Hepatic Uptake of Bilirubin and Its Conjugates by the Human Organic Anion Transporter SLC21A6*

Yunhai CuiDagger, Jörg König, Inka Leier, Ulrike Buchholz, and Dietrich Keppler

From the Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany

Received for publication, June 8, 2000, and in revised form, December 12, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bilirubin, the end product of heme catabolism, is taken up from the blood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of bilirubin and its conjugates into human hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing the recombinant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symbol SLC21A6) showed uptake of [3H]monoglucuronosyl bilirubin, [3H]bisglucuronosyl bilirubin, and [3H]sulfobromophthalein with Km values of 0.10, 0.28, and 0.14 µM, respectively. High-affinity uptake of unconjugated [3H]bilirubin by OATP2 occurred in the presence of albumin and was not mediated by another basolateral hepatic uptake transporter, human OATP8 (symbol SLC21A8). OATP2 and OATP8 differed by their capacity to extract substrates from albumin before transport. In comparison to the high-affinity transport by OATP2, OATP8 transported [3H]sulfobromophthalein and [3H]monoglucuronosyl bilirubin with lower affinity, with Km values of 3.3 and 0.5 µM, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a Ki value of 112 nM, but did not inhibit transport mediated by OATP8. Human OATP2 may play a key role in the prevention of hyperbilirubinemia by facilitating the selective entry of unconjugated bilirubin and its glucuronate conjugates into human hepatocytes.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bilirubin, the main bile pigment in most mammals, is the end product of heme catabolism (1). In the blood circulation, bilirubin is bound to serum albumin, which prevents its potential toxicity thought to be caused by the free ligand (2). Despite high-affinity binding to albumin, bilirubin is rapidly and selectively taken up into the liver (3, 4), biotransformed upon conjugation with glucuronate (5), and secreted into bile across the canalicular membrane of hepatocytes by an ATP-dependent conjugate export pump termed multidrug resistance protein 2 (transporter symbol ABCC2) (6, 7). In addition to a reduction of UDP-glucuronosyl transferase activity (8), impaired bilirubin uptake from the blood circulation into liver has been suggested to contribute to a subgroup of patients with Gilbert's syndrome (9), which is characterized by a mild unconjugated hyperbilirubinemia. Uptake of bilirubin by hepatocytes was considered to be a process mediated by specific membrane proteins, although passive diffusion has also been proposed as a possible mechanism (1, 3, 10). Because of its instability and low solubility in aqueous solution, hepatic uptake of bilirubin was studied predominantly by use of structurally related anionic substances like sulfobromophthalein (BSP)1 and indocyanine green (ICG) (3, 9, 11, 12). A transport protein for BSP with a Michaelis-Menten constant (Km) of 1.5 µM has been cloned from rat liver (13) and designated as organic anion-transporting polypeptide 1 (rat OATP1). Rat OATP1 belongs to a family of transport proteins (OATP family, symbol SLC21A) mediating the transport of organic anions including bile salts, steroid conjugates, thyroid hormones, prostaglandins, and BSP (14). For human OATP1 (SLC21A3), which is expressed at high levels in brain and kidney and at a low level in human liver, kinetic studies revealed only a moderate affinity for BSP with a Km value of 20 µM (15). In a search for additional OATP isoforms in human liver, we and other groups have recently cloned a new member of this transporter family, human OATP2 (also known as LST1 or OATP-C, gene symbol SLC21A6) (16-18). Most recently, we cloned an additional human liver OATP isoform termed OATP8 (gene symbol SLC21A8), which shares 80% identical amino acids with human OATP2 (19). Antibodies raised against both transport proteins localized them to the basolateral membrane of human hepatocytes (16, 19). Northern blot analyses demonstrated an apparently exclusive hepatic expression of both transporters (16, 19). The availability of cell lines stably expressing human OATP2 and OATP8 enabled us to answer the question of whether these two major human hepatic OATP family members are capable of transporting bilirubin and its conjugates from blood across the basolateral membrane into hepatocytes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Cell Lines-- HEK293 cells were cultured in minimum essential medium (Sigma) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C, 95% humidity, and 5% CO2 as described recently (16, 19). HEK-OATP2 cells (16) and HEK-OATP8 cells (19) permanently expressed high levels of human recombinant OATP2 and OATP8, respectively. The GenBankTM/European Molecular Biology Laboratory accession numbers for the sequences of OATP2 and OATP8 are AJ132573 and AJ251506, respectively.

