Proc. Nati Acad. Sci.
USA
Vol. 79, pp. 3983-3986, July 1982
Biochemistry
Catabolism of low density lipoproteins by perfused rabbit livers:
Cholestyramine promotes receptor-dependent hepatic
catabolism of low density lipoproteins
(hepatic low density lipoprotein receptor/low density lipoprotein metabolism/hypercholesterolemia/casein diet/high density lipoprotein
metabolism)
YU-SHENG CHAO, TING-TING YAMIN, AND ALFRED W. ALBERTS
Department of Biochemical Regulation, Merck Institute for Therapeutic Research, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065
Communicated by Alexander G. Bearn, April 1, 1982
ABSTRACT Rabbits fed a wheat starch/casein diet develop
a marked hypercholesterolemia accompanied by a decrease in the
number of EDTA-sensitive binding sites on plasma membrane
fractions of the liver for low density lipoproteins (LDL) and 13-
migrating very low density lipoproteins [Chao, Y.-S., Yamin, T.-
T. & Alberts, A. W. (1982)J. BioL Chem., in press]. Inclusion of
1% cholestyramine resin in this diet prevents the increase in
plasma cholesterol, increases the removal of LDL from plasma,
and increases the number of hepatic plasma membrane LDL-
binding sites. To determine the functional role of hepatic LDL-
binding sites in the catabolism of LDL, we studied the catabolism
of "MI-labeled LDL (125I-LDL) by in situ perfused rabbit livers in
a recirculating system. The rate of catabolism was measured from
the increment of nonprotein-bound radioiodine in the perfusate.
The receptor-dependent catabolism of LDL by the liver was cal-
culated from the difference of hepatic catabolism of 125I-LDL and
catabolism of LI-labeled cyclohexanedione-modified LDL, which
does not bind to LDL receptors. The data show that about 74%
of LDL catabolized by perfused livers from chow-fed rabbits is
through the receptor-dependent pathway and 26% is through the
receptor-independent pathway. In rabbits fed a cholesterol diet,
the hepatic catabolism of 125I-LDL is reduced, and the receptor-
dependent catabolism of 125I-LDL is abolished. In rabbits fed the
wheat starch/casein diet, the receptor-dependent catabolism of
125I-LDL is reduced by 40% when compared with hepatic catab-
olism in chow-fed rabbits. Perfused livers from rabbits fed the
wheat starch/casein diet supplemented with 1% cholestyramine
show a 5.4-fold increase of receptor-dependent catabolism of 125I-
LDL when compared with that of livers from rabbits fed the wheat
starch/casein diet alone. Thus, these studies demonstrate that the
change in the number of rabbit hepatic membrane LDL receptors
induced by dietary manipulation and drugs is correlated to the
functional rate of removal of LDL by the liver.
In cultured extrahepatic cells, low density lipoproteins (LDL)
are taken up by the cells after binding to the high-affinity re-
ceptor on the cellular surface. The activity of the LDL receptor
is regulated by the concentration of LDL apolipoprotein in the
culture medium (1). Several lines of evidence suggest that the
LDL receptor is involved in the catabolism of LDL in vivo.
Patients with homozygous familial hypercholesterolemia, who
have elevated plasma LDL concentration, lack the high-affinity
LDL receptor in their cultured skin fibroblasts (2). Shepherd
et al. (3) showed that in heterozygous familial hypercholester-
olemia, the removal of lmI-labeled LDL ('2I-LDL) from
plasma by the receptor-dependent pathway is reduced. Thomp-
son et al. (4) recently reported that in homozygous familial hy-
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3983
percholesterolemia, the removal of 125I-LDL by the receptor-
dependent pathway is essentially abolished.
Cholesterol, the major component of LDL, is metabolized
mainly by the liver. There is ample evidence that suggests that
a receptor similar to that found in extrahepatic tissue also exists
on the plasma membranes of the liver. Administration of phar-
macological doses of 17a-ethinyl estradiol to rats increases the
number of high-affinity binding sites for LDL on the liver mem-
branes (5), and the increased number of binding sites is accom-
panied by increased hepatic catabolism of 125I-LDL in isolated
perfused rat livers (6). A high-affinity binding site for LDL is
also present in cultured pig (7) and rabbit hepatocytes (8) and
in liver membranes prepared from dogs (9, 10) and rabbits (11).
The rabbit is a suitable animal model for the study of lipo-
protein metabolism. Recently, the Watanabe heritable hyper-
lipidemic (WHHL) rabbit line was discovered and found to be
a useful model for the study of lipoprotein metabolism in fa-
milial hypercholesterolemia (12). Homozygous WHHL rabbits
have greatly reduced numbers of LDL receptor in both cul-
tured fibroblasts (13) and hepatic membranes (11). Normal rab-
bits also can become hypercholesterolemic if they are fed either
a cholesterol diet (14) or a cholesterol-free wheat starch/casein
diet (15).
