Compartmentation of metabolism probed by [2-13C]alanine:: improved 13C NMR sensitivity using a CryoProbe detects evidence of a glial metabolon

The labelling of metabolites with the NMR active nucleus C-13 allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-C-13, N-15]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using C-13 NMR spectroscopy. The spectra were obtained using a CryoProbe(TM) optimised for C-13 detection (dual CryoProbe [C-13, H-1]) in which the receiver and transmitter coils are cooled to similar to20K 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 similar to17-fold using this device. A large proportion of alanine was initially metabolised over the first 20 min 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-C-13, N-15]aspartate. Using the increased sensitivity of the CryoProbe(TM), [2-C-13, N-15]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. (C) 2003 Elsevier Science Ltd. All rights reserved.