A Contradiction Between in vivo and in vitro Activities of Normal and Variant Glucose 6-Phosphate Dehydrogenase

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Abstract
The presumed “physiologic activity” of normal glucose 6-phos-phate dehydrogenase (G6PD), i.e. activity assayed in the presence of physiologic concentrations of ATP, 2,3-diphosphoglycerate, glucose 6-phosphate, NADP and NADPH in the normal red cells, is comparable to shunt pathway activity of intact normal red cells. In contrast, the presumed “physiologic activity” of G6PD variants associated with enzyme deficiency (Gd A® and Gd Mediterranean) is one order of magnitude higher than actual shunt pathway activity of the variant red cells. The difference of kinetic parameters of the enzyme in artificial buffer media and presumed physiologic medium in red cells cannot explain the discrepancy. The apparent discrepancy could be attributed either to (a) over-estimation of NADP and under-estimation of NADPH concentrations in the G6PD deficient red cells due to rapid oxidation of NADPH to NADP in preparation of hemolysate, or to (b) a large portion of NADP which accumulated in the G6PD deficient red cells binds with cellular components of intact red cells and is not available as substrate for G6PD. Whatever the mechanisms are, the balance of NADP and NADPH is a major factor for regulating shunt pathway activity, and the sensitivity of NADPH inhibition (i.e. Km for NADP versus Ki for NADPH) can be considered as an important factor in differentiating hemolytic severity of several Gd variants.