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 in vivo . 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 in vivo and 2 H label loss due to exchange reactions is required for DMI-based measurements of absolute metabolic rates. Here the deuterium KIE and label loss in vivo are investigated for glucose and acetate using a double substrate/double labeling strategy and 1 H-decoupled 13 C NMR in rat glioma cells and rat brain tissue metabolite extracts. The unique spectral patterns due to extensive 2 H– 13 C and 13 C– 13 C scalar couplings allow the identification of all possible metabolic products. The 2 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- 2 H 2 ]-glucose as the metabolic substrate. For [2- 2 H 3 ]-acetate, the 2 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 2 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 2 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 in vivo .