Glucuronidation of racemic O-desmethyltramadol, the active metabolite of tramadol

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 Km or S50 values were high: 1.2 mM ± 0.23 for 1R,2R-O-desmethyltramadol with UGT1A8 and 1.84 ± 1.2 and 4.6 ± 2.0 mM 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 24.6 pmol/mg/min, respectively. In intestinal microsomes, on the other hand, the two enantiomers were glucuronidated at similar rates, about 6 pmol/mg/min. The results shed new light on both tramadol metabolism and the substrate selectivity of the human UGTs.