*2.5. Characterization of Human UGTs Involved in Catalposide and 4-Hydroxybenzoic Acid Glucuronidation*

To identify the UGT enzymes responsible for formation of catalposide glucuronide (M4) from catalposide and 4-hydroxybenzoic acid glucuronide (M3) from 4-hydroxybenzoic acid (M2), 100 µL reaction mixtures containing human intestinal microsomes (40 µg protein), or human cDNA-expressed UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, or 2B17 (10 µg protein); 2 mM UDPGA; 0.025 mg/mL alamethicin; and 400 µM catalposide or 500 µM 4-hydroxybenzoic acid in 50 mM Tris buffer (pH 7.4) were incubated at 37 ◦C for 60 min. The reactions were stopped by addition of 100 µL methanol containing 30 ng/mL 4-methylumbelliferone (an internal standard). After vortex-mixing and centrifugation, 50 µL of each supernatant was diluted with an equal volume of water and a 5 µL aliquot was injected into the LC-HRMS system.

To explore the kinetics of the metabolism of catalposide to catalposide glucuronide, various concentrations of catalposide (10, 25, 100, 400, 800, 1200, 1600, and 2000 µM) were incubated in duplicate with 2 mM UDPGA, 0.025 mg/mL alamethicin, pooled human intestinal microsomes (40 µg protein), or human cDNA-expressed UGT1A8 or UGT1A10 supersomes (20 µg protein), to obtain *K*<sup>m</sup> and *V*max values.
