Sc110a

Approximately 55 mg of each sample was lysed in 600 μl ice-colded 80% methanol by Tissue-lyzer (QIAGEN Tissuelyzer, Germany) with stainless-steel beads (20 Hz for 90 s) (14 (link)). The lysate was transferred to new tubes and ultraphonic for 10 times (for a duration of 60 s each time with a 60-s interval between times) on ice, and the supernatant was collected by centrifugation (4°C, 11,180 g, 10 min). The pellet was extracted two more times following the same procedure. Pooled supernatants were centrifuged for 10 min (4°C, 11,180 g) to obtain the final extracts. Before proceeding to NMR analysis, the methanol in the final extracts was removed by a rotary evaporator (SC110A, Thermo, Germany). The remaining of the evaporated extracts were lyophilized and resuspended in 550 μl Na⁺/K⁺ buffer (0.15 M, 80% D₂O, 0.01071% TSP, pH 7.40) and cleaned by centrifugation (4°C, 11,180 g, 10 min); 500 μl of each sample was transferred to a 5-mm NMR tube for ¹H NMR detection.

Three-hundred microliters blood sample and 600 μL methanol were added into EP tube and placed at −20°C for 20 min. After centrifugation at 11,000 rpm for 30 min to take supernatant, methanol was removed with rotary evaporator (SC110A, Thermo, Germany). Then the powder sample was obtained with freeze-drying apparatus and redissolved in 0.55 mL Na⁺/K⁺ buffer (0.1 M, 50% D₂O, 0.001% TSP). After vortex shaking for 30 s, the sample was well mixed. After centrifugation (4°C, 12,000 rpm, 10 min), 0.5 mL of supernatant was taken and transferred to 5 mm nuclear magnetic tube for inspection. The 1D ¹H NMR spectra of all samples were collected on a Bruker AVIII 600 MHz NMR spectrometer (Bruker Biospin, Germany) equipped with an ultra-low temperature probe. The proton resonance frequency was 600.13 MHz and the experimental temperature was 298 K.