D9 betaine

Fasting blood samples were collected as soon as possible once recruited before any anticancer therapies. Serum concentrations of choline and betaine were assessed by high-performance liquid chromatography with online electro-spray ionization tandem mass spectrometry to measure the concentrations of choline, and betaine. Detailed protocol of choline measurement has been described previously [19 (link)]. Detection of serum betaine was similar to choline, except for the internal standard was d9-betaine (obtained from Sigma-Aldrich, St. Louis, USA). The coefficients of variation for the between-run assays were 4.91 and 6.21% for choline and betaine, respectively.

Blood containing EDTA as an anticoagulant was drawn at different time points and immediately centrifuged. Plasma was subsequently collected and frozen at − 80 °C until analysis. SAM, SAH, choline, d₉-choline, betaine, d₉-betaine, dimethylglycine (DMG), ethanolamine (EA), sarcosine, d₃-sarcosine, methionine, L-homocystine, dithiothreitol (DTT), creatine, d₃-creatine, guanidinoacetate (GAA), ¹³C₂-GAA and MMA were obtained from Sigma-Aldrich (Saint Louis, USA). d₃-SAM, d₆-dimethylglycine, d₄-ethanolamine, d₃-methionine, and d₈-DL-homocystine were purchased from CDN-isotope (Pointe-Claire, Canada). D₄-SAHwas bought by cayman chemical (Ann Arbor, USA).
Free choline, ethanolamine, creatine, GAA, and MMA were assayed after deproteinization by an LC–MS/MS method adapted from Holm et al. [19 (link)], Imbard et al. [20 (link)], and Cognat et al. [21 (link)]. The analytical system consisted of an Acuity UPLC I Class system (Waters, Milford, USA) coupled with a Xevo-TQD (Waters, Milford, USA) with an Atlantis HILIC analytical column (2.1 × 100 mm, 3 µm) (Waters, Milford, USA).
SAM, SAH, tHcy, Met, betaine, DMG, and sarcosine were measured using new LC–MS/MS developed in our laboratory (detailed in the Additional file 1).

Fasting serum was isolated and stored at −80 °C until analysis. Serum concentrations of TMAO, choline and betaine were quantified by high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (HPLC-MS/MS) (Agilent 6400 Series Triple Quad LCMS; CA, USA)20 (link). A volume of 60 μl of either the serum sample or standards was combined with 100 μl of acetonitrile containing 10 μM of internal standards [d9-choline, d9-betaine (Sigma-Aldrich, St. Louis, USA) and 9-TMAO (Toronto Research Chemicals Inc, Toronto, Canada)], and the sample was centrifuged at 13,000 × g for 10 min to precipitate the proteins. Finally, after an additional centrifugation step, the supernatant was analyzed after injection into a normal-phase silica column (2.1 mm × 100 mm, 5 μm) and equilibrated with 30% solution A (15 mmol/L ammonium formate in water, pH 3.0) and 70% solution B (acetonitrile) under isocratic elution with the flow rate of 0.2 mL/min. The coefficients of variation for the between-run assays were 6.0%, 4.91% and 6.21% for TMAO, choline and betaine, respectively.
Overnight fasting serum total cholesterol (TC), triglyceride (TG), LDL cholesterol (LDLc), HDL cholesterol (HDLc) and fasting blood glucose were measured by colorimetric methods using a Hitachi 7600-010 automated analyzer.