Acetic acid

Reagents: Folin-Ciocalteu, sodium persulfate, gallic acid (GA), BHT, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid, diammonium salt (ABTS), Trolox, ferrous chloride, and 2,2-diphenyl-1-picrylhydrazyl (DPPH), were purchased from Sigma-Aldrich (Athens, Greece); 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) was purchased from Fluka Chemica (Athens, Greece). Dimethylsulfoxide (DMSO), (Merck KGaA, Darmstaadt, Germany) was used as a solvent. Water was obtained from a Milli-Q water purification system (TGI Pure Water Systems, Topway Global, Greenville, SC, USA). Lichrosolv hypergrade for LC-MS acetonitrile was supplied by Merck (Darmstadt, Germany). Water (18.2 MΩ) was from a Milli-Q water system (Millipore, Bedford, MA, USA). Acetic acid was from LGC Standards (Middlesex, UK). Eighteen standard phenolic compounds were used (gallic acid, protocatechuic acid, HydroxyTyrosol, 4-hydroxybenzoic acid, chlorogenic acid, gentisic acid, caffeic acid, vanillic acid, syringic acid, p-coumaric acid, rutin, ferulic acid, trans-m-hydroxycinnamic acid, o-coumaric acid, salicylic acid, luteolin, eriodictyol and cinnamic acid) purchased from Sigma Aldrich.

SCFA was analyzed in the training cohort. 0.2 g of each fecal sample was separated. Six analytes were targeted for SCFA analysis including acetic acid (Dr. Ehrenstorfer, Germany), propionic acid (Dr. Ehrenstorfer, Germany), isobutyric acid (Supelco, United States), butyric acid (Dr. Ehrenstorfer, Germany), isovaleric acid (Sigma-Aldrich, United States), and valeric acid (Nu-Chek, United States). fecal samples were homogenized in 1.0 mL of ultrapure water that contained an internal standard, 2,2-dimethylbutyric acid (Dr. Ehrenstorfer, Germany). The supernatant was transferred into a new tube after centrifugation, followed 10 µL of 50% sulfuric acid and 0.5 g of sodium sulfate (Macklin, China) were added to the tube along with analytically pure diethyl ether (2 mL). The mixture was vortexed for 1 minute and then centrifuged at 5000 rpm (room temperature, 10 minutes). The ether layer was finally collected for gas chromatography with mass selective detection (5977B GC/MSD, Agilent Technologies, Santa Clara, CA, United States) measurement. The Gas chromatography mass spectrometry (GC/MS) data were acquired and analyzed using MassHunter Workstation software (Agilent Technologies) running on Windows 7 (Microsoft, Redmond, WA, United States). Final concentrations were calculated based on internal standards and are expressed as micromoles per gram of wet feces (μmol/g).