Cholic acid

Cecal contents (5 controls and 8 from L. paracasei treated mice) from trial 3 were used to quantify primary and secondary bile acids. Bile acids analysis was performed at Bioaster (Lyon, France). Samples were prepared from 50 to 60 mg of −80 °C frozen cecal content using aqueous methanol extraction process. Bile acids extracts were analysed by LC-MS/MS on a triple quadrupole Thermo Quantum Ultra (SN: TQU00665) combined to a Dionex Ultimate 3000 HPLC system (SN: 8074045 & 8087183). Samples were separated on a C18 column (2,7 μM, 150 × 2,1 mm) from Ascentis Express using a methanol/water gradient containing 5 mM ammonium acetate and 0,012% formic acid. All bile acid standards: LithoCholic acid, ChenoDeoxyCholic acid, DeoxyCholic acid, UrsoDeoxyCholic acid, Cholic acid, GlycoDeoxyCholic acid, GlycoursoDeoxyCholic acid, GlyChenoDeoxyCholic acid, Glychocolic acid, TauroLithoCholic acid, TauroDeoxyCholic acid, TauroCholic acid, were bought from Sigma Aldrich. Results of bile salt quantification were expressed in peak area, corrected for sample weight.

Faecal bile acid content was determined using a kit from Sigma-Aldrich as previously described⁽²⁵ (link)⁾. β-Nicotinamide adenine dinucleotide hydrate (NAD), nitroblue tetrazolium chloride (NBT), diaphorase, 3α-hydroxysterol dehydrogenase (3α-HSD) and cholic acid were obtained from Sigma-Aldrich. NAD, NBT, diaphorase and 3α-HSD were prepared in 0·01 m-phosphate buffer at pH 7·0. The reaction mixture included 40 µl of sample or standard with 4 µl of Triton X-100, 50 µl of NAD (2·5 mm), 50 µl of NBT (0·61 mm), 50 µl of diaphorase (625 U/l) and 50 µl of 3α-HSD (625 U/l). The mixture was incubated for 60 min at ambient temperature, after which 40 µl of phosphoric acid (1·33 m) were added to stop the reaction. The absorbance of each reaction mixture was measured at 530 nm. cholic acid in ethanol was used to generate a standard curve and the amount of faecal bile acid obtained was determined using the standard curve. Cholesterol was determined by the same assay as for liver described above.

Pure standards of all the studied BAs, namely cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA), and their respective glycine and taurine conjugated, were purchased from Sigma-Aldrich (ST. Louis, MO, USA). All the studied BAs were identified and quantified by high-pressure liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ES-MS/MS) using a previously validated method [21 (link)] suitable for BAs determination in plasma or serum after appropriate clean-up of pre-analytical procedures. Liquid chromatography analysis was performed using an Alliance HPLC system model 2695 from Waters combined with a triple quadruple mass spectrometer QUATTRO-LC (Micromass; Waters) using an electrospray interface. BAs were separated by elution gradient mode with a mobile phase composed of a mixture ammonium acetate buffer 15 mM, pH 8.0 (Solvent A) and acetonitrile: methanol = 75:25 v/v (Solvent B). All the chromatograms were acquired in electrospray negative ionization with the mass spectrometer operating in multiple reactions monitoring mode. Up to 15 BAs where identified and quantified in plasma with a limit of quantification suitable for their accurate analysis even in patients with low BAs concentration.

BAM8-22 was purchased from Tocris (Bristol, UK) and dissolved in 1× phosphate buffered saline (PBS). Cholic acid (CA), tauro-Cholic acid (TCA), glycol-Cholic acid (GCA), chenodeoxyCholic acid (CDCA), tauro-chenodeoxyCholic acid (TCDCA), glycol-chenodeoxyCholic acid (GCDCA), deoxyCholic acid (DCA), tauro-deoxyCholic acid (TDCA), lithoCholic acid (LCA), and tauro-lithoCholic acid (TLCA) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Protease inhibitor cocktail (PIC) was purchased from Bio Vision (Milpitas, CA 95035 USA).

BALF samples were treated with equal volumes of Sputolysin^® (Calbiochem, Darmstadt, Germany) and processed as described previously [16 (link),26 (link)]. Each BALF sample was analysed blinded to patient data for the presence of 12 principal bile acids compared to purified reference standards. Cholic acid (CA), chenodeoxyCholic acid (CDCA), deoxyCholic acid (DCA), lithoCholic acid (LCA), ursodeoxyCholic acid (UDCA), glycodeoxyCholic acid (GDCA), taurochenodeoxyCholic acid, (TCDCA), taurodeoxyCholic acid (TDCA), tauroCholic acid (TCA), glycoCholic acid (GCA), taurolithoCholic acid (TLCA) were purchased from Sigma-Aldrich (Buchs, Switzerland) and tauroursodeoxyCholic acid (TUDCA) was purchased from Calbiochem. All chemicals used were HPLC grade.