Uptake and reduction of α-lipoic acid by human erythrocytes

Objectives: The reducing capacity of erythrocytes has been used clinically as to estimate resistance to oxidant stress. In this work we targeted the antioxidant capacity of pyridine nucleotide disulfide reductases of these cells by measuring their ability to reduce the disufide α-lipoic acid. Methods: Erythrocyte reduction of α-lipoic acid and related disulfides was measured as reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) outside the cells. Results: 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. Conclusion: 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.