Quantitative Assessment of Neurometabolism by Deuterium and Proton NMR Spectroscopy Using [6,6'-²H₂]Glucose

Neurometabolic analysis is routinely performed using ¹³C NMR/MRS in conjunction with the administration of ¹³C-labeled tracers. Despite its widespread use, the low sensitivity of ¹³C NMR necessitates long acquisition times. To address this limitation, the present study introduces an indirect approach that uses a single ¹H 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'-²H₂]glucose for 10-90 min, and the ²H labeling of cortical metabolites was measured ex vivo using ²H NMR spectroscopy. After 90 min of [6,6'-²H₂]glucose infusion, resonances corresponding to [3-²H₂]lactate, [4-²H_x]glutamate, [4-²H_x]glutamine, and [3-²H]aspartate (∼2.60 ppm) were detected. In contrast, signals corresponding to [2-²H]aspartate (∼3.90 ppm), [2-²H]glutamate (∼3.76 ppm), and [3-²H]glutamate (∼2.09 ppm) were not detected, even after prolonged infusion, suggesting complete loss of the ²H 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 [4-²H_x]glutamate, [4-²H_x]glutamine, and [3-²H]aspartate, was 0.389 ± 0.031 μmol/g/min. The [4-²H_x]glutamate levels were also derived by subtracting the [4-¹H₂]glutamate resonance intensity from total [4-¹H₂]glutamate intensity estimated using the [3-¹H₂]glutamate intensity in unedited ¹H NMR spectra. The concentrations of [4-²H_x]glutamate determined from ¹H NMR spectra were comparable to those determined from ²H 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) [4-²H_x]glutamate levels compared with controls (2.99 ± 0.15 μmol/g, n = 7). This decrease was comparable to that observed with the direct ²H NMR measurements and was consistent with findings from ¹³C tracer-based studies.