NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions

Abstract

Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure 13C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional 1H-1H NMR spectra from 12C- and 13C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli. We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 μM. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling. © 2010 American Chemical Society.