N-Glucuronidation of Nicotine and Cotinine in Human: Formation of Cotinine Glucuronide in Liver Microsomes and Lack of Catalysis by 10 Examined UDP-Glucuronosyltransferases

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Abstract
Two predominant human glucuronide metabolites of nicotine result from pyridine nitrogen atom conjugation. The present objectives included determination of the kinetics of formation ofS(−)-cotinine N1-glucuronide in pooled human liver microsomes and investigation of the UDP-glucuronosyltransferases (UGTs) involved inN-glucuronidation of nicotine isomers andS(−)-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 N1-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 Kmand Vmax values of 5.4 mM and 696 pmol/min/mg, respectively, and the apparent intrinsic clearance value (Vmax/Km) was 9-fold less than that previously determined forS(−)-nicotine N1-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(−)-cotinineN1-glucuronide only averages 3-fold greater than forS(−)-nicotine N1-glucuronide. None of the UGTs examined catalyzed the N-glucuronidation ofS(−)-nicotine, R(+)-nicotine, andS(−)-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 theN-glucuronidation of both cotinine and nicotine.