In Vitro Characterization of Glucuronidation of Vanillin: Identification of Human UDP-Glucuronosyltransferases and Species Differences

Vanillin is a food flavoring agent widely utilized in foods, beverages, drugs, and perfumes and has been demonstrated to exhibit multiple pharmacological activities. Given the importance of glucuronidation in the metabolism of vanillin, the UDP-glucuronosyltransferase conjugation pathway of vanillin was investigated in this study. Vanillin glucuronide was identified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and a hydrolysis reaction catalyzed by -glucuronidase. The kinetic study showed that vanillin glucuronidation by HLMs and HIMs followed Michaelis-Menten kinetics and the kinetic parameters were as follows: 134.9 +/- 13.5M and 81.3 +/- 11.3M for K-m of HLMs and HIMs, 63.8 +/- 2.0nmol/min/mg pro and 13.4 +/- 2.0nmol/min/mg pro for V-max of HLMs and HIMs. All UDP-glucuronosyltransferase (UGT) isoforms except UGT1A4, 1A9, and 2B7 showed the capability to glucuronidate vanillin, and UGT1A6 exerted the higher V-max/K-m values than other UGT isoforms for the glucuronidation of vanillin when assuming expression of isoforms is similar in recombinant UGTs. Kinetic analysis using liver microsomes from six studied speices indicated that vanillin had highest affinity for the monkey liver microsomes enzyme (K-m=25.6 +/- 3.2M) and the lowest affinity for the mice liver microsomes enzyme (K-m=149.1 +/- 18.4M), and intrinsic clearance was in the following order: monkey>dog>minipig>mice>rat similar to human. These data collectively provided important information for understanding glucuronidation of vanillin. Copyright (c) 2012 John Wiley & Sons, Ltd.