Quantitative determination of glutamate turnover by 1H-observed, 13C-edited nuclear magnetic resonance spectroscopy in the cerebral cortex ex vivo: interrelationships with oxygen consumption.
The kinetics of glutamate 13C-4 label appearance from D-[1-13C]-glucose and 13C-4 label disappearance from steady state following D-12C-glucose incubation were quantified with 1H-observed, 13C-edited nuclear magnetic resonance (NMR) spectroscopy in the superfused brain slices under largely varying oxygen consumption. Label incorporation to and from glutamate C-4 were fitted into mono- or bicompartmental models in order to determine the respective rate constants and to assess the presence of plausible multiple pools. At a steady-state oxygen consumption of approximately 4 mumol/min/g dry weight, glutamate labelling could be fitted into a biexponential equation, suggesting that there were two compartments with a large difference in their rates (respective rate constants of 0.022 and 0.149) and pool sizes (relative contributions of 91.2 and 8.8%, respectively). Stimulation of oxygen consumption in the brain slice preparations with either 40 mM KCl by 59.5 +/- 10.3% or 5 microM carbonyl cyanide m-fluorophenyl hydrazone by 61.4 +/- 8.4% increased glutamate C-4 labelling rate constants to 0.058 +/- 0.009 and 0.054 +/- 0.006, respectively. In the stimulated slice preparation, glutamate labelling could only be fitted into a monoexponential equation. 13C-4 label disappearance, independent of oxygen uptake, could also only be fitted into a monoexponential equation. There was a close match between the rate constants of label disappearance and appearance in non-stimulated and carbonyl cyanide m-fluorophenyl hydrazone-stimulated slices. In the presence of 40 mM KCl label disappearance did not, however, increase. These data show that glutamate C-4 turnover from exogenous D-[1-13C]-glucose can be used as an index of oxidative metabolism in situ under steady-state conditions as well as when oxygen metabolism is strongly stimulated. The results are discussed with respect to the use of NMR spectroscopy as a means of mapping brain oxidative metabolism.
Lukkarinen
J
J
NMR Research Group, A.I. Virtanen Institute, University of Kuopio, Finland.
Oja
J M
JM
Turunen
M
M
Kauppinen
R A
RA
D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002247:Carbon Isotopes; D002540:Cerebral Cortex / Q000033:anatomy & histology / Q000378:metabolism*; D002952:Citric Acid Cycle; D004734:Energy Metabolism / Q000187:drug effects; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D019344:Lactic Acid / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008297:Male; D010101:Oxygen Consumption / Q000502:physiology*; D011522:Protons; D051381:Rats; D013268:Stimulation, Chemical
D002247:Carbon Isotopes; D011522:Protons; D001224:Aspartic Acid; D019344:Lactic Acid; D018698:Glutamic Acid; C000179:N-acetylaspartate