NMR Analysis of Plasma Metabolites

Plasma samples were thawed for 30 min at 4 °C and centrifuged for 5 min (4 °C, 12,100× g) to remove cells and precipitate. Aliquots of 400 μL of supernatant were mixed with 200 µL D₂O phosphate buffer (0.2 M Na₂HPO₄/NaH₂PO₄, pH 7.4, in 99% ²H₂O; Sigma-Aldrich, Germany) and subsequently recorded on a BRUKER AVIII-600 MHz NMR spectrometer (BrukerBioSpin, Germany) equipped with a CPP-TCI probe (Bruker BioSpin, Switzerland) at a temperature of 298.1 K. ¹H NMR spectra were acquired using a T₂-filtered one-dimensional Carr–Purcell–Meiboom–Gill (CPMG) [48 (link)] pulse sequence with water suppression, as previously described [9 (link),15 (link)]. Spectral acquisition was controlled using the TopSpin 3.1 software (Bruker BioSpin, Germany).