Role of apolipoprotein D in the transport of bilirubin in
plasma
Wolfram
Goessling1 and
Stephen D.
Zucker2
1 Department of Medicine, Brigham and Women's Hospital,
Boston, Massachusetts 02115; and 2 Division of Digestive
Diseases, University of Cincinnati Medical Center, Cincinnati, Ohio
45267
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ABSTRACT |
Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown
function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a
0.8:1 stoichiometry, we examined the contribution of this protein to
transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of
[14C]bilirubin reveals that 9% of unconjugated bilirubin
is associated with HDL, with the remaining pigment bound primarily to
serum proteins (i.e., albumin). The percentage of total plasma
bilirubin bound to HDL was found to increase proportionally with
bilirubin concentration. Affinity of human apo D for bilirubin was
determined by steady-state fluorescence quenching, with Scatchard
analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (Ka) of ~3 × 107 M
1. Incorporation of apo D into
phosphatidylcholine vesicles had no effect on
Ka, suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s
1 (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin
carrier of bilirubin in human plasma and further support the
proposition that the affinity of HDL for bilirubin is primarily the
result of binding to apo D.
high-density lipoproteins; human serum albumin; dissociation rate; cholesterol; cholic acid
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INTRODUCTION |
UNCONJUGATED
BILIRUBIN (UCB), the principal product of heme catabolism, is
efficiently cleared from the circulation by the liver. In patients with
impaired hepatic function, plasma bilirubin levels can increase
50-fold, producing yellow discoloration of the skin, sclerae, and
mucous membranes. Bennhold (3) first postulated more than
60 years ago that bilirubin, which exhibits minimal aqueous solubility
at physiological pH, is transported in the blood bound to serum
albumin. Electrophoretic analyses of human plasma by Ostrow et al.
(30) subsequently confirmed that circulating bilirubin is
associated with albumin. However, despite the high affinity of human
serum albumin (HSA) for bilirubin (6, 24,
35), the notion that albumin is vital for bilirubin transport was dispelled by the observation that individuals with analbuminemia, a rare genetic disorder associated with negligible serum
albumin concentrations, exhibit normal bilirubin clearance (15, 42). It is notable that, in both
analbuminemic humans (15) and rats (39),
bilirubin is associated predominantly with high-density lipoproteins
(HDL). This finding is not entirely unexpected, since Cooke and Roberts
(14) previously have shown that a proportion of serum
bilirubin is complexed with
-lipoproteins at pathophysiological
bilirubin concentrations. Although bilirubin is known to bind to
phospholipids (23, 28, 41), it
remains unclear why this pigment preferentially associates with HDL as opposed to other lipoprotein classes (e.g., low-density lipoproteins).
Apolipoprotein D (apo D), a 30-kDa glycoprotein of unknown
function, comprises roughly 1-2% of HDL protein
(26). apo D is structurally unrelated to other
apolipoproteins, exhibiting sequence homology with lipocalins, a family
of proteins that bind small hydrophobic ligands (44). On
the basis of molecular modeling analyses, Peitsch and Boguski
(31) hypothesized that apo D preferentially binds
heme-related compounds. These authors went on to show that bilirubin,
at supraphysiological concentrations, binds to purified apo D with a
stoichiometry of 0.8 moles of bilirubin per mole of protein. To extend
these observations and verify whether apo D confers the binding
specificity of HDL for bilirubin, we examined the contribution of
lipoproteins to the binding and transport of bilirubin in human plasma
and further characterized the affinity of apo D for this bile pigment.
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EXPERIMENTAL PROCEDURES |
Materials.
Bilirubin IX
, the physiological isomer of UCB, was obtained
from Porphyrin Products (Logan, UT). For all apo D binding analyses, bilirubin was further purified by phase extraction according to the
method of McDonagh and Assisi (27).
-[4-14C]aminolevulinic acid hydrochloride was
purchased from NEN Life Sciences (Boston, MA). Essentially fatty
acid-free HSA was obtained from Sigma Chemical (St. Louis, MO). All
glassware was washed in chloroform before use to eliminate potential
lipid contamination.
Preparation of radiolabeled UCB.
