The liver is responsible for the uptake, metabolism, and biliary excretion of a variety of small hydrophobic compounds, including unconjugated bilirubin. It generally is accepted that albumin, the most abundant serum protein, serves as the principal transporter of bilirubin as well as numerous other hydrophobic molecular species (1, 2, 3) . The existence of an albumin receptor on the hepatocyte surface was first postulated in 1981 to explain the dependence of hepatic oleic acid uptake on the concentration of the albumin-ligand complex, as opposed to the concentration of free ligand per se(4) . Serum albumin has since been shown to enhance the hepatocellular uptake of a variety of other organic anions, including bilirubin(5) , fatty acids(6) , taurocholate(7, 8) , and rose bengal(9, 10, 11) . The ``albumin receptor'' hypothesis also has received support from studies demonstrating saturable binding of albumin to isolated hepatocytes(4, 12, 13, 14) .

However, other investigators refute these findings (15, 16, 17, 18) and have further shown that the albumin receptor phenomenon is not unique to albumin in that similar uptake kinetics are observed with non-albumin-binding proteins(17, 18, 19) . Moreover, the binding of albumin to isolated hepatocytes and to rat liver plasma membranes is of relatively low affinity(20) , and attempts to identify a specific receptor for albumin on the liver cell surface have, to date, been unsuccessful(21) . In light of these observations, two additional theories have been proposed to explain the kinetics of albumin-bound ligand delivery to the liver. One such hypothesis suggests the induction of a reversible conformational change in the albumin molecule at the surface of the hepatocyte, which enhances albumin binding and facilitates the release of bound ligands(8, 10, 13) . Alternatively, an ``extended sinusoidal perfusion'' model has been developed (22, 23, 24) , which accounts for the facilitation of ligand uptake in the presence of albumin by postulating an unstirred water layer at the liver cell surface and by correcting for the free ligand concentration in the sinusoidal plasma. According to this theoretical model, which has come under criticism for its complexity(12, 25) , the rate of dissociation of a given ligand from albumin may, under certain conditions, serve as the rate-limiting step in the hepatocellular uptake process(26, 27) .

In an attempt to further delineate the mechanism(s) underlying the delivery of albumin-bound organic anions to the hepatocyte, we performed a systematic study of the kinetics of bilirubin transfer between serum albumin and both model and native hepatocyte plasma membrane vesicles. Despite the fact that albumin serves as the principal serum carrier for a host of hydrophobic organic anions, the limited available kinetic data on ligand dissociation from this protein have focused primarily on fatty acids(28, 29) , and relatively little information exists regarding the dissociation of the albumin-bilirubin complex(30, 31, 32) . Indeed, the precise determination of ligand off-rate constants from albumin is necessary in order to discriminate between the various models of hepatocellular uptake(27) . Moreover, while bovine serum albumin is employed routinely for in vitro studies of hepatocellular transport, there has been no analysis regarding the applicability of results obtained from one specific albumin species to uptake phenomena in general. Finally, we measure directly the transfer of bilirubin from albumin to isolated hepatocyte basolateral plasma membranes and provide kinetic evidence against the presence of an albumin receptor on the liver cell surface. Hence, this study contributes important information regarding the mechanism of organic anion delivery from the serum to the hepatocyte plasma membrane.

EXPERIMENTAL PROCEDURES

Materials

()

Preparation of Small Unilamellar Vesicles

Isolation of Basolateral Liver Plasma Membranes

Stopped-flow Measurement of Bilirubin Transfer from Serum Albumin to Acceptor Vesicles

As hepatocyte plasma membranes exhibit significant tryptophan fluorescence(37) , the rate of bilirubin transfer from serum albumin to membrane vesicles also was determined by monitoring changes in bilirubin fluorescence. It previously has been shown that bilirubin fluorescence intensity is markedly enhanced when bound to serum albumin(44, 51, 52) . While bilirubin also exhibits increased fluorescence on binding to membranes(53) , we found that bilirubin fluorescence is much less efficient when associated with phospholipid vesicles as compared with albumin. Thus, the spontaneous transfer of bilirubin from albumin to acceptor vesicles is reflected by a time-dependent diminution in bilirubin fluorescence intensity. Stopped-flow fluorometry was used to monitor the transfer process (excitation at 467 nm and emission at 525 nm) with a 500-nm long-pass emission filter to minimize light-scattering effects. Solutions of albumin-bound bilirubin were exposed to light from a standard 60-watt bulb for 5 min prior to initiation of the experiment in order to minimize any contribution from bilirubin photoisomerization (see ``Results'').

