvt. Ltd, Lalitpur, Nepal
Email: drkeyoor@gmail.com
Glucose-6-phosphate dehydrogenase deciency is the most common enzymatic defect of red blood cells,
which increases the vulnerability of erythrocytes to oxidative stress leading to hemolytic anemia. Since
its identication more than 60 years ago, much has been done with respect to its clinical diagnosis,
laboratory diagnosis and treatment. Association of G6PD is not just limited to anti malarial drugs, but
a vast number of other diseases. In this article, we aimed to review the history of Glucose-6-phosphate
dehydrogenase, the diagnostic methods available along with its association with other noncommunicable
diseases.
ABSTRACT
INTRODUCTION
Glucose-6-phosphate dehydrogenase (G6PD) deciency
is the most common enzymatic defect of red cells. G6PD
deciency increases the vulnerability of erythrocytes
to oxidative stress leading to hemolytic anemia. Sixty
years ago 3 enzyme deciencies that produce disease in
humans had been identied, all in human erythrocytes.
These enzymes were catalase, galactose-1-phosphate
uridyltransferase, and glucose-6-phosphate dehydrogenase.1
Although the technology was not so advance at that time,
a lot was accomplished regarding the diagnosis of G6PD
Gautam K1
1Department of Pathology, Samyak Diagnostic Pvt. Ltd, Lalitpur, Nepal
deciency, its genetic predisposition as well as the disease
management. Globally, 400 million people are estimated
to be affected with this enzymatic disorder of red blood
cells (RBCs), most of which are seen in the tropical and
subtropical zones of eastern hemisphere.2,3 Although a
large number of individuals are affected with this enzyme
deciency, fortunately only few present with a clinically
relevant symptom.
History
In the year 1926, Cordes reported the occurrence of
acute hemolysis in individuals treated for malaria
with 6-methoxy-8-aminoquinoline drugs, however the
mechanism of hemolysis could not be understood for
the next three decades.4 The discoveries that lead to the
Keywords:
G6PD;
Primaquine;
Neonatal Jaundice
1039
1035
recognition of G6PD deciency were the result of several
convergent events. The biochemical pathways through
which red cells metabolize sugar were established.5,6 The
tools available then seem incredibly primitive today, but
by 1950, virtually every step in red cell glycolysis was
established. Secondly, the development of isotopic methods
permitting the accurate estimation of red blood cell
survival was essential. It was the 51Cr method of labeling
erythrocytes, rst devised by Sterling and Gray to measure
the red cell mass and subsequently adapted for measuring
the cell survival, that set the stage for the studies that had
to be performed.7 Finally the circumstances surrounding
World War II and the Korean War had created the necessity
for the development of new, synthetic antimalarial drugs.
The funding required to carry out the studies materialized
from the United States Army. Volunteers serving sentences
in the Illinois State Penitentiary at Joilet were selected for
conducting the clinical investigation on understanding why
8-amino quinoline antimalarials caused hemolysis.6 When
normal volunteers were given 30 mg of primaquine daily,
some developed acute hemolytic anemia; most did not. Did
those who developed hemolytic anemia metabolized this
6-methozy-8-amino quinoline anitmalarial drug differently,
or did their red ells differ in some manner? Development
of 51Cr technique made it possible to answer this question.
When 51Cr labeled cells form a primaquine-sensitive subject
were transfused into a nonsensitive subject, primaquine
administration resulted in rapid destruction of the labeled
erythrocytes. But when 51Cr labeled cells from a non sensitive
subject were transfused into a primaquine sensitive subject,
they survived normally even when the host’s red cells were
being rapidly destroyed. These studies established clearly
that sensitivity to the hemolytic effect of primaquine was
due to an intrinsic defect of the erythrocyte.6
There were few morphological changes in the red cells of
subjects undergoing primaquine-induced hemolysis, but
Heinz bodies could be detected in the circulation before
the onset of hemolysis. Heinz bodies can be induced in
vitro by compounds such as phenylhydrazine or acetyl
phenlyhydrazine, and we were able to show that the pattern
of in vitro Heinze body formation was quite different in
sensitive and nonsensitive subjects.8 This made it possible,
for the rst time, to detect primaquine sensitivity in vitro.
The fact that iodoacetate and arsenite caused normal cells
to behave like primaquine sensitive cells focused attention
on the sulfhydryl groups of the erythrocyte. The amount of
glutathione in the cells was diminished, and the ability of
erythrocytes to maintain glutathione levels in the face of
oxidative stress was abnormal.9 Latter on Carson undertook
a study of the reduction of glutathione by hemolysates.
