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Limited studies have been carried out on the biotransformation of carboxyl nonsteroidal anti-inflammatory drugs (NSAIDs) in the liver. However, the role of the intestine in NSAID metabolism has not been investigated. In this report, the contribution of UDP-glucuronosyltransferases (UGTs) in the human gastrointestinal (GI) tract from five donors to the glucuronidation of the NSAIDs, RS-ketoprofen, S-naproxen, RS- and S-etodolac, was investigated. UGT activity and, for some donors, mRNA levels were evaluated. All NSAIDs were glucuronidated throughout the GI tract; however, glucuronidation was low in stomach and duodenum as compared to the remainder of the intestine. RT-PCR analysis demonstrated that the UGT1A isoforms, UGT1A3, 1A8, and 1A10, and UGT2B7 were expressed in the GI tract. Human recombinant UGT1A3, 1A9, 1A10 and 2B7 were actively involved in the glucuronidation of all NSAIDs while UGT1A7 and the intestine-specific UGT1A8 had no glucuronidating activity towards those compounds. Despite interindividual variations in both the levels of mRNA and the distribution of activity through the intestine, UGTs in the GI tract may contribute significantly to the first pass metabolism of orally administered NSAIDs.

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

Linoleic acid has recently been shown to be glucuronidated in vitro by human liver and intestinal microsomes and recombinant UGT2B7. In the present study, the dietary fatty acids (FA), phytanic acid (PA), and docosahexaenoic acid (DHA) have been used as substrates for human UDP-glucuronosyltransferases (UGTs). Both compounds were effectively glucuronidated by human liver microsomes (HLM; 1.25 +/- 0.36 and 1.12 +/- 0.32 nmol/mg x min for PA and DHA, respectively) and UGT2B7 (0.71 and 0.53 nmol/mg x min). Kinetic analysis produced relatively low K(m) values for PA with both HLM and UGT2B7 (149 and 108 microM, respectively). The K(m) for DHA glucuronidation by HLM (460 microM) was considerably higher than that for UGT2B7 (168 microM), suggesting the involvement in microsomes of other UGT isoforms in addition to UGT2B7. Glucuronidation of PA and DHA by gastrointestinal microsomes from 16 human subjects was determined. In general, both PA and DHA were glucuronidated by gastric and intestinal microsomes, and activity toward both substrates was lowest in the stomach, increased in the small intestine, and lower in the colon. However, there were large interindividual variations in UGT activity toward both substrates in all segments of the intestine, as has been seen with other substrates. Thus, PA and DHA are effective in vitro substrates for human liver, gastric and intestinal microsomes, and glucuronidation may play a role in modulating the availability of these FA as ligands for nuclear receptors.

We have demonstrated the subcellular localization of the human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A6, in endoplasmic reticulum (ER) and nuclear membrane from human hepatocytes and cell lines, by in situ immunostaining and Western blot. Double immunostaining for UGT2B7 and calnexin, an ER resident protein, showed that UGT2B7 was equally present in ER and nuclear membrane whereas calnexin was present almost exclusively in ER. Immunogold labeling of HK293 cells expressing UGT2B7 established the presence of UGT2B7 in both nuclear membranes. Enzymatic assays with UGT2B7 substrates confirmed the presence of functional UGT2B7 protein in ER, whole nuclei, and both outer and inner nuclear membranes. This study has identified, for the first time, the presence of UGT2B7 and UGT1A6 in the nucleus and of UGT2B7 in the inner and outer nuclear membranes. This localization may play an important functional role within nuclei: protection from toxic compounds and/or control of steady-state concentrations of nuclear receptor ligands. [Copyright &y& Elsevier]