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

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• 作者：Kathryn M. Brown ; Linda B. von Weymarn ; and Sharon E. Murphy
• 刊名：Chemical Research in Toxicology
• 出版年：2005
• 出版时间：December 2005
• 年：2005
• 卷：18
• 期：12
• 页码：1792 - 1798
• 全文大小：132K
• 年卷期：v.18,no.12(December 2005)
• ISSN：1520-5010

文摘

Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primarypathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and itsglucuronide 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 thecatalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown humanmetabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and itsstability under the conditions of the enzyme reactions was confirmed. The products of P4502A6-catalyzed [5-³H]cotinine metabolism were quantified by radioflow HPLC. The identificationof N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on threeunique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinineand 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 wasa 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. Theidentification 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 andP450 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.