Case - Citric Acid Cycle and Related Intermediates v1
Authors: Brunengraber Henri 
Published: May 2019

Summary: Analysis of the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) using gas chromatography mass spectrometry (GC-MS) is an analytical approach to identifying unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labelled molecules (eg. 2H- or 13C-labelled) and the naturally occurring most abundant molecules. The use of metabolic tracers (eg. 2H- or 13C-labelled) can offer additional information that can lead to the understanding of interrelationships between the pools of intermediary metabolism, such as with the CAC, and specific metabolic pathways. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions. The following SOP presents a relatively simple method that is sensitive for simultaneously measuring concentrations and heavy labelled molecules of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using GC-MS technology. Note: the following extraction procedure can also be used parallel analysis for acyl-CoA’s and lipids (for details on isolating and mass spectrometry assay procedures see, refs 4-6). References: 1. Yang, L., Kasumov, T., Kombu, R. S., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J. Biol. Chem. (2008) 283, 21988-21996 2. Yang, L., Kombu, R. S., Kasumov, T., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids. J. Biol. Chem. (2008) 283, 21978-21987 3. Kombu RS, Brunengraber H, and Puchowicz MA. Analysis of the Citric Acid Cycle Intermediates using Gas Chromatography-Mass Spectrometry In: Methods Molec Biol, “Metabolic Profiling: Methods and Protocols” (ed. Metz,TO), Humana Press, (2011) 708:14757 4. Zhang Y, Zhang S, Marin-Valencia I, Puchowicz MA. Decreased Carbon Shunting From Glucose Towards Oxidative Metabolism In Diet-Induced Ketotic Rat Brain. J Neurochem. (2015) 132(3): 301-12 5. Harris SR, Zhang GF, Sadhukhan S, Wang H, Shi C, Puchowicz MA, Anderson VE, Salomon RG, Tochtrop GP, Brunengraber H. Metabolomics and Mass Isotopomer Analysis as a Strategy for Pathway Discovery: Pyrrolyl and Cyclopentenyl Derivatives of the Pro-Drug of Abuse, Levulinate. Chem Res Toxicol. (2012) 26(2): 213-20 6. Zhang, G. F., Kombu, R. S., Kasumov, T., Han, Y., Sadhukhan, S., Zhang, J., Sayre, L. M., Ray, D., Gibson, K. M., Anderson, V. A., Tochtrop, G. P., and Brunengraber, H. Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAs. J. Biol. Chem. (2009) 284, 33521-33534