title: Glucuronidation of racemic O-desmethyltramadol, the active metabolite of tramadol.
abstract: O-Desmethyltramadol, the active metabolite of analgesic tramadol, is metabolised through glucuronidation. The present study was conducted to identify the human UDP-glucuronosyltransferases (UGTs) that catalyse the glucuronidation of O-desmethyltramadol, a racemic mixture of 1R,2R- and 1S,2S-enantiomers. We developed a fast and selective liquid chromatography-mass spectrometry method to separate, analyse and quantify the diastereomeric phenolic O-glucuronides of O-desmethyltramadol. To quantify O-desmethyltramadol glucuronidation, we biosynthesised both phenolic O-glucuronides of O-desmethyltramadol and verified their structure by mass spectrometry and nuclear magnetic resonance spectroscopy. Subsequently, the 16 human UGTs of subfamilies 1A and 2B were screened for O-desmethyltramadol glucuronidation activity. UGTs 1A7-1A10 exhibited a strict stereoselectivity, exclusively glucuroniding the 1R,2R-enantiomer. Similar though not strict enantioselectivity was exhibited by UGT2B15. UGT2B7, on the other hand, glucuronidated both O-desmethyltramadol enantiomers, with slight preference for 1S,2S-O-desmethyltramadol. Enzyme kinetic parameters were determined for the most active UGTs, 1A8 and 2B7. The apparent K(m) or S(50) values were high: 1.2mM±0.23 for 1R,2R-O-desmethyltramadol with UGT1A8 and 1.84±1.2 and 4.6±2.0mM for 1S,2S- and 1R,2R-O-desmethyltramadol enantiomers with UGT2B7, respectively. Glucuronidation analyses of O-desmethyltramadol with human liver microsomes exhibited stereoselectivity, favouring the 1S,2S-O-desmethyltramadol over 1R,2R-O-desmethyltramadol and yielding 62.4 and 24.6pmol/mg/min, respectively. In intestinal microsomes, on the other hand, the two enantiomers were glucuronidated at similar rates, about 6pmol/mg/min. The results shed new light on both tramadol metabolism and the substrate selectivity of the human UGTs.
authors.affiliation: Faculty of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, FI-00014, Finland.
authors.forename: Päivi
authors.initials: P
authors.lastname: Lehtonen
authors.forename: Taina
authors.initials: T
authors.lastname: Sten
authors.forename: Olli
authors.initials: O
authors.lastname: Aitio
authors.forename: Mika
authors.initials: M
authors.lastname: Kurkela
authors.forename: Katariina
authors.initials: K
authors.lastname: Vuorensola
authors.forename: Moshe
authors.initials: M
authors.lastname: Finel
authors.forename: Risto
authors.initials: R
authors.lastname: Kostiainen
mesh_terms: D000700:Analgesics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000145:classification / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D020033:Protein Isoforms; D021241:Spectrometry, Mass, Electrospray Ionization; D014147:Tramadol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000700:Analgesics; D020719:Glucuronides; D020033:Protein Isoforms; C080580:O-demethyltramadol; D014147:Tramadol; D014453:Glucuronosyltransferase