Sample preparation and 1H-NMR acquisition

Serum samples were immediately stored at −80°C after collection. At the time of NMR analysis, samples were thawed on ice. 150 μL of 100% D₂O buffer (40 mM TSP, 75 mM Na₂HPO₄, pH 7.4) were added to 500 μL of serum. Samples were filtered through a centrifugal filter (cut off 10 kDa) to remove macromolecules. After this, 550 μL of the mixture were transferred to a 5-mm NMR tube for analysis. ¹H-NMR spectra were acquired using a Bruker Avance II 600 MHz spectrometer equipped with a room temperature HCN inverse Z-gradient probe. at 37°C. A standard nuclear overhauser effect spectroscopy (NOESY) experiment [24] was acquired for each sample with a total of 64 accumulations and 72k data points over a spectral width of 20 ppm. A 4-second relaxation delay was included between free induction decays. The water presaturation pulse of 25 Hz was applied throughout the relaxation delays to improve solvent suppression. In addition, for assignment purposes, homonuclear 2D ¹H-¹H total correlation spectroscopy and 2D ¹H, ¹³C heteronuclear single quantum correlation spectra were acquired for selected samples. All spectra were multiplied by a line-broadening factor of 1 Hz and Fourier transformed. Spectra were automatically phased and baseline corrected, chemical shift referenced internally to the methyl group signal of alanine at 1.47 ppm using TOPSPIN 3.0 (Bruker Biospin).