Document Detail


Quantification of homocysteine-related metabolites and the role of betaine-homocysteine S-methyltransferase in HepG2 cells.
MedLine Citation:
PMID:  22653757     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Marek Kořínek; Václav Sístek; Jana Mládková; Petr Mikeš; Jiří Jiráček; Irena Selicharová
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-05-31
Journal Detail:
Title:  Biomedical chromatography : BMC     Volume:  27     ISSN:  1099-0801     ISO Abbreviation:  Biomed. Chromatogr.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2012-12-14     Completed Date:  2013-05-20     Revised Date:  2013-05-24    
Medline Journal Info:
Nlm Unique ID:  8610241     Medline TA:  Biomed Chromatogr     Country:  England    
Other Details:
Languages:  eng     Pagination:  111-21     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 John Wiley & Sons, Ltd.
Affiliation:
Apigenex s.r.o., Poděbradská 186/56, Prague 9, Czech Republic.
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MeSH Terms
Descriptor/Qualifier:
Betaine-Homocysteine S-Methyltransferase / metabolism*
Calibration
Chromatography, Liquid
Hep G2 Cells
Homocysteine / analogs & derivatives*,  analysis,  chemistry,  metabolism*
Humans
Linear Models
Reproducibility of Results
Sensitivity and Specificity
Tandem Mass Spectrometry
Chemical
Reg. No./Substance:
454-28-4/Homocysteine; EC 2.1.1.5/Betaine-Homocysteine S-Methyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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