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Glucose-6-Phosphate Dehydrogenase (G-6-PD)

Glucose-6-Phosphate Dehydrogenase (G-6-PD). Introduction. G6PD deficiency is an allelic abnormality which is inherited in an X-linked recessive fashion. G6PD deficiency is also known as "favism" since G6PD deficient individuals are also sometimes allergic to fava beans .

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Glucose-6-Phosphate Dehydrogenase (G-6-PD)

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  1. Glucose-6-Phosphate Dehydrogenase(G-6-PD)

  2. Introduction • G6PD deficiency is an allelic abnormality which is inherited in an X-linked recessive fashion. • G6PD deficiency is also known as "favism" since G6PD deficient individuals are also sometimes allergic to fava beans. • Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common human enzyme deficiency in the world; it affects an estimated 400 million people.

  3. When someone has G6PD deficiency, complications can arise; hemolytic anemia and prolonged neonatal jaundice are the two major pathologies associated with G6PD deficiency. • Both of these conditions are directly related to the inability of specific cell types to regenerate reduced nicotinamide adenine dinucleotide phosphate (NADPH); this reaction is normally catalyzed by the G6PD enzyme.

  4. In G6PD deficient individuals, anemia is usually caused by certain oxidative drugs, infections, or fava beans. • When any one of these agents, or their metabolites, enters a G6PD deficient red blood cell, hemoglobin becomes denatured, thus destroying its function as the principal oxygen carrying molecule. • In addition to being susceptible to hemolytic anemia, G6PD deficient individuals are also predisposed to prolonged neonatal jaundice, this can be a potentially serious problem as it can cause severe neurological complications and even death.

  5. Principle: • Glucose-6-phosphate dehydrogenase (G6PDH, D-glucose-6-phosphate) 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, which illustrated by the following equation: G-6-P + NADP+ G-6PDH 6-PG + NADPH +H+ • 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.

  6. Principle Cont. • Production of a second molar equivalent of NADPH by erythrocyte 6-phosphogluconate dehydrogenase (6-PGDH) according to the reaction : 6-PG + NADP+G-6PDH Ribulose-5- phosphate + NADPH + H+ + CO2 SpecimenWhole blood collected with EDTA, or acid citrate dextrose .

  7.  Stability and Storage: • Red cell G-6-PDH is stable in whole blood for one week refrigerated (2-8ºc), but is unstable in red cell hemolysates.

  8. Qualitative Method in G-6-DP Determination

  9. Principle: Glucose -6-phosphate dehydrogenase present in the red blood cell hemolysate, act on glucose -6-phosphate and reduces NADP to NADPH which, with the help of PMS, reduces blue colored 2,6 Dichlorophenol Indophenols into a colorless form. The reaction can be represented as: G-6-phosphate + NADP 6-phosphogluconic acid + NADPH NADPH + 2,6 Dichlorophenol indophenols (DCPIP)(Blue color) NADP + Reduced DCPIP (colorless) • Rate of declorization is directly proportional to the activity of G-6-PD.

  10. Note: • Fresh blood sample should be use since refrigeration reduces the enzyme activity. • Heparin sample should not be use as interfere with enzyme reaction. • Avoid exposure of substrate vial to the light (it is photosensitive).

  11. Procedure: Step1: Preparation of red cell hemolysate: • Purified water : 2.5ml • Fresh blood : 0.05ml • Mix well and allow standing for 5min at R.T. Step2: Assay of the enzyme: • Add 1mi of the hemolysate (step 1) to the vial of solution 1 and mix gently. • Add immediately about 1ml of reagent 3. • Seal the vial with aluminum foil and incubate in water bath at 37ºc.

  12. Procedure Cont. Observe: • the time taken for the color change from initial deep blue to reddish purple. Follow up to a maximum of 6 hours with 30 min intervals. Results: • Normal : 30-60 min. • G-6-PD deficient (heterozygous males, homozygous female): 140min-24hr • G-6-Pdcarriers (heterozygous females): 90min-several hours.

  13. Quantitative Method in G-6-DP Determination

  14. Principle 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.

  15. Procedure 1- SAMPLE PREPARATION • Wash 0.2 ml of blood with 2 ml aliquots of 0.9% NaCl solution. • Centrifuge after each wash for 10 min at around 3000 rpm. • Repeat 3 times. • Suspend the washed centrifuged erythrocytes in 0.5 ml of solution 4 and let stand for 15 min at +4°C and then centrifuge again. • Use the supernatant in the assay within 2 hours.

  16. Procedure 2- Enzyme assay A-Add the following reagents Hemolysate 15 µl R1 (buffer) 1000 µl R2 (NADP) 30 µl B- Mix incubate 5 min at 370C C- Add 15 µl R3 (Substrate G6P) Mix and read the initial absorbance at 340 nm against air(A1) D- Read again after 1, 2 and 3 minutes.(A2, A3, A4)

  17. Calculation: To determine G-6-PDH activity, do the following calculations: • ΔA per min =( Afinal- Ainitial ) • G-6-PDH activity is expressed as U/1012 erythrocyte (RBC)or as U/g hemoglobin (Hb).

  18. G-6-PDH (U/1012 RBC) • G-6-PDH (U/1012 RBC) =( ΔA per min X60571) / erythrocyte count • G-6-PDH(U/g Hb) = ΔA per min X60571 X100/Hb (g/dl) • Example • ΔA = 0.011 • RBCs count 5.3 x 10 12 • Hb = 15 • G-6-PDH (U/1012 RBC) = 0.011 x 60571/5.3 = 125 • G-6-PDH(U/g Hb)= 0.011 x 60571 x 100/15 = 4.442

  19. Note: • If anemia and/or leukocytosis is present: Use Buffy coat free blood sample for assay (platelets and WBCs marked activity in this enzyme) • Normal range: G-6-PDH (U/1012 RBC): (245-299) G-6-PDH (U/g Hb): (6.97-20.5)

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