Trent Kolter
BCHM450-002
Case Study 1
An African-American man in the midst of a drug-induced acute hemolytic crisis is
admitted to the hospital. He has a history of neonatal jaundice and you suspect G6PDH
deficiency. You order the fluorescent spot test, which comes back negative. Is it possible
that the result is a false negative? Why?
A fluorescent spot test is a semi-quantitative test used in the diagnosis of G6PDH deficiencies,
however, false negatives are possible and make the test somewhat unreliable. The fluorescent
spot method consists of shining UV light on a blood sample and looking for NADPH within the
blood to fluoresce. A positive test is indicated by no fluorescence, showing the erythrocytes are
deficient in the G6PDH enzyme and are incapable of reducing NADP+ to NADPH (Nantakomol,
2013). A false negative may ensue with females who are heterozygous for the disorder and have
some non-deficient copies of the G6PDH enzyme, or within fully deficient G6PDH patients that
are in an acute hemolytic crisis. While in an acute hemolytic crisis, older erythrocytes containing
more deficient forms of the enzyme are lysed from the oxidative stress and replaced by new red
blood cells. The new red blood cells have higher levels of the G6PDH enzyme and can sustain
the oxidative stress better through some production of NADPH, causing a blood sample taken
from a period of crisis to fluoresce (Frank, 2005).
Females heterozygous for the G6PDH gene were found to have two distinct populations of
erythrocytes, one that contains normal G6PDH (no mutant enzyme), and the other that
contains mutant G6PDH (and no wild-type). How can this be explained?
Because the G6PDH gene is an X-linked trait, women can either be homozygous or
heterozygous for the G6PDH deficiency, while males can only be hemizygous. Heterozygous
females may have a milder form of the deficiency due to the ability to have some erythrocytes
with the mutant enzyme and some erythrocytes with the wild type enzyme (Frank, 2005). This
aspect can be attributed to X Barr Body inactivation. Because females have two X-chromosomes
and only one functional copy is required, one of the chromosomes becomes inactive. Usually, the
inactivated chromosome becomes a fixed decision after implantation of the embryo and before
the stem cells begin to differentiate, but this process is considered leaky and possesses room for
variability (Chen & Prchal, 2007). Because blood cells are derived from several different
predecessors that may have different inactivation of either the maternal or paternal X Barr body,
hematopoietic cells may result in a mosaic presentation of the affected or non-affected X
chromosome (Chen & Prchal, 2007).
Most mutations in the G6PDH gene are single amino acid substitutions, and very few large
deletions, frameshift mutations, and nonsense mutations have been described. In addition,
mutations in the eight-amino-acid substrate-binding site have not been described. What
might be the explanation for this?
There are over 400 G6PDH gene mutations that result in a G6PDH deficiency, but few of these
deletions are large deletions, frameshift, or nonsense mutations. Most of the mutations are single
amino acid substitutions that result in a less functional versions but still semi-productive
enzymes. There are 5 different classifications of the deficiency based on the level of enzyme
activity and the resulting intensity of the disease (NORD, 2017). Mutations leading to the
G6PDH deficiencies are often small because life is still sustainable with a partially functional
enzyme (NORD, 2017). In the case of frameshifts, large deletions, of nonsense mutations, the
enzyme would most likely be completely non-functional and the body would be in a chronic
state of hemolytic anemia and put too much stress on the kidneys. Major mutations such as these
would result in an early death that does not allow the mutated genes to be passed on to progeny.
Mutations to the eight-amino-acid substrate-binding site have not been described because any
patients born with this mutation probably would not survive long enough to be studied. Any
mutations that were found close to the binding site severely affected enzyme folding and activity
and significantly reduced the enzyme’s stability (Boonyuen et al., 2016). A mutation directly to
the binding site would completely distort the folding of the enzyme rendering it completely non-
functional and would lead to rapid degradation of the unstable protein. Again, this form of
complete inability to withstand oxidative stress would overexert the kidneys and the rest of the
body as it struggles through hemolytic anemia. This type of constant strain would not be
sustainable for the human body.
