© 1997 American Heart Association, Inc.
Articles |
Dietary Fish Oil
Influence on Lesion Regression in the Porcine Model of Atherosclerosis
From the Department of Anatomy and Cell Biology, University of Western Ontario (London), Canada (M.L.B., K.A.R.), and Department of Medicine, Division of Endocrinology, Metabolism, and Nutrition, Duke University Medical Center, Durham, NC (K.F.K., J.R.G.).
Correspondence to Dr Kem A. Rogers, Department of Anatomy and Cell Biology, University of Western Ontario, London, ON Canada N6A 5C1. E-mail krogers{at}julian.uwo.ca
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Abstract |
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Abstract We examined the influence of dietary fish oil on lesion regression in a porcine model of atherogenesis. Thirty-two female Yucatan miniature pigs were fed an atherogenic diet for 8 months. A no-regression group (n=8) was killed to determine the extent of atherosclerosis at 8 months. Three regression groups were switched to normal minipig chow supplemented with either MaxEPA fish oil (FO group, n=8), a control oil with the ratio of polyunsaturated to monounsaturated to saturated fatty acid matched to that of the fish oil (CO group, n=8), or no oil supplement (NO group, n=8) for a further 4 months. Plasma cholesterol levels reached between 15 and 20 mmol/L during the atherogenic phase and returned to normal (2 mmol/L) within 2 months of the beginning of the regression diet. Compared with the NO group, fish oil supplementation during the regression phase caused a decrease in VLDL and HDL cholesterol and an increase in LDL cholesterol. Similarly, the control oil also caused a decrease in VLDL cholesterol; however, in contrast to the FO group, HDL cholesterol increased and LDL cholesterol was unchanged. FO LDL, which had decreased levels of 20:4 (n-6 fatty acid) and increased levels of 18:3, 20:5, and 22:6 (n-3 fatty acids), was shown to be twice as susceptible to copper-mediated oxidation as CO LDL particles. Morphological examination of the major blood vessels revealed a significant reduction in lesion area in the ascending and thoracic aorta as well as the carotid artery after the regression diet; however, there was no significant difference between the fish oil and control oil groups in any of the vessels measured. Therefore, despite increased LDL, decreased HDL, and an increased susceptibility to in vitro oxidation of LDL, fish oil supplementation of a regression diet did not influence lesion regression.
Key Words: dietary fish oil • miniature swine • lipoprotein • atherosclerosis • regression
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Introduction |
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It has been shown that populations consuming moderate to large quantities of fish have a low frequency of cardiovascular disease compared with groups consuming a standard Western diet. It has been suggested that the presence of n-3 fatty acids in the fish oils of these diets may be responsible for this observation.1 2 3 n-3 fatty acids have been shown to interfere with n-6 fatty acid metabolism, resulting in a reduction in certain proatherogenic eicosanoids (ie, thromboxane A2 and leukotriene B4) in favor of n-3 fatty acid metabolites, which are relatively inactive.4 5 6
Numerous investigators have explored the possibility that n-3 fatty acids from fish oils promote the regression of atherosclerosis in animal models; however, the results have been conflicting. Studies in rabbits and swine7 8 9 suggest that fish oils promote lesion regression, while studies by one group in primates10 11 concluded that fish oils had no effect. The variation in these studies may be due to the choice of dietary control for the fish oil. It has been clearly demonstrated that the P/S ratio of the diet directly influences the development of atherosclerosis12 13 14 ; increasing polyunsaturated and monounsaturated fatty acids at the expense of saturated fatty acids reduces the extent of lesion formation. Studies showing a positive effect of fish oil on lesion regression either have not used a control oil7 or have used a control oil with a P/S ratio significantly lower than that of fish oil.8 9
Studies examining the influence of dietary fish oil on the progression of atherosclerosis have also produced varied results, again primarily because of the design of the control diet. Our laboratory has recently shown that when an appropriate control oil is used, fish oils do not influence the development of lesions in the porcine model of atherosclerosis.15 Using the same dietary controls and the same animal model, in this study we examined the influence of n-3 fatty acids on the regression of atherosclerotic lesions.
