Abstract
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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.023234.
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ABBREVIATIONS: MRP/Mrp, multidrug resistance-associated protein; LC, liquid chromatography; MS/MS, liquid chromatography tandem mass spectrometry; AQUA, absolute quantification; RT, real-time; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SIL, stable isotope labeled; ANOVA, analysis of variance; q, quantitative.
- Received July 2, 2008.
- Accepted October 1, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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