Biosynthesis of [3H]Bilirubin-- [3H]Bilirubin was obtained biosynthetically in rats in a procedure similar to the one described by Crawford et al. (20). Two Harlan Sprague-Dawley rats were given an intravenous or intraportal injection of 500 µCi of [3,5-3H]delta-aminolevulinic acid (25.9 GBq/mmol; NEN Life Sciences, Boston, MA) at a dose of 83 and 42 MBq/kg body weight, respectively. [3H]Bilirubin was isolated from bile by hydrolysis of its glucuronides and extraction with chloroform (20). The purity of the [3H]bilirubin was confirmed by HPLC, and the specific radioactivity was 120,000-140,000 dpm/nmol (2.0-2.3 GBq/mmol) for the material obtained from both rats. In all experiments, [3H]bilirubin was protected from exposure to light.

Synthesis of [3H]Monoglucuronosyl Bilirubin and [3H]Bisglucuronosyl Bilirubin-- Bilirubin glucuronides were prepared using recombinant UDP-glucuronosyl transferase 1A1 and UDP[1-3H]glucuronic acid (0.56 TBq/mmol; Biotrend, Köln, Germany) together with unlabeled bilirubin as described previously (6, 7) and purified using radio-HPLC.

Uptake Studies-- For uptake assays, cells were seeded in 6-well plates (coated with 0.1 mg/ml poly-D-lysine) at a density of 1.5-2 × 106 cells/well and cultured with 10 mM sodium butyrate for 24 h. Before the uptake experiments, cells were washed with uptake buffer (142 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 5 mM glucose, and 12.5 mM HEPES, pH 7.3). The transport assay was started by the addition of 1 ml of uptake buffer containing 3H-labeled substrate (18.5-37 MBq/ml) to the cells. 3H-labeled substrates were obtained from NEN Life Sciences.

[3H]BSP (0.5 TBq/mmol) was obtained by custom synthesis (Hartmann Analytic, Köln, Germany); its purity (>98%) was confirmed by reverse-phase HPLC analysis on a C18 Hypersil column (5-µm particles; Shandon, Runcorn, United Kingdom) using two different systems. The isocratic elution was performed with 45% methanol/55% water containing 50 mM NaH2PO4 and 5 mM Na2SO4 at pH 2.8. The linear gradient elution was performed from 100% buffer A (45% methanol/55% water containing 2 mM tetrabutylammonium hydroxide at pH 6.0) to 100% buffer B (90% methanol/10% water containing 2 mM tetrabutylammonium hydroxide at pH 6.0). The specific radioactivity of [3H]BSP did not change during repeated HPLC analyses, indicating that 3H exchange was below detectability. [3H]BSP strictly co-chromatographed with unlabeled BSP during HPLC. Moreover, the unlabeled BSP and the 3H-labeled BSP were analyzed by nanoelectrospray mass spectrometry as described by Lehmann and Kaspersen (21). The isotopic pattern of molecular ions of unlabeled BSP and [3H]BSP was very similar, and quantitative evaluation indicated the following relative amounts: unlabeled BSP, 63.1%; singly labeled [3H]BSP 27.4%; and doubly labeled [3H]BSP, 9.5%. These data correspond to a specific radioactivity of 0.5 TBq/mmol.

For inhibition studies, inhibitors were included in the uptake buffer. After incubation at 37 °C, transport was stopped at different time points by the addition of 1 ml of cold uptake buffer. Cells were subsequently washed three times with uptake buffer and lysed with 1 ml of 0.2% SDS in water. Aliquots (250 µl) of the lysate were counted for radioactivity. Protein content was determined by the Lowry method using 100 µl of lysate.

Uptake Studies with [3H]Bilirubin-- Due to high background binding of [3H]bilirubin to the poly-D-lysine-coated plastic dishes, uptake of [3H]bilirubin into transfected cells was measured in cell suspension. Cells were cultured with 10 mM butyrate for 24 h as described previously (16). For uptake assays, cells were detached from culture flasks by knocking, washed twice with uptake buffer, and resuspended in uptake buffer at a density of 3 × 106 cells/ml. [3H]Bilirubin was diluted with human serum albumin (HSA; Sigma; fatty acid-free) in uptake buffer (75,000-100,000 dpm/ml). Unlabeled bilirubin was added to give the desired final concentrations. Uptake was started by mixing 1 ml of cell suspension with 1 ml of bilirubin/albumin solution to give a final radioactivity of 37,500-50,000 dpm/ml and stopped at different time points by centrifugation of the mixture at 13,000 rpm for 10 s. Cell pellets were washed twice with 1 ml of uptake buffer containing HSA and lysed in 2 ml of 0.2% SDS in water. Aliquots (300 µl) of the lysate were counted for radioactivity. To determine the nonspecific binding of [3H]bilirubin, cells were incubated with [3H]bilirubin in the presence of HSA for 1 min at 4 °C. Cell-associated radioactivity measured under this condition was used as a blank and subtracted from all other values. No differences between this method and the method using adherent cells were observed for other substrates like BSP.