Measurement of the Rate of Bilirubin Transfer from Phospholipid Vesicles to Serum Albumin

Kinetic Analysis of Bilirubin Transfer Data

Figure 1: Kinetic model of bilirubin transfer between serum albumin and membrane vesicles. This schematic diagram outlines the kinetic models for the collisional transfer and the diffusional transfer of bilirubin between serum albumin and membrane vesicles used in this study. Alb, B, and V represent the free aqueous concentrations of albumin, bilirubin, and vesicles, respectively. AlbB, BV, and AlbBV reflect the concentrations of the albumin-bilirubin, bilirubin-vesicle, and albumin-bilirubin-vesicle complexes. Rate constants are depicted by a k with subscripted numbers.

where [V] is the free vesicle phospholipid concentration; s is the monolayer surface area per phospholipid molecule; and [Alb], [AlbB], and [VB] are the concentrations of free albumin, the albumin-bilirubin complex, and the vesicle-bilirubin complex, respectively. The rate constant for the collisional transfer of bilirubin from albumin to acceptorvesicles can then be described by (55) .

Similarly, for a diffusional process, if the bilirubin free monomer concentration ([B]) is assumed to reach steady-state equilibrium rapidly,

and the overall transfer rate constant can be expressed as follows(55) .

When the ratio of albumin to vesicles is maintained constant, the collisional model () predicts a linear increase in the bilirubin transfer rate coincident with the albumin (or vesicle) concentration, while a constant rate would be anticipated for a diffusional mechanism of transfer (), regardless of the absolute donor or acceptor concentration.

RESULTS

The quenching of the steady-state fluorescence of human serum albumin (HSA), rat serum albumin (RSA), or bovine serum albumin (BSA) by unconjugated bilirubin, after correcting for inner filter effects, was linear up to a 1:1 molar ratio of bilirubin to albumin. Hence, bilirubin concentrations within this range were utilized in all transfer experiments.

Kinetic Analysis of Bilirubin Transfer from Serum Albumin to Small Unilamellar Phosphatidylcholine Vesicles

Figure 2: Effect of acceptor vesicle concentration on bilirubin transfer from rat serum albumin. Data from a series of experiments measuring the transfer of bilirubin from rat serum albumin to small unilamellar phosphatidylcholine vesicles are presented. The tryptophan fluorescence of albumin was monitored over a 5-s interval, during which time a steady-state signal was attained. The time-dependent re-emergence of fluorescence reflects the transfer of unconjugated bilirubin (4 µM) from an equimolar concentration of RSA to increasing concentrations of acceptor vesicles (0-8.5 mM phospholipid). Each curve represents the mean of 8-10 repetitive stopped-flow injections performed at 25 °C and is fitted by a single exponential function (solid lines).

Figure 3: Effect of increasing acceptor vesicle concentration on the rate of bilirubin transfer from albumin. Bilirubin transfer from rat serum albumin (1:1 molar ratio) to small unilamellar phosphatidylcholine acceptor vesicles was monitored by changes in the intrinsic tryptophan fluorescence of albumin. The transfer rate constant is plotted versus the concentration of acceptor vesicle phospholipid (1.0-9.5 mM), with the RSA concentration maintained at 4 µM. Each point represents the mean ± S.D. of three separate sets of experiments performed at 25 °C. The solid line was generated from the best fit parameters (r = 0.976) for the diffusional model of bilirubin transfer (). These data are inconsistent with the collisional transfer model, which predicts a linear increase in rate.