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Methods |
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Animals and Diet
Thirty-two female Yucatan miniature swine (Charles River) were fed an atherogenic diet16 for 8 months (minipig chow supplemented with 1.5 wt% cholesterol and 15 wt% beef tallow). The atherogenic diet consisted of 34% energy in the form of fat (calculated from information provided by the manufacturer of the minipig chow and from the known quantities of added fat). After the atherogenic phase, the pigs were rank ordered and block randomly assigned into four experimental groups based on their plasma cholesterol values at 8 months. The first group (n=8, NR) was killed to determine the extent of atherosclerosis at 8 months. The remaining pigs were switched to the normal low-fat, cholesterol-free minipig chow supplemented with either MaxEPA fish oil concentrate (0.5 mL·kg-1·d-1; R.P. Scherer; n=8, FO group) or the same dose of a control oil (a mixture of beef tallow and corn and safflower oils) with the same P/S ratio (Table 1
) as the fish oil (n=8, CO group). The
regression diet with the oil supplements was composed of 10.6% energy
from fat. A fourth group received no oil supplement (n=8, NO group;
4.9% energy from fat). These regression diets were fed for a further 4
months. The fish oil was divided into aliquots and stored at 4°C
under nitrogen. The daily oil supplements were administered orally to
each pig via a syringe at the time of feeding. These precautions were
taken to minimize the oxidation of fish oil, which is known to occur at
room temperature in the presence of oxygen. All procedures were carried
out in accordance with University of Western Ontario regulations
governing the use of animals in research.
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Lipoprotein Cholesterol and Triglyceride
Profiles
Baseline lipoprotein profiles were determined from fasting blood
samples before the atherogenic diet. Thereafter, fasting blood samples
were analyzed bimonthly. Full lipoprotein analyses were
carried out on 20-mL blood samples taken from each animal after a 12-
to 16-hour fast. The blood samples were collected retro-orbitally into
25-mL syringes containing a solution of Na2EDTA (final
concentration, 5 mmol/L in blood). The plasma portion of each
blood sample was then isolated by centrifugation at
3000 rpm for 20 minutes at 4°C with a Sorvall HS-4 rotor. From the
plasma portion, the VLDL (d<1.006 g/mL), IDL
(d=1.006 to 1.019 g/mL), LDL (d=1.019 to 1.063
g/mL), and HDL (d=1.063 to 1.21 g/mL) lipoprotein fractions
were separated by sequential
ultracentrifugation17 with a Beckman Ti
50.4 rotor and Beckman 6.0-mL polypropylene "QuickSeal" tubes.
Absolute concentrations of cholesterol and
triglyceride were measured with the
high-performance CHOD-PAP and GPO-PAP kits, respectively, from
Boehringer Mannheim Canada Ltd.
LDL Fatty Acid Profiles
The LDL fraction of terminal fasting blood samples was separated
from the plasma and concentrated with an additional
ultracentrifugation. The samples were stored under
nitrogen at 4°C. Total fatty acid profiles were determined on all
terminal blood samples. A Folch extraction18 was used to
remove the lipid fraction, and fatty acid methyl esters were prepared
by the use of boron trifluoride/methanol according to the
American Oil Chemists Society Official Method Ce 1b-89. Individual
fatty acids were identified with a Hewlett-Packard 5890 Series II gas
chromatograph equipped with a split/splitless injection port, a
flame ionization detector, and a 30-m Econo-cap capillary column
(Alltech Associates, Inc) 0.32 mm in diameter with a 0.25-µm
carbowax stationary phase. Helium was the carrier gas and nitrogen was
the makeup gas, with a solvent split ratio of 1:70. The injector
temperature was 200°C, the detector was 250°C, and the oven was
programmed as follows: 150°C for 8 minutes, 3°/min to 190°C, and
hold for 15 minutes. Individual fatty acids and retention times were
verified by injection of appropriate fatty acid methyl ester
standards.
In Vitro Oxidation of LDL
With LDL from terminal blood samples, in vitro oxidation was
carried out according to the protocol of Kleinveld et al19
to measure the formation of conjugated dienes. Briefly, samples of LDL
(50 to 200 µg protein/mL; determined by the method of Lowry et
al20 as modified by Markwell et al21 )
containing 10 µmol/L Na2EDTA were incubated with
copper sulfate (5.0 µmol/L) in PBS. The kinetics of conjugated
diene production at 30°C was determined by continuous
monitoring of the change in absorbance at 234 nm over a period of 3
hours. The lag phase (minutes) of the oxidation curve was determined
from the intersection of the initial readings with the slope of the
exponential plot. The maximal rate of oxidation was determined by
calculating the slope of the exponential plot.
