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

In smokers, the primary pathway of nicotine metabolism is P450 2A6-catalyzed 5′-oxidation. The nicotine Δ ^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 [5-³H]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 Δ^5′(1′)- iminium ion was monitored. The K_m and V_max values for P450 2A6 were 144 ± 15 μM 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 μM. P450 2A13 was a somewhat better catalyst of nicotine Δ ^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.