Further support for a diffusional mechanism of transfer is derived from a plot of the bilirubin transfer rate versus the albumin:vesicle phospholipid concentration ratio (Fig. 4), which is well described by . Moreover, when the molar ratio of albumin (donor) to phospholipid vesicles (acceptor) is held constant, the first-order transfer rate remains unchanged over a wide range of albumin concentrations (Fig. 4, inset), as predicted by the diffusional model of transfer. Under these experimental conditions, the collisional model () predicts a linear increase in rate. Collectively, these data suggest that the spontaneous transfer of bilirubin from serum albumin to small unilamellar vesicles occurs via aqueous diffusion. Bilirubin transfer from serum albumin to dansyllabeled (0.2 mol %) acceptor vesicles also was monitored by the time-dependent changes in dansyl fluorescence. Transfer rates were identical to those obtained measuring the tryptophan fluorescence of albumin, confirming that the observed fluorescence changes reflect the transfer of bilirubin from albumin to acceptor vesicles and are not the result of conformational changes in the albumin molecule(59) .

Figure 4: Variations in the bilirubin transfer rate with the donor:acceptor molar ratio. The rate constant for bilirubin (0.25-6 µM) transfer from RSA (1:1 molar ratio) to small unilamellar vesicles (0.5-11 mM phospholipid), as measured by changes in albumin fluorescence at 25 °C, is plotted against the concentration ratio of albumin to vesicle phospholipid. Each point represents the mean ± S.D. of three separate sets of experiments. The diffusional model of bilirubin transfer () provides an excellent fit (r = 0.994) of the data (solid line). In the inset, the concentration of albumin and acceptor vesicles was varied while maintaining a 1:1800 molar ratio of albumin to phospholipid. Under these conditions, the bilirubin transfer rate remains constant over a 24-fold range in albumin concentration, as predicted by the diffusional model (solid line), but not the collisional model (broken line).

A concern regarding the use of the above kinetic equations is that they are based on the premise that the concentration of bilirubin remains low, such that the free donor and acceptor concentrations do not change appreciably during the transfer process(55) . While this assumption is quite reasonable with respect to acceptor vesicle phospholipid, which is present at a 250-5000 molar excess of bilirubin, it may not hold under conditions where the albumin donor is present at a 1:1 molar ratio with bilirubin. However, the relatively weak fluorescence signal of serum albumin and the markedly higher binding affinity of albumin for bilirubin as compared with the acceptor vesicles necessitated the use of a 1:1 ratio of bilirubin to albumin for the majority of experiments. To examine the influence of the bilirubin:albumin molar ratio on bilirubin transfer kinetics, we varied this ratio over a 5-fold range while maintaining the donor albumin and acceptor vesicle concentrations constant (Fig. 5A). A 44-fold increase in the free serum albumin concentration (calculated to be 55 nM at a 1:1 bilirubin:albumin molar ratio) resulted in a minimal increase in the transfer rate, which was not statistically significant (p = 0.20). Additional studies demonstrated a statistically insignificant effect of the bilirubin:albumin molar ratio on the kinetics of bilirubin transfer over a wide range of acceptor concentrations (Fig. 5B). Hence, our data indicate that changes in the molar ratio of bilirubin to albumin that occur over the course of a given transfer experiment have little impact on the transfer rate and, if anything, would be expected to skew the results in favor of the collisional model, due to an increase in the donor:acceptor ratio over time.

Figure 5: Influence of the bilirubin:albumin molar ratio on the bilirubin transfer rate. In A, the rate of bilirubin (0.6-3.0 µM) transfer from RSA (3 µM) to small unilamellar phosphatidylcholine acceptor vesicles (9 mM phospholipid) was determined by monitoring changes in albumin fluorescence. The rate constant is plotted against the albumin:bilirubin molar ratio, with each bar representing the mean ± S.D. of three separate sets of experiments performed at 25 °C. A 5-fold increase in the ratio of albumin to bilirubin did not result in a significant increment in the rate constant (p = not significant). In B, an identical experimental approach was used to examine the effect of the albumin:bilirubin molar ratio on the kinetics of bilirubin transfer from RSA to acceptor vesicles. The concentration of albumin is the same as indicated in A. The transfer rate constant is plotted against the albumin:vesicle phospholipid (PL) concentration ratio at two molar ratios of albumin to bilirubin (, 1:1; , 2:1). There is no significant difference in the rate constant at each albumin:phospholipid molar ratio (p = not significant), and the overall kinetic behavior of the transfer process is identical.

