Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism

Chem Res Toxicol. 1999 Jul;12(7):639-45. doi: 10.1021/tx990015e.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Baculoviridae / metabolism
  • Chromatography, Liquid
  • Cotinine / analogs & derivatives
  • Cotinine / metabolism*
  • Cricetinae
  • Cytochrome P-450 CYP2A6
  • Cytochrome P-450 Enzyme System / metabolism*
  • In Vitro Techniques
  • Mass Spectrometry
  • Mesocricetus
  • Microsomes, Liver / enzymology
  • Mixed Function Oxygenases / metabolism*


  • hydroxycotinine
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP2A6
  • Cotinine