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 R 2 larger than 0.99. Limits of quantification were in the range of 0.02–0.91 ng/10 6 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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通过 LC-MS/MS 同时定量 IPEC-J2 和 PIEC 细胞中蛋氨酸相关代谢物和辅因子

蛋氨酸循环是为包括甲基化和氧化还原反应在内的许多基本生物过程提供底物的关键途径。在这里，我们展示了一种快速、灵敏的液相色谱-串联质谱 (LC-MS/MS) 方法，用于量化甲硫氨酸代谢的代谢物和辅因子。分析物包括甲硫氨酸、S-腺苷甲硫氨酸、S-腺苷高半胱氨酸、5'-脱氧-5'-（甲硫基）腺苷、高半胱氨酸、胱硫醚、半胱氨酸、谷胱甘肽、5-甲基四氢叶酸、维生素 B6、叶酸和维生素 B12。在 R 2 大于 0.99 的所有分析物中都获得了线性。定量限分别在0.02–0.91 ng/10 6 细胞的范围内。所有分析物的回收率都处于低水平，细胞裂解物中的中高浓度范围为 74% 至 117%，准确度范围为 93.5% 至 123.4%。日内和日间精密度低于相对标准偏差的 20%。该方法专门用于测定猪小肠上皮细胞系和猪髂动脉内皮细胞系中这些分析物的细胞内浓度。当细胞面临缺乏、中等或过量的甲硫氨酸时，它可以证明甲硫氨酸中间体的浓度变化。该方法有望有助于理解营养物质对蛋氨酸代谢的调控机制。该方法专为测定这些分析物在猪小肠上皮细胞系和猪髂动脉内皮细胞系中的细胞内浓度而设计。当细胞面临缺乏、中等或过量的甲硫氨酸时，它能够证明甲硫氨酸中间体的浓度变化。该方法有望有助于理解营养物质对蛋氨酸代谢的调控机制。该方法专为测定这些分析物在猪小肠上皮细胞系和猪髂动脉内皮细胞系中的细胞内浓度而设计。当细胞面临缺乏、中等或过量的甲硫氨酸时，它可以证明甲硫氨酸中间体的浓度变化。该方法有望有助于理解营养物质对蛋氨酸代谢的调控机制。