Kinetics of Bilirubin Transfer from Small Unilamellar Donor Vesicles to Albumin

Figure 6: Bilirubin transfer from phospholipid vesicles to albumin: influence of the acceptor concentration. The rate of bilirubin (0.5 µM) transfer from dansyl-labeled small unilamellar donor vesicles (100 µM phospholipid) to bovine serum albumin (0-1.25 mM) was determined by monitoring the time-dependent changes in dansyl fluorescence at 25 °C. The first-order transfer rate constant is plotted versus the BSA concentration, with each point representing the mean ± S.D. of three separate sets of experiments. Lines were generated from best fit parameters for the collisional (broken line) and diffusional (solid line) models of bilirubin transfer. The diffusional model (r = 0.982) produces a significantly better fit of the data as compared with the collisional model (r = 0.521).

Activation Energies for Bilirubin Dissociation from Human, Rat, and Bovine Serum Albumins

Figure 7: Bilirubin transfer from albumin to acceptor vesicles: Arrhenius plot. The rate of bilirubin transfer from human (), rat (), or bovine () serum albumin to small unilamellar phosphatidylcholine acceptor vesicles was measured over a temperature range of 10-40 °C by the time-dependent changes in albumin fluorescence. The concentrations of serum albumin, bilirubin, and vesicle phospholipid were 2 µM, 2 µM, and 5 mM, respectively. The natural log of the transfer rate constant (k) is plotted against inverse temperature (K), and the activation energies for the dissociation of bilirubin from each albumin species were determined from the slope of the linear fits. The thermodynamic behavior of BSA was found to differ significantly from that of RSA and HSA.

Bilirubin Transfer from Rat Serum Albumin to Model and Native Membrane Vesicles as Measured by Changes in Bilirubin Fluorescence

Figure 8: Changes in albumin-bound bilirubin fluorescence following stopped-flow mixing. Bilirubin (4 µM) complexed to rat serum albumin in a 1:1 molar ratio was rapidly mixed with an identical RSA:bilirubin solution using stopped-flow techniques. Bilirubin fluorescence was recorded at 25 °C over a time interval of 30 s. The upper curve was obtained under conditions where the bilirubin:albumin solution was maintained in constant darkness prior to stopped-flow injection and is well described by a single exponential function (solid line) with a rate constant of 0.43 s. The lower curve was recorded following a 5-min exposure to broad spectrum visible light, which abolishes completely the time-dependent changes in bilirubin fluorescence.

In contrast to the results obtained by monitoring tryptophan fluorescence, the transfer of bilirubin from serum albumin to acceptor vesicles, when measured by changes in bilirubin fluorescence, was best described with a double exponential function. The fast rate constant derived from the second-order fit correlated closely with the first-order rate constant obtained for the identical experiments using albumin fluorescence (Fig. 9). We postulate that the slow component of transfer represents bleaching of the bilirubin molecule, as both the amplitude and rate were diminished (although not entirely eliminated) by decreasing the width of the excitation slit. Alternatively, the slow phase may reflect conformational changes in the bilirubin molecule occurring at the acceptor vesicle.

Figure 9: Transfer of bilirubin from albumin to vesicles as monitored by changes in albumin and bilirubin fluorescence. Stopped-flow fluorescence recordings of bilirubin (4 µM) transfer from rat serum albumin (4 µM) to small unilamellar phosphatidylcholine vesicles (8.5 mM phospholipid) are displayed. Each curve represents the mean of 15 repetitive injections performed at 25 °C and is normalized to a scale of 0-10. In the upper panel, the time-dependent re-emergence of the intrinsic tryptophan fluorescence of RSA results from the transfer of bilirubin from albumin to acceptor vesicles. The lower panel depicts bilirubin dissociation from RSA as measured by the decrease in bilirubin fluorescence following dissociation from albumin and binding to acceptor vesicles. The rate constant obtained from the single exponential fit of the upper curve and the fast rate constant from the double exponential fit of the lower curve (solid lines) are identical.

