Towards dynamic metabolic network measurements by multi-dimensional NMR-based fluxomics

Novel technologies for measuring biological systems and methods for visualizing data have led to a revolution in the life sciences. Nuclear magnetic resonance (NMR) techniques can provide information on metabolite structure and metabolic dynamics at the atomic level. We have been developing a new method for measuring the dynamic metabolic network of crude extracts that combines [C-13(6)]glucose stable isotope labeling of Arabidopsis thaliana and multi-dimensional heteronuclear NMR analysis, whereas most conventional metabolic flux analyses examine proteinogenic amino acids that are specifically labeled with partially labeled substrates such as [2-C-13(1)]glucose or 10% [C-13(6)]glucose. To show the validity of our method, we investigated how to obtain information about biochemical reactions, C-C bond formation, and the cleavage of the main metabolites, such as free amino acids, in crude extracts based on the analysis of the C-13-C-13 coupling pattern in 2D-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated C-13-C-13 fine couplings at the C2 position of amino acids. As another approach, f1-f3 projection of the HCACO spectrum also helps in the analysis of C-13-C-13 Connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [C-13(6)]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric (CO2)-C-12 assimilation. (C) 2007 Elsevier Ltd. All rights reserved.