Glucose-6-Phosphate Dehydrogenase Deficiency

 Inherited disease characterized by hemolytic anemia caused by the

inability to detoxify oxidizing agents
 Defect in an enzyme called Glucose-6-phosphate Dehydrogenase
 Most common disease producing enzyme abnormality in humans
 Affects more than 400 million individuals worldwide
 Many individuals are asymptomatic
 X-linked and is a family of deficiencies caused by more than 400 different
mutations in the gene coding for G6PD
 Highest prevalence in the Middle East, tropical Africa, Asia and parts of
the Mediterranean

Hexose Monophosphate (HMP) Shunt

 Also called Pentose Phosphate Pathway, (PPP) Phosphogluconate Shunt,
Warburg-Dickens Pathway
 alternate route for the metabolism of glucose
 occurs exclusively in the cytosol of the cell
 Major sites of HMP shunt
 Liver
 Red Blood Cell
 Steroidogenic Tissues (Adrenal Gland, Testis, Ovary)

2 major functions of the HMP Shunt

1. Formation of NADPH
-for synthesis of fatty acids, steriods
-maintaining reduced glutathione for antioxidant activity
2. Synthesis of ribose
-bio synthesis of nucleotide and nucleic acid formation

2 phases of the HMP shunt

1. Irreversible Oxidative Reactions
 Glucose-6-Phosphate undergoes Dehydrogenation and Decarboxylation
 formation of ribulose-5-phosphate and intermediate products CO2 and
NADPH
 Dehydrogenation of Glucose-6-Phosphate
 irreversible oxidation of glucose-6-phosphate to 6-
phosphogluconolactone catalyzed by G6PD enzyme with NADP+ as
coezyme
 produces one molecule of NADPH
 Formation of Ribose-6-Phosphate
 Hydrolysis of 6-Phosphogluconolactone producing 6-
phosphogluconate
 6-phosphogluconate is Decarboxylated producing now Ribulose-6-
phosphate and the second NADPH molecule

2. Reversible Non-oxidative Reaction

 Ribulose-5-phosphate also undergo several reactions to produce:
 Ribose-5-phosphate (nucleic acid synthesis)
 Fructose-6-phosphate and Glyceraldehyde-3-phosphate (glycolysis
pathway catalyze the interconversion of sugars containing 3 to 7
carbons
 initiated when the cell needs more NADPH than ribose-5-phosphate
 Conversion of ribulose-6-phosphate to either ribose-6-phosphate for
nucleotide synthesis or to fructose-6-phosphate or glyceraldehyde-3-
phosphate which can go directly to glycolysis
  Ribose-5-Phosphate reacts with Xylulose-5-Phosphate it produces
Sedoheptulose-7-Phosphate and Glyceraldehyde-3-Phosphate
Glucose-6-Phosphate Dehydrogenase
 rate-limiting step
 primary control point
 catalyzes the 1st NADPH of the pathway
 stimulated by NADP and inhibited by NADPH
 increased levels by insulin
NADPH (Nicotinamide Adenine Dinucleotide Phosphate)
-Coenzyme of Glutathione Reductace

Uses of NADPH:

Glutathione
Also known as GSH
Antioxidant
- Molecule that inhibits the oxidation of other molecules
- Terminate chain reactions by removing free radical
intermediates, and inhibit other oxidation reactions
- They do this by being oxidized themselves, so antioxidants are
often reducing agents such as thiols, ascorbic acid, or
polyphenols
Chemical Reaction:
GS–SG + NADPH + H+ → 2 GSH + NADP+
oxidized reduced
Gluconolactone Hydrolase
 Undergo spontaneous hydrolysis
 Hydrolase merely accelerate the process
6-Phosphogluconate Dehydrogenase
 Catalyzes the 2nd NADPH formation
 Yields 3-keto-6-phosphogluconate that rapidly decarboxylates to
release ribulose-5-phosphate
The combined effect of HMP shunt to glutathione metabolism is responsible
for protecting intracellular proteins from oxidative stress
Five Classifications of G6PD Deficiency
• Class 1: severe enzyme deficiency; with chronic nonspherocytic
hemolytic anemia
– less than 10% of normal activity ; uncommon, occur across
populations
• Class 2: G6PD Mediterranean; severe enzyme deficiency; with
intermittent hemolysis
– less than 10% of normal activity; more common in asian and
mediterranean populations
• Class 3: African descent; moderate to mild enzyme deficiency;
hemolysis with stressors
– 10-60% of normal activity
• Class 4: very mild or no enzyme deficiency ; normal activity
– at least 60% of normal activity
– no clinical manifestation
• Class 5: increased enzyme activity
– no clinical significance
Case belongs to class 3 since hemolysis only occurred in the presence of
stressors

