Stimulation of Fecal Steroid Excretion After Infusion of
Recombinant Proapolipoprotein A-I
Potential Reverse Cholesterol Transport in Humans
Mats Eriksson, MD, PhD; Lars A. Carlson, MD, PhD;
Tatu A. Miettinen, MD, PhD; Bo Angelin, MD, PhD
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Background—Apolipoprotein (apo) A-I is the major protein component of HDL, a cholesterol transport particle that
protects against atherosclerosis. Apo A-I is believed to promote reverse cholesterol transport, transferring cholesterol
from peripheral cells to the liver for subsequent elimination. To test this hypothesis in humans, we measured fecal
steroid excretion before and after the intravenous infusion of human proapo A-I (precursor of apo A-I)
liposome complexes.
Methods and Results—Four 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, 139% and 130%, 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.
Conclusions—Infusion 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.
(Circulation. 1999;100:594-598.)
Key Words: apolipoproteins n atherosclerosis n cholesterol n lipoproteins n metabolism
T
he risk of developing coronary heart disease is directly
related to plasma concentrations of total and LDL cholesterol and inversely related to HDL cholesterol levels.1,2
Although the mechanisms for regulation of plasma LDL
cholesterol and its role in the process of atherogenesis are
relatively well understood,3,4 it is still unclear how HDL may
exert its protective actions.5–7 HDL comprises a mixture of
particles that differ in size and composition and undergo
continuous metabolic changes in the circulation.5 Nascent
HDL is formed in the liver and intestine, where its major
apolipoprotein, apoA-I, is secreted as a propeptide
(proapoA-I) that is rapidly cleaved to mature apoA-I.1,7 The
findings that infusion of HDL particles or apoA-I liposomes,
as well as overexpression of the human gene for apoA-I,
inhibits the disease process in animal models of atherosclerosis8 –13 support the concept of a important role for apoA-I in
the prevention of atherogenesis.
See p 576
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.5–7,14,15 HDL particles, as
well as apoA-I liposomes, can remove cholesterol from
cultured cells, including cholesteryl ester–loaded macrophage
foam cells.15,16 After esterification by the enzyme lecithincholesterol 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 receptors5–7,17 or directly via specific HDL “docking” sites that recognize apoA-I.18 –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
Received January 11, 1999; revision received April 25, 1999; accepted May 14, 1999.
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 [email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
594
Eriksson et al
Proapo A-I Infusion and Cholesterol Excretion
595
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
Patients
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Four male patients (S.G., G.G., L.M., and I.N.) were studied. They
were 43, 61, 43, and 42 years old and weighed 87, 85, 97, and 80 kg,
respectively. All had phenotypical heterozygous familial hypercholesterolemia,3 and the first 2 were brothers carrying the FH-Helsinki3
mutation. Patients S.G. and L.M. had coronary heart disease; all
patients were in stable clinical condition on unchanged therapy
(b-blockers in S.G., G.G., L.M.) during the study. The study was
approved by the Ethics Committee of the Karolinska Institute, and
informed consent was obtained from all subjects.
Experimental Procedure
The patients were studied before and after the intravenous infusion of
a large amount of cholesterol-free, proapoA-I liposomes. All lipidlowering 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 z
kg21 z d21 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-mm 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 –34 Fecal neutral sterols
(cholesterol, coprostanol, and coprostanone) and fecal bile acids
were measured by quantitative gas-liquid chromatography.35–37
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
Figure 1. Plasma levels of apoA-I (A) and HDL cholesterol (B) in
response to infusion of 4 g proapoA-I/liposome complexes in
individual patients.
Data Analysis
Means6SEM were calculated, and changes were evaluated by paired
t test or ANOVA. Logarithmic transformations were used when
appropriate.
Results
The patients were carefully supervised, including continuous
ECG and blood pressure monitoring during the infusion. No
adverse reactions were observed. All laboratory safety parameters were repeatedly within normal limits before and
during follow-up for 15 days.
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
596
Circulation
August 10, 1999
Lipoprotein, Apolipoprotein, and Sterol Measurements
9 Days Before and 9 Days After the Infusion of 4 g of
Proapo A-I Liposomes
Before
Infusion
After
Infusion
Cholesterol
430633
406618
Triglycerides
106616
8969
LDL cholesterol
344631
329614
Plasma, mg/dL
HDL cholesterol
4167
3966
Apo A-I
140610
134613
Apo B
242617
244612
12.4563.74
17.2864.59*
Fecal excretion, mg z kg21 z d21
Neutral sterols
Bile acids
7.2361.88
9.3862.62†
19.7065.41
26.3366.75*
D8-Lathosterol
0.16560.064
0.16860.060
D7-Lathosterol
1.68360.584
1.61060.578
Total steroids
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Serum, mg/mg cholesterol
Values are mean6SEM.
*Significantly different from basal period, P,0.02; †P,0.05 (2-sided paired
t test for log-transformed values).
between 9% and 34% above initial values at 24 hours
(P,0.001).
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 (18% to
180%; mean, 135%) 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 z kg21 z
d21, 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).
To test the possibility that the phospholipid complexes had
an independent stimulatory effect on cholesterol excretion,
Figure 2. Fecal excretion of bile acids and neutral steroids 9
days before (left bars) and 9 days after (right bars) infusion of
proapoA-I liposomes. Individual mean levels are shown for 2
periods; individual changes in total steroid excretion (mg/d) are
indicated above each pair of bars. Increase in response to treatment was significant for bile acids (P,0.03), neutral sterols
(P,0.03), and total steroid excretion (P,0.02).
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 (214% and 210% 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% (112.8 mg z kg21 z d21) when infused with proapoA-I
liposomes, displayed only a 2% increase (10.3 mg z kg21 z
d21) after the administration of phosphatidylcholine. Similarly, L.M.’s cholesterol excretion increased 16% (15.2 mg z
kg21 z d21) after proapoA-I complexes and only 2% (10.8
mg z kg21 z d21) after the control infusion. Safety monitoring
did not reveal any abnormalities, and serum lathosterol levels
were not affected (not shown).
Discussion
In this open study, we were able to observe an enhanced
cholesterol excretion in response to the infusion of proapoA-I
Eriksson et al
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liposomes in humans. The dose of proapoA-I administered
corresponds to '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 1B). Although
no detailed kinetic evaluation was performed in the present
study, it is of interest to note that the initial rate of lowering
of apoA-I clearly was more rapid in the 2 patients with low
initial apoA-I levels (Figure 1A). This may reflect a more
rapid clearance of the apolipoprotein in those subjects, a
phenomenon known to be linked to lower plasma apoA-I
levels.1,2,5–7,38 – 40
The 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 7.361.5%
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 –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
Proapo A-I Infusion and Cholesterol Excretion
597
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 ester–loaded 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.5–7,17–22 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 totalbody 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
This study was supported by grants from the Swedish Medical
Research Council (03X-7137), the Ax:son Johnson and Osterman
Foundations, the Foundation of Old Servants, and a grant-in-aid from
UCB Pharma, Belgium. We thank C. Roobol, J. Gobert, and E.
Wülfert at UCB Pharma, Belgium, for the provision of proapoA-I
liposomes and helpful discussions; Catharina Sjöberg, Lilian
Larsson, and Pia Hoffström for technical assistance; and Mats
Rudling for performing fast protein liquid chromatography analyses.
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Circulation. 1999;100:594-598
doi: 10.1161/01.CIR.100.6.594
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