N-glucuronidation of nicotine and cotinine in human: Formation of cotinine glucuronide in liver microsomes and lack of catalysis by 10 examined UDP-glucuronosyltransferases

Two predominant human glucuronide metabolites of nicotine result from pyridine nitrogen atom conjugation. The present objectives included determination of the kinetics of formation of S (−)-cotinine N 1-glucuronide in pooled human liver microsomes and investigation of the UDP-glucuronosyltransferases (UGTs) involved in N- glucuronidation of nicotine isomers and S (−)-cotinine by use of recombinant enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15). Quantification was by radiochemical high-performance liquid chromatography with use of radiolabeled substrates. S (−)-Cotinine N 1-glucuronide formation in human liver microsomes was proven by comparing the chromatographic behaviors and electrospray ionization-mass spectral characteristics of the metabolite with a synthetic reference standard. This glucuronide was formed by one-enzyme kinetics with K m and V max values of 5.4 mM and 696 pmol/min/mg, respectively, and the apparent intrinsic clearance value ( V max /K m ) was 9-fold less than that previously determined for S (−)-nicotine N 1-glucuronide (0.13 versus 1.2 μl/min/mg) using the same pooled microsomes. This comparison of values is consistent with the observation that on smoking cigarettes, although the average S (−)-cotinine plasma levels usually far exceed S (−)-nicotine levels, the urinary recovery of S (−)-cotinine N 1-glucuronide only averages 3-fold greater than for S (−)-nicotine N 1-glucuronide. None of the UGTs examined catalyzed the N- glucuronidation of S (−)-nicotine, R (+)-nicotine, and S (−)-cotinine, including UGT1A3 and UGT1A4, the only isoforms known to catalyze many substrates at a tertiary amine. Also, neither S (−)-nicotine or S (−)-cotinine affected enzyme inhibition of trifluoperazine, a UGT1A4 substrate. It would appear that the same, as yet unexamined, UGT catalyzes the N- glucuronidation of both cotinine and nicotine.