(Circulation. 1999;100:594-598.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
From the Center for Metabolism and Endocrinology, Department of Medicine, and the Center for Nutrition and Toxicology, Novum, Karolinska Institute at Huddinge University Hospital, Huddinge (M.E., B.A.); King Gustaf V Research Institute, Karolinska Hospital, Stockholm (L.A.C.); and the Department of Medicine, University of Helsinki (T.A.M.).
Correspondence to Dr Bo Angelin, CME M63, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail bo.angelin{at}medhs.ki.se
| Abstract |
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Methods and ResultsFour subjects with heterozygous familial
hypercholesterolemia were studied under
standardized conditions. The fecal excretion of bile acids and neutral
sterols was determined for 9 days before and 9 days after an
intravenous infusion of recombinant human proapo A-I (4 g
protein) liposome complexes. Plasma apoA-I and HDL
cholesterol levels increased transiently (mean peak
concentrations were 64% and 35% above baseline, respectively) during
the first 24 hours. Mean lipoprotein lipid and apolipoprotein levels
were not different during the 2 collecting periods, however. Serum
lathosterol, a precursor of cholesterol whose concentration
reflects the rate of cholesterol synthesis in vivo, was
also unchanged. The fecal excretion of cholesterol (neutral
sterols and bile acids) increased in all subjects (mean increase, +39%
and +30%, respectively), corresponding to the removal of
500 mg/d
excess cholesterol after infusion. Control infusions with
only liposomes in 2 of the patients did not influence lipoprotein
pattern or cholesterol excretion.
ConclusionsInfusion of proapoA-I liposomes in humans promotes net cholesterol excretion from the body, implying a stimulation of reverse cholesterol transport. This mechanism may prove useful in the treatment of atherosclerosis.
Key Words: apolipoproteins atherosclerosis cholesterol lipoproteins metabolism
| Introduction |
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ApoA-I is believed to play a major role in the process of reverse cholesterol transport, ie, the transfer of cholesterol from peripheral tissues to the liver.5 6 7 14 15 HDL particles, as well as apoA-I liposomes, can remove cholesterol from cultured cells, including cholesteryl esterloaded macrophage foam cells.15 16 After esterification by the enzyme lecithin-cholesterol acyltransferase, cholesterol can be delivered to steroidogenic tissues, such as the liver, adrenal glands, and gonads. This may occur either via exchange with triglyceride-rich lipoproteins or LDL and subsequent tissue uptake by lipoprotein receptors5 6 7 17 or directly via specific HDL "docking" sites that recognize apoA-I.18 19 20 21 The liver is of critical importance in maintaining reverse cholesterol transport, because it is the only organ from which substantial net excretion of cholesterol can occur, either directly or after conversion to bile acids.22 23 If transfer of cholesterol from peripheral tissues to the liver could be stimulated and a net increase in cholesterol excretion achieved, this might have important implications in the treatment of atherosclerosis. To evaluate whether such a stimulation of reverse cholesterol transport could be obtained in humans, we studied the effect of infusion of recombinant human proapoA-I24 on the fecal excretion of cholesterol and bile acids in hypercholesterolemic subjects.
| Methods |
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Experimental Procedure
The patients were studied before and after the
intravenous infusion of a large amount of
cholesterol-free, proapoA-I liposomes. All lipid-lowering
drugs were withdrawn
6 weeks before the patients entered the study,
which was performed at the metabolic ward on an outpatient
basis, with daily visits. The patients were given a standardized,
natural-type diet adjusted to keep their weight and
cholesterol and fat intake stable.25 26 This
diet contains
3.5 mg · kg-1 ·
d-1 of cholesterol; fat content is
35%. Nine days before infusion, fecal collections were started in
3-day portions, and the elimination of cholesterol from the
body was determined. Chromic oxide was given during the whole study
period to normalize for variations in fecal flow.27 At the
time of infusion, fecal collections were stopped, the patients were
given a capsule of red stain, and complete fecal collection for another
9 to 12 days was restarted when the stain appeared in the feces.
ProapoA-I Liposome Infusion
During the infusion, 200 mL of recombinant human proapoA-I
soybean phosphatidylcholine liposomes containing 4 g of the
proapolipoprotein was given intravenously over 20
minutes. ProapoA-I liposomes (UCB SA, Pharma Sector) were prepared from
recombinant human proapoA-I,28 which was associated with
phosphatidylcholine exactly as described previously.24 One
hour before infusion, 40 vials, each containing 100 mg recombinant
proapoA-I associated with 125 mg of soybean phosphatidylcholine, were
dissolved by the addition of 5 mL NaHCO3 (20
mmol/L; pH 8.0) per vial. The vials were gently shaken until all
particulate material was dissolved, after which the solutions from all
vials were mixed and filtered through a 0.22-µm filter. The loss of
liposome material is 2% to 5%; the characteristics of this
"synthetic HDL" have been described in detail.24 29
For the control experiments, an identical protocol was followed, but
proapoA-I was excluded from the liposome preparation.
Assays
Measurements of plasma lipoprotein lipids, apoA-I and B, and
serum levels of cholesterol precursors and plant sterols
were performed repeatedly during the whole
study.25 26 30 31 32 33 34 Fecal neutral sterols
(cholesterol, coprostanol, and coprostanone) and fecal bile
acids were measured by quantitative gas-liquid
chromatography.35 36 37 Complete blood
counts, liver and kidney function tests, fasting glucose and insulin,
albumin, electrolytes, and thyroid hormones were checked
repeatedly during the study and for 15 days thereafter. Sera obtained
immediately before infusion and 1, 2, 4, 8, and 15 days thereafter were
analyzed for antibodies against recombinant proapoA-I and total
Escherichia coli protein by ELISA
assays.24
Data Analysis
Means±SEM were calculated, and changes were evaluated by paired
t test or ANOVA. Logarithmic transformations were used when
appropriate.
| Results |
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Two of the patients (S.G. and L.M., who both had coronary
disease) had low basal plasma apoA-I levels. In response to the
proapoA-I liposome infusion, plasma apoA-I levels increased in all
patients (Figure 1A
). After 1 hour, total
apoA-I concentrations were between 39% and 84% (mean, 64%) above
initial values (P<0.005; paired t test).
Thereafter, plasma apoA-I levels fell gradually, but they still were
between 9% and 34% above initial values at 24 hours
(P<0.001).
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The clear increase in apoA-I was accompanied by a less marked elevation
of HDL cholesterol levels, as determined after
ultracentrifugation (Figure 1B
). Peak levels
(+8% to +80%; mean, +35%) were observed at 6 hours
(P<0.05) or 12 hours (P<0.02), and in all
patients except L.M., HDL cholesterol generally had
returned to baseline levels within 24 hours. Separation of lipoproteins
by fast protein liquid
chromatography31 (not shown)
confirmed the relatively modest changes in HDL cholesterol.
Protein analysis of these lipoprotein fractions by SDS-PAGE
demonstrated a moderately increased apoA-I in HDL within the first 12
hours (not shown).
Thus, the drastic expansion of the apoA-I pool was associated with
relatively moderate changes in lipoprotein lipid levels. As seen in the
Table
, there were no significant
differences in the mean levels of total, LDL, or HDL
cholesterol during the 2 periods when
cholesterol elimination was measured. However, when the
fecal excretion of bile acids and neutral sterols during 9 days after
the infusion was compared with the baseline measurements, remarkable
increases were observed in all 4 patients (Figure 2
). The mean excretion of bile acids
increased by 30% and that of neutral sterols by 39%, corresponding to
2.15 and 4.83 mg · kg-1 ·
d-1, respectively (Table
). The results
thus imply that during >1 week after the infusion, a mean of
500
mg/d excess cholesterol was being removed. The excretion
was not measured for more than 12 days in any individual, so we cannot
determine how prolonged this stimulation was. There was no change in
serum lathosterol levels in response to the infusion
(Table
).
|
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To test the possibility that the phospholipid complexes had an independent stimulatory effect on cholesterol excretion, we repeated the study in 2 of the patients, G.G. and L.M., with infusion of liposomes prepared without proapoA-I. In response to the intravenous administration of 5 g phosphatidylcholine, plasma HDL cholesterol levels increased slightly within 1 hour, from 19 to 27 mg/dL in L.M. and from 43 to 50 mg/dL in G.G. This level remained for 12 and 6 hours, respectively, after which lipoprotein levels were similar to baseline. There was no concomitant increase in plasma apoA-I concentration; instead, there was a tendency toward reduced levels (-14% and -10% after 1 and 6 hours, respectively). Treatment with pure liposomes did not affect the fecal excretion of cholesterol, either as neutral sterols or as bile acids. G.G., whose cholesterol excretion increased by 49% (+12.8 mg · kg-1 · d-1) when infused with proapoA-I liposomes, displayed only a 2% increase (+0.3 mg · kg-1 · d-1) after the administration of phosphatidylcholine. Similarly, L.M.'s cholesterol excretion increased 16% (+5.2 mg · kg-1 · d-1) after proapoA-I complexes and only 2% (+0.8 mg · kg-1 · d-1) after the control infusion. Safety monitoring did not reveal any abnormalities, and serum lathosterol levels were not affected (not shown).
