Quantifying Plasma TMAO Levels

Blood samples were collected via radial or femoral access before heparinization using vacutainer tubes containing EDTA. Samples were maintained at 4°C, processed within 3 h, and then stored at −80°C until further analysis. Plasma levels of TMAO were quantified by stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry using an API 3200 triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA) with a d9-(trimethyl)-labeled internal standard as described previously (13 (link)). The estimated glomerular filtration rate (mL/min per 1.73 m²) was calculated using the Modification of Diet in Renal Disease study equation (14 (link)).