Erythrocyte reduction of α-lipoic acid and related disulfides was measured as reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) outside the cells.
Lipoic acid-dependent DTNB reduction by human erythrocytes required d-glucose and consumed NADPH, but not NADH. This activity was inhibited by carmustine and phenylarsine oxide, as expected if α-lipoic acid is reduced by the glutathione and thioredoxin reductase systems. Reduction of hydroxyethyl disulfide, which provides an estimate of total erythrocyte disulfide reduction capacity, was similar to that of α-lipoic acid. Erythrocytes incubated with α-lipoic acid also reduced extracellular ferricyanide, although rates of dehydroascorbate reduction were several-fold greater, probably because intracellular GSH can recycle ascorbate but not α-lipoic acid in erythrocytes.
These results show that α-lipoic acid-dependent DTNB reduction provides a simple method to selectively assess the capacity of pyridine nucleotide disulfide reductases of human erythrocytes. When coupled with other non-destructive assays, such as reduction of hydroxyethyl disulfide and ferricyanide, this assay provides a comprehensive approach to assessing erythrocyte reducing capacity in a variety of clinical conditions associated with oxidant stress.