In vitro correction of erythrocyte glucose 6-phosphate dehydrogenase (G6PD) deficiency

Abstract

A high-yield procedure based on hypotonic lysis and isotonic resealing of erythrocytes was used in order to achieve entrapment of homogeneous human glucose 6-phosphate dehydrogenase and of other metabolically related enzyme proteins (hexokinase and 6-phosphogluconate dehydrogenase). Erythrocytes from normal subjects and from individuals affected by glucose 6-phosphate dehydrogenase deficiency (glucose 6-phosphate dehydrogenase Mediterranean) were used for such studies. The following properties of the variously loaded erythrocyte suspensions were investigated: (a) hexose monophosphate shunt activity, (b) glycolytic activity, (c) intracellular concentrations of glucose 6-phosphate, 6-phosphogluconate, NADP, and NADPH. These parameters were evaluated on the erythrocyte suspensions both in the resting state and under oxidative stimulation by methylene blue. In the glucose 6-phosphate dehydrogenase-deficient cells, complete normalization of hexose monophosphate shunt activity and of the other metabolic properties under study was achieved by restoring the intracellular glucose 6-phosphate dehydrogenase activity up to the levels of normal erythrocytes. Specifically, hexokinase became the rate-limiting step of the overall glucose oxidation pathway under methylene blue stimulation, this reproducing the situation found in normal erythrocytes. The extent of Heinz bodies formation upon incubation with acetylphenylhydrazine confirmed the full metabolic competence of the glucose 6-phosphate dehydrogenase-reconstituted Mediterranean erythrocytes compared with the unloaded ones.