The hepatic LDL receptor in normal rabbits can be regulated
by the addition of dietary constituents to the animal diet. Feed-
ing rabbits a cholesterol diet caused reduced activity of hepatic
LDL receptor (16). Slater et al. (17) have demonstrated that
cholestyramine resin, a bile acid sequestrant, increases the he-
patic uptake of LDL by promoting the activity of hepatic LDL
receptor. We have shown that the hepatic EDTA-sensitive
binding sites for 125I-LDL is down-regulated in rabbits fed a
wheat starch/casein diet and that this binding site can be in-
duced when cholestyramine is included in the diet (18).
In the current study, we investigated the catabolism of 1"I-
LDL by perfused rabbit livers and the regulation of the activity
of hepatic LDL receptor in rabbits fed different diets.
METHODS
Animals and Diets. Male New Zealand White Rabbits weigh-
ing 1.5-2.0 kg were used. Blood donors weighed 3.5-4 kg.
Cholesterol-fed rabbits were fed milled rabbit chow supple-
mented with 0.5% cholesterol and 10% corn oil (wt/wt) for 2
Abbreviations: LDL, low density lipoproteins; 1251-LDL, "2I-labeled
LDL; HDL, high density lipoproteins; 125I-,B3VLDL, 125I-labeled ,-
migrating very low density lipoproteins; WHHL rabbit, Watanabe her-
itable hyperlipidemic rabbit; CHD-LDL, 1,2 cyclohexanedione-mod-
ified LDL; 125I-CHD-LDL, 125I-labeled CHD-LDL; FCR, fractional
catabolic rate.
3984 Biochemistry: Chao et aL
months. The composition of the wheat starch/casein diet is as
described (18).
Preparation of Lipoproteins. LDL (1.02 < p < 1.063 g/ml)
and high density lipoproteins (HDL) (1.063 < p < 1.21 g/ml)
were isolated from the plasma of chow-fed rabbits by sequential
ultracentrifugation (19) and labeled with 1"I by a modification
(20) of McFarlane's method (21). Unreacted 1"I was removed
by dialysis for 24 hr against 50 mM Tris/25 mM NaCl/0.5 mM
CaCl2, pH 8.0. Cyclohexanedione (CHD)-modified LDL
(CHD-LDL) was prepared essentially as described by Mahley
et aL (22). On agarose gel electrophoresis (pH 8.6), the modified
LDL migrated more rapidly as a homogenous band than did
native LDL.
Catabolism of Lipoproteins by Perfused Rabbit Livers.
Liver perfusion was carried out in pairs of rabbits by the in situ
perfusion method for rat livers of Mortimore (23) and modified
by Hamilton et al. (24) with a "lung" of silastic tubing for gas
exchange (25). The apparatus was scaled up to accommodate
rabbits. Liver donors were lightly anesthesized by intravenous
injection of Nembutal (Abbott; 0.5 mg/kg of body weight). The
peritoneal cavity was opened, and a ligature was placed around
the vena cava between the liver and kidney. The portal vein was
then cannulated with a Quik-Cath catheter (14 gauge x 5.1 cm,
Travenol Laboratories, Dallas, TX). The thorax was opened, and
another catheter was placed in the inferior vena cava through
the right atrium. The ligature placed around the vena cava be-
tween the liver and kidney was tied off. The liver was first
flushed with 200 ml of perfusate with washed (four times) rabbit
erythrocytes at a hemotocrit of 10% in Krebs-Ringer bicarbon-
ate buffer with glucose (100 mg/dl) and then was perfused with
100 ml of perfusate containing rabbit erythrocytes at hematocrit
22% in a recirculating system at 37°C at a perfusion rate of 50
ml/min. The perfusate was equilibrated with 95% 02/5% CO2
at pH 7.4. The 1"I-labeled lipoproteins were added to the per-
fusate after a "recovery" period of 15 min. The rate ofcatabolism
of 1"I-labeled lipoproteins by the liver was measured by the
production of trichloroacetic acid-soluble 1"I in perfusate-
plasma (20). Because the production of trichloroacetic acid-sol-
uble "I in the perfusate-plasma is curvilinear, fractional ca-
tabolic rate (FCR) of '"I-LDL by perfused livers was calculated
from the period of constant production of total trichloroacetic
acid-soluble '25I from 125I-LDL (from 0.5 to 1.5 hr of perfusion).
