期刊
BIOMEDICAL CHROMATOGRAPHY
卷 27, 期 1, 页码 111-121出版社
WILEY-BLACKWELL
DOI: 10.1002/bmc.2755
关键词
homocysteine; BHMT; LC-MS; MS; HepG2; metabolites
类别
资金
- Grant Agency of the Czech Republic [P207/10/1277]
- Academy of Sciences of the Czech Republic [RVO:61388963]
We optimized and validated a rapid and sensitive liquid chromatographytandem 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 betainehomocysteine S-methyltransferase (BHMT), HepG2 cells and HepG2 cells that were stably transfected with BHMT (BHMTHepG2) 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. Copyright (c) 2012 John Wiley & Sons, Ltd.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据