© 1999 American Heart Association, Inc.
Original Contributions |
Effects of Alcohol and Cholesterol Feeding on Lipoprotein Metabolism and Cholesterol Absorption in Rabbits
From the Department of Animal Sciences (M.A.L.), Purdue University, West Lafayette, Ind; Divisions of Atherosclerosis, Nutrition and Lipid Research (B.W.P., R.T.K., G.S.), and Endocrinology, Diabetes and Metabolism (R.E.O.), Department of Medicine, Washington University; and Purina Mills, Inc (D.H.), St. Louis, Mo.
Correspondence to Dr Gustav Schonfeld, Department of Internal Medicine, Washington University School of Medicine, Box 8046, 660 S Euclid Ave, St. Louis, MO 63110. E-mail Gschonfeld{at}imgate.wustl.edu
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Abstract |
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Abstract—Alcohol fed to rabbits in a liquid formula at 30% of calories increased plasma cholesterol by 36% in the absence of dietary cholesterol and by 40% in the presence of a 0.5% cholesterol diet. The increase was caused almost entirely by VLDL, IDL, and LDL. Cholesterol feeding decreased the fractional catabolic rate for VLDL and LDL apoprotein by 80% and 57%, respectively, and increased the production rate of VLDL and LDL apoprotein by 75% and 15%, respectively. Alcohol feeding had no effect on VLDL apoprotein production but increased LDL production rate by 55%. The efficiency of intestinal cholesterol absorption was increased by alcohol. In the presence of dietary cholesterol, percent cholesterol absorption rose from 34.4±2.6% to 44.9±2.5% and in the absence of dietary cholesterol, from 84.3±1.4% to 88.9±1.0%. Increased cholesterol absorption and increased LDL production rate may be important mechanisms for exacerbation by alcohol of hypercholesterolemia in the cholesterol-fed rabbit model.
Key Words: cholesterol • alcohol • diet • cholesterol absorption • lipoproteins • apoproteins • atherosclerosis
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Introduction |
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The effects of alcohol on cholesterol and lipoprotein metabolism have substantial public health implications. It is generally believed that moderate alcohol consumption reduces coronary heart disease risk because observational studies show that it is associated with elevated HDL cholesterol levels and decreased cardiovascular events.1 2 3 However, alcohol has many biological actions, and adverse effects on lipid metabolism and cardiovascular disease risk are also well-known, especially in individuals with familial predisposition to hyperlipidemia.4 5 Thus, the results of alcohol consumption are likely to reflect a balance that might be positive or negative in a given individual. Clearly, a more complete understanding of the interaction between alcohol and lipid metabolism is needed.
To investigate the effects of alcohol under controlled dietary conditions, we previously reported the application of cholesterol- and alcohol-containing liquid formula diets that are palatable and allow good weight gain and gastrointestinal function in rabbits.6 7 This work showed that instead of protecting against atherosclerosis, ethanol feeding at 20% to 30% of calories in the presence of dietary cholesterol (0.5% by weight) increased both plasma lipoproteins and aortic atherosclerotic lesions. In the presence of dietary cholesterol, plasma VLDL and LDL doubled when alcohol was substituted for carbohydrates at 30% of calories, and aortic arch cholesterol content increased fourfold. The current work was performed to investigate potential mechanism(s) responsible for these adverse alcohol-induced changes. The kinetics of VLDL and LDL were determined by tracer methods after varying periods of dietary treatment, and cholesterol absorption was measured at the end of the experiment. The results suggest that alcohol induced increased cholesterol absorption. As a result fractional catabolic rates for VLDL and LDL decreased and production of LDL increased. These changes probably account for the adverse effects of alcohol on lipoprotein metabolism and atherosclerosis in the cholesterol-fed rabbit.
