TMAO Measurement

Participants were asked to fast ≥12 hours and avoid heavy physical activity and smoking for the 2 hours before the exam. Blood was drawn by venipuncture and stored at 4°C. Within 90 minutes of collection, blood samples were separated through centrifugation, aliquoted into airtight vials, flash‐frozen, and stored at −70°C.

TMAO was quantified using liquid chromatography/stable‐isotope dilution/multiple‐reaction monitoring/mass spectrometry by the University of North Carolina Nutrition Obesity Research Center.11, 12 Using 3 volume units of internal standard‐spiked acetonitrile (40 μmol/L of TMAO‐d9, DLM‐4779‐1; Cambridge Isotope Laboratories, Inc., Tewksbury, MA), samples were extracted for TMAO. Samples were chromatographed on an Atlantis HILIC Silica 3‐μm 4.6×50 mm column (Waters Corporation, Milford, MA) in junction with a Waters ACQUITY UPLC system. The 5‐minute sequencing time consisted of a gradient of 5% A for 0.05 minutes, to 15% A at 0.40 minutes, to 20% A at 1.00 minutes, to 30% A at 2.00 minutes, to 45% A at 2.55 minutes, to 55% A at 2.60 minutes, at 55% A for 0.90 minutes, to 5% A at 3.55 minutes, at 5% A for the remainder of the sequence. Mobile phase A was composed of 10% acetonitrile and 90% water with 10 mmol/L of ammonium formate and 0.125% formic acid; mobile phase B was 90% acetonitrile and 10% water with 10 mmol/L of ammonium formate and 0.125% formic acid. Flow rate was kept constant at 1 mL/min, and the column manager was set at 40°C for the duration of the sequence. A Waters TQ detector equipped with an electrospray ionization probe operating in positive‐ion mode was used for mass spectrometric analysis. TMAO specificity was maintained by monitoring ion transitions from precursor to product ions for both TMAO (75→58) and the internal standard, TMAO‐d9 (85→66). Quantification was achieved by using a calibration curve constructed from the peak area ratios of TMAO to its internal standard. Assay quality assurance was monitored by routine analysis of pooled quality control (QC) plasma at 2 concentrations of TMAO: a low quality control (LQC) consisted of normal pooled human plasma with endogenous TMAO and a high quality control (HQC) prepared by spiking a stock solution of TMAO into pooled LQC plasma sample for a final concentration of 50.0 μmol/L. Two of each QC sample were extracted simultaneously with CARDIA samples and standards per each assay. Coefficients of variation were 6.1% for TMAO, 5.8% for LQC, and 4.11% for HQC. Limits of detection and quantification for TMAO were 0.03 and 0.06 μmol/L, respectively; the linear range was 0.06 to 500 μmol/L.13