2.5. NMR Spectroscopy

Lyophilized brain samples were thawed and reconstituted in deuterium oxide (D₂O) containing 0.02% sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TMSP) and 0.25% dioxane and used as internal standards for quantification of ¹H and ¹³C spectra, respectively. The sample pH was adjusted to 6.95–7.05. ¹H spectra and proton-decoupled ¹³C-NMR spectra were obtained on a Bruker AVANCE III 950 MHz NMR spectrometer at the NMR Center, University of Maryland, Baltimore. Fully relaxed ¹H NMR spectra (64 scans) were acquired using a 90° pulse angle and acquisition time of 2.7 s, and a relaxation delay of 20 s. Chemical shifts for the ¹H-spectra are reported relative to the TMSP peak at 0.0 ppm. The ¹³C-spectra were acquired at 25 °C using a 35° pulse angle, 25 KHz spectra width, and 64 K data points, using an acquisition time of 0.99 s, with a 3 s relaxation time. The average number of scans per sample was 9925. All ¹³C-spectra were corrected for nuclear Overhauser effects (nOe) and for relaxation time [30,33] by using combined nOe and a 25.7 s relaxation time as correction factors for each isotopomer [30,33]. A line broadening width of 5 Hz was used. Chemical shifts values are reported relative to the internal standard dioxane peak at 67.4 ppm. Peak assignments were made by comparison to spectra from pure standard ¹³C compounds and to literature values. All ¹³C-NMR peaks were corrected for natural abundance. Data are reported as mean ± SEM nmol ¹³C incorporated/mg protein.