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13 C‐NMR isotopomer distribution analysis: a method for measuring metabolic fluxes in condensation biosynthesis
Author(s) -
Puccetti Caterina,
Aureli Tommaso,
Manetti Cesare,
Conti Filippo
Publication year - 2002
Publication title -
nmr in biomedicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.278
H-Index - 114
eISSN - 1099-1492
pISSN - 0952-3480
DOI - 10.1002/nbm.795
Subject(s) - isotopomers , metabolic flux analysis , chemistry , nuclear magnetic resonance spectroscopy , carbon 13 nmr , molecule , analytical chemistry (journal) , stereochemistry , metabolism , organic chemistry , biochemistry
13 C NMR spectroscopy associated with the use of 13 C‐enriched substrates is a powerful tool to investigate intracellular metabolism because of the wealth of information contained in the distribution of isotopes in key metabolites. A new method of using 13 C label distribution measurements in carbon skeletons of metabolites to estimate metabolic fluxes through biochemical reaction networks is presented here. This method can be applied to metabolite synthesis occurring by condensation reactions of the type n A → B, where n is the number of precursor A molecules needed to synthesize one molecule of product B. NMR isotopomer distribution analysis (NMR‐IDA) involves the introduction of a 13 C‐enriched precursor, and measurements of the 13 C positional enrichments at just one carbon atom position of the product B via 13 C NMR spectroscopy. Information on isotopomer distribution is obtained, and data are analyzed according to a mathematical model based on multinomial probability expressions to obtain the best fit between theoretical and experimental 13 C label distribution. The use of the NMR‐IDA method allows for estimation of two key parameters representing the fractional flux of 13 C‐enriched tracer A molecules to total precursor A pool and the fraction of product B synthesized in the presence of a 13 C‐enriched source, respectively. A practical example of NMR‐IDA application to fatty acid synthesis from [(1,2 13 C 2 )acetyl]‐ L ‐carnitine in cultured primary astrocytes is also presented. Copyright © 2002 John Wiley & Sons, Ltd.