Absolute Metabolite Quantification Using Pure Shift NMR: Toward Quantitative Metabolic Profiling of Aqueous Biological Samples

Accurate quantification of metabolites by nuclear magnetic resonance (NMR) is of prime importance in the field of health sciences for understanding the metabolic pathways of the investigated system, to address the mechanisms of action of diseases, and improving their diagnosis, treatment, and prognosis. Unfortunately, the absolute quantitative analysis of complex samples is still limited by sensitivity and resolution issues that are intrinsic to this technique. Ultrahigh-resolution pure shift methods have especially shown to be suitable for interpreting mixtures of metabolites in biological samples. Here, we introduce a robust analytical protocol based on the use of a pure shift library of calibration reference spectra to fit the fingerprint of each metabolite of interest and determine its concentration. The approach based on the SAPPHIRE pulse sequence enhanced with a block for solvent suppression has been validated through the results of a series of model mixtures, exhibiting excellent trueness (slope values in the range of 0.93-1.02) and linearity (R2 > 0.996) in a total time (a few hours) that is fully compatible with metabolomics studies. Furthermore, we have successfully applied our method to determine the absolute metabolite concentrations in a lymphoma extracellular medium, which improves metabolomic protocols reported to date by providing a quantitative and highly resolved vision of metabolic processes at play.

Automated summary

Accurate quantification of metabolites by NMR is crucial for understanding metabolic pathways and addressing diseases. A new analytical protocol using pure shift library of calibration reference spectra has been developed to determine metabolite concentrations. This method, validated through model mixtures, provides quantitative and high-resolution vision of metabolic processes in a time compatible with metabolomics studies. Furthermore, it has been successfully applied to determine absolute metabolite concentrations in a lymphoma extracellular medium.