Assessing the Reductive Capacity of Cells by Measuring the Recycling of Ascorbic and Lipoic Acids

Most mammalian cells cannot synthesize vitamin C, or ascorbic acid, and thus must have efficient mechanisms for its intracellular recycling. Ascorbate can be recycled from both its oxidized forms using electrons from several intracellular reducing co-factors, including GSH and the reduced pyridine nucleotides. Methods have been developed to assess the ability of intact cells to recycle ascorbate, which include assay of extracellular ferricyanide reduction and measurement of the ability of the cells to reduce dehydroascorbic acid to ascorbate. Lipoic acid, a disulfide containing medium chain fatty acid, is also taken up by cells and reduced to dihydrolipoic acid, which can be measured upon efflux from the cells using Ellman’s reagent. Together, these assays provide an estimate of the ability of different cell types to recycle ascorbate and to generate intracellular reducing equivalents required to maintain the redox status of the cells.