Gas chromatography-mass spectrometry based O stable isotope labeling of Krebs cycle intermediates.
Eylem, Cemil Can
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New technologies permit determining metabolomic profiles of human diseases by fingerprinting metabolites levels. However, to fully understand metabolomic phenotypes, metabolite levels and turnover rates are necessary to know. Krebs cycle is the major hub of energy metabolism and cell signaling. Traditionally, C stable isotope labeled substrates were used to track the carbon turnover rates in Krebs cycle metabolites. In this study, for the first time we introduce H[O] based stable isotope marker that permit tracking oxygen exchange rates in separate segments of Krebs cycle. The chromatographic and non-chromatographic parameters were systematically tested on the effect of labeling ratio of Krebs cycle mediators to increase selectivity and sensitivity of the method. We have developed a rapid, precise, and robust GC-MS method for determining the percentage of O incorporation to Krebs cycle metabolites. The developed method was applied to track the cancer-induced shift in the Krebs cycle dynamics of Caco-2 cells as compared to the control FHC cells revealing Warburg effects in Caco-2 cells. We demonstrate that unique information could be obtained using this newly developed O-labeling analytical technology by following the oxygen exchange rates of Krebs cycle metabolites. Thus, O-labeling of Krebs cycle metabolites expands the arsenal of techniques for monitoring the dynamics of cellular metabolism. Moreover, the developed method will allow to apply the O-labeling technique to numerous other metabolic pathways where oxygen exchange with water takes place.
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