Unconjugated Bilirubin Exhibits Spontaneous Diffusion through
Model Lipid Bilayers and Native Hepatocyte Membranes*
Stephen D.
Zucker
§,
Wolfram
Goessling¶, and
Alison G.
Hoppin
From the
Division of Digestive Diseases, University
of Cincinnati Medical Center, Cincinnati, Ohio 45267-0595, the
¶ Department of Medicine, Brigham and Women's Hospital, Boston,
Massachusetts 02115, and the
Division of Pediatric
Gastroenterology, Massachusetts General Hospital,
Boston, Massachusetts 02114
 |
ABSTRACT |
The liver is responsible for the clearance and
metabolism of unconjugated bilirubin, the hydrophobic end-product of
heme catabolism. Although several putative bilirubin transporters have
been described, it has been alternatively proposed that bilirubin
enters the hepatocyte by passive diffusion through the plasma membrane.
In order to elucidate the mechanism of bilirubin uptake, we measured
the rate of bilirubin transmembrane diffusion (flip-flop) using
stopped-flow fluorescence techniques. Unconjugated bilirubin rapidly
diffuses through model phosphatidylcholine vesicles, with a first-order rate constant of 5.3 s
1 (t1/2 = 130 ms). The flip-flop rate is independent of membrane cholesterol
content, phospholipid acyl saturation, and lipid packing, consistent
with thermodynamic analyses demonstrating minimal steric constraint to
bilirubin transmembrane diffusion. The coincident decrease in pH of the
entrapped vesicle volume supports a mechanism whereby the bilirubin
molecule crosses the lipid bilayer as the uncharged diacid. Transport
of bilirubin by native rat hepatocyte membranes exhibits kinetics
comparable with that in model vesicles, suggesting that unconjugated
bilirubin crosses cellular membranes by passive diffusion through the
hydrophobic lipid core. In contrast, there is no demonstrable flip-flop
of bilirubin diglucuronide or bilirubin ditaurate in phospholipid vesicles, yet these compounds rapidly traverse isolated rat hepatocyte membranes, confirming the presence of a facilitated uptake system(s) for hydrophilic bilirubin conjugates.
 |
INTRODUCTION |
Unconjugated bilirubin is the principal degradation product of
heme metabolism. Although the physiologic isomer, bilirubin IX
, is a
dicarboxylic acid, the molecule has minimal aqueous solubility at
physiologic pH (1, 2) due to the formation of intramolecular hydrogen
bonds (3). For this reason, bilirubin undergoes biotransformation in
the liver to more polar conjugates prior to secretion in the bile.
However, while unconjugated bilirubin is not hydrophilic, neither can
it be characterized as lipophilic (1), since this compound is equally
insoluble in apolar solvents (e.g. n-hexane,
1-pentanol). These unique physical-chemical properties have generated
controversy regarding the mechanism of bilirubin uptake by the liver.
Based on the spontaneous leakage from multilamellar liposomes (4), it
has been proposed that bilirubin is able to diffuse through cellular
membranes (5, 6). On the other hand, in vivo (7) and whole
organ (8, 9) studies indicate that hepatic bilirubin uptake is
saturable and occurs against a concentration gradient, findings that
support a protein-mediated transport mechanism. To date, four putative
bilirubin transporters have been identified in liver cells:
BSP/bilirubin-binding protein, organic anion-transporting polypeptide,
bilitranslocase, and organic anion-binding protein. Each of these
proteins facilitates uptake of the hydrophilic organic anion,
sulfobromophthalein (BSP),1 a
process that is competitively inhibited by bilirubin (10, 11).
However, despite the substantial body of evidence in support of
protein-mediated bilirubin uptake, none of the aforementioned candidate
proteins has been directly shown to transport unconjugated bilirubin.
This is due, in part, to the use of BSP as a surrogate for bilirubin,
based on the long standing assumption that these two compounds share a
common transporter (12, 13). However, the cellular uptake of bilirubin
and BSP can be dissociated (14, 15), suggesting distinct transport
mechanisms. While it has been argued that the unique "permeability"
of hepatocytes to bilirubin confirms the presence of a specific
transporter (12, 16), other studies have demonstrated nonsaturable and
non-energy-dependent bilirubin uptake (17), indicative of a
diffusional process. Moreover, fibroblasts transfected with cDNA
for the bilirubin-conjugating enzyme, UDP-glucuronosyltransferase, do
not express any of the candidate transporters yet are effectively able
to take up and conjugate bilirubin (18).
In an attempt to reconcile these disparate results and to further
delineate the mechanism whereby bilirubin traverses cellular membranes,
we devised a stopped-flow fluorescence system to facilitate detailed
kinetic analysis of bilirubin flip-flop in model and isolated native
membrane vesicles. Our data suggest that unconjugated bilirubin is
capable of rapid, spontaneous diffusion through lipid bilayers,
findings with important implications for bilirubin clearance by the liver.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Nigericin, valinomycin, and essentially fatty
acid-free bovine and human serum albumin were purchased from Sigma.
Bilirubin IX
(purity >99.5% by absorbance in chloroform,
453 = 62,000 M
1·cm
1) and bilirubin
conjugate (ditaurate·2Na) were obtained from Porphyrin Products
(Logan, UT). Grade 1 egg lecithin (phosphatidylcholine) was purchased
from Lipid Products (Surrey, United Kingdom), and cholesterol was
obtained from Nu-Chek Prep (Elysian, MN).
Dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, and
N-(5-dimethylaminonaphthalene-1-sulfonyl) dipalmitoyl-L-
-phosphatidylethanolamine (dansyl-PE)
were purchased from Avanti Polar Lipids (Birmingham, AL). The
fluorescent probes 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine)
and Cascade Blue-conjugated bovine serum albumin as well as
anti-Cascade Blue antibodies were obtained from Molecular Probes, Inc.
(Eugene, OR).
Preparation and Loading of Unilamellar Phospholipid
Vesicles--
Small unilamellar vesicles were prepared by probe
sonication using a modification (19) of the technique of Barenholz
et al. (20) A chloroform solution of phosphatidylcholine was
evaporated to dryness under argon atmosphere, solubilized in ether, and
re-evaporated to form a uniform film. The lipids then were desiccated
overnight under vacuum to remove traces of ether, suspended in 0.1 M potassium phosphate solution at pH 7.4 (unless otherwise
stated), and sonicated until clear. For the preparation of
fluorescence-labeled vesicles, dansyl-PE was added to the
lecithin-containing chloroform solution so as to comprise 1 mol % of
total phospholipid. Large unilamellar vesicles were prepared using a
modification (21) of the injection method of Kremer et al.
(22) Constituent phospholipids were suspended in ethanol and slowly
injected into a magnetically stirred aqueous solution of 0.1 M potassium phosphate (pH 7.4), with vesicle size regulated
by the concentration of phospholipid in the injected ethanol. Cascade
Blue-conjugated bovine serum albumin (cbBSA) was entrapped at the time
of vesicle preparation by injecting the ethanolic phospholipid solution
into phosphate buffer containing 50 µM cbBSA. The vesicle
suspension (5 ml) was subjected to dialysis against 100 volumes of
phosphate buffer three successive times to remove retained ethanol, and
unentrapped cbBSA was separated from the vesicles by elution on a
Sepharose 4B column (42 × 2.5 cm). Mean vesicle hydrodynamic
diameter was determined by quasielastic light scattering (21).
Isolation of Membrane Vesicles from Rat Liver--
Rat liver
microsomal membranes were isolated from fasted male Sprague-Dawley
rats, as described previously (21), and protein concentration was
quantified by the Bio-Rad assay. Hepatocyte basolateral (bLPM) and
canalicular liver plasma membranes were prepared by the method of Meier
and Boyer (23), with appropriate enrichment documented by enzymatic
assay (21), and by Western blotting with polyclonal antibodies to the
-subunit of the plasma membrane Na+/K+
ATPase and with monoclonal antibodies to the canalicular ecto-ATPase (generously provided by Dr. R. Green). Membranes were loaded with cbBSA
(50 µM) by homogenization in the presence of the probe, followed by exhaustive washes.
