Nicotine 5'-oxidation and methyl oxidation by P450 2A enzymes

• Published in: Drug metabolism and disposition Vol. 33; no. 8; pp. 1166 - 1173
• Main Authors: MURPHY, Sharon E, RAULINAITIS, Vytautas, BROWN, Kathryn M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.08.2005
• Subjects: Aryl Hydrocarbon Hydroxylases - metabolism; Biological and medical sciences; Chromatography, High Pressure Liquid; Cotinine - metabolism; Cytochrome P-450 CYP2A6; Humans; In Vitro Techniques; Kinetics; Medical sciences; Microsomes, Liver - metabolism; Mixed Function Oxygenases - metabolism; Nicotine - analogs & derivatives; Nicotine - metabolism; Nitrosamines; Pharmacology. Drug treatments; Smoking; Steroid Hydroxylases - metabolism; Tritium; Alkaloid; Enzyme; Cytochrome P450; Oxidation; Metabolism; Pharmacokinetics; Nicotine
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.105.004549

In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5'-oxidation. The nicotine Delta(5'(1'))-iminium ion product of this reaction is further metabolized to cotinine by aldehyde oxidase. Previous investigators have reported kinetic parameters for cotinine formation using human liver cytosol as a source of aldehyde oxidase. Using [5-(3)H]nicotine and radioflow high-performance liquid chromatography analysis, we determined kinetic parameters for nicotine 5'-oxidation by P450 2A6 and the closely related human extrahepatic P450 2A13 as well as the rodent P450s 2A3, 2A4, and 2A5. The formation of both cotinine and nicotine Delta(5'(1'))-iminium ion was monitored. The K(m) and V(max) values for P450 2A6 were 144 +/- 15 muM and 1.30 +/- 0.05 pmol/min/pmol, respectively. Previously reported K(m) values for cotinine formation by P450 2A6 in the presence of cytosol were much lower, ranging from 11 to 45 muM. P450 2A13 was a somewhat better catalyst of nicotine Delta(5'(1'))-iminium formation, with 2-fold lower K(m) and 2-fold higher V(max) values than P450 2A6. The rat P450 2A3 and the mouse P450 2A5, which are 85 and 84% identical to P450 2A6, were much more efficient catalysts of nicotine 5'-oxidation. P450 2A4 was not an efficient catalyst of nicotine metabolism. Whereas 5'-oxidation was the major pathway of nicotine metabolism for all five P450 2A enzymes, these enzymes also catalyzed methyl oxidation. Nornicotine, the product of this reaction was detected as 5 to 15% of the total nicotine metabolites. Nornicotine is the amine precursor to the esophageal carcinogen N'-nitrosonornicotine. Therefore, methyl oxidation of nicotine by P450 2A6 or P450 2A13 followed by nitrosation of nornicotine are possible endogenous pathways of N'-nitrosonornicotine formation.