UCB was radiolabeled biosynthetically by infusing the metabolic
precursor,
-[4-14C]aminolevulinic acid, into male
Sprague-Dawley rats (16, 29). [14C]bilirubin was isolated from a 6-h collection of bile
by hydrolysis of glucuronides, precipitation with lead acetate,
extraction with chloroform, and subsequent recrystallization
(16). The specific activity of the bilirubin prepared in
this manner ranged between 35 and 75 Ci/mol.
Determination of the binding distribution of bilirubin in human
plasma.
Blood from nonfasted healthy volunteers was drawn into tubes containing
5% (vol/vol) 0.2 M EDTA, and plasma was isolated by centrifugation at
265 g for 15 min. The phlebotomy protocol and consent
procedure were approved by the Brigham and Women's Hospital Human
Research Committee (Protocol no. 96-07907). Plasma samples were treated
with ascorbic acid (6 mM) to prevent bilirubin oxidation and with
NaN3 (0.01% wt/vol) to inhibit microbial growth. Because of poor aqueous solubility at physiological pH (6), UCB
was solubilized in 0.1 M potassium phosphate (pH 12) (51).
In a standard experiment, a 3 mM stock solution of UCB was dissolved in
alkaline buffer and spiked with enough [14C]bilirubin to
produce 10,000-12,000 dpm/µl. A small (18 µl) aliquot of the
bilirubin stock solution was added to 3.6 ml of plasma, producing a
final bilirubin concentration of 15 µM. Although this manipulation
caused minimal alteration of the pH of the plasma sample (
0.02 pH
units), an equal volume (18 µl) of 0.1 M potassium phosphate (pH 1.9)
was immediately added to correct the pH to baseline.
Samples of human plasma were incubated with radiolabeled bilirubin for
15 min at room temperature and then adjusted to a density of 1.21 g/ml
with solid KBr (0.325 g/ml) to facilitate resolution of plasma
lipoprotein [very low-density lipoprotein, low-density lipoprotein
(LDL), HDL] and protein components by density gradient centrifugation
(11). Following centrifugation (197,000 g × 48 h) at 17°C, sequential 200-µl fractions were harvested
and 50-µl aliquots were diluted into 5 ml of Ecoscint (National
Diagnostics, Atlanta, GA) and counted. The average recovery was
99.8 ± 6.0% of added counts. Fraction density was determined
using a Mettler/Paar DMA 45 density meter (Anton Paar, Graz, Austria).
Protein was quantified by the method of Lowry et al. (11,
25), phospholipid by the assay technique of Bartlett
(2), and cholesterol by the enzymatic method of Allain et
al. (1).
Purification of human HDL and apo D.
apo D was purified from human plasma according to the method of
McConathy and Alaupovic (26). One liter of plasma was
adjusted to a density of 1.27 g/ml with solid KBr and subjected to
centrifugation at 140,000 g for 24 h at 17°C. The
lipoprotein layer was harvested and diluted with isotonic saline
containing 10 mM Tris and 0.1% sodium azide (pH 7.4) to achieve a
density of 1.12 g/ml. Following repeat centrifugation at 140,000 g, the resultant HDL was dialyzed exhaustively,
lyophilized, and delipidated. The precipitated protein was dissolved in
1 mM potassium phosphate (pH 8.0) containing 8 M urea and twice eluted
on a hydroxyapatite column (25 × 1 cm). Polyclonal rabbit
anti-apo D serum subsequently was produced by injection of the purified
protein (ICN, Aurora, OH) and was used for Western blot analysis. Blots
were developed using the Renaissance enhanced chemiluminescence
detection system (NEN Life Science).
Determination of apo D affinity for bilirubin.
A variety of fluorescence quenching analyses were performed to
facilitate the determination of the affinity of apo D for UCB. For each
of these studies, commercial bilirubin was further purified by phase
extraction (27) to avoid potential competition with contaminating lipid species. Serial 2-µl aliquots of purified UCB
(500 µM in DMSO) were added to a stirred cuvette containing 3 ml of a
10 µM solution of apo D in 0.1 M potassium phosphate (pH 7.40). The
steady-state fluorescence (excitation: 280 nm, emission: 332 nm) of apo
D at 25°C was recorded following the addition of each aliquot using
an Aminco-Bowman II fluorescence spectrophotometer. All readings were
corrected for the added volume of DMSO and for inner filter effects
resulting from bilirubin absorption (51). The affinity
constant (Ka) was determined by Scatchard
analysis, according to the method of Levine (24).
