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Before analysis, plasma samples were thawed at 4°C. Quality control (QC) samples were prepared by collecting an equal volume from each plasma sample. For protein precipitation, 150 µL of cold methanol was added to 50 mL of plasma and vortexed for 10 min. After centrifugation at 21,300 g for 15 min at 4°C, 100 µL of the supernatant was transferred to a 2.0 mL polypropylene tube. The samples were evaporated using a speed vacuum at 45°C for 2 h. Following reconstitution with 200 µL of methanol and vortexing for 10 min, the residue was centrifuged at 21,300 g at 4°C. The samples were then transferred to an autosampler vial for analysis. , For PP sample preparation, 50 μL of saliva was mixed with 100 μL of methanol (1:1, v/v). The mixture was vortexed for 5 min and then quenched at −20°C for 2 h. After quenching, the sample was centrifuged at 21,300 × g for 15 min. Subsequently, 100 μL of the supernatant was transferred to a 2.0 mL E-tube and dried using a SpeedVac at 45°C and 5.1 Torr for 3 h. The dried sample was reconstituted with 100 μL of water (1:1, v/v) and vortexed for 5 min. Finally, 50 μL of the reconstituted sample was transferred to an LC vial for LC analysis. , Urine samples were thawed at 4°C before analysis. For each subject, 50uL of urine samples were prepared. Quality control (QC) samples were mixed with equal amounts of urine from all participants. To precipitate proteins, 100uL of cold acetonitrile was added to each urine sample. The samples were mixed using a vortex mixer for 15 min and then centrifuged at 16,000 g for 10 min at 4°C. The protein-removed samples were transferred to new 2 mL tubes and dried for 3 hours at 45°C using a speed vacuum. For reconstitution, 150uL of cold DIW was added to the residual sample and mixed using a vortex mixer for 10 min. The resulting 150uL of sample was transferred to a vial, and the analysis was started.

Ultra-high performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UHPLC-QTOF/MS) was utilized for untargeted metabolic profiling. Thermo Scientific Dionex UltiMate 3000 UHPLC (Dionex Softron GmbH, Germering, Germany) with a Waters ACQUITY UPLC® BEH C18 column (100 mm × 2.10 mm, 1.7 mm, 130 Å; Waters, Milford, MA, USA) was used to separate plasma samples. The temperature of the column oven was 50°C, and the autosampler was 4°C. Injection volume and flow rate were 2 uL and 0.2 mL/min, respectively. For positive ionization mode, the mobile phase consisted of 0.1% formic acid in deionized water (A) and 0.1% formic acid in methanol (B). The gradient was as fol- lows: 0.0–0.5 min, 1% B; 0.5–1.0 min, 65% B; 1.0–4.0 min, 90% B; 4.0–12.0 min, 99% B; 12.0–22.0 min, held at 99% B; 22.0–22.5 min, 1% B; and 22.5–26.5 min, 1% B. For negative ionization mode, the mobile phases comprised 0.02% acetic acid in deionized water (A) and 0.02% acetic acid in acetonitrile (B). The gradient was as follows: 0.0–0.5 min, 1% B; 0.5–1.0 min, 65% B; 1.0–4.0 min, 90% B; 4.0–19.0 min, 90% B; 19.0–19.5 min, 99% B; 19.5–22.5 min, held at 99% B; 22.5–23.0 min, 1% B; and 23.0–28.5 min, 1% B. , UHPLC analysis was performed using an ACQUITY UPLC® BEH C18 column (100 × 2.10 mm, 1.7 μm, 130 Å) at a column oven temperature of 40°C. The mobile phase consisted of 0.1% formic acid in water (Pump A) and 0.1% formic acid in acetonitrile (Pump B). The autosampler temperature was maintained at 8°C. The injection volume was 10 μL in positive mode and 5 μL in negative mode. The flow rate was maintained at 0.3 mL/min throughout the analysis. The gradient program was as follows: 1% B from 0 to 2 min, increased to 10% B at 6.5 min and maintained at 10% B until 10 min, followed by an increase to 30% B at 16 min and to 99% B at 18.5 min. The mobile phase was maintained at 99% B until 20.5 min, decreased to 1% B at 20.6 min, and maintained at 1% B until the end of the analysis at 23.5 min. , Untargeted metabolic profiling was conducted using ultra-high performance liquid chromatography quadrupole time-of-flight/mass spectrometry (UHPLC-QTOF/MS). Chromatographic separation was performed using a Bruker Elute UHPLC (Dionex Softron GmbH, Germering, Germany) equipped with a Waters ACQUITY UPLC® BEH C18 column (100 mm × 2.10 mm, 1.7 um, 130 Å; Waters, Milford, MA, USA). The column oven and auto-sampler were maintained at 40°C and 4°C, respectively. The mobile phases consisted of DIW for A and acetonitrile for B. In positive ionization mode, 0.1% formic acid was added to both mobile phases. While in negative ionization mode, 0.1% acetic acid was added to both mobile phases. The gradient was consistently applied in both modes: 1% B, 0.0–0.5 min; 1–20% B, 0.5–2.5 min; 20–30% B, 2.5–4.0 min; 30–60% B, 4.0–11.5 min; 60–80% B, 11.5–13.0 min; 80% B, 13.0–18.0 min; 80–1% B, 18.0–18.5 min; and 1% B, 18.5–22.0 min. The injection volume and flow rate were 2 uL and 0.15 mL/min, respectively.

MS analysis was performed on a Bruker Compact™ mass spectrometer (Bruker Dal- tonics GmbH & Co., KG, Bremen, Germany) equipped with an electrospray ionization source. The MS parameters were set as follows: mass scan range, 50–1000 m/z; nebulizer gas pressure, 0.8 bar; dry gas flow rate, 10.0 L/min; dry gas temperature, 200 ◦ C; capillary voltage, ±4500 V; and end-plate offset, −500 V. , Mass spectrometric analysis was performed using a timsTOF Pro² (Bruker) mass spectrometer equipped with an electrospray ionization (ESI) source operated in both positive and negative ionization modes. Data were acquired in full-scan mode over a mass-to-charge ratio (m/z) range of 50–1,000. The dry gas temperature was set to 220°C, with a dry gas flow rate of 10 L/min and a nebulizer gas pressure of 2.2 bar. The end plate offset was set to 500 V. The capillary voltage was set to 4,500 V in positive mode and 4,000 V in negative mode. , The MS analysis was conducted using an Impact II mass spectrometer (Bruker Daltonics GmbH & Co. KG, Bremen, Germany) equipped with an electrospray ionization source, operating in both positive and negative modes. The MS parameters were set as follows: the mass scan range, 50– 1000 mass-to-charge ratio (m/z); the nebulizer gas pressure, 2.2 bar; the dry gas flow rate, 10.0 L/min; the dry gas temperature, 220°C; the capillary voltage, ±4,500 V; and the end plate offset, 500 V.