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

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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.

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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).

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Acknowledgments

We thank Laura Bandura for assistance with cell growth. This work was supported by the Kentucky Challenge for Excellence (to ANL). NMR spectra were recorded at the JG Brown Cancer Center NMR Suite supported by the Brown Foundation and NSF EPSCoR grant EPS-0132295 for NMR instrumentation.

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Authors and Affiliations

  1. JG Brown Cancer Center, University of Louisville, 529 South Jackson St., Louisville, KY, 40202, USA

    Andrew N. Lane

  2. Department of Chemistry, University of Louisville, 2320 S. Brook St., Louisville, KY, 40208, USA

    Teresa W. -M. Fan

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  1. Andrew N. Lane
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  2. Teresa W. -M. Fan
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Correspondence to Andrew N. Lane.

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Lane, A.N., Fan, T.W.M. Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY. Metabolomics 3, 79–86 (2007). https://doi.org/10.1007/s11306-006-0047-x

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  • DOI: https://doi.org/10.1007/s11306-006-0047-x

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