The rate of bilirubin transfer from rat serum albumin to isolated rat bLPM was measured at various concentrations of donors and acceptors in order to determine kinetically whether a receptor for albumin is present on the hepatocyte surface. Plasma membrane preparations were 30-40-fold enriched in ouabain-sensitive Na/K-ATPase activity as compared with whole liver homogenate. Consistent with data obtained by other investigators (36, 38) , contamination of bLPM with microsomal and canalicular plasma membranes was less than 9 and 5%, respectively. When the rate constant for the fast component of transfer is plotted versus the albumin concentration, with the plasma membrane vesicle concentration held constant, we observed that the bilirubin transfer rate remained constant in the face of increasing albumin levels (Fig. 10A), which is inconsistent with a collisional mechanism of transfer. Moreover, for both small unilamellar phosphatidylcholine and isolated bLPM vesicles, a plot of the rate constant versus acceptor phospholipid (after correction for membrane surface area), at constant donor albumin concentration, is well described by the diffusional model of transfer (Fig. 10B). The decline in the bilirubin transfer rate with increasing phospholipid concentration is incompatible with the collisional model. A receptor for albumin, however, would be expected to exhibit collision-mediated kinetics since the transfer process necessitates direct contact between the membrane-bound receptor and the albumin molecule. Hence, our data indicate that the delivery of RSA-bound bilirubin to rat basolateral liver plasma membranes occurs via aqueous diffusion and mitigate against the presence of a receptor for albumin on the hepatocyte surface.

Figure 10: Comparison of bilirubin transfer rates from albumin to model vesicles and to isolated basolateral plasma membranes. The rate of bilirubin transfer from RSA (1:1 molar ratio) to small unilamellar phosphatidylcholine acceptor vesicles () or to isolated rat basolateral liver plasma membrane vesicles () was determined by monitoring changes in bilirubin fluorescence at 25 °C. In A, the rate constant for the fast component of bilirubin transfer is plotted against the albumin concentration, with the acceptor bLPM concentration maintained at 0.5 mg/ml. We observed that the transfer rate remained constant over a 10-fold range on RSA concentration. Alternatively, B presents a plot of the fast transfer rate at constant albumin concentration (2 µM) versus acceptor phospholipid (PL) concentration for both small unilamellar (0.6-9.5 mM phospholipid) and bLPM (0.25-1.0 mg/ml) vesicles. The relative phospholipid content of the vesicles was estimated using 2.86 10 m mol as the surface area per mol of phospholipid for bLPM (74) , as compared with a value of 4.45 10 m mol for small unilamellar vesicles(75) . The solid lines indicate the best fit parameters for the diffusional model of bilirubin transfer. As an increase in the number of collisions per unit time occurs with increasing numbers of donors or acceptors, the collisional transfer model cannot account for a constant or declining rate.

DISCUSSION

This study represents the first systematic investigation of the spontaneous movement of bilirubin between serum albumin and membrane vesicles and provides strong evidence for a diffusional mechanism of bilirubin transfer from all species of albumin studied (i.e. human, rat, and bovine). The findings were confirmed both for the forward (albumin to membrane) and reverse (membrane to albumin) processes and by utilizing three distinct experimental approaches: albumin, bilirubin, and membrane probe (dansyl) fluorescence. A novel aspect of this study is that, with the exception of dansyl-PE, which we previously have shown does not impact bilirubin-membrane binding(33) , transfer rates were determined utilizing the intrinsic optical properties of the native compounds. Hence, fluorescent probes, which potentially can alter ligand physicochemical properties, were not required for the performance of these experiments.

