Studies on the Glucaric Acid Pathway in the Metabolism of D-Glucuronic Acid in Mammals. IV. Fluorometric Method for the Determination of D-Glucaric Acid in Serum

In connection with the biochemical studies on D-glucaric acid, a normal constituent of human urine which is derived from D-glucuronolactone, we developed a colorimetric method for the quantitative determination of this acid. Thus, D-glucaric acid separated from mammalian urines using ion-exchange column chromatography was oxidized with periodic acid to give glyoxylic acid, which was further converted into the intensely colored 1, 5-diphenyl-formazan and determined colorimetrically. During the course of our studies on D-glucaric acid a much more highly sensitive method was required for the accurate estimation of normal serum level of D-glucaric acid in mammals. The present paper deals with the fluorometric determination of D-glucaric acid which is based on the condensation of glyoxylic acid derived from D-glucaric acid with 4'-hydrazino-2-stilbazole to yield a highly fluorescent product using a modification of the procedure for the determination of α-oxo acids originally reported by Mizutani, et al.(Fig. 1).