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.
Footnotes
-
This work was supported by a Canadian Institutes of Health Research operating Grant MOP-36513 (to E.M.H.) and a Health Services Utilization Research Council of Saskatchewan Research Fellowship (to O.G.).
- Abbreviations used are::
- UGT
- UDP-glucuronosyltransferase
- UDPGA
- UDP-glucuronic acid
- HPLC
- high-performance liquid chromatography
- ESI
- electrospray ionization
- HLM
- human liver microsomes
- Received January 14, 2002.
- Accepted June 3, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|