The flux from glucose to glutamate in the rat brain in vivo as determined by 1H-observed, 13C-edited NMR spectroscopy.
The rate of incorporation of carbon from [1-13C]glucose into the [4-CH2] and [3-CH2] of cerebral glutamate was measured in the rat brain in vivo by 1H-observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 60-min infusion of [1-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved [4-13CH2] resonances of glutamate and glutamine increased exponentially during the infusion and attained a steady state in approximately 20 min with a first-order rate constant of 0.130 +/- 0.010 min-1 (t1/2 = 5.3 +/- 0.5 min). The appearance of the [3-13CH2] resonance in the POCE difference spectrum lagged behind that of the [4-13CH2] resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26.6 +/- 4.1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intracellular pH as determined by 31P NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate, the rate of glutamate labeling provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be approximately 1.4 mumols g-1 min-1. These experiments demonstrate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1H-observed, 13C-decoupled NMR spectroscopy.
Fitzpatrick
S M
SM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Hetherington
H P
HP
Behar
K L
KL
Shulman
R G
RG
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002247:Carbon Isotopes; D002952:Citric Acid Cycle; D005947:Glucose / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D008956:Models, Chemical; D010758:Phosphorus; D051381:Rats; D011919:Rats, Inbred Strains
D002247:Carbon Isotopes; D005971:Glutamates; D010758:Phosphorus; D018698:Glutamic Acid; D006859:Hydrogen; D005947:Glucose