serum albumin acts as a shuttle to … manuscript reference # jlr/2012/031336 serum albumin acts as...
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Manuscript reference # JLR/2012/031336
SERUM ALBUMIN ACTS AS A SHUTTLE TO ENHANCE
CHOLESTEROL EFFLUX FROM CELLS
Sandhya Sankaranarayanan, Margarita de la Llera-Moya, Denise Drazul-Schrader,
Michael C. Phillips, Ginny Kellner-Weibel, George H. Rothblat
Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The
Children's Hospital of Philadelphia, Philadelphia, PA 19104
Running title: Albumin enhances cholesterol efflux
Contact address of the corresponding author:
George H. Rothblat, 3615 Civic Center Blvd, Abramson Research Center, 1102 C, Children's Hospital of Philadelphia Philadelphia, PA 19104 Email: [email protected]
Phone: 215 590 0585
Fax: 215 590 0583
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Abbreviations: ABCA1, ATP binding cassette transporter A1; ABCG1, ATP binding
cassette transporter G1; ACAT, acyl-CoA cholesterol acyltransferase; AcLDL,
acetylated LDL; apo A-I, apolipoprotein A-I; apo-B, apolipoprotein B; CAD, coronary
artery disease; DPBS, Dulbecco’s phosphate-buffered saline; MEM, Minimum Essential
Medium Eagle; Egg PC, egg L-α-phosphatidylcholine; FC, free cholesterol; GLC, gas
liquid chromatography; HSA, human serum albumin; BSA, bovine serum albumin;
LCAT, lecithin-cholesterol acyltransferase; LPDS, lipoprotein-depleted sera; NEM, n-
ethylmaleimide; RBC, red blood cells; RCT, reverse cholesterol transport; rHDL,
reconstituted HDL; SR-BI, scavenger receptor class B type I; SUV, small unilamellar
vesicle.
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Abstract
An important mechanism contributing to cell cholesterol efflux is aqueous transfer in
which cholesterol diffuses from cells into the aqueous phase and becomes incorporated
into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles
transferring cholesterol to cholesterol acceptors which act as cholesterol sinks. We
have examined whether particles in serum can enhance cholesterol efflux by acting as
shuttles. This task was accomplished by incubating radiolabeled J774 cells with
increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in
serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red
blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in
fractional efflux in excess of that predicted by the addition of the individual efflux values
of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated
with cells and LDL. When different components of LPDS were used as shuttles,
albumin produced synergistic efflux, while Apo A-I did not. A synergistic effect was also
obtained when RBC were used as the sink and albumin as shuttle. The previously
observed negative association of albumin with coronary artery disease might be linked
to reduced cholesterol shuttling which would occur when serum albumin levels are low.
Supplementary key words: cholesterol efflux, aqueous diffusion, shuttle, albumin,
coronary artery disease, LDL.
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Introduction
There are a number of mechanisms that have been shown to contribute to the
flux of cholesterol molecules between cells and extracellular particles. These include
pathways mediated by scavenger receptor class B type I (SR-BI), ATP binding cassette
transporter A1 (ABCA1), ATP binding cassette transporter G1 (ABCG1) and
unmediated aqueous transfer. All of these mechanisms have been extensively studied
and there are a number of reviews that discuss them in detail (1-3). The earliest
cholesterol flux mechanism that was identified was aqueous transfer in which there is a
desorption of free cholesterol (FC) molecules from a donor, such as a cell membrane,
into the aqueous phase followed by diffusion of the molecules and their subsequent
collision with and incorporation into an acceptor particle (such as a lipoprotein) (4). This
diffusional movement of FC can be enhanced by what has been described as a “shuttle
and sink” mechanism in which some compounds present in the aqueous phase can
serve as FC shuttles, enhancing the flux of cholesterol between donor and acceptor
particles (“sinks”) (5-6). Perhaps the best examples of shuttles are cyclodextrins which
act as catalysts that bind FC, greatly increasing the bidirectional flux of FC through the
aqueous phase (6). Although the studies using cyclodextrins clearly demonstrated the
feasibility of shuttles and sinks enhancing FC flux, there was no evidence that
synergistic efflux could be the result of the interaction of physiologically relevant
particles and that such a process was taking place in serum.
