Immunochemical and LC-MS/MS Detection of Protein Adduction Resulting from Metabolic Activation of Columbin In Vitro and In Vivo.

Columbin (CLB) is a diterpenoid furanolactone compound occurring in some herbal medicines. Administration of CLB has been reported to induce liver injury. The reported CLB hepatotoxicity is suggested to require metabolism to a cis -enedial intermediate. We successfully detected hepatic protein adduction resulting from the metabolic activation of CLB and found that the intermediate reacted with lysine residues or lysine/cysteine residues to produce the corresponding pyrroline derivative or pyrrole derivative. The detection was achieved by proteolysis- and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods. Furthermore, we prepared a polyclonal antibody approach which allowed us to detect the protein adduction in the forms of protein immunoblot as well as tissue- and cell-based immunostaining. The antibody technique verified the protein adduction detected by LC-MS/MS.