Fig 1. Graphic design of the workflow for urines preparation and ¹H-NMR spectra processing.

Urine samples were centrifuged and 700 μl of the supernatant were added to 0.2 mL of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) sodium salt 10 mM in deuterated water. The pH values of the solutions were adjusted to 7.0 by means of NaOH 1M and the samples were subjected to ¹H-NMR spectroscopy. The spectra obtained by ¹H-NMR were aligned and baseline-adjusted (normalization) and the signals were assigned to a specific metabolite by comparing their chemical shift and multiplicity with dedicated databases and libraries. Quantification of the molecules was achieved after the calculation of the area under each peak by means of a rectangular integration. The added TSP, at a known concentration, was employed as internal standard.