Bbiorefcode 2 0 0

NMR spectra were acquired on a Bruker Avance III HD 500 NMR spectrometer (University of Aveiro, Portuguese NMR Network, Aveiro, Portugal) operating at 500.13 MHz for ¹H observation, at 298 K. One-dimensional (1D) 1H spectra were recorded with 32 k points, 7002.80 Hz spectral width, a 2 s relaxation delay, and 512 scans, using the pulse programs “noesypr1d” and “zg” for aqueous and lipidic samples, respectively. Spectral processing was carried out in TopSpin 4.0.3 (Bruker BioSpin, Rheinstetten, Germany), and consisted of cosine multiplication (ssb 2), zero-filling to 64 k data points, manual phasing, baseline correction, and calibration to TSP-d4/TMS signals (0 ppm). Two-dimensional (2D) NMR spectra, namely, 1H-1H TOCSY, J-resolved, and 1H-13C HSQC spectra, were also recorded for selected samples to aid metabolite identification. Signal assignment was based on matching 1D and 2D spectral information to reference spectra available in Chenomx 9.0 (Edmonton, AB, Canada), BBIOREFCODE-2–0–0 (Bruker Biospin, Rheinstetten, Germany), and HMDB.

For NMR analysis, dried aqueous and lipid extracts were dissolved, respectively, in 600 μL of deuterated phosphate buffer (PBS 100 mM, pH 7.4) containing 0.1 mM 3-(trimethylsilyl) propionic acid (TSP-d₄), and 600 μL of deuterated chloroform containing 0.03% tetramethylsilane (TMS). After transferring 550 μL of each sample to 5 mm NMR tubes, analysis was performed on a Bruker Avance III HD 500 NMR spectrometer (University of Aveiro, Portuguese NMR Network) operating at 500.13 MHz for ¹H observation, at 298 K. Standard 1D ¹H spectra (pulse programs ‘noesypr1d’ and ‘zg’ for aqueous and lipid samples, respectively) were recorded with 32 k points, 7002.80 Hz spectral width, a 2 s relaxation delay and 512 scans. Spectral processing in TopSpin 4.0.3 (Bruker BioSpin, Rheinstetten, Germany) comprised cosine multiplication (ssb 2), zero-filling to 64 k data points, manual phasing, baseline correction and calibration to TSP-d₄/TMS signals (δ 0 ppm). Two-dimensional NMR spectra, namely, ¹H-¹H TOCSY, J-resolved and ¹H-¹³C HSQC spectra, were also recorded for selected samples to aid metabolite identification. Signal assignment was based on matching 1D and 2D spectral information to reference spectra available in Chenomx 9.0 (Edmonton, AB, Canada), BBIOREFCODE-2–0–0 (Bruker Biospin, Rheinstetten, Germany) and HMDB.

At the time of NMR analysis, the dried aqueous extracts were suspended in 600 μL of deuterated phosphate buffer (PBS 100 mM, pH 7) containing 0.1 mM of 3-(trimethylsilyl)propionic acid (TSP-d₄), and 550 μL of each sample were transferred to 5 mm NMR tubes. All samples were analyzed in a Bruker Avance III HD 500 NMR spectrometer (University of Aveiro, Portuguese NMR Network) operating at 500.13 MHz for ¹H observation, at 298 K. Standard 1D ¹H spectra with water presaturation (pulse program ‘noesypr1d’, Bruker library) were recorded with 32 k points, 7002.801 Hz spectral width, a 2 s relaxation delay and 2048 scans. Spectral processing in TopSpin 4.0.3 (Bruker BioSpin, Rheinstetten, Germany) comprised cosine multiplication (ssb 2), zero-filling to 64 k data points, manual phasing and baseline correction, and calibration to the TSP-d₄ signal (δ 0 ppm). Two-dimensional NMR spectra, namely ¹H-¹H TOCSY, J-resolved and ¹H-¹³C HSQC spectra, were also recorded for selected samples to aid metabolite identification. Metabolite assignment was based on matching 1D and 2D spectral information to reference spectra available in Chenomx (Edmonton, Canada), BBIOREFCODE-2–0–0 (Bruker Biospin, Rheinstetten, Germany) and HMDB^{28 (link)}.