Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY
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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.
Keywordsmetabolomics isotopomer analysis indirect detection
phosphate buffered saline
gas chromatography mass spectrometry
total correlation spectroscopy
- AXP, GXP, UXP, CXP
mixed adenosine, guanine, cytosine and uridine phosphates (X=M, D or T).
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