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Background  Metabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems.    Methodology/Principal Findings  The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000  1 H and  13 C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in  Arabidopsis  T87 cultured cells and  Bombyx mori , we systematically detected >450 HSQC peaks in each  13 C-HSQC spectrum derived from model plant,  Arabidopsis  T87 cultured cells and the invertebrate animal model  Bombyx mori . Furthermore, for the first time, efficient  13 C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts.    Conclusions/Significance  Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in  B. mori  by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of  13 C atoms of given metabolites on development-dependent changes in the 56 identified  13 C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism.

Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways