Metabolomics

, Volume 3, Issue 2, pp 79–86

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

Authors

    • JG Brown Cancer CenterUniversity of Louisville
  • Teresa W. -M. Fan
    • Department of ChemistryUniversity of Louisville
Article

DOI: 10.1007/s11306-006-0047-x

Cite this article as:
Lane, A.N. & Fan, T.W.-. Metabolomics (2007) 3: 79. doi:10.1007/s11306-006-0047-x
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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

metabolomicsisotopomer analysisindirect 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)

Copyright information

© Springer Science+Business Media, LLC 2007