Sample preparation for NMR analysis

NMR-based metabolomics analysis of plasma samples was performed according to previously described methods⁽,21^,22⁾ after slight modification. In brief, plasma samples were thawed and filtered by ultracentrifuge (10 000 g, 4°C) using prewashed (eight consecutive washes with 0·5 ml MQ water at 4000 g and 36°C to remove glycerol) nanosep centrifugal filters with a 3 kDa cutoff (Pall Life Science). Next, 250 µl of the filtrate were transferred into a clean microfuge tube and the final volume was made up to 600 µl by the addition of 150 µl phosphate buffer (0·4 mol/l, pH 7·0), 45 µl ²H₂O, 125 µl MQ water and 30 µl sodium 3-(trimethylsilyl)(2,2,3,3-²H₄)propionate (5·8 mmol/l) (Cambridge Isotope Laboratories) as an internal standard. Finally, 560 µl of the mixture were transferred to an NMR tube (5 mm) and capped for analysis.