Mechanism-guided untargeted-to-targeted lipidomics identifies phosphatidylcholine 38:4 in rat bile as an Abcb4/Mdr2 inhibition marker.

Abstract

Drug-induced liver injury can occur when the canalicular phospholipid floppase multidrug resistance protein 3 (Mdr2 in rodents) is inhibited, but there is still a lack of early biomarkers to detect this risk. In this study, bile duct-cannulated rats were dosed with multidrug resistance protein 3/Mdr2 inhibitor itraconazole (ITZ; 100 mg/kg/d for 3 days) to assess phospholipid changes via an untargeted-to-targeted lipidomics workflow. Untargeted profiling of bile and liver samples identified 1347 and 2475 tentative lipids, of which 221 and 404 were phosphatidylcholines (PCs) in bile and the liver, respectively. Unsupervised principal component analysis revealed strong treatment effects on bile PCs. A volcano plot indicated a selective, but not global, reduction in biliary PCs after ITZ treatment. Among these, PC 38:4 stood out as the most consistently decreased bile species. Structural elucidation using multistage collision-induced dissociation/mass spectrometry³ fragmentations confirmed its identity as arachidonyl PC 18:0/20:4. Subsequent absolute quantitation showed that bile PC 38:4 remained stable in controls (10.5 ± 1.02 μM; 5.6% CV) but declined rapidly after the first dose of ITZ (6.89 ± 1.50 μM at 0-4 hours) and continued to decrease to 4.22 ± 0.958 μM by day 3, a 2.7-fold decrease. Conversely, hepatic PC 38:4 showed a modest, yet significant increase (∼1.2-fold). Plasma bile acids remained unaffected, supporting a mechanism involving Mdr2 rather than the bile salt export pump. These findings identify PC 38:4 (18:0/20:4) as a sensitive and mechanistically relevant marker of Mdr2 inhibition. Monitoring PC 38:4 in nonclinical species may enable early, transporter-specific drug-induced liver injury risk assessment during drug development. SIGNIFICANCE STATEMENT: Untargeted-to-targeted lipidomics workflows identified phosphatidylcholine 38:4 as a sensitive, specific, and mechanistically linked biomarker of Mdr2 inhibition in rats. Multistage collision-induced dissociation/mass spectrometry³ fragmentation further confirmed the identity as arachidonyl phosphatidylcholine 18:0/20:4. Its rapid and specific decline in the presence of the Mdr2 inhibitor itraconazole offers a potential new tool for early detection of human multidrug resistance protein 3-related liver injury risk during drug development.