The affinity of apo D for bilirubin was also determined by measuring
the equilibrium distribution of UCB between apo D and HSA. For these
studies, UCB (0.25 mM) was solubilized in 50 mM potassium phosphate (pH
12) and 10-µl aliquots were added to 1 ml of 0.1 M potassium
phosphate (pH 7.4) containing varying ratios of apo D to HSA.
Partitioning was quantified by recording steady-state bilirubin
fluorescence (excitation: 467 nm, emission: 525 nm) and fitting the
data to the expression
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(1)
|
where Iapo D and IHSA indicate the
fluorescence intensity of bilirubin in the presence of apo D or HSA
alone, Iobs is the observed bilirubin fluorescence at a
defined molar ratio of [apo D]:[HSA], Ka is
the affinity constant, and [apo D] and [HSA] are the
concentrations of apo D and HSA, respectively. The total protein concentration ([apo D] + [HSA]) was maintained constant to control for inner filter effects, and all data were corrected for intrinsic protein fluorescence. Based on Eq. 1, the slope of a plot of
(IHSA
Iapo D)/(Iobs
I apo D) vs. [apo D]/[HSA] provides a measure of the
relative affinity (Kaapo
D/KaHSA) of the proteins for
bilirubin. Samples were maintained in the dark and only briefly exposed
to light for all fluorescence measurements to minimize the risk of
bilirubin photodegradation. We further measured bilirubin binding by
the fluorimetric titration of apo D (10 µM) in 0.1 M potassium
phosphate (pH 7.4) with increasing concentrations of bilirubin
(excitation: 467 nm, emission: 525 nm). With this approach, the
affinity of apo D for bilirubin was derived from a linear least-square
plot, according to the method of Cogan et al. (13).
Preparation of phospholipid vesicles.
Small unilamellar vesicles were prepared by sonication according to the
method of DiCorleto and Zilversmit (17). A chloroform solution of egg phosphatidylcholine (Lipid Products, Surrey, UK) was
evaporated to dryness under argon atmosphere, solubilized in diethyl
ether, and then reevaporated to form a uniform film. The lipids were
desiccated overnight under vacuum to remove all traces of solvent and
then suspended in 0.1 M potassium phosphate (pH 7.4). The lipid
suspension was sonicated under argon atmosphere in a bath sonicator
(Laboratory Supplies, Hicksville, NY) until clear. apo D was
incorporated into small unilamellar vesicles by sonication in the
presence of phosphatidylcholine at a phospholipid-to-apo D molar ratio
of 500:1.
Stopped-flow analysis of bilirubin dissociation.
The rate of bilirubin dissociation from isolated HDL, HSA, and apo D
was determined from the time-dependent changes in intrinsic protein
fluorescence (excitation: 280 nm, emission: 360 nm), reflecting bilirubin transfer from the donor particle to a large molar excess of
small unilamellar phosphatidylcholine acceptor vesicles
(49). Studies of bilirubin dissociation from HSA and apo D
employed an Aminco-Bowman II fluorescence spectrophotometer equipped
with an SLM-Aminco MilliFlow reactor (mixing time: 20 ms) using a
320-nm cut-on emission filter to minimize light-scattering effects.
Because of the rapid rate of bilirubin dissociation from HDL, an
Applied Photophysics (Leatherhead, UK) fluorescence spectrophotometer with SPF-17 stopped-flow device (mixing time: 0.7 ms) was utilized. The
rate constant for bilirubin dissociation from HDL, HSA, and apo D was
determined by fitting the observed fluorescence intensities to both
single and double exponential functions, with fit quality assessed by
regression analysis of variance (51). In the case of a
double exponential fit, an average rate (kav)
for bilirubin dissociation was calculated by the expression
(38)
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(2)
|
where A is the amplitude and k the rate constant for
the fast and slow components.
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RESULTS |
Distribution of bilirubin in human plasma.
