Characterization of Multiple Products of Cytochrome P450 2A6-Catalyzed Cotinine Metabolism

The primary metabolite of nicotine in smokers is cotinine. Cotinine is further metabolized to trans-3ʹ-hydroxycotinine, the major urinary metabolite of nicotine in tobacco users. It was recently reported that cytochrome P450 2A6 catalyzes the conversion of cotinine to trans-3ʹhydroxycotinine. In this work, we report that P450 2A6 metabolizes cotinine not only to trans3ʹ-hydroxycotinine but also to 5ʹ-hydroxycotinine, norcotinine, and a fourth as yet unidentified metabolite. The products ofbaculovirus-expressed P450 2A6 [methyl-3H]cotinine metabolism were analyzed by radioflow HPLC. Three 3H-labeled metabolites were detected and were present in approximately equal amounts. The identities of two of the metabolites were confirmed to be 5ʹ-hydroxycotinine and trarts-3ʹ-hydroxycotinine by LC/MS/MS and LC/MS analysis and comparison to standards. The third product was not identified. A fourth product of P450 2A6Catalyzed cotinine metabolism was detected by LC/MS. It was identified by cochromatography with a standard and MS and MS/MS data to be norcotinine. An attempt was made to further characterize the unidentified 3H-labeled metabolite by comparison to the cotinine metabolites generated by hamster liver microsomes. Hamster liver microsomes contain a P450, 2A8, which is closely related to P450 2A6, and have previously been shown to metabolize cotinine to three hydroxylated products, trans-3ʹ-hydroxycotimne, 5ʹ-hydroxycotinine, and N-(hydroxymethyl)norcotinine. We were unable to confirm that N-(hydroxymethyl)norcotinine was the unidentified cotinine metabolite generated by P450 2A6.