UDP-Glucuronosyltransferase 1A6 Is the Major Isozyme Responsible for Protocatechuic Aldehyde Glucuronidation in Human Liver Microsomes Hui-Xin Liu, Yong Liu, Jiang-Wei Zhang, Wei Li, Hong-Tao Liu, and Ling Yang Laboratory of Pharmaceutical Resource Discovery (H.-X.L., Y.L., J.-W.Z., W.L., H.-T.L., L.Y.), Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; and Graduate University of Chinese Academy of Sciences, Beijing, China (H.-X.L., J.-W.Z., W.L., H.-T.L.) Received January 28, 2008; accepted May 9, 2008 ABSTRACT: Glucuronidation is an important pathway in the metabolism of protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, PAL). How- ever, 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 microsomes (HLMs), human intestine microsomes (HIMs), and 12 recombinant UGT (rUGT) isozymes to identify what kinds of metabolites are present and which human UGT isozymes are involved. Two metabolites (M-1 and M-2) were detected in reactions catalyzed by HLMs, HIMs, rUGT1A6, and rUGT1A9 and were identified as monoglucuronides by liquid chromatography- mass spectrometry. A kinetic study showed that PAL glucuronida- tion 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 M for M-1 and 336.7  15.3, 494.3  48.7, 211.4  13.4, and 238.5  26.2 M for M-2, respectively. The PAL glucuronidation activity was significantly correlated with UGT1A6 activity rather than with UGT1A9 activity from 15 individual HLMs. A chemical inhibition study showed that the IC 50 for phenylbutazone inhibition of PAL glucuronidation was similar in HLMs (61.9  7.9 M) compared with rUGT1A6 (45.3  7.7 M). In contrast, androsterone inhibited rUGT1A9-catalyzed and HLM-catalyzed PAL glucuronidation with IC 50 values of 27.1  3.8 and >500 M, respectively. In combination, we identified UGT1A6 as the major isozyme responsible for PAL glucuronidation in HLMs. Glucuronidation represents a major phase II reaction of numerous xenobiotic as well as endogenous compounds (King et al., 2000). By adding the glucuronosyl group of UDP-glucuronic acid (UDPGA), which is catalyzed by a superfamily of UDP-glucuronosyltransferases (UGTs), such compounds become more hydrophilic and are therefore more readily excreted. Currently, at least 17 human UGTs have been identified and are classified into the two subfamilies, UGT1 and UGT2, based on sequence homologies (Radominska-Pandya et al., 1999; Mackenzie et al., 2005). The majority of UGTs, as with other xenobiotic-metabolizing enzymes, display broad and overlapping sub- strate specificities. But a few specific reactions have been described for certain UGT isoforms, e.g., the glucuronidation of bilirubin by UGT1A1 (Bosma et al., 1994), trifluoperazine by UGT1A4 (Di Marco et al., 2005), serotonin by UGT1A6 (Krishnaswamy et al., 2003), propofol by UGT1A9 (Ebner et al., 1993), and 3'-azido-3'-deoxythy- midine by UGT2B7 (Court et al., 2003). UGTs are not limited to the liver, and some extrahepatic tissues are known to exhibit significant activities. For example, UGT1A7, 1A8, and 1A10 are specifically expressed in the gastrointestinal tract with little or no activity in the liver (Strassburg et al., 1999). Salvia miltiorrhiza has been widely used in China for the treatment of coronary heart disease, cerebrovascular disease, bone loss, hepati- tis, hepatocirrhosis, and chronic renal failure (Sugiyama et al., 2002; Ding et al., 2005; Chang et al., 2006). PAL (Fig. 1) has been consid- ered as one of the major active constituents of S. miltiorrhiza (Ye et al., 2003). A number of pharmacological studies showed that PAL possessed biological activities such as reducing inflammation, reduc- ing atherosclerosis, improving the microcirculation, and inhibiting the aggregation of platelets (Han et al., 2005; Zhou et al., 2005). In general, the water-soluble curative components of S. miltiorrhiza including PAL are obtained by water extraction from the medicinal herb and then are orally administrated. This class of components was called water-soluble phenolic acids and was likely to go through phase II conjugation reactions (Baba et al., 2004; Konishi et al., 2005). Phase II reactions lead to the formation of a covalent linkage between a functional group either on the parent compound or on one intro- duced as a result of a phase I reaction (Iyanagi, 2007). The conjuga- tion reactions, sulfation, methylation, and glucuronidation, catalyzed This work was supported by the National Natural Science Foundation of China (30630075 and 30772608) and by the Dalian Institute of Chemical Physics Inno- vation Fund of Chinese Academy of Sciences and Ph.D. Exploring Fund (S200628). Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.020560. ABBREVIATIONS: UDPGA, uridine-5'-diphosphoglucuronic acid; UGT, UDP-glucuronosyltransferase; PAL, protocatechuic aldehyde; ST, sulfo- transferase; COMT, catechol-O-methyltransferase; HLM, human liver microsome; HIM, human intestinal microsome; rUGT, recombinant UGT; SAM, S-adenosyl-L-methionine; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; HPLC, high-performance liquid chromatography. 0090-9556/08/3608-1562–1569$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 8 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 20560/3363129 DMD 36:1562–1569, 2008 Printed in U.S.A. 1562 at Shields Library - UC Davis on August 15, 2014 dmd.aspetjournals.org Downloaded from