Topspin 3

1D ¹H NMR spectroscopy was used to analyze the degree of substitution as well as the uronic acid composition of the alginates. The samples were subjected to mild acid hydrolysis as previously described [53 ] to reduce the viscosity prior to the NMR analysis. 6–10 mg of the samples were then dissolved in 600 µL D₂O (99.9%) and added 5 µL 3-(Trimethylsilyl)propionic 2,2,3,3-d₄ acid (TSP, Sigma-Aldrich, Oslo, Norway) as an internal standard and 15 µL Triethylenetetraamine-hexaacetic acid (TTHA, Sigma Aldrich) as a chelating agent for residual divalent ions. The latter was not added to samples already added EDTA (analysis of leaked alginate). 1D ¹H spectra were recorded at 90 °C on a Bruker Ascend 400 MHz Avance III HD spectrometer, equipped with a 5-mm SmartProbe z-gradient probe and SampleCase (Bruker BioSpin AG, Fällanden, Switzerland). The spectra were recorded using TopSpin 3.2 software (Bruker BioSpin, Fällanden, Switzerland) and processed and analyzed with TopSpin 3.5 software (Bruker BioSpin).

Samples were received in MeOH and transferred to pre-weighed scintillation vials, rinsing the ampoules with MeOH (3 × 0.5 mL). The solvent was evaporated under a stream of dry N₂, weighed, dissolved in CD₃OD (750 µL, 99.5 atom % D; Cambridge Isotope Laboratories, Andover, MA, USA), and transferred to NMR tubes (5 mm i.d.). ¹H NMR spectra were acquired on a Bruker DRX-500 NMR spectrometer at 500.13 MHz (64 scans, 20 °C) using Icon NMR in Bruker TopSpin 3.2, and data were processed with TopSpin 3.6.2.

To analyze metabolites, one-dimensional ¹H-NOESY NMR spectrum was obtained at 298 K on a Bruker ASCEND III 600 spectrometer equipped with a cryoprobe. The NOESY pulse sequence (noesygppr1d) was applied with presaturation to suppress the residual water signal. ¹H-NMR spectrum for each sample consisted of 256 scans with following parameters: spectral width = 12,019.2 Hz, spectral size = 65,536 points, pulse width (90) = 12.2 μs, relaxation delay (RD) = 5.0 s, and a mixing time of 10 ms.
For quantitative profiling of CSF samples, spectra were processed and analyzed with Bruker topspin 3.1 (Bruker GmbH, Karlsruhe, Germany) and Chenomx NMR suite 7.7 (Chenomx Inc., Edmonton, Canada). Each free induction decay (FID) was zero-filled to 64,000 points and transformed with line broadening (LB) = 0.3 Hz. NMR spectra were initially phased in Bruker topspin 3.1 and the final baseline was corrected using Chenomx NMR suite 7.7. The baseline model was built in each spectrum using the algorithm of multipoint baseline correction. The chemical shifts were referenced against the resonance of TSP as 0.0 ppm. Metabolites were identified using the database stored in Chenomx NMR suite 7.7 and were quantified from the comparison of the internal standard (TSP). A total of 31 metabolites identified from the CSF samples. The identified metabolites were evaluated in ¹H-¹³C HSQC and 2D ¹H-TOCSY spectra.