Tmao d9

TMAO and IS quantitative analysis were performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Briefly, serum samples were mixed with deuterated internal standard (TMAO-d₉ or IS-d_4, Sigma-Aldrich, Saint-Louis, MO, USA) and proteins were precipitated using cold methanol/acetonitrile (50/50 v/v). After centrifugation and dilution, supernatants were injected in an UFLC chromatographic system (Shimadzu, Kyoto, Japan) connected to a SCIEX QTRAP^® 5500 mass spectrometer (SCIEX, Toronto, ON, Canada). Chromatographic separation was performed using a Pursuit^®pentafluorophenyl (PFP) column (Agilent technologies, Santa Clara, CA, USA) in gradient conditions, and using electrospray source in positive (TMAO) or negative (IS) mode. Multiple Reaction Monitoring (MRM) transitions were m/z 76→58 and m/z 85→66 for TMAO and TMAO-d₉, respectively, and m/z 212→132 and m/z 216→136 for IS and IS-d₄, respectively. All coefficients of variation (CV) were lower than 5% in validation assays. TMAO and IS determination was performed in all AUD patients and HCat the inclusion in the study, and only in 12 AUD patients at the end of alcohol withdrawal due to low sample quantity.

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.