Kořínek Marek, Sístek Václav, Mládková Jana, Mikeš Petr, Jiráček Jiří, Selicharová Irena
Apigenex s.r.o., Poděbradská 186/56, Prague 9, Czech Republic.
Biomed Chromatogr. 2013 Jan;27(1):111-21. doi: 10.1002/bmc.2755. Epub 2012 May 31.
We optimized and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of six metabolites of homocysteine metabolism: homocysteine, methionine, cysteine, S-adenosylmethionine, S-adenosylhomocysteine and betaine. The detection limits for these metabolites were in the nanomolar range, and the intra- and inter-day precisions were lower than 20% of the relative standard deviations. The method was specifically designed for the determination of the intracellular concentrations of the metabolites in cultured cells. To study the role of betaine-homocysteine S-methyltransferase (BHMT), HepG2 cells and HepG2 cells that were stably transfected with BHMT ((BHMT) HepG2) were treated with homocysteine or with a specific inhibitor of BHMT, and metabolite levels were subsequently measured. Severely compromised methyl group metabolism in the HepG2 cells, which is typical of cancer-derived cells, prevented clear evaluation of the changes caused by the external manipulations of homocysteine metabolism. However, the ease of handling these cells and the almost unlimited source of experimental material supplied by cells in permanent culture allowed us to develop a reliable methodology. The precautions concerning intracellular metabolite determinations using LC-MS/MS in cultured cells that are expressed in this work will have global validity for future metabolomics studies.
我们优化并验证了一种快速灵敏的液相色谱-串联质谱法（LC-MS/MS），用于定量同型半胱氨酸代谢的六种代谢物：同型半胱氨酸、蛋氨酸、半胱氨酸、S-腺苷甲硫氨酸、S-腺苷同型半胱氨酸和甜菜碱。这些代谢物的检测限在纳摩尔范围内，日内和日间精密度低于相对标准偏差的20%。该方法专为测定培养细胞中代谢物的细胞内浓度而设计。为了研究甜菜碱-同型半胱氨酸S-甲基转移酶（BHMT）的作用，用同型半胱氨酸或BHMT的特异性抑制剂处理HepG2细胞和稳定转染BHMT的HepG2细胞（（BHMT）HepG2），随后测量代谢物水平。HepG2细胞中严重受损的甲基代谢是癌症来源细胞的典型特征，这使得难以清晰评估同型半胱氨酸代谢的外部操作所引起的变化。然而，处理这些细胞的便利性以及永久培养的细胞提供的几乎无限的实验材料来源，使我们能够开发出一种可靠的方法。本文中阐述的在培养细胞中使用LC-MS/MS测定细胞内代谢物的注意事项对未来的代谢组学研究具有普遍的有效性。
