Urinary ascorbic acid--HPLC determination and application as a noninvasive biomarker of hepatic response.

Abstract

A high-performance liquid chromatograph (HPLC) procedure has been developed for the determination of rat urinary ascorbic acid, a major metabolite of the hepatic glucuronic acid pathway. The presence of EDTA and HCl effectively inhibited degradation of ascorbic acid during the collection of urine specimens. The reliability of the procedure was demonstrated by its high recovery (90%), specificity (characteristic absorption maximum and discrimination from isoascorbic acid), and reproducibility (2-3% coefficient of variation). The usefulness of this assay as an indicator of hepatic response was demonstrated in preliminary experiments where increases in urinary ascorbic acid excretion were detected in male rats treated with PCB 126 (3,3',4,4',5-pentachlorobiphenyl) or PCB 105 (2,3,3',4,4'-pentachlorobiphenyl). The HPLC measurement also showed that the two PCB congeners differed markedly in their potency in stimulating urinary ascorbic acid excretion. For example, 10 micrograms/kg bw/day of PCB 126 was sufficient to cause a fourfold increase in urinary ascorbic excretion while 5000 micrograms/kg bw/day of PCB 105 was required for a sevenfold increase. In response to the administration of PCB 105 or PCB 126, urinary ascorbic acid appeared to increase to the same extent as increases in hepatic ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyltransferase (UGT) activities, and to a much higher extent than changes in liver weight and hematological and serum clinical chemical parameters. The sensitivity and specificity, the ease in obtaining timed specimens, and the noninvasive nature make this assay a useful biomarker of hepatic response in dose-finding and various acute and chronic studies.