The determination of allantoin, a possible indicator of oxidant status, in human plasma

Because uric acid may be oxidized to allantoin by various reactive oxygen species, it is postulated that uric acid functions as an antioxidant in human bodily fluids. The measurement of allantoin concentrations may therefore be useful for the quantitation of oxidant generation in humans. We develop a reliable assay for the quantitation of human plasma allantoin levels. The method consists of rapid and selective separation of allantoin from uric and glyoxylic acid (which interfere in the assay) using anion-exchange solid-phase extraction prior to conversion of allantoin to glyoxylic acid and derivatization with 2,4-dinitrohenylhydrazine. Determination of glyoxylate-2,4-dinitrophenylhydrazone is achieved by using reversed-phase high-performance liquid chromatography with gradient elution and ultraviolet detection at 360 nm. The analytical performance of the method is satisfactory, with a day-to-day coefficient of variation of 8.9% and a within-batch coefficient of variation of 7.2%. The method is applied to the measurement of allantoin concentrations in the plasma of 99 apparently healthy men, and a preliminary reference range is reported. Clinical studies can now be performed to evaluate allantoin as a possible indicator of free radical damage in vivo.