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 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.

Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation.

Testosterone and epitestosterone are secreted mainly as glucuronide metabolites and the urinary ratio of testosterone glucuronide to epitestosterone glucuronide, often called T/E, serves as a marker for possible anabolic steroids abuse by athletes. UDP-glucuronosyltransferase (UGT) 2B17 is the most important catalyst of testosterone glucuronidation. The T/E might be affected by drugs that interact with UGT2B17, or other enzymes that contribute to testosterone glucuronidation. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used by sportsmen and we have examined the effect of two NSAIDs, diclofenac and ibuprofen, on testosterone and epitestosterone glucuronidation in human liver microsomes. In parallel, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. Both diclofenac and ibuprofen inhibited testosterone glucuronidation in microsomes, as well as UGT2B15 and UGT2B17. Interestingly, UGT2B15 was more sensitive than UGT2B17 to the two drugs, particularly to ibuprofen. Human liver microsomes lacking functional UGT2B17 exhibited significantly higher sensitivity to ibuprofen, suggesting that UGT2B15 plays a major role in the residual testosterone glucuronidation activity in UGT2B17-deficient individuals. Nonetheless, a minor contribution of other UGTs, particularly UGT1A9, to testosterone glucuronidation in such individuals cannot be ruled out at this stage. The epitestosterone glucuronidation activity of human liver microsomes was largely insensitive to ibuprofen and diclofenac. Taken together, the results highlight potential interactions between NSAIDs and androgen glucuronidation with possible implications for the validity of doping tests.

UDP-glucuronosyltransferases in conjugation of 5alpha- and 5beta-androstane steroids.

We have examined the glucuronidation of androsterone (5alpha-androstane-3alpha-ol-17-one), etiocholanolone (5beta-androstane-3alpha-ol-17-one), 5alpha-androstane-3alpha-,17beta-diol (5alpha-diol), and 5beta-androstane-3alpha-, 17beta-diol (5beta-diol) by 19 recombinant human UDP-glucuronosyltransferases (UGTs). The results reveal large differences in stereo- and regioselectivity between UGT2B7, UGT2B15, and UGT2B17. UGT2B7 conjugated all four androgens at the 3-OH but not at the 17-OH that is available in both diols. UGT2B7 exhibited a higher glucuronidation rate toward the steroids with a flat backbone, androsterone and 5alpha-diol, compared with etiocholanolone and 5beta-diol, which have a bent backbone. UGT2B17 readily glucuronidated androsterone and, particularly, etiocholanolone at the 3-OH, but in the two diols it exhibited high preference for the 17-OH and low glucuronidation rate at the 3-OH. UGT2B15 did not glucuronidate any of the studied four androgens at the 3-OH, but it did conjugate both diols at the 17-OH, with a clear preference for 5alpha-diol. Of the UGT1A subfamily, only UGT1A4 catalyzed the glucuronidation of androsterone and 5alpha-diol at measurable rates, even if low. UGT2A1 and UGT2A2 glucuronidated most compounds in this study, but mostly at rather low rates. An exception was the glucuronidation of etiocholanolone by UGT2A1 that revealed a very low substrate affinity in combination with very high V(max) value. The results shed new light on the substrate selectivity of individual UGTs in steroid glucuronidation. In addition they bear implications for doping analyses and its dependence of genetic polymorphism because testosterone is a precursor in the biosynthesis of these four androgens, whereas the contribution of UGT2B17 to their glucuronidation varies greatly.

UDP-glucuronosyltransferases (UGTs) 2B7 and UGT2B17 display converse specificity in testosterone and epitestosterone glucuronidation, whereas UGT2A1 conjugates both androgens similarly.

Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K(m) of this reaction is significantly lower than the K(m) of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

Prominent but reverse stereoselectivity in propranolol glucuronidation by human UDP-glucuronosyltransferases 1A9 and 1A10.

Propranolol is a nonselective beta-adrenergic blocker used as a racemic mixture in the treatment of hypertension, cardiac arrhythmias, and angina pectoris. For study of the stereoselective glucuronidation of this drug, the two propranolol glucuronide diastereomers were biosynthesized, purified, and characterized. A screen of 15 recombinant human UDP-glucuronosyltransferases (UGTs) indicated that only a few isoforms catalyze propranolol glucuronidation. Analysis of UGT2B4 and UGT2B7 revealed no significant stereoselectivity, but these two enzymes differed in glucuronidation kinetics. The glucuronidation kinetics of R-propranolol by UGT2B4 exhibited a sigmoid curve, whereas the glucuronidation of the same substrate by UGT2B7 was inhibited by substrate concentrations above 1 mM. Among the UGTs of subfamily 1A, UGT1A9 and UGT1A10 displayed high and, surprisingly, opposite stereoselectivity in the glucuronidation of propranolol enantiomers. UGT1A9 glucuronidated S-propranolol much faster than R-propranolol, whereas UGT1A10 exhibited the opposite enantiomer preference. Nonetheless, the Km values for the two enantiomers, both for UGT1A9 and for UGT1A10, were in the same range, suggesting similar affinities for the two enantiomers. Unlike UGT1A9, the expression of UGT1A10 is extrahepatic. Hence, the reverse stereoselectivity of these two UGTs may signify specific differences in the glucuronidation of propranolol enantiomers between intestine and liver microsomes. Subsequent experiments confirmed this hypothesis: human liver microsomes glucuronidated S-propranolol faster than R-propranolol, whereas human intestine microsomes glucuronidated S-propranolol faster. These findings suggest a contribution of intestinal UGTs to drug metabolism, at least for UGT1A10 substrates.