Comprehensive NMR Metabolite Profiling

¹H-NMR analysis has been described in detail previously [31 (link)]. In short, spectra were recorded at 800 MHz with a Bruker Advance III HD spectrometer with a 3-mm TCI cryoprobe. NMR data were recoded using the Bruker pulse sequence “zgespe”. A total of 128 scans were collected into 64 k data points. Data processing was performed with TopSpin 3.2p16 (Bruker BioSpin) and MatLab (MathWorks Inc., Natick, MA, USA), using TSP-d4 for referencing. In total 237 peaks were manually aligned and integrated peak-by-peak, and these variables represent ∼70 metabolites. A variable could also reflect more than one metabolite. Only variables of interest were identified.
For annotation Chenomx NMR suite 8.31 (Chenomx Inc., Edmonton, AB, Canada), the Human Metabolome Database [37 (link)] and an in-house implementation of the statistical total correlation spectroscopy (STOCSY) routine [38 (link)] were used.