Immunoblot Analysis-- Preparation of crude membrane fractions from transfected cells and immunoblot analysis were performed as described previously (16, 19). The polyclonal antibody ESL (16) was used to detect recombinant human OATP2.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sulfobromophthalein, Monoglucuronosyl Bilirubin, and Bisglucuronosyl Bilirubin Are High-affinity Substrates for Human OATP2-- BSP, a widely used anionic model compound for studies on uptake into the liver, is a high-affinity substrate for OATP2 with a Km value of 140 nM (Fig. 1). In comparison with OATP8, OATP2 showed a 24-fold higher affinity for BSP (Table I). In addition, monoglucuronosyl bilirubin and bisglucuronosyl bilirubin were identified as high-affinity substrates for OATP2 with nanomolar Km values. Monoglucuronosyl bilirubin was also a good substrate for human OATP8 (Table I). Kinetic properties of additional substrates for OATP2 and OATP8 are summarized in Table I. Despite the remarkable difference in their affinities for BSP, human OATP2 and OATP8 showed similar affinities for 17beta -glucuronosyl estradiol (E217beta G).


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Fig. 1.   BSP uptake mediated by human OATP2. A, [3H]BSP transport into cells was measured at a concentration of 1 µM using HEK293 cells transfected with human OATP2 (HEK-OATP2) or with vector alone (HEK-Co). B, concentration dependence of [3H]BSP uptake. Uptake of [3H]BSP into HEK-OATP2 cells (black-square) and HEK-Co cells () was measured at concentrations between 10 and 210 nM. The net uptake rates () were calculated by subtracting values obtained with HEK-Co cells from those obtained with HEK-OATP2 cells. Data are the means ± S.D. from two triplicate experiments.

                              
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Table I
Kinetic constants for substrate transport mediated by OATP2 and OATP8
Transport studies with 3H-labeled substrates were performed in the absence of HSA using HEK293 cells stably expressing recombinant human OATP2 or human OATP8 (GenBankTM/European Molecular Biology Laboratory data bank accession numbers AJ132573 and AJ251506, respectively). Transport into control vector-transfected cells served as a blank. Michaelis-Menten constants (Km) are given as means ± S.D. (n = 6).

OATP2, but not OATP8, Transports Substrates in the Presence of Albumin-- An important aspect with regard to BSP uptake mediated by both transporters is the differential influence of HSA. As shown in Fig. 2A, HSA, in a 20-fold molar excess, did not significantly affect OATP2-mediated BSP uptake but abolished OATP8-mediated BSP uptake. One-third of the respective Km value (50 nM for OATP2 and 1 µM for OATP8) was chosen as the BSP concentration in these experiments. A similar influence of HSA was observed when BSP uptake was measured for both transporters at a constant BSP concentration (1 µM) with varying HSA concentrations (Fig. 2B). HSA caused only a minor decrease of OATP2-mediated BSP uptake. Uptake assays using 1 µM E217beta G as substrate showed that 5 µM HSA did not change the uptake rate with OATP2. The OATP2-mediated uptake was not chloride-dependent in the presence or absence of HSA because replacement of chloride in uptake buffer by gluconate had no effect on the rate of uptake. It is well known that many organic anions bind to HSA in the blood circulation, and a particular role of HSA in the hepatic uptake of organic anions has been proposed (22). It is therefore conceivable that a hepatic transport protein like OATP2 has the ability to extract substrates from HSA. For OATP8, the interaction with HSA is different (Fig. 2). We have identified four lipophilic organic anions (monoglucuronosyl bilirubin, BSP, E217beta G, and dehydroepiandrosterone 3-sulfate) as 3H-labeled substrates for OATP8. These experiments, however, were performed without HSA in the buffer (Table I). The fact that OATP8 is not capable of extracting substrates from HSA raises the question of the physiological function of OATP8 in the basolateral hepatocyte membrane. From the data obtained thus far, it is feasible that OATP8 functions predominantly under circumstances where the binding capacity of albumin for lipophilic organic anions is exceeded.