Analysis of Bilirubin Transmembrane Diffusion--
The flip-flop
rate for unconjugated bilirubin was determined from fluorescence
recordings of bilirubin equilibration between a suspension of
dansyl-PE-labeled small unilamellar phosphatidylcholine vesicles and
bovine serum albumin (Fig. 1A). The binding of bilirubin to
dansyl-PE-labeled vesicles causes a reduction in fluorescence intensity
due to resonance energy transfer between bilirubin and the dansyl
moiety (19). Since unconjugated bilirubin is poorly soluble at neutral
pH, incorporation into phospholipid vesicles was accomplished by
dissolving bilirubin in alkaline (pH 12) potassium phosphate (19). The
addition of a small aliquot (
1%, v/v) of the bilirubin solution to a
suspension of phospholipid vesicles buffered at pH 7.4 in 0.1 M potassium phosphate caused no detectable alteration in
pH. Alternatively, when indicated, bilirubin was dissolved in dimethyl
sulfoxide (Me2SO), and a small aliquot (
1%, v/v) was
added to the vesicle suspension (24). At this low concentration, Me2SO has no discernible affect on membrane physical
properties (25, 26), and the use of either method to solubilize
bilirubin yielded identical kinetic results. All steps were performed
in diminished light to minimize bilirubin photodegradation.
An Aminco-Bowman II fluorescence spectrophotometer equipped with an
SLM-Aminco MilliFlow stopped-flow reactor (mixing time: 3 ms)
facilitated the rapid mixing of a suspension of dansyl-labeled vesicles
containing bound bilirubin with an equal volume of a bovine serum
albumin solution. Bilirubin equilibration is manifest by a
time-dependent reemergence of dansyl fluorescence
(excitation, 340 nm; emission, 525 nm), reflecting the transfer of
bilirubin from the vesicles to BSA (27). The time course was analyzed by fitting the fluorescence curves to the function,
|
(Eq. 1)
|
where t is time, ki is the
ith order rate constant, Ai is the
amplitude, and C is a constant term. Quality of fit was
assessed by regression analysis, and an F-test was applied to determine
if the inclusion of an additional term provided a statistically
significant improvement in curve fit (19, 21). Due to the rapid rate of
bilirubin solvation (19), analysis of bilirubin-membrane dissociation
was performed with an Applied Photophysics (Leatherhead, UK)
fluorescence spectrophotometer equipped with an SPF-17 stopped-flow
device (mixing time: 0.7 ms).
An alternative method utilized for monitoring bilirubin flip-flop
involved the preparation of a suspension of bilirubin-containing phosphatidylcholine vesicles, which were mixed, using stopped-flow techniques, with a solution of free cbBSA. Bilirubin binding to cbBSA
induces a concentration-dependent decrease in Cascade Blue fluorescence (excitation, 380 nm; emission, 430 nm), and flip-flop rates were calculated from the time course of the quenching of cbBSA.
The rate of bilirubin transmembrane diffusion also was determined from
the quenching of cbBSA entrapped within model or native membrane
vesicles (Fig. 1B). Membranes
were preincubated with anti-Cascade Blue antibodies in order to
completely neutralize all residual unincorporated probe; therefore, the
quenching of Cascade Blue fluorescence is predicated on bilirubin
traversing the acceptor membrane and binding to the entrapped cbBSA.
For these studies, bilirubin was incorporated into unlabeled small unilamellar phosphatidylcholine donor vesicles or, alternatively, solubilized in 0.1 M potassium phosphate (pH 7.4)
containing Me2SO (20%, v/v). In order to minimize
light-scattering effects, experiments utilizing isolated native
membrane vesicles employed an emission wavelength of 450 nm with a
440-nm cut-on filter.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic illustration of experimental
systems utilized for studying bilirubin transmembrane diffusion.
A depicts the experimental schema for monitoring bilirubin
equilibration between donor vesicles, composed of phosphatidylcholine
(dark circles) with 1 mol % dansyl-phosphatidylethanolamine
(light circles) and BSA. At time 0 (t0), bilirubin-containing vesicles are rapidly
mixed with a solution of BSA, using stopped-flow techniques. The
equilibration rate is determined from the time-dependent
re-emergence of dansyl fluorescence caused by the spontaneous transfer
of bilirubin from the vesicles to albumin. The resultant fluorescence
curve reflects both rapid solvation (Dissociation) from the
external hemileaflet of the bilayer and slower transmembrane diffusion
(Flip-flop) of bilirubin from the inner to the outer
hemileaflet. Bilirubin flip-flop across unlabeled (nonfluorescent)
phospholipid vesicles was monitored by substituting cbBSA for BSA and
recording the quenching of Cascade Blue fluorescence by bilirubin.
B illustrates an alternative method for measuring bilirubin
flip-flop rates in which cbBSA is entrapped within acceptor vesicles.
Bilirubin flip-flop is reflected in the time-dependent
quenching of Cascade Blue fluorescence following mixing of
cbBSA-containing vesicles with bilirubin-incorporated donor
vesicles.
|
|
Kinetic Analysis of Bilirubin Transmembrane Diffusion--
We
previously have shown that the equilibration of bilirubin between
unilamellar phospholipid vesicles and BSA occurs via aqueous diffusion
(27). Assuming that bilirubin flip-flop is significantly slower than
membrane dissociation, the equilibration rate (R) can be
described by the expression (27, 28),
|
(Eq. 2)
|
where kff represents the flip-flop rate
constant, koffBSA represents the
dissociation rate constant from BSA, Ka represents the association constant, and [BSA] and [V] represent the
concentrations of bovine serum albumin and vesicle phospholipid,
respectively. Based on this equation, a plot of R
versus [V]/[BSA] intersects the y axis at
kff and asymptotically approaches
koffBSA at high molar ratios of
phospholipid to BSA. Values for the various kinetic parameters were
determined by measuring the bilirubin equilibration rate over a range
of phospholipid:albumin molar ratios and fitting the data to Equation 2.
Preparation of Bilirubin Diglucuronide--
Bilirubin
diglucuronide (BDG) was isolated from bile enriched by the intravenous
administration of 20 mM unconjugated bilirubin in 3.2%
bovine serum albumin to anesthetized male Sprague-Dawley rats (29). BDG
was extracted with chloroform/ethanol and NaCl-saturated acidified
glycine and isolated by thin layer chromatography. The purity of the
BDG preparation (>95%) was measured by reverse-phase high pressure
liquid chromatography (30), with the initial mobile phase consisting of
a 35% solution of 1% acetic acid and 65% 25 mM sodium
acetate in methanol/chloroform (95:5, v/v). After 30 s, the sodium
acetate solution was increased in a linear fashion to 100% over 8 min,
followed by a linear decrease to 80% over 5 min. The flow rate was
maintained constant at 1 ml/min.
Effect of Bilirubin on Membrane Vesicle pH--
In order to
determine the effect of bilirubin on the internal pH of membrane
vesicles, 0.5 mM pyranine, a hydrophilic pH-sensitive fluorescent probe, was dissolved in 25 mM HEPES/KOH (pH
7.4) and entrapped within small unilamellar phosphatidylcholine
vesicles by sonication (31). Unentrapped pyranine was removed from the medium by elution on a Sephadex G-25 (coarse grade) column (40 × 1 cm). Pyranine-loaded vesicles were diluted to a concentration of 0.5 mM phospholipid, and 2 ml of total volume was placed in a
temperature-controlled, stirred cuvette. The internal pH of the
vesicles, as reflected by the fluorescence intensity of the entrapped
pyranine, was monitored continuously at 25 °C using steady-state
techniques. Excitation (405 nm) and emission (520 nm) wavelengths were
selected so as to minimize bilirubin inner filter effects. At these
wavelengths, pyranine fluorescence intensity correlates inversely with
pH such that an increase in hydrogen ion concentration manifests as a
corresponding increase in probe fluorescence.
