Assignment of resonance in the 1H spectrum of rat brain by two-dimensional shift correlated and J-resolved NMR spectroscopy

Abstract

Two-dimensional shift-correlated (COSY) and J-resolved NMR spectroscopy was used to identify and assign resonance in the aliphatic region (0.8 to 4.5 ppm) of the 1H spectrum of acid extracts and tissue of rat brain. The chemical shift and spin-spin coupling constants of several resonances, which could not be resolved in one-dimensional spectra of tissue, were determined. These properties, together with the appropriate multiplet structure and scalar coupling patterns observed in 2D J-resolved and COSY spectra, were used to assign the resonances of lactate, threonine, alanine gamma-amino butyrate, N-acetyl aspartate, aspartate, glutamate, glutamine, taurine, and myo-inositol. The single peak observed at 1.33 ppm in the 1D 1H spectrum of excised brain tissue, which is generally assigned to lactate, was shown instead to consist of two overlapping peaks in 2D COSY contour plots. The intensity of both resonances increased considerably after death. The second resonance was assigned to threonine and the assignment was confirmed by ion-exchange chromatography. Several cross peaks were identified in 2D COSY spectra of intact brain tissue that were not present in similar spectra of the acid extract. The nonmetabolite resonances were assigned to macromolecules. Analysis of 2D COSY contour plots in conjunction with 1D spectra revealed that the total creatinine (creatinine + phosphocreatine) resonance at 3.0 ppm was overlapped by both GABA and macromolecules. This macromolecule resonance was present in a 2D COSY spectrum of the rat brain obtained in situ with a surface coil following cardiac arrest, indicating that its presence in the 2D spectrum of excised brain tissue did not arise from tissue disruption.