When it was found that hemolysates from primaquine
sensitive men could not utilize glucose-6-phosphate to
reduce glutathione, the more expensive NADPH was
added as the hydrogen donor. This substrate could reduce
glutathione in hemolysates from primaquine sensitive
men, making it apparent that the primary defect was in the
glucose-G-phosphate utilizing enzyme, G6PD.10
Genetics and Classication
G6PD enzyme is encoded by a human X-linked gene (Xq2.8)
consisting of 13 exons and 12 introns, spanning nearly 20
kb in total; the rst exon is noncoding, while the remaining
12 range from 120 to 235 bp. It has over 400 allelic variants
know. These variants have been biochemically characterized
based on: (a) the different residual enzyme activities, (b)
electrophoretic mobility patterns, and (c) physiochemical
or kinetic properties.11 G6PD variants are grouped into ve
classes based on WHO guidelines (Table 1).
Pathophysiology
Red cells are vulnerable to injury by oxidants. Abnormalities
in the hexose monophosphate shunt or glutathione
metabolism resulting from decient or impaired enzyme
function reduce the ability of red cells to protect themselves
against oxidative injuries, leading to hemolytic disease. As
noted in Figure 1, G6PD reduces NADP to NADPH while
oxidizing glucose-6-phosphate. NADPH then provides
reducing equivalents needed for conversion of oxidized
glutathione to reduced glutathione, which protects against
Table 1 : Classication of G6PD deciencies following the WHO recommendations
Class Residual enzymatic
activity Protein location Clinical symptoms Frequency and geo-
graphical distribution Type of mutation
I<1% or not detectable Dimer interface Struc-
tural NADP
Chronic hemolytic ane-
mia
Rare and without a pre-
cise geographical distri-
bution
G6PD-Buenos Aires,
G6PD Durban
II <10% Dimer interface NADP
binding site
Acute hemolytic anemia
fava beans and drug de-
pendent
Frequent and distributed
throughout the world
G6PD-Mediterranean
G6PD-Cassano G6PD-
Santamaria
III 10-60% Scattered throughout the
whole enzyme
Occasionally acute he-
molytic anemia
Very frequent in malaria
areas
G6PD-A- G6PD-Seattle
G6PD-Canton G6PD-
Rignano
IV 60-90% normal activity Neutral protein site Asymptomatic Undened frequency G6PD-Montalbano
G6PD-Orissa
V>110% increased activity Neutral protein site or
promoter mutations Asymptomatic Undened frequency Not reported
Glusoce-6-phosphate Dehydrogenase
1036
oxidant injury by catalyzing the breakdown of compounds
such as H2O2. In simpler terms, the disposal of H2O2, a
potential oxidant, is dependent on the adequacy of reduced
glutathione (GSH), which is generated by the action of
NADPH. The synthesis of NADPH is dependent on the
activity of G6PD.11
Clinical spectrum of G6PD deciency
The clinical spectrum of G6PD deciency has been identied
in a vast number of conditions. Clinical manifestations
depend on the degree of the enzyme deciency, which
in turn is determined by the characteristics of the G6PD
variant. Majority of individuals are asymptomatic and do
not have hemolysis in the steady state. They have neither
anemia, evidence of increased red blood cell destruction,
nor an alteration in blood morphology, although a modest
shortening of RBC survival can be demonstrated by isotopic
techniques. However, episodes of acute hemolysis with
hemolytic anemia may be triggered by medications, certain
foods, and acute illnesses, especially infection.12
Drug induced hemolysis
The hemolytic effect of primaquine in G6PD decient
individuals were already established. But later on it was
realized that the spectrum of drugs that caused hemolysis
was much more than that had been anticipated. Transfusing
labeled red cells from a few primaquine sensitive donors
into a larger number of nonsensitive recipients and then
challenging the sensitive cells with a variety of drugs made
it possible to quantitate the extend to which each drug
produced hemolysis.6,13,14 Table 2 lists drugs that have been
shown to be capable of producing clinically signicant
hemolytic anemia in doses that are normally used. Table 3
indicates qhich drugs can be given safely to most patients
with G6PD deciency.15,16
Foods inciting hemolysis
Certain foods can induce hemolysis in a G6PD decient
individual. Intravascular hemolysis induced by ingestion
of fava beans, known to as favism, is a well know cause
and is seen predominantly in young males age 1 to 5 years.
Symptoms include headache, back pain, chills, fever
within 24 hours of ingestion, followed by hemoglobinuria
and jaundice. An important thing to be noted is that all
patient with favism are G6PD decient, but many G6PD
decient individuals can eat fava beans with impunity.
Thus, the deciency is a necessary but not sufcient cause
of hemolysis. It has been suggested that the glycosides
divicine and isouramil are the components of the bean
responsible for a hemolysis, but there is no rigorous proof
that this is the case, and the additional factor that makes a
minority of G6PD decient individuals develop hemolysis
when they ingest the bean is still unknown, although it is
probably inherited.