Flip-flop of bilirubin from the outer to the inner bilayer hemileaflet
was monitored by changes in pyranine fluorescence following the
addition of a small (10-µl) aliquot of unconjugated bilirubin. To
facilitate distinction between fluorescence changes induced by
bilirubin absorbance versus those due to alterations in the pH of the entrapped volume, vesicles were pH-clamped by pretreatment with 3 µl (1 µg/mg phospholipid) of an ethanolic solution of the proton/potassium ionophore, nigericin (31). Control experiments were
performed to document that the small volume of added ethanol (0.15%,
v/v) did not alter the permeability of the vesicles to protons and that
there was no effect of the bilirubin vehicle on pyranine fluorescence,
either in the presence or absence of nigericin. Flip-flop of bilirubin
from the inner to the outer hemileaflet of the membrane bilayer was
examined by adding a 10-µl aliquot of bovine serum albumin dissolved
in 25 mM HEPES/KOH (pH 7.4) to bilirubin-containing
vesicles, producing a final BSA concentration of 5 µM.
 |
RESULTS |
In order to determine whether unconjugated bilirubin is able to
spontaneously traverse a membrane bilayer, we studied the equilibration
of bilirubin between 1 mol % dansyl-PE-labeled small unilamellar
phosphatidylcholine vesicles and bovine serum albumin (Fig.
2). The resultant fluorescence curve is
best described by a double exponential function, suggesting that the
equilibration process consists of two kinetically distinct events. We
postulate that the fast phase reflects bilirubin dissociation from the
external hemileaflet of the vesicle bilayer, while the slow phase,
which is readily resolved using a longer sampling interval (Fig. 2, inset), represents bilirubin flip-flop from the inner to the
outer hemileaflet of the phospholipid bilayer. In support of this
hypothesis, the rate constant for the fast component
(kfast) is identical to previously reported
bilirubin off-rates from small unilamellar phosphatidylcholine vesicles
(19). The finding that the ratio of the amplitudes of the fast
versus slow phases of transfer
(Afast:Aslow) is
identical to the outer:inner surface area of the vesicles (1.8:1) further supports the contention that the slower process corresponds to
transmembrane diffusion from the internal to the external hemileaflet of the membrane bilayer.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Bilirubin equilibration between
dansyl-labeled vesicles and bovine serum albumin. A representative
fluorescence curve obtained after mixing a suspension of 1 mol % dansyl-PE-labeled small unilamellar phosphatidylcholine vesicles
containing unconjugated bilirubin (0.5 µM) with a
solution of bovine serum albumin is displayed. The
time-dependent reemergence of fluorescence reflects
bilirubin equilibration between the donor vesicles (100 µM phospholipid) and BSA (50 µM). The
tracing, which represents the mean of six stopped-flow
injections performed at 25 °C, was generated by obtaining a total of
200 fluorescence readings over a time interval of 50 ms, followed by an
additional 200 readings during the ensuing 500 ms. The data are
normalized to a scale of 0 to 1 and fit to both single (dotted
line) and double (solid line) exponential functions.
The double exponential equation provides the best fit of the data,
supporting the presence of two distinct kinetic components to the
equilibration process: Afast = 0.202 ± 0.003, kfast = 222 ± 5 s 1,
Aslow = 0.112 ± 0.001, kslow = 10.8 ± 0.4 s 1 (± S.E.), where A is the amplitude and k is the rate
constant. In the inset, the slow phase of bilirubin transfer
is isolated from the fast phase by recording fluorescence intensity
every 200 ms over a 20-s interval. The curve reflects the mean of 10 stopped-flow injections performed at 25 °C and is best described by
a single exponential (solid line), with a first-order rate
constant of 3.7 ± 0.4 s 1.
|
|
A fluorescence system utilizing phosphatidylcholine vesicles loaded
with cbBSA also was employed to verify that the slow component of
bilirubin transfer represents bilirubin transmembrane flip-flop. Small
unilamellar phosphatidylcholine donor vesicles were preincubated with
bilirubin and then rapidly mixed with a suspension of cbBSA-loaded acceptor vesicles (Fig. 3,
left panel). Alternatively, when bilirubin was
solubilized in phosphate buffer containing 20% Me2SO (in
the absence of donor vesicles), a similar flip-flop signal was
observed, since bilirubin must traverse the acceptor vesicle bilayer in order to quench cbBSA fluorescence. While the curve obtained in the
presence of Me2SO equilibrates at a slightly higher base
line due to the enhanced aqueous solubility of bilirubin, the finding that the equilibration rate constant is unaffected by the presence or
absence of donor vesicles indicates that diffusion of bilirubin across
the acceptor membrane is rate-limiting. Control experiments demonstrate
that neither vesicles nor Me2SO have a direct effect on
cbBSA fluorescence (Fig. 3, left panel).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Detection of bilirubin flip-flop with cascade
blue-labeled bovine serum albumin. In the left panel,
bilirubin flip-flop is monitored by the quenching of cbBSA entrapped
within phospholipid vesicles. In these experiments, bilirubin (2 µM) is bound to small unilamellar phosphatidylcholine
donor vesicles (SULV) or alternatively solubilized in 0.1 M potassium phosphate (pH 7.4) containing 20% dimethyl
sulfoxide (DMSO) and then mixed with an equal volume of
cbBSA-loaded acceptor vesicles. Each curve represents an average of
five stopped-flow injections conducted at 25 °C. Similar kinetics
are observed in the presence or absence of donor vesicles, since
bilirubin must traverse the acceptor membrane bilayer to quench the
entrapped cbBSA. No change in cbBSA fluorescence is observed in the
presence of small unilamellar vesicles (or Me2SO) but in
the absence of bilirubin (Control). The
right panel displays the results of experiments in which
bilirubin equilibration is monitored by the quenching of free
(unentrapped) cbBSA. A characteristic flip-flop curve is obtained when
vesicles (SULV) serve as the bilirubin donor, reflecting
bilirubin diffusion across the donor bilayer. However, when bilirubin
is dissolved in Me2SO, equilibration is instantaneous
(DMSO), since there is no intervening membrane to impede
access of bilirubin to the albumin, and the bilirubin on-rate for cbBSA
exceeds the mixing time of the stopped-flow apparatus. Again, no
quenching of cbBSA is observed in the absence of bilirubin
(Control).
|
|
The transfer of bilirubin from unlabeled phosphatidylcholine donor
vesicles to free cbBSA (Fig. 3, right
panel) exhibits kinetics comparable with cbBSA-loaded
acceptor vesicles, although, in this case, the fluorescence signal
reflects bilirubin flip-flop from the inner to the outer hemileaflet of
the donor vesicles. This hypothesis is supported by the
nearly instantaneous decline in fluorescence that occurs when bilirubin
is solubilized in 20% Me2SO, since there is no intervening
membrane to impede bilirubin access to cbBSA, and the association rate
of bilirubin for cbBSA is more rapid than can be resolved by the
stopped-flow apparatus. Hence, the quenching of cbBSA by bilirubin is
nearly complete before the first data point is recorded. These findings
confirm that a flip-flop signal is observed only when bilirubin and
cbBSA are separated by a membrane bilayer. Moreover, the similarity in
the equilibration rate between unlabeled (Fig. 3, right
panel) and dansyl-labeled (Fig. 2, inset)
vesicles indicates that the presence of the dansyl-PE probe does not
affect bilirubin flip-flop kinetics.
Transmembrane Diffusion of Bilirubin Conjugates--
We examined
whether the non-hydrogen-bonded, water-soluble bilirubin derivative,
bilirubin ditaurate (BDT), is able to diffuse spontaneously through
phospholipid bilayers. Since BDT is less efficient than unconjugated
bilirubin at quenching cbBSA fluorescence (Fig.
4, inset), a 10-fold higher
concentration was utilized to ensure that a flip-flop signal would be
readily detectable. In contradistinction to unconjugated bilirubin,
bilirubin ditaurate causes no significant attenuation in the
fluorescence of entrapped cbBSA over a 150-s time course (Fig. 4),
indicating that spontaneous diffusion of BDT across phosphatidylcholine
bilayers is extremely slow. In control experiments, 20%
Me2SO had no effect on the rate of BDT flip-flop, verifying
that Me2SO does not alter membrane permeability to this
bilirubin conjugate. Results identical to BDT were obtained with the
principal physiologic bilirubin conjugate, BDG, while mesobilirubin
XIII
(kindly provided by Dr. D. Lightner), a bilirubin isomer that
retains internal hydrogen bonding (26), exhibited a flip-flop signal
identical to that of bilirubin IX
. These findings suggest that
internal hydrogen bonds are permissive to bilirubin transmembrane
diffusion.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 4.