Little research has been done on the shuttle/sink mechanism since the initial
reports. However, a recent publication by Hoang, et al. (7) studying the flux of
cholesterol in human sera after infusion of reconstituted HDL (rHDL) yielded data that
were consistent with a shuttle/sink mechanism. The results of this study indicated that
although rHDL are good cholesterol acceptors, they have only a limited capacity to store
cholesterol. This property of rHDL would result in rHDL performing the role of a
cholesterol shuttle. In contrast, the data from this infusion study indicated that the
plasma reservoir with a large capacity to hold cholesterol are the apoB-containing LDL
and VLDL, thus playing the role of cholesterol sinks. In the present study we have
examined the possibility that a synergistic enhancement of cholesterol flux occurs
through the interaction of components that are present in serum. For these studies we
have employed J774 mouse macrophages, labeled with [3H]-cholesterol as cholesterol
donors, plasma components as shuttles, and LDL, small unilamellar vesicles (SUV) or
red blood cells (RBC) as cholesterol sinks. To investigate synergistic effects we
quantitated the FC efflux from cells incubated with a constant amount of LDL, or other
potential sinks, and increasing concentrations of lipoprotein-depleted sera (LPDS), or
components present in LPDS. Synergy was measured as the difference in fractional
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efflux in excess of that predicted by the addition of the individual efflux values of sink
and shuttle alone.
Materials and Methods
Materials
Tissue culture plasticware was purchased from Thermo Fisher (Rochester, NY) and
Corning (Corning, NY). Minimum essential medium Eagle (MEM), RPMI 1640, and
Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Mediatech Cellgro
(Manassas, VA). MEM buffered with 10 mM HEPES (pH 7.4) was prepared in the
laboratory using HEPES purchased from BioWhittaker (Walkersville, MD). Fetal bovine
serum (FBS), gentamicin, trypsin EDTA, acyl-CoA cholesterol acyltransferase (ACAT)
inhibitor (Sandoz 58-035), Cpt-cAMP, ovalbumin, and fatty acid-free human serum
albumin (HSA) were all purchased from Sigma-Aldrich (St. Louis, MO). Fatty acid-free
BSA was purchased from Millipore (Kankakee, IL) and egg L-α-phosphatidylcholine
(egg PC) was from Avanti Polar Lipids (Alabaster, AL). Red blood cells were purchased
from Innovative Research, Inc (Novi, MI) and ([1, 2-3H]-cholesterol was from Perkin
Elmer Analytical Sciences (Waltham, MA). All other reagents and organic solvents
were purchased from Fisher Scientific (Pittsburgh, PA).
Preparation of lipoproteins and lipoprotein depleted serum (LPDS)
Human LPDS, HDL, LDL, and VLDL were isolated by sequential ultracentrifugation from
serum obtained with approved consent from healthy, normolipemic individuals as
previously described (HDL, d = 1.06-1.21; LDL, d = 1.01-1.06; and VLDL, d = 0.95-1.01)
(8). To obtain acetylated low density lipoprotein (acLDL), LDL was modified using
acetic anhydride, as previously described (9). Apo A-I was purified from delipidated
HDL using ethanol/diethyl ether followed by anion-exchange chromatography on a Q-
Sepharose column. The apo A-I fractions were pooled, dialyzed against 5 mM
NH4HCO3, lyophilized, and stored at -20ºC. The apo A-I was resolubilized in 6 M
guanidine hydrochloride and dialyzed extensively against saline (0.15 M NaCl, pH 7.4)
prior to incubating with the cells. Prior to use, LPDS, HDL, LDL, and VLDL were all
extensively dialyzed against 0.15 M NaCl (pH 7.4) and sterilized by filtration using a
0.45 µm Millipore filter. Egg PC small unilamellar vesicles (SUV) were made as
previously described (10).
