Evidence for the metabolic activation of higenamine to quinone methide and ortho-quinone metabolites in vitro and in vivo using liquid chromatography tandem mass spectrometry

Abstract

Higenamine (HG), a naturally occurring benzyltetrahydroisoquinoline alkaloid, has been revealed a variety of biological activities and is extensively utilized in dietary supplements. Currently, HG is under investigation in phase I clinical trials, however, the liver metabolism of HG has so far not been fully elucidated. The present study aimed to identify reactive metabolites of HG using ultrahigh-performance liquid chromatography-tandem mass spectrometry. Four glutathione (GSH) conjugates (M1-M4) and four cysteine conjugates (M5-M8) derived from reactive metabolites of HG were detected in GSH/cysteine-fortified mouse/human microsomal incubations. The cysteine conjugates were chemically synthesized for structural elucidation using manganese dioxide as the oxidizing agent. The reactive metabolites of HG were identified as quinone methide, hydroxyquinone methide, and ortho-quinone based on the fragmentation patterns of cysteine conjugates. Multiple CYP450 enzymes including CYP2D6, CYP3A4, and CYP2E1 were mediated in the formation of quinone methide, with the major role assigned to CYP2D6. While the oxidation of catechol to ortho-quinone metabolite and the subsequent isomerization into hydroxyquinone methide were independent of CYP450 isoforms. In addition, these electrophilic metabolites were found to react with biliary GSH and cysteine residues of hepatic protein in HG-treated mice. The in vitro and in vivo evidence of the metabolic activation of HG to quinone methide and ortho-quinone metabolites raised health concerns regarding the consumption of HG-containing supplements.

Keywords: CYP450; Glutathione conjugates; Higenamine; LC-MS/MS; Metabolic activation; Protein modification.