Transmembrane diffusion of unconjugated
bilirubin and bilirubin ditaurate. The flip-flop of unconjugated
bilirubin (2 µM) and bilirubin ditaurate (20 µM) across unilamellar phosphatidylcholine vesicles (1 mM phospholipid) was monitored by the
time-dependent quenching of entrapped cbBSA. A solution of
unconjugated bilirubin (UCB) solubilized in 20%
Me2SO or BDT dissolved in 0.1 M potassium
phosphate, was mixed with a suspension of cbBSA-loaded vesicles, using
stopped-flow techniques. Each curve reflects the average of six
stopped-flow injections performed at 25 °C. The inset
demonstrates that unconjugated bilirubin (circles) and
bilirubin ditaurate (squares) quench the fluorescence of
free cbBSA (5 µM) in a linear manner up to a 1:1 molar
ratio of bilirubin to albumin. Each point reflects the mean ± S.D. of three experiments and, following correction for bilirubin inner
filter effects, is normalized to a scale of 0-1.
|
|
Kinetic Analysis of Bilirubin Flip-flop--
In order to determine
the rate of bilirubin transmembrane diffusion, equilibration of
bilirubin between dansyl-PE-labeled small unilamellar
phosphatidylcholine vesicles and BSA was studied over a broad range of
donor and acceptor concentrations, and the measured first-order rate
constants were plotted against the phospholipid:BSA molar ratio (Fig.
5). It is notable that individual
transfer curves (Fig. 5, inset) constitute approximately
one-third of the total change in fluorescence signal from base line (0 µM BSA), consistent with the internal:external surface
area of the vesicles. According to Equation 2, at low ratios of
phospholipid to BSA, the equilibration rate (R) approaches
kff, the value of which is calculated to be 5.3 ± 0.6 s
1 (t1/2 = 130 ± 15 ms; ±S.E.) based on the best fit of the data. Conversely, at high
ratios of phospholipid to albumin, R asymptotically
approaches koffBSA. The value of
0.16 s
1 obtained from the curve fit is consistent with
previously reported bilirubin off-rates from BSA (27). Comparable
results were obtained when bilirubin equilibration between unlabeled
(nonfluorescent) phosphatidylcholine vesicles and cbBSA was examined
(data not shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Influence of the
phospholipid:albumin molar ratio on the bilirubin equilibration
rate. The rate of bilirubin (2 µM) equilibration
between small unilamellar phosphatidylcholine vesicles containing 1 mol
% dansyl-PE and BSA was measured at 25 °C over a range of
phospholipid (PL) and albumin concentrations. The
first-order rate constant is plotted against the molar ratio of
phospholipid to albumin, with each point reflecting the mean of five
stopped-flow injections. The curve (solid line)
is generated from best-fit (p < 0.0005) parameters for
Equation 2: kff = 5.3 ± 0.6 s 1, koffBSA = 0.16 ± 0.18 s 1,
KaBSA/KaV = 778 ± 1000 (±S.E.). The inset displays fluorescence
recordings of bilirubin equilibration between dansyl-labeled vesicles
(1 mM phospholipid) and increasing concentrations of BSA
(0-20 µM) obtained at 200-ms intervals. Each curve
reflects the average of six stopped-flow injections and is fit to a
single exponential function (solid line).
|
|
While it previously has been shown that membrane lipid packing and
cholesterol content are key determinants of bilirubin solvation (21),
we found that neither vesicle hydrodynamic diameter (Table I), nor phospholipid:cholesterol ratios
up to 50 mol % (data not shown) significantly altered the rate of
bilirubin flip-flop. There also was no difference in the flip-flop rate
for vesicles composed of dioleoylphosphatidylcholine or
dipalmitoylphosphatidylcholine as compared with egg
phosphatidylcholine, suggesting that acyl chain length and saturation
have little impact on the flip-flop process. To investigate why
membrane composition and lipid packing do not alter the rate of
bilirubin flip-flop, we determined thermodynamic activation parameters
by examining bilirubin equilibration between small unilamellar
phosphatidylcholine vesicles and cbBSA over a temperature range of
10-40 °C (19). From an Arrhenius plot of the data (Fig.
6), the free energy of activation
(
G
) at 25 °C was calculated to be 16.9 kcal/mol, the principal determinant of which is the activation enthalpy
(
H
= 12.6 kcal/mol), with only a minor
entropic contribution (T
S
= -4.3 kcal/mol). These findings suggest a relatively modest energy
barrier to bilirubin transmembrane diffusion, with minimal steric
constraints.
View this table:
[in this window]
[in a new window]
|
Table I
Effect of vesicle size on the rate of bilirubin flip-flop
The effect of membrane lipid packing on bilirubin flip-flop was
determined by comparing the first-order rate constant for bilirubin
equilibration between unilamellar phosphatidylcholine vesicles (small
versus large) and cbBSA (10 µM). The mean
hydrodynamic diameter ± S.D. of the vesicle preparations (1 mM phospholipid) was determined by quasielastic light
scattering. Equilibration rate constants, which reflect the mean ± S.D. of four separate sets of 10 stopped-flow injections performed
at 25 °C, were not statistically different between the two groups of
vesicles (p = 0.72).
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Bilirubin transmembrane diffusion: Arrhenius
plot. The natural log of the first-order rate constant
(k) for bilirubin (2 µM) equilibration between
small unilamellar phosphatidylcholine vesicles (1 mM
phospholipid) and cbBSA (10 µM) is plotted against
inverse temperature (K 1). Each point
represents the mean ± S.D. of three experiments. Thermodynamic
activation parameters are calculated from the slope of the plot
(r2 = 0.939).
|
|
Bilirubin Flip-flop in Isolated Rat Liver Membranes--
Since
long-chain fatty acid flip-flop has been shown to be slower in native
as compared with model membranes (32), we determined bilirubin
flip-flop rates from the time-dependent quenching of cbBSA
entrapped within isolated native rat hepatocyte membrane vesicles.
Under the conditions utilized, bilirubin off-rates (21) are over 40 times faster than transmembrane flip-flop; hence, solvation from the
surface of the donor vesicle is not rate-limiting. Rat liver microsomes
initially were utilized as the bilirubin acceptor, with vesicle latency
and adequate entrapment of cbBSA confirmed by mannose-6-phosphatase
assay and treatment with anti-Cascade Blue antibodies. The rate of
bilirubin flip-flop across microsomal membranes was found to be
identical to that for phosphatidylcholine vesicles (Fig.
7).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 7.
