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.
Li
Na
N
Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Pfizer Global Research & Development, St. Louis Laboratories, Pfizer Inc., St. Louis, Missouri, USA.
Zhang
Yiqun
Y
Hua
Fengmei
F
Lai
Yurong
Y
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid; D004285:Dogs; D005260:Female; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007223:Infant; D008099:Liver / Q000378:metabolism*; D008252:Macaca fascicularis; D008253:Macaca mulatta; D008297:Male; D008875:Middle Aged; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D053719:Tandem Mass Spectrometry
D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C000715087:ABCC2 protein, human