NMR Sample Preparation for Biofluids

NMR samples were prepared by thawing aliquots of the stored fluids followed by the addition of a suitable buffer. Plasma and serum samples were mixed with a buffer of pH = 7.4 (0.14 M Na₂HPO₄, 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 4% NaN₃ in H₂O) according to protocols reported elsewhere Buffer used for urine samples prepared in 100% D₂O and pH = 7.4 (1.5 M KH₂PO₄, 0.05 mM DSS and 4% NaN₃ 2 mM). 300 μL of plasma and serum samples were mixed with 240 μL buffer and 60 μL D₂O, while 540 μL of urine samples were mixed with 60 μL buffer (Suarez-Diez et al., 2017 (link)). The final step before NMR experiments was the transfer of 550 μL in a 5 mm NMR tube (Bruker BioSpin GmbH).