Interindividual Variability in Hepatic Organic Anion-Transporting Polypeptides and P-Glycoprotein (ABCB1) Protein Expression: Quantification by Liquid Chromatography Tandem Mass Spectroscopy and Influence of Genotype, Age, and Sex

Chemicals and Reagents.

The ProteoExtract native membrane protein extraction kit was procured from Calbiochem (Temecula, CA). The protein quantification bicinchoninic acid (BCA) kit and the in-solution trypsin digestion kit were purchased from Pierce Biotechnology (Rockford, IL). Synthetic signature peptides (Table 1) for OATP1B1, OATP1B3, OATP2B1, and P-gp were obtained from New England Peptides (Boston, MA). The corresponding stable isotope-labeled (SIL) internal standards were obtained from Thermo Fisher Scientific (Rockford, IL). High-performance liquid chromatography–grade acetonitrile was purchased from Fischer Scientific (Fair Lawn, NJ), and formic acid was purchased from Sigma-Aldrich (St. Louis, MO). All reagents were analytical grade.

Human Liver Samples and Hepatocytes.

Sixty-four liver tissue samples from the human liver bank of the School of Pharmacy, University of Washington (UW), were used. Most of the subjects were Caucasian [except one Asian male (HL165) and three Black males not of Hispanic origin (HL104, 105, and 137)]; the subjects’ ages ranged from 7 to 70 years and comprised 30 females and 34 males. Procurement (approved by the UW Human Subjects Division), characteristics, and storage of these samples have been previously described (Paine et al., 1997), and additional details are provided in Supplemental Table 1. Most of these livers were obtained from organ donors who met with accidental death (e.g., trauma from vehicular accidents, subarachnoid hemorrhage, cerebrovascular accident) and were harvested from breathing donors who were perfused with UW solution for organ transplant purposes. Most of the subjects were not taking any chronic medication but did receive medication in the intensive care unit before tissue harvest. Cryopreserved human hepatocytes (seven batches of individual and five batches of pooled hepatocytes) (Table 2) were procured commercially from Celsis IVT (London, UK), Xenotech (Lenexa, KS), Life Technologies (Carlsbad, CA), and BD Gentest (San Jose, CA).

Membrane Protein Extraction and Total Protein Quantification.

The liver tissue (∼100 mg) was processed, as we have described before, to isolate total membrane proteins (Calbiochem) (Deo et al., 2012; Prasad et al., 2013). Briefly, the tissue was homogenized in 2 ml of extraction buffer I (ProteoExtract native membrane protein extraction kit) containing protease inhibitor cocktail (10 µl) of the kit and incubated with gentle shaking for 10 minutes. The resultant homogenate was centrifuged at 16,000g for 15 minutes, and the supernatant was discarded. The pellet was resuspended in 1 ml of extraction buffer II of the kit with 10 µl of protease inhibitor cocktail. The latter was incubated with gentle shaking for 30 minutes at 4°C followed by centrifugation at 16,000g for 15 minutes at 4°C. Total isolated membrane protein concentration (i.e., the supernatant) was determined using the BCA protein assay kit. The supernatant was diluted to a working concentration of 2 µg of protein per microliter or lower. Similar to the tissues, the pellet of cryopreserved hepatocytes (2–5 × 10⁶ cells) were processed as discussed except the cells were washed twice with washing solution of the kit before the addition of 2 ml of extraction buffer I. The remaining procedure was the same as described for the liver tissue.

Purification of Human Pgp.

Human Pgp from crude membranes of High Five insect cells was purified as described previously (Sauna et al., 2006) with some modifications. Briefly, the crude membranes were solubilized with n-dodecyl-β-d-maltoside (DDM) (1.00% w/v) in the presence of 20% glycerol. Solubilized proteins were subjected to metal affinity chromatography (Ni-NTA resin; Qiagen Inc, Valencia, CA) in the presence of 0.51 mM DDM; purified P-gp was eluted with 200 mM imidazole. The eluate was further purified by gel filtration chromatography using Superdex S-200 column to remove imidazole. The fractions containing P-gp were then concentrated using Amicon ultrafiltration concentrators with 100 KDa cutoff (EMD Millipore, Billerica, MA) to ∼0.5–1.5 mg/ml and stored at −70°C. The protein concentration was quantified using the BCA protein assay kit as per the manufacturer’s instructions.

