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20 μl of serum was aliquoted to 96-well plate. 80 μl of MeOH was added to each well and vortexed for 10 minutes. After vortexing, plates were incubated in 4 ºC for 2 hours to precipitate protein. After protein precipitation, centrifuge at 3300g for 20 minutes. 12.5 μl of sample was transeferred into new 96-well plate. 87.5 μl of 80 % MeOH and 100 μl of internal standard solution in 20 % MeOH was added. , 20 μl of serum was aliquoted to 96-well plate. 80 μl of MeOH was added to each well and vortexed for 10 minutes. After vortexing, plates were incubated in 4 ºC for 2 hours to precipitate protein. After protein precipitation, centrifuge at 3300g for 20 minutes. 10 μl of sample was transeferred into new 96-well plate. 90 μl of 80 % MeOH and 100 μl of internal standard solution in 20 % MeOH was added. , For stool metabolite extraction and NMR sampling, 40 mg of each fecal sample was transferred into an Eppendorf tube. Specifically, 800 μL of 0.2 M deuterated phosphate buffer (pH = 7.4, 2 mM sodium azide) was added and the sample was ground using a homogenizer pestle. The homogenized solution was vigorously vortexed for 10 min and incubated at 4 °C for 1 h. After incubation, centrifugation was performed at 3000 rpm for 10 min followed by 12700 rpm for 10 min. A mixture of 540 μL of supernatant and 60 μL of 1 mM TSP-d4 (3-(trimethylsilyl) propionic 2,2,3,3-d4 acid sodium salt (98% atom %)) in deuterium oxide was transferred into the NMR tube , For urine NMR sampling, all urine samples were centrifuged at 12700 rpm for 10 min. Specifically, 100 μL of each supernatant was transferred into an Eppendorf tube, and 500 μL of 0.2 M deuterated phosphate buffer (pH = 7.0, 2 mM sodium azide) was added. The pH was adjusted to 7.0 ± 0.1 and 540 μL of the mixture and 60 μL of 5 mM TSP-d4 in deuterium oxide were mixed and transferred into an NMR tube.

To quantify the levels of TMAO in plasma, targeted profiling was performed on Agilent 1290 Infinity LC and Agilent 6495 Triple Quadrupole MS systems equipped with an Agilent Jet Stream ESI source (Agilent Technologies, Palo Alto, CA, USA). Chromatic separation of plasma TMAO was carried out using an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm; Waters) at 25 °C. The initial flow rate was set as 0.2 mL/min and 2 μL of samples were injected for single measurement. The mobile phases used were 0.1 % (v/v) formic acid in water (A) and 0.1 % (v/v) formic acid in methanol (B). The linear gradient of mobile phases was as follows: 0–2 min, isocratic 5 % B; 2–3 min, 5–95 % B; 3–6 min, isocratic 95% B; 6–6.1 min, 95–5 % B; 6.1–9 min, and isocratic 5 % B. Phenylalanine-13C6 and Leucine-13C6 were used as internal standards. Pooled QC samples were analyzed for every 11 samples, and coefficients of variation (CVs) of QC samples were calculated to check the reproducibility of the results.

Dynamic multiple reaction monitoring (dMRM) mass spectrometry mode was used for the analysis of TMAO, phenylalanine-13C6 and leucine-13C6 MS/MS at m/z 76 → 58, m/z 172 → 126, and m/z 138 → 91, respectively. The calibration curve of TMAO was obtained by using TMAO standard solutions of various concentrations.

For stool metabolite extraction and NMR sampling, 40 mg of each fecal sample was transferred into an Eppendorf tube. Specifically, 800 μL of 0.2 M deuterated phosphate buffer (pH = 7.4, 2 mM sodium azide) was added and the sample was ground using a homogenizer pestle. The homogenized solution was vigorously vortexed for 10 min and incubated at 4 °C for 1 h. After incubation, centrifugation was performed at 3000 rpm for 10 min followed by 12700 rpm for 10 min. A mixture of 540 μL of supernatant and 60 μL of 1 mM TSP-d4 (3-(trimethylsilyl) propionic 2,2,3,3-d4 acid sodium salt (98% atom %)) in deuterium oxide was transferred into the NMR tube , For urine NMR sampling, all urine samples were centrifuged at 12700 rpm for 10 min. Specifically, 100 μL of each supernatant was transferred into an Eppendorf tube, and 500 μL of 0.2 M deuterated phosphate buffer (pH = 7.0, 2 mM sodium azide) was added. The pH was adjusted to 7.0 ± 0.1 and 540 μL of the mixture and 60 μL of 5 mM TSP-d4 in deuterium oxide were mixed and transferred into an NMR tube.

1D-NMR: One-dimensional 1H NMR spectra of serum were acquired at 298 K on a Bruker Avance III HD 800 MHz NMR spectrometer (Bruker BioSpin, Germany) with a Bruker 5 mm CPTCI Z-GRD probe.