Compartmentation of cerebral glutamate in situ as detected by 1H/13C n.m.r.
Incorporation of 13C label from either [1-13C]glucose to glutamate C-4 and lactate C-3 or from [2-13C]acetate to glutamate C-4 was monitored in situ in a superfused brain slice preparation by using 1H-detected/13C-edited (1H/13C) n.m.r. spectroscopy. The fractional enrichments of both metabolites were determined by this means in both brain slices and acid extracts of the preparations in order to assess their 1H-n.m.r. detectabilities. The 1H/13C satellite resonances from glutamate C-4 and lactate C-3 in brain tissue were followed from 4 min onwards in the presence of 5 mM [1-13C]glucose. Fractional enrichment of glutamate C-4 in the slice preparations was higher than in their acid extracts throughout the incubation of 100 min; at 30 min the enrichment was 15.9 +/- 0.6% in the slice preparations and 10.6 +/- 0.9% in extracts and at 100 min 24.5 +/- 1.7% compared with 19.7 +/- 0.4%, respectively. In contrast, lactate C-3 reached a steady-state fractional enrichment of approx. 43% by 15 min and there was no difference between the values determined in the slice preparations and the acid extracts. There was a significant difference between the glutamate C-4 fractional enrichments in the brain slices (7.4 +/- 0.6%) and extracts (5.1 +/- 0.3%) after 60 min of incubation with [2-13C]acetate. Thus 13C label from both glucose and exogenous acetate enters a pool of glutamate that is more amenable to 1H n.m.r. detection than total acid-extracted brain biochemical glutamate, whereas lactate is labelled with full 1H n.m.r. visibility. The results are discussed in the light of the biochemical factors that affect glutamate 1H-n.m.r. susceptibility and thus its n.m.r. visibility.
Kauppinen
R A
RA
Department of Biochemistry and Biotechnology, A.I. Virtanen Institute, University of Kuopio, Finland.
Pirttilä
T R
TR
Auriola
S O
SO
Williams
S R
SR
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002247:Carbon Isotopes; D002451:Cell Compartmentation; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006168:Guinea Pigs; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D011522:Protons
D002247:Carbon Isotopes; D005971:Glutamates; D011522:Protons; D018698:Glutamic Acid