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OtherDRUG METABOLISM AND DISPOSITION

Hepatic Uptake of Bromosulfophthalein-Glutathione in Perfused Eisai Hyperbilirubinemic Mutant Rat Liver: A Multiple-Indicator Dilution Study

Wanping Geng, Andreas J. Schwab, Tohru Horie, Carl A. Goresky and K. Sandy Pang
Journal of Pharmacology and Experimental Therapeutics February 1998, 284 (2) 480-492;

Abstract

The hepatocellular uptake of the glutathione conjugate of bromosulfophthalein (BSPGSH) was examined in Eisai hyperbilirubinemic rats (EHBR; originating from Sprague-Dawley rats), which lacked the ATP-dependent canalicular transport for non-bile acid organic anions, a trend common to other mutant rat strains (TR− and GY, originating from Wistar rats). Single-pass perfused rat liver experiments were conducted with BSPGSH (26–257 μM) using the multiple indicator dilution technique. The steady-state extraction ratio of BSPGSH was close to zero due to lack of biliary excretion. After the introduction of a bolus dose containing vascular (51Cr-labeled red blood cells), interstitial (125I-labeled albumin and [14C]sucrose) and cellular space (D2O) indicators and [3H]BSPGSH into the portal vein, the outflow dilution profile of [3H]BSPGSH was found to display a protracted declining profile (tailing) at low input BSPGSH concentrations; the tail disappeared at higher BSPGSH concentrations. When data were fitted with the barrier-limited model of Goresky as used previously for BSPGSH for the Sprague-Dawley rat (SDR), model fitting was found to evoke an additional “deep pool” within the hepatocyte to account for the “tail” component. The deep pool became evident for the EHBR because biliary excretion of BSPGSH was absent and the rate of return from the deep pool was slow. The concentration of BSPGSH within the deep pool was estimated to be 12 ± 8 times that in the cytosol. The binding of BSPGSH to EHBR S9 (effective binding concentration of 53 μM and a binding association constant KAof 2.4 × 104 M−1), however, was found to be lower than that of SDR S9 and could not account for the late-in-time data. The influx permeability-surface area product was concentration dependent and decreased from 0.27 to 0.01 ml·sec−1·g−1 with increasing BSPGSH concentration; the throughput component, or the portion of the dose that goes through the liver without entering the hepatocyte, increased with increasing concentration. The trends were characteristic of carrier-mediated transport and were similar to those found for the uptake of BSPGSH in SDR.

Biliary excretion of drugs or their usually more polar conjugates involves consecutive processes of net sinusoidal uptake, intracellular trafficking through the cytoplasm and excretion across the apical or canalicular membrane. At the sinusoidal membrane, specialized transport systems for bile acids (Hagenbuch et al., 1991; Hagenbuch and Meier, 1994), organic anions (Jacquemin et al., 1991,1994; Kullak-Ublick et al., 1994) and cations (Bossuytet al., 1996; Gründemann et al., 1994) have been discovered. Equally important is the host of canalicular transporters that contribute to the formation of concentrated bile. Although the canalicular membrane accounts for only 15% of the total surface area of the hepatocyte cell membrane (Evans, 1980), it is highly specialized for excretion, a process facilitated by the presence of specific transporters.

The canalicular transporters belong to a superfamily of proteins occurring in all cellular organisms and are characterized by an ABC in their primary structure. To date, three subfamilies have been identified of ABC transporters that mediate concentrative transport of endobiotics and xenobiotics on the canalicular membrane (for reviews, see Gatmaitan and Arias, 1995; Meier et al., 1997;Müller and Jansen, 1997). One subfamily of the ABC transporters, the P-glycoproteins, are coded by various multidrug-resistant genes in humans (MDR) and mice (Mdr) and are found to transport a wide variety of substrates, which are mostly lipophilic, neutral, or positively charged, including phosphatidylcholine and phospholipids (Lomri et al., 1996; Oude Elferink et al., 1997). A second ABC transporter that is responsible for ATP-dependent bile acid transport has been purified and functionally reconstituted (Müller et al., 1991; Ruetz et al., 1987; Sippel et al., 1990). A third distinct ABC system mediates the concentrative transport of a variety of non-bile acid organic anions (Büchler et al., 1996; Paulusmaet al., 1996; Müller et al., 1996b). This transport system was named cMOAT. Separate and distinct sinusoidal and canalicular transport proteins have been implicated in the transport of the important tripeptide, GSH (Fernandez-Checa et al., 1992).

The molecular basis of cMOAT was disclosed recently. cMOAT bears sequence homology with an MRP found to be associated with lung cancer cells (Büchler et al., 1996) and appears to be an isoform found exclusively in the canalicular membrane of hepatocytes, denoted as cMrp (Büchler et al., 1996) or mrp2 (Müller et al., 1996b) in the rat and cMRP (Kartenbecket al., 1996) or MRP2 (Müller et al., 1996b; Paulusma et al., 1996) in humans. Although MRP1 is present in many other tissues (Müller et al., 1996a), MRP2 is located on the lateral membrane of the liver (Mayer et al., 1995).

The presence of cMOAT was prompted by the serendipitous discovery of several strains of jaundiced mutant rats. They are the TR− and GY rats originating from the Wistar strain (Jansen et al., 1985; Kuipers et al., 1988; Kitamura et al., 1990; Nishida et al., 1992) and the EHBR (EHBR/Eis) from the Sprague-Dawley strain (Hosokawaet al., 1992). In TR− rats, the defect is due to a single-nucleotide deletion in the gene coding for cMRP, resulting in reduced mRNA levels and absence of the 190-kDa cMOAT protein in the canalicular membrane (Paulusma et al., 1996). A similar reduction in protein as well as mRNA levels were shown in EHBR (Ito et al., 1996; Kartenbeck et al., 1996), where the genetic defect occurs due to the substitution of one nucleotide, which results in the introduction of a premature stop codon (Ito et al., 1997). These mutant rats manifest predominantly conjugated hyperbilirubinemia and lack the hepatic excretion of various non-bile acid organic anions. It was recently shown that cMrp is absent in EHBR and TR− rats (Kobayashi et al., 1990; Mayer et al., 1995; Müller et al., 1996b) and that cMRP was absent in a patient with Dubin-Johnson syndrome (Kartenbeck et al., 1996;Müller et al., 1996b). The phenotype of these mutant rats follows autosomal recessive inheritance and is similar to that of the mutant Corriedale sheep and human Dubin-Johnson syndrome.