Infection
Infection is the most common cause of acute hemolysis in
G6PD-deceinet persons, although the exact mechanism
by which this occurs is unknow. Leukocytes may release
oxidants during phagocytosis that cause oxidative stress
to the erythrocytes; however, this explanation alone would
not account for the variety of infections associated with
hemolysis in G6PD decient persons. The most common
infections agents causing hemolysis include Salmonella,
Escherichia coli, beta-hemolytic streptococci, rickettsial
infections, viral hepatitis, and inuenza A.17
Neonatal Jaundice
Anemia and jaundice are often rst noted in neonates
with severe G6PD deciency. The principle cause of
neonatal icterus in G6PD decient infants is the inability
of the liver to adequately conjugate bilirubin. This
problem is compounded when the infant also inherits the
UDP glucuronosyl transferase promoter polymorphism
that is associated with Gilbert disease. As noted,
hyperbilirubinemia is likely secondary to impairment of
bilirubin conjugation and clearance by the liver leading to
indirect hyperbilirubinemia. G6PD deciency can lead to
an increased risk and earlier onset of hyperbilirubinemia,
which may require phototherapy or exchange transfusion.
In certain populations, hyperbilirubinemia secondary to
G6PD deciency results in an increased rate of kernicterus
and death, whereas in other populations this has not been
observed. This may reect genetic mutations specic to
different ethnic groups.
Table 2: Drugs and chemicals to be avoided by persons
with G6PD deciency
Acetanilide
Diaminodiphenyl sulfone
Furazolidone (Furoxone)
Glibenclamide
Henna (Lawsone)
Isobutyl nitrate
Methylene blue
Naphthalene
Niridazole (Ambilhar)
Nitrofurantoin
Phenazopyridine
Phenylhydrazine
Primaquine
Sulfacetamide
Sulfanilamide
Sulfapyridine
Thiazolesulfone
Trinitrotoluene
Urate oxidase
Gautam K et al.
1037Glusoce-6-phosphate Dehydrogenase
Table 3: Drugs that probably can be safely given in normal
therapeutic doses to G6PD-decient patients without nons-
pherocytic hemolytic anemia
Acetaminophen
Acetophenetidin
Acetylsalicylic acid
Aminopyrine
Antazoline
Antipyrine
Ascorbic acid
Benzhexol
Chloramphenicol
Chlorguanidine
Chloroquine
Colchicines
Diphenyldramine
Isoniazid
L-dopa
Menadione sodium bisulfate
p-Aminobenzoic acid
p-Aminosalicylic acid
Phenylbutazone
Phenytoin
Probenecid
Procainamide hydrochloride
Pyrimethamine
Quinine
Spectromycin
Sulfacytine
Sulfadiazine
Sulfametazine
Sulfamethoxazole
Sulfamethoxypyridazine
Sulsoxazole
Tiaprofenic acid
Trimethoprim
Tripelennamine
Vitamin K
Congenital nonspherocytic hemolytic anemia and
chronic hemolysis
Chronic hemolysis is not characteristic of most individuals
with G6PD deciency, but those with severe deciency can
have chronic hemolysis with or without chronic anemia.
Variants that produce chronic hemolytic anemia are referred
to as class I variants. These individuals have such severe
G6PD deciency that they may have hemolysis even in the
absence of oxidant injury from medications or illnesses.18,19
These individuals may also be referred to as having
congenital nonspherocytic hemolytic anemia. The term
nonspherocytic is somewhat of a misnomer, since these
individuals may have spherocytes on the pheripheral
blood smear. However, this term is useful in distinguishing
individuals with G6PD deciency, in whom spherocytes are
relatively infrequent at baseline, from those with hereditary
spherocytosis, in whom spherocytes are abundant. The
severity of hemolysis varies, causing mild hemolysis to
transfusion dependent anemia. Exposure to oxidative stress
can cause acute hemoysis in these persons.20
Cardiovascular disease
The clinical effects of G6PD deciency on the heart remains
largely unexplored despite it being the most common
known enzyme deciency in the world. Although studies
suggest that G6PD deciency may decrease superoxide
production in failing myocardium and that G6PD deciency
may decrease the risk of developing coronary heart disease,
recent studies in mice indicate increased oxidative stress
in G6PD decient failing myocardium and that G6PD
deciency adversely affects the development of heart
failure.
The effects of G6PD deciency on the development and
progression of heart failure in human patients could be
explored by screening hypertensive patients for G6PD
deciency and then following these patients over an
extended period to see whether g6PD deciency affects
the development of heart failure in these patients. Another
study could examine G6PD decient patients who have
already developed heart failure to determine whether G6PD
deciency positively or negatively affects prognosis. Thus
Figure 1: : Role of glucose-6-phosphate dehydrogenase
in defense against oxidant injury.
