D9 choline

Glucose, peptone, yeast extract, KCl, NaCl, Na₂HPO₄, KH₂PO₄, MgSO₄ × 7H₂O, CaCl₂ × 6H₂O, ZnSO₄ × 7H₂O, NaHCO₃, ammonium formate, hemin, bile salts, Tween 80, vitamin K1, resazurin, L-cysteine, choline, choline-d9, L-carnitine, betaine, γ-butyrobetaine, TMAO, TMA, 3,3-dimethyl-1-butanol (DMB), gallic acid, chlorogenic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, ammonia and ethyl bromoacetate were purchased from Sigma-Aldrich/Millipore (St. Louis, MO, USA). Choline-d₉, L-carnitine-d₉, betaine-d₉, TMAO-d₉ and TMA-d₉ (internal standards; IS) were also purchased from Sigma-Aldrich. Acetonitrile and water (LC-MS grade) as well as dimethyl sulfoxide (DMSO; reagent grade) were purchased from VWR International (Suwanee, GA, USA). Fecal samples from two different healthy donors (ID: 0105-0003-11 and 0128-0001-01) were obtained from OpenBiome (Cambridge, MA, USA). OpenBiome collects samples from rigorously screened donors, for which the health histories, clinical data, pathogen screen results and 16S rDNA sequences are available. Samples are processed in sterile 12.5% glycerol and 0.9% saline buffer at 2.5 mL of buffer per gram of stool, and filtered through a 330 µm filter to remove large particulate matter, and frozen at −80 °C until use.

Fasting serum samples were isolated and stored at − 80 °C until analysis. Serum TMAO and choline were assessed using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (HPLC-MS/MS) (Agilent 6400 Series Triple Quad LCMS; CA, USA) [29 (link)], using multi-reaction monitoring (MRM) functions. 100 μl of acetonitrile containing 10 μM of internal standards [d9-TMAO (Toronto Research Chemicals Inc., Toronto, Canada), d9-choline (Sigma-Aldrich, St. Louis, USA)] was added to 60 μl of either the serum sample or standards. The samples were then centrifuged at 13,000×g for 10 min to precipitate the proteins. Finally, the remaining supernatant was injected 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 a flow rate of 0.2 mL/min. Ten pairs of duplicate control samples were randomly interspersed to assess laboratory precision. The coefficients of variation for the between-run assays were 6.0 and 4.9% for TMAO and choline, 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.