The binding distribution of [14C]bilirubin in human
plasma was determined by KBr density gradient centrifugation. This
technique was found to provide excellent resolution of plasma LDL, HDL, and protein fractions (Fig. 1), the
latter consisting primarily of HSA (8). The results of
experiments analyzing the distribution of physiological (15 µM)
concentrations of UCB in human plasma are displayed in Fig.
2. We found that 89.2 ± 0.4% of
added 14C-labeled UCB was associated with the protein
fraction of human plasma, whereas 9.1 ± 0.4% was bound to HDL
and 1.5 ± 0.1% to LDL. Since UCB is associated with serum HDL in
analbuminemic rats (19), we also examined the distribution
of [14C]UCB in rat plasma and found it to be similar to
that in human plasma, with 87.7 ± 0.4%, 10.9 ± 0.3%, and
1.4 ± 0.1% bound to protein, HDL, and LDL, respectively.

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Fig. 1.
Density gradient centrifugation of human plasma.
Human plasma was subjected to KBr density gradient centrifugation, and
serial 200-µl fractions were harvested. A: total protein
concentration (mg/ml) of the fractions ( ) and protein
concentration in mg/ml × 10 1 ( ).
The dashed line indicates the density of the fractions measured at
17°C. Fractions corresponding to low-density lipoprotein (LDL),
high-density lipoprotein (HDL), and human serum albumin (HSA) are
identified. B: cholesterol concentration, with the two main
peaks corresponding to plasma LDL and HDL. C: phospholipid
concentration, with the small peak in fractions 1-4
reflecting very low-density lipoprotein (VLDL) and chylomicrons.
All experiments were performed in triplicate. Values are means ± SD.
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Fig. 2.
Distribution of unconjugated bilirubin (UCB) in human
plasma. 14C-labeled UCB ( ) was added to human
plasma at a concentration of 15 µM. Density gradient centrifugation
was performed, and sequential 200-µl fractions were analyzed for
radioactivity. Each point represents the mean ± SD of 3 separate
experiments. Inset: [14C]UCB (10 µM) was
preincubated with a solution of 2.4 mg/ml HSA ( ) or 2.4 mg/ml
isolated human HDL ( ) solubilized in 0.15 M NaCl, 0.1 M
Tris · HCl, 1 mM EDTA (pH 7.4) and then subjected to density
gradient centrifugation. The peaks for HDL- and HSA-bound bilirubin
were found to correlate with the corresponding peaks identified in
human plasma. In the absence of a bilirubin carrier ( ),
a gradual slope upward is observed, reaching a maximum at a density of
1.23 g/cm3.
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To confirm the binding of bilirubin to HDL, we preincubated a
suspension of HDL (isolated from human plasma) or a solution of HSA
with [14C]UCB and then performed density gradient
centrifugation (Fig. 2, inset). The principal peaks of
radioactivity coincide with those of plasma HDL and protein,
respectively. We postulate that the smaller high-density peak observed
in the HDL-bound bilirubin sample represents unbound (aggregated)
bilirubin. This hypothesis is supported by experiments in which
centrifugation of [14C]UCB was performed in the absence
of carrier particles (Fig. 2, inset). Further validation of
the centrifugation technique was achieved by examining the distribution
of [14C]cholic acid and [14C]cholesterol in
human plasma (Fig. 3). Our results
correlate closely with previous reports (8,
21, 34) and confirm that the observed plasma
distribution patterns are ligand specific.

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Fig. 3.
Plasma distribution of cholic acid and cholesterol. Density
gradient centrifugation was performed on samples of human plasma
preincubated with 1 µM [14C]cholic acid
( ) or 2 nM [14C]cholesterol ( )
under the same conditions as outlined in Fig. 2.
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Effect of concentration and pH on the distribution of bilirubin in
plasma.
Since serum bilirubin levels are markedly elevated under certain
pathological conditions, we utilized density gradient centrifugation to
examine the effect of bilirubin concentration on its plasma distribution. In these experiments, [14C]UCB (15-900
µM) was added directly to samples of human plasma (Fig.