There is dramatic impairment of biliary excretion of various organic anions in these mutant (TR−, GY and EHBR) rat strains. The organic anions include the sulfate and glucuronides of bile acids (Jansen et al., 1985; Kuipers et al., 1988), bilirubin glucuronide (Nishida et al., 1992), indocyanine green (Hosokawa et al., 1992; Sathirakulet al., 1993), GSH and cysteine adducts (Oude Elferinket al., 1989Kitamura et al., 1990; Geng et al., 1995b; Müller et al., 1996a) and drug glucuronide conjugates (Shimamura et al., 1994, 1996;Takenaka et al., 1995a, 1995b). BSPGSH has been used repeatedly as a model substrate for the study of this canalicular transporter because cleavage of the GSH moiety to the cysteinylglycinyl or cysteinyl adducts seldom occurs, and even if it does, the extent is only minimal (Sano et al., 1992; Snel et al., 1993; Sorrentino et al., 1989; Zhao et al., 1993). Avid excretion and an extremely high bile/liver concentration ratio of 1600, presumably due to the formation of micelles, have been reported for BSPGSH in liver perfusion experiments (Geng et al., 1995b). Saturable uptake and slow efflux as well as high plasma and tissue binding are other characteristics found to be associated with BSPGSH (Geng et al., 1995b).

In the present investigation, we examined the handling of BSPGSH by the perfused EHBR liver with the MID technique to assess quantitatively the transport processes for BSPGSH. Because the microcirculation and polarity are maintained in the perfused liver preparation, the kinetics of basolateral and canalicular transport are assessed without disruption of cellular integrity and bioenergetics, thus allowing evaluation of the impact of the mutation on the functionality of the organ. In addition, the MID technique exploits tracer methodology and enables investigation of the transport and removal processes to be studied simultaneously. Through analysis of the dilution profiles of [3H]BSPGSH and comparison with those of a set noneliminated reference indicators, we examined the uptake of BSPGSH in absence of excretion in the EHBR and compared this with the observations in the SDR (Geng et al., 1995b). The difference in tissue binding of BSPGSH between these two rat strains also was studied.

Experimental Procedures

Materials.

Unlabeled bromosulfophthalein, reduced GSH, GST (rat) and bovine serum albumin (25% in Tyrode’s buffer) were obtained from Sigma Chemical (St. Louis, MO). [3H]GSH (specific activity, 1.22 Ci/mmol) was obtained from DuPont Canada (Markham, Ontario, Canada). Unlabeled BSPGSH and [3H]BSPGSH were synthesized from GSH and [3H]GSH, respectively, as previously described (Snel et al., 1993; Whelan et al., 1970). After purification, the purity of BSPGSH, determined by HPLC (Snel et al., 1993), was >95%, whereas the radiochemical purity of [3H]BSPGSH was >96% as found by TLC (silica gel in a solvent system of 1-butanol/water/acetic acid, 75:25:10, v/v/v) and >93% as found by HPLC. All reagents used were of glass-distilled HPLC grade or the highest purity available (Fisher Scientific, Mississauga, Ontario, Canada).

Rat liver perfusion.

Male EHBR (289 ± 19 g; liver weights, 14 ± 1 g) were provided by Eisai Co. Ltd. (Tsukuba City, Ibaraki, Japan) and were kept under artificial lighting on a 12:12-hr light/dark cycle. The animals were fed ad libitum(Purina Rat Chow) and allowed free access to water. Before surgery, the animals were anesthetized with intraperitoneal administration of sodium pentobarbital (50 mg/kg). The surgical procedure and the perfusion apparatus were identical to those described previously (Geng et al., 1995b). The artificial perfusate contained 20% washed outdated human RBC (Red Cross, Toronto, Canada), 1% bovine serum albumin, 3% dextran T-40 (Pharmacia Fine Chemicals, Piscataway, NJ) and 17 mM glucose (Travenol Labs, Deerpark, IL) in KHB solution buffered to pH 7.4. Because the perfusion medium was identical in composition to that used for SDR liver perfusion, the binding of BSPGSH to albumin should be identical to that observed previously (Genget al., 1995); namely, there were two classes of binding sites: n1 = 0.5,KA 1 = 1 × 105 M−1;n2 = 5,KA 2 = 3.5 × 103 M−1, wheren1 and n2 are the numbers of sites for classes I and II, andKA 1 andKA 2 are the corresponding binding association constants. The common bile duct was cannulated with polyethylene PE-50 tubing (Becton Dickinson, Parsippany, NJ), and the portal vein was cannulated with an intravenous catheter/needle unit (Vascular Access; Becton Dickinson, Sandy, UT). The perfusate entered the liver through the portal vein and exited through the hepatic vein at a rate of 12 ml/min (0.85 ± 0.07 ml·min−1·g−1) in a single-pass fashion (nonrecirculating); the hepatic artery was ligated.

Preliminary experiments showed that a steady state was reached within 90 min (>200 μM) or 120 min (<150 μM) of perfusion. Single-pass perfusion with unlabeled BSPGSH (one concentration was used for each preparation, ranging from 26 to 257 μM) was carried out for 150 min. During steady state, outflow samples were collected at 4-min intervals before injection of the MID dose. The mean of three perfusate plasma samples from the reservoir was taken to determine the input concentration, CIn; the average of at least three constant values was used for determination of the steady-state output plasma concentration of unlabeled BSPGSH, COut. Bile was collected at 30-min intervals before and at 10-min intervals after MID dose injection. These samples were collected for confirmation that disappearance of BSPGSH from plasma was zero and biliary excretion had not occurred. At the end of the perfusion experiment, the liver was perfused with 25 ml of ice-cold KHB, removed, weighed quickly and homogenized with an equal volume of KHB. The resulting liver samples were stored at −20°C.

MID study.

Injection of the MID dose was carried out during steady state (at 90 or 120 min after the onset of perfusion), as described previously (Geng et al., 1995b). The injection mixture (0.23 ml) contained [3H]BSPGSH (0.64 ± 0.12 μCi), unlabeled BSPGSH (at the same concentration as in the perfusate), a vascular indicator (51Cr-labeled washed human RBC, 0.57 ± 0.25 μCi), interstitial indicators [125I-labeled albumin (3.3 ± 0.8 μCi) and [14C]sucrose (0.6 ± 0.08 μCi)] and an accessible water space indicator (D2O, 0.14 ± 0.012 ml), in a composition otherwise identical to that of the perfusate. This was rapidly introduced into the portal veinvia an electronically controlled HPLC injection valve (Valco Instruments, Houston, TX). Simultaneously, serial outflow samples (at successive 1-, 2- and 3-sec intervals for a total of 180 sec) were collected using a homebuilt fraction collector. In all experiments, the Hct of the blood perfusate and the dose were determined by use of an Hct centrifuge (model MB Microhematocrit Centrifuge, IEC, Fisher Scientific).