Comparison of bilirubin diffusion in model
vesicles versus hepatic microsomes. The changes
in fluorescence associated with equilibration of bilirubin (2 µM) between small unilamellar donor vesicles (1 mM phospholipid) and cbBSA-loaded phosphatidylcholine
acceptor vesicles (upper panel) or 0.5 mg of
protein/ml cbBSA-loaded rat liver microsomes (lower
panel) were recorded at 25 °C. Each curve represents the
average of six stopped-flow injections. At comparable membrane
phospholipid concentrations, the rate of bilirubin flip-flop is similar
between model and native vesicles, with rate constants of 0.071 ± 0.003 s 1 and 0.077 ± 0.004 s 1
(±S.E.), respectively.
|
|
While these results confirm that microsomes are permeable to
bilirubin, since bilirubin gains access to the entrapped cbBSA only by
traversing the microsomal membrane, the possibility that microsomal
bilirubin transport is more rapid than in model vesicles with the
observed rate limited by bilirubin diffusion across the donor vesicle
is not excluded. For this reason, flip-flop also was monitored by the
equilibration of bilirubin between isolated native rat liver membranes
and free cbBSA (Fig. 8). Experiments were
conducted over a range of membrane phospholipid and cbBSA concentrations, since the measured rate is a function of the molar ratio of phospholipid to albumin (Equation 2). Equilibration rates for
native hepatocyte membranes were found to be equivalent to or lower
than in model vesicles at all phospholipid:albumin molar ratios. Since
model vesicles contain no transport proteins, these data indicate that
the rate of bilirubin uptake by rat liver membranes does not exceed
spontaneous diffusion. The convergence of rates at low
phospholipid:cbBSA molar ratios further suggests that
kff is similar for model and native membranes,
consistent with the hypothesis that bilirubin transport across
hepatocyte membranes occurs via spontaneous diffusion. We postulate
that the divergence in equilibration rates at high ratios of
phospholipid to cbBSA is due to variations in the binding affinity of
the membrane preparations for bilirubin. While it also is conceivable
that these differences reflect the effect of membrane lipid composition
or vesicle size on bilirubin diffusion, this possibility seems unlikely
in light of our demonstration that membrane hydrodynamic diameter, acyl chain saturation, and cholesterol content do not alter flip-flop kinetics (see above).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Bilirubin flip-flop in model and native
hepatocyte membranes. The rate of bilirubin (2 µM)
equilibration between membrane vesicles and free cbBSA was measured
over a range of phospholipid (PL):albumin molar ratios.
Small unilamellar phosphatidylcholine vesicles ( , short
dashed line), microsomal membranes ( ,
solid line), basolateral liver plasma membranes
( , dotted line), or canalicular liver plasma
membranes ( , long dashed line), at
concentrations ranging from 25 to 500 µM phospholipid,
were utilized as the bilirubin donor. Each point reflects the mean ± S.D. of five sets of experiments performed at 25 °C, with lines
generated from best fit parameters for Equation 2.
|
|
In order to establish whether water-soluble bilirubin conjugates are
able to traverse hepatocyte membranes, we measured the rate of
bilirubin diglucuronide equilibration between small unilamellar phosphatidylcholine vesicles (Fig. 9,
upper left panel), rat liver microsomes (Fig. 9, upper right
panel), basolateral liver plasma membranes (Fig. 9,
lower left panel), or canalicular
plasma membranes (Fig. 9, lower right
panel) and cbBSA. In contrast with model membranes, both BDG
and BDT (data not shown) readily traverse microsomal membranes at a
rate that is 5-fold faster than unconjugated bilirubin. These compounds
also exhibit rapid diffusion across bLPM and canalicular liver plasma
membranes, confirming the presence of transporters for bilirubin
conjugates in these hepatocyte membrane fractions.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 9.
Transport of unconjugated bilirubin and
bilirubin diglucuronide by hepatocyte membranes. Representative
fluorescence tracings of the equilibration of unconjugated bilirubin
(UCB) and BDG between small unilamellar phosphatidylcholine
vesicles (1 mM phospholipid) or isolated rat liver membrane
fractions (0.5 mg/ml protein) and free cbBSA (10 µM) are
displayed. Each curve reflects the average of 10 stopped-flow
injections performed at 25 °C and is normalized to a scale of 0-1.
The rate of flip-flop of unconjugated bilirubin (2 µM)
across model vesicles (upper left
panel) is similar to that measured in isolated rat liver
microsomal membranes (upper right
panel), basolateral plasma membranes (lower
left panel), and canalicular plasma membranes
(lower right panel). In contrast,
bilirubin diglucuronide (20 µM) exhibits no spontaneous
flip-flop in model vesicles but rapidly traverses native hepatocyte
membranes. At these donor and acceptor concentrations, the first-order
rate constant for BDG flip-flop in microsomal membranes of 0.29 ± 0.02 s 1 (±S.E.) is 5 times faster than unconjugated
bilirubin (k = 0.058 ± 0.002 s 1).
|
|
Mechanism of Bilirubin Transmembrane Diffusion--
Increased
uptake of unconjugated bilirubin by basolateral liver plasma membranes
in the presence of an inwardly directed potassium gradient and the
potassium ionophore, valinomycin, has been cited as evidence for a
bilirubin-specific electrogenic transporter (24). We examined the
effect of valinomycin on bilirubin transport by preincubating isolated
rat bLPM with 10 µM valinomycin (or the ethanol vehicle)
prior to recording bilirubin equilibration, using cbBSA as the
bilirubin acceptor. Initial experiments were performed in the
absence of a potassium gradient
(Kin+ = Kout+) by suspending the
membranes and cbBSA in 0.1 M potassium phosphate, 0.15 M sucrose, 10 mM HEPES (pH 7.4). In the
presence of valinomycin, bilirubin equilibration was best described by
a single exponential function, while the equilibration curve was best
fit by a double exponential equation in the absence of valinomycin
(Fig. 10). In the latter experiments,
the rate constant for the fast component of transfer (1.8 ± 0.8 s
1) is essentially identical to the first-order rate
constant measured in the presence of valinomycin (1.9 ± 0.3 s
1), suggesting that the fast process represents
bilirubin flip-flop. In the absence of potassium, (0.25 M
sucrose, 10 mM HEPES), bilirubin equilibration fits a
double exponential function irrespective of whether the vesicles are
pretreated with valinomycin (data not shown), demonstrating that
abrogation of the slow component requires the presence of both
potassium and valinomycin.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of valinomycin on bilirubin
flip-flop. The equilibration of bilirubin (2 µM)
between isolated rat bLPM (0.125 mg/ml protein) and cbBSA (20 µM) was monitored after preincubating the membranes in
the presence (+ val) or absence ( val) of
valinomycin (10 µM). Each curve reflects the average of
six stopped-flow injections performed at 25 °C and is normalized to
a scale of 0-1. When no valinomycin is present, the fluorescence
signal is best fit by a double exponential function (solid
line), while a single exponential best describes the
curve generated in the presence of valinomycin (dashed
line). Similar results are obtained when the flip-flop of
bilirubin across small unilamellar phosphatidylcholine vesicles
(100 µM phospholipid) is examined under identical
conditions (inset).
|
|
We subsequently examined the effect of a potassium gradient
on the rate of bilirubin equilibration between rat bLPM and free cbBSA.
Since these experiments monitor bilirubin flip-flop from the inner to
the outer hemileaflet of the membrane bilayer, we applied an outwardly
directed potassium gradient by preparing rat bLPM in 0.1 M
potassium phosphate, 0.15 M sucrose, 10 mM
HEPES (pH 7.4) and diluting 1:200 in 0.25 M sucrose, 10 mM HEPES (pH 7.4). This vesicle suspension then was mixed
with an equal volume of 0.25 M sucrose, 10 mM
HEPES (pH 7.4) containing cbBSA. Findings were no different from those
observed in the presence of equimolar potassium, indicating that it is
not a potassium gradient per se that drives bilirubin
uptake. Taken together, these data support the existence of two
distinct phases of bilirubin uptake, the slower of which is enhanced by
the combined presence of valinomycin and potassium. Our results are
consistent with those of Pascolo et al. (24) and could be
construed as evidence for electrogenic transport. However, when similar
experiments are performed using model vesicles composed of
phosphatidylcholine in place of native bLPM, identical results are
obtained (Fig. 10, inset). These findings strongly suggest
that the enhanced uptake of bilirubin in the presence of potassium and
valinomycin is not due to a specific electrogenic transport system.
Alternatively, based on our kinetic data, we postulate that the uptake
of bilirubin by bLPM occurs via spontaneous transmembrane diffusion of
the uncharged bilirubin diacid (BH2). At pH 7.4, a
significant proportion of bilirubin is present as the mono- and dianion
(2). If our hypothesis is correct, bilirubin uptake would necessitate
the acquisition of a proton(s) at the external hemileaflet and
subsequent release of a proton(s) at the internal hemileaflet of the
membrane bilayer, in a manner analogous to fatty acid flip-flop (31).
