n-methyl-n-(tert-butyldimethylsilyl)trifluoroacetamide and ammonium-acetate

13C-enrichment analysis of glutamate in the extracellular fluid (GLU(ECF): 2-3 microM) by gas-chromatography/mass-spectrometry (GCMS) was combined with in vivo NMR observation of whole-brain GLU (approximately10 mM) to study neurotransmitter uptake. Brain GLU C5 was 13C-enriched by intravenous [2,5-13C]glucose infusion. GLU(ECF) was collected by microdialysis from the cortico-striatal region of awake rats. The 13C-enrichment of basal dialysate GLU C5 during 0.75-1.25 hr of infusion was 0.263 +/- 0.01, very close to the enrichment of whole-brain GLU C5. The result strongly suggests that dialysate GLU consists predominantly of neurotransmitter GLU. For selective 13C-enrichment of neurotransmitter GLU, the whole-brain 13C-enrichment was followed by [12C]glucose infusion to chase 13C from the small glial GLU pool. This leaves [5-13C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. The uptake of synaptic [5-13C]GLU(ECF) into glia and metabolism to glutamine (GLN) were monitored in vivo by NMR observation of [5-13C,15N]GLN formed during 15NH4Ac infusion. The rate of GLN synthesis, derived from neurotransmitter GLU(ECF) (which provided 80-90% of the substrate) was 6.4 +/- 0.44 micromol/g/hr. Hence, the observed rate represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.

Topics: Acetamides; Acetates; Animals; Brain; Brain Chemistry; Carbon Isotopes; Chromatography, High Pressure Liquid; Dicarboxylic Acids; Extracellular Fluid; Fluoroacetates; Gas Chromatography-Mass Spectrometry; Glucose; Glutamic Acid; Infusions, Intravenous; Intracellular Fluid; Magnetic Resonance Spectroscopy; Microdialysis; Models, Biological; Neuroglia; Neurotransmitter Agents; Nitrogen Isotopes; Organosilicon Compounds; Pyrrolidines; Rats; Trifluoroacetic Acid