Metabolomics

, Volume 3, Issue 2, pp 79–86

Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY

Article

Abstract

Isotopomer analysis is a very powerful technique for determining site enrichment with stable isotopes. Such information helps determine the relative flux through metabolic pathways. We have developed 1H NMR detection methods to isotopomer analysis of human rhabdomyosarcoma cells grown in the presence of uniformly 13C-labeled glucose. We show that TOCSY can be used both to identify the isotopomer distributions in a substantial number of key compounds and to determine the site-specific enrichment with good precision. Effects of differential relaxation have been specifically addressed. We have identified and quantified isotopomer distributions in Ala, Lactate, (glycolysis markers), nucleotide riboses (pentose phosphate markers), Asp, Glu and Gln (citric acid cycle and anaplerosis markers) as well as in nucleotide pyrimidine rings. Due to the high sensitivity of proton experiments, a reasonable throughput was achieved using a cold probe on only 3–5 mg dry cell weight. This methodology can be applied to biological system using different labeled precursors to examine their metabolic phenotypes and their response to external perturbations.

Keywords

metabolomics isotopomer analysis indirect detection 

Abbreviations

PBS

phosphate buffered saline

TCA

trichloracetic acid

GC–MS

gas chromatography mass spectrometry

TOCSY

total correlation spectroscopy

AXP, GXP, UXP, CXP

mixed adenosine, guanine, cytosine and uridine phosphates (X=M, D or T).

Supplementary material

11306_2006_47_MOESM1_ESM.doc (1.6 mb)
ESM 1 (DOC 1,642 kb)

