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A LC-MS/MS Method for Concurrent Determination of Nicotine Metabolites and Role of CYP2A6 in Nicotine Metabolism in U937 Macrophages: Implications in Oxidative Stress in HIV + Smokers

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Abstract

Nicotine, the major constituent of tobacco, is predominantly metabolized by liver CYP2A6 into cotinine and many other compounds, including nicotine-derived nitrosamine ketone (NNK), which is known to cause oxidative stress. We have recently shown that CYP2A6 is highly expressed in U937 monocyte-derived macrophages. In this study we investigated the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages. To study nicotine metabolism, we developed a highly sensitive LC-MS/MS method for simultaneous quantitative determination of nicotine, cotinine, and NNK. The LC-MS/MS analysis was carried out by multiple reaction monitoring mass transitions with m/z of 163.2/130.1, 177.4/98.3, and 208.4/122.1 for nicotine, cotinine, and NNK, respectively. The calibration curves were linear within 3.3–1028.1 ng/ml for nicotine and 0.3–652.6 ng/ml for cotinine and NNK. This novel method was then applied to quantify nicotine metabolites, cotinine and NNK, in nicotine-treated U937 macrophages. Cotinine and NNK initially formed at 30 min, followed by a peak at 2–3 h. The role of CYP2A6 in nicotine metabolism in U937 macrophages was further confirmed by using CYP2A6-selective inhibitor, tryptamine, which significantly decreased cotinine (70%) and completely inhibited NNK formations. Finally, we showed that nicotine-treated macrophages increase the formation of oxidant at 30–60 min, which is consistent with the initial formation of cotinine and NNK. In conclusion, we have developed a new LCMS/MS method for concurrent determination of nicotine metabolites and analyzed the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages, which may have implications in viral replication among HIV + smokers.

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The authors declare that they have no competing interests or conflicts of interest.

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Correspondence to Santosh Kumar.

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Mengyao Jin and Ravinder Earla Equal contributions.

This work is supported by NIH grants DA031616-01.

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Supplement Figure 1

LC-MS/MS multiple reaction monitoring (MRM) chromatograms of A. nicotine, B. cotinine, C. NNK, and D. ritonavir in U937 macrophages. Blank, lower limit of quantification, and upper limit of quantification for nicotine, cotinine, and NNK are shown from top to bottom in the left column. Ritonavir is shown in the right column, except for blank. The intensity (cps) is presented in Y-axis and time (min) is presented in X-axis. (DOC 7735 kb)

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Jin, M., Earla, R., Shah, A. et al. A LC-MS/MS Method for Concurrent Determination of Nicotine Metabolites and Role of CYP2A6 in Nicotine Metabolism in U937 Macrophages: Implications in Oxidative Stress in HIV + Smokers. J Neuroimmune Pharmacol 7, 289–299 (2012). https://doi.org/10.1007/s11481-011-9283-6

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