Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H‐31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1 H and 31 P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1 H and 31 P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1 H and 31 P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3 J PH and 4 J PH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90° Gaussian pulse on the 31 P channel was introduced to increase the resolution in the F 1 -domain and make the method even faster. The procedure was then applied to unambiguously assign the 1D 31 P and 1 H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31 P and 1 H methods lead to different results is discussed, and the use of 1 H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31 P spectrum is proposed.