Cell culture J774 mouse macrophage stock cells were maintained in RPMI growth medium
supplemented with 10% FBS and gentamicin. For experiments, J774 cells were plated
in RPMI growth medium in 24-well plates at a density of 150,000 cells/well for 24 h.
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Cholesterol-normal cells were labeled for 24 h with 1 Ci/ml [3H]-cholesterol in medium
supplemented with 5% FBS and 2 g/ml ACAT inhibitor. In experiments using
cholesterol-enriched cells, J774 cells were incubated with the same labeling medium
plus 25 g/ml acLDL. After labeling plus or minus cholesterol enrichment, the cells
were washed 2x with MEM HEPES and equilibrated with RPMI medium containing
0.2% BSA and cAMP (0.3 mmol/L) for 18 h. After this equilibration period, the cells
were washed 2x with MEM-HEPES buffer and incubated with MEM-HEPES media
containing exogenous acceptors alone or in combination (shuttle and sink) for 4 h, or as
indicated in the figures, to measure the labeled FC efflux. Aliquots of media were
collected to measure the release of radiolabeled cholesterol. J774 cell monolayers at
time zero (t0) were washed 2x with DPBS and cholesterol was extracted using
isopropanol. The % efflux is calculated based on the [3H]-cholesterol in the cells at t0.
The ACAT inhibitor was used in all phases of experiments (from labeling to efflux
phase) to ensure pools of radiolabeled CE would not be present since this could
complicate the determination of the fractional release of labeled cholesterol from the
cells.
Protein and cholesterol mass determination
At the end of the efflux experiment, the cell monolayers were washed with DPBS and
cell lipid was extracted using 0.5 ml of 2-propanol containing 5 µg/ml of cholesteryl
methyl ether (CME; Sigma, St. Louis, MO) as an internal standard for gas liquid
chromatography (GLC) analysis. A fraction of the extracted lipid was used to measure
total cholesterol radioactivity incorporated into cellular lipids using scintillation counting.
The remaining lipid was prepared for GLC to measure cholesterol mass as previously
described (11). The extent of loss of cell FC mass was calculated based on the
cholesterol content of the cells at t0, prior to the efflux phase. Cell protein was
measured by the method described by Markwell et al. (12). Cell proteins were
measured before the incubation period (t0) and after the incubation period (samples)
and there were no statistically significant changes in cell growth between the end of
equilibration period and at the end of efflux period.
Statistical analysis
All statistical analyses were performed using Prism (4.0) software, GraphPad Inc. (San
Diego, CA). Data were presented as mean + SD. Statistical significance was
determined by unpaired t test unless otherwise indicated. Significance was assessed at
P ≤ 0.05.
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Results and Discussion
The Shuttle and Sink Mechanism
The earliest mechanism demonstrated to play a role in the movement of FC
molecules between cells and lipoproteins was termed aqueous diffusion. In this
proposed mechanism molecules of FC desorb from the cell membrane and are then
either incorporated into a phospholipid-containing acceptor, such as a lipoprotein, or re-
incorporated into the plasma membrane (4, 10, 13). This physicochemical mechanism
is ubiquitous and contributes to the exchange of FC between cells and lipoproteins as
well as the exchange of FC between lipoproteins (14). Because of the limited solubility
of FC in an aqueous environment the FC flux between cells and acceptors by aqueous
transfer is relatively inefficient, although a number of recent studies have demonstrated
that aqueous transfer does contribute significantly to total cell FC efflux (2, 15). The
fractional contribution of aqueous transfer to total efflux is determined by the level of
other competing flux pathways present in the cell. The inefficiency associated with
aqueous transfer could be enhanced if a cholesterol carrier were present that had the
ability to loosely associate with FC and speed the flux between cholesterol donor and
acceptor. A number of years ago this concept led us to propose a model termed
“shuttle and sink” in which a cell would act as a FC donor, and a lipoprotein or
phospholipid vesicle would serve as a FC acceptor (i.e. “Sink”) (6, 16). Since these
prior studies used non-physiological compounds as shuttles and sinks (cyclodextrins
and phospholipid vesicles) we initiated the present study to determine if there are
compounds in serum that acted as shuttles, increasing the movement of FC between
cells and lipoproteins which serve as sinks.
