Detection of trimethyllysine and trimethylamine N-oxide

Fasting serum blood samples were collected and frozen at –80^°C until analysis to avoid oxidation or modification. None of the participants performed any exercise prior to blood collection. Levels of TML and TMAO in serum were detected by liquid chromatography-mass spectrometry (LC-MS). Isotopically labeled TML-d9 (sc-475693, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TMAO-d9 (sc-475042, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as internal standards. 150 μl of TML-d9 (20 μg/ml, dissolved in ultrapure water) and 500 μl of TMAO-d9 (1 ug/ml, dissolved in ultrapure water) were added to 200 ml of pure methanol, then vortexed and mixed to obtain the internal standard dilution working solution (ISWS). 50 μl of serum sample was placed in a 96-well plate, and 400 μl of ISWS was added to each sample, vortexed and mixed for 15 min, followed by centrifugation at 3,000 g for 15 min at 4^°C. 150 μl supernatant was then transferred to the 96-well plate and tested on the machine.

Samples were separated on a 1.7 μm (2.1 × 50 mm) column (ACQUITY UPLC BEH Amide, Waters, Milford, MA, USA) through the high-performance liquid chromatography (UltiMate 3000, Dionex, Sunnyvale, CA, USA) using an elution gradient of two mobile phases (A) 10 mM ammonium acetate 0.1% formic acid and (B) 10 mM ammonium acetate 95% acetonitrile 0.1% formic acid. Quantification was performed with a triple quadrupole mass spectrometry (TSQ Vantage, Thermo Scientific, Waltham, MA, USA) with positive electrospray ionization in the multiple reaction monitoring mode where the precursor ion (parent ion) of the target substance was first screened by the quadrupole and the fragment ions of the precursor ion were filtered through QQQ to select a desired characteristic fragment ion for quantification. The retention times of TML and TMAO were 2.54 and 1.5 min, respectively. The MRM-transitions monitored were for TML m/z 189 > 84, for TML-d9 m/z 198 > 84, for TMAO m/z 76 > 58, and for TMAO-d9 m/z 85 > 66. The peak area of each analyte was corrected by the peak area of the internal standards compound, and the concentrations of TML and TMAO in the serum sample were calculated semi-quantitatively by the ratio of the peak area of each analyte to the internal standards compound, and further quantified according to the standard concentration.

The same volume of serum was drawn from each sample, pooled and mixed, which is the quality control (QC) sample. During the testing process, every 12 samples were followed by a QC sample. The samples and the machine can be considered stable if the relative standard deviation (RSD) of QC samples were below 15%. And the RSD of TMAO and TML in the QC sample was 6.51 and 3.71%, respectively.