References

  1. Anousis N., Carvalho R.A., Zhao P.Y., Malloy C.R., Sherry A.D. (2004). Compartmentation of glycolysis and glycogenolysis in the perfused rat heart. NMR Biomed. 7, 51–59CrossRefGoogle Scholar
  2. Birkemeyer C., Luedemann A., Wagner C., Erban A., Kopka J. (2005). Metabolome analysis: the potential of in vivo labeling with stable isotopes for metabolite profiling. Trends Biotechnol. 23, 28–33PubMedCrossRefGoogle Scholar
  3. Bollard M.E., Stanley E.G., Lindon J.C., Nicholson J.K., Holmes E. (2005). NMR-based metabonomic approaches for evaluating physiological influences on biofluid composition. NMR Biomed. 18, 143–162PubMedCrossRefGoogle Scholar
  4. Burgess S.C., Carvalho R.A., Merritt M.E., et al. (2001). C-13 isotopomer analysis of glutamate by J-resolved heteronuclear single quantum coherence spectroscopy. Anal. Biochem. 289, 187–195PubMedCrossRefGoogle Scholar
  5. Carvalho R.A., Jeffrey F.M.H., Sherry A.D., Malloy C.R. (1998). C-13 isotopomer analysis of glutamate by heteronuclear multiple quantum coherence total correlation spectroscopy (HMQC-TOCSY). FEBS Lett. 440, 382–386PubMedCrossRefGoogle Scholar
  6. Carvalho R.A., Zhao P., Wiegers C.B., et al. (2001). TCA cycle kinetics in the rat heart by analysis of C-13 isotopomers using indirect H-1 C-13 detection. Am. J. Physiol. Heart Circ. Physiol. 281, H1413–H1421PubMedGoogle Scholar
  7. Chatham J.C., Bouchard B., Des Rosiers C. (2003). A comparison between NMR and GCMS C-13-isotopomer analysis in cardiac metabolism. Mol. Cell. Biochem. 249, 105–112PubMedCrossRefGoogle Scholar
  8. Cline G.W., LePine R.L., Papas K.K., Kibbey R.G., Shulman G.I. (2004). C-13 NMR isotopomer analysis of anaplerotic pathways in INS-1 cells. J. Biol. Chem. 279, 44370–44375PubMedCrossRefGoogle Scholar
  9. Des Rosiers C., Lloyd S., Comte B., Chatham J.C. (2004). A critical perspective of the use of C-13-isotopomer analysis by GCMS and NMR as applied to cardiac metabolism. Metab. Eng. 6, 44–58PubMedCrossRefGoogle Scholar
  10. Fan T.W.-M., Bandura L.L., Lane A.N., Higashi R.M. (2005). Metabolomics-edited transcriptomics analysis of se anticancer action in human lung cancer cells. Metabolomics 1, 325–339CrossRefGoogle Scholar
  11. Fan T.W.M., Colmer T.D., Lane A.N., Higashi R.M. (1993). Determination of metabolites by H-1-NMR and Gc—analysis for organic osmolytes in crude tissue-extracts. Anal. Biochem. 214, 260–271PubMedCrossRefGoogle Scholar
  12. Fan T.W.M., Higashi R.M., Lane A.N., Jardetzky O. (1986). Combined use of H-1-NMR and GC–MS for metabolite monitoring and in vivo H-1-NMR assignments. Biochim. Biophys. Acta 882, 154–167PubMedGoogle Scholar
  13. Fan T.W.M., Lane A.N., Higashi R.M. (2004). The promise of metabolomics in cancer molecular therapeutics. Curr. Opin. Mol. Ther. 6, 584–592PubMedGoogle Scholar
  14. Foxall P.J.D., Parkinson J.A., Sadler I.H., Lindon J.C., Nicholson J.K. (1993). Analysis of biological-fluids using 600 MHz proton NMR-spectroscopy—application of homonuclear 2-dimensional J-resolved spectroscopy to urine and blood-plasma for spectral simplification and assignment. J. Pharm. Biomed. Anal. 11, 21–31PubMedCrossRefGoogle Scholar
  15. Griffin J.L., O’Donnell J.M., White L.T., Hajjar R.J., Lewandowski E.D. (2000). Postnatal expression and activity of the mitochondrial 2-oxoglutarate-malate carrier in intact hearts. Am. J. Physiol. Cell Physiol. 279, C1704–C1709PubMedGoogle Scholar
  16. Heijne W.H.M., Lamers R., van Bladeren P.J., et al. (2005). Profiles of metabolites and gene expression in rats with chemically induced hepatic necrosis. Toxicol. Pathol. 33, 425–433PubMedGoogle Scholar
  17. Henry P.G., Oz G., Provencher S., Gruetter R. (2003a). Toward dynamic isotopomer analysis in the rat brain in vivo: automatic quantitation of C-13 NMR spectra using LC model. NMR Biomed. 16, 400–412CrossRefGoogle Scholar
  18. Henry P.G., Tkac I., Gruetter R. (2003b). H-1-localized broadband C-13 NMR spectroscopy of the rat brain in vivo at 9.4 T. Magn. Reson. Med. 50, 684–692CrossRefGoogle Scholar
  19. Katz-Brull R., Seger D., Rivenson-Segal D., Rushkin E., Degani H. (2002). Metabolic markers of breast cancer: enhanced choline metabolism and reduced choline-ether-phospholipid synthesis. Can. Res. 62, 1966–1970Google Scholar
  20. Khairallah M., Labarthe F., Bouchard B., et al. (2004). Profiling substrate fluxes in the isolated working mouse heart using C-13-labeled substrates: focusing on the origin and fate of pyruvate and citrate carbons. Am. J. Physiol. Heart Circ. Physiol. 286, H1461–H1470PubMedCrossRefGoogle Scholar
  21. Lean C.L., Bourne R., Thompson J.F., et al. (2003). Rapid detection of metastatic melanoma in lymph nodes using proton magnetic resonance spectroscopy of fine needle aspiration biopsy specimens. Melanoma Res. 13, 259–261PubMedCrossRefGoogle Scholar
  22. Lewandowski E.D., Johnston D.L., Roberts R. (1991). Effects of inosine on glycolysis and contracture during myocardial-ischemia. Circ. Res. 68, 578–587PubMedGoogle Scholar
  23. Lindon J.C., Holmes E., Nicholson J.K. (2004). Metabonomics: systems biology in pharmaceutical research and development. Curr. Opin. Mol. Ther. 6, 265–272PubMedGoogle Scholar
  24. Lloyd S.G., Zeng H.D., Wang P.P., Chatham J.C. (2004). Lactate isotopomer analysis by H-1 NMR spectroscopy: consideration of long-range nuclear spin-spin interactions. Magn. Reson. Med. 51, 1279–1282PubMedCrossRefGoogle Scholar
  25. London R.E., Allen D.L., Gabel S.A., DeRose E.F. (1999). Carbon-13 nuclear magnetic resonance study of metabolism of propionate by Escherichia coli. J. Bacteriol. 181, 3562–3570PubMedGoogle Scholar
  26. Lu D.H., Mulder H., Zhao P.Y., et al. (2002). C-13 NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS). Proc. Natl. Acad. Sci. U.S.A. 99, 2708–2713PubMedCrossRefGoogle Scholar
  27. Marin S., Lee W.N.P., Bassilian S., et al. (2004). Dynamic profiling of the glucose metabolic network in fasted rat hepatocytes using 1,2-C-13(2) glucose. Biochem. J. 381, 287–294PubMedCrossRefGoogle Scholar
  28. Puccetti C., Aureli T., Manetti C., Conti F. (2002). C-13-NMR isotopomer distribution analysis: a method for measuring metabolic fluxes in condensation biosynthesis. NMR Biomed. 15, 404–415PubMedCrossRefGoogle Scholar
  29. Vanzijl P.C.M., Chesnick A.S., Despres D., et al. (1993). In-vivo proton spectroscopy and spectroscopic imaging of (1-C-13)-glucose and its metabolic products. Magn. Reson. Med. 30, 544–551CrossRefGoogle Scholar
  30. Vizan P., Boros L.G., Figueras A., et al. (2005). K-ras codon-specific mutations produce distinctive metabolic phenotypes in human fibroblasts. Can. Res. 65, 5512–5515CrossRefGoogle Scholar
  31. Zwingmann C., Chatauret N., Leibfritz D., Butterworth R.F. (2003). Selective increase of brain lactate synthesis in experimental acute liver failure: results of a H-1–C-13 nuclear magnetic resonance study. Hepatology 37, 420–428PubMedCrossRefGoogle Scholar
  32. Zwingmann C., Richter-Landsberg C., Leibfritz D. (2001). C-13 isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes. Glia 34, 200–212PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.JG Brown Cancer CenterUniversity of LouisvilleLouisvilleUSA
  2. 2.Department of ChemistryUniversity of LouisvilleLouisvilleUSA

Personalised recommendations