Shuttling by LPDS
The experimental design that allowed us to identify and quantitate the
contribution of shuttles to efflux employed human LDL as a sink and human lipoprotein-
deficient serum (LPDS) as a source of potential shuttles. The fractional efflux of FC
from [3H]-cholesterol labeled J774 cells produced by each of these two components,
alone and in combination, was determined. Any efflux obtained from the combination of
LDL and LPDS that was greater than the additive values of the LDL and LPDS alone
resulted from synergy, attributable to the presence of a shuttle/s in the LPDS (see Fig.
1). This synergy was dependent on the LPDS dose (Fig. 2) and can be expressed
either as the actual fractional efflux of the shuttle component (Fig.2, Panel A) or as the
percent of the total efflux (Fig.2, Panel B). Even though the absolute synergistic efflux
values are relatively small (1-3.5% depending on LPDS concentration), the contribution
of this efflux to the total efflux is much larger (1-25% depending on LPDS concentration,
Fig.2, Panel B). In related experiments the LDL was replaced with human VLDL which
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also served as a cholesterol sink, although the level of synergy was less than that
obtained with LDL.
A series of experiments was conducted in an effort to identify the compound/s in
serum that contributed to the synergy illustrated in Fig. 1 and 2. A likely candidate was
HDL, and when added as a potential shuttle at concentrations ranging from 2.5 to 50 µg
/ ml low levels of shuttling was produced at concentrations between 2.5 and 15 µg /ml,
but no significant shuttling was evident at higher levels of HDL (Fig. 3). The presence of
shuttling at low HDL concentrations and the loss of this effect as HDL was increased
may be attributed to the high cholesterol efflux efficiency of HDL. Thus, as HDL
concentration is increased, and total efflux is elevated, the relative contribution of
shuttling to total efflux becomes progressively less.
Since apolipoproteins could potentially serve as shuttles we replaced the LPDS
with increasing concentrations of human apo A-I ranging from 2 to 10 µg / ml.
Interestingly, rather than a synergistic response, we observed a consistent reduction in
total efflux so that the efflux obtained in a system with apo A-I and LDL was less than
the additive values of apo A-I and LDL alone (average 36% reduction, range 26-42%,
data not shown). The reason for this less than additive value is not clear, but it may be
related to a possible interaction of apo A-I with either the LDL sink or the donor cells
since these J774 cells expressed ABCA1. A somewhat similar phenomenon was
reported by Zhao and Marcel (17) in which cholesterol efflux from fibroblasts to media
containing both serum albumin and Lp2A-I HDL was less than the sum of the efflux
induced by each component alone. Since it has been demonstrated that the activity of
LCAT can influence the flux of FC between cells and lipoproteins by shifting the
FC/phospholipid ratio of acceptor lipoproteins and thus shifting the FC equilibrium
between cells and lipoproteins (18-22), we treated the LPDS with n-ethylmaleimide
(NEM) at a concentration that inhibited 95% of the LCAT activity present in LPDS.
When added to the cell system at increasing concentration the LPDS shuttling capacity
was not changed by LCAT inhibition (data not shown).