4). The percentage of UCB associated with
the protein fraction of plasma was found to decrease with increasing
bilirubin concentration (Fig. 4, inset). This decline in the
percentage of albumin-associated bilirubin coincides with an increase
in the relative amount of bilirubin bound to HDL, which rose to a
maximum of 19% at 900 µM UCB. On the basis of the observation that
the risk of bilirubin neurotoxicity is increased in the setting of
acidosis (7), we also determined the effect of pH on the
binding distribution of UCB in human plasma. Plasma pH was adjusted
between 6.8 and 8.0 before the addition of radiolabeled bilirubin (15 µM), and density gradient centrifugation subsequently was performed.
No significant effect of pH on the distribution of UCB was observed (data not shown).

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Fig. 4.
Distribution of bilirubin in human plasma: influence of bilirubin
concentration. Samples of human plasma were preincubated with
[14C]UCB at concentrations of 15 µM ( ), 120 µM ( ), and 900 µM ( ), corresponding
to bilirubin-to-albumin molar ratios of 1:60, 1:5, and 1.5:1,
respectively. The plasma samples were subjected to density gradient
centrifugation for 48 h at 17°C, and 200-µl aliquots were
sequentially harvested and counted. In the inset, the
percentages of total counts associated with HDL ( ) and HSA
( ) are plotted as functions of the bilirubin:albumin
molar ratio.
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Determination of apo D binding affinity for bilirubin.
To examine whether the selective binding of bilirubin by HDL is due to
the presence of apo D, we isolated apo D from human plasma (Fig.
5) and prepared rabbit anti-apo D
antiserum. Samples of human and rat plasma were then separated by
density gradient centrifugation, and the various lipoprotein and
protein fractions were harvested, subjected to SDS-PAGE, and blotted
with anti-apo D antiserum (Fig. 6). The
highest concentrations of apo D were identified in the HDL fractions of
both human and rat plasma. The affinity of purified apo D for UCB was
measured using resonance energy transfer techniques. The addition of
increasing concentrations of UCB to a solution of apo D causes
progressive quenching of the intrinsic tryptophan fluorescence of the
protein (Fig. 7). Scatchard analysis of
the quenching curve (Fig. 7, inset) demonstrates a single,
high-affinity bilirubin binding site, with a Ka
of 2.6 ± 0.5 × 107 M
1.

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Fig. 5.
SDS-PAGE of human apolipoprotein D (apo D). A 10-µg sample of apo
D isolated from human plasma was subjected to SDS-PAGE on a 14%
polyacrylamide gel and stained with Coomassie blue.
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Fig. 6.
Western blot of human and rat plasma. Fractions
corresponding to VLDL, LDL, HDL, and protein were isolated from human
and rat plasma, and 10 µg total protein was subjected to SDS-PAGE on
a 14% gel (top). Following transfer onto nitrocellulose,
apo D was probed with rabbit antiserum to human apo D. Bands were
quantified by densitometry, and the percentage of total apo D in each
fraction was determined (bottom).
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Fig. 7.
Bilirubin binding to apo D as determined by the quenching of apo D
fluorescence. The affinity of UCB for purified apo D (10 µM) was
determined from the quenching of apo D steady-state fluorescence by
serial aliquots of UCB. Each point represents the mean ± SD of 3 experiments conducted at 20°C and is corrected for the added volume
and for bilirubin inner filter effects. a.u., Arbitrary units.
Inset: Scatchard plot of UCB binding to apo D, where Q
reflects the ratio of apo D-bound bilirubin to total apo D and R is the
molar ratio of total bilirubin to apo D. The affinity constant
(Ka) of 2.6 ± 0.5 × 107
M 1 was determined from the slope
(r2= 0.714) of the plot, according to the method
of Levine (24).
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To confirm these findings, the affinity of apo D for UCB was also
calculated from the equilibrium binding distribution of bilirubin
between apo D and HSA (Eq. 1), as measured by bilirubin steady-state fluorescence (Fig. 8). The
ratio of the affinity constants
(Kaapo D/KaHSA)
is derived from the slope of a plot of bilirubin fluorescence intensity
vs. the apo D:HSA molar ratio (Fig. 8, inset). With the use of a consensus value for KaHSA of
1.1 × 108 M
1 (6,
12, 24, 46), the affinity
constant of apo D for UCB is calculated to be 1.2 ± 0.3 × 107 M
1, which is similar to the
results obtained by Scatchard analysis.

