Quantification of Plasma TMAO and MPO

Blood samples were collected via radial or femoral access before the interventional procedure using vacutainer tubes containing ethylenediaminetetraacetic acid (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. (Wang et al., 2014a (link)). Plasma MPO was measured by enzyme-linked immunosorbent assay using a commercial kit (DMYE00B, R&D Systems, United States), according to procedures recommended by the manufacturer. Duplicates of 20 randomly selected samples and all samples were measured to evaluate the intra-assay coefficient variation of TMAO and MPO, respectively. The estimated glomerular filtration rate (eGFR; ml/min per 1.73 m²) was calculated using the Modification of Diet in Renal Disease study equation (Levey et al., 2009 (link)).