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

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.