The pentose phosphate pathway is a major source of cellular NADPH; however, another
cellular enzyme activity also produces NADPH. What is it and what reaction does it
catalyze?
The pentose phosphate pathway typically generates NADPH through the enzyme glucose-6-
phosphate dehydrogenase. When this enzyme is deficient, erythrocytes experience acute
hemolytic anemia because they cannot generate NADPH to combat the oxidative stress.
Meanwhile other tissues within the body, such as the liver, of an induvial with a G6PDH
deficiency still actively combat oxidative stress. This contrast can be attributed to a lack of
mitochondria. Mitochondria transport the molecule citrate from their matrix into the cellular
cytosol when the cell has high levels of ATP, and the citric acid cycle slows down. The secretion
of citrate triggers the cell to start forming pyruvate to generate acetyl-CoA for fatty acid
synthesis. During this time a cycle is generated in which cytosolic citrate triggers the formation
of oxaloacetate to malate to pyruvate then pyruvate is transported back into the mitochondrial
matrix and acetyl-CoA is generated and citrate is released to continue the cycle. Throughout this
cycle, malate is converted to pyruvate via malic enzyme which generates CO2 and reduces
NADP+ to NADPH. Because other cell types possess malic enzyme and can undergo fatty acid
synthesis, NADPH will continue to be regenerated even in the absence of a functional version of
G6PDH. Erythrocytes lack mitochondria and malic enzyme; therefore, they will be unable to
undergo the regeneration of NADPH under oxidative stress and will lyse in response.
Is iron-deficiency anemia a hemolytic anemia? Briefly describe the underlying mechanism
of iron-deficiency anemia.
Iron-deficiency anemia and hemolytic anemia both result in an inability to oxygenate the body
adequately, but iron-deficiency anemia is not hemolytic and operates through a different
mechanism. Hemolytic anemia may occur from a G6PDHH deficiency when the erythrocytes do
not form enough NADPH to combat oxidative stress and lysis in response. Hemolytic anemia
may also occur in response to an overactive spleen, medications, or bacterial/viral infections that
cause the blood cells to burst. The blood cells are destroyed via phagocytosis faster than they can
be replenished leading to a deficient amount of erythrocytes to oxygenate the body (Johns
Hopkins, 2020). Iron-deficiency anemia results from inadequate supplies of iron causing a lack
of hemoglobin formation in erythrocytes. Immature erythrocytes acquire iron during their
reticulocyte stage and synthesize hemoglobin. Hemoglobin consists of four heme subunits, each
containing a cofactor of iron. The iron cofactors have a high affinity for oxygen and allow
hemoglobin in the R conformation to pick up oxygen then deliver oxygen to depleted tissues by
changing to the T conformation. When iron stores in the body are deficient hemoglobin synthesis
will be significantly reduced and fewer erythrocytes will be able to transport oxygen throughout
the body (Meisenberg & Simmons, 2017). Therefore, iron-deficiency anemia can be attributed to
a reduced number of formed erythrocytes or insufficiently formed erythrocytes, but the reduction
in erythrocytes is not due to destruction of red blood cells in response to oxidative stress.
The enzyme catalase, like glutathione peroxidase, can dispose of harmful cellular peroxides.
Both enzymes are found in erythrocytes. Do you think catalase plays a major role in the
disposal of peroxides in the erythrocytes of G6PDH-deficient patients undergoing a
hemolytic crisis? Under conditions when the patient is not in a crisis?
