title: N-glucuronidation of trans-3'-hydroxycotinine by human liver microsomes.
abstract: trans-3'-Hydroxycotinine is the major nicotine metabolite excreted in the urine of smokers and other tobacco or nicotine users. On average, about 30% of the trans-3'-hydroxycotinine in urine is present as a glucuronide conjugate. The O-glucuronide of trans-3'-hydroxycotinine has been isolated from smokers urine and appears to be the major glucuronide conjugate of trans-3'-hydroxycotinine in urine. However, nicotine and cotinine are both glucuronidated at the nitrogen atom of the pyridine ring. We report here that human liver microsomes catalyze both the N-glucuronidation and the O-glucuronidation of trans-3'-hydroxycotinine. The N-glucuronide was purified by HPLC, and its structure was confirmed by NMR. Both N- and O-glucuronidation of trans-3'-hydroxycotinine were detected in 13 of 15 human liver microsome samples. The ratio of N-glucuronidation to O-glucuronidation was between 0.4 and 2.7. One sample only catalyzed N-glucuronidation, and one sample did not catalyze either reaction. The rates of N-glucuronidation varied more than 6-fold from 6 to 38.9 pmol/min/mg protein; rates of O-glucuronidation ranged from 2.8 to 23.4 pmol/min/mg protein. The rate of trans-3'-hydroxycotinine N-glucuronidation by human liver microsomes correlated well with both the rate of nicotine and the rate of cotinine N-glucuronidation. trans-3'-Hydroxycotinine O-glucuronidation correlated with neither nicotine nor cotinine N-glucuronidation. These results suggest that the same enzyme(s) that catalyzes the N-glucuronidation of nicotine and cotinine may also catalyze the N-glucuronidation of trans-3'-hydroxycotinine in the human liver but that a separate enzyme catalyzes trans-3'-hydroxycotinine O-glucuronidation.
authors.affiliation: Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota Cancer Center, Minneapolis, Minnesota 55455, USA.
authors.forename: Gwendolyn E
authors.initials: GE
authors.lastname: Kuehl
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Murphy
mesh_terms: D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003367:Cotinine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D020719:Glucuronides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D020719:Glucuronides; D014535:Uridine Diphosphate Glucuronic Acid; C001381:hydroxycotinine; D003367:Cotinine