Binding to intracellular proteins.

The EHBR liver was perfused with ice-cold KHB for 3 to 5 min and then homogenized with 3 volumes of ice-cold KHB with a homogenizer (Ultra-turrax T25; Janke & Kunkel IKA-Labortechnik, Staufen im Breisgau, Germany). The liver homogenate was centrifuged at 9000 × g (M2-J Centrifuge; Beckman Canada, Mississauga, ON, Canada) for 20 min at 4°C. After centrifugation, the supernatant was removed, and bulk BSPGSH and [3H]BSPGSH were added to this. The final concentrations of BSPGSH in the supernatant (S9) varied from 3.5 to 526 μM. Tissue protein binding was examined through ultrafiltration (Centricon, Amicon; 10,000 molecular weight cutoff) of a 0.5-ml sample of S9 at 1000 × g (M2-J Centrifuge, Beckman) for 20 min at room temperature.

The radioactivity in the S9 supernatant before ultrafiltration (Ct) and in the ultrafiltrate (Ct,u) was measured. The concentration of bulk BSPGSH in the S9 fraction was assayed by ultraviolet light spectroscopy as described previously (Snel et al., 1993). Assuming only one class of binding sites for binding of BSPGSH to S9 proteins, equation 1 was used for estimation of the binding parameters:Ct,b=Ct−Ct,u=n[Pt]Ct,uKD+Ct,u Equation 1where Ct and Ct,bare the total and bound concentrations in S9, respectively, Ct,u is the unbound concentration in S9 water,KD is the dissociation binding constant, n is the number of binding sites and [Pt] is the molar concentration of the binding protein. Because the molecular weight of the binding protein is unknown, the molar concentration of binding protein could not be calculated, nor could the number of binding sites be resolved (Genget al., 1995b). However, the productn[Pt], or the effective concentration of binding sites in S9, and the dissociation binding constant may be obtained.

Assays.

Quantification of 51Cr and125I radiolabels in blood perfusate (25–100 μl) was carried out by two-channel gamma counting with crossover correction (Cobra II; Canberra-Packard Canada Ltd., Mississauga, Ontario, Canada). Assays of [14C]sucrose and [3H]BSPGSH were carried out according to Geng et al. (1995b). All plasma samples (50–200 μl) were made up to a constant volume of 200 μl with blank plasma for constant quench, and 200 μl of a saturated urea solution (1 g/ml) was added to the plasma sample for the release of [3H]BSPGSH from its tight binding to albumin. After the further addition of 800 μl of acetonitrile, the3H and 14C radioactivities in 1 ml of supernatant were determined by triple-channel (3H, 125I and14C) counting (model 5801; Beckman Instruments, Brea, CA) in 5 ml of ReadyProtein (Beckman). Previous studies had demonstrated that 125I-labeled albumin, normally precipitated with acetonitrile, persisted here as small125I-labeled peptide fragments due to the use of urea (Geng et al., 1995b; Snel et al., 1993). D2O in plasma samples (80 μl) was assayed by Fourier transform infrared spectrometry (model 1600; Perkin Elmer Canada, Rexdale, Ontario, Canada) over the frequency interval from 2300 to 2700 cm−1, as previously described (Panget al., 1991). Assay of the MID dose was performed in a similar fashion, after dilution of the blood or plasma dose 1:10 (v/v) with blank blood perfusate or blank plasma, respectively.

Unlabeled BSPGSH in plasma, S9 fraction and bile.

The concentration of unlabeled BSPGSH in plasma samples was determined directly by UV spectroscopy at 580 nm after alkalinization of the plasma samples with 100 μl of 0.1 N NaOH. The bile samples were examined by HPLC (Snel et al., 1993). The concentration of BSPGSH in S9 was determined spectrophotometrically, after deproteinization with acetonitrile (Geng et al., 1995b).

Data treatment.

The steady-state extraction ratio, E, was used to monitor the disappearance of bulk BSPGSH from plasma. E was calculated from:E=CIn−COutCIn Equation 2where CIn and COutare the steady-state input and output plasma concentrations of BSPGSH, respectively. For the EHBR which lacks the canalicular transporter for BSPGSH biliary excretion, E was found to be virtually zero.

Splining, transit times and superposition.

For the MID portion of the experiment, the outflow radioactivity for each indicator was expressed as a fraction of radioactivity of injected dose per milliliter of blood. The concentration of radiolabels at the end of the collection (180 sec) was <0.1% of peak values. The resulting outflow profiles were approximated with cubic spline functions (De Boor, 1978). From these, recoveries were calculated as the product of the time integrals of the fractional recovery and blood flow. Integrals of the product of fractional recovery and time (AUMC) and fractional recovery (AUC) were calculated similarly (Geng et al., 1995b). The ratio AUMC/AUC gave the mean transit time.

From the fractional outflow recovery curve of the vascular reference (the labeled RBC curve), the transfer function was deconvolved of the injection and collection system of the outflow profile for the sham MID experiment conducted in absence of a liver. A linear flow-limited transformation of the deconvolved RBC curve was then carried out to generate a calculated first pattern for each diffusible reference through selection of trial values for the ratio of the extravascular to vascular distribution spaces and of t0, the common large-vessel transit time (Geng et al., 1995b). The resulting curve was convolved with the system transfer function. The generated diffusible reference curve (for labeled albumin, sucrose or D2O) was compared with that obtained experimentally, and the space parameter ratios (γ) for albumin, sucrose or D2O were refined repetitively until a best fit was obtained. These are the ratios of the stationary space to sinusoidal plasma volume, in which the stationary space is the Disse space or Disse space plus intracellular water space. With these values in hand, a similar process was used to gain best-fit values for influx and efflux coefficients for BSPGSH in EHBR.

Modeling.

