NMR metabolomics. (i) Sample preparation.

Each study sample was given a unique identifier and subsequently randomized. We added five Red Cross human plasma samples as external controls and two empty vials as extraction blanks. All samples were thawed at room temperature, and their individual masses were recorded. Samples were prepared by adding 1 ml of liquid chromatography-mass spectrometry (LC-MS)-grade water (Chromasol) per gram of cecal material, obtaining a 1:1 (wt/vol) ratio. The samples were then vortex mixed for 20 min and centrifuged in a cold AccuSpin 3R centrifuge for 30 min at 3,500 × g at 4°C. The supernatants were collected, and 1-ml aliquots were centrifuged for 30 min at 22,000 × g in a high-speed microcentrifuge inside a cold 4°C room.

NMR samples were prepared by mixing 675 μl of the final cecal extract supernatant and 75 μl of sodium phosphate buffer (1 M) in D₂O, with DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) as an internal standard (Cambridge Isotope Laboratories), which provides a final pH of 7.4 and a final DSS concentration of 0.33 mM. A total of 580 μl of each NMR sample was transferred to a 5-mm SampleJet NMR tube. The remaining 170 μl was pooled, and five internal controls were created and randomized with the study samples, the external controls, and the extraction blanks. Three additional NMR tubes containing sodium phosphate buffer were inserted at the beginning, middle, and end of the NMR run as buffer blank quality controls.