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Fig. 2.   Effect of HSA on BSP uptake mediated by OATP2 and OATP8. A, uptake of [3H]BSP was measured at 50 nM for OATP2 and 1 µM for OATP8 in the presence of 1 µM (OATP2) or 20 µM (OATP8) HSA (BSP:HSA = 1: 20 in both cases). No significant effect of albumin on [3H]BSP uptake was observed for OATP2, whereas OATP8-mediated [3H]BSP uptake was completely abolished by the addition of albumin. B, uptake of 1 µM [3H]BSP measured with OATP2 and OATP8 in the presence of increasing concentrations of HSA ranging from 0.2 to 5 µM. HSA had a much greater effect on the OATP8-mediated [3H]BSP uptake than on the OATP2-mediated uptake (p < 0.01). Data are the means ± S.D. (n = 6).

Indocyanine Green Inhibits Transport Mediated by OATP2 but not Transport Mediated by OATP8-- We tested a number of organic anions for their ability to inhibit uptake by human OATP2 and OATP8. ICG did not affect OATP8-mediated E217beta G transport at concentrations up to 10 µM (Fig. 3A) but inhibited OATP2-mediated uptake in a competitive manner with a Ki value of 112 nM. Although BSP and ICG were thought to be taken up into hepatocytes by the same mechanism (3), patients with apparently normal hepatic BSP uptake but delayed ICG clearance have been reported (11). Our studies raise the possibility that such patients may have a deficiency in OATP2, leading to delayed ICG clearance, but normal OATP8, which may function in BSP uptake under this condition.


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Fig. 3.   Inhibition of OATP2- and OATP8-mediated transport. Uptake of 17beta -glucuronosyl [3H]estradiol (E217beta G; 2 µM) by HEK293 cells transfected with OATP2 or OATP8 was measured in the presence of different inhibitors and in the absence of HSA. A, uptake of [3H]E217beta G mediated by OATP2 was potently inhibited by ICG, whereas ICG exerted no significant inhibition on OATP8-mediated uptake. B, both OATP2 and OATP8 were inhibited by pravastatin (25 µM), rifamycin SV (50 µM), and rifampicin (75 µM). Data are the means ± S.D. from two triplicate experiments.

Unlike ICG, the drugs pravastatin, rifamycin SV, and rifampicin inhibited both OATP2- and OATP8-mediated uptake of [3H]E217beta G (Fig. 3B). The HMG-CoA reductase inhibitor pravastatin was a competitive inhibitor of OATP2-mediated transport of E217beta G with an inhibition constant of 53 µM. This is in line with a recent study showing that pravastatin is a substrate for human OATP2 with a Km value of about 30 µM (17).

Unconjugated Bilirubin Is Transported by OATP2 in the Presence of Albumin-- The fact that BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin are high-affinity substrates for human OATP2 and the fact that ICG inhibits OATP2-mediated uptake competitively at nanomolar concentrations suggested that OATP2 might be the long-sought uptake transporter for unconjugated bilirubin in the basolateral hepatocyte membrane. We therefore measured [3H]bilirubin uptake by OATP2-transfected HEK293 cells (HEK-OATP2) at a concentration of 1 µM in the presence of 2 µM HSA. The calculated free bilirubin concentration under this condition, using the dissociation constant for HSA and bilirubin (12), is about 25 nM. As shown in Fig. 4A, [3H]bilirubin was taken up by HEK-OATP2 cells time-dependently at 37 °C. The uptake rate into HEK-OATP2 cells (8.5 pmol·min-1·mg protein-1) differed from that into vector-transfected HEK293 cells (1.5 pmol·min-1·mg protein-1) by a factor of 5.7 (p < 0.01). When the uptake was determined at 4 °C, no significant difference in uptake rates was detected between OATP2-expressing cells and control cells. Uptake of bilirubin mediated by recombinant human OATP2 was concentration-dependent as shown in Fig. 4B. In these experiments, the HSA concentration was kept constant at 20 µM so that the bilirubin:HSA ratio did not exceed 0.5. Because of the uncertainty of calculated free bilirubin concentrations (12), the Km value for free unconjugated bilirubin could only be estimated and was about 160 nM. For comparison, we investigated whether OATP8 would also transport unconjugated bilirubin. At the same [3H]bilirubin and HSA concentrations and under the same conditions used for OATP2 (Fig. 4), no significant uptake of [3H]bilirubin was detected with HEK293 cells expressing human OATP8 (data not shown).