Chemical Analysis. Protein was determined by a modified
procedure of Lowry with bovine serum albumin as standard
(26). Serum cholesterol concentration was determined by the
cholesterol oxidase method with the Agent assay reagent from
Abbott Laboratories.
RESULTS
Rabbits fed a wheat starch/casein diet developed marked hy-
percholesterolemia. After 30 days on the diet, the average
serum cholesterol level of eight rabbits was about 175 mg/dl.
In a previous study, serum cholesterol increased from an av-
erage of 50 mg/dl to 175 mg/dl with =76% of the total cho-
lesterol in the LDL fraction (1.006 < p < 1.063 g/ml) (18). The
rate of appearance of trichloroacetic acid-soluble '25I in liver
perfusates is shown in Fig. 1. In livers from chow-fed rabbits,
production of nonprotein-bound 1"I increased linearly with
time in the perfusate after a 15-min latent period (Fig. 1A). This
latent period is similar to that observed in isolated perfused rat
liver studies (6). After 4 hr of perfusion, 9% of the added '"I-
LDL was catabolized by the perfused chow-fed rabbit livers.
It has been shown that modification of the arginyl-residues
of LDL by 1,2-cyclohexanedione abolishes its ability to bind to
high-affinity sites for LDL in cultured human fibroblasts (22)
Proc. Natl. Acad. Sci. USA 79 (1982)
12 - A -B
N
8-0X
0~~~~~~~~~~
F
0 1 2 3 4 1 2 3 4
Perfusion, hr
FIG. 1. Production of nonprotein-bound 1251I from livers of chow-
fed (A) or cholesterol-fed (B) rabbits during perfusion with rabbit 125I-
LDL (o) or rabbit '25I-CHD-LDL (o). The mean serum cholesterol con-
centrations for chow-fed and cholesterol-fed rabbits are 58 and 1,257
mg/dl, respectively. For studies of catabolism of 1251I-LDL, data show
the mean values (± SD) of perfused livers (eight) from chow-fed rabbits
and livers (three) from cholesterol-fed rabbits. For studies of catabo-
lism of '25I-CHD-LDL, individual data from a paired experiment are
shown. Pool size of rabbit LDL or CHD-LDL apolipoproteins in the
perfusate in these experiments was between 0.1 and 0.3 mg.
and that the removal of "I-labeled CHD-LDL (125I-CHD-
LDL) in humans (3) and in rabbits (17) is only by the receptor-
independent pathway. Thus, we studied the rate of catabolism
of lipoproteins by the hepatic receptor-independent pathway
by measuring the rate of catabolism of CHD-LDL. After 4 hr,
about 3% of the 125I-CHD-LDL in the perfusates was catabo-
lized by the receptor-independent liver from chow-fed rabbits
(Fig. 1A).
Shepherd et al have shown that rabbits fed a diet containing
cholesterol resulted in a 90% suppression ofreceptor-mediated
LDL uptake into liver, kidney, heart, lung, spleen, and gonads
(27). Kovanen et al. (16) also have demonstrated that the hepatic
LDL receptor was suppressed in rabbits fed a cholesterol diet.
To study the regulation of hepatic LDL receptor in rabbits, we
studied the catabolism of 125I-LDL by perfused livers from rab-
bits that had been fed a cholesterol diet and whose serum cho-
lesterol concentrations were >1,000 mg/dl. The rate of catab-
olism of 125I-LDL by perfused livers from cholesterol-fed
rabbits was markedly reduced, whereas the hepatic catabolism
of 125I-CHD-LDL was not altered by the cholesterol feeding
(Fig. 1B).
Rabbits fed a wheat starch/casein diet develop hypercholes-
terolemia accompanied by suppression of the hepatic EDTA-
sensitive binding sites for 125I-LDL and 125I-labeled ,-migrat-
ing very low density lipoproteins ('"I-P-VLDL) (18). The rate
of catabolism of 125I-LDL (receptor-dependent and receptor-
independent) was reduced by 40% by the livers from rabbits
fed this diet (Fig. 2A) compared to that seen in chow-fed rabbits
(Fig. 1A).
Supplementing the wheat starch/casein diet with 1% cho-
lestyramine has resulted in lower levels of serum cholesterol
(the average serum cholesterol level of eight rabbits was 45
mg/dl) and increased the rate of removal of 125I-LDL from
plasma and the induction of hepatic EDTA-sensitive binding
sites for 125I-LDL and 125I-,/3VLDL (18). The rate ofcatabolism
of 125I-LDL was markedly increased in livers from rabbits fed
the wheat starch diet supplemented with 1% cholestyramine
(Fig. 2B). The increased hepatic catabolism of 125I-LDL by cho-
lestyramine treatment is specifically for the receptor-depen-
dent catabolism because the hepatic catabolism of 125I-CHD-
LDL was not affected in this system (Fig. 2B).
