Absolute Difference of Hepatobiliary Transporter Multidrug Resistance-Associated Protein (MRP2/Mrp2) in Liver Tissues and Isolated Hepatocytes from Rat, Dog, Monkey, and Human

We previously reported that hepatobiliary transporter multidrug resistance-associated protein (MRP2/Mrp2) is considered to be the major cause of the interspecies differences detected by efflux of fluorescent substrates in isolated hepatocytes. In the present study, the interspecies differences of MRP2/Mrp2 were first evaluated by quantitative real-time polymerase chain reaction and Western blotting. The mRNA levels were able to distinguish the difference among species with a rank order comparable with the corresponding activities observed, whereas the extents of the differences remained unknown. The cross-reactions of MRP2/Mrp2 protein of different species with anti-human MRP2 polyclonal antibody were found by Western blotting. However, because of the unknown binding affinity of antibody to MRP2/Mrp2 protein across species and lack of purified MRP2/Mrp2 proteins for calibration, the immunoblotting assay was excluded from the absolute quantification of MRP2/Mrp2 protein for multiple species. By using our newly developed liquid chromatography-tandem mass spectrometry quantification method, we were able to measure the absolute amount of MRP2/Mrp2 in liver tissues and isolated hepatocytes across species. Freshly isolated hepatocytes conserved MRP2/Mrp2 protein levels that are comparable with those in the liver tissues. The amount of Mrp2 in rat liver was approximately 10-fold higher than that in other species. Moreover, a significant loss of Mrp2 protein in the membrane fraction of rat cryopreserved hepatocytes was observed. Thus, the absolute differences of MRP2/ Mrp2 levels in various species were determined, for the first time, by direct quantification. The results could potentially fill the translational gaps of in vitro/in vivo or preclinical species to human extrapolation of hepatobiliary elimination mediated by MRP2/ Mrp2.