A scheme describing the kinetic events underlying the disposition of BSPGSH is shown in figure1. As a starting point, the model adopted previously for interpretation of the uptake and excretion of BSPGSH in perfused liver studies of SDR (Geng et al., 1995b) was used (fig. 1A). The rate constants (k1 andk−1), which are related to the cellular volume, Vcell, have been defined previously (table 1); alternatively, these are expressed as the rate coefficients (k12 andk21), which are defined with respect to the compartments from which the fluxes originate. The sequestration coefficient, kseq ork20, was set to zero to reflect the dysfunctional canalicular excretion of BSPGSH in EHBR. Although there is no binding of BSPGSH to RBC, binding of BSPGSH to proteins occurs within both the plasma and interstitial spaces where protein concentrations are assumed to be the same as are the unbound and bound concentrations of BSPGSH (Geng et al., 1995b). Because albumin is excluded from part of the Disse space (Goresky, 1964), the space of distribution for bound BSPGSH is diminished accordingly. The unbound BSPGSH in the Disse space exchanges with that in the hepatocellular compartment, denoted by the appropriate transfer coefficients for sinusoidal entry into (k12) and efflux from (k21) hepatocytes. Correction of the rate coefficients for binding to plasma and cellular proteins, respectively, and multiplication by the appropriate compartmental volumes will yield the permeability-surface area products for transport (PinS or PoutS) (table 1).

Figure 1
Figure 1

Schematic representation of BSPGSH uptake at the level of a single sinusoid for SDR (A), whose rate constants for influx (k1), efflux (k−1) and sequestration (kseq) have been defined in reference to Vcell, the accessible cell water volume (see table 1), and for EHBR (B), for which excretion is absent and there was segregation of a shallow pool and a deep pool within the hepatocyte. The sinusoidal transfer coefficients are denoted by k12 andk21, respectively, and the rate constants between the cellular and deep pools by k23and k32 (see table 1 for definition). However, elimination (denoted by dashed line) could hypothetically occur from the shallow pool (with rate constantk20 >0) or from the deep pool (with rate constant k30 >0). Equilibria are assumed between bound and unbound forms of BSPGSH in plasma and tissue.cp,u, cp,b,cD,u, cD,b,ch,u and ch,b are the unbound and bound concentrations of BSPGSH in the sinusoid, Disse space and cell, respectively.

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Table 1

Interrelationships between influx, efflux and sequestration coefficients and their physical equivalents

In contrast to the case of SDR, the model for the [3H]BSPGSH data for EHBR required an additional “deep pool” within the hepatocyte (fig. 1B) because a protracted tail persisted in the data, especially for lower-input concentrations of BSPGSH. The kinetic analysis is presented of the events underlying the disposition of BSPGSH in the single-pass perfused EHBR rat liver preparation (fig. 1B), although the biological nature of the deep pool could not be appraised from the data. The parameters describing the transfer coefficients between the “shallow” (cytosolic) and deep pools within the cell are denoted by k23and k32. As before,k20 was assumed to be zero.

The presence of two intracellular pools may be interpreted in different ways. In the first instance, the pools may represent different labeled species that occupy the same physical space, such as drug in cytosolic solution and drug bound to intracellular proteins. In this case, the efflux permeability-surface area product is obtained from:PoutS=k21 Vcell/ft Equation 3Alternatively, they may represent different physical locations within the cell. In the latter case, the cellular volume Vcell has to be partitioned according to the ratio k23/k32, and the outflow permeability-surface area product is obtained from:PoutS=k21 Vcellft1+k23k32 Equation 4A quantitative analysis of the [3H]BSPGSH outflow profiles was carried out with a model (see ). To evaluate the outflow profiles, the dispersion of the injected bolus by the injection apparatus and the inflow and outflow catheters must be considered, as described previously in detail (Chiba et al., 1998; Geng et al., 1995b). Because the outflow profile of sucrose served as the reference curve, a catheter-corrected outflow profile was obtained by deconvolving the transfer function of the injection apparatus and the inflow and outflow catheters,Ccath(t) (Bassingthwaighte, 1967; Beck et al., 1985). The latter was obtained from the outflow profile of sham experiments in the absence of a liver, performed by injecting a tracer into the inflow and outflow catheters.

The calculated BSPGSH outflow profile,CBSPGSH(t), is obtained through convolution of the liver transport function for BSPGSH,hBSPGSH (t), with the outflow profile obtained from the apparatus in the absence of a liver,Ccath(t):CBSPGSH(t)=(Ccath*hBSPGSH)(t) Equation 5For BSPGSH that was removed in SDR, the organ transport function for a one-pool system (fig. 1A) is given in the (see equationEA9). In the absence of removal (k20 = 0), the transport function for BSPGSH, assuming two intracellular pools in liver, is given by equation EA11 in the . The theoretical reference transport function,href(t), then may be appropriately related to that for sucrose,hSuc(t) (equation EA7, ) to describe the extracellular behavior of BSPGSH, with use of the parameter γrel. The latter is obtained through linear superposition of the outflow profiles of the nonmetabolized indicators RBC, albumin and sucrose as previously described and outlined (equations EA8 and EA3 in the ).

Equation 5 was fitted to the data through variation of the influx and efflux coefficients (fuk1θ′ and k′−1) and γrel for the one-pool model ork12, k21,k23 and k32 and γrel for the two-pool model by use of a least-squares procedure as described previously (Chiba et al., 1998; Geng et al., 1995b; Schwab, 1984). Fitted parameters were obtained by a weighted least-squares procedure (nonlinear regression analysis) from the International Mathematical and Statistical Library (IMSL; Houston, TX). The classic weighted least-squares approach to parameter estimates was used as the criterion for fitting. Weighting was applied according to counting statistics noise, assuming an estimated error proportional to the square root of the magnitude of the observation (Landaw and DiStefano, 1984). The jacobian matrix (matrix of sensitivities) obtained from the fitting program was used to calculate variances and covariances of the fitted parameters. The square roots of the variances that were calculated for each experiment represented the standard deviations of the fitted parameters or the uncertainty in the determination of the parameters for the experiment.

Results

Tissue binding of BSPGSH and tissue concentrations.

The intracellular binding of BSPGSH was explored over tissue concentrations ranging from 3.5 to 526 μM, in duplicate. The optimized fitting indicated that there was only one class of binding sites for BSPGSH in S9 fraction (fig. 2A). The effective binding concentration, n[Pt], and the dissociation constant, KD , were obtained from a nonlinear regression procedure. After correction for the dilution of S9 for the study of tissue binding, the resulting binding constants were: KA , the binding association constant, or 1/KD , was 2.4 × 104 M−1, and the binding capacity, n[Pt], was 53 μM. These constants revealed a weaker binding of BSPGSH to EHBR liver S9 proteins in relation to those from SDR (Geng et al., 1995b). The unbound fraction of BSPGSH in EHBR liver homogenate (ft = Ct,u/Ct) was found to increase from 0.15 to 0.56 for BSPGSH S9 concentrations of 3.5 to 526 μM. These unbound tissue fractions were much higher than those observed at comparable BSPGSH concentrations in the liver S9 fraction for the SDR (Geng et al., 1995b).

