Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum

The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the S.tag (TM), a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305 +/- 248 (BCRP), 66 +/- 70 (MRP2), and 275 +/- 205 (Pgp) fmoles per cm(2). Hepatic levels were 2.6 +/- 0.9 (BCRP), 19.8 +/- 10.5 (MRP2), and 26.1 +/- 10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8 mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3 mg crude membrane per cm(2) mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo-in vitro extrapolation and in silica prediction of drug absorption and elimination, thus supporting drug development. (C) 2011 Elsevier Inc. All rights reserved.