Compartmentation of metabolism probed by [2-[formula omitted]]alanine: improved [formula omitted] NMR sensitivity using a CryoProbe detects evidence of a glial metabolon

Abstract

The labelling of metabolites with the NMR active nucleus 13C allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-13C, 15N]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using 13C NMR spectroscopy. The spectra were obtained using a CryoProbe™ optimised for 13C detection (dual CryoProbe [13C, 1H]) in which the receiver and transmitter coils are cooled to ∼20K to reduce contributions to noise in the signal obtained. Compared with conventional inverse geometry probe, the signal-to-noise ratio (S/N) was increased by ∼17-fold using this device. A large proportion of alanine was initially metabolised over the first 20min by glial cells, as indicated by the relative importance of the glial, only enzyme pyruvate carboxylase to the labelling pattern of glutamate, with the ratio of pyruvate carboxylase to pyruvate dehydrogenase derived glutamate being 0.25, and exported [2-13C, 15N]aspartate.Using the increased sensitivity of the CryoProbe™, [2-13C, 15N]aspartate was also detected in the extracts of cerebral tissue. This metabolite could only have been derived via the pyruvate carboxylase pathway, and given the large export of the metabolite into the superfusion buffer suggests the occurrence of a ‘metabolon’ arrangement of enzymes within glial cells.