title: Identification of N-(hydroxymethyl) norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.
abstract: Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primary pathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and its glucuronide conjugate account for up to 50% of the nicotine metabolites excreted by smokers. Minor metabolites of cotinine excreted by smokers include norcotinine and cotinine N-oxide, each of which account for <5% of the nicotine dose. It has been reported that P450 2A6 is the catalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human metabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and its stability under the conditions of the enzyme reactions was confirmed. The products of P450 2A6-catalyzed [5-3H]cotinine metabolism were quantified by radioflow HPLC. The identification of N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on three unique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14 and 8% of the total cotinine metabolites, respectively. N-(Hydroxymethyl)norcotinine was a product of cotinine metabolism by the extrahepatic P450, 2A13, but it was a minor one. The major product of P450 2A13-catalyzed cotinine metabolism was 5'-hydroxycotinine, which was formed at twice the rate of trans-3'-hydroxycotinine. The identification of all cotinine metabolites formed by both enzymes was confirmed by LC/MS/MS analysis. Kinetic parameters for cotinine metabolism were determined for P450 2A6 and P450 2A13. This work has confirmed that the major metabolite of cotinine in smokers, trans-3'-hydroxycotinine, is only a minor metabolite of P450 2A6-catalyzed cotinine metabolism.
authors.affiliation: The Cancer Center and the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Brown
authors.forename: Linda B
authors.initials: LB
authors.lastname: von Weymarn
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Murphy
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003367:Cotinine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism
chemical_list: C401641:5'-hydroxycotinine-N-oxide; C089287:N-hydroxymethylnorcotinine; D003577:Cytochrome P-450 Enzyme System; D003367:Cotinine