Urine Sample Preparation for NMR Analysis

Urine samples were stored at -80 °C until analysis [1 (link)]. Samples were filtered to remove protein with Centrifree Ultrafiltration devices (Millipore, Bilerica, MA). Filters were washed 5 times to remove glycerol. Samples were filtered by centrifugation at 6000 rpm for 2–3 h at 4 °C. For NMR analysis, 1 mL of filtrate was mixed with 0.5 mL of 0.2 M sodium phosphate buffer. The solution was placed on ice for 10 min and then centrifuged at 7000 g at room temperature for 10 min. 500 μL of the supernatant was withdrawn and combined with 50 μL of 1 mM 3-(trimethylsilyl) propionic acid (TSP, Sigma-Aldrich, St. Louis, MO, USA) [9 ]. The pH of the solution was measured, then the solution was transferred to 5 mm NMR tubes for analysis.