Role of Glutathione in RBCs

 principal reducing agent in erythrocytes and the essential cofactor in the
glutathione peroxidase reaction.
 cellular defense against the oxidizing effects of H2O2
 normally pressent in the form of Reduced Glutathione (GSH)
a tripeptide (glutamyl-cysteinyl-glycine)
 Glutathione Peroxidase (GPx) utilizes GSH as the electron donor in the
process of reducing H2O2 to H2O while simultaneously generating GSSG
 In the course of reactions protecting hemoglobin from oxidation, GSH is
oxidized, forming oxidized glutathione (GSSG)

 for a continuous supply of GSH, a system for recovery of reduced

glutathione needed
 Such a system is provided by glutathione reductase
 Glutathione Reductase converts (reduces) GSSG to GSH using NADPH
as electron donor
Glutathione is recycled

 Reduced glutathione plays a very important role in the survival of the red
blood cells. (prevents oxidation of membrane)
 In G6PD Deficiency, there is decrease production of NADPH
-GSSG is not converted to its reduced form
-accumulation of ROS and H2O2 is not detoxified
-Oxidation -> membrane Deterioration
-Leading to HEMOLYSIS
Signs and Symptoms
Asymptomatic
• Most individuals who have inherited a G6PD mutation do not show
clinical manifestations
• However, some patients with G6PD deficency develop hemolytic
anemia if they are exposed to certain triggers
o bacterial and viral infections
o painkillers and fever-reducing drugs
o antibiotics (especially those that have "sulf" in their
names)
o antimalarial drugs (especially those that have
"quine" in their names)
Symptomatic
• Prolonged neonatal jaundice • Dark colored urine
• Hemolytic anemia, in • Fever
response to:
• Weakness
– Illness
• Dizziness
– Certain drugs, foods,
• Confusion
chemicals
• Splenomegaly, hepatomegaly
• Diabetic ketoacidosis
• Tachycardia
• Acute kidney failure (very
severe crises) • Heart murmur

Factors that precipitate haemolytic anemia in patients with G6PD

deficiency
Most individuals who inherited a G6PD mutation do not show manifestations,
however some patients with G6PD deficiency develop haemolytic anemia if
they are treated with an oxidant drug, ingest fava beans, or contract a severe
infection.

Oxidant drugs: commonly used drugs that produce haemolytic anemia in

patients with G6PD include antibiotics, antimalarials, and analgesics.

Sulfonamides • Cotrimoxazole
• Sulfacetamide • Ciprofloxacin
• Sulfanilamide • Norfloxacin
• Sulfamethoxazole • Niridazole
• Sulfasalazine Antimalarials
• Sulfisoxazole • Primaquine
• Sulfadiazine • Pamaquine
• Mafenide • Chloraquine
Non-Sulfa Antibiotics • Dapsone
• Chloramphenicol • Chloroproguanil
• Nalidixic acid Antipyretics/Analgesics
• Nitrofuratoin • Aspirin (Acetylsalicylic Acid)
• Isoniazid • Phenazopyridine
• Furazolidone • Acetanilide
• Dapsone • Phenacetin
Infection: Infection is the most common precipitating factor of hemolysis in
G6PD deficiency.
• Inflammatory response to infection results in the generation of free
radicals in macrophages, which can diffuse into the RBC and cause
oxidative damage
Clinical Correlation
 Patient Jay R
 At 2 days old
- Bilirubin climbed to 17mg/dL
- Preparations for exchange transfusions were made but cancelled when
it when subsequent bilirubin levels became lower
- Hemoglobin level fell to 13g/dL
- Reticulocyte count is 5 to 10%
Family History
Older brother – anemia and darkening of urine:
1. during respiratory infection
2. when UTI was treated with a sulfonamide of unknown type
Parents – both well
Maternal Uncle – had intermittent jaundice and
anemia
The child developed normally
Steady-state haemoglobin of 10.5 g/Dl
Reticulocyte of 10%
 At 6 years old
- Dark urine in the course of a respiratory infection
- Haemoglobin declined to 5.4 g/dL
- Transfusion of 1 U of packed red cells
- Subsequently, haemoglobin rose to the usual level

 At 14 years old
- Anemia was re-evaluated
- Red cells were profoundly deficient in G6PD activity
 - Has continued to get along quite well clinically
- Mildly jaundiced at times during infections:
- hemolytic anemia
- fall in hgb concentration
- darkening of the urine
- has been cautioned to seek medical attention
properly
- Has dealt well with infections up to now

