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Analysis of the Citric Acid Cycle Intermediates Using Gas Chromatography-Mass Spectrometry

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Metabolic Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 708))

Abstract

Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.

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References

  1. Krebs, H. A. (1940) The citric acid cycle and the szent-gyorgyi cycle in pigeon breast muscle. Biochem J 34, 775–779.

    PubMed  CAS  Google Scholar 

  2. Des, R. C., Fernandez, C. A., David, F., Brunengraber, H. (1994) Reversibility of the mitochondrial isocitrate dehydrogenase reaction in the perfused rat liver. Evidence from isotopomer analysis of citric acid cycle intermediates. J Biol Chem 269, 27179–27182.

    Google Scholar 

  3. Yang, L., Kasumov, T., Kombu, R. S., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., Brunengraber, H. (2008) Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J Biol Chem 283, 21988–21996.

    Article  PubMed  CAS  Google Scholar 

  4. Yang, L., Kombu, R. S., Kasumov, T., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., Brunengraber, H. (2008) 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 283, 21978–21987.

    Article  PubMed  CAS  Google Scholar 

  5. Reszko, A. E., Kasumov, T., Pierce, B. A., David, F., Hoppel, C. L., Stanley, W. C., Des, R. C., Brunengraber, H. (2003) Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver. J Biol Chem 278, 34959–34965.

    Article  PubMed  CAS  Google Scholar 

  6. Brunengraber, H., Roe, C. R. (2006) Anaplerotic molecules: current and future. J Inherit Metab Dis 29, 327–331.

    Article  PubMed  Google Scholar 

  7. Kasumov, T., Sharma, N., Huang, H., Kombu, R. S., Cendrowski, A., Stanley, W. C., Brunengraber, H.. (2009) Dipropionylcysteine ethyl ester compensates for loss of citric acid cycle intermediates during post ischemia reperfusion in the pig heart. Cardiovasc Drugs Ther 23, 459–469.

    Article  PubMed  CAS  Google Scholar 

  8. Beylot, M., Soloviev, M. V., David, F., Landau, B. R., Brunengraber, H. (1995) Tracing hepatic gluconeogenesis relative to citric acid cycle activity in vitro and in vivo. Comparisons in the use of [3-13C]lactate, [2-13C]acetate, and alpha-keto[3-13c]isocaproate. J Biol Chem 270, 1509–1514.

    Article  PubMed  CAS  Google Scholar 

  9. Schwenk, W. F., Berg, P. J., Beaufrere, B., Miles, J. M., Haymond, M. W. (1984) Use of t-butyldimethylsilylation in the gas chromatographic/mass spectrometric analysis of physiologic compounds found in plasma using electron-impact ionization. Anal Biochem 141, 101–109.

    Article  PubMed  CAS  Google Scholar 

  10. Weckwerth, W., Fiehn, O. (2002) Can we discover novel pathways using metabolomic analysis?. Curr Opin Biotechnol 13, 156–160.

    Article  PubMed  CAS  Google Scholar 

  11. Katz, J., Wals, P., Lee, W. N. (1993) Isotopomer studies of gluconeogenesis and the krebs cycle with 13c-labeled lactate. J Biol Chem 268, 25509–25521.

    PubMed  CAS  Google Scholar 

  12. Yang, L., Kasumov, T., Yu, L., Jobbins, K., David, F., Previs, S., Kelleher, J., Brunengraber, B. (2006) Metabolomic assays of the concentration and mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates. Metabolomics 2, 85–94.

    Article  CAS  Google Scholar 

  13. Chang, H. C., Maruyama, H., Miller, R. S., Lane, M. D. (1966) The enzymatic carboxylation of phosphoenolpyruvate. 3. Investigation of the kinetics and mechanism of the mitochondrial phosphoenolpyruvate carboxykinase-catalyzed reaction. J Biol Chem 241, 2421–2430.

    PubMed  CAS  Google Scholar 

  14. Deng, S., Zhang, G. F., Kasumov, T., Roe, C. R., Brunengraber, H. (2009) Interrelations between C4 ketogenesis, C5 ketogenesis, and anaplerosis in the perfused rat liver. J Biol Chem 284, 27799–27807.

    Article  PubMed  CAS  Google Scholar 

  15. 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., Brunengraber, H. (2009) Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAS. J Biol Chem 284, 33521–33534.

    Article  PubMed  CAS  Google Scholar 

  16. Watson, D. (1993) Chemical Derivatization in Gas Chromatography. In: Baugh, P., Ed.. Gas Chromatography: A Practical Approach, Oxford University Press, New York, NY, 133–153.

    Google Scholar 

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Acknowledgments

This work was supported, in whole or in part, by National Institutes of Health Roadmap Grant R33DK070291 and Grant R01ES013925. This work was also supported by a grant from the Cleveland Mt. Sinai Health Care Foundation. We acknowledge the Mouse Metabolic Phenotyping Center (MMPC) at Case Western Reserve University where many of these procedures were developed.

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Correspondence to Michelle A. Puchowicz .

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Kombu, R.S., Brunengraber, H., Puchowicz, M.A. (2011). Analysis of the Citric Acid Cycle Intermediates Using Gas Chromatography-Mass Spectrometry. In: Metz, T. (eds) Metabolic Profiling. Methods in Molecular Biology, vol 708. Humana Press. https://doi.org/10.1007/978-1-61737-985-7_8

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  • DOI: https://doi.org/10.1007/978-1-61737-985-7_8

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-984-0

  • Online ISBN: 978-1-61737-985-7

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