Investigation of resveratrol metabolism using liquid chromatography-mass spectrometry.

摘要

This thesis describes the drug development investigations of in vitro and in vivo metabolism of resveratrol as a dietary cancer chemopreventive agent using liquid chromatography-mass spectrometry.; In the present study, human liver-derived in vitro experimental systems have been used extensively for the evaluation of drug metabolism. Cryopreserved human and rat hepatocytes were compared to human liver microsomes in the evaluation of resveratrol metabolism. Differentiated Caco-2 cell monolayers were used in the investigatation of the intestinal metabolism of resveratrol. In vivo studies using urine, serum, plasma, and tissues provided an overall indication of drug metabolism resulting from multiple organ systems. All of the metabolism profiles including metabolite structures were obtained using HPLC with photodiode array (DAD) UV detection, connected on-line with electrospray mass spectrometry (LC-DAD-MS) or electrospray tandem mass spectrometry (LC-UV-MS-MS). Research results from the human, rat, and mouse experiments indicate that traps-resveratrol-3-O-glucuronide is the primary metabolite of resveratrol in human liver and rat urine, and that traps-resveratrol-3-O-glucuronide and traps-resveratrol-3-sulfate are both significant metabolites in human serum, rat plasma and lung tissue, mouse serum, and formed by rat hepatocytes and human Caco-2 cells. It is important to note that no Phase I metabolites of resveratrol such as oxidations, reductions or hydrolyses were detected in any of these systems.; Recombinant human enzymes including specific cytochrome P450, UDP-glucuronosyltransferases (UGT), and sulfotransferases (SULT) were used for phenotyping and inhibition studies. UGT1A1, UGT1A9 and SULT1A3, SULT1E were the major UGT and SULT isoforms responsible for human resveratrol metabolism. Using a new LC-MS-MS method for the evaluation of inhibition of multiple CYP isozyme, traps-resveratrol was identified as a marginal inhibitor of CYP3A4, a weak inhibitor of CYP2C19. traps-Resveratrol-3-sulfate did not inhibit any of the five CYP isozymes investigated.; An LC-DAD-MS and LC-UV-MS-MS method for quantitative analysis of cis- and traps-resveratrol was developed and applied to the analysis of blueberry and bilberry samples. traps-Resveratrol was detected in High Bush Michigan blueberries, northern North American wild blueberries and European bilberries, which shows that blueberry and bilberry may serve as alternative dietary sources for the antioxidant resveratrol. No cis-resveratrol was detected. (Abstract shortened by UMI.)