Received for publication July 3, 2008.
Revised September 30, 2008.
Accepted for publication October 1, 2008.

Absolute Difference of Hepatobiliary Transporter MRP2/Mrp2 in Liver Tissues and Isolated Hepatocytes from Rat, Dog, Monkey and Human

Abstract

Previously, we have reported that hepatobiliary transporter MRP2/Mrp2 is considered as the major cause of the interspecies differences detected by fluorescent substrates efflux in isolated hepatocytes. In the present study, the interspecies differences of MRP2/Mrp2 were firstly evaluated by qRT-PCR and western blotting. The mRNA levels were able to distinguish the difference among species with the rank order comparable to the corresponding activities observed, while the extents of the differences remained unwarranted. The cross reactions of MRP2/Mrp2 protein of different species with anti-human MRP2 polyclonal antibody were found by western blotting. However, due to 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 LC-MS/MS 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 the MRP2/Mrp2 protein levels that are comparable to 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. Taken together, the absolute differences of MRP2/Mrp2 level 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.

Key words: ABC transporters, hepatobiliary transport, hepatocytes, isolated hepatocytes, mass spectrometry