Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.
Anal Biochem. 2013 May 15;436(2):151-9. doi: 10.1016/j.ab.2013.01.027. Epub 2013 Feb 8.
The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography-mass spectrometry methods to determine (13)C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [(13)C]glucose or [(13)C]glutamine. Once steady-state labeling was reached, (13)C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.
三羧酸循环（TCA）参与需氧细胞中有机酸的完全氧化为二氧化碳。它不仅使用来自糖酵解的乙酰辅酶 A，还使用蛋白质、脂肪酸和核酸的分解产物。因此，三羧酸循环通过中心代谢产生许多碳通量，以产生还原剂，并将产生的电子转移到需氧电子传递系统，在那里通过氧化磷酸化形成能量。尽管三羧酸循环在需氧生物和组织中起着至关重要的作用，但其中间体（有机酸）缺乏直接的同位素标记信息，使得其代谢通量的定量相当近似。这是本研究旨在填补的主要技术空白。在这项工作中，我们建立并验证了液相和气相色谱-质谱法，分别使用定时多反应监测和单离子监测模式来确定 TCA 循环中涉及的有机酸的（13）C 标记。使用培养有 [(13)C]葡萄糖或 [(13)C]谷氨酰胺的玉米胚胎产生标记样品。一旦达到稳定标记状态，就提取和纯化（13）C 标记的有机酸。当将我们的质谱方法应用于这些提取物时，成功确定了七种主要有机酸的质量同位素丰度。
