2 methylbutyric acid

SCFAs were extracted and analyzed according to a modified version of the method described by Zhao et al.^{45 (link)}. Briefly, each sample was thoroughly mixed using a vortex for 5 min. An aliquot of 0.5 ml of the mixed fecal solution was taken. Its pH was adjusted to 2–3 by adding 150 μl of orthophosphoric acid (16% v/v), after which it was kept at room temperature for 10 min with occasional shaking. The suspension was transferred into a polypropylene tube and centrifuged at 4 °C for 5 min at 10,000 rpm. The supernatant was transferred to a chromatographic vial for gas chromatography analyses, and 2-methyl-butyric-acid (#109959, Sigma-Aldrich, USA) was added to each vial in order to reach a final concentration of 0.001 M. 2-methyl-butyric-acid was added as an internal standard (IS) to correct for injection variability between samples and minor changes in the instrument response. All vials were stored at − 20 °C before GC analysis. The feces sediment was dried at 60 °C for 5–7 days, and its weight determined.

SCFAs were extracted from 22 fecal samples (two from each of 11 participants, at T0 and T1) and analyzed as previously described (36 (link)). Briefly, each sample was thoroughly mixed using a vortex for 5 min. An aliquot of 0.5 mL of the mixed fecal solution was taken. Its pH was adjusted to 2–3 by adding 150 μL of orthophosphoric acid (16% v/v), after which it was kept at room temperature for 10 min with occasional shaking. The suspension was transferred into a polypropylene tube and centrifuged at 4°C for 5 min at 10,000 rpm. The supernatant was transferred to a chromatographic vial for gas chromatography analyses, and 2-methyl-butyric-acid (#109959, Sigma-Aldrich, USA) was added to each vial, to reach a final concentration of 0.001 M. To correct for injection variability between samples and minor changes in the instrument response, 2-methyl-butyric-acid was added as an internal standard. All the vials were stored at −20°C before gas chromatography mass spectrometry (GC–MS) analysis. The fecal sediment was dried at 60°C for 5–7 days, and its weight determined.

Acetic acid (C2), propionic acid (C3), butyric acid(C4), isobutyric acid (C4; 2-methylpropionic acid), 2-methylbutyric acid (C5), isovaleric acid (C5; 3-methylbutyric acid), valeric acid (C5; pentanoic acid), 2,2-dimethylpropionic acid (C5), caproic acid (C6;hexanoic acid), 2,2-dimethylbutyric acid(C6), 2-ethylbutyric acid (C6), and 2-methylvaleric acid (C6; 2-methylpentanoic acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isotope-labeled internal standards, including Acetic acid-d₃ (C2-2,2,2-d₃), propionic acid-d₆ (C3-d₆), butyric acid-d₇ (C4-d₇), valeric acid-d₄ (C5-2,2,3,3-d₄), and caproic acid-d₅ (C6-5,5,6,6,6-d₅), were purchased from Sigma-Aldrich (St. Louis, MO). All of the stock solutions were prepared in water and stored at −20 °C. 4-Acetoamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Triphenylphosphine (TPP), 2,2’-dipyridyl disulfide (DPDS), and other reagents, including mobile phase solvents, were from Sigma-Aldrich or J. T. Bakers (Center Valley, PA, USA).