Peptide Selection, Trypsin Digestion of Membrane Proteins, and Sample Preparation.

Two unique signature peptides (not present in any other known protein) were selected for quantification of each transporter (Table 1) based on previously reported criteria (Kamiie et al., 2008) and literature reports (Zhang et al., 2011; Balogh et al., 2012). Briefly, peptides with transmembrane regions, single nucleotide polymorphisms (SNPs), posttranslational modifications, or those susceptible to degradation were not selected. Continuous R and K sequences (RR, RK, KR, and KK) were excluded to avoid the miscleavages by trypsin. The length of selected peptides was between 9 and 16 amino acid residues. Selected signature peptides were NVTGFFQSFK/YVEQQYGQPSSK (OATP1B1), NVTGFFQSLK/ IYNSVFFGR (OATP1B3), VLAVTDSPAR/SSPAVEQQLLVSGPGK (OATP2B1), and NTTGALTTR/IATEAIENFR (P-gp). The corresponding peptides containing labeled [¹³C₆¹⁵N₂]-lysine and [¹³C₆¹⁵N₄]-arginine residues were used as the internal standards.

Trypsin digestion conditions were optimized for time (24 hours) and protein:trypsin ratio (25:1; w/w), and 20 µl of 2.0 µg/µl (or lower concentration) of tissue membrane preparation was incubated with 4 µl of dithiothreitol (100 mM) and 4 µl of iodoacetamide (200 mM) in 10 µl of ammonium bicarbonate digestion buffer (50 mM, pH 7.8). The protein samples were digested by trypsin in a final volume of 60 µl at 37°C for 24 hours, and the reaction was quenched by 20 µl of SIL peptide internal standard cocktail (prepared in 70% acetonitrile in water containing 0.1% formic acid) and 10 µl of the neat quenching solvent. The samples were centrifuged at 4000g for 5 minutes. For calibration standards, the working solution (10 µl) of the standard cocktail was added in the last step instead of the neat quenching solvent.

Analytical Method Parameters.

Agilent 6460A triple-quadrupole mass spectrometer coupled to Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA) operated in electrospray ionization positive ionization mode was used for liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis of the signature peptides. Approximately 2 µg or less of the trypsin digest (5 µl) was injected onto the column (Kinetex 2.6 µm, C18, 100 × 3 mm, Phenomenex, Torrance, CA) and eluted at 0.4 ml/min. The mobile-phase gradient conditions were 97% A (water containing 0.1% v/v formic acid) and 3% B (acetonitrile containing 0.1% v/v formic acid) held for 4 minutes, followed by seven steps of linear gradient of mobile phase B concentration of 3%–12.5%, 12.5%–18%, 18%–19.5%, 19.5% –20%, 20%–35%, 35%–50%, and 50%–90% over 4–8 minutes, 8–11 minutes, 11–13.5 minutes, 13.5–16 minutes, 16–18 minutes, 18–18.4 minutes, and 18.4–18.6 minutes, followed by the washing step using 90% mobile phase B for 1.6 minutes, and re-equilibration for 4.8 minutes. The doubly charged parent to singly charged product transitions for the analyte peptides and their respective SIL peptides were monitored using optimized LC-MS/MS parameters (Table 1).

The calibration curve standards were prepared by spiking peptide standards into the extraction buffer II of the membrane protein extraction kit. Seven calibration concentrations ranging from ∼0.2 to 20.0 fmol (on column) were used. The analytical method was validated for accuracy, interday and intraday precision, and stability (freeze and thaw, bench-top, and autosampler conditions) as we have described previously (Prasad et al., 2013). The quality control samples prepared by spiking extraction buffer II or pooled human liver membrane were quantified after every 12–15 samples. In addition, the reliability of surrogate peptide-based LC-MS/MS protein quantification was validated using the only purified transporter available to us, namely P-gp. Four different concentrations of P-gp protein standard (2.2, 4.4, 17.7, and 35.3 fmol, on-column) were prepared as quality control samples and were then processed, as were the membrane extracts of the liver tissues. The data were processed by integrating the peak areas generated from the reconstructed ion chromatograms for the analyte peptides and their respective internal standards using the MassHunter software (Agilent Technologies). For quantification of samples or standards, peak response from the two transitions from each peptide was averaged.

