Heptanoic acid

All the HPLC-grade (methanol, acetonitrile, hexane) and analytical-grade (orthophosphoric acid, ethanol, ethyl acetate, sodium hydroxide, sodium thiosulfate) reagents were acquired from Merck (Darmstadt, Germany). The standards used to identify and quantify the phenolic compounds (o-coumaric acid, p-hydroxyphenylacetic acid, hydroxytyrosol, tyrosol, vanillic acid, syringic acid, vanillin, hydroxytyrosol acetate, p-coumaric acid, ferulic acid, tyrosol acetate, pinoresinol, luteolin, apigenin, methyl-luteolin) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The standards used to identify and quantify the volatile compounds (4-methyl-2-pentanol, ethanol, ethyl propanoate, pentanal, 4-methylpentan-2-one, ethyl-2-methylbutyrate, butyl acetate, hexanal, 2-methylbutan-1-ol, 3-methylbutan-1-ol, (E)-2-hexenal, 3-octanone, octanal, (E)-2-heptenal, 2-heptanol, 1-hexanol, nonanal, (E)-2-nonenal, (E)-2-hexenol, acetic acid, propanoic acid, 1-octanol, butanoic acid, heptanoic acid) were purchased from Merck. Trolox, fluorescein, and 2,20-Azobis (2-amidinopropane) dihydrochloride (AAPH) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Acetic acid was purchased from Thermo Fisher Scientific (Fair Lawn, NJ). Propionic acid, isobutyric acid, butyric acid, 2-methylbutyric acid, isovaleric acid, valeric acid, 2-methylpentanoic acid, 3-methylpentanoic acid, isocaproic acid, caproic acid, 2-methylhexanoic acid, 4-methylhexanoic acid, heptanoic acid, hexanoic acid-6,6,6-d₃ internal standard, N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), and methoxyamine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO).

Cecal digesta (∼150 mg) collected from rats were weighed and diluted at 1:3 (w/w) with deionized water containing 1.68 mM heptanoic acid/L as an internal standard (Sigma Chemical Co., St. Louis, MO, United States). Acetate, butyrate, propionate, and the total SCFA (including minor SCFA) were measured using a gas chromatography (GC) system based on our previous published methods (Wang et al., 2017 (link)).

Fermentation supernatant (FS) samples were homogenized in three volumes of internal standard solution (heptanoic acid, 1.68 mmol/L) (Sigma-Aldrich) and centrifuged at 3000× g for 10 min. The supernatant was then distilled and 0.3 µL was injected into a gas chromatograph (Hewlett-Packard 5890 Series II A, Wilmington, DE, USA) equipped with a flame ionization detector and a capillary column (Zebron ZB-FFAP, 30 m × 0.53 mm i.d., 1-µm film, SGE, Phenomenex, Torrance, CA, USA). Helium was used as the carrier gas; the initial oven temperature was 120 °C and was increased at 30 °C/min to 190 °C; the injector temperature was 210 °C and the detector temperature was 210 °C. A standard SCFA mixture containing acetate, propionate, and butyrate (Sigma-Aldrich) was used for calculations, and the results are expressed as µmol/g of sample [31 (link)].

Frozen stool samples were extracted for short chain and branched chain fatty acids (SCFA and BCFA, respectively) using acidified water (pH 2.5), as described previously [25 (link)]. Peak areas were normalized to the total signal and represented as a percentage of total SCFA. Commercial standards of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, caproic acid and heptanoic acid (Sigma, St. Louis, MO, USA) were used to confirm compound identities.

1,3,5-Tris(4-phenylboronic acid) benzene (BLDpharm, 98%), heptanoic acid (Sigma-Aldrich, ≥99%), octanoic acid (Sigma-Aldrich, ≥ 99%), methyl octanoate (TCI, >99%), 1,2,4-trichlorobenzene (Sigma-Aldrich, ≥99%), 1-phenyloctane(Thermo scientific, 99%) and dimethylsulfoxide (Sigma-Aldrich, ≥99%) were used directly without further purification. Solutions of 1,3,5-tris(4-phenylboronic acid) benzene were prepared by dissolving the solid compound in dimethylsulfoxide at a ratio of 1 mg/ml. The DMSO stock solution was further diluted using a specific solvent listed above to generate a concentration series (the amount of DMSO in each solution is less than 1.1%V/V) for the STM experiments. For a few STM experiments, OA was dried by stirring over freshly activated molecular sieves (Carl Rot) for 48 h and was stored in a round bottom flask over anhydrous sodium sulfate.

Faecal samples were thawed at 4°C, pooled, homogenised, and then subsampled for analysis.
For the determination of SCFA, weighed portions were diluted at 1:3 (w/w) with deionised
water containing 1·68 mmol heptanoic acid/l as an internal standard (Sigma Chemical Co.),
and processed for SCFA analysis using GC as described previously⁽⁸⁾. Total SCFA concentration was calculated as the sum of acetic, propionic,
butyric, isobutyric, caproic, isovaleric and valeric acid concentrations. Total
branched-chain fatty acids concentration was calculated as the sum of isobutyric and
isovaleric acid concentrations. Phenol and p-cresols were measured in the
faeces by using vacuum microdistillation and HPLC⁽²²⁾. Faecal NH₃ concentration was measured by using the indophenol
blue method⁽²³⁾. Aqueous extracts of the faeces were prepared by diluting 1 g faeces with
4 ml of distilled water, homogenised and centrifuged (4500 rpm, 4°C)⁽²⁴⁾, and total apparent NOC were measured by chemical denitrosation with HBr and
chemiluminescence detection of the released nitric oxide using a thermal energy analyser
(TEA)^(25,26). Concentrations were calculated by comparing the TEA response of a faecal
water sample with the response of an N-nitrosodipropylamine standard
(16·6 μg/ml), and values were expressed as total apparent NOC (ng/ml)⁽²⁷⁾.