Morphological Analysis of Lesion Area
The perfusion fixation of the vessels and their subsequent
preparation and staining with the lipophilic dye Sudan IV have been
described elsewhere.15 22 Lesion extent was measured by
capturing the images on a computer and tracing the vessel perimeter to
determine vessel area. The percentage area stained with Sudan IV was
then determined with the computer imaging program JAVA (Jandel
Scientific).
Statistical Analysis
To determine whether significant differences existed in the
lipoprotein profiles and weights among the groups, ANOVA with repeated
measures on the time factor was applied separately to the atherogenic
and regression phases of the experiment. For the regression phase, the
8-month lipoprotein values were used as covariates. For all other
parameters, a one-way ANOVA with a post hoc Scheffé
test was used to determine the significance of differences among the
groups. In all cases, P
.05 was considered statistically
significant.23
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Results |
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All animals remained healthy throughout the experiment. At 8 months, there was no significant difference among the groups with respect to weight (average weight, 42 kg; range, 39 to 45 kg). Similarly, at 12 months, the end of the regression phase, there was also no significant weight difference (average weight, 56 kg; range, 55 to 59 kg).
The total plasma and lipoprotein fraction cholesterol
levels for the NR group are shown in Fig 1. Total plasma
cholesterol levels increased steadily over the first 6
months, and by 8 months the levels had reached a steady state. The
majority of the cholesterol was carried by the LDL and IDL
fractions, and the changes in cholesterol levels over time
were due primarily to increases within these fractions.
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Fig 2 shows the total plasma cholesterol
concentrations for those groups of animals placed on the regression
diet. In all cases, the total plasma cholesterol levels
increased steadily to 15 to 20 mmol/L during the induction phase,
returned to baseline (
2.4 mmol/L) within 2 months on the
regression diet, and remained at that level for the duration. The
general shape of this curve was also typical for the lipoprotein
fractions.
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Effects of fish oil and control oil supplements on lipoprotein
cholesterol and triglyceride levels are
summarized in Table 2. The total plasma
cholesterol levels were not significantly altered in the
regression groups, nor were the IDL cholesterol levels.
VLDL cholesterol was significantly lowered in both the CO
and FO groups (23% and 40%, respectively). Although there was a
significant increase in LDL cholesterol in the FO group
compared with the CO group, there was no difference between the NO
group and either the FO or CO group. HDL cholesterol levels
increased 29% in the CO group and decreased 6% in the FO group.
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More than 70% of the plasma triglyceride was carried in the VLDL fraction, with the remainder distributed equally among the other lipoprotein fractions. The plasma triglyceride levels increased only slightly over the course of the induction phase, and these changes were due to increases in the VLDL fraction. The total plasma and IDL triglyceride levels were not significantly affected by the oil supplements. VLDL triglyceride was reduced by both the MaxEPA and control oil supplementation; however, only the former was significant. In addition, only the MaxEPA-supplemented group had a significant increase (33%) in the LDL triglyceride levels.
The terminal LDL protein level in the FO group was 23% higher than the CO group (0.31±0.02 and 0.24±0.02 mg/mL, respectively). However, there were no differences between the protein levels of the NO compared with the FO and CO groups. There were also no significant differences among the regression groups with respect to the cholesterol/protein or cholesterol/triglyceride ratios.
The results of fatty acid analysis of the terminal LDL samples
are shown in Table 3. With the exception of 20:3 n-6, a
minor component, the fatty acid compositions of LDL from both the NR
and NO groups were identical. The addition of the control oil
supplement to the regression diet caused significant increases in 18:0
and 18:2 n-6 and significant decreases in 16:1, 18:1, and 18:3 n-3
fatty acids. In contrast, the addition of the fish oil supplement to
the regression diet caused a significant reduction in 18:1 and
significant increases in 20:5 and 22:6 fatty acids. Compared with the
CO group, there was significantly less 18:0 and 20:4 n-6 and
significantly more 16:1, 18:3, 20:5, and 22:6 n-3 in the LDL fatty
acids of the FO group.
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The time interval before exponential conjugated diene
production occurs when lipoproteins are exposed to copper ions
is a measure of the susceptibility of an LDL particle to oxidation; the
shorter this lag time, the more easily the particle is oxidized. There
were no significant differences among the terminal LDL lag times for
the NR, NO, and CO groups (Table 4). However, the
terminal LDL particles from the MaxEPA-fed pigs showed a significantly
shorter lag time than the other groups. There were no significant
differences in the rate and maximum conjugated diene production
in the terminal LDL particles among any of the regression groups.
