Avance 600 mhz nmr

EDTA plasma samples were removed from -20°C storage and allowed to thaw, after which they were filtered through a 3,000 MW cutoff filter (Amicon, 0.5 mL capacity, Milipore) to remove insoluble lipid particles and proteins. If necessary, an appropriate amount of Milli-Q water was added to the filtrate to achieve a volume of 199 μL, followed by 8 μL of potassium phosphate buffer (1 M, pH 6.1), and 23 μL of internal standard containing 5 mM DSS-d6, 0.2% NaN₃ in 99.8% D₂O. A final volume of 180 μL was transferred to a 3 mm NMR tube. Samples were stored at 4°C until NMR data acquisition. NMR spectra were acquired at 25°C using the Bruker noesypr1d experiment on a Bruker Avance 600 MHz NMR spectrometer. ¹H NMR spectra were acquired with a spectral width of 12 ppm, a total acquisition time of 2.5 s, 8 dummy scans, 32 transients and water saturation during the prescan delay (2.5 s) and mixing time (100 ms) to minimize impact of water on the spectrum as previously described [21 (link)]. NMR spectra were zero-filled to 128k data points, Fourier Transformed with a 0.5-Hz line broadening, and manually phased and baseline corrected using Chenomx NMR Suite v8.0 Processor (Chenomx Inc., Edmonton, Canada). Profiler was used to quantify metabolites using the concentration of the reference signal (DSS-d6) as previously described [21 (link)]. Concentrations are reported in micromol/L (μM).

Lyophilized cecal and nopal extracts were diluted in 260–270 μl of phosphate buffer (pH 6.8, 10mM) and centrifuged (10k g, 4°C, 10 min). Serum samples were prepared for NMR analysis as previously described [21 (link)]. All metabolomics samples were prepared using 3 mm Bruker NMR tubes with 3-(trimethylsilyl)-1-propanesulfonic acid-d6 (DSS-d6) as an internal standard. Data acquisition was within 24 hours of sample preparation. NMR spectra were acquired as previously described [22 (link)] using a Bruker Avance 600 MHz NMR equipped with a SampleJet autosampler at 25°C. NMR spectra were processed and profiled as previously described [22 (link)] using Chenomx NMR Suite v7.6 Processor (Chenomx Inc., Edmonton, Canada). Corrections for serum sample dilution were made where appropriate using a dilution factor that was calculated by dividing the final volume of sample by the initial volume of serum used for sample preparation. The concentrations of serum samples are reported in micro-molar concentration (μM). The concentrations of cecal and nopal sample metabolites are reported in mmol/g dry weight.

First, 5.5 g of Lupasol WF (25,000 g/mol) was dissolved in 70 mL tert-butanol and heated to 80 °C. Then, 21 g of potassium carbonate was added. Over a period of 2–3 min, 53 mL of bromohexane was added. The reaction was allowed to proceed for ca. 24 h, after which the potassium carbonate was removed via filtration. 15 mL of iodomethane was added and stirred at 60 °C in a closed container for 24 h. The polymer was then precipitated and washed with hexanes. Nuclear magnetic resonance (NMR) analysis was recorded on Bruker Avance 600 MHz NMR. CDCl₃ was used as the solvent, referenced to residual solvent peak (7.26 ppm ¹H, 77.15 ¹³C). ¹H acquired with 32 scans, d1 = 2 s. ¹H NMR of N-hexyl, N-methyl PEI in CDCl₃ (d, ppm): 0.89 ppm (2H), 1.32 (3.7H), 1.81 (1H), 3.45 to 4.54 (5.2H). Peaks assigned as terminal methyl from hexyl chain (0.9), interior CH₂ groups (1.3), b-CH₂ groups (1.8), a-CH₂ groups and methyl ammoniums, backbone peaks (3.4 to 4.5) [21 (link)].

The stoichiometries of SULF/PDADMA coacervates were determined using solution ¹H nuclear magnetic resonance (NMR) spectroscopy. NMR samples were prepared by dissolving the dry PEC in a solution of KBr in D₂O. This allowed the number of protons on both the small molecules and polyelectrolytes to be measured. The KBr concentrations used in NMR sample preparation were different from one to another because the CSCs required to fully dissociate the PECs were different. For SULF1/PDADMA, SULF2/PDADMA, and SULF3/PDADMA, 10 mg of the dry PEC was dissolved in 1.0 M KBr in D₂O, whereas 10 mg of dry SULF4/PDADMA was dissolved in 3.0 M KBr in D₂O. An AVANCE 600-MHz NMR (Bruker) was used to acquire the spectra. NMR spectra are shown in fig. S2.

After collection, specimens were stored refrigerated and/or frozen until transport on dry ice to the site of analysis, where they were stored at -80°C until sample preparation. Once defrosted, samples were filtered using washed Amicon Ultra-0.5 mL centrifugal filters with a cut-off of 3000 MW (Millipore, Billerica, MA) to remove lipids and proteins. When needed, filtrate volume was adjusted to 207 μL when preparing for 3mm NMR tubes or 585 μL when preparing for 5mm NMR tubes with Type I ultrapure water from Millipore Synergy UV system (Millipore, Billerica, MI). Samples were prepared for analysis by the addition of 23 μL or 65 μL of internal standard containing approximately 5 mmol/L of DSS-d6 [3-(trimethylsilyl)-1-propanesulfonic acid-d6], 0.2% NaN₃, in 99.8% D₂O to 207 μL or 585 μL of CSF filtrate, respectively. The pH of each sample was adjusted to 6.8 ± 0.1 by adding small amounts of NaOH or HCl. A 180 or 600 μL aliquot was subsequently transferred to 3 mm or 5mm Bruker NMR tubes, respectively, and stored at 4 ^oC until NMR acquisition (within 24 hours of sample preparation). NMR spectra were acquired as previously described [12 (link)] on a Bruker Avance 600-MHz NMR equipped with a SampleJet autosampler using a NOESY-presaturation pulse sequence (noesypr) at 25°C.