Figure 2
Figure 2

Tissue binding of BSPGSH to EHBR liver S9. Nonlinear regression of BSPGSH protein binding was performed, and binding conformed to one class of cytosolic binding site. The binding constants denote lesser binding of BSPGSH to EHBR liver proteins in comparison to controls (SDR).

The BSPGSH concentrations in S9 at the end of the EHBR liver perfusion experiments ranged from 159 to 276 μM (220 ± 38 μM), and the corresponding unbound fractions in tissue (ft) varied from 0.12 to 0.23. The tissue unbound concentration of BSPGSH in the perfusion experiments were approximated by taking the product of ft and Ct (ranging from 47 to 120 μM; mean, 83 ± 23 μM).

Lack of removal of BSPGSH.

Among the perfusion experiments where different bulk plasma concentrations of BSPGSH (26–257 μM) were used, the steady-state hepatic extraction ratios of unlabeled BSPGSH was ∼0 (fig. 3). The radiolabel in the outflowing perfusate was mainly [3H]BSPGSH, as found by TLC. Only trace amounts of metabolites of BSPGSH were found in bile with HPLC. Cleavage products were not found in liver homogenates (data not shown). In contrast, the extraction ratio of [3H]BSPGSH, estimated as (1 − integral recovery in plasma), was 0.26 ± 0.11 at the lower concentration of BSPGSH; the lack of complete recovery in outflow plasma was attributed to accumulation of [3H]BSPGSH in the liver, whose efflux into hepatic venous plasma was slow, as found earlier (Geng et al., 1995b).

Figure 3
Figure 3

The steady state extraction ratios of BSPGSH in EHBR in the present single-pass studies (virtually zero in value) are compared with those in SDR (data of Geng et al., 1995b).

MID studies.

Recoveries of radiolabeled51Cr-RBC, 125I-albumin, [14C]sucrose and D2O in hepatic venous samples were complete, within experimental errors. Representative outflow profiles for the labeled substances injected into the portal vein of the liver, expressed as fractional recovery of dose per milliliter for each species, are shown in figure4.

Figure 4
Figure 4

Fractional recoveries of [3H]BSPGSH and the noneliminated reference indicators, with different input concentrations of BSPGSH in linear (left) and semilogarithmic (right) presentation, with a lower (top) and higher (bottom) input concentration of BSPGSH. Note the presence of tailing of the outflow profile of [3H]BSPGSH at low BSPGSH bulk concentration. Saturation of the uptake process was evident because there was less separation between the [14C]sucrose and [3H]BSPGSH curves at the higher bulk concentrations of BSPGSH.

The labeled RBC emerged first and reached the highest and earliest peak; their outflow curve had the steepest upslope, and the downslope decayed most rapidly (fig. 4, left). The [125I]albumin curve rose slightly less quickly and decayed with a slightly reduced slope; it showed a lower and later peak. The [14C]sucrose curve showed, in comparison to the labeled albumin curve, a slightly delayed upslope, a lower and later peak and a more prolonged downslope. The greatest dispersion was seen with D2O, whose upslope and downslope were much delayed and whose peak occurred much later with a lower magnitude due to its permeation of the cellular as well as vascular and interstitial spaces.

Good superposition of the noneliminated reference indicator curves onto the RBC curve was found for nonsequestered references (data not shown), confirming their flow-limited distribution (Goresky, 1964). The values for γ and t0 for the present experiments are given in table 2 and were compared with those for SDR (data from Geng et al., 1995b). The estimated t0 (1.4 ± 0.7 sec) was lower than that reported previously for normal rats, suggesting a lower large-vessel volume for EHBR. The mean transit times for each of the indicators and the volumes of the Disse space and the cellular water space were calculated for the nonsequestered reference indicators (table 2). The sinusoidal blood volume (Vsin) and the Disse space of albumin or sucrose were similar (P > .05) to those for SDR, as were the space ratios γAlb, γSuc and γD2O (table 2). The average value of θ (Vcell/Vp) in the EHBR rat was not different from that for SDR (P > .14).

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Table 2

Comparison of the mean transit times of noneliminated references and their distribution volumes in the MID studies in EHBR and SDR

BSPGSH behavior.

The initial upslopes in the outflow profiles for [3H]BSPGSH were slightly delayed with respect to those for the labeled RBC and albumin, reached a lower magnitude and decayed more slowly (fig. 4, right). A characteristic tailing curve pattern was observed at lower bulk BSPGSH concentrations, whereas at a higher bulk concentration of BSPGSH, the [3H]BSPGSH curve was of a higher magnitude and exhibited a downslope similar to that of sucrose (fig. 4, right), suggesting saturation of the process underlying uptake and an earlier return to the vascular space.

One- vs. two-pool model.

The calculated BSPGSH outflow profile obtained through convolution of the liver transport function for BSPGSH, hBSPGSH(t), for the one-pool model (equation EA9, ) with the outflow profile obtained from the apparatus in the absence of a liver,Ccath(t), failed to describe the late-in-time [3H]BSPGSH outflow data (fig. 5). In contrast, when the liver transport function for the two-pool model (equation EA11,) was used, a much improved fit was obtained (fig. 5). The fitted curves, based on the two-pool model, were further segregated into the throughput and returning components (fig.6). A greater throughput component (proportion of the dose traversing the organ without entering the liver cell) existed with the higher BSPGSH concentration (fig.7). The concentration dependence observed for EHBR was similar to that observed for SDR.

Figure 5
Figure 5

Fits of the [3H]BSPGSH curve to the one-pool (see fig. 1A) and two-pool (see fig. 1B) with absence of sequestration. Note the superior fit of the data to the two-pool model.

Figure 6
Figure 6

Segregation of the [3H]BSPGSH fitted curve according to the throughput component, the portion of dose which never left the vasculature, and the returning component, the portion of dose which entered the hepatocyte and returned at a later time.

Figure 7
Figure 7

The throughput component was higher with increasing logarithmic average plasma unbound concentration of BSPGSH,Ĉp,u, for EHBR. The trend was similar to that previously observed for SDR (data from Geng et al., 1995b) and suggests saturation of the uptake process.

