Regio- and Isoform-Specific Glucuronidation of Psoralidin: Evaluation of 3-O-Glucuronidation as a Functional Marker for UGT1A9

In th is study, we aimed to determine the glucuronidation potential of psoralidin in humans and to perform validation on use of psoralidin-3-O-glucuronidation as afunctional marker for UGT1A9. Glucuronidation kinetics was determined using human liver microsomes (HLMs), human intestine microsomes (HIM), and expressed UDP-glucuronosyltransferase (UGT) enzymes. The chemical structures of metabolites were determined by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analyses. Validation of psoralidin-3-O-glucuronidation as a UGT1A9 marker was performed using combined approaches including reaction phenotyping, chemical inhibition, activity correlation analysis, and determination of relative activity factor (RAF). HLM and UGT1A9 generated two monoglucuronides (9-O-glucuronide and 3-O-glucuronide) from psoralidin, whereas HIM, UGT1A1, UGT1A7, and UGT1A8 generated one only (9-O-glucuronide). Formation of 3-O-glucuronide in HLM was markedly inhibited by the UGT1A9-selective inhibitors magnolol and niflumic acid. Further, psoralidin-3-O-glucuronidation was strongly correlated with propofol-glucuronidation in a group of nine individual HLMs (r = 0.978, p < 0.001). Strong correlation was also observed between psoralidin-3-O-glucuronidation and the UGT1A9 protein levels measured by Western blotting (r = 0.944, p < 0.001). Moreover, UGT1A9 was responsible for 99.6% of psoralidin-3-O-glucuronidation in HLM based on the RAF approach. In conclusion, psoralidin was subjected to efficient glucuronidation, generating one or two monoglucuronides depending on UGT isozymes. Also, psoralidin-3-O-glucuronidation was an excellent in vitro marker for UGT1A9.