Chenodeoxycholic acid

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).

EAM-2201 was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Acetaminophen, N-acetylserotonin, alamethicin, chenodeoxycholic acid, coumarin, diclofenac, dimethyl sulfoxide (DMSO), 7-hydroxycoumarin, midazolam, mycophenolic acid, naloxone, naloxone 3-β-d-glucuronide, NADPH, phenacetin, trifluoperazine, Trizma base and uridine 5′-diphosphoglucuronic acid (UDPGA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ¹³C₂,¹⁵N-Acetaminophen, bufuralol, N-desethylamodiaquine, 1′-hydroxybufuralol, d₉-1-hydroxybufuralol, 4-hydroxydiclofenac, 4-hydroxymephenytoin, 1′-hydroxymidazolam, [S]-mephenytoin and ultrapooled human liver microsomes (150 donors) were purchased from Corning Life Sciences (Woburn, MA, USA). N-Acetylserotonin β-d-glucuronide, chenodeoxycholic acid 24-acyl-β-glucuronide, mycophenolic acid β-d-glucuronide and SN-38 glucuronide were obtained from Toronto Research Chemicals (Toronto, ON, Canada). SN-38 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Acetonitrile, methanol and water were of liquid chromatography–mass spectrometry (LC-MS) grade and purchased from Fisher Scientific Co. (Fair Lawn, NJ, USA). All other chemicals were of the highest quality available.

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.

All chemicals and solvents were of the highest purity commercially available. Standard bile acids (sodium salts), including glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurocholic acid (TCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and cholic acid (CA), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Three different oxgall powder samples were obtained from Sigma-Aldrich (Product code B3883), Oxoid (Product code LP005, Basingstoke, Hampshire, UK) and BD Difco (Product code 212820, Sparks, MD, USA). Acetonitrile and methanol (HPLC grade) were supplied by Tedia (Fairfield, OH, USA). The derivatization reagents p-bromophenacylbromide and N, N-diisopropylethylamine, and the ion-pairing reagent tetrabutylammonium hydrogen sulfate were also purchased from Sigma-Aldrich.