The estimated parameters based on the two-pool model are summarized (table 3). The influx coefficient,k12, governing the value of the first term of the right side of equation EA11 is linked to influx and varied in a concentration-dependent manner with BSPGSH concentration (table 3). The involvement of transporters for BSPGSH became evident in the plots of PinS vs.Ĉp,u, the logarithmic average plasma unbound concentration of BSPGSH (fig.8A); the latter is defined in relation to the unbound input (CIn,u) and unbound output (COut,u) concentrations, as follows:C^p,u=CIn,u−COut,uln(CIn,u/COut,u) Equation 6The influx permeability-surface area product (PinS) was found to decrease with increase of the logarithmic average unbound plasma concentration of BSPGSH (fig. 8A). Because θ and γrel are constants (tables 2and 3), values of k1 also decreased in a corresponding fashion (fig. 8B). Much scatter, however, existed for PinS for EHBR (fig. 8). When the data were regressed against the logarithmic unbound concentrationĈp,u (equation 6), values ofVmax (0.47 ± 0.41 μmol·sec−1·ml−1cellular water space) and Km (0.07 ± 2.6 μM) were obtained for BSPGSH entry into the EHBR liver. These may be unreliable due to the high degree of scatter. The parameters were, nevertheless, similar to those observed for those for the SDR: Vmax (0.79 ± 0.24 μmol·sec−1·ml−1cellular water space) and Km (1.48 ± 0.78 μM) as found by Geng et al. (1995b).

View this table:
Table 3

Optimized parameters derived from fitting of the [3H]BSPGSH data from EHBR to the two-pool model

Figure 8
Figure 8

Plots of the influx permeability-surface area product (A) and the influx rate constant, k1(B), as functions of the logarithmic average plasma unbound concentration of BSPGSH, Ĉp,u, for EHBR and SDR (data of Geng et al., 1995b).

Efflux of BSPGSH and tissue partitioning.

The interpretation of the efflux permeability-surface area product, PoutS, depends on whether the deep pool shares the same space as the shallow pool or whether the deep pool represents some other distinct space. For this reason, two sets of efflux permeability-surface area products and tissue-partitioning ratios (PinS/PoutS) were estimated with equations 3 and 4. Values for PoutS associated with the shallow and deep pools being a common space were similar to those for influx (PinS), but these were substantially lower when the shallow and deep pools were viewed as separate spaces (table 3). The tissue-partitioning (PinS/PoutS) values of BSPGSH for the former case varied from 2.3 to 0.03 with increasing BSPGSH concentration but were dramatically higher (from 29 to 0.2) when the deep pool was a separate, discrete space. The trend for higher values was similar to that observed for the ratios of the unbound tissue to unbound plasma concentration, which ranged from 43 to 1 (table 3).

Discussion

The handling of BSPGSH, a model substrate that enters and leaves the liver cell through carrier-mediated systems at both the sinusoidal and canalicular membranes, was presently studied in the EHBR perfused liver preparation. In bile, only trace amounts of BSPGSH or its cleavage metabolites were found by HPLC, confirming the impairment of ATP-dependent biliary transport of the GSH conjugates. Compared with normal SDR, an apparent steady state was reached only slowly in EHBR (after 2 hr at <150 μM and ≈1 hr at >200 μM BSPGSH). Although the apparent steady-state hepatic extraction ratio of unlabeled BSPGSH was almost equal to zero, an apparent loss of [3H]BSPGSH from plasma within the sampling time for MID was observed at the lower BSPGSH concentrations. The loss could be accounted for by the temporary storage in liver inasmuch as efflux of BSPGSH is known to be extremely slow (Geng et al., 1995b).

Apart from the lack of biliary excretion in EHBR rats, both similarities and differences in trans-sinusoidal transfer and hepatic distribution were found. Of note, the blood volume, Disse spaces for albumin and sucrose, Disse-sinusoidal space ratios (γ) and accessible intracellular water space in the EHBR liver were found to be similar to those in controls (table 2). The t0 was, however, smaller, and the reason for the difference is not apparent because there was no difference observed among the physiological volumes. The measured protein concentration in S9 (156 ± 23 mg/g liver, n = 10 livers) in EHBR liver was consistently lower than that in normal SDR (200–250 mg/g liver), and the binding of BSPGSH in EHBR liver tissue also differed from controls (Geng et al., 1995b). In EHBR, only one class of binding site was identified in the liver S9 fraction (KA = 2.4 × 104 M−1 and an effective binding concentration of 53 μM). In control SDR, tissue binding showed tighter binding of BSPGSH with two classes of binding sites (2.5 × 105 and 4.8 × 103 M−1; effective binding concentrations of 29 and 657 μM) (Geng et al., 1995b). The binding proteins likely represent GSH transferase B (ligandin or Y protein), and to a lesser extent, fatty acid-binding protein (Sorrentino et al., 1989). Because of lower binding capacity and affinity, the tissue unbound fractions of BSPGSH (0.15–0.56 for the tissue S9 concentration range of 3.5–526 μM) were significantly higher than those at comparable concentrations in SDR; the latter varied from 0.1 to 0.3 when the S9 BSPGSH concentrations ranged from 3 to 522 μM (Geng et al., 1995b). Reduced tissue binding of indocyanine green to ligandin was similarly observed in EHBR livers (Sathirakul et al., 1993), and changes in plasma protein binding also have been noted in vivo (Nadai et al., 1994). However, there was no change in the cytosolic binding of dibromosulfophthalein or for other sulfate/glucuronide conjugates (Takenaka et al., 1995a). The alteration in cytosolic binding appears to be substrate specific.

Modeling of the outflow dilution data of [3H]BSPGSH in EHBR with a one-pool system in the liver, as described previously for the MID data obtained from SDR liver perfusions (Geng et al., 1995b), was unable to fully describe the late-in-time data (fig. 5). The tail normally has been construed as a slowly returning component due to the presence of an extra deep pool (Schwab et al., 1990). The ratiok23/k32 was found to be 12 ± 8 (table 3), suggesting that this deep pool contained much higher BSPGSH concentrations than those in the cellular pool. The deep pool cannot be readily associated with liver-binding proteins because the effective binding concentration for BSPGSH (53 μM) was actually lower than that found for controls (Geng et al., 1995b). An equally plausible explanation of the data would be the slow intracellular diffusion (Luxon and Weisiger, 1993); however, this view is incompatible with relatively high unbound fractions inside the cells. Thus, the physiological meaning of this extra deep pool is not evident.

