Quantification of Fecal Short-Chain Fatty Acids

SCFAs levels were determined in the fecal supernatants by means of gas chromatography, as described by Moris et al. [36 (link)]. Briefly, cell free-supernatants (250 μl) from fecal homogenates, prepared as indicated above, were mixed with 100 μl methanol (Merck, Darmstadt, Germany), 50 μl internal standard solution (2-ethylbutyric 1.05 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA), and 50 μl of 20% v/v formic acid (Sigma-Aldrich, St. Louis, MO, USA). The mix was then centrifuged, and the supernatant was used to quantify SCFAs in a system composed of a 6890NGC injection module connected to a flame injection detector (FID) and a mass spectrometry detector (MS, 5973N) (Agilent Technologies, Madrid, Spain).