SPECTROPHOTOMETRIC DETERMINATION OF DEHYDROASCORBIC ACID IN BIOLOGICAL SAMPLES

We describe a method for accurately and precisely measuring dehydroasc orbic acid in perchloric acid extracts prepared from human plasma, lym phocytes, and mammalian cells. Samples were assayed by spectrophotomet rically monitoring the kinetics of the concentration-dependent absorba nce changes of dehydroascorbic acid with phosphate-methanol-containing buffers. The lowest detectable dehydroascorbate concentration using t his assay is estimated to be below 0.1 mu mol/liter, Total analysis ti me is less than 10 min and allows the simultaneous measurement of nume rous samples. The calibration curve is linear (r > 0.995) over the ran ge 0-200 mu mol/liter. The dehydroascorbic acid concentrations measure d in supplemented samples agree with known concentrations. Interferenc e of ascorbic acid and 2,3-diketogulonic acid with this assay was excl uded. The correlation with a highly specific chromatographic procedure gave comparable results over the range of physiologically relevant co ncentrations. The procedure avoids the most commonly applied method of measuring the native ascorbic acid, then reducing the dehydroascorbic acid, and finally measuring the total ascorbic acid and determining d ehydroascorbic acid by the difference. Stabilization of ascorbic acid during assay was achieved by addition of desferrioxamine. (C) 1994 Aca demic Press, Inc.