Two-Dimensional Statistical Recoup ling for the Identification of Perturbed Metabolic Networks from NMR Spectroscopy

The development of Statistical Total Correlation Spectroscopy (STOCSY), a representation of the autocorrelation matrix of a spectral data set as a 2D pseudospectrum, has allowed more reliable assignment of one- and two-dimensional NMR spectra acquired from the complex mixtures that are usually used in metabolomics/metabonomics studies, thus, improving precise identification of candidate biomarkers contained in metabolic signatures computed by multivariate statistical analysis. However, the correlations obtained cannot always be interpreted in terms of connectivities between metabolites. In this study, we combine statistical recoupling of variables (SRV) and STOCSY to identify perturbed metabolite systems. The resulting Recoupled-STOCSY (R-STOCSY) method provides a 2D correlation landscape based on the SRV clusters representing physical, chemical, and biological entities. This enables the identification of correlations between distant clusters and extends the recoupling scheme of SRV, which was previously limited to the association of neighboring clusters. This allows the recovery of only meaningful correlations between metabolic signals and significantly enhances the interpretation of STOCSY. The method is validated through the measurement of the distances between the metabolites involved in these correlations, within the whole metabolic network, which shows that the average shortest path length is significantly shorter for the correlations detected in this new way compared to metabolite couples randomly selected from within the entire KEGG metabolic network. This enables the identification without any a priori knowledge of the perturbed metabolic network. The R-STOCSY completes the recoupling procedure between distant clusters, further reducing the high dimensionality of metabolomics/metabonomics data set and finally allows the identification of composite biomarkers, highlighting disruption of particular metabolic pathways within a global metabolic network. This allows the perturbed metabolic network to be extracted through NMR based metabolomics/metabonomics in an automated, and statistical manner.