(Received for publication, July 15, 1994; and in revised form, October 20, 1994)
From the
Unconjugated bilirubin is transported in the plasma bound
primarily to serum albumin, from which it is taken up and metabolized
by the liver. To better characterize the mechanism of bilirubin
delivery to the hepatocyte, stopped-flow techniques were utilized to
study the kinetics of bilirubin transfer between serum albumin and both
model phospholipid and native hepatocyte plasma membrane vesicles. The
transfer process was best described by a single exponential function,
with rate constants of 0.93 ± 0.04, 0.61 ± 0.03, and 0.10
± 0.01 s (±S.D.) at 25 °C for
human, rat, and bovine serum albumins, respectively. The observed
variations in rate with respect to donor and acceptor concentrations
provide strong evidence for the diffusional transfer of free bilirubin.
Thermodynamic analysis suggests that the binding site on bovine serum
albumin demonstrates higher specificity for the bilirubin molecule than
that on human or rat serum albumin, which exhibit similar binding
characteristics. Kinetic analysis of bilirubin transfer from rat serum
albumin to isolated rat basolateral liver plasma membranes indicates
that the delivery of albumin-bound bilirubin to the hepatocyte surface
occurs via aqueous diffusion, rather than a collisional process,
thereby mitigating against the presence of an ``albumin
receptor.''
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.
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'').
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, B
V, and Alb
B
V 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
[V
B] 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.
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.
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.
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).
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.
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.
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 (T
S
) 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 = k
V
[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) .