Uptake and reduction of alpha-lipoic acid by human erythrocytes.
OBJECTIVE
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 disulfide alpha-lipoic acid.


METHODS
Erythrocyte reduction of alpha-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 alpha-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 alpha-lipoic acid. Erythrocytes incubated with alpha-lipoic acid also reduced extracellular ferricyanide, although rates of dehydroascorbate reduction were several-fold greater, probably because intracellular GSH can recycle ascorbate but not alpha-lipoic acid in erythrocytes.


CONCLUSIONS
These results show that alpha-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.
Department of Medicine, Vanderbilt University School of Medicine, 715 Preston Research Building, Vanderbilt University School of Medicine, Nashville, TN 37232-6303, USA. james.may@vanderbilt.edu
James M
JM
May
Zhi-chao
ZC
Qu
Deanna J
DJ
Nelson
D001205:Ascorbic Acid / Q000494:pharmacology; D004136:Dinitrobenzenes / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D008063:Thioctic Acid / Q000493:pharmacokinetics*
D004136:Dinitrobenzenes; D009243:NAD; D008063:Thioctic Acid; D005978:Glutathione; C010936:2,4-dinitrothiocyanatobenzene; D001205:Ascorbic Acid