Diagnostic Procedures
Heinz bodies
 Purple, blue inclusions
 Single or multiple
 Attached to the inner surface of the RBC
 Alters rigidity
 Formed by oxidant damage to the hemoglobin
 Not revealed by routine staining
 Visualized by using a supravital stain, Heinz body prep
 Also seen in patients with unstable forms of hemoglobin
 Heat denaturation, and high performance liquid chromatography can
be used to rule out G6PD deficiency’s

Bite Cells
 When a macrophage of the spleen identifies a RBC with a Heinz body
 Removes the precipitate and a small piece of the membrane
 Characteristic bite cell

Rapid Fluorescent Spot Test: Beutler Test

• Recommended screening test
• Visually identifies NADPH produced by G6PD under ultraviolet light
• NADPH fluoresces under UV light, when converted from NADP in the
presence of G6PD
• NADP does not fluoresce under UV light
• Lack of fluorescence indicates G6PD Deficiency
• When the blood spot does not fluoresce, the test is positive
 • It can be falsely negative in patients who are actively hemolysing
• Therefore, it only be done 2–3 weeks after a hemolytic episode
Genotypic Testing
 Useful for population screening, family studies, females, and prenatal
diagnosis
 Uses primers to check for mutation in the G6PD gene and is useful in
the diagnosis of homozygously-deficient male, hemizygously-deficient
female, and heterozygously-deficient female patients
Rapid Point of Care Tests
 Qualitative enzyme chromatographic tests
 A rapid test for the qualitative detection of G6PD enzyme activity in
human venous whole blood.
 BinaxNow G6PD
o G6PD – normal (negative) result: distinct color change to
black/brown
o G6PD – deficient (positive) result: no color change
 CareStart G6PD RDT
o G6PD – normal (negative) result: distinct purple color
o G6PD – deficient (positive) result: no color change or very faint
purple color

Cytochemical Assays
 Used to assess the G6PD status of individual erythrocytes and can be
used to detect all forms of G6PD deficiency
 Technically difficult and time consuming

Other laboratory tests

CBC & reticulocyte count – increased reticulocyte means increased
bone marrow production in response to anemia
Liver enzymes – to exclude other causes of jaundice
Lactate dehydrogenase – elevated in hemolysis and is a marker of
hemolytic severity
Haptoglobin – decreased in hemolysis
Coombs’ test – direct antiglobulin test – should be negative
– hemolysis in G6PD is not immune mediated
Urinalysis – for hematuria
What complications of G6PD deficiency make DNA Analysis best for
identification of carriers and patient diagnosis?
  While testing for enzyme activity should be performed when patients
are in remission, results may be falsely negative during acute
hemolysis
 DNA analysis is preferably done over quantitative enzymatic method
 Identification of carriers and patient diagnosis

Treatment
 Prevention is the most important measure
o Avoidance of oxidant stressors – drugs and foods that cause
hemolysis
o Vaccination against common pathogens (hepa A) may prevent
infection induced attacks
 Blood transfusions – in the acute phase of hemolysis or in dialysis in
acute renal failure
o Important symptomatic measure as the transfused red calls are
not G6PD deficient
 Splenectomy – some patients benefit from the removal as it is an
important site of red cell destruction
 Folic acid supplement – used in any disorder with a high red cell
turnover

G6PD deficiency and Malaria

Normal G6PD
• Malaria is transmitted to humans by infected mosquitoes where it
incubates in the liver.
• The malaria parasites (Plasmodium) are then released into the blood
stream where they infect red blood cells.
• The parasites then grow and replicate in the red blood cell for 10 to 14
days until the RBC bursts.
• When this happens, several poisons are released into the blood stream
which causes the high fever, chills and sweats.
G6PD deficient
• When an infected RBC dies before the parasite is ready, the malaria
parasite dies as well and it does not have the chance to produce the
poisons.
• The growth of malaria parasite is naturally impaired upon first passage
from normal to G6PD deficient RBC (less favorable environment to the
parasite).
• In addition, cells infected with the Plasmodium parasite are cleared
more rapidly by the spleen. This phenomenon might have given G6PD
deficiency carriers an evolutionary advantage.
Summary
o HMP shunt produces NADPH which is important for reduction of
glutathione

o Reduced Glutathione is an antioxidant that has the ability to convert

free radicals such as hydrogen peroxide into water
o A deficiency in the enzyme G6PD causes hemolysis of RBC due to the
accumulation of hydrogen peroxide in the circulation which decreases
their lifespan
o There are 5 classifications of G6PD deficiency

o Diagnostic studies involves the use of blood smear

o Heinz bodies are specific cells found in the smear of affected

individuals
o Newborn Screening