Stool collected at home was immediately frozen at −20°C and later transported to the hospital in the frozen state. The stool collected at the hospital and the stool transported to the hospital at −20°C were immediately brought to a freezer with −70°C. Within one month, S.T.A.R. (Stool Transport and Recovery; Roche, Basel, Switzerland) buffer solution was added to the samples (stool/S.T.A.R. ratio 1/3) and vortexed to a homogenous suspension and stored at −80°C.
Stool samples were analysed for short-chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) by gas chromatography. The Thermo Focus with a flame ionization detector (Thermo Fisher, Waltham, Massachusetts, USA) was used. Helium was used as carrier gas, and the column used was 30 m-long Stabilwax-DA from Restek with polyethylene glycol (PEG) as the stationary phase. The chromatography conditions were as follows: gas pressure 0.6 bar; injector temperature 210°C; start temperature 80°C; temperature program 0°C/min for 3 min, 23.3°C/min for 3 min, and 25°C/min for 2 min; end temperature 200°C; and detector temperature 230°C [27 (link)]. In addition to the individual fatty acids, the difference between propionic and butyric acids (PmB), which has been shown to be a potential biomarker for IBS, was used in the analyses [28 (link)].
Stool samples were analysed for short-chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) by gas chromatography. The Thermo Focus with a flame ionization detector (Thermo Fisher, Waltham, Massachusetts, USA) was used. Helium was used as carrier gas, and the column used was 30 m-long Stabilwax-DA from Restek with polyethylene glycol (PEG) as the stationary phase. The chromatography conditions were as follows: gas pressure 0.6 bar; injector temperature 210°C; start temperature 80°C; temperature program 0°C/min for 3 min, 23.3°C/min for 3 min, and 25°C/min for 2 min; end temperature 200°C; and detector temperature 230°C [27 (link)]. In addition to the individual fatty acids, the difference between propionic and butyric acids (PmB), which has been shown to be a potential biomarker for IBS, was used in the analyses [28 (link)].