Quantifying Fecal Short-Chain Fatty Acids

Stool SCFAs were measured using a gas chromatography-mass spectrometry (GC-MS; Gas chromatograph (type 7890A, Agilent, USA) equipped with a mass selective detector (type 5975C, Agilent, USA). The measurement details were described in our previous study (30 (link)) and Supplementary File 1. The standards, including acetate-d3, propionate-d5, butyrate-d7, valerate-d9, isobutyric acid, 2-methylbutyric acid, and 3-methylbutyric acid (isovaleric acid), were purchased from Sigma (St. Louis, MO, USA). A set of diluted standards was prepared (1,000 μg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL, 0.1 μg/mL, and 0.01 μg/mL) with ethyl acetate before analysis. Data analysis was performed with ChemStation (Agilent, CA, USA, v E.02.02.1432) and Chroma TOF software (LECO, St. Joseph, MI, USA, v 4.34). SCFA concentrations in fecal samples were reported in milligrams per gram (mg/g) of feces.