Nmr processor

¹H-NMR spectra at 25°C were collected using a Bruker Avance 600 MHz spectrometer (Bruker, Billerica, MA, USA) using the noesypr1d pulse sequence with parameters as previously described [36] (link). Spectra were manually phase- and baseline-corrected using Chenomx NMR Processor (v. 8.1, Chenomx, Edmonton, AB, USA). Metabolite concentrations for each sample type (liver or serum) were quantified by a single researcher using Chenomx Profiler (v. 8.1, Chenomx, Edmonton, AB, USA). The quantification relies on the internal standard (DSS-d₆) to determine each metabolite's concentration using the Chenomx 600 MHz Library (v. 10), which allows for both absolute quantification and the ability to quantify many compounds within the spectrum [37] (link).

Approximately 50 mg of frozen liver from non-injected fish (one fish processed per tank per condition) were homogenized in 250 μL of cold acetonitrile with an ultrasound homogenizer on ice. A 250-μL cold water was then added and the solution was homogenized by sonication on ice. After a 5-minute 10 000 × g centrifugation at 4°C, 400 μL of the supernatant containing hydrophilic metabolites were transferred to a borosilicate tube and evaporated 1 hour under a nitrogen stream. The samples were stored at −80°C until further analysis.
Just before ¹H NMR analysis, samples were resuspended in 900 μL of D₂O and 100 μL of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS, internal calibrator, 0.5 mM final concentration). Approximately 700 μL were transferred to a 5-mm NMR tube. The ¹H-NMR spectra were recorded on a Bruker Advance III 400 MHz spectrometer at 298K. Analyses were performed with the noesypr1d pulse sequence. One-dimensional spectra were obtained after 128 scans of 64 000 data points. The spectral width was set at 12 KHz, the acquisition time at 6.6 seconds and the recycle delay at 1 second per scan. Correction and calibration of the spectra were performed using the Chenomx NMR Processor (Chenomx Inc., Edmonton, AB, Canada) and analyzed using the Chenomx NMR Profiler (Chenomx Inc., ) and the Human Metabolome Database (HMDB) (Wishart et al., 2018 (link)).