Shuttling by Albumin
Another component of LPDS that could be responsible for FC shuttling is
albumin. A number of studies have demonstrated that some FC can associate with
albumin (17, 23), and albumin can serve as a cholesterol acceptor, although the
fractional efflux produced by the incorporation of albumin in the media is less than that
obtained with HDL or LPDS (17, 24-25). It has been demonstrated that albumin
mediates the bidirectional flux of cellular FC and functions as a low affinity, high
capacity cholesterol transporter (17). These properties of albumin are similar to those of
cyclodextrins which have been shown to be efficient shuttles moving FC between
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donors and acceptors. Data obtained with albumin demonstrate synergy when
expressed as % synergy (Fig. 4, Panel A) or % of total efflux (Fig. 4, Panel B), similar to
that obtained with LPDS (compare Fig. 2 to Fig. 4). There was no difference in synergy
when BSA was compared to human serum albumin (HSA) (data not shown); however,
replacement of BSA with ovalbumin demonstrated the specificity of serum albumin
since no synergy was produced by ovalbumin (data not shown). This result could be
anticipated since Zhao and Marcel (17) previously demonstrated that ovalbumin could
not stimulate the efflux of FC from fibroblasts when compared to HSA.
Even though there are no specific binding sites for FC on albumin (26), and the
association of FC with purified albumin is limited, removal of albumin from plasma by
immunoaffinity chromatography reduces total efflux by as much as 40% (23). Thus
purified albumin added to the culture medium alone is a relatively poor promoter of
efflux, whereas the presence of lipoproteins with albumin, as is the case with whole
plasma, enhances efflux. In addition to studies in which cell cholesterol efflux to
albumin was directly measured, we also conducted an experiment to determine the
change in fractional efflux upon removal of albumin from LPDS. The removal of
albumin resulted in a 22% reduction in total efflux and a 91% reduction in the efflux
attributed to synergy (Details are given as supplemental data).
LDL served as the cholesterol sink in the experiment, presented in Figs 1-4.
Since LDL contains FC, the shuttling capacity of albumin contributes to both the influx of
cholesterol into cells as well as the efflux of FC, thereby altering FC exchange. Under
these conditions movement of isotopic cholesterol out of the donor cells would be
generally matched by the influx of unlabeled cholesterol into the cells (27-29). This
bidirectional flux results in the appearance of [3H]-cholesterol in the media, but no
change in cell cholesterol mass. This was the case since a number of attempts to
detect changes in the cholesterol content of donor J774 cells incubated with albumin
and LDL were unsuccessful. To measure the ability of albumin, acting as a shuttle, to
influence cell cholesterol mass it was necessary to provide a cholesterol sink that
lacked cholesterol, thus eliminating the influx arm of the bidirectional flux system. In the
present study LDL was replaced with SUV which served as a cholesterol-free sink. In
such a system reductions in cell FC mass could be measured after a short incubation
time (6 h), as shown in Table 1. Using both FC normal and enriched cells there was a
significant loss of cell cholesterol mass with the combination of albumin as a shuttle and
SUV as a sink, illustrating that the shuttling capacity of albumin can contribute to
movements of cholesterol mass, if sufficient FC gradients are established. Under the
experimental conditions used in this experiment significant reductions in donor cell
cholesterol mass were not observed with SUV or albumin alone (Table 1).
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The data collected using either LDL or SUV as sinks are consistent with the role
of albumin acting as a shuttle and enhancing the aqueous diffusion of FC, and not
influencing flux via a receptor-mediated process. To assess the capacity of albumin to
function as a shuttle, we conducted an experiment in which radiolabeled J774 cells
were incubated with HSA or LPDS and the conditioned media collected was used to
measure influx to fresh cells (Experiment details are given as supplemental data).
Cholesterol influx from either LPDS or HSA was efficient and the rates over the time-
course of this experiment were 2.5% [3H]-cholesterol per h for LPDS and 1.8% per h for
HSA (see supplemental figure 1). These results indicate that, in the absence of other
proteins, albumin promotes cholesterol movement into cells and is responsible for a
substantial portion of the influx promoted by LPDS.
To document that the flux of FC between cells could be increased by albumin we
used RBC as a cholesterol sink and determined if the presence of albumin resulted in a
greater fractional efflux of FC from J774 cells as donors and RBC as acceptor. As
shown in Table 2 the fractional efflux of cholesterol from J774 was greater with the
combination of albumin plus RBC than RBC alone, consistent with a model in which
albumin affects the FC movement by binding cholesterol and increasing the
concentration of the sterol in the aqueous phase, and further illustrates that albumin can
enhance movement of FC, not only between cells and lipoproteins, but also between
cells.
