title: Characterization of nicotine and cotinine N-glucuronidations in human liver microsomes.
abstract: The nicotine and cotinine N-glucuronidations in human liver microsomes were characterized. The Eadie-Hofstee plots of nicotine N-glucuronidation in human liver microsomes were clearly biphasic, indicating the involvement of multiple enzymes. The apparent K(m) and V(max) values were 33.1 +/- 28.1 micro M and 60.0 +/- 21.0 pmol/min/mg and 284.7 +/- 122.0 micro M and 124.0 +/- 44.0 pmol/min/mg for the high- and low-affinity components, respectively, in human liver microsomes (n = 4). However, the Eadie-Hofstee plots of cotinine N-glucuronidation in human liver microsomes were monophasic (apparent K(m) = 1.9 +/- 0.3 mM, V(max) = 655.6 +/- 312.3 pmol/min/mg). The nicotine and cotinine N-glucuronidations in the recombinant human UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells that are commercially available were determined. However, no recombinant UGT isoforms showed detectable nicotine and cotinine N-glucuronides (the concentrations of nicotine and cotinine were 0.5 and 2 mM, respectively). Nicotine and cotinine N-glucuronidations in pooled human liver microsomes were competitively inhibited by bilirubin as a substrate for UGT1A1 (K(i) = 3.9 and 3.3 micro M), imipramine as a substrate for UGT1A4 (K(i) = 6.1 and 2.7 micro M), and propofol as a substrate for UGT1A9 (K(i) = 6.0 and 12.0 micro M). The nicotine N-glucuronidation (50 micro M nicotine) in 14 human liver microsomes was significantly (r = 0.950, P < 0.0001) correlated with the cotinine N-glucuronidation (0.2 mM cotinine), indicating that the same isoform(s) is involved in both glucuronidations. Furthermore, weak correlations between imipramine N-glucuronidation and nicotine N-glucuronidation (r = 0.425) or cotinine N-glucuronidation (r = 0.517) were observed. In conclusion, the involvement of UGT1A1 and UGT1A9 as well as UGT1A4 in nicotine and cotinine N-glucuronidations in human liver microsomes was suggested, although the contributions of each UGT isoform could not be determined conclusively.
authors.affiliation: Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan. nmiki@kenroku.kanazawa-u.ac.jp
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.forename: Eriko
authors.initials: E
authors.lastname: Tanaka
authors.forename: Jun-Tack
authors.initials: JT
authors.lastname: Kwon
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D003367:Cotinine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D008862:Microsomes, Liver / Q000737:chemistry / Q000201:enzymology*; D009538:Nicotine / Q000737:chemistry / Q000378:metabolism*; D014535:Uridine Diphosphate Glucuronic Acid / Q000032:analysis / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D014535:Uridine Diphosphate Glucuronic Acid; D009538:Nicotine; D003367:Cotinine