Quantitative Assessment of Neurometabolism by Deuterium and Proton NMR Spectroscopy Using [6,6′- ² H ₂ ]Glucose

Neurometabolic analysis is routinely performed using 13 C NMR/MRS in conjunction with the administration of 13 C-labeled tracers. Despite its widespread use, the low sensitivity of 13 C NMR necessitates long acquisition times. To address this limitation, the present study introduces an indirect approach that uses a single 1 H NMR acquisition to measure deuterium labeling by exploiting labeling at specific sites, thereby providing a novel and more efficient strategy for assessing neurometabolism. In this study, male SD rats were infused with [6,6’- 2 H 2 ]glucose for 10–90 min, and the 2 H labeling of cortical metabolites was measured ex vivo using 2 H NMR spectroscopy. After 90 min of [6,6′- 2 H 2 ]glucose infusion, resonances corresponding to [3- 2 H 2 ]lactate, [4- 2 H x ]glutamate, [4- 2 H x ]glutamine, and [3- 2 H]aspartate (∼2.60 ppm) were detected. In contrast, signals corresponding to [2- 2 H]aspartate (∼3.90 ppm), [2- 2 H]glutamate (∼3.76 ppm), and [3- 2 H]glutamate (∼2.09 ppm) were not detected, even after prolonged infusion, suggesting complete loss of the 2 H label during the second turn of the tricarboxylic acid cycle. The cerebral metabolic rate of glucose oxidation, estimated by fitting an exponentially saturating curve to the pooled labeling of [4- 2 H x ]glutamate, [4- 2 H x ]glutamine, and [3- 2 H]aspartate, was 0.389 ± 0.031 μmol/g/min. The [4- 2 H x ]glutamate levels were also derived by subtracting the [4- 1 H 2 ]glutamate resonance intensity from total [4- 1 H 2 ]glutamate intensity estimated using the [3- 1 H 2 ]glutamate intensity in unedited 1 H NMR spectra. The concentrations of [4- 2 H x ]glutamate determined from 1 H NMR spectra were comparable to those determined from 2 H NMR. Moreover, when applied to 5xFAD mice (2.53 ± 0.43 μmol/g, n = 7), a model of Alzheimer’s disease, this indirect approach revealed significantly reduced ( p = 0.028) [4- 2 H x ]glutamate levels compared with controls (2.99 ± 0.15 μmol/g, n = 7). This decrease was comparable to that observed with the direct 2 H NMR measurements and was consistent with findings from 13 C tracer-based studies.