Quantification of Ascorbic Acid and Glutathione

The estimation of the total ascorbate (AsA and DHA), reduced ascorbate (AsA), and dehydroascorbate (DHA) was carried out using the method of Gossett et al. [19 (link)]. In this method, the reduction of Fe³⁺ to Fe²⁺ with ascorbic acid in an acidic medium resulted in the formation of a red chelate between Fe²⁺, and 2,2′-bipyridyl was read at 525 nm. Dehydroascorbate was determined by subtracting AsA from AsA and DHA. The ascorbate content was calculated using a series of standards of L-ascorbate (Sigma Alrdich, MO, USA). GSH and glutathione disulfide (GSSG) were quantified by following the 5,5′-dithiobis (2-nitrobenzoic acid) method using glutathione reductase and 2-vinylpyridine and were measured at 415 nm according to the protocol of Griffith [20 (link)]. The GSH content was measured by subtracting the GSSG content from total glutathione content using the standard curve that was prepared with different concentrations of GSH.