ENZYMATIC METHOD TO DETERMINE DEHYDROASCORBIC ACID IN BIOLOGICAL SAMPLES AND IN BREAD DOUGH AT VARIOUS STAGES OF MIXING

An enzymatic method is described for measuring L-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 hour using three column chromatography steps. The enzyme catalyzes the reduction of dehydroas corbic acid by glutathione, and the ascorbic acid product is measured spectrophotometrically at 265 nm. The assay is a fast (about 2 min) an d 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 dehydro ascorbic acid/g or ml sample [513.8(Abs - 0.013) x factor for dilution of extract]. The formula was derived from a calibration graph using p ure L-dehydroascorbic acid standards. Pure L-dehydroascorbic acid was prepared from pure L-ascorbic acid, and the ascorbate oxidase used to catalyze the reaction was removed by ultrafiltration. Commercial L-deh ydroascorbic acid (Aldrich) was unsuitable for use as a standard becau se the purity was only 56-67% in comparison to laboratory-prepared L-d ehydroascorbic acid. The sensitivity of the assay was such that when d ehydroascorbic acid was added to healthy human blood plasma during ext raction with perchloric acid at the level of 7.5 nmol/ml plasma the de hydroascorbic acid could be measured with complete recovery. Low level s 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 1 0-fold. Dehydroascorbic acid increased rapidly during mixing of bread dough containing added L-ascorbic acid. (C) 1996 Academic Press Inc.