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 ²H 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 ¹H-decoupled ¹³C NMR in rat glioma cells and rat brain tissue metabolite extracts. The unique spectral patterns due to extensive ²H-¹³C and ¹³C-¹³C scalar couplings allow the identification of all possible metabolic products. The ²H 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 [6,6-²H₂]-glucose as the metabolic substrate. For [2-²H₃]-acetate, the ²H 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 ²H 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 ²H 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.