Isohexanoic acid

HPLC-grade SCFA standards (>99% pure), including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, isohexanoic acid, and 1,3-butanediol (the internal standard), were purchased from Sigma-Aldrich (St. Louis, MO, USA). The SCFAs in feces were determined using a gas chromatograph with some modification. Briefly, 0.5 g of feces was placed in a 10 mL centrifuge tube and mixed with 10 mL of ultrapure water. After homogenization and centrifugation, the fecal supernatant was diluted with ultrapure water and filtered through a 0.22 μm filter. Filtrates were placed in headspace vials and injected into the Agilent 8890B gas chromatography system coupled to an Agilent 5977B/7000D mass selective detector with an inert electron impact (EI) ionization source at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The Dionex ICS-3000 ion chromatography system with an auto-sampler was used. The column used was the HP-FFAP (30 m × 0.25 mm × 0.25 µm) capillary column. The injector was split at 1:10. The carrier gas was nitrogen at a flow rate of 1.0 mL/min. Peaks corresponding to each SCFA were quantified using standard curves.

Fecal SCFA analysis was conducted using gas chromatography-mass spectrometry (GC-MS). An Agilent HP-INNOWax GC-MS (Agilent Technologies, Inc., Santa Clara, CA, USA) was used for quantification. To 50 mg of sample, 50 μL of 15% phosphoric acid (China National Pharmaceutical Group Co., Ltd., Beijing, China), 100 μL of 125 μg/mL internal standard solution (isohexanoic acid, >98%; Sigma), and 400 μL of ether (China National Pharmaceutical Group) were added and homogenized for 1 min and centrifuged at 4°C at 12,000 rpm for 10 min, and the supernatant was taken for the test. Acetic acid (AA, >99.5%), propionic acid (PA, >99.0%), isobutyric acid (IBA, >99.0%), butyric acid (BA, >99.0%), isovaleric acid (IVA, >99.0%), and valeric acid (VA, >98.0%) were purchased from Sigma. Caproic acid (CA, ≥99.5%) was purchased from Aladdin Shanghai Biochemical Technology (Shanghai, China).

Sweet tea was collected from Sichuan Mu Jiang Ye Ke Tea Co., Ltd. (Chengdu, China). Dextran sulfate sodium salt (DSS, molecular weight 36–50 kDa); salicylazosulfapyridine (SASP); and standards for SCFAs, including acetic acid, propanoic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid, and isohexanoic acid, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The commercial kits for myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), TNF-α, transforming growth factor-β (TGF-β), IL-10, IL-6, IL-1β, LPS, and urine fecal occult blood were obtained from Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). The rabbit polyclonal antibodies specific to Zonula occludens-1 (ZO-1), Occludin, G-protein-coupled receptor 43 (GPR43), G-protein-coupled receptor 109A (GPR109A), histone deacetylase 3 (HDAC3), and NF-κB p65 were obtained from Abcam (Shanghai) Trading Co., Ltd. (Shanghai, China).