Glucuronidation of d-Luciferin In Vitro: Isoform Selectivity and Kinetics Characterization

Background d -luciferin is one of the most commonly used substrates in bioluminescence imaging for real-time monitoring of sophisticated biological processes in models of human biology or disease in vitro and in vivo. d -luciferin is rapidly cleared from blood circulation after being exogenously delivered in vivo and the presence of phenolic groups indicates that glucuronide conjugation is a possible metabolic pathway for the compound. Objectives This study aimed to characterize the glucuronidation pathway of d -luciferin in human liver microsomes (HLM) and human intestine microsomes (HIM). Methods HLM and HIM were employed to catalyze the glucuronidation of d -luciferin in vitro. The activity of recombinant uridine-diphospho-glucuronosyltransferase (UGT) isoforms towards d -luciferin glucuronidation was screened. Chemical inhibition assay and kinetic analysis was combined to determine the UGT isoforms responsible for the formation of d -luciferin glucuronide in HLM and HIM. Results d -luciferin could be catalyzed to form one mono-glucuronide which was characterized as 6′- O -glucuronide in HLM and HIM. UGT1A1, 1A3, 1A6, 1A8, 1A9 and 1A10 participated in the formation of d -luciferin glucuronide, with UGT1A1 exhibiting the highest catalytic activity. Both chemical inhibition assays and kinetic analysis showed that UGT1A1 and UGT1A3 played important roles in d -luciferin-6′- O -glucuronidation in HLM and HIM, with UGT1A6 also giving a non-negligible contribution to this biotransformation in HLM. Conclusions UGT1A1, UGT1A3 and UGT1A6 were responsible for 6′- O -glucuronidation of d -luciferin in HLM, while UGT1A1 and UGT1A3 were the major contributors to this biotransformation in HIM.