1038
the development of heart failure should be examined in
G6PD decient patients. Overall, G6PD deciency may
decrease the rate of cardiovascular disease development
among humans through its effect on atherogenesis.21
Meloni has concluded that subjects with G6PD decient
phenotype are less prone to coronary heart disease (CHD).
They suggest that such a protective effect may be ascribable
to a reduced 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (HMG-CoA R) activity, a statin-like effect, as well
as to a downregulation in NADPH oxidase activity with a
consequent reduction in oxygen free radical production.22
G6PD activity in Diabetic patients
Diabetes mellitus is a common and complicated disease.
Activities of enzyme G6PD is important in preventing
its complications. Unsuitable control of blood glucose
decreases G6PD activity and increases diabetes mellitus
complications. This issue itself aggravates diabetic injury
due to inappropriate antioxidation process. Simultaneous
dyslipidemia and obesity may intensity the effect of
hyperglycemia and oxidative stress. G6PD activity level can
reect the gylcemic control, and even predict subsequent
complications while they are not present Literature has
shown that G6PD mean activity in diabetics is signicantly
lower than non diabetics. Various studies have concluded
that reduced activity of G6PD is a risk factor for DM. Since
diabetic patients simultaneously suffer from other endocrine
problems like dyslipidemia, it is worthy to evaluate the
impact of such associated abnormalities on G6PD activity.
Comparing G6PD activity between dyslipidemic and non
dyslipidemic patients within diabetics and comparing
G6PD activity between non diabetic and dyslipidemic
diabetic patients, the researchers showed that its activity
was signicantly lower in dyslipidemic patients.23
On the other hand studies have also concluded that G6PD
deciency showed a trend for protection against diabetes
with proliferative diabetic retinopathy. Further experimental
and clinical studies are necessary for a better understanding
of the mechanism by which G6PD deciency may affect
diabetes and its retinal vascular complications.24
Laboratory Tests
Enzymatic evaluation: The WHO recommends the diagnosis
of G6PD deciency utilizing universal tests, mainly based
on the generation of NADPH from NADP.
Semiquantitative assays are as follows: (i) uorescent spot
test, which is rapid, simple, sensitive and inexpensive. This
test method is used in countries where G6PD deciency is
both frequent and malaria endemic, before starting treatment
with antimalarial drugs, such as primaquine. A variant of
the spot test, not requiring the sue of an ultraviolet lamp
but a naked eye evaluation, allows the larger population
screening in the tropical areas. (ii) other screening tests
available which determine the NADPH concentration
indirectly, by measuring the reduced methemoglobin levels
produced after NADPH oxidation. (iii) Finally, the Heinz
body examination and GSH stability test may be employed
to distinguish G6PD decient from normal individuals.
For a biochemical denitive diagnosis, a quantitative analysis
is mandatory done by spectrophootometry. Diagnostic issues
can arise when G6PD activity is measured after or during
an episode of acute hemolysis, or in the presence of high
blood reticulocyte count, being the reticulocytes activity
about ve times higher than that of old RBCs, resulting in
a false negative result. Because protein synthesis is absent
in RBCs, the activity of G6PD, and of other enzymes
gradually decreases during RBCs aging, which will be
selectively destroyed. If any acute intravascular hemolytic
G6PD dependent anemia is suspected, any potentially
dangerous drugs must be discontinued and the test should
be rerun 10-15 days after, or later, if the patient has been
transfused. In these cases, genetic analysis or family study,
when available, can improve the diagnostic tool. For a
complete diagnostic assessment of the G6PD deciency
rate, the following laboratory parameters are also important:
RBCs and reticulocyte counts, total and indirect plasma
bilirubin, plasma iron and lactate dehydrogenase levels,
serum haptoglobin and urine hemoglobin concentration.11
Molecular diagnosis
Molecular analysis may be useful for population screening,
family studies, or parental diagnosis, although this approach
is not used routinely. For a correct laboratory practice, the
molecular diagnosis of G6PD deciency should employ two
analytical steps: 1) a rst screening level, to research the
most frequent mutations belonging to a specic geographical
area. In this case, a PCR coupled to RFLP represents a
rapid valid, and reliable molecular screening approach; 2) a
second level, based on the whole gene sequencing, nalized
to the identication of the less frequent, or novel, mutation.
25
DNA based test for the screening of the most frequent
mutations in a specic geographical area can be used as
an alternative tool to the biochemical assay. The costs for
chemicals dedicated to molecular test are comparable to
those used for the enzyme assay. In the future, more advance
systems should be utilized to improve the efciency of the
molecular assay.11
CONCLUSION
G6PD deciency is one of the most common enzyme defect
of red cells which has been studied for more than 50 years
since its identication. Point of care testing has helped in
screening individuals prior to administration of malarial
drugs. Molecular testing has been important in identifying
the different variants which have clinical signicance.
Gautam K et al.
1039
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Glusoce-6-phosphate Dehydrogenase