Identification of 5-Hydroxycotinine in the Plasma of Nicotine-Treated Mice: Implications for Cotinine Metabolism and in Vivo

Two oxidation products of cotinine, 5-hydroxycotinine (5-HC) and co-tinine N-oxide (CNO), were identified for the first time in vivo in the plasma of C57BL/6 mice after injection of nicotine (1 mg/kg) or exposure to an e-cigarette (e-cig) containing 2.4% nicotine. Liquid chromatography-mass spectrometry was used to separate 3-hydrox-ycotinine (3-HC), 5-HC, and CNO and to quantify each by the sensitive direct detection of their parent ion with m/z of 193.097. In nicotine -in-jected mice, 5-HC was as abundant as 3-HC 15 minutes postinjection, and CNO was readily detectable. In e-cig-exposed mice with plasma nicotine levels resembling that of human smokers, plasma 5-HC and CNO, as well as 3-HC, were readily quantifiable at the end of the 4 -hour exposure time. In nicotine-injected mice, the combined concen-tration of 3-HC plus 5-HC plus CNO, all formed from cotinine by CYP2A5, was higher (P < 0.01) in females than in males, although the male-female difference in cotinine plasma level did not reach statisti-cal significance. The result highlights the importance of considering these three oxidation products of cotinine in examining cotinine me-tabolism and disposition. Coumarin 7-hydroxylase activity, a specific marker of CYP2A5, measured in the hepatic microsomes of untreated mice showed that females have higher activity (P < 0.001) than males (N 5 8 per sex). The abundance of plasma 5-hydroxycotinine in nico-tine-treated mice raises intriguing questions about the site of its origin (hepatic or possibly kidney CYP2A5) and the routes of its disposition because urinary excretion of 5-HC has not been detected by liquid chromatography with tandem mass spectrometry in mice and is con-troversial in human smokers.

Automated summary

This study identified two oxidation products of cotinine, 5-hydroxycotinine and cotinine N-oxide, for the first time in the plasma of mice. The results highlight the importance of considering these oxidation products in studying cotinine metabolism and disposition.