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Fig. 8.
Binding distribution of UCB between HSA and apo D. The intrinsic
fluorescence of 2.5 µM UCB was measured in the presence of varying
concentrations of HSA and apo D, maintaining the total protein
concentration constant at 24 µM. Each point represents the mean ± SD of 3 experiments performed at 25°C
(r2 = 0.994). Inset: inverse of
the fractional fluorescence is plotted against the molar ratio of apo
D:HSA. The slope of the plot (0.11 ± 0.01) reflects the ratio of
the association constants
(Kaapo D/KaHSA).
[apo D] and [HSA], concentrations of apo D and HSA, respectively;
IHSA, Iapo D, and Iobs,
fluorescence intensity of bilirubin in the presence of HSA alone, apo D
alone, or observed fluorescence intensity at a defined molar ratio of
[apo D] to [HSA], respectively.
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When a solution of apo D is titrated with increasing concentrations of
UCB, an approximately linear rise in bilirubin fluorescence intensity
is observed, with saturation occurring as the molar ratio of
bilirubin:apo D approaches 1:1 (Fig. 9).
An apparent dissociation constant (Kd) of
25 ± 19 nM was derived from a linear least-square plot of the
data (Fig. 9), using the method of Cogan et al. (13). The
calculated Kd corresponds to a
Ka of 4.0 × 107
M
1, which closely correlates with affinity constants
obtained by the previously outlined methods. The calculated number of
binding sites (n) is 0.8 ± 0.2, consistent with a 1:1
binding stoichiometry.

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Fig. 9.
Affinity of apo D for bilirubin as determined by fluorimetric
titration. A: steady-state bilirubin fluorescence was
recorded at 25°C following the addition of increasing concentrations
of UCB to a solution of 10 µM apo D ( ) or to buffer alone
( ). Data generated in the presence of apo D after
correction for the contribution of free bilirubin fluorescence are
shown as . B: linear least-square plot of
the data (r2 = 0.989), where
P0 is the total protein concentration,
R0 is the total bilirubin concentration, and reflects the fraction of free binding sites on the apo D molecule
(13).
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Although apo D has no identifiable membrane-spanning domains
(31, 44), the protein is associated primarily
with lipid structures (i.e., cell membranes, lipoproteins) in vivo
(47). For this reason, we examined whether the affinity of
the protein for bilirubin is altered by a lipid environment. Following
the incorporation of apo D into small unilamellar phosphatidylcholine vesicles, we determined the binding affinity for UCB by competitive binding against HSA. The measured Ka for
membrane-associated apo D (2.5 × 107
M
1) is not significantly different from values obtained
for free apo D, whereas the affinity constant for phosphatidylcholine
(PC) vesicles alone is ~0.08% that of HSA
(KaPC = 9.0 × 104 M
1). These results suggest that the affinity of apo D for
UCB is not altered by a lipid environment.
Stopped-flow analysis of bilirubin dissociation from HDL and apo D.
There is evidence that bilirubin clearance from the plasma is limited
by the rate of dissociation from serum albumin (45, 50). To assess whether HDL-bound bilirubin may be cleared
more efficiently than albumin-bound bilirubin, we compared the rate of
dissociation of UCB from isolated human HDL with that from HSA using
stopped-flow techniques. The average rate of UCB dissociation from HDL
(Eq. 2) was found to be ~240-fold faster than from HSA (Fig. 10), suggesting that HDL is the
more efficient bilirubin donor. Regression analysis of the fluorescence
tracings reveals that bilirubin dissociation from HDL is best described
by a double exponential function (Fig.
11). The rate constant of 215 ± 14 s
1 [half-time (t1/2) = 3.2 ± 0.2 ms] for the fast component of dissociation is
identical to previously reported off rates for UCB from small unilamellar phospholipid vesicles (51). On the basis of
these findings, we postulate that the fast component of dissociation from HDL represents bilirubin solvation from surface phospholipids. We
further propose that the slow component reflects bilirubin dissociation
from apo D. The 1:1 ratio of the amplitudes of the fast and slow
components of bilirubin dissociation suggests that approximately half
of HDL-bound bilirubin is associated with apo D. The dissociation of
UCB from purified apo D was best described by a single exponential
function (Fig. 11, inset), with a first-order rate constant
of 5.4 ± 1.1 s
1 (t1/2 = 129 ± 26 ms).