Physiological Significance
Although albumin does not have specific binding sites for cholesterol (26) it can
solubilize small amounts of sterol; however, because of the high concentrations of
albumin in plasma the contribution of this protein to efflux from cells is considerable.
This was demonstrated in an early study by Fielding and Moser (23) who showed that
the removal of albumin from plasma by immunoaffinity resulted in a reduction of
approximately 40% in cholesterol efflux from fibroblasts. A detailed study by Zhao and
Marcel (17) indicated that albumin could participate in the bidirectional flux of
cholesterol between fibroblasts and lipoproteins, although this ability to stimulate efflux
is less than that obtained with HDL or reconstituted HDL (rHDL) particles. The ability of
albumin to solubilize FC would allow it to function as a shuttle, similar to that shown to
operate when cyclodextrins increase the flux of cholesterol between donor cells and
acceptors such as lipoproteins particles (30). Although there are a number of pathways
that can participate in cell FC flux, albumin acting as a shuttle exerts its effect by
enhancing aqueous diffusion, as illustrated by the synergy observed when SUV or RBC
served as FC acceptors. Since aqueous diffusion of FC is ubiquitous and can play an
important role in cell cholesterol flux (15), the enhancement of aqueous diffusion by
albumin could have a significant impact on cholesterol turnover in vivo. This might not
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be a major factor at normal serum albumin levels (4.0-5.6 g/dl) where the synergistic
potential of albumin might be saturated and variations within this normal range will not
influence the contribution of albumin to synergistic efflux. However, in hypoalbuminemic
individuals with albumin levels <3.5 gm/dl, changes in serum albumin levels may reduce
synergistic cholesterol efflux. There have been numerous reports suggesting that lower
serum albumin concentrations in humans may be associated with increased risk of
coronary artery disease (CAD) (31-32), and a number of hypotheses have been
proposed to explain this association including: arterial inflammation, antioxidant effects,
control of blood pressure and association with HDL concentrations (33). It is possible
that the association of albumin with CAD is a reflection of the ability of albumin to shuttle
FC between a variety of acceptors. Such shuttling would increase the movement of FC
down its concentration gradient between the numerous cholesterol pools present in both
plasma and tissues, thereby facilitating the restoration of steady-state levels as
cholesterol is metabolized. A particularly important cholesterol pool that could be
influenced by the shuttling capability of albumin is RBC cholesterol. The cholesterol
concentration of RBC is roughly equivalent to that found in plasma lipoproteins (34) and
it has been recently shown that RBC plays a role in reverse cholesterol transport (34).
FC in the membrane of RBC rapidly exchanges with other pools of cholesterol and this
transport is via an aqueous diffusion mechanism (35-37). Thus, both the flux of
cholesterol between plasma lipoproteins and RBC, as well as the delivery of RBC
cholesterol to hepatocytes (34) is enhanced by the ability of albumin to serve as a
cholesterol shuttle.
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Acknowledgment
We thank Margaret L. Nickel for her excellent technical expertise. The studies
described in this paper were supported by NIH grant HL22633 (SS, GR, MCP, GW,
MM).
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Figure legends
Figure 1. Cholesterol efflux from J774 cells when LPDS is used as a shuttle and
LDL as a sink
Radiolabeled J774 cells were incubated with increasing concentrations of LPDS alone
or in combination with 25 µg/ml LDL. Cholesterol efflux was measured after 4h.
Synergy, expressed as % efflux / 4 h was calculated by subtracting the individual efflux
values obtained by incubating the cells with LPDS or LDL alone from that of the total
efflux obtained from the combination of LPDS and LDL. The synergy obtained was
LPDS concentration dependent. % LDL efflux, open bar; % LPDS efflux, hatched bar;
% synergy efflux, solid bar. Data are presented as mean ± SD (n=9).