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Fig. 4.   Bilirubin as a substrate for human OATP2. A, uptake of [3H]bilirubin was measured at 1 µM bilirubin in the presence of 2 µM HSA. Cell-associated radioactivity after incubation of cells with [3H]bilirubin for 1 min at 4 °C was used as a blank and subtracted from all other values. The uptake rate into HEK-OATP2 cells was 8.5 pmol·min-1·mg protein-1 (n = 10) and differed significantly from the uptake rate into vector-transfected HEK293 cells (1.5 pmol·min-1·mg protein-1; n = 5). B, concentration dependence of bilirubin uptake. Uptake of [3H]bilirubin at different concentrations was measured at a constant HSA concentration of 20 µM. The estimated free bilirubin concentration ranged from 5 to 25 nM. Measurements of [3H]bilirubin uptake at each concentration were performed at 1 min (blank) and 10 min; the rate of uptake was calculated from the difference between these two measurements (n = 5). C, inhibition of [3H]bilirubin uptake by BSP. Uptake of 10 µM [3H]bilirubin was measured in the presence of 20 µM HSA (n = 5), and the uptake was calculated as described in B; for inhibition studies, 5 µM BSP was included into the uptake buffer. All experiments were reproduced at least twice. Data are the means ± S.D. Asterisks indicate a significant difference compared with controls (p < 0.01). Inset in A, immunoblot with the polyclonal antibody ESL (16) of membranes (20 µg of protein) membranes (20 µg of protein) from HEK-OATP2 cells (left lane) or HEK-Co cells (right lane). The arrow points to the fully glycosylated OATP2 at 90 kDa.

Bilirubin, which was examined as a complex with HSA, inhibited the uptake of E217beta G and BSP by human OATP2, with 50% inhibition at 5 µM with E217beta G as substrate and at 20 µM with BSP as substrate. Uptake of [3H]bilirubin into HEK-OATP2 cells was strongly inhibited by BSP (Fig. 4C), demonstrating mutual inhibition of BSP and bilirubin uptake.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolar Km values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 µM (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6, 7).


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Fig. 5.   Bilirubin uptake and conjugate export by human hepatocytes. Bilirubin (B) is taken up across the basolateral membrane by human OATP2 (SLC21A6; Fig. 4A) and conjugated with glucuronate (GA) by UDP-glucuronosyl transferase 1A1 (UGT1A1), resulting in monoglucuronosyl bilirubin (BGA) and bisglucuronosyl bilirubin (B(GA)2) (5). The excretion of BGA and B(GA)2 is mediated by the apical ATP-dependent conjugate export pump, multidrug resistance protein 2 (symbol ABCC2) (6, 7).

Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver.

    ACKNOWLEDGEMENTS

We thank W. D. Lehmann (Deutsches Krebsforschungszentrum, Spectroscopy, Heidelberg, Germany) for analysis of the labeled and unlabeled BSP by nanoelectrospray mass spectrometry, J. M. Crawford (University of Florida, Department of Pathology, Gainesville, FL) and A. F. McDonagh (University of California, Division of Gastroenterology, San Francisco, CA) for advice on the preparation of [3H]bilirubin, K. Bode and M. Donner (Deutsches Krebsforschungszentrum, Division of Tumor Biochemistry, Heidelberg, Germany) for help during the biosynthesis of [3H]bilirubin, and G. Jedlitschky (Deutsches Krebsforschungszentrum, Division of Tumor Biochemistry, Heidelberg, Germany) for critical reading of the manuscript.

    FOOTNOTES

* The work was supported in part by Deutsche Forschungsgemeinschaft through SFB601 and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel.: 49-6221-422400; Fax: 49-6221-422402; E-mail: y.cui@dkfz-heidelberg.de.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M004968200

    ABBREVIATIONS

The abbreviations used are: BSP, sulfobromophthalein; E217beta G, 17beta -glucuronosyl estradiol; HSA, human serum albumin; ICG, indocyanine green; OATP, organic anion-transporting polypeptide; HPLC, high pressure liquid chromatography.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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