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Fig. 10.
Comparison of bilirubin dissociation from HDL vs.
HSA. The dissociation of UCB (0.5 µM) from 50 µg protein/ml HDL
(left) is compared with that from 0.5 µM HSA
(right). Dissociation rates are derived from the
time-dependent increase in protein fluorescence associated with the
transfer of bilirubin to phosphatidylcholine acceptor vesicles (1 mM
phospholipid). Each curve reflects the average of 5 stopped-flow
injections at 25°C. Solvation of bilirubin from HSA is best described
by a single exponential function (solid line) with a rate constant of
0.90 ± 0.08 s 1 (t1/2 = 770 ± 68 ms), whereas dissociation from HDL is best fit by 2 exponentials (see Fig. 11).
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Fig. 11.
Stopped-flow analysis of bilirubin dissociation from HDL and apo
D. The rate of dissociation of UCB (0.5 µM) from HDL (50 µg
protein/ml) was determined under experimental conditions identical to
those in Fig. 10. The fluorescence curve represents the average of 12 stopped-flow injections performed at 25°C and is fit to both single
(dashed line) and double (solid line) exponential functions. The data
are best described by two exponentials, with rate constants of 215 ± 14 s 1 and 19 ± 2 s 1.
Inset: fluorescence curve generated by the transfer of
bilirubin (2 µM) from free apo D (2 µM) to small unilamellar
phosphatidylcholine vesicles (2 mM phospholipid). The curve is best
described by a single exponential function (solid line) with a
first-order rate constant of 5.4 ± 1.1 s 1.
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DISCUSSION |
The present study demonstrates that HDL is the principal carrier
of non-albumin-bound bilirubin in plasma, consistent with previous
observations in analbuminemic humans (4) and rats (19). Although the amount of UCB partitioning into HDL
under normal physiological conditions is only 9% of total plasma
levels, we show that the relative contribution of HDL to UCB binding
increases at higher molar ratios of bilirubin to albumin, as can occur
in various disease states. Our findings are concordant with prior work
demonstrating the formation of
-lipoprotein-bilirubin complexes in
human plasma when bilirubin concentrations approach the saturation limit of albumin (14). The substantial bilirubin carrying
capacity of HDL is reflected in the normal bilirubin transport
and clearance observed in analbuminemic individuals (4,
20, 43).
From the results of plasma distribution studies, we conclude that the
affinity of HDL for UCB exceeds that of LDL by an order of magnitude, a
phenomenon that cannot be explained by lipid binding alone
(23, 28, 41). The observed
second-order dissociation kinetics suggest the existence of two
distinct populations of bilirubin associated with the HDL particle. We
postulate that the fast component reflects bilirubin bound to surface
phospholipids, a hypothesis that is supported by the similarity in the
off-rate constant to that for bilirubin solvation from small
unilamellar vesicles (51). We further propose that the
slow phase of bilirubin dissociation from HDL is the result of binding
to apo D, as evidenced by the close correspondence of the rate constant
with that for bilirubin solvation from purified apo D. Taking into
account the measured binding affinity of apo D and phospholipids for
bilirubin, as well as the bilirubin binding distribution in plasma,
our data correlate closely with the affinity of HDL for UCB
estimated from studies of analbuminemic rats (39).