Similar parameters for PinS and γrel were obtained for EHBR when data were fitted to the one-pool model, although the tail component was not fitted well (fig. 5). Alternatively, when the two-pool model was applied to fit the SDR data, good fits were obtained, and there was virtually no change in the values of PinS and γrel between the one- and two-pool models (comparison not shown). In contrast, the incorporation of the additional deep pool fully described the outflow profile of [3H]BSPGSH in the EHBR perfused livers. Discrete patterns of influx and efflux of BSPGSH were obtained in EHBR rats in the absence of excretion. With appraisal of the outflow dilution profile of [3H]BSPGSH in relation to that for the theoretical reference, the influx parameters, characterized by the influx permeability-surface area product (or influx clearance/g of liver), PinS (fig. 8A), or the influx rate constant, k1 (fig. 8B), were similar to those for SDR (Geng et al., 1995b), and the pattern is characteristic of a carrier-mediated process (Geng et al., 1995a). The absence of change in the influx rate constant between SDR and EHBR livers was also observed for the dual thromboxane A2 synthetase and 5-lipoxygenase inhibitor, 6-hydroxy-5,7-dimethyl-2-methyamino-4(3-pyridylmethylbenzothiazole), E3040 (Takenaka et al., 1995b).

The data for EHBR were, however, consistent with those for the SDR. In SDR, biliary excretion reduces the amount of labeled BSPGSH available for return to the vasculature. When the return rate of this material is very low, the tail was much smaller and was not consistently present. The presence of the deep pool will be concealed and the one-pool model was considered adequate to describe the SDR data (Geng et al., 1995b). If this hypothesis is correct, the only difference between the two systems should be excretion at the canalicular membrane. This is illustrated in figure9, which depicts the influence of biliary excretion on the shape of a BSPGSH outflow profile for EHBR at low BSPGSH concentration. Keeping the parameters the same as for the EHBR curve, inclusion of excretion from the shallow pool (k20 = 0.1 sec−1) in the two-pool system predicted an outflow profile with a much smaller tail, resembling the data observed for SDR. In contrast, the correspondence of the predicted outflow profile to SDR data was not as good if excretion was assumed to occur from the “deep pool” (k30 = 0.1 sec−1). Hence, in the absence of the ATP-dependent biliary excretion mechanism, as is the situation in EHBR mutant rats, BSPGSH will accumulate in hepatocytes, revealing the presence of the deep pool.

Figure 9
Figure 9

Plots of the[3H]BSPGSH outflow data and fitted curve based on a two-pool model for the EHBR against a background set of data obtained with SDR (Geng et al., 1995b) at comparable [3H]BSPGSH concentrations (25 μM) in single-pass perfused rat liver preparations (12 ml/min). The [3H]BSPGSH curve for EHBR was modified by the incorporation of excretion in the shallow (k20 = 0.1 sec−1) or deep (k30 = 0.1 sec−1) pool. Note the similarity in shapes between the SDR data and the predicted curve based on excretion from the shallow pool.

There existed remaining ambiguity in the assessment of BSPGSH efflux (k−1, k21 or PoutS) and its equilibrium tissue partitioning for EHBR due to the uncertainty regarding the deep pool, whether this is physically part of, or separate from, the shallow pool. The values for PoutS would indeed differ between the one- and two-pool models (comparison not shown). Although the equilibrium partitioning between the deep and shallow pools (k23/k32) is known, the volume of the deep pool appears to be unattainable from the data. Values of PoutS associated with a common space for the shallow and deep pools are, however, too high and are incompatible with previous characteristics observed for BSPGSH, which is seldom detected in the hepatic venous blood/plasma after administration of bromosulfophthalein (Chen et al., 1984;Zhao et al., 1993). This has been attributed to the inherently slow efflux of BSPGSH (Geng et al., 1995b). Moreover, the partitioning of BSPGSH in S9 was high (see table 3), as was that for SDR (Geng et al., 1995b). For the above reasons, that separate spaces exist for the distribution of BSPGSH in liver appears to be a reasonable assumption.

In summary, the sinusoidal blood volume, Disse space and accessible cellular water space in EHBR livers were found to be similar to those for control rats (table 2). The fitting procedure evoked an additional pool that became apparent only in EHBR because biliary excretion of BSPGSH was absent. The deep pool appeared to be a space distinct from that for the shallow pool in view of the low level of return of BSPGSH and the tissue-to-plasma concentration ratios (table 3). The entry of BSPGSH into the liver cell of EHBR rats, given the scatter observed in the data, was found to be similar to that of controls. These findings confirm the notion that the mutation present in EHBR has only eliminated canalicular transport without causing other major effects on cellular function.

Acknowledgments

The authors thank André Simard for preparation of the figures.

Mathematical Description of the Model

To evaluate the experimentally obtained outflow profiles, the dispersion of the injected bolus by the injection apparatus and the inflow and outflow catheters must be considered, as previously described in detail (Chiba et al., 1998; Genget al., 1995b). The experimental sucrose curve,CSuc(t), is the convolution of the organ sucrose transport function (catheter-corrected outflow profile or impulse response),hSuc(t), with the outflow profile obtained from the apparatus in the absence of a liver,Ccath(t):CSuc (t)=(Ccath*hSuc)(t) Equation A1where * is the convolution operator. Similarly, for RBC,CRBC (t)=(Ccath*hRBC)(t) Equation A2The transport functions of the reference tracers,hRBC(t) andhSuc(t), were computed from the experimental data for CRBC(t),CSuc(t) andCcath(t) through deconvolution using an algorithm obtained from the National Simulation Resource in Mass Transport and Exchange (University of Washington, Seattle, WA) (Bassingthwaighte, 1967; Beck, et al., 1985).

The parameters of linear superposition according to the flow-limited model of Goresky (Chiba et al., 1998; Goresky, 1964; Goreskyet al., 1973, 1992; Schwab et al., 1990), the interstitial-to-vascular distribution spaces, γSuc, and the common large-vessel transit time,t0, were found by first calculating the RBC transport function, hRBC(t), through deconvolution as mentioned above and then calculating the organ sucrose transport function,hSuc(t), from the organ RBC transport function, hRBC(t), according to the following equation:hSuc (t)=11+γSuc hRBCt−t01+γSuc+t0 Equation A3A calculated sucrose outflow profile then is determined through convolution according to equation EA1. A similar procedure is used to calculate the corresponding outflow profile for labeled albumin,CAlb(t). Both calculated profiles then are fitted simultaneously to the experimental outflow profiles,CSuc(t) andCAlb(t) according to a nonlinear least-squares procedure, to obtain optimal values fort0 and γSuc.

