Quantification of Serum Choline and TMAO

Serum choline and TMAO levels were measured according published methods [5 (link)]. In brief, serum samples were prepared for analysis by precipitating proteins with 4 volumes of ice-cold methanol spiked with 2.5 μM deuterium-labeled choline and deuterium-labeled TMAO internal standards. Samples were centrifuged at 18,213×g at 4 °C for 3 min. The recovered supernatants were diluted 1:1 in uHPLC-grade water prior to screening. After sample preparation, identification and quantitation of TMAO and choline were performed using a uHPLC (Thermo Scientific/Dionex 3000) coupled to a high-resolution mass spectrometer (Thermo Scientific Q Exactive). Liquid chromatography separation was achieved on a Dikma Bio-Bond C4 column (150 mm × 2.1 mm; 3-μm particle size) using a 7 min isocratic gradient (50:50 methanol [MeOH] − water, 5 mM ammonium formate, and 0.1% formic acid). Quantitation of TMAO (76.0762) and d9-TMAO (85.1318) was performed via targeted MS/MS in positive mode using the following fragments masses: TMAO (58.0659) and d9-TMAO (68.1301). Quantitation of choline (104.1075) and d9-choline (113.1631) was performed in positive mode with full-MS scan by monitoring their exact masses.