Deoxycholic 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.

GPMVs were isolated from cultured HUVEC as previously described^{18 (link),69 (link)}. Briefly, cells were cultured up to 70% confluency, washed twice with GPMV buffer (150 mM, NaCl, 2 mM, 10 mM HEPES, CaCl2, pH 7.4) and incubated with GPMV vesiculant chemical mix (GPMV buffer plus 25 mM PFA and 2 mM DTT) for 1 h at 37 °C. Subsequently, cells were placed at 4 °C and treated with 500 µM deoxycholic acid (Sigma–Aldrich) for 30 min to cause phase separation. GPMVs were then collected from the supernatant and transferred into a plastic vessel containing CHIM-L preclicked to Alexa Fluor 488 and FAST DiI, all used at a final concentration of 5 µM. Next day, GPMVs present at the bottom of the vessel were imaged at room temperature after transferring the vesicles into poly-l-lysine 0.1 % (w/v) (Sigma–Aldrich) coated eight-well μ-slide glass bottom dishes (Ibidi). The respective GMPV isolations were performed in three independent experiments.

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).

Cellfectin II reagent, pFastBac1 vector, E. coli DH10Bac cells, Sf900II SFM, and Gibco fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, USA). Spodoptera frugiperda Sf9 insect cells were obtained from the China Center for Type Culture Collection (Wuhan, China). Rabbit anti-human UGT2B7 polyclonal antibodies were purchased from ProteinTech Group, Inc. (Chicago, USA). Rabbit anti-UGT1A Polyclonal antibodies were obtained Institute of Genetics and Developmental Biology Chinese Academy of Sciences (Beijing, China). Rabbit anti-human UGT2B7 polyclonal antibodies were purchased from ProteinTech Group, Inc. (Chicago, USA).
Zidovudine and quercetin (chemical purity >98.5%) were purchased from National Institute for Food and Drug Control (Beijing, China). Zidovudine O-glucuronide was purchased from Toronto Research Chemicals (Toronto, Canada). Uridine 5-diphosphoglucuronic acid (UDPGA), alamethicin, paraformaldehyde (PFA) and deoxycholic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Nonidet P-40 and Tween-20 were purchased from Amresco Inc. (Solon, OH, USA). Tris-HCl, NaCl, EDTA, MgCl₂, perchloric acid and KH₂PO₄ were purchased from Sinopharm Chemical Regent Co. (Beijing, China). Chromatographic grade methanol was purchased from Tedia, Co. (Fairfield, OH, USA).

As previously described [39 (link),40 (link)], bacteria were inoculated onto the MRS agar medium containing 5 g/L taurodeoxycholic acid (Sigma-Aldrich, Saint Louis, MO, USA) and incubated at 37 °C for 5 d. The taurodeoxycholic acid was hydrolysed by bile salt hydrolase into deoxycholic acid precipitates, the size of which reflected the enzyme activity.