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Simultaneous Quantification of Methionine-Related Metabolites and Co-factors in IPEC-J2 and PIEC Cells by LC–MS/MS

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Abstract

The methionine cycle is a key pathway to provide substrates for many basic biological processes including methylation and redox reactions. Here, we demonstrated a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantifying the metabolites and co-factors of the methionine metabolism. The analytes included methionine, S-adenosylmethionine, S-adenosylhomocysteine, 5′-deoxy-5′-(methylthio)adenosine, homocysteine, cystathionine, cysteine, glutathione, 5-methyltetrahydrofolate, vitamins B6, folic acid and vitamin B12. Linearities were obtained in all of the analytes with R2 larger than 0.99. Limits of quantification were in the range of 0.02–0.91 ng/106 cells, respectively. The recoveries of all of the analytes spiked at low, medium and high concentrations in cell lysates ranged from 74 to 117% and the accuracies ranged from 93.5 to 123.4%. The intra-day and inter-day precisions were lower than 20% of the relative standard deviations. This method was specifically designed for determining the intracellular concentrations of these analytes in the porcine small intestinal epithelial cell lines and the pig iliac artery endothelial cell lines. It enables the demonstration of changes in the concentrations of methionine intermediates when the cells are faced with deficient, moderate or excessive methionine. This method is expected to facilitate the understanding of the regulatory mechanism of nutrients on methionine metabolism.

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Acknowledgements

The authors greatly appreciate the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University for the LC–MS/MS usage.

Funding

This study was supported by the Fundamental Research Funds for the Central Universities of China (no. 2662018JC009 and no. 2662017PY017); National key Research and Development project of China (no. 2017YFD0502004); China Agriculture Research System (no. CARS-36); Hubei Provincial Creative Team Project of Agricultural Science and Technology (no. 2007-620).

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Authors and Affiliations

  1. Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Fangrui Zuo, Jian Peng & Hongkui Wei

  2. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, People’s Republic of China

    Jian Peng

  3. State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Qiongyao Gu & Shengqing Li

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  1. Fangrui Zuo
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  2. Qiongyao Gu
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  4. Hongkui Wei
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Correspondence to Hongkui Wei or Shengqing Li.

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Zuo, F., Gu, Q., Peng, J. et al. Simultaneous Quantification of Methionine-Related Metabolites and Co-factors in IPEC-J2 and PIEC Cells by LC–MS/MS. Chromatographia 83, 361–371 (2020). https://doi.org/10.1007/s10337-019-03852-4

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