As it previously has been shown that dispersion of a transmembrane pH
potential is limited by slow counterdiffusion of potassium ions (33),
we propose that the slow phase of bilirubin equilibration reflects
gradual dissipation of the proton gradient generated by bilirubin
flip-flop and that valinomycin abolishes this process by collapsing the
bilirubin-induced pH gradient. Based on this postulated mechanism,
bilirubin uptake should cause a decrease in the pH of the entrapped
volume as bilirubin anions acquire protons from the external medium and
release them into the vesicle interior.
In order to test this hypothesis, we incorporated pyranine, a
water-soluble pH-sensitive probe, within small unilamellar
phosphatidylcholine vesicles and monitored pH through changes in
pyranine fluorescence. The addition of bilirubin to pyranine-loaded
vesicles induces a sharp decline in pyranine fluorescence intensity,
despite preincubation of the vesicles with 1 µg nigericin/mg
phospholipid to dissipate any transbilayer pH gradient (Fig.
11, left panel).
Since the vesicles are effectively pH-clamped and the bilirubin vehicle
does not alter pyranine fluorescence, the decrease in fluorescence
intensity does not reflect changes in vesicle pH but rather is due to
bilirubin inner filter effects and/or resonance energy transfer. When
BSA subsequently is added to the vesicle suspension, a sharp increase in fluorescence is observed, probably reflecting decreased quenching of
the entrapped pyranine and shifts in the bilirubin absorbance spectrum
associated with binding to BSA (34). Despite these pronounced spectral
effects, fluorescence changes resulting from alterations in the pH of
the entrapped vesicle volume can be distinguished from those caused by
bilirubin absorbance. In the absence of nigericin, the
addition of bilirubin to pyranine-loaded vesicles also induces an
abrupt decline in pyranine fluorescence (Fig. 11, middle
panel). However, the fluorescence signal equilibrates at a
higher intensity as compared with identical studies performed in the
presence of nigericin (Fig. 11, left panel). As
pyranine fluorescence intensity correlates inversely with pH, these
findings are indicative of a decrease in the internal pH of the
vesicles, an effect that is promptly reversed by nigericin. The
subsequent addition of BSA brings the fluorescence signal back to base
line. In contrast, when BSA is added to a suspension of pyranine-loaded
vesicles prior to treatment with nigericin (Fig. 11,
right panel), the fluorescence signal
equilibrates at a level below base line, consistent with an increase in
vesicle pH. This bilirubin-induced pH gradient is dissipated by the
addition of nigericin, which allows the vesicle pH to drop back to base
line levels. These data indicate that flip-flop of bilirubin from the
external to internal bilayer hemileaflet results in acidification,
while movement of bilirubin out of the vesicle causes alkalinization of
the entrapped volume, findings that are consistent with transbilayer
diffusion of the bilirubin diacid.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 11.
Bilirubin effects on vesicle pH. A
representative series of experiments examining the effect of bilirubin
(5 µM) on the fluorescence of pyranine-loaded small
unilamellar phosphatidylcholine vesicles (0.5 mM
phospholipid) is displayed. In the left panel,
vesicles were pretreated with nigericin (NIG), at a
concentration of 1 µg/mg phospholipid. Injection of unconjugated
bilirubin (UCB) induces a sharp decrease in fluorescence
intensity (A), while the subsequent addition of 5 µM BSA causes an increase in pyranine fluorescence
(D). When bilirubin is added to pyranine-loaded vesicles in
the absence of nigericin (middle panel), a lesser
decline in fluorescence intensity (B) is observed,
indicating acidification of the entrapped vesicle volume, as supported
by the abrupt decrease in fluorescence (A) induced by
nigericin. Injection of BSA is again associated with a rapid rise in
fluorescence to the level (D) previously observed. In the
right panel, following the addition of bilirubin, the
fluorescence signal again settles at a level (B) consistent
with acidification of the internal vesicle volume. Additionally, when
BSA is added prior to nigericin, the fluorescence signal
equilibrates below base line (C), indicative of
alkalinization of the entrapped volume. Gradual dissipation of the
proton gradient is observed before the addition of nigericin, which
immediately collapses the pH potential and returns the fluorescence
intensity to base line (D).
|
|
 |
DISCUSSION |
Our results indicate that unconjugated bilirubin is able to
diffuse spontaneously through phospholipid bilayers. The first-order rate constant for bilirubin flip-flop (5.3 s
1), although
40 times slower than solvation from the membrane surface (19), exceeds
the bilirubin dissociation rate from human and bovine serum albumin by
over 5- and 50-fold, respectively (27). These findings are consistent
with the concept of dissociation-limited uptake, originally proposed by
Weisiger (35), in which the rate-limiting step in bilirubin clearance
is solvation from serum albumin. The value obtained for the flip-flop
rate constant for unconjugated bilirubin was determined from the fit of
the equilibration data to Equation 2. While one cannot exclude the
existence of alternative mathematical models that may potentially yield
different kinetic parameters, the model utilized in the derivation of
Equation 1 (28) is straightforward, and the parameters
(koffBSA,
KaBSA/KaV)
obtained from the fit of the data correspond closely with published values (27, 36).
Our results are discordant with the findings of Noy et al.
(37) that the rate of bilirubin transmembrane diffusion
exceeds bilirubin solvation from unilamellar
dioleoylphosphatidylcholine vesicles (k > 70 s
1). The log-order discrepancy between the rate constant
for bilirubin flip-flop determined by these authors and the present
study is unlikely to reflect differences in the phospholipid content of the vesicles, since we demonstrate that the rate of bilirubin diffusion
across dioleoylphosphatidylcholine vesicles is the same as in vesicles
composed of phosphatidylcholine. However, Noy et al. derive
the rate of bilirubin flip-flop from a single exponential fit of
bilirubin equilibration curves, while we found that bilirubin transfer
is best described by a double exponential function. This raises the
possibility that these authors may not have examined a long enough time
course to identify a flip-flop signal or that their fluorescence system
was unable to resolve the two components of equilibration. Indeed, the
value for the bilirubin dissociation rate determined by these authors
is intermediate between the off-rate and flip-flop rate constants
obtained in our studies. Moreover, we have shown that it is imperative
to examine equilibration rates over a range of
phospholipid:albumin ratios in order to determine the flip-flop rate
constant (Equation 2).
Thermodynamic analyses indicate a moderate energy barrier to bilirubin
flip-flop, composed primarily of an enthalpic component. Spectroscopic
studies have shown that the negatively charged propionate groups of
bilirubin form ion pairs with the quaternary ammonium of sphingomyelin
(38); hence, we postulate that the activation enthalpy reflects the
energy required to disrupt this carboxylate/quaternary ammonium ion
interaction (since phosphatidylcholine and sphingomyelin possess an
identical head group). In contrast to long-chain fatty acids (32),
unconjugated bilirubin traverses hepatocyte plasma membranes at a rate
similar to that for phosphatidylcholine vesicles. This finding is
consistent with the small entropic contribution to the free energy of
activation, suggesting few steric or orientation constraints to
bilirubin transmembrane diffusion. The lack of an effect of
cholesterol, lipid packing, phospholipid acyl chain length, or
saturation on the bilirubin flip-flop rate supports this hypothesis.
Based on these data, the plasma membrane does not appear to pose a
significant barrier to the diffusion of bilirubin into cells.
The proposition that bilirubin is able to freely enter all cells of the
body raises questions as to the mechanism underlying the specificity of
bilirubin clearance by the liver. While our studies do not exclude the
presence of specific sinusoidal membrane transporter(s) that may be
particularly important at low plasma bilirubin concentrations, our
findings are entirely consistent with a diffusional uptake mechanism
for unconjugated bilirubin. Despite the large body of kinetic evidence
supporting the existence of protein-mediated bilirubin transport and
the identification of several putative bilirubin transport proteins in
liver cells (12, 39, 40), none of the candidate transporters has been shown to facilitate bilirubin uptake. The only study to directly examine bilirubin transport (by organic anion-transporting polypeptide) reveals no enhancement in bilirubin uptake by transiently transfected HeLa cells over nontransfected controls (15). Evidence that bilirubin
is transported by bilitranslocase is limited to the induction of
hyperbilirubinemia by injection of polyclonal anti-bilitranslocase antibodies into rats (41), while support for a role for
BSP/bilirubin-binding protein is derived from partial inhibition of
bilirubin uptake by pretreatment of HepG2 cells (42) and cultured
hepatocytes (10) with a monospecific antibody. These latter findings
provide the most convincing proof of the existence of protein-mediated bilirubin transport, although antibody-inhibitable uptake accounts for
less than one-half of total uptake. We speculate that the noninhibitable component of bilirubin uptake represents spontaneous transmembrane diffusion and that high affinity binding to glutathione S-transferase (the principal cytosolic bilirubin binding
protein in the hepatocyte) and rapid bilirubin conjugation by
UDP-glucuronosyltransferase provide sufficient driving force for
efficient bilirubin clearance by the liver (43).
