NMR Serum Sample Preparation

Each serum sample (100 μL) was mixed with 500 μL of deuterium oxide (²H₂O) (ISOTEC, Sigma-Aldrich, St. Louis, MO, USA) and pipetted into a 5-mm (outer diameter) NMR tube (Wilmad-LabGlass, Vineland, NJ, USA) for NMR analysis [23 (link)]. Solution-state NMR analyses were performed at a proton resonance frequency of 400 MHz (9.4 Tesla) using an ECZ^™ NMR spectrometer (JEOL Ltd., Tokyo, Japan) interfaced with a probe (digital auto-tunable type [NM-03812RO5S]) and equipped with Delta^™ NMR processing and control software, version 5.3.2 (JEOL Ltd.). The field was locked to the ²H resonance of the ²H₂O solvent. One-dimensional proton NMR signals were automatically acquired at a probe temperature of 30°C using the program supplied by JEOL that supported the macro function in Delta^™. Free induction decay (FID) data were acquired using a single pulse with a 2.0-s relaxation delay between repeated pulse sequences. The strong signal arising from free water was suppressed using DANTE presaturation. Other conditions were as follows: radio-frequency pulse width, 2.93 μs; acquisition time, 1.636 s; repetition time, 3.636 s; spectral width, 10,016 Hz; number of data points, 16,384; and number of steady-state transients, 400. The FID data were saved in JEOL Delta format (JDF).