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Abstract:
In recent years, the analysis of intracellular fluxes and levels of metabolites gained importance, since the complete understanding of an organism cannot be achieved only through genome and proteome analysis. A popular strategy is flux analysis, e.g. keeping track of isotopes on their way through metabolism. However, the labeled substrates make this method inconvenient for investigations at larger scales due to their high costs. An alternative strategy is the measurement of intracellular pools of metabolites. Due to the anionic character of many intracellular molecules anion-exchange chromatography was chosen for our approach. In this work, an adherent MDCK cell line, used for the production of influenza vaccines, is examined for intracellular intermediates of the energy metabolism, especially the pathways glycolysis and citric acid cycle. Clear differences in the metabolite profiles are expected before and after viral infection. Moreover, media composition, e.g. with and without serum, will probably have an influence. Here we present first results on various sample preparation and separation methods for the parallel analysis with two anion-exchange chromatography systems. Major problems for the analysis of intracellular metabolites are quenching, washing, and extraction procedures. Different strategies and methods were investigated, but the diverse properties of the metabolites complicated the finding of an optimal method. The analysis was performed on two different anion exchange chromatography systems. One system (DX-320, Dionex, Idstein, Germany) is designed for the separation of inorganic ions, organic acids, and energy phosphates using conductivity and UV for detection. The other system (DX-600, Dionex) is suitable for the analysis of sugars and sugar phosphates using a Pulsed Amperometric Detector (PAD). In standard runs, most of the intermediate metabolites of glycolysis and TCA could be separated and quantified even in concentrations below the micromolar range. To assure the identification of the peaks, extraction samples were spiked with standards and measured again. After first extractions, it was possible to detect and quantify several metabolites. Further work will include an optimization of the quenching and extraction procedures, the validation of the quantification method and the identification of unknown peaks. Moreover, variations in metabolite composition in different physiological states will be investigated as well as between diverse adherent and suspension cell lines.