Much of the kinetic data supporting the existence of specific bilirubin
transporters are derived from studies that utilize hydrophilic organic
anions, such as BSP and BDG, as surrogate markers for unconjugated
bilirubin (10, 11, 44). While unconjugated bilirubin competitively
inhibits BSP transport (10, 11), hepatocellular uptake of bilirubin can
be dissociated from BSP (14, 15), supporting the presence of distinct
transport mechanisms. We have further demonstrated a marked difference
in the uptake kinetics for unconjugated bilirubin and bilirubin
diglucuronide in hepatic microsomes (Fig. 9), suggesting that these two
compounds also are unlikely to share identical transport mechanisms, at
least within this membrane compartment. These findings raise questions as to whether uptake kinetics for hydrophilic organic anions
can be appropriately extrapolated to unconjugated bilirubin.
Protein-mediated transport has been inferred from the increased uptake
of unconjugated bilirubin by isolated rat basolateral liver plasma
membranes in the presence of valinomycin and an inwardly directed
potassium gradient (24). While this finding has been construed as
evidence for electrogenic uptake, it is equally consistent with a model
of bilirubin transport across biologic membranes, originally proposed
by Wennberg (5, 45), in which unconjugated bilirubin traverses the
hydrophobic membrane core as the uncharged diacid. We have shown that
bilirubin induces acidification of the entrapped volume of phospholipid
vesicles, supporting a mechanism whereby bilirubin anions acquire a
proton(s), diffuse through the lipid bilayer, and subsequently release
hydrogen ions into the vesicle interior (31). Since the dissipation of
a transmembrane proton gradient is limited by the slow counterflow of
potassium ions (46), we propose that increased uptake of bilirubin by bLPM in the presence of potassium and valinomycin results from rapid
dissipation of a bilirubin-generated pH gradient, as opposed to
electrogenic transport of charged bilirubin species.
The UDP-glucuronosyltransferases are a family of enzymes that convert
hydrophobic endo- and xenobiotics (e.g, bilirubin, drugs) into
water-soluble glucuronides. Topographical studies of
UDP-glucuronosyltransferase indicate that the active site is oriented
toward the lumen (47, 48) of the endoplasmic reticulum, and it has long
been postulated that specific transport systems exist to facilitate the
movement of polar glucuronides back across the microsomal membrane into the cytosol. Although uptake of bilirubin diglucuronide by hepatocyte basolateral and canalicular plasma membranes has been well
characterized (49), our demonstration of rapid BDG transport across
microsomal membranes provides some of the first direct evidence for the
presence of a glucuronide transporter in the endoplasmic reticulum
(50). Based on these findings, we propose that unconjugated bilirubin gains access to active site of UDP-glucuronosyltransferase via spontaneous flip-flop across the microsomal membrane, while bilirubin glucuronide export is carrier-mediated.
In contrast with newborns who are at significant risk for the
development of kernicterus (bilirubin encephalopathy), adults are
resistant to the neurotoxic effects of bilirubin. The basis for this
observation has been presumed to reside in the lipid composition of the
adult blood-brain barrier, which renders it impermeant to bilirubin.
Our findings regarding the relative ease with which bilirubin traverses
membranes composed of a variety of lipid species shed doubt on this
hypothesis. UDP-glucuronosyltransferase activity has been identified in
brain microvessels (51) and choroid plexus (52), and we speculate that
glucuronidation of bilirubin to form membrane-impermeant bilirubin
conjugates is an essential mechanism to prevent bilirubin entry into
the adult brain. In the newborn, hepatic UDP-glucuronosyltransferase
activity is extremely low, approaching adult levels only after several days of life (53). Assuming that expression of
UDP-glucuronosyltransferase in brain capillary endothelial cells
parallels that in the hepatocyte, there exists a period during early
postnatal development when the blood-brain endothelium is unable to
effectively conjugate bilirubin, permitting access of unconjugated
bilirubin to the central nervous system.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Dr. Richard Green
for assistance with Western blot analyses, Emma Bootle for the
performance of stopped-flow experiments, and Drs. David Lightner and
Martin Carey for insightful criticism and support.
 |
FOOTNOTES |
*
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.
This work was supported by National Institutes of
Health Research Grant DK-51679 (to S. D. Z.), a Charles H. Hood
Foundation Child Health Research Award (to S. D. Z.), a Harvard
Digestive Diseases Center Pilot/Feasibility Grant (to S. D. Z.), and
a BASF Foundation Postdoctoral Research Award (to W. G.).
Preliminary reports of this work have been published in abstract form
(54, 55).
§
To whom correspondence should be addressed: Division of Digestive
Diseases, University of Cincinnati Medical Center, 231 Bethesda Ave.
(ML 0595), Cincinnati, OH 45267-0595. Tel.: 513-558-5244; Fax:
513-558-1744.
 |
ABBREVIATIONS |
The abbreviations used are:
BSP, sulfobromophthalein;
BDT, bilirubin ditaurate;
BDG, bilirubin
diglucuronide;
BSA, bovine serum albumin;
cbBSA, Cascade Blue-labeled
BSA;
bLPM, basolateral (sinusoidal) liver plasma membranes;
dansyl-PE, N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoyl-L-
-phosphatidylethanolamine;
pyranine, 8-hydroxypyrene-1,3,6-trisulfonic acid.
 |
REFERENCES |
-
Brodersen, R.
(1979)
J. Biol. Chem.
254,
2364-2369[Abstract]
-
Ostrow, J. D.,
Mukerjee, P.,
and Tiribelli, C.
(1994)
J. Lipid. Res.
35,
1715-1737[Medline]
[Order article via Infotrieve]
-
Bonnett, R.,
Davies, J. E.,
Hursthouse, M. B.,
and Sheldrick, G. M.
(1978)
Proc. R. Soc. Lond. B Biol. Sci.
202,
249-268[Medline]
[Order article via Infotrieve]
-
Hayward, D.,
Schiff, D.,
Fedunec, S.,
Chan, G.,
Davis, J. P.,
and Poznansky, M. J.
(1986)
Biochim. Biophys. Acta
860,
149-153[Medline]
[Order article via Infotrieve]
-
Wennberg, R. P.
(1988)
Pediat. Res.
23,
443-447[Abstract]
-
Schmid, R.
(1972)
N. Engl. J. Med.
287,
703-709[Medline]
[Order article via Infotrieve]
-
Scharschmidt, B. F.,
Waggoner, J. G.,
and Berk, P. D.
(1975)
J. Clin. Invest.
56,
1280-1292[Medline]
[Order article via Infotrieve]
-
Stollman, Y. R.,
Gartner, U.,
Theilmann, L.,
Ohmi, N.,
and Wolkoff, A. W.
(1983)
J. Clin. Invest.
72,
718-723[Medline]
[Order article via Infotrieve]
-
Paumgartner, G.,
and Reichen, J.
(1976)
Clin. Sci. Mol. Med.
51,
169-176[Medline]
[Order article via Infotrieve]
-
Stremmel, W.,
and Berk, P. D.
(1986)
J. Clin. Invest.
78,
822-826[Medline]
[Order article via Infotrieve]
-
Kullak-Ublick, G. A.,
Hagenbuch, B.,
Stieger, B.,
Wolkoff, A. W.,
and Meier, P. J.
(1994)
Hepatology
20,
411-416[Medline]
[Order article via Infotrieve]
-
Sottocasa, G. L.,
Passamonti, S.,
Battiston, L.,
Pascolo, L.,
and Tiribelli, C.