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Methods |
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Animals and Diet
Forty female New Zealand White rabbits were housed individually during a 6-week study. The animals were divided into 4 groups at the beginning of the trial and fed liquid diets as previously described in detail.7 All diets were isonitrogenous and isocaloric with alcohol replacing carbohydrate. The control diet (L) contained 18% fat and 56% carbohydrate and was free of cholesterol. Fats were derived from vegetable sources and were 16% saturated, 58% monounsaturated, and 26% polyunsaturated. In the other diet groups alcohol was included at 30% of calories (LA), cholesterol at 0.5% by weight (LC), or both were included (LAC). The diets were obtained as powders from Purina Test Diet. Diets and water were available ad libitum, except during determinations of kinetics as described below. Body weight was recorded weekly and feed consumption was monitored daily. Fasted venous plasma samples were obtained from a marginal ear vein every week between 8 AM and 9 AM. Lipids and lipoproteins were determined by Lipid Research Clinics methodology.8 Lipoproteins from fresh plasma obtained from 2 animals/group were analyzed by fast protein liquid chromatography (FPLC) using two 25-mL Superose 6 columns (Pharmacia Biotech) connected in series and eluted with 0.15 mol/L NaCl containing 1 mmol/L EDTA, pH 8.0, at 0.5 mL/min.
Cholesterol Absorption Experiments
4-[14C]cholesterol (51
mCi/mmol) and 5,6-[3H]sitostanol (47 Ci/mmol)
were purchased from Dupont New England Nuclear. Radiolabeled sitostanol
was repurified by thin-layer chromatography in 98:2
chloroform:acetone on silica gel G. Unlabeled anhydrous
cholesterol (product C8503, <0.1% water) was
purchased from Sigma. Percent cholesterol absorption was
measured by continuous feeding of
[14C]cholesterol and
[3H]sitostanol for 7 days at the end of a
6-week dietary treatment. Tracers were taken up in ethanol, and 1-mL
aliquots were added to 4 kg feed in a blender for L and LA diets. For
the diets containing cholesterol, tracers dried from 1 mL
of the ethanolic solution were dissolved in 12 mL chloroform containing
5.9 g natural cholesterol and allowed to dry overnight
in a Petri dish. The solid mixture was then added to 4 kg formula and
mixed thoroughly in a blender. The animals received a daily dose of
0.08 µCi [14C]cholesterol and 0.2
µCi [3H]sitostanol. Stools from day 6 and day
7 were collected for separate analyses, and each lot was
vigorously mixed in 100 mL water with a Waring blender for 5 minutes.
An aliquot of 4 mL stool suspension was saponified in 9.3 mL ethanol
and 0.64 mL 45% KOH at 65°C for 45 minutes, and sterols were
extracted 3 times with petroleum ether and counted. Percent
cholesterol absorption was calculated as
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Kinetic Analyses
After a 12-hour fast one rabbit from each treatment group was
sacrificed and bled through cardiac puncture at 2 weeks and 6 weeks of
diet treatment. VLDL (d<1.006) and LDL (1.019<d<1.063) were isolated
by sequential ultracentrifugation9 and
dialyzed against 0.15 mol/L NaCl containing 1 mmol/L EDTA, pH 7.4,
for 24 hours. Lipoprotein composition was determined by measuring free
and total cholesterol and phospholipids using enzymatic
assays (WAKO Chemicals USA, Inc) and total protein.10
Lipoproteins were labeled with either 125I or
131I by the method of McFarlane,11
separated from free iodine by Sephadex G25
chromatography, and injected within 24 hours of
iodination. At either baseline, 2 weeks, or 6 weeks, 2 rabbits from
each treatment group were fasted for 12 hours and then injected with
2.5 µCi 131I-VLDL and 5 µCi
125I-LDL by the marginal ear vein. Blood samples
were removed at times 0, 10, 20, and 40 minutes, and at 1, 2, 4, 8, 12,
24, 48, 72, 96, and 120 hours. To minimize the concentration of
intestinally synthesized lipoproteins rabbits were fasted at least 12
hours before any bleeding period. VLDL and LDL were isolated by
ultracentrifugation, and trichloroacetic acid
precipitable radioactivity was determined using narrow gamma counter
windows. ApoB12 accounted for 82% of the precipitated
VLDL disintegrations per minute (dpm) and 95% of precipitated LDL dpm.