The specificity of the cholestyramine effects was studied by
determining the catabolism of 125I-HDL by the perfused rabbit
livers. HDL was prepared from rabbit plasma and was free of
 Biochemistry: Chao et al. Proc. Natl. Acad. Sci. USA 79 (1982) 3985
Table 1. FCR of 125I-LDL by perfused rabbit livers
Receptor- Receptor-
20
4-'1 Total, independent, dependent,
Diets % hr-l* % hr-1t % hr-1t
16- Chow 3.1 0.8 2.3
Cholesterol 0.8 0.8 0
Cd12- Wheat starch/casein 2.2 0.8 1.4
0
Wheat starch/casein
with cholestyramine 9.8 0.8 9.0
* Total FCR is estimated from the period of constant production of total
trichloroacetic acid-soluble 125I from 125I-LDL.
t Receptor-independent FCR is estimated from the period of constant
J production of total trichloroacetic acid-soluble 125I from 125I-CHD-
12 3
L_
4 0 hr1
Perfusion, 2
I
3
I_
4
LDL.
* Receptor-dependent FCR is calculated from the difference of total
FCR and receptor-independent FCR.
FIG. 2. Production of nonprotein-bound 125I during perfusion of
livers from rabbits fed a wheat starch/casein diet (A) and from rabbits ceptor-dependent pathway and 26% is through the receptor-
fed the wheat starch/casein diet supplemented with 1% cholestyra- independent pathway. Feeding the wheat starch/casein diet
mine resin (B), with rabbit 125I-LDL (o) and rabbit 125I-CHD-LDL resulted in 40% reduction of receptor-dependent catabolism of
(o). Rabbits were fed the diets for 30-40 days. Their mean cholesterol LDL. Inclusion of 1% cholestyramine in the wheat starch/ca-
concentration was 176 mg/dl for the group fed the wheat starch/ca-
sein diet and 35 mg/dl for the group fed the wheat starch/casein diet sein diet caused increased hepatic catabolism of 125I-LDL. Fur-
supplemented with 1% cholestyramine. For studies of catabolism of thermore, only the receptor-dependent catabolism of 125I-LDL
125I-LDL, mean values (±SD) of perfused livers (four) from rabbits fed increased 5.4-fold, whereas the receptor-independent catabo-
the wheat starch/casein diet and livers (six) from rabbits fed the wheat lism was not changed. The data also show that only the receptor-
starch/casein diet supplemented with cholestyramine are shown. For dependent catabolism of 125I-LDL by rabbit livers is subject to
studies of '25I-CHD-LDL, individual data from a paired experiment are regulation by the dietary manipulations of the animal.
shown. Pool size of rabbit LDL or CHD-LDL apolipoproteins in the
perfusate was between 0.1 and 0.3 mg. Our data from a different methodology are in reasonable
agreement with those of Slater et al (17). They demonstrated
apolipoprotein E as judged by visual inspection of an overloaded that, in rabbits fed a chow diet, human 125I-LDL is cleared from
10% polyacrylamide gel that had been electrophoresed in the plasma by a receptor-dependent pathway and that the rate of
presence of 0.2% sodium dodecyl sulfate. The catabolic rate of clearance is stimulated by the inclusion of cholestyramine in the
125I-labeled HDL was essentially identical in perfused livers diet. Furthermore, the major site of accumulation of label and
from rabbits fed either the wheat starch/casein diet alone or the the only organ where accumulation was stimulated by choles-
diet supplemented with 1% cholestyramine (Fig. 3). tyramine is the liver.
Rabbits fed the wheat starch/casein diet develop hypercho-
DISCUSSION lesterolemia and down-regulation of the hepatic EDTA-sensi-
tive binding site for LDL and f-VLDL, yet the EDTA-resistant
Table 1 summarizes the FCR of 125I-LDL by perfused rabbit binding site and the nonspecific binding site are not affected
livers determined from the data in Figs. 1-3. In chow-fed rab- by the diet (18). Whether the observed receptor-dependent
bits, -74% of LDL catabolized by the liver is through the re- catabolism of 15I-LDL by the perfused livers from the wheat
starch/casein-fed rabbits represents the catabolism through the
EDTA-resistant binding site or through the small amounts of
12 residual EDTA-sensitive binding site remains to be determined.
