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-3'-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-3'-hydroxycotinine. In this work, we report that P450 2A6 metabolizes cotinine not only to trans-3'-hydroxycotinine but also to 5'-hydroxycotinine, norcotinine, and a fourth as yet unidentified metabolite. The products of baculovirus-expressed P450 2A6 [methyl-(3)H]cotinine metabolism were analyzed by radioflow HPLC. Three (3)H-labeled metabolites were detected and were present in approximately equal amounts. The identities of two of the metabolites were confirmed to be 5'-hydroxycotinine and trans-3'-hydroxycotinine by LC/MS/MS and LC/MS analysis and comparison to standards. The third product was not identified. A fourth product of P450 2A6-catalyzed 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 (3)H-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-3'-hydroxycotinine, 5'-hydroxycotinine, and N-(hydroxymethyl)norcotinine. We were unable to confirm that N-(hydroxymethyl)norcotinine was the unidentified cotinine metabolite generated by P450 2A6.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Cancer Center, Minneapolis, Minnesota 55455, USA.
S E
SE
Murphy
L M
LM
Johnson
D A
DA
Pullo
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000378:metabolism; D002853:Chromatography, Liquid; D003367:Cotinine / Q000031:analogs & derivatives / Q000378:metabolism*; D006224:Cricetinae; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*
C001381:hydroxycotinine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D003367:Cotinine