The kinetics of VLDL and LDL turnover were analyzed with the
SAAM II compartmental modeling program (SAAM Institute, University of
Washington). The model used (Figure 1
)
represents the minimal structural complexity that was both
necessary and sufficient to account for the turnover of
radioiodinated lipoproteins in all 4 groups of rabbits. It
is similar to those previously used in New Zealand White
rabbits.13 14 VLDL was assumed to consist of both a fast
(V1) and a slow (V2)
compartment restricted to the plasma, and the total VLDL fractional
catabolic rate (FCR) was calculated as the average of fast and slow
components weighted by the fraction passing through those pathways. LDL
was also modeled as a 2-compartment system including a plasma
compartment and a nonplasma compartment. Production rates for
VLDL and LDL (mg/dL per hour) were calculated as the product of
each compartment mass (mg/dL) and the FCR. The precision for estimation
of model parameters, computed as mean fractional standard
deviation, was 0.043 (range, 0.015 to 0.110) for VLDL and 0.071 (range,
0.035 to 0.158) for LDL.
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Statistics
The data were analyzed with the General Linear Model of
the Statistical Analysis System (SAS Institute). A split-plot
design was used with diet as the whole-plot factor and week of sampling
as the subplot factor. For cholesterol absorption studies
cholesterol and alcohol were analyzed as main
effects and replications on successive days within individual rabbits
as a random effect. Statements of significance were based on
P<0.05 unless otherwise noted.
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Results |
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All dietary groups gained weight (Figure 2A
) and had adequate food consumption
(Figure 2B) on the liquid formula diets. After 6 weeks
alcohol-treated animals weighed 11% less than those not receiving
alcohol and consumed an average of 17% less feed during the
experimental period. Despite slightly reduced intake, alcohol was
associated with an increase in plasma cholesterol levels
(Figure 3). The effect of alcohol was
seen at 4 weeks and thereafter in cholesterol-fed animals,
and between week 4 and week 6 plasma cholesterol on the LAC
diet was 40% higher than on LC (P=0.03 by repeated measures
ANOVA). During the same period plasma cholesterol was
increased by 36% or 42 mg/dL (P=0.005) in animals not
receiving dietary cholesterol. The lipoprotein response to
the diets was determined by fast protein liquid
chromatography at both 2 and 6 weeks (Figure 4). Most of the increase in plasma
cholesterol during cholesterol feeding, both in
the presence and absence of alcohol, was because of increases in VLDL,
LDL, and intermediate fractions between them.
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Table 1 gives changes in total plasma
lipid concentration and percentage composition over time with each of
the diets. Alcohol, either in the presence or absence of dietary
cholesterol, had no effect on total lipid composition.
However, as expected, cholesterol feeding resulted in
cholesteryl ester enrichment at the expense of
triglycerides regardless of whether alcohol was
present. Table 2 shows lipid
composition data for isolated VLDL and LDL. By analysis of
variance using all of the data there was a strong overall effect of
cholesterol feeding to increase the percentage of
lipoprotein cholesterol and to decrease lipoprotein
triglyceride (both P<0.0001) with no effect on
phospholipid. In contrast, the effect of alcohol depended on whether
cholesterol was present in the diet. In the presence of
cholesterol, alcohol had no effect on lipid composition.
However, in the absence of cholesterol, alcohol increased
the percentage of cholesterol (P=0.01) and
decreased the percentage of triglyceride in VLDL and LDL
(P=0.002).
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The kinetics of VLDL and LDL apoB turnover were investigated in
animals consuming a chow diet at baseline and after 2 or 6 weeks of
treatment with liquid formulas containing cholesterol or
alcohol or both (Figure 5, Tables 3 and 4).
