title: In vivo microdialysis and gas-chromatography/mass-spectrometry for 13C-enrichment measurement of extracellular glutamate in rat brain.
abstract: Extracellular glutamate (GLU(ECF)) was collected by microdialysis from the corticostriatal region of awake rats, at the basal level and after elevation by perfusion of GLU uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid. Concurrently, [2,5-(13)C]glucose was infused intravenously to 13C-enrich brain GLU predominantly at C5. The 13C enrichment of GLU(ECF) was measured, after tert-butyldimethylsilylation, by gas-chromatography/mass-spectrometry. Excellent signal-to-noise ratios of the analyte signals at three selected ion-pairs were achieved at approximately 20 pmol. The fractional 13C enrichment of basal dialysate GLU C5, collected during 0.75-1.25 h of [2,5-(13)C]glucose infusion, was 0.263+/-0.01, very close to the enrichment of whole-brain (predominantly intracellular) GLU C5 measured in parallel NMR study. The result strongly suggests that the dialysate GLU consists predominantly of neurotransmitter GLU, which was 13C-enriched in, and released from, neurons by exocytosis and had diffused to the dialysis probe; the label is undiluted by 12C-GLU(ECF) present before the enrichment. Hence, our result supports the view, proposed on the basis of Ca(2+)- and tetrodotoxin-sensitivity of dialysate GLU, that basal dialysate GLU in awake non-stimulated brain mainly represents neurotransmitter GLU. Isotope labeling provides a novel method for determining the extent to which dialysate GLU reflects synaptic GLU(ECF), and for measuring its turnover under physiological or pathological conditions.
authors.affiliation: Mass Spectrometry Facility, University of California, Riverside, CA, USA.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Kondrat
authors.forename: Keiko
authors.initials: K
authors.lastname: Kanamori
authors.forename: Brian D
authors.initials: BD
authors.lastname: Ross
mesh_terms: D000081:Acetamides; D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005110:Extracellular Space / Q000378:metabolism*; D005463:Fluoroacetates; D008401:Gas Chromatography-Mass Spectrometry; D018698:Glutamic Acid / Q000378:metabolism*; D007262:Infusions, Intravenous; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D017551:Microdialysis / Q000379:methods*; D017646:Organosilicon Compounds / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014269:Trifluoroacetic Acid / Q000494:pharmacology
chemical_list: D000081:Acetamides; D002250:Carbon Radioisotopes; D005463:Fluoroacetates; D017646:Organosilicon Compounds; D018698:Glutamic Acid; C059151:N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; D014269:Trifluoroacetic Acid