Glucuronidation of oxidized fatty acids and prostaglandins

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) B 1 and E 2 , and 15and 20-hydroxyeicosatetraenoic acids (15and 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 PGB 1 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 K m values of 37.9 and 45.8 M, respectively. PGB 1 was glucuronidated by UGT1A1 with a K m of 26.3 M. The K m values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB 1 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. —Little, J. M., M. Kurkela, J. Sonka, S. Jäntti, R. Ketola, S. Bratton, M. Finel, and A. Radominska-Pandya. Glucuronidation of oxidized fatty acids and prostaglandins B 1 and E 2 by human hepatic and recombinant UDP-glucuronosyltransferases. J. Lipid Res. 2004. 45: 1694–1703. Supplementary key words uridine diphosphate-glucuronosyltransferase • prostaglandins • hydroxyeicosatetraenoic acid • human liver • human intestine • liquid chromatography-mass spectrometry Glucuronide conjugation is one of the major pathways of biotransformation of steroid and thyroid hormones, bilirubin, bile acids, retinoids, xenobiotics, and several classes of drugs (1–5). The majority of endogenous compounds that are substrates for UDP-glucuronosyltransferases (UGTs) contain a carboxylic function. Bilirubin, bile acids, retinoids, and the recently identified new substrates for glucuronidation, Fatty acids (FAs) (6–10), all form acyl glucuronides. Acyl glucuronides may bind to albumin and other proteins to form adducts that can act as antigens to stimulate an immunoreaction (11). This covalent binding has been demonstrated for a number of carboxylic acid-containing drugs (11). However, carboxyllinked glucuronides of endogenous compounds have not been shown to form similar adducts. Glucuronidation is considered a detoxification pathway; however, glucuronidation of some endogenous/ exogenous compounds (e.g., morphine, lithocholic acid, and estrogens) (12–14), can result in an increase in biological activity and/or toxicity. Although glucuronidation in general may terminate the biological activity of some compounds, it, like hydroxylation and sulfation, may also be involved in the bioactivation of compounds to toxins, mutagens, and carcinogens. To date, 52 individual UGT gene products have been identified from different species. They are classified into two families, UGT1A and UGT2B (15, 16), based on the similarity of their DNA sequences (15, 17). The isoforms in family 1A are derived from a single gene locus that extends over 100 kb on human chromosome 2 (18–20). In contrast, UGT2B isoforms are encoded by separate genes clustered on human chromosome 4 and exhibit differAbbreviations: AA, arachidonic acid; GlcUA, glucuronic acid; HETE, hydroxyeicosatetraenoic acid; HI, human intestine; HL, human liver; 13-HODE, 13-hydroxyoctadecadienoic acid; LA, linoleic acid; LCMS, liquid chromatography-mass spectrometry; OFA, oxidized fatty acid; 13-OXO, 13-oxooctadecadienoic acid; PG, prostaglandin; UDPGlcUA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase. 1 To whom correspondence should be addressed. e-mail: [email protected] Manuscript received 15 March 2004 and in revised form 16 June 2004. Published, JLR Papers in Press, July 1, 2004. DOI 10.1194/jlr.M400103-JLR200 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom