© 1998 American Heart Association, Inc.
Original Contributions |
Potencies of Lipoproteins in Fasting and Postprandial Plasma to Accept Additional Cholesterol Molecules Released From Cell Membranes
From the Atherosclerosis Research Unit, Department of Medicine (B.H.C., J.P.S., K.H.), the Department of Pediatrics (F.F.), and the Clinical Research Center (B.H.S.C.), University of Alabama at Birmingham; and the H.E. Moore Heart Research Foundation, University of Illinois, Champaign (B.E.D.).
Correspondence to Byung Hong Chung, PhD, Atherosclerosis Research Unit, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294. E-mail Chung{at}aru.dom.uab.edu
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Abstract |
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Abstract—To investigate the role of various lipoproteins in plasma to promote cholesterol efflux from cell membranes, potencies of lipoproteins in normolipidemic fasting and postprandial (PP) plasmas to accept additional cholesterol molecules from cell membranes were determined. We used red blood cells (RBCs) and lipoproteins in fresh blood as donors and acceptors of cell membrane cholesterol, respectively. When fresh fasting plasma (n=24) containing active lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer proteins (CETP) was incubated with a 3-fold excess of autologous RBCs at 37°C for 18 hours, plasma cholesterol levels increased by 19.6% (38.5±14.2 mg/dL) owing to an exclusive increase in the CE level. Very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions retained 48.1%, 26.3%, and 25.6% of the net cholesterol mass increase in fasting plasma, resulting in 91%, 8%, and 21% increases in their cholesterol contents, respectively. The PP plasma was 1.3-fold more potent than fasting plasma in promoting cholesterol efflux from RBCs by associating excess cholesterol with chylomicrons, resulting in a 356% increase in the cholesterol content of chylomicrons. These increases in lipoprotein cholesterol content indicate that chylomicrons were about 3.9x, 44x, and 17x more potent than fasting VLDL, LDL, and HDL, respectively, in accepting additional cholesterol molecules released from RBCs. The capacity of PP plasma to promote cholesterol efflux from RBCs was significantly correlated with plasma cholesterol levels (r=0.60, P<0.005), triglycerides (r=0.68, P<0.001), chylomicrons (r=0.90, P<0.001), VLDL (r=0.65, P<0.001), and LDL (r=0.47, P<0.025) but not with the levels of HDL (r=-0.34, P<0.20). In fasting plasma containing a low level of VLDL and HDL, isolated chylomicrons supplemented to the plasma were
9x more potent than HDL in boosting
the capacity of plasma to promote cholesterol efflux from
RBCs. This study indicates that chylomicrons in PP plasma are the most
potent ultimate acceptors of cholesterol released from cell
membranes and that a low HDL level is not a factor that limits the
ability of PP plasma to promote cholesterol efflux from
cell membranes. Our data obtained from an in vitro system suggest that
PP chylomicrons may play a major role in promoting reverse
cholesterol transport in vivo, since the transfer of
cholesterol from cell membranes to chylomicrons will lead
to the rapid removal of this cholesterol by the liver. HDL
in vivo may promote reverse cholesterol transport by
enhancing the rapid removal of chylomicrons from the circulation, since
the rate of clearance of chylomicrons is positively correlated with the
HDL level in plasma.
Key Words: chylomicrons • HDL • lecithin:cholesterol acyltransferase • cholesteryl ester transfer proteins • reverse cholesterol transport
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Introduction |
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Reverse cholesterol transport (RCT) in vivo occurs through the plasma compartment. Although high density lipoprotein (HDL) in plasma may play a major role in promoting RCT,1 2 the role of other lipoproteins in the process of RCT is not clear. Studies in cultured cells and tissues in vitro3 4 and isotopic studies in vivo5 indicate that unesterified cholesterol (UC) moves constantly between lipoproteins and cells and among different lipoprotein fractions in plasma. Thus, the concentration of UC in cell membranes and in various plasma lipoproteins in vivo may come to equilibrium. Glomset and Norum1 first demonstrated that the equilibrium of UC between cell membranes and lipoproteins can be disturbed by an enzyme, lecithin:cholesterol acyltransferase (LCAT), which can lower the levels of UC on the lipoprotein surface by converting it into cholesterol ester (CE) and subsequently trapping the CEs in the cores of lipoproteins. Thus, LCAT-mediated generation of a UC gradient between cell membranes and lipoproteins allows a net transfer of cholesterol from cell membranes into lipoproteins. A major feature of the role of HDL in RCT promotion is its ability to serve as a major substrate for LCAT.1 It was originally postulated that cellular UC accepted by lipoproteins and subsequently esterified by LCAT on HDL would be carried by HDL to the liver for excretion.1 However, in humans and animals having cholesteryl ester transfer protein (CETP) activity in plasma, a major portion of CE formed on HDL by LCAT may be transferred to apoB-containing lipoproteins.6 In rabbits,
70% of HDL CE was cleared from the plasma after its transfer into
VLDL and LDL.7 The plasmas from both humans and
rabbits have high CETP activities,8 9 but the
extent of clearance of LCAT-generated CEs through apoB-containing
lipoproteins in humans has not been quantified. LCAT and CETP
activities in human plasma have thus been envisaged as part of a
well-regulated sequence of reactions by which cellular
cholesterol can be converted into lipoprotein CE for its
transport to the liver.10 11 Although RCT in vivo
in humans will require not only HDL and LCAT but also apoB-containing
lipoproteins and CETP, RCT in vitro, however, has been commonly
evaluated in cultured cells by measuring the ability of HDL to release
cellular cholesterol without fully evaluating the effect of
the levels of various apoB-containing lipoproteins and the activities
of LCAT and CETP on the release of cellular
cholesterol. Earlier studies of interacting cultured cells with diluted human plasma containing active LCAT and CETP showed that the ability of plasma to promote cellular cholesterol efflux differed among plasmas obtained from normolipidemic and various hyperlipidemic subjects.11 12 The abnormality consistently associated with plasma's having a defective ability to promote net cholesterol transport from cultured cells was shown to be largely a result of an increased influx of cholesterol to the cells from plasma.11 A number of recent studies13 14 15 have shown that the ability to promote efflux of radiolabeled cholesterol on cultured hepatocytes by sera obtained from humans, normal mice, and transgenic mice and rats expressing human apoA-1 was best correlated with the HDL levels in sera. In human sera, the parameters associated with apoB containing lipoproteins were not correlated with its ability to promote cholesterol efflux from cultured cells.13 Castro and Fielding,16 however, reported that the presence of postprandial (PP) lipoproteins in human plasma increases its ability to promote net cholesterol transport from cultured cells by increasing the extent of LCAT and the rate of transfer of CE to PP VLDL and LDL. Because cultured cells often have a low level of cellular cholesterol, the low level of effluxable cholesterol in cultured cells may make it difficult to measure the potencies of plasma to promote cholesterol efflux.
The objective of this study was to determine the influence of the activities of LCAT and CETP and the levels of lipoproteins in fasting and PP plasmas on the capacity of plasma to promote cholesterol efflux from cell membranes and the potencies of each lipoprotein class in the plasma to accommodate additional cholesterol molecules released from cell membranes. To achieve this objective, we developed a simple experimental system that used red blood cells (RBCs) and lipoproteins in fresh whole blood as donors and acceptors of cell membrane cholesterol, respectively, and the lipoprotein cholesterol autoprofiler method, developed in our laboratory,17 18 as a tool to directly quantify the association of membrane-derived cholesterol mass among major lipoprotein fractions in fasting and PP plasma. RBCs are rich in cholesterol, and in vitro and in vivo studies have indicated that UC on RBC membranes is in equilibrium with UC on lipoproteins19 and perhaps also with that on arterial wall cells. The RBCs from animals on an atherogenic diet are enriched with UC relative to phospholipids,20 and RBCs from normolipidemic subjects have been shown to act as exceptionally potent acceptors of cholesterol from cholesterol-loaded, cultured macrophages.21 Although RBCs, unlike nucleated cells, do not synthesize or metabolize cholesterol, studies of cholesterol efflux from RBC membranes into plasma should provide pertinent information about the flux of cholesterol from arterial walls into plasma in vivo.
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Methods |
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Human Subjects and Fasting and PP Blood
Healthy adult men and women (25 to 51 years of age) consuming ad libitum diets were recruited to obtain both fasting and PP blood. Interested volunteers underwent a screening examination at the University of Alabama at Birmingham (UAB) General Clinical Research Center (GCRC). The examination included a medical history, measurement of body weight and height, a brief physical examination, and measurement of fasting plasma lipids. Subjects having dyslipoproteinemia or those who were on medication or had any chronic illness were excluded as study subjects. The experimental protocol was approved by the UAB Institutional Review Board. To obtain PP lipemic blood samples, subjects, who fasted overnight (12 hours), were given a meal rich in fat (polyunsaturated to saturated fat ratio, 2.49). The fatty meals consisted of 15% of calories from protein, 20% from carbohydrate, and 65% from fat and contained 600 mg cholesterol. The meals were calculated on the basis of 50 g of fat per square meter of body surface and were prepared in the research kitchen of the UAB GCRC. Samples of fasting blood (40 mL) and PP lipemic blood (80 mL) were obtained from the participants just before the meal and 4 to 5 hours after the meal.
Treatment of Blood and Plasma Samples
Blood samples were collected in tubes containing EDTA (0.1%)
and were placed in an ice bath immediately after collection. The blood
samples were spun at 1000 rpm for 10 minutes in a precooled (4°C),
low-speed centrifuge. After this centrifugation
step, about two thirds of the plasma in the centrifuge tube was
separated from RBCs, and one third of the plasma was trapped within the
packed RBCs. The upper plasma fraction was then transferred to another
chilled tube and then divided into 2 aliquots. An aliquot of plasma and
blood enriched with a 2-fold excess of RBCs was incubated for 18 hours
in a 37°C water bath (Forma Scientific Co), while the other aliquot
of plasma was kept in an ice bath (4°C). Because even slight
agitation of blood samples results in the hemolysis of RBCs, the blood
samples were placed in a 37°C water bath without agitation. In a
separate experiment, multiple aliquots of RBC-enriched blood were
incubated at 37°C, and 2 aliquots of RBC-enriched blood were then
withdrawn after 3, 6, 9, 12, 15, and 18 hours of incubation. Plasma was
separated from RBCs by centrifugation of blood samples
at 3000 rpm for 30 minutes and kept in an ice bath until
analysis. To determine the effect of adding an additional
acceptor of cholesterol in plasma on its ability to promote
cholesterol efflux, preisolated chylomicrons or HDL,
discoidal complexes of apoA-I and dimyristoylphosphatidylcholine
(DMPC), apo A-I, or DMPC liposomes, which were dialyzed against
isotonic buffered saline, were added to fasting blood before separation
of the plasma from RBCs. Blood samples containing the isotonic,
buffered saline were used as controls. After incubation, the
RBC-enriched blood samples were centrifuged at 3000 rpm for 30
minutes to separate the plasma from RBCs. The levels of
triglycerides (TGs), total cholesterol (TC),
and UC in unincubated control plasma and plasma incubated with or
without RBCs were measured by using enzymatic assay kits obtained from
Boehringer Mannheim (kit Nos. 236691 and 348292) and Merck Co
(kit No. 14106), respectively. Lipoprotein cholesterol and
TG profiles of control and incubated fasting and PP sera were examined
by the lipoprotein autoprofiler method developed in this
laboratory.17 The lipoprotein autoprofiler method
involves a short, single-spin (150 minutes) density gradient
ultracentrifugal separation of plasma lipoproteins in a swing-out rotor
(AH 650 Sorvall rotor) and continuous flow monitoring of
cholesterol or TG levels in the effluents collected from
the density gradient tubes after on-line mixing of the effluent with
enzymatic assay cholesterol or TG reagent. Levels of plasma
TC and distribution of cholesterol or TG among VLDL, LDL,
and HDL density fractions were calculated after deconvolution of
lipoprotein cholesterol profiles as described
previously.18 The levels of
cholesterol associated with chylomicrons were determined by
subtracting cholesterol levels in the VLDL density peak of
fasting plasma profiles from those of PP plasma profiles.
Capacities of Plasma to Promote LCAT, CETP, or RCT
Reaction
The extent of the LCAT reaction in plasma in the absence or
presence of RBCs was determined by measuring the increase in the plasma
CE levels after an 18-hour incubation at 37°C. Because we observed
that the CETP reaction in whole plasma resulted in a net increase of
cholesterol mass, mostly in the VLDL density fraction, with
a net loss of cholesterol mass from LDL and/or HDL, the
extent of the CETP reaction in plasma in the absence or presence of
RBCs was determined by measuring the increase in the levels of
cholesterol in the VLDL density fraction. To determine the
distribution of LCAT-derived CE and the redistribution of preexisting
CEs among lipoproteins after the plasma incubation, fasting plasma
containing a trace amount of 3H labeled UC was
incubated at 37°C for 18 hours in the absence of RBCs. A portion of
the radiolabeled plasma was kept in an ice bath as a control. At the
end of the 18-hour incubation, control and incubated plasma samples
were subjected to single-spin density gradient
ultracentrifugation to separate the major plasma
lipoprotein fractions.18 After quantitative
fractionation of VLDL, LDL, and HDL fractions in the density gradient
tubes, the levels of total radioactivity, TC mass, or both were then
determined. The ratio of 3H-labeled UC to
3H-labeled CE in each lipoprotein fraction was
then determined after extraction of the lipids and separation of UC and
CE by thin-layer chromatography with a mixture of
chloroform/hexane (3:1, vol/vol) as a developing solvent. The UC to CE
mass ratio was determined by measuring TC and UC levels in each
lipoprotein fraction by the enzymatic methods described previously. The
net increase in plasma TC mass after incubation of plasma with RBCs was
used as a measure of the plasma capacity for RCT.
Statistical Analysis
Quantitative variables were expressed as mean±SD. The
paired Student's t test was applied to compare the levels
of lipoproteins in fasting and PP plasma and in control, unincubated
plasma and plasma incubated at 37°C in the presence or absence of
RBCs.22 The SigmaPlot computer program (Jandel
Scientific) was used to obtain linear correlation coefficients between
the plasma capacity of RCT and the level of plasma
cholesterol, TG, or lipoprotein cholesterol or
the extent of LCAT or CETP reaction in plasma; regression lines for
different data sets; and values for testing the significance of the
null hypothesis.
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Results |
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Levels of TGs, TC, UC, and CE in Fasting and PP Plasma Kept in an Ice Bath or Incubated at 37°C in the Absence or Presence of Autologous RBCs
Table 1
shows the levels of TG, TC,
UC, and CE in fasting and PP plasma kept in an ice bath or incubated at
37°C overnight with or without autologous RBCs. The high-fat meal
resulted in a marked (147%) increase in plasma TGs in PP plasma, with
a minimal (2.3%) increase in plasma cholesterol.
Incubation of fasting and PP plasmas at 37°C for 18 hours in the
absence of RBCs resulted in the esterification of 47.9% (19.5 mg/dL)
and 52.0% (25.8 mg/dL), respectively, of UC in plasma by the activity
of endogenous LCAT. Thus, CE levels in fasting and PP
plasmas were increased by 13.3% and 18.9%, respectively (Table 1).
Inclusion of RBCs during incubation of fasting plasma resulted in a
19.6% (38.5 mg/dL) increase in plasma TC level by the efflux of
cholesterol on RBC membranes into plasma. The levels of
cholesterol released from RBCs into PP plasma (24.6% of
TC, or 49.4 mg/dL) were 1.3-fold higher than those released into
fasting plasma (Table 1). The UC levels in fasting and PP plasmas
decreased slightly after their incubation with RBCs; thus, the net
increase in plasma TC level was due exclusively to an increase in
levels of CE (Table 1). These data indicate that LCAT activity is
essential for the efflux of cholesterol from RBC membranes
into plasma. Czarnecka and Yokoyama23 recently
reported that in the absence of LCAT, the cholesterol
influx rate from lipoproteins into RBCs is equal to the efflux rate
from RBCs, resulting in no change in the levels of
cholesterol on lipoproteins and RBCs. The LCAT reaction was
shown to reduce the influx of cholesterol from lipoproteins
to RBCs and thus, induce the net cholesterol efflux from
RBCs through a nonspecific cholesterol-exchange mechanism.
We observed that inclusion of an inhibitor of LCAT, DTNB,
in plasma during its incubation with RBCs virtually inhibited the net
increases in plasma cholesterol levels (data not shown). It
should be noted that the levels of UC esterified in fasting and PP
plasmas by LCAT in the presence of RBCs were much (>2-fold) greater
than in the absence of RBCs, with a minimal decrease in plasma UC
levels (Table 1). These data indicate that provision of RBCs as an
exogenous source of UC in plasma can increase the extent of LCAT
reaction without a change in LCAT levels in plasma by replenishing the
plasma UC that was esterified by LCAT. About 56% of UC that is
esterified in plasma in the presence of RBCs was estimated to be
derived from RBCs (Table 1).
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Changes in Lipoprotein Cholesterol and TG Levels After
Incubation of Fasting and PP Plasma at 37°C in the Absence or
Presence of RBCs
Figure 1 shows
representative lipoprotein cholesterol
profiles of fasting and PP plasmas obtained from a normolipidemic
subject with a rapid chylomicron response to a fatty meal (profiles A
and D). The effect of incubating these fasting and PP plasmas in the
absence or presence of RBCs on the change in lipoprotein
cholesterol profiles is also shown in Figure 1 (profiles B,
C, E, and F). The mean levels of cholesterol in the VLDL,
LDL, and HDL fractions in control and incubated fasting and PP plasmas
from all study subjects (n=24) are summarized in Table 2. As Figure 1 and Tables 1
and 2 show, a
fatty meal resulted in a significant increase in
cholesterol levels in the VLDL density fraction
(P<0.05), without a significant increase in levels of
plasma TC. The levels of LDL and HDL cholesterol in PP
plasma were consistently lower than those in fasting plasma
(Figure 1 and Table 2), indicating that PP lipemia caused the transfer
of a small amount of cholesterol from LDL and HDL into PP
chylomicrons. The incubation of fasting plasma in the absence of RBCs
resulted in a substantial net increase of cholesterol mass
in the VLDL (+37.5%, or +7.8 mg/dL) and HDL (4.9%, or +2.4 mg/dL)
fractions. This change resulted from a net decrease in
cholesterol mass in the LDL fraction (-7.1%) due to the
activities of endogenous LCAT and CETP (Table 2). The LCAT-
or CETP-mediated increase in cholesterol levels in the VLDL
density fraction (+70.3%, or +20.6 mg/dL) and the decrease of
cholesterol levels in the LDL fraction (-17.0%, or -21.1
mg/dL) in PP plasma were >2.5x greater than those occurring in
fasting plasma (Table 2 and Figure 1).
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To further examine whether the increase in levels of
cholesterol mass in the VLDL density fraction after
incubation of plasma occurred a result of the transfer of LCAT-derived
CE or the transfer of preexisting LDL CEs into VLDL, fasting plasma
containing a trace amount of 3H-labeled UC was
incubated overnight, and the distribution of
3H-labeled CEs formed from
[3H]UC by LCAT and CE masses among various
lipoproteins in plasma was examined. This study showed that 46% to
49% of [3H]UC or UC mass in fasting plasma was
esterified after its incubation at 37°C (data not shown). VLDL, LDL,
and HDL fractions retained 24.4%, 48.7%, and 26.9%, respectively, of
total [3H]CE formed in plasma; however, most
(>80%) of the net CE mass increase in plasma was associated with the
VLDL fraction (Table 3). Although the LDL
fraction accepted 48.7% of LCAT-derived
3H-radiolabeled CE, the LDL CE mass in the
incubated plasma was less than that in unincubated, control plasma
(Table 3). These data indicate that the increased
cholesterol mass in the VLDL fraction was mainly due to the
CETP-mediated transfer of preexisting, unlabeled CEs from LDL or HDL
into VLDL, and the level of preformed CEs transferred from LDL to VLDL
or HDL is greater than the level of the new LCAT-derived CEs
incorporated into LDL.
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The presence of RBCs during incubation of fasting and PP plasmas
resulted in significant net increases of cholesterol mass
in all lipoprotein fractions (Figure 1 and Table 2). Because the net
increase of cholesterol mass in plasma or lipoproteins
after incubation of plasma with RBCs was due exclusively to an increase
in CE levels, a product of the LCAT reaction (Table 1), and because
HDL is a primary and perhaps exclusive substrate of LCAT in
plasma,24 the above data indicate that RBC
cholesterol after its efflux into plasma and subsequent
esterification on HDL by LCAT is transferred into the VLDL and LDL
density fractions by plasma CETP activity. When lipoprotein
cholesterol levels in unincubated fasting plasma (control)
were compared with those incubated with RBCs, the VLDL, LDL, and HDL
fractions retained 48.1%, 26.3%, and 25.6%, respectively, of the net
cholesterol mass increase in fasting plasma. Thus, the
cholesterol content of VLDL, LDL, and HDL fractions in
control fasting plasma increased by 91.3%, 8.2%, and 20.7%,
respectively, after incubation of fasting plasma with RBC (Table 2).
The above data indicate that the potency of VLDL in fasting plasma to
accept additional cholesterol molecules from RBCs is 11x
greater than that of LDL and 4x greater than that of HDL (Table 2).
In PP plasma, most (82.6%) of the RBC cholesterol released
into plasma was associated with the VLDL density fraction, containing
VLDL and chylomicrons (Table 2). When the levels of
cholesterol associated with chylomicrons in unincubated PP
plasma and PP plasma incubated with RBCs were determined by subtracting
cholesterol levels in the VLDL density fraction of fasting
plasma from those of PP plasma, the levels of chylomicron
cholesterol in control PP plasma and PP plasma incubated
with RBCs were calculated to be 8.5 and 30.3 mg/dL, respectively (Table 2). These data indicate that the cholesterol content of
chylomicrons in control plasma was increased by 356% after incubation
of PP plasma with RBCs. The change in cholesterol content
of lipoproteins in fasting and PP plasma after their incubation with
RBCs (Table 2) indicates that PP chylomicrons are 3.9x, 44x, and
17x more potent than are fasting VLDL, LDL, and HDL, respectively, in
accepting additional cholesterol molecules released from
RBCs.
Because the CETP reaction in plasma also mediates reverse transfer of
TGs from TG-rich lipoproteins (VLDL and chylomicrons) to CE-rich
lipoproteins (LDL and HDL),25 the lipoprotein TG
profiles of fasting and PP plasmas kept in an ice bath or incubated at
37°C for 18 hours in the absence or presence of RBCs were further
examined. As Figure 2 shows, TGs in fresh
fasting and PP plasmas were associated mostly with the VLDL density
fraction. Incubation of fasting and PP plasmas resulted in the transfer
of 45% of VLDL TGs in fasting plasma and 26% of VLDL and
chylomicron TGs in PP plasma into LDL and HDL fractions. The absolute
levels of TGs transferred from the VLDL into the LDL and HDL fractions
was 2x greater in PP plasma than in fasting plasma (98 versus 45
mg/dL) (Figure 2). More than 90% of TG removed from the VLDL density
fraction was associated with the LDL fraction (Figure 2). Although
CETP-mediated transfer of CE from LDL and HDL into the VLDL density
fraction in the presence of RBC is about 2x greater than that in the
absence of RBCs (Table 2), the extent of TG transfer from the VLDL into
LDL and HDL fractions was only minimally affected by the presence of
RBCs during incubation (Figure 2). These data support the findings of
other studies that have shown that the exchange of TGs and CEs between
TG-rich lipoproteins and CE-rich lipoproteins is not an equimolar
exchange.26 It should also be noted that the
levels of LDL and HDL TGs in fresh PP plasma are substantially (20% to
60%) higher than those in fasting plasma (Figure 2), indicating that
the CETP-mediated transfer of TGs from PP chylomicrons into LDL and HDL
may occur during PP lipemia in vivo. It should also be noted that the
level of cholesterol released from RBCs into PP plasma was
1.3-fold greater than that released into fasting plasma (Table 1), but
the net increase in cholesterol content of PP LDL (3.6%)
and HDL (7.7%) was much less than the increase in fasting LDL (8.2%)
and HDL (20.7%) (Table 2). These data indicate that the appearance of
chylomicrons in plasma lowers the ability of LDL and HDL to accept
additional cholesterol molecules released from cell
membranes, which is likely due to the greater CETP-mediated enrichment
of TGs in the cores of LDL and HDL in PP plasma than in fasting plasma
(Figure 2).
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In a further study, the time-course release of cholesterol
from RBCs into PP plasma, obtained from a subject with a normal
chylomicron response, and the distribution of RBC-derived
cholesterol among VLDL, LDL, and HDL density fractions were
examined (Figure 3). The efflux of
cholesterol from RBCs into PP plasma occurred continually
during an entire 18-hour incubation period, with the greatest rate of
efflux during the initial 3-hour incubation period (Figure 3). The rate
of cholesterol efflux after the 3-hour incubation period
was nearly steady up to 15 hours of incubation (Figure 3). Efflux then
started to plateau after 15 hours of incubation (Figure 3). The
increase in net plasma cholesterol level after the first 3
hours of incubation of RBC-enriched blood was associated with an
increase in cholesterol levels in all lipoprotein
fractions. VLDL, LDL, and HDL retained 50%, 33%, and 17% of RBC
cholesterol released into plasma, respectively (Figure 3).
After the 3-hour incubation period, the increase in plasma
cholesterol level, by the efflux of RBC
cholesterol into plasma, was associated with the increase
in cholesterol levels in the VLDL density fraction, with
little or no further change in the cholesterol levels in
the LDL and HDL fractions (Figure 3). These data indicate that LDL and
HDL fractions in PP plasma are more rapidly saturable than are TG-rich
VLDLs and chylomicrons with cholesterol released from RBC
membranes.
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We further examined the levels of RBC cholesterol released
into plasma after incubation of whole blood with or without RBC
enrichment. Owing to the danger of hemolysis, blood and RBC-enriched
blood samples remained stationary during their incubation at 37°C for
18 hours. Thus, a portion of plasma in whole blood was not directly in
contact with or exposed to RBCs during incubation owing to the settling
of RBCs to the bottom of the tubes; most plasma in RBC-enriched blood
should be exposed directly to RBCs during incubation, because all
plasma was trapped in packed RBCs. This study showed that the net
increase in plasma cholesterol (29 mg/dL) level after
incubation of whole blood was substantially less than that after
incubation of RBC-enriched blood (42 mg/dL). It should be noted that
the net increase in plasma cholesterol level after
incubation of whole blood was mostly associated with the increase in
cholesterol levels in the VLDL fraction, without fully
saturating LDL and HDL with RBC-derived cholesterol (Figure 4).
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Relationship Between the Capacity of PP Plasma to Promote
Cholesterol Efflux From RBC Membranes and Levels of Plasma
TC, TGs, or Lipoproteins or Plasma LCAT and CETP Activities
Because humans are predominantly in the PP lipemic state during
the day as a result of regular meals, the relationship between the
potency of PP plasma to promote cholesterol efflux from RBC
membranes and the levels of plasma cholesterol, TGs, and
lipoproteins was further determined. As the scatterplots of Figure 5 (top) show, the levels of
cholesterol released from RBCs into PP plasma were
correlated significantly with plasma TC levels (r=0.60,
P<0.005), TGs (r=0.68, P<0.001),
chylomicrons (r=0.90, P<0.001), VLDL
(r=0.65, P<0.001), and LDL
(r=0.50, P<0.025) but not with the level of
HDL (r=-0.32, P<0.20). The levels of
cholesterol released from RBCs into PP plasma were also
correlated significantly with the levels of CE formed by LCAT in
plasma, in either the absence or presence of RBCs (Figure 5, bottom,
upper panel). Because the increase of cholesterol mass in
plasma after its incubation with RBCs was exclusively due to an
increase in CE levels (Table 1), the levels of cholesterol
released from RBCs into plasma were correlated much more closely with
the levels of CEs formed in the presence of RBCs than those formed in
their absence (r=0.94, P<0.001 versus
r=0.42, P<0.05) (Figure 5, bottom, upper panel).
It should be noted that the levels of UC that were esterified in plasma
among individuals varied narrowly in the absence of RBCs but varied
widely in the presence of RBCs (Figure 5, bottom, upper panel). This
suggests that depletion of plasma UC may be a factor limiting the
extent of LCAT in the system without RBCs.
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Because the CETP reaction in plasma resulted in a net increase of
cholesterol mass primarily in the VLDL fraction by the
transfer of CEs from LDL or HDL (Table 2), the increase in VLDL
cholesterol mass after incubation of PP plasma at 37°C in
the absence or presence of RBCs was used as a measure of the extent of
plasma CETP activity. The amount of cholesterol transferred
from RBCs to PP plasma was correlated significantly with the extent of
the CETP reaction in either the absence or presence of RBCs (Figure 5, bottom, lower panel); however, the capacity of plasma to promote
cholesterol efflux from RBCs was correlated more closely
with CETP activity measured in the presence of RBCs than that measured
in their absence (Figure 5, bottom, lower panel).
Cholesterol Efflux Promoted by Fasting Plasma
Supplemented With HDL, Chylomicrons, Discoidal Complexes of ApoA-I and
DMPC, ApoA-I, or DMPC Liposomes
Since the capacity of PP plasma to promote cholesterol
efflux from RBCs was correlated most closely with chylomicron levels
and inversely related to HDL levels, the effect of supplementing fresh
fasting plasma with preisolated HDL and chylomicrons on the potencies
of plasma to promote cholesterol efflux was further
determined (Figure 6). Isolated HDL was
supplemented to fasting plasma sample containing a relatively low HDL
cholesterol level (34 mg/dL) to produce a 50% increase in
its HDL cholesterol level, whereas isolated chylomicrons
were supplemented to fasting plasma containing a relatively high HDL
cholesterol level (67 mg/dL) to result in a chylomicron TG
level of 200 mg/dL. Supplementation of isolated HDL to fasting
plasma containing a low HDL level, which is mildly
hypertriglyceridemic, had no enhancing
effect on the capacity of plasma to promote cholesterol
efflux from RBCs (Figure 6, top). The amount of cholesterol
transferred from RBCs to HDL-supplemented plasma was somewhat less than
that transferred to control plasma (46 mg/dL versus 44 mg/dL) (Figure 6, top). Supplementation of fasting plasma containing a low level of
VLDL with preisolated chylomicrons markedly increased the capacity of
plasma to promote the efflux of cholesterol from RBCs
(Figure 6, bottom). By bringing the chylomicron TG or
cholesterol level in fasting plasma to 200 mg/dL (TG) or 11
mg/dL (cholesterol), the capacity of fasting plasma to
efflux cholesterol from RBCs was increased 1.7-fold
(Figure 6, bottom).
|
In a further study, the capacity of fasting plasma containing an
abnormally low HDL level (hypoalphalipoproteinemic plasma) to efflux
cholesterol from RBCs was compared with that of plasma
containing a normal HDL level (Figure 7, top). As lipoprotein cholesterol profiles in Figure 7 (top)
show, the HDL level in hypoalphalipoproteinemic plasma (9 mg/dL) is
6x lower than that in control plasma (60 mg/dL) (profiles A and C),
but the level of cholesterol transferred from RBCs into
hypoalphalipoproteinemic plasma (47 mg/dL) was substantially greater
than that transferred into control plasma (38 mg/dL) (profiles A
through D). This suggests that HDL is not rate limiting, even at
relatively low levels. The major portion (77%) of
cholesterol transferred from RBCs to
hypoalphalipoproteinemic plasma was associated with apoB-containing
VLDLs and LDLs (Figure 7, top). Because the HDL as well as the VLDL or
TG level is low in hypoalphalipoproteinemic plasma, the effect of
supplementing it with preisolated HDL or chylomicrons on its capacity
to promote cholesterol efflux from RBCs was further
examined (Figure 7, bottom). Isolated HDL was supplemented to
hypoalphalipoproteinemic plasma to produce a 4-fold increase in its HDL
level, whereas chylomicrons were supplemented to result in a
chylomicron TG level of 100 mg/dL. This study showed that
supplementation of hypoalphalipoproteinemic plasma with either
preisolated HDL or chylomicrons can boost the capacity of plasma to
promote cholesterol efflux from RBCs (Figure 7, bottom),
although supplementation of HDL to
hypertriglyceridemic plasma had no such
enhancing effect (Figure 6, top). We observed that the ability of HDL
to increase the capacity of plasma to efflux cholesterol
from RBCs diminished as the levels of plasma TGs or TG-rich
lipoproteins (VLDL or chylomicrons) increased (data not shown),
suggesting that high TG levels in plasma may inhibit the ability of
HDLs to accommodate additional CE molecules derived from RBCs.
|
When levels of HDL and chylomicrons supplemented to
hypoalphalipoproteinemic plasma and the net increase in
cholesterol levels of HDL- and chylomicron-supplemented
plasma after its incubation with RBCs were determined (Figure 7, bottom), the levels of cholesterol transferred from RBCs to
the control hypoalphalipoproteinemic plasma increased by 12 mg/dL by
supplementation with 32 mg HDL cholesterol per deciliter of
plasma (profiles A through D) or by 17 mg/dL by supplementation with 5
mg chylomicron cholesterol per deciliter (Figure 7, bottom;
profiles A, B, E, and F). These data indicate that the
cholesterol efflux–boosting potency per chylomicron
particle was 9x greater than that of HDL.
Because cholesterol-free, reconstituted complexes of apoA-I
and phospholipids have been shown to be more effective than HDL in
promoting cholesterol efflux from cultured
cells,27 the effect of supplementing fresh,
fasting plasma with apoA-I, DMPC liposomes, or discoidal complexes of
apoA-I and DMPC on the capacity of plasma to promote
cholesterol efflux from RBCs was further examined (Figure 8). Supplementation of fasting plasma
with apoA-I/DMPC complexes or DMPC liposomes markedly increased the
capacity of plasma to promote cholesterol efflux from RBCs
by associating excess cholesterol with the HDL density
fraction (Figure 8, profiles E through H); however, apoA-I supplemented
to plasma had little or no effect in boosting the ability of plasma to
promote cholesterol efflux from RBCs (Figure 8; profiles C
and D). We observed that the extent of the LCAT reaction in plasma
supplemented with DMPC liposomes or apoA-I/DMPC complexes was much
higher than that in control plasma; thus, the net
cholesterol mass increase in plasma by supplementation with
these liposomes or complexes was exclusively due to an increase in
plasma CE levels (data not shown). We observed that control DMPC
liposomes were recoverable in the LDL region of the density gradient
tubes (data not shown), but the increase in potencies of DMPC
liposome–supplemented plasma to promote cholesterol efflux
from RBCs was mostly due an increased association of RBC-derived
cholesterol with HDL (Figure 8, profiles F and H). Because
HDL in plasma is known to disintegrate phospholipid liposomes,
resulting in the production of large, phospholipid-enriched
HDLs,28 the increase in potency of plasma to
promote cholesterol efflux from RBCs after its
supplementation with DMPC liposomes may occur through increases in the
phospholipid content of the HDL fraction. Fournier et
al15 29 have reported recently that a major
factor determining the capacity of human sera to promote
cholesterol efflux from cultured cells is HDL phospholipid
content and composition.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have shown in this study that fresh, fasting and PP plasmas in the presence of active LCAT and CETP are very effective in promoting efflux of a measurable amount of cholesterol from RBCs. The levels of cholesterol released from RBCs into plasma can be increased by supplementing it with chylomicrons, native or reconstituted HDL, or phospholipids (Figures 6
, 7, and 8), indicating
that the limiting factor for efflux of cholesterol from RBC
membranes into plasma is the capacity of plasma lipoproteins to
accommodate additional molecules of cholesterol and not the
depletion of cholesterol that can be released from RBC
membranes. Murphy30 has previously shown that
35% of RBC cholesterol can be removed by fresh plasma.
Because 1 mL of packed RBCs contains 1.3 mg
cholesterol31 and because incubation
of fresh plasma with a 3-fold excess of RBCs resulted in a 38.6
mg/dL increase in plasma cholesterol mass (Table 1), the
amount of RBC cholesterol effluxed into plasma under our
experimental conditions was estimated to be 10% of the TC on RBC
membranes.
Fielding and Moser32 have previously reported
that HDL contains the major cholesterol efflux–promoting
activity of plasma. However, much (>82%) of the RBC
cholesterol released into PP plasma was associated with
TG-rich VLDLs and chylomicrons after esterification (Table 2),
indicating that RBC membrane cholesterol, after its efflux
into plasma and subsequent esterification on HDL by LCAT, is
predominantly transferred to TG-rich VLDLs and chylomicrons by the
activity of CETP in plasma. Our in vitro data show that the number of
RBC-derived cholesterol molecules accepted by a particle of
VLDL or chylomicrons in PP plasma was 12x to 71x greater than that
accepted by a particle of LDL or HDL in PP plasma (Table 2). The higher
capacity of TG-rich VLDLs and chylomicrons to accept additional
cholesterol molecules derived from RBCs is likely due to
CETP-mediated bidirectional movement of TGs and CEs between TG-rich
chylomicrons and VLDLs and CE-rich LDLs and
HDLs.25 Lipoprotein TG profiles of fasting and PP
plasma incubated with or without RBCs revealed that the CETP reaction
in the presence of active LCAT resulted in the transfer of many TG
molecules from TG-rich lipoproteins into LDL and HDL, resulting in
significant enrichment of the LDL and HDL cores with TG (Figure 2). We
observed that the number of TG molecules transferred from TG-rich
lipoproteins to LDL or HDL during LCAT and CETP reactions in plasma is
always greater than the number of CE molecules transferred from LDL and
HDL fractions to TG-rich lipoproteins (data not shown). Because the
molecular volume of TG is 1.5x greater than that of
CE,33 the CETP reaction in plasma will cause
TG-rich VLDLs and chylomicrons to become poor in core lipid but will
cause LDL and HDL to become lipid-rich particles. Thus, CETP reactions
in plasma will likely enhance the ability of TG-rich lipoproteins to
accommodate additional CEs derived from cellular
cholesterol but will limit this ability of LDL and HDL.
Undisrupted RCT in vivo would require rapid hydrolysis of LDL and HDL
TGs transferred from TG-rich lipoproteins by hepatic lipase so that the
CETP-mediated transfer of LCAT-derived CEs from HDL to TG-rich
lipoproteins occurs continually. Hirano et al34
reported that the reduction of hepatic lipase activity in
hyperalphalipoproteinemic subjects was associated with increased
atherosclerotic disease, despite markedly higher levels of plasma HDL.
This may provide evidence that defective removal of HDL TGs, accepted
from TG-rich lipoproteins by CETP activity, could impair RCT in vivo
and thus increase the risk of developing
atherosclerosis.
Because the increase in net cholesterol mass that occurred
in plasma after RBC incubation was due exclusively to an increase in CE
levels (Table 1), the capacity of plasma to promote
cholesterol efflux from RBCs was closely correlated with
the extent of the LCAT reaction in plasma in the presence of RBCs
(Figure 5). The capacity of plasma to promote cholesterol
efflux from RBCs was correlated significantly with the levels of total
plasma TGs, cholesterol, chylomicrons, VLDL, and LDL but
not with the levels of HDL. This significant, positive correlation is
likely due to the fact that all lipoproteins in plasma can ultimately
accept CE molecules derived from RBCs. The stronger, positive
correlation between plasma capacity to promote cholesterol
efflux and levels of TG-rich lipoproteins than between plasma capacity
and LDL levels may be due to the higher capacity of TG-rich
lipoproteins to accommodate additional CE cholesterol
molecules than does LDL. The inverse relationship found between plasma
capacity to promote cholesterol efflux and HDL levels,
though not statistically significant, is likely due to low levels of
TG-rich lipoproteins in those plasmas containing high levels of
HDL.
The extent of LCAT and CETP reactions or the capacity of plasma to
promote cholesterol efflux from RBCs can be increased
significantly by supplementing plasma with preisolated chylomicrons
without changing LCAT and CETP levels (Figures 6 and 7). These data
suggest that the capacity of plasma to support LCAT and CETP reactions
or RCT in vivo may be influenced by the level of lipoproteins that can
accept many additional CE molecules formed by LCAT, such as
chylomicrons and VLDL. Plasma LCAT and CETP rates were reported to be
higher in hypertriglyceridemic subjects
than in normolipidemic subjects and were related positively with VLDL
levels or negatively with HDL
levels.13 35 36 37
Our in vitro data indicate that the cholesterol content of
chylomicrons can be increased by 356% by acceptance of CEs
derived from RBCs, but such accumulation of CEs in PP chylomicrons may
not occur in vivo, since PP chylomicrons are rapidly removed by the
liver before such accumulation can occur. However, because humans will
be mostly PP lipemic during the day owing to consumption of regular
meals, LCAT- and CETP-mediated transformation of UC on cell membranes
into chylomicron CEs may occur throughout the PP lipemic period in
vivo. A number of studies38 39 40 have shown PP
chylomicronemia to be accompanied by a significant net decrease in LDL
and HDL CE level as well as total plasma CE levels or a significant
shift in the distribution of CEs from LDL and HDL to PP chylomicrons.
The PP decreases in LDL and HDL CE levels were proportional to the
increases of plasma TGs in PP plasma or the amount of fat
ingested.40 Our current data also show that PP
lipemia causes a small, net decrease in plasma CE levels and of
cholesterol in LDL and HDL fractions and a significant net
increase of cholesterol in the VLDL fraction (Tables 1 and 2). These observations may provide evidence that CETP-mediated transfer
of CEs from LDL and HDL and possibly, of CEs derived from cell
membranes into PP chylomicrons, occurs in vivo.
It is well recognized that high levels of HDL in plasma protect
against the development of
atherosclerosis.41 Although the
antiatherogenic effect of HDL has been attributed to its ability to
mediate the efflux of excess cholesterol from
peripheral cells and its delivery to the
liver,1 whether high levels of HDL in plasma
promote the exit of cholesterol from cell membranes more
than do low levels in vivo is not currently clear. Osono et
al42 reported recently that the rate of
centripetal cholesterol flux from peripheral
organs to the liver in transgenic mice expressing CETP was independent
of HDL cholesterol concentration in plasma. Our in vitro
data indicate that the capacity of plasma to promote
cholesterol efflux from RBCs was not defective in plasma
containing an abnormally low level of HDL (Figure 7) and is not
correlated with plasma HDL levels (Figure 5); this suggests that a low
HDL level in plasma may not be a factor that limits the ability of
plasma to promote cholesterol efflux from the cell membrane
in vivo.
Because chylomicrons are the most potent, ultimate acceptors of
cholesterol transferred from cell membranes into plasma
(Table 2), delayed clearance of cholesterol-enriched
chylomicrons will probably lower the rate of RCT in vivo. Quarfordt et
al43 recently reported that enrichment of
chylomicrons with CEs enhances apoE-mediated uptake of chylomicrons by
the liver, suggesting that CETP-mediated enrichment of chylomicrons
with CEs in vivo may be an important physiological
process in regulating chylomicron removal. Because chylomicron
remnants, formed at the endothelial surface by
lipoprotein lipase, may be atherogenic,44 45
delayed clearance of cholesterol-rich chylomicrons could
enhance the development of atherosclerosis. Several
case-control studies have shown that the levels or residence times of
PP chylomicrons and their remnants were significantly higher in
patients with coronary heart disease than in normal
subjects.46 47 48 49 Because the rate of clearance of
PP chylomicrons is known to be directly correlated with HDL levels in
plasma,50 transport of cholesterol
derived from arterial walls to the liver through PP
chylomicrons will be faster in individuals with a high HDL level than
in those with a low HDL level. Thus, HDL levels in plasma can influence
the extent of RCT by influencing the clearance rate of
chylomicrons.
Our data show that the amounts of cholesterol
released from RBCs into PP plasma are highly correlated with the extent
of CETP reactions in plasma (Figure 2). High CETP activity has often
been considered to be a proatherogenic factor,51
but hypertriglyceridemic mice expressing a
CETP transgene were shown to be protected against
atherosclerosis.52 It is probable
that high CETP activity will be an antiatherogenic factor when
CE-enriched PP chylomicrons, which carry the product of CETP, are
removed rapidly by the liver, but will be proatherogenic when the
removal of CE-enriched chylomicrons by the liver is delayed. Thus, the
effect of CETP on atherogenesis may depend on the metabolic
context, as suggested by Zhong et al.53
In summary, the current study indicates that any lipoprotein in plasma can ultimately accept cholesterol derived from cell membranes through the activities of LCAT and CETP in vivo, which supports the concept that lipoproteins can act as a "sink" for cell cholesterol.54 Because chylomicrons, among all lipoproteins in PP plasma, are the most potent acceptors of cholesterol released from cell membranes and because chylomicrons appear periodically in circulating blood after each meal and are mostly cleared by the liver (with a clearance rate at least 70 to 580 times faster than that of endogenous lipoproteins VLDL, LDL, and HDL),55 the rapidly clearing chylomicrons should play an important role in promoting RCT in vivo.
|
Acknowledgments |
|---|
This study was supported by a grant from the National Livestock and Meat Board (B.H.C.), by US Public Health Service grant (NIH HL 50719) (B.H.C., J.P.S.), and by GCRC grant RR0032 (B.E.D.). The authors thank Drs G.M. Anantharamaiah and William Bradley for helpful discussions.
Received October 10, 1997; accepted February 18, 1998.
|
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M. Guerin, P. Egger, C. Soudant, W. Le Goff, A. van Tol, R. Dupuis, and M. J. Chapman Cholesteryl ester flux from HDL to VLDL-1 is preferentially enhanced in type IIB hyperlipidemia in the postprandial state J. Lipid Res., October 1, 2002; 43(10): 1652 - 1660. [Abstract] [Full Text] [PDF]
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A. E. Christian, H.-S. Byun, N. Zhong, M. Wanunu, T. Marti, A. Fürer, F. Diederich, R. Bittman, and G. H. Rothblat Comparison of the capacity of {beta}-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins J. Lipid Res., August 1, 1999; 40(8): 1475 - 1482. [Abstract] [Full Text]
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G. H. Rothblat, M. de la Llera-Moya, V. Atger, G. Kellner-Weibel, D. L. Williams, and M. C. Phillips Cell cholesterol efflux: integration of old and new observations provides new insights J. Lipid Res., May 1, 1999; 40(5): 781 - 796. [Abstract] [Full Text]
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