Genotyping and Genotype-Dependent Changes on Protein Expression.

Genotyping data were kindly provided by Dr. Yvonne Lin, Department of Pharmaceutics, University of Washington. Briefly, genomic DNA was extracted from liver tissues. The genotype analysis was done using Affymetrix DMET Plus Array (Santa Clara, CA) according to the manufacturer’s protocol. Individual genotypes were resolved, and the minor allele frequencies were determined using the Affymetrix DMET Plus console (version 1.1) using the Dynamic Genotype Boundaries algorithm. Unpaired t test was used to compare protein expression observed in two genotypic groups. When comparing protein expression in three or more groups, the Bonferroni multiple comparison correction was applied.

PBPK Simulations.

The effect of SLCO1B1 polymorphism on the pharmacokinetics of OATP1B1 substrates rosuvastatin and repaglinide was predicted using the population-based ADME simulator of Simcyp (version 12.0, SimCYP Ltd, Sheffield, UK). The rosuvastatin and repaglinide parameters of Simcyp library file were used as such except that the relative expression factor (see Supplemental Table 2) of OATP1B1 was varied for a given genotype or haplotype compared with the default SimcyP value for wild-type (set at 1). The 90% confidence intervals (CIs) of mean relative expression factors were used to estimate population variability in each group (see Supplemental Table 2). Expression of other transporters (i.e., OATP1B3, OATP2B1, BCRP, and sodium-taurocholate cotransporting polypeptide (NTCP)) was assumed constant for all simulations. Data for 16 subjects (70 kg; age 18–65 years, proportion of females: 0.5) given a single dose of rosuvastatin (20 mg) or repaglinide (0.50 mg) were simulated (four trials × 4 subjects).

Analytical Methods.

The calibration curves were linear across the calibration range ∼0.2–20.0 fmol (on column). The lower limit of quantification was between 0.1 and 0.3 fmol (on-column) for all the peptides (except YVEQQYGQPSSK, which had a lower limit of quantification of 1.2 fmol). Accuracy and precision in the quantification of the quality control samples were acceptable (CV <25%) at three different concentrations. Trypsin digestion of the transporters was maximized by monitoring the time at which there was no further increment in the yield of the peptide (data not shown). All the peptides were stable during sample preparation (i.e., when exposed to three cycles of freeze and thaw, at bench-top for 6 hours, and in an autosampler for 48 hours).

Using the purified P-gp, our peptide-based LC-MS/MS approach was able to recover the concentration of P-gp in the quality control samples with an accuracy of 124.0% ± 11.2% and 74.7% ± 4.1% using the P-gp surrogate peptides 1 and 2 (Table 1), respectively. For the remaining transporters, since purified protein standards are not available, the analytical method was based on the assumption that the proteins are completely digested by trypsin to their corresponding peptides and there is a complete extraction of membrane proteins from the tissue homogenate. Although we used two peptides to quantify each protein, only one peptide for OATP1B1, peptide 1, was detectable with acceptable sensitivity. For the remaining transporters, we observed a minor (up to 1.5-fold) but systematic difference (P < 0.05, paired t test, Table 3) in protein quantification between the two different signature peptides. We assume that this difference was due to different degrees of trypsin digestion. Therefore, the results reported here are based on the peptides that yielded higher protein expression, namely, NVTGFFQSFK (OATP1B1), NVTGFFQSLK (OATP1B3), VLAVTDSPAR (OATP2B1), and NTTGALTTR (P-gp). Nevertheless, our final conclusions about the effect of genotype, age, and sex on transporter expression were the same irrespective of the peptide used (see later).

Interindividual Variability in Transporter Expression in Human Livers and Cryopreserved Human Hepatocytes.