Radioiodinated VLDL and LDL obtained from an animal with a
similar feeding history were injected, and parameters of
the model of Figure 1
were determined. The independent relation
of cholesterol and alcohol feeding to lipoprotein FCR or
production rate (PR) was determined in a statistical model
using kinetic data from 0-, 2-, and 6-week time points. As expected,
cholesterol feeding had substantial effects on
apolipoprotein kinetics. VLDL FCR declined 80% at 6 weeks
(P<0.0001 for an independent effect of
cholesterol on VLDL FCR over time) and VLDL PR increased
75% (P=0.03). Cholesterol feeding reduced LDL
FCR 57% at 6 weeks (P<0.0001) and increased LDL PR 15%
(P=0.03).
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The independent effects of alcohol were determined similarly. Alcohol had no statistically significant effect on either VLDL FCR or VLDL PR. However, alcohol increased LDL FCR slightly by 7.3% at 6 weeks (P=0.001 for an overall effect) and increased LDL PR more substantially by 55% (P=0.01).
Percent cholesterol absorption was measured by including
[14C]cholesterol and
[3H]sitostanol (a nonabsorbable phytosterol) in
each diet during week 6 of the feeding experiment in 4 groups of 5
rabbits each (Table 4). Duplicate measurements of the isotope
ratio were made in stool collected on days 6 and 7 of isotope
administration and compared with the administered material. Evaluating
all dietary groups together in a single statistical model, both
cholesterol (P<0.0001) and alcohol
(P=0.0008) independently altered cholesterol
absorption. The interaction between cholesterol and alcohol
feeding was not statistically significant (P=0.20), showing
that alcohol was effective whether or not cholesterol was
also present in the diet. As expected, cholesterol
feeding alone reduced the efficiency of cholesterol
absorption from 84.3±1.4% to 34.4±2.6% (P<0.0001).
Alcohol alone increased cholesterol absorption from
84.3±1.4% to 88.9±1.0% when no dietary cholesterol was
given (P=0.029). In the presence of dietary
cholesterol, cholesterol absorption was
increased from 34.4±2.6% to 44.9±2.5% (P=0.018).
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Discussion |
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Alcohol had substantial effects in the cholesterol-fed rabbit model. After 4 weeks total plasma cholesterol was increased in animals fed alcohol plus cholesterol and by 6 weeks the difference was 43% above the level in animals fed cholesterol alone (Figure 3
). The lipoproteins that
accumulated were principally VLDL, LDL, and intermediate-density
particles as judged by FPLC analysis (Figure 4). As
expected, cholesterol feeding resulted in
cholesterol enrichment of VLDL and LDL at the expense of
triglyceride (Table 2). Alcohol had no effect on
lipoprotein composition in the presence of dietary
cholesterol, but it also increased cholesterol
and decreased triglyceride in the absence of dietary
cholesterol. Our previous work showed that adding dietary alcohol to the cholesterol-fed rabbit substantially increased aortic intimal lesions.3 One reason for this is that alcohol increased plasma cholesterol levels. The present work documents the mechanisms that might be important. Because absorption of dietary cholesterol is not complete,15 increased intestinal absorption caused by alcohol would exacerbate the experimental hypercholesterolemia and produce the elevated plasma levels observed. Very little previous work has been reported on the relation of alcohol to cholesterol absorption. In rats, Klurfeld and collaborators16 observed a 74% increase in the amount of cholesterol tracer found in plasma 3 days after a single oral tracer dose when the diet contained alcohol. However, cholesterol absorption was not quantitated, and a corresponding decrease in fecal sterols was not observed; thus, the difference in plasma cholesterol tracer level might have been caused by alteration in plasma cholesterol turnover or pool size as well as a change in absorption. In contrast, using human subjects, Crouse and Grundy17 found no difference in percent cholesterol absorption caused by chronic consumption of alcohol at 19% of calories in human subjects on a very low cholesterol diet. Our data establish that, in the rabbit, chronic ethanol at 30% of calories resulted in significant increases in the efficiency of intestinal cholesterol absorption. The effect was observed whether or not dietary cholesterol was present, although it was quantitatively greater in the presence of dietary cholesterol, in which cholesterol absorption efficiency was increased by 30.5% of the initial value. Because cholesterol is soluble in alcohol, the transfer of solid cholesterol to intestinal bile salt micelles might have been increased in the presence of alcohol. However, it is also possible that alcohol might have had a direct effect on enterocyte function or lymphatic flow. Previous workers found that acute, but not chronic, ethanol administration increased the rate of lymph flow and triglyceride absorption in the rat.18 Similar studies with respect to cholesterol absorption have not been performed. Future studies of the effect of alcohol on cholesterol absorption should consider the time course as well as the amount of alcohol given.