Glutathione peroxidase and catalase detoxify glutathione after its sulfhydryl group relieves
oxidative stress by donating electrons to free radicals. Glutathione peroxidase and catalase use
NADPH to reduce glutathione so it can continue preventing oxidative stress (Zam & Belal,
2020). During a hemolytic crisis of a G6PDH-deficient patient, catalase would not play a major
role in the disposal of erythrocytes because catalase is still an NADPH dependent enzyme like
glutathione peroxidase. Because erythrocytes do not have an alternative mechanism for
regenerating NADPH other than glucose-6-phosphate dehydrogenase in the pentose pathway, all
NADPH dependent enzymes would cease to function during an induced crisis after initial
amounts of NADPH are depleted (Thuraisingham & Adu, 2014). G6PDH-deficient patients are
often encouraged to avoid reactive oxidative stressors that induce a hemolytic crisis such as
medications, fava beans, and infections (Elyassi & Rowshan, 2009). Normal red blood cells have
to deal with oxidative stress induced by the autooxidation of unsaturated fats which generates
free radicals (Loftsson, 2014). Naturally, catalase and glutathione peroxidase reduce the
peroxides formed from this reaction using NADPH. If patients are not in a hemolytic crisis,
catalase would be able to function at limited capacity with the NADPH molecules that were
available, and the erythrocytes would not be destroyed from the oxidative stress. Younger
erythrocytes would be able to handle this mild form of oxidative stress because they have less
deficient forms of the G6PDH enzyme and can still regenerate NADPH more consistently
(Frank, 2005).
References
Boonyuen, U., Chamchoy, K., Swangsri, T., Saralamba, N., Day, N. P., & Imwong, M. (2016).
Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase
(G6PDH) variants, G6PDHViangchan and G6PDHViangchan+Mahidol: Decreased
stability and catalytic efficiency contribute to the clinical phenotype. Molecular genetics
and metabolism, 118(2), 84–91. https://doi.org/10.1016/j.ymgme.2016.03.008
Chen, G. L., & Prchal, J. T. 2007. X-linked clonality testing: interpretation and limitations.
Blood, 110(5), 1411–1419. https://doi.org/10.1182/blood-2006-09-018655
Elyassi, A., Rowhan, M. 2009. Perioperative Management of the Glucose-6-Phosphate
Dehydrogenase Deficient Patient: A Review of Literature. Retrieved from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749581/
Frank, J.E. 2005. Diagnosis and Management of G6PDH Deficiency. American Family
Physician. 1;72(7):1277-1282. Retrieved from:
https://www.aafp.org/afp/2005/1001/p1277.html
Johns Hopkins. 2020. Hemolytic Anemia. Retrieved from
https://www.hopkinsmedicine.org/health/conditions-and-diseases/hemolytic-anemia
Meisenberg, G., Simmons, W.H. 2017. Lipid metabolism. Principals of Biochemistry. Elsevier
Inc. 4 th edition.
Nantakomol, D., Paul, R., Palasuwan, A., Day, N. P., White, N. J., & Imwong, M. (2013).
Evaluation of the phenotypic test and genetic analysis in the detection of glucose-6-
phosphate dehydrogenase deficiency. Malaria journal, 12, 289.
https://doi.org/10.1186/1475-2875-12-289
NORD. 2017. Glucose-6-Phosphate Dehydrogenase Deficiency. National Organization for Rare
Disorders. Retrieved from: https://rarediseases.org/rare-diseases/glucose-6-
phosphatedehydrogenase-deficiency/
Loftsson, T. 2014. Drug Stability for Pharmaceutical Scientists. Retrieved from:
https://www.sciencedirect.com/topics/biochemistry-genetics-and-
molecularbiology/autooxidation
Thuraisingham, R., Adu, D. 2014. Glucose-6-phosphate Dehydrogenase Deficiency. Manson's
Tropical Infectious Diseases (23).
Zam, W., Belal, L., 2020. Ex Vivo Study of Laban's Role in Decreasing Hemolysis Crisis in
G6PDH-Deficient Patients. The Journal of Nutrition and Metabolism. 2020. 1-5.
https://doi.org/10.1155/2020/8034672
