Identification of N-(Hydroxymethyl) Norcotinine as a Major Product of Cytochrome P450 2A6,but Not Cytochrome P450 2A13-Catalyzed Cotinine Metabolism

摘要

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.