a)
-4'A
The mechanisms by which cholestyramine promotes hepatic
LDL catabolism in rabbits fed the wheat starch/casein diet is
not known. Because the wheat starch/casein diet is known to
0
8 - decrease the fecal excretion of bile acids in rabbits (15), the ad-
,-,o dition of a bile acid sequestrant to the diet may promote the
04
excretion of bile acids, thereby increasing the demand for cho-
lesterol for bile acid synthesis in the liver. This demand can be
80/ 0
supplied either by an increased level of de novo synthesis or
4 0 from plasma LDL. In in vitro studies, we have demonstrated
that cholestyramine induces the hepatic EDTA-sensitive bind-
0 ing site for 125I-LDL to a level that is the same as that of liver
membranes from rabbits fed chow diet (18). Moreover, the dis-
A.0/ III sociation constant for 125I-LDL binding to liver membranes
1 2 3 4 from rabbits fed chow diet is the same as that of liver mem-
Perfusion, hr branes from the cholestyramine-treated rabbits. Yet the hepatic
receptor-dependent catabolism of 1"I-LDL by the cholestyr-
FIG. 3. Production of nonprotein-bound 125I during perfusion of amine-treated rabbits is 3 times greater than that by the chow-
livers from rabbits fed either a wheat starch/casein diet alone (o) or fed rabbits. Because the rabbits used in this study were different
one supplemented with 1% cholestyramine (W). Individual data from
a paired experiment are shown. The serum cholesterol concentrations
from those used in previously reported binding studies, the
for the wheat starch/casein-fed and the cholestyramine-treated rab- discrepancy between the two studies could be attributed to in-
bits are 276 and 88 mg/dl, respectively. Pool size of rat HDL apolip- dividual variations between rabbits. In rats treated with 17a-
oprotein is 0.5 mg. ethinyl estradiol, the increased hepatic catabolism of '25I-LDL
3986 Biochemistry: Chao et aL
is accompanied with increased binding capacity of 125I-LDL to
liver membranes (5, 6). Whether cholestyramine has any effect
on the intracellular degradation of LDL-e.g., promotes the
recycling of LDL receptor and increases the levels of choles-
terol 7a-hydroxylase or 3-hydroxy-3-methylglutaryl-CoA re-
ductase, etc.-in this system needs investigation.
Recently, WHHL rabbits have been used as the animal
model for the study of lipoprotein metabolism in familial hy-
percholesterolemia (12). The homozygous WHHL rabbits re-
semble their human counterparts in having elevated plasma
LDL cholesterol (450-855 mg/dl) (13) and having a reduced
number of the classic LDL receptor in their fibroblasts in cul-
ture (13). Furthermore, the EDTA-sensitive binding site for
125I-LDL and 125I-f-VLDL is reduced in liver membranes of
the WHHL rabbits (11). The receptor-dependent degradation
of 125I-LDL is also lacking in cultured hepatocytes of the
WHHL rabbits (28). However, the total degradation of '"I-
LDL by the cultured hepatocytes from the WHHL rabbits is
somewhat increased rather than decreased (28). Because 6% of
the LDL receptor activity remained in the WHHL rabbit fi-
broblasts (13) and small amounts of residual EDTA-sensitive
binding remained in the liver membranes from WHHL rabbits
(11), it would be of interest to investigate if the homozygous
WHHL rabbits respond to cholestyramine treatment by in-
creasing the hepatic catabolism of LDL.
Cholesterol is excreted from the body by conversion to bile
acids in the liver. Thus, a therapeutically effective means of
lowering levels of plasma cholesterol is by increasing the con-
version of LDL cholesterol to bile acids. This can be accom-
plished by increasing the hepatic uptake of LDL through the
hepatic LDL receptor (29). Recently, several pharmacological
agents have been reported that are capable of inducing the he-
patic LDL receptor in experimental animals: pharmacological
doses of 17a-ethinyl estradiol in rats (5, 6), large doses of 3-hy-
droxy-3-methyglutaryl-CoA reductase inhibitors like mevinolin
and compactin (30, 31) in swine (7), cholestyramine in rabbits
(17), and a combination of mevinolin and colestipol in dogs (9).
Recent observations that mevinolin also reduces plasma choles-
terol levels in rabbits fed the wheat starch/casein diet (32) sug-
gest that mevinolin alone or in combination with cholestyramine
induces hepatic LDL receptor levels in this model.
Our results show that cholestyramine lowers plasma choles-
terol levels in rabbits with endogenous induced hypercholes-
terolemia by promoting the hepatic catabolism of 125I-LDL and
suggest that the rabbit is a suitable animal model for studying
the mechanism of action of hypocholesterolemic agents.
We thank Joan Kiliyanski for her secretarial assistance in preparing
this manuscript.
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