(1996)
J. Hepatol.
24,
36-41[Medline]
[Order article via Infotrieve]
-
Berk, P. D.,
Potter, B. J.,
and Stremmel, W.
(1997)
Semin. Liver. Dis.
7,
165-176
-
Gartner, U.,
Stockert, R. J.,
Levine, W. G.,
and Wolkoff, A. W.
(1982)
Gastroenterology
83,
1163-1169[Medline]
[Order article via Infotrieve]
-
Kanai, N.,
Lu, R.,
Bao, Y.,
Wolkoff, A. W.,
and Schuster, V. L.
(1996)
Am. J. Physiol.
270,
F319-F325[Abstract/Free Full Text]
-
Baldini, G.,
Passamonti, S.,
Lunazzi, G. C.,
Tiribelli, C.,
and Sottocasa, G. L.
(1996)
Biochim. Biophys. Acta
856,
1-10
-
Iga, T.,
Eaton, D. L.,
and Klaassen, C. D.
(1979)
Am. J. Physiol.
236,
C9-C14[Abstract/Free Full Text]
-
Seppen, J.,
Tada, K.,
Hellwig, S.,
Bakker, C. T. M.,
Prasad, V. R.,
Roy-Chowdhury, N.,
Roy-Chowdhury, J.,
Bosma, P. J.,
and Oude Elferink, R. P. J.
(1996)
Biochem. J.
314,
477-483[Medline]
[Order article via Infotrieve]
-
Zucker, S. D.,
Storch, J.,
Zeidel, M. L.,
and Gollan, J. L.
(1992)
Biochemistry
31,
3184-3192[Medline]
[Order article via Infotrieve]
-
Barenholz, Y.,
Gibbes, D.,
Litman, B. J.,
Goll, J.,
Thompson, T. E.,
and Carlson, R. D.
(1977)
Biochemistry
16,
2806-2810[Medline]
[Order article via Infotrieve]
-
Zucker, S. D.,
Goessling, W.,
Zeidel, M. L.,
and Gollan, J. L.
(1994)
J. Biol. Chem.
269,
19262-19270[Abstract/Free Full Text]
-
Kremer, J. M. H.,
Esker, M. W. J.,
Pathmamanoharan, C.,
and Wiersema, P. H.
(1977)
Biochemistry
16,
3932-3935[Medline]
[Order article via Infotrieve]
-
Meier, P. J.,
and Boyer, J. L.
(1990)
Methods Enzymol.
192,
534-545[Medline]
[Order article via Infotrieve]
-
Pascolo, L.,
del Vecchio, S.,
Koehler, R. K.,
Bayon, J. E.,
Webster, C. C.,
Mukerjee, P.,
Ostrow, J. D.,
and Tiribelli, C.
(1996)
Biochem. J.
316,
999-1004[Medline]
[Order article via Infotrieve]
-
Anchordoguy, T. J.,
Carpenter, J. F.,
Crowe, J. H.,
and Crowe, L. M.
(1992)
Biochim. Biophys. Acta
1104,
117-122[Medline]
[Order article via Infotrieve]
-
Nogales, D.,
and Lightner, D. A.
(1995)
J. Biol. Chem.
270,
73-77[Abstract/Free Full Text]
-
Zucker, S. D.,
Goessling, W.,
and Gollan, J. L.
(1995)
J. Biol. Chem.
270,
1074-1081[Abstract/Free Full Text]
-
Nichols, J. W.,
and Pagano, R. E.
(1982)
Biochemistry
21,
1720-1726[Medline]
[Order article via Infotrieve]
-
Blanckaert, N.,
Gollan, J. L.,
and Schmid, R.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
2037-2041[Abstract]
-
Spivak, W.,
and Carey, M. C.
(1985)
Biochem. J.
255,
787-805
-
Kamp, F.,
and Hamilton, J. A.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
11367-11370[Abstract]
-
Kleinfeld, A. M.,
Chu, P.,
and Romero, C.
(1997)
Biochemistry
36,
14146-14158[CrossRef][Medline]
[Order article via Infotrieve]
-
Kamp, F.,
and Hamilton, J. A.
(1993)
Biochemistry
32,
11074-11086[Medline]
[Order article via Infotrieve]
-
Reed, R. G.
(1977)
J. Biol. Chem.
252,
7483-7487[Abstract]
-
Weisiger, R. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
1563-1567[Abstract]
-
Leonard, M.,
Noy, N.,
and Zakim, D.
(1989)
J. Biol. Chem.
264,
5648-5652[Abstract/Free Full Text]
-
Noy, N.,
Leonard, M.,
and Zakim, D.
(1992)
Biophys. Chem.
42,
177-188[CrossRef][Medline]
[Order article via Infotrieve]
-
Yang, B.,
Morris, M. D.,
Xie, M.,
and Lightner, D. A.
(1991)
Biochemistry
30,
688-694[Medline]
[Order article via Infotrieve]
-
Wolkoff, A. W.
(1996)
Semin. Liver Dis.
16,
121-127[Medline]
[Order article via Infotrieve]
-
Berk, P. D.,
and Noyer, C.
(1994)
Semin. Liver Dis.
14,
331-343[Medline]
[Order article via Infotrieve]
-
Sottocasa, G. L.,
Tiribelli, C.,
Luciani, M.,
Lunazzi, G. C.,
and Gazzin, B.
(1979)
in
Function and Molecular Aspects of Biomembrane Transport (Quagliariello, E., Palmieri, F., Papa, S., and Klinkenberg, M., eds), pp. 451-458, Elsevier/North-Holland Biomedical Press, Amsterdam
-
Stremmel, W.,
and Diede, H. E.
(1990)
J. Hepatol.
10,
99-104[Medline]
[Order article via Infotrieve]
-
Tiribelli, C.,
and Ostrow, J. D.
(1993)
Hepatology
17,
715-736[Medline]
[Order article via Infotrieve]
-
Torres, A. M.,
Lunazzi, G. C.,
Stremmel, W.,
and Tiribelli, C.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8136-8139[Abstract/Free Full Text]
-
Bratlid, D.
(1990)
Clin. Perinatol.
17,
449-465[Medline]
[Order article via Infotrieve]
-
Deamer, D. W.,
and Nichols, J. W.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
165-168[Abstract]
-
Shepherd, S. R. P.,
Baird, S. J.,
Hallinan, T.,
and Burchell, B.
(1989)
Biochem. J.
259,
617-620[Medline]
[Order article via Infotrieve]
-
Jansen, P. L. M.,
Mulder, G. J.,
Burchell, B.,
and Bock, K. W.
(1992)
Hepatology
15,
532-544[Medline]
[Order article via Infotrieve]
-
Oude Elferink, R. P. J.,
Meijer, D. K. F.,
Kuipers, F.,
Jansen, P. L. M.,
Groen, A. K.,
and Groothuis, G. M. M.
(1995)
Biochim. Biophys. Acta
1241,
215-268[Medline]
[Order article via Infotrieve]
-
Banhegyi, G.,
Braun, L.,
Marcolongo, P.,
Csala, M.,
Fulceri, R.,
Mandl, J.,
and Benedetti, A.
(1996)
Biochem. J.
315,
171-176[Medline]
[Order article via Infotrieve]
-
Ghersi-Egea, J. F.,
Minn, A.,
and Siest, G.
(1988)
Life Sci.
42,
2515-2523[CrossRef][Medline]
[Order article via Infotrieve]
-
Leininger-Muller, B.,
Ghersi-Egea, J. F.,
Siest, G.,
and Minn, A.
(1994)
Neurosci. Lett.
175,
37-40[CrossRef][Medline]
[Order article via Infotrieve]
-
Wishart, G. J.
(1978)
Biochem. J.
174,
485-489[Medline]
[Order article via Infotrieve]
-
Zucker, S. D.,
and Hoppin, A. G.
(1996)
Hepatology
24,
132 (abstr.)
-
Goessling, W.,
and Zucker, S. D.
(1997)
Hepatology
26,
385 (abstr.)
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.