| Discussion |
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75% to 100% of the calculated plasma apoA-I pool,
and the plasma levels reached therefore indicate distribution in the
extravascular space as well. In agreement with this contention, the
apparent volume of distribution of radiolabeled proapoA-I liposomes was
calculated to be larger than plasma volume when 4 patients were infused
with 1.6 g of an identical preparation of proapoA-I in a previous
study.24 The finding of a more sustained HDL
cholesterol increase after a lower proapoA-I load in that
study may be related to the primary selection of subjects with low
initial HDL cholesterol levels. Accordingly, patient L.M.
of our study, who had low initial HDL cholesterol,
displayed 40% higher levels for >1 week after the infusion (Figure 1BThe degree of stimulation of cholesterol excretion in response to proapoA-I infusion observed in our study was surprisingly large. Analyses from 3-day stool collections, as performed here, have been shown to give reliable information on fecal elimination of cholesterol.35 36 The daily variation in fecal steroid excretion was recently reported to be 7.3±1.5% from the highest and lowest values in 21 subjects.41 We did not study a parallel control group, but the virtually unchanged excretion of cholesterol before and after the infusion of pure liposomes in 2 of the subjects provides a strong argument against the possibility that the change observed in response to proapoA-I was due to technical errors. Obviously, only a limited number of male patients with heterozygous familial hypercholesterolemia were investigated, and generalization of these results therefore has to be done with great caution. Experiments in other groups of subjects, possibly including patients with biliary diversion,42 will be of importance to further confirm our present results.
Two major objections to our interpretation that the increase in cholesterol elimination reflects an enhanced reverse cholesterol transport should be discussed. First, because apoA-I liposomes may interact with liver cell membranes,16 17 18 they may actually extract cholesterol from the liver and instead create an increased demand for cholesterol in the hepatocyte, resulting in an enhanced biosynthesis of cholesterol,22 23 43 which could result in elevated net excretion of fecal steroids. Although we cannot completely exclude this possibility, we find it less likely because measurements of serum lathosterol concentrations, which provide a good indication of changes in hepatic and overall body cholesterol synthesis,35 44 45 did not show any differences between the 2 study periods. The remote possibility that the apoA-I infusion influenced the intestinal absorption of cholesterol cannot be completely excluded. However, it should be mentioned that serum campesterol, which reflects dietary cholesterol absorption,32 34 was not changed in any of the subjects (not shown).
A second major concern is whether the apolipoprotein moiety of the
proapoA-I liposomes is the active factor in promoting
cholesterol excretion or whether the soybean
phosphatidylcholine liposome component may actually promote reverse
cholesterol transport equally well. It is known that
phospholipid complexes without apolipoproteins can extract free
cholesterol from cellular membranes,16 17 and
they may also serve to deliver this cholesterol to
hepatocytes. However, in experiments with cholesteryl
esterloaded human macrophages incubated with liposomes
containing proapoA-I or only phosphatidylcholine, the former extracted
70% of the cholesteryl esters, whereas the latter caused a
cholesteryl ester egress of only 20%.29 To directly
address this question, we restudied 2 patients who were given liposomes
without proapoA-I. Only minor, if any, changes in
cholesterol excretion were observed. Thus, although some
changes in cholesterol transfer may well occur in response
to apolipoprotein-free liposomes, it is clear that apoA-I plays a major
role in promoting the effects observed in the present study.
In conclusion, a pronounced increase in body cholesterol
excretion was observed after the intravenous infusion of
recombinant proapoA-I liposome complexes. Because the dietary intake of
cholesterol was stable and there was no detectable
upregulation of body cholesterol synthesis, these results
strongly suggest the possibility of stimulating reverse
cholesterol transport in humans. Although such estimations
are obviously subject to considerable error, it is interesting to note
that
5 g of excess cholesterol appears to have been
removed after the administration of 4 g of proapoA-I in liposome
form. This may indicate that each apoA-I molecule is utilized several
times in cholesterol transport, lending support to the
importance of reutilization of HDL apolipoproteins in reverse
cholesterol transport.5 6 7 17 18 19 20 21 22 The recent
finding of an increased biliary output of cholesterol in
mice that overexpress the HDL receptor SR-BI in the
liver21 indicates that this pathway of
cholesterol delivery may be an important mechanism
explaining the coupling between HDL and cholesterol
excretion. Finally, it is of interest to relate the amount of excess
cholesterol removed in our patients to the total-body
stores of cholesterol, which have been estimated to be
100 g.46 Although we cannot identify the precise source
of the excess excreted cholesterol, it is tempting to
speculate that repeated treatments with proapoA-I liposomes may
actually reduce cholesterol in the arterial
wall to some extent. Animal experiments give some reason for optimism
for this view, but clinical trials will obviously be necessary to
evaluate the antiatherogenic potential of such therapy.
| Acknowledgments |
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Received January 11, 1999; revision received April 25, 1999; accepted May 14, 1999.
| References |
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