apo D is structurally unrelated to other apolipoproteins, and the
precise function of this protein remains obscure. It is classified as a
member of the lipocalin family of binding proteins (36), a
characteristic feature of which is the presence of a binding pocket
formed by eight antiparallel strands. Insecticyanin, a lipocalin
isolated from the tobacco hornworm, Manduca sexta, binds
biliverdin IX
with high specificity and is believed to be important
for protective coloration (18, 32). On the
basis of the high degree of homology between insecticyanin and apo D, including the topology of the binding pocket, Peitsch and Boguski (31) originally proposed that apo D is capable of binding
heme-related compounds. These authors also showed that bilirubin IX
binds to apo D at a molar ratio of 0.8:1, albeit at supersaturating bilirubin concentrations. Our studies extend the observations of these
investigators, confirming the high-affinity binding of UCB by apo D
(Ka
3 × 107
M
1) and further support a role for this protein in
bilirubin transport. It is notable that the affinity of apo D for UCB
is similar to that measured for lipocalin-type prostaglandin D synthase
(5). Because prostaglandin D synthase is the most abundant
protein in human cerebrospinal fluid, it has been proposed that this
molecule may act as a scavenger for harmful hydrophobic compounds
(5). On the basis of the high levels of expression of apo
D in the brain (44), it is conceivable that apo D may
serve a similar function.
There is evidence suggesting that solvation from HSA is the
rate-limiting step in the hepatocellular uptake of UCB
(48, 50). Since bilirubin dissociation from
HDL (and apo D) is significantly faster than from HSA, it appears that
HDL is the more proficient bilirubin donor. The increased rate of
appearance of circulating bilirubin in the bile of analbuminemic rats
compared with control animals supports this contention
(39). It is notable that the risk of bilirubin-induced
neurotoxicity (kernicterus) in newborns correlates most closely with
the concentration of nonalbumin-bound bilirubin in the plasma
(10). The observation that HDL is the principal nonalbumin
carrier of UCB suggests that HDL-bound bilirubin may be an important
mediator of bilirubin toxicity, perhaps as a result of increased
availability for uptake into the central nervous system. This
hypothesis is supported by the higher levels of UCB in the brains of
analbuminemic rats infused with HDL-bound vs. albumin-bound bilirubin
(40). Although it has been reported that the risk of
kernicterus is increased in the setting of acidosis (37),
we found no effect of pH on the binding distribution of bilirubin in
human plasma.
Serum albumin concentrations in the early neonatal period are dependent
on gestational age, varying from a mean of 1.9 g/dl before 30 wk
gestation to 3.1 g/dl at term (9). One study revealed a
mean serum albumin level of 2.5 g/dl (range: 0.8-4.0 g/dl) for infants with gestational ages ranging from 26 to 42 wk (mean: 33 wk)
admitted to the neonatal intensive care unit (33).
Although serum levels of apo D also appear to correlate with
gestational age, variations are much less pronounced compared with
albumin, with a mean value of 3.7 ± 1.4 mg/dl at birth
(22). Compared with average adult values for serum albumin
(4.2 g/dl) and apo D (12 mg/dl), neonatal serum has a significantly
lower total bilirubin binding capacity. On the basis of the results of
our centrifugation studies, the reduced serum albumin levels in
newborns would be expected to result in higher levels of HDL-bound
bilirubin, potentially increasing the availability of bilirubin for
uptake into the brain. Support for this hypothesis is derived from the
work of Cooke and Roberts (14), who demonstrated the
presence of
-lipoprotein-bilirubin complexes in the serum of a
jaundiced neonate but not in normal controls.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the technical assistance of Drs. Alison
Hoppin, Xiaoyang Qi, and Gregory A. Grabowski in the performance of
fluorescence analyses. We also thank Dr. J. Donald Ostrow for helpful
advice regarding bilirubin purification and Drs. Richard M. Green, John
L. Gollan, and Martin C. Carey for their valuable comments and criticisms.
 |
FOOTNOTES |
Preliminary reports of this work have been published in abstract form
(Hoppin AG et al. Gastroenterology 108: A1086, 1995; Goessling W and Zucker SD, Hepatology 26: 385A, 1997).
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-51679 (S. D. Zucker), a
Charles H. Hood Foundation Child Health Research Award (S. D. Zucker), a postdoctoral award from the BASF-Foundation, Germany (W. Goessling), and the Alfried Krupp von Bohlen und Halbach-Stiftung, Germany (W. Goessling).
Address for reprint requests and other correspondence: W. Goessling, Dept. of Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail:
wgoessling{at}bics.bwh.harvard.edu).
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. §1734 solely to indicate this fact.
Received 9 June 1999; accepted in final form 18 March 2000.
 |
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