A critical assessment of methods of measuring metabolite concentrations by NMR spectroscopy

Many NMR studies are made by scientists and clinicians in order to measure metabolite levels. There is a growing need to obtain absolute quantification of these metabolites. We have therefore reviewed the methods that are available and those which are being developed, for studies in vivo and on isolated tissues. We describe the following methods and assess their advantages and limitations: (1) Conventional spectroscopy and area ratios, (2) Tissue extracts, (3) External capillary, (4) Internal endogenous marker, (5) Internal exogenous marker, (6) External marker in bath, (7) Reference to internal 1H water concentration, (8) Gradient-localized spectroscopy and (9) Fourier series analysis localization. The relevant biological and technical aspects associated with quantification of metabolite concentrations are also discussed. This includes NMR visibility, origin of the NMR signals, definitions of concentration, influence of the RF coil, data collection and data analysis.