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

Mengyao Jin, Ravinder Earla, Ankit Shah, Rajya L. Earla, Raeesa Gupte, Ashim K. Mitra, Anil Kumar, Santosh Kumar

Research output: Contribution to journalArticle

32 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)289-299
Number of pages11
JournalJournal of NeuroImmune Pharmacology
Volume7
Issue number1
DOIs
StatePublished - Mar 1 2012
Externally publishedYes

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Nicotine
Oxidative Stress
Cotinine
Nitrosamines
Macrophages
HIV
Ketones
Oxidants
Calibration
Tobacco

All Science Journal Classification (ASJC) codes

  • Neuroscience (miscellaneous)
  • Immunology and Allergy
  • Immunology
  • Pharmacology

Cite this

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. / Jin, Mengyao; Earla, Ravinder; Shah, Ankit; Earla, Rajya L.; Gupte, Raeesa; Mitra, Ashim K.; Kumar, Anil; Kumar, Santosh.

In: Journal of NeuroImmune Pharmacology, Vol. 7, No. 1, 01.03.2012, p. 289-299.

Research output: Contribution to journalArticle

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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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