Our yield of total membrane protein was 3.7 ± 1.1 mg/100 mg tissue. The expression of OATPs and P-gp in tissue samples was comparable to that observed in the cryopreserved hepatocytes (Table 3). Similar to human liver tissue, the expression of these transporters in hepatocytes were in the order of OATP1B1 > OATP2B1 > OATP1B3 > P-gp. Interindividual variability, calculated as the -fold range in transporter expression (i.e., maximum or minimum expression) was 5- to 8-fold (see Fig. 1). Expression of all the transporters was independent of age (Fig. 2) or sex (Fig. 3).

Effect of Genotype on Hepatic Transporter Protein Expression.

Reported SNPs of all transporters (except SLCO2B1) were observed in our liver bank (Table 4). Among the OATPs, only OATP1B1 expression was genotype-dependent. When analyzed individually (without regard to haplotypes), livers homozygous for the nonsynonymous SLCO1B1 SNPs, c.388A>G or c.463C>A, or the synonymous SNP, c.597C>T, expressed 1.8-, 2.1-, and 1.7-fold higher OATP1B1 protein than those with the corresponding wild-type allele, respectively (P < 0.0001) (Fig. 4). Moreover, livers that were heterozygous for c.463CA had higher OATP1B1 expression than the wild-type livers (P < 0.0001), and the expression in the former was comparable to that in homozygous livers, c.463AA (Fig. 4). The expression of OATP1B1 in livers carrying a single c.388AG, c.597CT, c.571TT, c.571TC, or -1187GA allele was not significantly different from the wild-type livers. Only one liver in our tissue bank was homozygous for the functionally relevant SNP c.521T>C. The expression of OATP1B1 in livers with c.521TT versus 521TC was not significantly different (Fig. 4).

In agreement with other studies (Kalliokoski et al., 2008a; Nies et al., 2013), we observed significant linkage disequilibrium among the above SNPs. Therefore, we examined the expression of OATP1B1 based on SLCO1B1 haplotypes of the three previously described key variants (c.388A>G, c.463C>A, and 521T>C; Fig. 5A). Hepatic OATP1B1 protein expression was significantly higher (P < 0.0001) in livers with SLCO1B1 haplotypes *14/*1a and *14/*14 compared with those harboring the reference allele (i.e., *1a/*1a), *15/*1a or *1b/*1a (Fig. 5B). The carriers of haplotypes *14/*1a and *14/*14 had, respectively, 1.9- and 2.2-fold higher OATP1B1 expression than those carrying the reference allele. Furthermore, livers homozygous for c.388A>G independently increased OATP1B1 protein expression even when carriers of both 463AC and 463AA were excluded (Fig. 5C). SNPs c.388A>G and c.521T>C show the opposite effect on the pharmacokinetics of a number of drugs (Niemi et al., 2004; Kameyama et al., 2005; Hartkoorn et al., 2010; Rodrigues et al., 2011; Schipani et al., 2012; Sortica et al., 2012; Nies et al., 2013). In agreement with these observations, when homozygous alleles of 388A>G were excluded from analysis, we observed a modest but statistically significant (P < 0.05) decrease in OATP1B1 expression in individuals with c.521TC versus c.521TT (Fig. 5C). An independent effect of SNP c.521CC (*5) could not be examined because of its low frequency in our liver bank.

Subjects harboring SLCO1B3 haplotype (c.334G>T, c.699A>G, c.1557G>A, and c.1833A>G) did not affect protein expression. Genotype-dependent protein expression could not be investigated for P-gp because of the large number of haplotypes, each with a limited sample size. Individually, none of the ABCB1 SNPs significantly affected protein expression (Table 4).

Quantitative Impact of OATP1B1 Polymorphism on Pharmacokinetics of Rosuvastatin and Repaglinide.

Utilizing the protein expression data (Figs. 4 and 5; see Supplemental Table 2), we predicted the impact of the SLCO1B1 genotypes on the human pharmacokinetics of OATP1B1 substrates rosuvastatin and repaglinide. In agreement with the expression data, the various genotypes and haplotypes of OATP1B1 had marked effects on the predicted plasma concentration-time profiles of the OATP1B1 substrates rosuvastatin and repaglinide (Fig. 6), translating into reductions in area under the curve (AUC) of up to 40% (Fig. 7) compared with the respective wild-type and reference allele.