The increase in efficiency of intestinal cholesterol
absorption may be a fundamental mechanism of action for alcohol in the
rabbit model. The lag time of 4 weeks before an alcohol-induced
increase in plasma cholesterol could be observed in the
cholesterol-fed animal is consistent with an effect
on cholesterol absorption, because whole body
cholesterol turnover is slow and the pool of body
cholesterol is large.19 The most pronounced
apoprotein kinetic change observed with alcohol feeding was increased
LDL PR (55% increase in the absence of cholesterol and
92% increase in the presence of dietary cholesterol, Table 4 and Figure 5). Previous
human studies have found a positive association between increased LDL
PR and increased cholesterol absorption.20 21
It is possible, therefore, that increased LDL PR as a result of alcohol
in the rabbit might be caused by increased cholesterol
absorption. However, our work does not distinguish between this
possibility and a direct effect of alcohol on lipoprotein
metabolism. Increased cholesterol absorption
would be expected to reduce hepatic expression of LDL receptors, but
the LDL receptor status of our rabbits is unclear. In previous work we
reported a 41% reduction in hepatic LDL receptor mRNA with the
cholesterol plus alcohol diet compared with a diet
containing cholesterol without alcohol,7
suggesting that alcohol decreases LDL receptor expression. However,
here we find a 7% increase in LDL FCR, suggesting a slight increase in
LDL receptor expression. Further work will be needed to clarify this
question because mRNA changes do not necessarily reflect LDL receptor
function and changes in affinity of lipoproteins for the LDL receptor
may make kinetic studies of fractional catabolic rate difficult to
interpret.22
Additional insights into mechanisms of hyperlipoproteinemia in the rabbit follow from the apolipoprotein kinetic studies. Cholesterol feeding alone decreased VLDL and LDL apolipoprotein FCR and increased PR. Thus, both altered degradation and production of apolipoproteins are important in the pathogenesis of dietary hypercholesterolemia in the rabbit. No overall effects of alcohol were observed on VLDL apoprotein metabolism. Despite this negative result, significant increases in LDL PR were observed both in the presence and absence of alcohol. This is consistent with the ability of alcohol to increase turnover of lipoproteins4 but suggests a more complex mechanism. Increased LDL production in the absence of increased VLDL production could be caused by reduced removal of VLDL and IDL in the lipoprotein cascade or increased direct secretion of IDL and LDL. The former possibility might be associated with concomitant activation of lipoprotein lipase. The lipoprotein kinetic studies are limited in that only overall effects over time are analyzed statistically. Potential effects at 2 weeks and 6 weeks could not be determined with statistical certainty because only 2 animals per time point were used. Likewise, kinetic testing of alcohol-induced lipoproteins in nonalcohol-treated animals were not performed.
In summary, the data indicate that alcohol added to the cholesterol-containing diet accelerates the absorption of cholesterol, leading to alteration of whole body cholesterol and lipoprotein metabolism. LDL apoB PR is prominently increased, resulting in increased hypercholesterolemia and enhanced atherosclerosis.7
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Acknowledgments |
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This work was supported by NIH grants AA09988 (G.S.) and HL50420 (R.E.O.).
Received February 25, 1998; accepted August 17, 1998.
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