Received for publication March 4, 2008.
Revised May 8, 2008.
Accepted for publication May 9, 2008.

UDP-Glucuronosyltransferase 1A6 Is the Major Isozyme Responsible for Protocatechuic Aldehyde Glucuronidation in Human Liver Microsomes

Abstract

Glucuronidation is an important pathway in the metabolism of protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, PAL). However, the metabolites and primary UDP-glucuronosyltransferase (UGT) isozymes responsible for PAL glucuronidation remain to be determined in human. Here, we characterized PAL glucuronidation by human liver micorsomes (HLMs), human intestine microsomes (HIMs) and 12 recombinant UGT (rUGT) isozymes to identify what kinds of metabolites are and which human UGT isozymes involved. Two metabolites (M-1 and M-2) were detected in reactions catalyzed by HLMs, HIMs, rUGT1A6 and rUGT1A9, and identified as mono-glucuronides by LC-MS. A kinetic study showed that PAL glucuronidation by rUGT1A6, rUGT1A9, HIMs, and HLMs followed Michaelis-Menten kinetics. The K_m values of HLMs, HIMs, rUGT1A6 and rUGT1A9 for PAL glucuronidation were as follows: 432.7 ± 24.5, 626.9 ± 49.2, 367.5 ± 25.1 and 379.9 ± 42.5 µM for M-1; 336.7 ± 15.3, 494.3 ± 48.7, 211.4 ± 13.4, and 238.5 ± 26.2 µM for M-2, respectively. The PAL glucuronidation activity was significantly correlated with UGT1A6 activity rather than UGT1A9 activity from 15 individual HLMs. Chemical inhibition studies showed that the IC₅₀ for phenylbutazone inhibition of PAL glucuronidation was similar in HLMs (61.9 ± 7.9 µM) compared with rUGT1A6 (45.3 ± 7.7 µM). In contrast, androsterone inhibited rUGT1A9-catalyzed and HLMs-catalyzed PAL glucuronidation with IC₅₀ values of 27.1 ± 3.8 µM and >500 µM, respectively. In combination, we identified UGT1A6 was the major isozyme responsible for PAL glucuronidation in HLMs.

Key words: liver microsomes, phase II drug metabolism, UDP glucuronyltransferases