Quantification of homocysteine-related metabolites and the role of betaine-homocysteine S-methyltransferase in HepG2 cells

0391512 - ÚOCHB 2014 RIV GB eng J - Článek v odborném periodiku
Kořínek, M. - Šístek, V. - Mládková, Jana - Mikeš, P. - Jiráček, Jiří - Selicharová, Irena
Quantification of homocysteine-related metabolites and the role of betaine-homocysteine S-methyltransferase in HepG2 cells.
Biomedical Chromatography. Roč. 27, č. 1 (2013), s. 111-121. ISSN 0269-3879. E-ISSN 1099-0801
Grant CEP: GA ČR(CZ) GAP207/10/1277
Institucionální podpora: RVO:61388963
Klíčová slova: homocysteine * BHMT * LC-MS/MS * HepG2 * metabolites
Kód oboru RIV: CE - Biochemie
Impakt faktor: 1.662, rok: 2013

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.
Trvalý link: http://hdl.handle.net/11104/0220544