A corollary of the aqueous diffusion model of transfer () is that as the donor:acceptor ratio nears zero, the transfer rate approaches the dissociation rate (k) from the donor (i.e. albumin)(55) . Conversely, as the ratio of donor to acceptor increases toward infinity, the bilirubin transfer rate approaches that for the dissociation from acceptor vesicles (k). Since the off-rate from membrane vesicles (33) is more rapid than from serum albumin, this model provides an explanation for the observed decline in the bilirubin transfer rate with increasing numbers of acceptor vesicles ( Fig. 3and 10B). Hence, kinetic parameters for diffusion-mediated ligand transfer should be derived only in the context of the donor:acceptor molar ratio, dissociation rates, and relative binding affinities for the specific ligand under analysis. This is the likely explanation of the disparate results for the dissociation rate constant for bilirubin from BSA obtained by Noy et al.(32) . The high albumin:phospholipid (dioleoylphosphatidylcholine) molar ratio of 1.7:1 utilized in that study would be expected to result in an overestimate of the bilirubin off-rate due to the significant contribution of dissociation from the acceptor vesicles. Indeed, their reported rate constant for bilirubin transfer from BSA is 18-fold higher than was observed in this study and by other investigators(64) .

A comparison of the bilirubin transfer rate constants for human, rat, and bovine serum albumins indicates markedly different dissociation kinetics for BSA as compared with HSA and RSA. These results are supported by thermodynamic analysis, which reveals a high activation enthalpy (H) and an increase in the activation entropy (TS) for bilirubin dissociation from BSA. Since bilirubin transfer from each albumin species to membrane acceptors occurs through the aqueous phase and the end point of bilirubin dissociation is identical (i.e. free aqueous bilirubin), the measured differences in the activation energies are a direct reflection of the bilirubin-albumin interaction. Thus, it appears that BSA-bound bilirubin is held in a rigid conformation, such that the steric constraints are even greater than for aqueous bilirubin, supporting the existence of a specific, high-affinity binding site. Conversely, the binding of bilirubin to human and rat serum albumins appears to be much less rigid, so that less energy is required for bilirubin dissociation, resulting in faster rates of transfer. More important, these data indicate that, despite the routine use of BSA in the majority of transport and uptake studies (5, 6, 8, 10, 11, 15-18, 26, 65), rat serum albumin more closely mimics the physical properties of human albumin, at least with regard to bilirubin binding. Moreover, theoretical models of hepatic uptake frequently use estimates of ligand dissociation rates that are derived from studies of BSA(66) , and this may result in a significant underestimation for bilirubin (or potentially an overestimation for other ligands) of the dissociation rate from human or rat albumin.

The observed kinetics of bilirubin transfer from rat serum albumin to isolated rat basolateral liver plasma membranes indicate that the delivery of bilirubin to the hepatocyte occurs via aqueous diffusion and does not require direct interaction between the albumin-bilirubin complex and the plasma membrane. The fact that bilirubin transfer from albumin to bLPM does not occur via a collisional mechanism provides strong evidence against the hypothesis that adsorption of albumin to the hepatocyte surface facilitates the release of bound bilirubin(13) . Hence, these studies suggest that the limited binding of albumin to the basolateral plasma membrane does not dramatically enhance ligand dissociation and represents, at best, an insignificant proportion of net bilirubin delivery to the hepatocyte. The demonstration of saturation kinetics with increasing albumin concentration offers a potential explanation for the albumin receptor phenomenon. Indeed, our data confirm that the ratio of albumin to acceptor membranes, rather than bilirubin to albumin, is the principal determinant of the bilirubin transfer rate.

