Tsp d4

After all subjects had fasted for 10 hours, venous blood samples were drawn into vacuum tubes and stored at room temperature for 30 min before centrifugation (1,200 ×g for 10 min). Plasma samples were collected in vacuum tubes, stored at −80 ℃, and transported on dry ice. Samples were collected in the blood collection room of the hospital. Plasma samples were prepared and tested using Bruker IVDr Standard Operating Procedures (19 (link)) at ProteinT Biotechnology Co., Ltd. (Tianjin, China). Samples were thawed at room temperature, and 400-µL plasma samples were mixed thoroughly with 400 µL buffer (phosphate buffer pH 7.4 containing TSP-D4; Bruker, Rheinstetten, Germany), of which 600 µL was transferred to a 5-mm NMR tube for analysis.
The test was performed on a 600 MHz NMR AVANCE III HD spectrometer equipped with a BBI probe head and a SampleJet autosampler, adjusted to 6 ℃ during the test (Bruker Biospin). Before acquisition of sample data, each sample was automatically tuned and shimmed. The free induction decay signals (FIDs) were presented in the form of a Fourier-transformed spectrum, and automatic phase and baseline correction were performed in Toppin software as Bruker IVDr. The concentrations of metabolites were expressed as mmol/L (Figure S1).

LAK2 (25 mg as formate) was dissolved in 0.6 mL of deuterium oxide containing 0.05% 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid, sodium salt (TSP-d₄, Sigma-Aldrich). Nuclear magnetic resonance (NMR) measurements were performed using an AVANCE III HD 500 MHz NMR spectrometer equipped with a QCI CryoProbe (Bruker, Billerica, MA, USA) at 298 K. The ¹H- and ¹³C-NMR chemical shifts were recorded relative to the internal reference TSP-d₄. The molecular structure of LAK2 was determined based on the NMR spectra of one-dimensional (1D)-¹H, 1D-¹³C, ¹H–¹H COSY, NOESY, ¹H–¹³C edited-HSQC, ¹H–¹³C HMBC, 1,1-ADEQUATE, and ¹H–¹⁵N HMBC. Instrument operation, data processing, and data analysis were performed using the TopSpin software 3.6 (Bruker).

Serum samples and cell supernatants were prepared and determined at ProteinT Biotechnology Co. Ltd. (Tianjin, China) in accordance with the Bruker Standard Operating Procedure for In Vitro Diagnostic Studies (IVDr SOP). Briefly, the 300 µL serum sample (thawed at room temperature) was mixed with 300 µL buffer (phosphate buffer pH 7.4, containing TSP-D4; Bruker Corp, Billerica, MA, USA), and the resulting 600 µL mixture was transferred to a 5 mm NMR tube for analysis. Detection was performed on a 600 MHz NMR Avance III HD spectrometer equipped with a BBI probe and SampleJet autosampler, which was adjusted at 6 °C during detection (Bruker Biospin, Rheinstetten, Germany). Each sample was automatically tuned and homogenized prior to collection. Free induction decays were presented in spectral form after Fourier transform and automatically phased and baseline-corrected in Topspin software (Bruker Biospin, Rheinstetten, Germany) as Bruker IVDr. The metabolite concentration is expressed as mmol/L.