Isobutyrate

Methanol (MeOH), acetonitrile and formic acid were of HPLC grade. Analytical grade of NaOH, propan-1-ol, pyridine, hexane and propylchloroformate (PCF) were purchased as well from Sigma-Aldrich (Saint Quentin Fallavier, France). Deionized water comes from a Milli-Q Elix system fitted with a LC-PaK and a MilliPak filter at 0.22 μm (Merck Millipore, Guyancourt, France). The following bile acid standards: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), lycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), and taurolithocholic acid (TLCA) and ammonium acetate were purchased from Sigma Chemical (St. Louis, MO, USA). All tryptophan reference, isotope labeled metabolites and SCFAs (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, acetate-D3, butyrate-13C2 and valerate-D9) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The stock solutions of bile acids, tryptophan metabolite and SCFAs were prepared separately in methanol at the concentration of 10 mmol/l.

Prior to analysis, 100 mg frozen stool sample was mechanically homogenized using 10% isobutanol and ceramic beads. Formate, acetate, propionate, butyrate, isobutyrate, and valerate, in addition to the amino acids alanine, L-arginine, L-cystine, L-glutamic acid, L-leucine, L-lysine, L-serine, L-threonine, L-tyrosine, L-valine, and L-histidine, were obtained from Sigma-Aldrich (St. Louis, MO, USA) for generation of calibration curves. Samples and calibration standards were derivatized with chloroFormate isobutyl. Metabolites were extracted and derivatized as previously described (Kulecka et al., 2023 (link)).
Gas chromatographic analysis was performed using an Agilent 7000D Triple Quadrupole mass spectrometer coupled with a 7890 GC System with G4513A autosampler (Agilent Technologies, Santa Clara, CA, USA). MS data were analyzed using MassHunter software (Agilent Technologies, Santa Clara, CA, USA).

In each digesta sample, the main concentrations of SCFAs was separated and quantified by using a gas chromatograph (CP3800, Varian) with capillary column 30 m × 0.53 mm × 1 μm film thickness (HP-FFAP) and flame ionization detector, 250°C according to previous study (Zamora-Gasga et al., 2014 (link)). Standard samples (e.g., acetate, propionate, butyrate, isovalerate, isobutyrate, and valerate, Supelco, Sigma-Aldrich Trading Co., Ltd., Shanghai, China) were used. Briefly, 0.7 g of sample was collected in a centrifuge tube, and was mixed with ultrapure water (1.5 ml) for 30 min and centrifuged (1,000 g, 15 min). The supernatant (1 ml) was added 0.2 mL of 25% (w/v) metaphosphate solution (Tianjin Kemiu Chemical Reagent Co., Ltd., Tianjing, China) and 23.3 μl of 210 mmol/l crotonic acid solution (Sigma-Aldrich Trading Co., Ltd., Shanghai, China), and the mixture was incubated for 30 min and then centrifuged at 8,000 g for 10 min. The supernatant (0.3 ml) was mixed with 0.9 ml of chromatographic methanol (Thermo Fisher Scientic Inc., Waltham, MA, United States) at 8,000 g for 5 min. One milliliter of the supernatant was subjected to capillary gas chromatography (CP3800, Varian).