Figure 2, Panel A and B. Synergy obtained from J774 cells when LPDS is used as
a shuttle and LDL as a sink
Radiolabeled J774 cells were incubated with increasing concentrations of LPDS alone
or in combination with 25 µg/ml LDL and cholesterol efflux was measured after 4h.
Synergy was calculated as the actual fractional efflux (difference between efflux values
from the combination of LPDS+LDL and the additive value of LPDS and LDL added
alone). Data are presented as the % efflux that is attributed to synergy (squares, Panel
A) or the % contribution of synergy to total efflux (circles, Panel B). Both methods for
expressing synergy were LPDS concentration dependent. Data are presented as mean
± SD (n=9). The error bars are contained within the symbols.
Figure 3. Cholesterol efflux from J774 cells when HDL is used as a shuttle and
LDL as a sink
Cholesterol efflux from radiolabeled J774 cells was measured after incubating them with
different concentrations of HDL (2.5-50 µg/ml) alone or in combination with 25 µg/ml
LDL for 4 h. Synergy, expressed as % efflux was calculated by subtracting the
individual efflux values obtained by incubating the cells with HDL or LDL alone from that
of the combination of HDL and LDL. Data are presented as mean ± SD (n=3). % LDL
efflux, open bar; % HDL efflux, hatched bar; % synergy efflux, solid bar. Synergy was
obtained at the lower concentration of HDL (2.5-15 µg/ml), but was lacking when HDL
concentration was high.
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Figure 4. Synergy obtained from J774 cells when BSA is used as a shuttle and
LDL as a sink
Radiolabeled J774 cells were incubated with different concentrations of BSA alone or in
combination with 25 µg/ml LDL for 4 h to measure cholesterol efflux. Synergy was
calculated as the actual fractional efflux (absolute difference between efflux values
when cells were incubated with BSA+LDL and when BSA and LDL were added
separately). Data are expressed as either the % efflux that was synergistic (squares,
Panel A) or as the percent of the total efflux that can be attributed to synergy (circles,
Panel B). Synergy expressed by either method was BSA concentration dependent.
Data are mean ± SD (n=6). The error bars are contained within the symbols.
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Table 1. Effect of albumin on the reduction of cholesterol mass from J774 cells when incubated with SUV
Media
supplements
Normal cells Cells enriched with AcLDL
(25 µg/ml)
µg cholesterol/mg protein µg cholesterol/mg protein
SUV (200 µg/ml)
14.7 ± 0.6
53.9 ± 0.2
BSA (1 mg/ml) 15.1 ± 0.4 50.6 ± 3.7
BSA+SUV 12.8 ± 0.3* 40.8 ± 3.8*
J774 cells were incubated for 6 h with media supplemented with either, 200 µg/ml of
SUV, 1.0 mg/ml BSA or a combination of BSA plus SUV. Total cholesterol from cell
monolayers was measured using GLC and is expressed as µg cholesterol/mg protein.
*Significantly different (p ≤ 0.05, n=3) compared to their corresponding t0 value (t0 for
normal cells = 15.6 ± 0.6 µg cholesterol/mg protein; t0 for cells enriched with 25 µg/ml of
AcLDL = 54.1 ± 2.6 µg cholesterol/mg protein).
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Table 2. Effect of albumin on exchange of cholesterol between J774
cells and RBC
Media supplements % [3H]-cholesterol efflux / 2 h
RBC alone 1.3 ± 0.8
BSA alone 0.2 ± 0.2
BSA + RBC* 2.5 ± 0.4
Radiolabeled J774 cells were incubated for 2 h with media supplemented with either 5 x
106 RBC, 1.5 mg /ml BSA or a combination of BSA plus RBC. % efflux is based on the
[3H]-cholesterol in the J774 cells at time 0(t0). *Statistically different than BSA or RBC
alone (p ≤ 0.05, n=6).
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