Glucose 6-phosphate dehydrogenase (G6PD)

Glucose 6-phosphate dehydrogenase

(G6PD)



Glucose-6-phosphate dehydrogenase (G6PDH), catalyzes the first step in the pentose phosphate shunt,



oxidizing glucose-6-phosphate (G-6-P) to:



6-phosphogluconate (6-PG) and reducing NADP to NADPH.

 G-6-P + NADP⁺ 6-PG + NADPH + H^{G-6PDH +}

- is an alternative oxidative pathway for glucose rather than .glycolysis.

- results in no ATP production (not like glycolysis).

- is the major pathway for NADPH production.

- produces Ribose-5-phosphate for nucleotide synthesis (required for DNA, RNA & others)

Pentose Phosphate Pathway (PPP)

or Hexose Monophosphate Pathway (HMP)

Glucose 6-phosphate has 3 fates.

Pentose Phosphate Pathway (PPP)

or Hexose Monophosphate Pathway (HMP)

as a source for NADPH

Glucose 6-phsphate Dehydrogenase

First enzyme in the pathway

Glucose

Pentose phosphate pathway (or hexose monophosphate pathway) . including RBCs occurs in the cytosol of many cells of the body

NADPH 2

are produced for each one molecule

of glucose

1- Role in fatty acid synthesis (reductive agent)

2- Role in antioxidant mechanisms (part of glutathione system)

3- Role in oxygen-dependent phagocytosis by WBCs 4- Role in synthesis of nitric oxide (NO)

Uses of NADPH in normal cellular

metabolism

Hydrogen peroxide (H₂O₂) is one member of the family of reactive oxygen species (ROS).

ROS are formed continuously from aerobic metabolism of drugs and environmental toxins or diminished

antioxidants. All these lead to oxidative stress.

ROS cause damage to DNA, protein and unsaturated lipids of the cells including cell membranes..

They are implicated in cancer, chronic inflammatory disease and aging.

Role of NADPH in antioxidant mechanisms

Peroxidation of RBC membrane lipid

→↑↑ membrane fragility → Hemolysis

The function G6PD in the red cell is to generate NADPH → reduced glutathione → protect the RBCs from the oxidative damage by H₂O₂

Definition

• It is an inherited disease characterized by hemolytic anemia caused by inability to detoxify oxidized agents.

• G6PD deficiency is the most common disease producing enzyme anomalies in humans, affecting more than 400 million individuals worldwide.

The highest prevalence in the Middle East, tropical Africa & Asia.

• G6PD Deficiency is caused by different mutations in gene coding for G6PD, only few of them causes the clinical symptoms of the disease.

Glucose 6-phosphate dehydrogenase deficiency (G6PD deficiency)

• Deficiency of G6PD occurs in all cells of affected individual.

• It is severe in RBCs because the only pathway to form NADPH in RBCs is pentose phosphate pathway (using G6PD).

Decreased amounts of reduced glutathione due to decreased production of NADPH

Precipitating factors in G6PD deficiency:

Some of patients with G6PD develop hemolytic anemia if they are exposed or ingest any of the followings oxidizing agents:

1-Oxidant drugs:

Antibiotics : e.g. sulfamethoxazole Antimalarias : e.g. primaquine

Antipyretics : e.g. acetanilid 2- Favism:

The hemolytic effect of ingesting of fava beans is not observed in all individuals with G6PD deficiency but all patients with favism have G6PD deficiency

PD activity 6

Measurement of G

 NADP is reduced by G-6-PDH in the presence of G-6-P. The rate of formation of NADPH is

directly proportional to the G-6-PDH activity and is measured spectrophotometrically as an

increased in absorbance at 340nm.

Specimen collection and storage

 Whole blood collected with EDTA, heparine or acid citrate dextrose .Red cell G-6-PDH is stable in whole blood for one week refrigrated (2-8ºc).

Procedure

1.

prepare reaction mixture:

• Add 0.01ml blood directly to vial containing G-6-PDH assay solution and mix throughly to completely suspend erythrocytes, lat stand at room tempreture(18-25ºc) for 5-10min.

• Add 2.0ml G-6-PDH substrate solution

directly to vial and mix gently by inverting several times.

• Transfer contents of vial to cuvet.

 Place cuvet in constant tempreture cuvet

compartment or water bath and incubate for approximatly 5min to attain therma; equilibrium.

 Read and record absorbance (A1) of test at 340 nm vs water or ptassium dichromate solution. This is initial A .(if using awater bath or incubator ,return cuvet to it)

 Exactly 5min later, again read and record (A2), this is final A.

 To determine G-6-PDH activity do the following calculation.

Calculation

 = ΔA per min X 4839 / Hb (g/dl) X TCF Where:

 Hb (g/dl) = hemoglobin concentration determined for each specimen

 TCF = temperature correction factor (1 at 30ºc)

Reference

 Lippincott’s Illustrated Biochemistry

 Essential Hematology(Hoffbrand, Pettit & Moss)

Case Study

 21 yrs. male medical student with malaria

 Treated with primaquine

 Four days later:

 Black colored urine

 Low RBC count

 Elevated reticulocyte count

 RBC with Heinz bodies

 Low hemoglobin

 Elevated serum bilirubin

 Pt recovered in a few days

On

admission

10 days later

Normal range

Hemoglobin, g/dl 9.2 14.5 14 - 18

Red blood cells  10¹²/l 3.5 5 5

Reticulocytes, % 12 4 0.5 - 1.5

Bilirubin, mol/l 340 23 2 - 14

Red cell glucose-6-

phosphate dehydrogenase, units per gram hemoglobin

? ? 13 - 19



QUESTIONS

1. Determine red cell glucose-6-phosphate dehydrogenase activity in units/gm Hb.

2. What is the cause of changes in the laboratory findings?

3. What is your diagnosis for such a case?

4. What are the functions of glucose-6- phosphate dehydrogenase in different tissues?

5. What are the chemical characteristics of drugs which would be expected to bring on a hemolytic crisis?

6. From your knowledge in vitamins how could you treat this case?