Uptake, release and sequestration of BSPGSH were evaluated by use of the barrier-limited space-distributed variable transit time model developed by Goresky et al. (1973). This model allows the determination of the mass transfer coefficients (table 1) by comparing the outflow profile of BSPGSH under study with appropriate reference indicators that are not taken up by hepatocytes. The calculated BSPGSH outflow profile, CBSPGSH(t), is obtained through convolution of the liver transport function for BSPGSH, hBSPGSH(t), with the outflow profile obtained from the apparatus in the absence of a liver,Ccath(t):CBSPGSH(t)=(Ccath*hBSPGSH)(t) Equation A4BSPGSH does not distribute into RBC and is partly excluded from the interstitial space, so none of the experimental reference indicators are directly usable for the modeling process, and a theoretical reference transport function was constructed, as followshref (t)=11+γref hRBCt−t01+γref+t0 Equation A5where γref is the ratio of the extravascular to the vascular distribu-tion space of BSPGSH. The value of this ratio isγref=fuγSuc+(1−fu)γAlb Equation A6Because sucrose was used as a reference indicator, the appropriate reference transport function was calculated as follows (Chiba et al., 1998):href(t)=11+γrel hSuct−t01+γref+t0 Equation A7whereγrel=1+γref1+γSuc−1 Equation A8Expressions for thehBSPGSH(t) have been previously found as follows (Schwab et al., 1990), for one intracellular pool:hBSPGSH (t)=e−k12(t−t0) href(t)+e−(k21+k20)(t−t0) Equation A9·∫0t−t0 href (τ+t0)e(−k12+k21+k20)τ ∑n=1∞ (k12k21 τ)n(t−t0−τ)n−1n!(n−1)! dτ where k12 =fuk1θ/(1 + γref) and k21 =ft k−1 are transfer coefficients for entry into and efflux from the hepatocytes, andk20 = ftkseq, is the cellular sequestration coefficient. The ratio of cellular to extracellular distribution spaces for BSPGSH, θ′, is obtained as follows (Chiba et al., 1998):θ′=θ(1+γref) Equation A10The analogous expression for two intracellular pools is (Schwabet al., 1990):hBSPGSH (t)=e−k12(t−t0) href(t)+es1(t−t0) ∫0t−t0 href(τ+t0)e−(k12+s1)τ Equation A11·∑n=1∞k12k21[s1+k32]τs2−s1n(t−t0−τ)n−1n!(n−1)! dτ +es2(t−t0) ∫0t−t0 href(τ+t0)e−(k12+s2)τ ·∑n=1∞k12k21[s2+k32]τs2−s1n(t−t0−τ)n−1n!(n−1)! dτ+es2(t−t0) ∫0t−t0 href(τ+t0)e−(k12+s1)τ  ∫τt−t0 e(s2−s1)h ∑n=1∞k12k21[s1+k32]τs2−s1n(h−t)n−1n!(n−1)! ·∑n=1∞k12k21[s2+k32]τs2−s1n (t−t0−h)n−1n!(n−1)! dh dτ where k23 andk32 are transfer coefficients for exchange between the shallow and the deep pool, ands1 and s2 are the roots of the following quadratic equation in s:s2+(k21+k23+k32+k20)s+k32(k21+k20) Equation A12However, numerical evaluation of equation EA11 with convolution according to equation EA4 is not practicable because the required computer time becomes excessive. We therefore used a finite-difference method that yields directly CBSPGSH(t) from href(t) and transfer coefficients. When applied to the situation with one intracellular pool, this method yields results that are, with reasonable accuracy, identical to those obtained with direct evaluation of equation EA11.

CBSBGSH(t) then was fitted to the experimental data by varying the parametersk12, k21,k23, k32 and γrel. Because biliary excretion of BSPGSH is absent in EHBR, k20 was set to zero. From the fitted value of k12 =fuk1θ/(1 + γref), k1 was calculated using θ′ and fu. Finally, the influx permeability-surface area product, PinS, was obtained as follows:PinS=k12VSuc(1+γrel)/fu Equation A13The value of the efflux permeability-surface area product, PoutS, depends on whether the two intracellular pools are assumed to occupy the same physical space or separate physical spaces. In the first instance, which would represent a situation in which two different species of BSPGSH are present inside the cells (e.g., unbound and proteins bound), the efflux permeability-surface area product is obtained from:PoutS=k21Vcell/ft Equation A14In the other case, assuming that diffusion of BSPGSH between different parts of the cell is slow, the cellular volume Vcell must to be partitioned according to the ratio k23/k32. The efflux permeability-surface area product then is obtained from:PoutS=k21 Vcellft1+k23k32 Equation A15For a compound that is not eliminated, the tissue-partitioning ratio is given by the following:PinSPoutS=Ct,uC^p,u Equation A16

Footnotes

  • Send reprint requests to: Dr. K. S. Pang, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S 2S2. E-mail: pang{at}phm.utoronto.ca

  • ↵1 This work was supported by the National Institutes of Health, US Public Health Service (Grant GM-38250), Medical Research Council of Canada (Grant MT-11228) and Fast Foundation.

  • Abbreviations:
    EHBR
    Eisai hyperbilirubinemic rat
    SDR
    Sprague-Dawley rat
    MID
    multiple indicator dilution
    ABC
    ATP-binding cassette
    TLC
    thin-layer chromatography, HPLC, high-performance liquid chromatography, E, extraction ratio
    AUC
    area under the curve
    Hct
    hematocrit
    RBC
    red blood cells
    BSPGSH
    bromosulfophthalein glutathione conjugate
    GSH
    glutathione
    GST
    glutathioneS-transferase
    KHB
    Krebs-Henseleit bicarbonate solution
    MRP
    multidrug-resistance protein
    cMOAT
    canalicular multispecific organic anion transporter
    • Received May 8, 1997.
    • Accepted October 20, 1997.

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OtherDRUG METABOLISM AND DISPOSITION

Hepatic Uptake of Bromosulfophthalein-Glutathione in Perfused Eisai Hyperbilirubinemic Mutant Rat Liver: A Multiple-Indicator Dilution Study

Wanping Geng, Andreas J. Schwab, Tohru Horie, Carl A. Goresky and K. Sandy Pang
Journal of Pharmacology and Experimental Therapeutics February 1, 1998, 284 (2) 480-492;
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