NMR Spectroscopic Analysis of Metabolites

¹H NMR spectra were obtained using a 700-MHz Bruker AVII spectrometer, operating at a 16.4T equipped with a ¹H (13C/15N) TCI cryoprobe. Sample temperature was maintained at 298K, and spectra were acquired using a 1D NOESY experiment (brain) and CPMG experiment (liver) as described previously (Radford-Smith et al., 2022 (link)). Topspin 2.1 (Bruker, Germany) was used for data processing: to manually phase the spectra, correct baselines, and reference the chemical shifts to the lactate CH₃ doublet resonance at δ = 1.33 ppm. All spectra of the same tissue were overlayed and automatically binned with 0.02 ppm-sized bins, although some compounds were manually binned. Each bin was normalized to the sum of all integrals in the spectrum of each sample and data exported to R v3.3.1 and Microsoft Excel for statistical analysis and metabolite identification. Metabolites were assigned by comparing spectral peaks to the Human Metabolome Database (Wishart et al., 2018 (link)) and by spiking in known compounds.