Fecal Short-Chain Fatty Acids Quantification

Fecal SCFAs extraction was performed following De Baere et al.,^{66 (link)} protocol. SCFAs concentrations were measured using high-performance liquid chromatography UltiMate 3000 UHPLC system (HPLC-UV) (Thermo Scientific, Breda, The Netherlands) with external calibration standards curve method as previously described in details.^{67 (link)}Briefly, calibration standards were prepared at concentrations ranging from 0.5 mM to 50 mM for acetic acid (AA), butyric acid (BA), propionic acid (PA), and succinic acid (SA) as internal standard (all purchased from Sigma-Aldrich, St. Louis, MO, USA). After chromatographic separation testing on a Hypersil Gold aQ column (150 × 4.6 mm i.d.) with a 3 μm particle (Thermo Scientific, Breda, The Netherlands), HPLC-UV was performed on thermostated and guard column protected HPLC columns using UV detection at 210 nm. The mobile phase consisted of 20 mM of sodium dihydrogen phosphate (Merck, Darmstadt, Germany) in HPLC water (pH 2.2) (Merck) (A) and HPLC grade acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) (B).
HPLC-UV data were processed using Chromeleon version 6.8 software (Thermo Fisher Scientific, MA, USA). SCFAs concentration were calculated using mathematical equation: SCFA (AA, BA, PA) = (organic acid in fecal sample × 6 × 10⁻³)/(succinic acid in fecal sample × fecal sample mass) × 1000 [mmol/kg]. All measurements were done in triplicate.