It recently has been suggested that ligand dissociation from albumin may represent the rate-limiting step in hepatocellular uptake(26, 27, 67) . Conditions necessary for dissociation-limited uptake(27, 68) include the following: 1) the majority of the ligand in the serum must be albumin-bound (i.e. high-affinity binding); 2) ligand extraction by the liver must exceed the unbound fraction; and 3) the rate of ligand influx into the hepatocyte must surpass the rate of rebinding to albumin within the liver sinusoids. The first two conditions have been well established for bilirubin(18) . Indirect evidence that the latter condition also may be operational comes from evidence that the rate constant for the uptake of several organic anions (including bilirubin) by rat liver is at least 1-2 s(18, 27) , which is significantly higher than the bilirubin dissociation rate of 0.6 s from RSA. For a dissociation-limited process, the uptake velocity is given by the following equation: v = kV[B], where k is the off-rate constant for bilirubin from albumin, V is the volume (0.15 ml/g of liver) of the hepatic sinusoids(15, 69) , and [B] is the bound bilirubin concentration(68) . Assuming an average liver weight of 30 g/kg of rat (67) , calculated rates of bilirubin uptake based on our value for the dissociation rate constant from RSA are 15-fold higher than those measured in intact animals(70, 71) . However, these calculations are based on the estimated sinusoidal volume and on the assumption that all plasma/sinusoidal bilirubin is available for uptake by hepatocytes, which may not be the case due to the binding of bilirubin by erythrocytes. ()In fact, rate constants obtained for the uptake of BSA-bound bilirubin by the isolated, perfused rat liver(18, 65) , measurements that are independent of the sinusoidal volume and the bilirubin concentration, correspond closely to our calculated rate for bilirubin dissociation from BSA. Thus, our data provide support for the concept of dissociation-limited organic anion uptake by the liver.

The design of this study does not permit direct determination of the rate of bilirubin transport across the plasma membrane. However, the observation that bilirubin movement from acceptor vesicles back to albumin contributes to the measured transfer rate suggests that the transmembrane movement of bilirubin is either exceedingly slow (such that bilirubin uptake into the vesicles is negligible) or remarkably fast (so that flip-flop essentially is instantaneous) as compared with the time scale for albumin dissociation. A variety of studies employing model phospholipid vesicles indicate that the rate of transmembrane flip-flop of fatty acids(72, 73) , bile acids(73) , and bilirubin (32, 37) exceeds that for the dissociation from albumin. However, since these studies all were performed using model phospholipid vesicles, the influence of native membrane lipid composition and protein content on the rate of ligand flip-flop is unknown(73) . If the spontaneous transmembrane movement of bilirubin is rapid with respect to the rate of dissociation from albumin, then bilirubin would be predicted to partition into all tissues in contact with the plasma compartment. Under these circumstances, targeting of bilirubin to the liver would be predicated on rapid hepatic metabolism (glucuronidation) and biliary excretion or potentially on the existence of high-affinity intracellular binding sites (e.g. glutathione S-transferase). Conversely, if bilirubin flip-flop across the plasma membrane is slow relative to albumin dissociation, hepatic targeting likely would be determined by the presence of transport proteins on the hepatocyte surface (e.g. organic anion transporter). In either case, our findings support the concept that dissociation from albumin may be rate-limiting in the plasma clearance of bilirubin and, potentially, of other small hydrophobic compounds(26, 27, 68) .

FOOTNOTES

*

This work was supported by National Institutes of Health Research Grants DK-02047, DK-36887, DK-43955, and DK-34854, a Harvard Digestive Diseases Center pilot/feasibility project grant (to S. D. Z.), and by the Alfried Krupp von Bohlen und Halbach-Stiftung Scholarship Foundation (to W. G.). Preliminary reports of this work have been published in abstract form (Zucker, S. D., Storch, J., and Gollan, J. L.(1990) Hepatology12, 1003 (abstr.); Zucker, S., Storch, J., Zeidel, M., and Gollan, J.(1992) Gastroenterology102, 914 (abstr.); Zucker, S. D., Zeidel, M. L., and Gollan, J. L.(1993) Gastroenterology104, 1026 (abstr.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Div. of Gastroenterology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-5828; Fax: 617-730-5807.

()

The abbreviations used are: dansyl-PE, N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoyl-L--phosphatidylethanolamine; bLPM, basolateral (sinusoidal) liver plasma membrane(s); HSA, human serum albumin; RSA, rat serum albumin; BSA, bovine serum albumin.

()

S. D. Zucker, W. Goessling, and J. L. Gollan, unpublished observations.

ACKNOWLEDGEMENTS

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