Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey.
Hacettepe University, Hacettepe University, Vocational School of Health Services, Ankara, Turkey.
Anal Chim Acta. 2021 Apr 15;1154:338325. doi: 10.1016/j.aca.2021.338325. Epub 2021 Feb 16.
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.
新技术可以通过指纹代谢物水平来确定人类疾病的代谢组学特征。然而，为了充分了解代谢组学表型，有必要了解代谢物水平和周转率。三羧酸循环是能量代谢和细胞信号的主要枢纽。传统上，使用 C 稳定同位素标记的底物来跟踪三羧酸循环代谢物中的碳周转率。在这项研究中，我们首次引入了基于 H[O]的稳定同位素标记物，该标记物可跟踪三羧酸循环中不同片段的氧交换率。系统地测试了色谱和非色谱参数对标记比的影响，以提高方法的选择性和灵敏度。我们开发了一种快速、精确和强大的 GC-MS 方法，用于确定 O 掺入三羧酸循环代谢物的百分比。该方法用于跟踪与对照 FHC 细胞相比，Caco-2 细胞中三羧酸循环动力学的癌症诱导变化，揭示了 Caco-2 细胞中的瓦伯格效应。我们证明，通过跟踪三羧酸循环代谢物的氧交换率，可以使用这种新开发的 O 标记分析技术获得独特的信息。因此，三羧酸循环代谢物的 O 标记扩大了监测细胞代谢动力学的技术手段。此外，所开发的方法将允许将 O 标记技术应用于许多其他发生与水的氧交换的代谢途径。
