Characterization of Kinetic Isotope Effects and Label Loss in Deuterium-Based Isotopic Labeling Studies.

Deuterium metabolic imaging (DMI) is a novel, 3D, magnetic resonance (MR)-based method to map metabolism of deuterated substrates . The replacement of protons with deuterons could potentially lead to kinetic isotope effects (KIEs) in which metabolic rates of deuterated substrates are reduced due to the presence of a heavier isotope. Knowledge of the extent of KIE and H label loss due to exchange reactions is required for DMI-based measurements of absolute metabolic rates. Here the deuterium KIE and label loss are investigated for glucose and acetate using a double substrate/double labeling strategy and H-decoupled C NMR in rat glioma cells and rat brain tissue metabolite extracts. The unique spectral patterns due to extensive H-C and C-C scalar couplings allow the identification of all possible metabolic products. The H label loss observed in lactate, glutamate, and glutamine of rat brain was 15.7 ± 2.6, 37.9 ± 1.1, and 41.5 ± 5.2% when using [6,6-H]-glucose as the metabolic substrate. For [2-H]-acetate, the H label loss in glutamate and glutamine was 14.4 ± 3.4 and 13.6 ± 2.2%, respectively, in excellent agreement with predicted values. Steady-state H label accumulation in the C4 position of glutamate and glutamine was contrasted by the absence of label accumulation in the C2 or C3 positions, indicating that during a full turn of the tricarboxylic acid cycle all H label is lost. The measured KIE was relatively small (4-6%) for both substrates and all measured metabolic products. These results pave the way for further development of quantitative DMI studies to generate metabolic flux maps .