Short Communication GLUCURONIDATION OF THE ASPIRIN METABOLITE SALICYLIC ACID BY EXPRESSED UDP-GLUCURONOSYLTRANSFERASES AND HUMAN LIVER MICROSOMES

Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatographytandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies. Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used in the treatment of pain and inflammation and in the secondary prevention of cardiovascular disease. Several studies have suggested that aspirin may be an effective chemopreventive agent against cancer, especially colorectal cancer (for review, see Baron and Sandler, 2000). The rate of elimination of aspirin and its metabolite salicylic acid is likely a major factor determining drug efficacy among users. In the body, aspirin is rapidly deacetylated to form salicylic acid (Hutt et al., 1986). Salicylic acid is further metabolized by conjugation, with either glycine or glucuronic acid, or by oxidation. The glycine conjugate, salicyluric acid, is the most abundant metabolite in the urine of individuals dosed with aspirin. However, the glucuronide conjugates of salicylic acid, salicylic acid phenolic glucuronide (SAPG), and salicylic acid acyl glucuronide (SAAG), account for between 0.8% and 42% of the administered aspirin dose (Hutt et al., 1986). The wide range in urinary levels of SAPG, SAAG, and salicyluric acid among individuals dosed with aspirin may, in part, explain differences in therapeutic response within a population. Glucuronidation activity may be a critical determinant in aspirin efficacy; however, the enzymes catalyzing this conjugation have yet to be identified and characterized. Polymorphisms in many of the UGT enzymes have been identified (Ciotti et al., 1997; Lévesque et al., 1997; Lampe et al., 1999, 2000; Lévesque et al., 1999; Balram et al., 2002; Gagné et al., 2002; Huang et al., 2002; Villeneuve et al., 2003; Ehmer et al., 2004; Iwai et al., 2004; Verlaan et al., 2004; Yamanaka et al., 2004). Therefore, variability in glucuronidation activity among aspirin users is probably a result of specific expression levels and/or functional polymorphisms present in the UGTs catalyzing the conjugation of salicylic acid. Among aspirin users, carrying a UGT1A6 variant allele has been associated with decreased risk of colorectal adenoma (Bigler et al., 2001; Chan et al., 2005). Consistent with these findings, Ciotti et al. (1997) demonstrated that UGT1A6 was able to catalyze the glucuronidation of salicylic acid, with expressed UGT1A6*2 demonstrating a 2-fold lower salicylic acid glucuronidation compared with UGT1A6*1. We aimed to identify the UGT enzymes catalyzing the glucuronidation of salicylic acid. We measured the ability of individually expressed UGTs to catalyze the glucuronidation of salicylic acid using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and measured the apparent kinetic parameters of salicylic acid glucuronidation catalyzed by selected UGTs. This is the first study to systematically screen all UGTs for salicylic acid glucuronidation activity and to identify the role of individual UGTs in the catalysis of glucuronidation in human liver microsomes. This research was supported by Grant R01 CA94954 from the National Institutes of Health (to J.D.P.). G.E.K. was supported partially by National Cancer Insitute Training Grant T32 CA77116. Presented in part at the American Association for Cancer Research Annual Meeting, Orlando, FL, 27–31 Mar, 2004