3 phosphoadenosine 5 phosphosulfate paps

Catalposide (purity, 98%) was obtained from Aobious Inc. (Gloucester, MA, USA). Alamethicin, 3-phosphoadenosine-5-phosphosulfate (PAPS), and uridine 5′-diphosphoglucuronic acid (UDPGA) were from Sigma-Aldrich Co. (St. Louis, MO, USA). 4-hydroxybenzoic acid and 4-hydroxybenzoic acid glucuronide were purchased from Toronto Research Chemicals (North York, ON, Canada). Pooled human intestinal microsomes; pooled human liver S9 fractions; human cDNA-expressed UGTs 1A1/3/4/6/7/8/9/10 and 2B4/7/15/17 supersomes; human cDNA-expressed CESs 1b, 1c, and 2 supersomes; cryopreserved human hepatocytes; and hepatocyte purification kits were obtained from Corning Life Sciences (Woburn, MA, USA). Human cDNA-expressed SULT 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, 1E1, and 2A1 supersomes were purchased from Cypex Ltd. (Dundee, UK). Methanol (HPLC grade) was from Burdick & Jackson Inc. (SK Chemicals, Ulsan, Korea), and all other chemicals were of the highest quality available. Calibration mixtures for Exactive MS [ProteoMass LTQ/FT-hybrid ESI positive mode Cal Mix (MSCAL5) and negative mode Cal Mix (MSCAL6)] were obtained from Supelco (Bellefonte, PA, USA).

Sulfation of the tested compounds by individual sulfotransferases was examined using cytosolic fractions from Escherichia coli expressing recombinant human SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, 1E1, or 2A1 (Cypex, Dundee, UK). The incubations were performed in 0.2 mL of potassium phosphate–HCl buffer (pH 7.4; 50 mM) containing 5 mM MgCl₂, 10 mM dithiothreitol, 50 µM 2,3-dehydrosilybin A or B (in 0.1% (v/v) DMSO), 50 µg/mL E. coli cytosol protein, and 120 µM 3′-phosphoadenosine 5′-phosphosulfate (PAPS; Sigma-Aldrich, St. Louis, MO, USA). Control samples were prepared by incubating the tested compounds in the absence of PAPS and/or the cytosolic fraction. All samples were incubated for 30 min at 37 °C and 300 rpm in a Thermomixer Comfort (Eppendorf, Hamburg, Germany), and then stored at –80 °C until their analysis by UHPLC-MS.

Sulfation of the tested compounds was examined using human liver and intestinal cytosols, as well as by using individual human sulfotransferases. Human liver cytosol pooled from 50 donors of mixed gender (H0610.C) and human intestinal cytosol pooled from 13 donors of mixed gender (H0610.IC) were obtained from Sekisui XenoTech (Kansas City, KS, USA). Cytosolic fractions from Escherichia coli expressing recombinant human SULT1A1*1, SULT1A1*2, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, SULT1E1, and SULT2A1 were obtained from Cypex (Dundee, UK). The incubations were performed in 0.2 mL of potassium phosphate–HCl buffer (pH 7.4; 50 mM) containing 5 mM MgCl₂, 10 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA), 50 µM tested compounds or silymarin in 0.1% (v/v) DMSO, 100 µg/mL tissue cytosol protein or 50 µg/mL E. coli cytosol protein, and 120 µM 3’-phosphoadenosine 5’-phosphosulfate (PAPS; Sigma-Aldrich, St. Louis, MO, USA). Control samples were prepared by incubating the tested compounds in the absence of PAPS or the cytosolic fractions. After incubation for 30 min at 37 °C and 300 rpm using a Thermomixer Comfort (Eppendorf, Germany), the samples were stored at –80 °C until their analysis by UHPLC-MS.