Ultrashield plus nmr spectrometer

One dimensional proton nuclear magnetic resonance (¹H‐NMR) spectroscopy analysis was performed using a 600 MHz Bruker Ultrashield Plus NMR spectrometer (Bruker BioSpin Ltd., Canada). We used ChenomX NMR Suite 7.1 software (ChenomX Inc., Edmonton, Alberta, Canada) for profiling NMR spectra to identify and quantify metabolites in a non‐targeted approach (Weljie et al., 2006 (link)). DSS (4,4‐dimethyl‐4‐silapentane‐1‐sulfonic acid) was added to samples as an internal standard (Wishart et al., 2009 (link)). Two hundred microliters of plasma was filtered using a 5 times prewashed 3 KDa filter (NanoSep microcentrifuge) at 12,000 × g for 1 h at 4ºC followed by a rinse using 100 μl D₂O. The filtrates were adjusted to 400 μl by adding 80 µl of phosphate buffer (0.5 M NaH₂PO₄ buffer solution at pH 7.0) containing 2.5 mM 2,2‐dimethylsilapentane‐5‐sulfonate (DSS, final concentration 0.5 mM), 10 µl sodium azide (1 M NaN₃), and D₂O. All samples were adjusted for pH 7.0 ± 0.04 at room temperature.

¹H-NMR spectroscopic analysis was performed in one-dimensional mode for all samples using a 600-MHz Bruker Ultrashield Plus NMR spectrometer (Bruker BioSpin Ltd., Milton, ON, Canada). Details of the pulse sequence can be found in the data acquisition section of the supplement (see Additional file 1). Chenomx NMR Suite 7.1 software (Chenomx Inc., Edmonton, AB, Canada) was used to profile the ¹H-NMR spectra for metabolite identification and quantification using a nontargeted profiling approach in the profiler module [12 (link)]. We used 4,4-dimethyl-4-silapentane-1-sulfonic acid as an internal standard for metabolite quantification [13 (link)].

The sample preparation protocol and NMR spectral acquisition are presented in the Supplementary Methods and have previously been described in detail^{12 (link)–14 (link)}. Briefly, high resolution one-dimensional ¹H NMR spectra of filtered serum samples were obtained on a 600 MHz Bruker Ultrashield Plus NMR spectrometer (Bruker BioSpin Ltd., Canada). The spectra were profiled in Chenomx NMR Suite 7.5 software (Chenomx Inc., Edmonton, AB, Canada)^{15 (link)}. The concentration of internal standard (0.5 mM) was used to determine the concentrations of detected metabolites. All samples and ¹H NMR spectra were randomized prior to analysis to avoid progressive bias.