title: Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography-tandem mass spectrometry in cultured tumor cells and liver biopsies.
abstract: By means of liquid chromatography-tandem mass spectrometry we showed recently, that the chromosomal deletion or inactivation of the methylthioadenosine phosphorylase (MTAP) gene led to the accumulation of 5'-deoxy-5'-(methylthio)adenosine (MTA) in cancer cells. Here, we expanded the method to other key intermediates of the methionine and polyamine pathways to further elucidate the molecular consequences of a lack of MTAP activity. Employing multiple-reaction monitoring, limits of detection and lower limits of quantification in the range of 2.5-100 and 5.0-500 nM, respectively, were achieved according to the guidelines of the FDA, thus enabling the direct measurement of the metabolites in biological samples without prior enrichment and derivatization with an analytical repeatability of 1-3%. Relative standards deviations for quadruplicate 80% methanol extractions of metabolites from cultured tumor cells ranged from 1.1 to 25.5%, while the combined methodological and biological variability in metabolite concentrations in 10 liver biopsies was 11.8-51.4%. The method enabled the demonstration of changes in the concentration of intermediates of the methionine and polyamine metabolism other than MTA in hepatocellular carcinoma specimens lacking the enzyme MTAP compared to normal liver tissue.
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str 9, 93053 Regensburg, Germany. axel.stevens@klinik.uni-regensburg.de
authors.forename: Axel P
authors.initials: AP
authors.lastname: Stevens
authors.forename: Katja
authors.initials: K
authors.lastname: Dettmer
authors.forename: Georgi
authors.initials: G
authors.lastname: Kirovski
authors.forename: Keijiro
authors.initials: K
authors.lastname: Samejima
authors.forename: Claus
authors.initials: C
authors.lastname: Hellerbrand
authors.forename: Anja K
authors.initials: AK
authors.lastname: Bosserhoff
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Oefner
mesh_terms: D002138:Calibration; D006528:Carcinoma, Hepatocellular / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry* / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D008715:Methionine / Q000032:analysis* / Q000378:metabolism; D011073:Polyamines / Q000032:analysis* / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D011073:Polyamines; D008715:Methionine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D000432:Methanol