Enzymatic Method to Determine Dehydroascorbic Acid in Biological Samples and in Bread Dough at Various Stages of Mixing

Abstract

An enzymatic method is described for measuringL-dehydroascorbic acid in perchloric acid extracts of biological samples. The enzyme used in the assay was glutathione dehydrogenase (glutathione:dehydroascorbate oxidoreductase), which was purified from wheat flour using three column chromatography steps. The enzyme catalyzes the reduction of dehydroascorbic acid by glutathione, and the ascorbic acid product is measured spectrophotometrically at 265 nm. The assay is a fast (about 2 min) and simple two-step procedure. First, a mixture of extract and buffer is set to zero absorbance against air in a spectrophotometer. Second, a solution of glutathione dehydrogenase and glutathione is added and the absorbance after 2 min is used in a formula to calculate nmol dehydroascorbic acid/g or ml sample [513.8(Abs − 0.013) × factor for dilution of extract]. The formula was derived from a calibration graph using pureL-dehydroascorbic acid standards. PureL-dehydroascorbic acid was prepared from pureL-ascorbic acid, and the ascorbate oxidase used to catalyze the reaction was removed by ultrafiltration. CommercialL-dehydroascorbic acid (Aldrich) was unsuitable for use as a standard because the purity was only 56–67% in comparison to laboratory-preparedL-dehydroascorbic acid. The sensitivity of the assay was such that when dehydroascorbic acid was added to healthy human blood plasma during extraction with perchloric acid at the level of 7.5 nmol/ml plasma the dehydroascorbic acid could be measured with complete recovery. Low levels of dehydroascorbic acid were detected in fresh fruits and vegetables. When samples were ground for several minutes before extraction with perchloric acid, the dehydroascorbic acid levels increased more than 10-fold. Dehydroascorbic acid increased rapidly during mixing of bread dough containing addedL-ascorbic acid.