Reduced nicotinamide adenine dinucleotide and the reduction of oxidized glutathione in human erythrocytes

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Abstract
The ability of reduced nicotinamide adenine dinucleotide (NADH), generated through the activity of lactic acid dehydrogenase, to support the reduction of endogenous oxidized glutathione in intact human erythrocytes and in hemolysates was investigated. Rapid initial oxidation of endogenous reduced glutathione was effected with methyl phenylazoformate. Freshly obtained normal erythrocytes and erythrocytes deficient in glucose-6-phosphate dehydrogenase activity were unable to regenerate reduced glutathione upon incubation with lactate. Only normal erythrocytes were capable of reducing oxidized glutathione after preincubation with glucose, inosine, or a medium which promoted the synthesis of increased amounts of intracellular NAD. This regeneration of reduced glutathione could be explained by the generation of reduced nicotinamide adenine dinucleotide phosphate through the metabolism of accumulated phosphorylated intermediates of glycolysis. Hemolysates prepared from both normal erythrocytes and from erythrocytes deficient in glucose-6-phosphate dehydrogenase activity were able to reduce oxidized glutathione in the presence of added lactate and NAD. The results obtained indicated either an inability of the intact erythrocyte to utilize the NAD at the concentrations attained or an altered behavior of the system for the regeneration of reduced glutathione after lysis of the cell.