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To examine the extent of lesion formation, the aorta was divided into
ascending, thoracic, and abdominal segments. The Sudan IV–positive
areas stain red and indicate regions of lipid infiltration (lesion
formation). After the induction phase, the ascending aorta showed
intense staining around the origins of the coronary arteries.
The staining extended from this region to cover almost the entire area
superior to the sinus of Valsalva. More than 50% of the surface area
of the ascending aorta was covered with lesion (Table 5). After the regression phase, the intensity of the
staining had diminished, with only small patches of deeply staining
areas remaining around the coronary ostia. Areas of the arch
that were intensely stained after the induction phase were almost
normal after regression, and the total area stained was reduced by
50%.
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The thoracic and abdominal segments of the aorta are shown in Fig 3. After the induction phase, the thoracic aorta had
lesions over essentially the entire arch area and the area extending
from the arch to the region of the ductus scar. In addition, lesions
were found in the areas surrounding the intercostal ostia (Fig 3A,
upper vessel). On average, 20% of the surface area of the thoracic
aorta stained after the induction phase. In contrast, the lipid
infiltration of the abdominal aorta had a more patchy distribution (Fig 3A, lower vessel), covering, on average, 17% of the surface area.
Regions distal to the branch points of the various vessels were
positive, as well as an area extending diagonally from the left renal
artery to the trigone region. After the regression phase, the arch of
the thoracic aorta was essentially clear, with the exception of lipid
staining remaining around the ductus scar and intercostal ostia (Fig 3B, upper vessel). On average, the percentage of the vessel surface
stained was reduced by >50% after the regression diet. In contrast to
the thoracic segment, in the abdominal aorta (Fig 3B, lower vessel)
there was no reduction in the percentage of surface area stained.
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Before regression, lesion formation in the carotid artery was confined
to regions of the bicarotid trunk, the origin of the right subclavian
branch, and the proximal portion of the left carotid branch. Only 6%
of the carotid artery surface was stained. After regression, there was
a reduction in staining in all of the lesion-prone regions, and the
total surface area stained was reduced by 75% (Table 5
).
Lesions were confined mainly to the regions proximal to and surrounding
the branches of the common iliac artery. There were some lesions
covering the flow dividers and areas distal to the ostia in the femoral
artery. After regression, the intensity of staining in these areas was
reduced; however, the percent of the vessel surface stained was not
significantly reduced after the regression phase (Table 5).
The right coronary, left anterior descending (including the
left common coronary), and left circumflex coronary
arteries were examined separately. Before regression, the Sudan IV
staining had a patchy distribution, with lesions surrounding and
extending distally to the ostia. Lesions extended over most of the left
common coronary and surrounded the ostia in the left anterior
descending artery. Lesions in the right coronary showed a much
less confluent distribution than the left coronary artery. The
left circumflex coronary artery had lesions at the branch
points. None of the coronary arteries examined showed a
statistically significant reduction in the percentage of the vessel
surface stained with Sudan IV after regression (Table 5).
There were no significant differences in the degree of lesion regression in any of the vessels examined when the FO group was compared with the CO group.
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Discussion |
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This study was designed to test the effects of n-3 fatty acids from fish oils on the regression of atherosclerotic lesions in the Yucatan miniature pig. The P/S ratio of the diet is known to have an effect on plasma cholesterol,24 25 which in turn has an effect on atherosclerosis.26 27 Therefore, a control oil was used that matched the P/S ratio of the fish oil. Thus, any effects observed would be primarily a result of the presence or absence of n-3 fatty acids in the diet. Atherosclerotic lesions were induced by dietary means alone; after 8 months, the atherogenic agents were removed and the normal (regression) diets supplemented with fish oil or a control oil to promote lesion regression. Atherosclerosis was evaluated by examining the percentage of the surface area of the major arteries covered by lesion.
The ranges of cholesterol (15 to 20 mmol/L) and triglyceride (0.5 to 0.7 mmol/L) concentrations observed during the progression phase of this study are consistent with results we have previously obtained15 and those found by other investigators using the same model.16 Within 2 months of the removal of the atherogenic stimulus, the plasma cholesterol levels returned to baseline values. These effects have also been seen in other regression studies using swine, with8 9 and without28 fish oil supplements.
The decrease in VLDL and increase in LDL cholesterol and triglyceride levels seen in our fish oil group are consistent with changes caused by n-3 fatty acids in humans.29 Other swine regression studies using fish oil found significant decreases in the total plasma, VLDL, and LDL cholesterol levels in groups supplemented with fish oil compared with those supplemented with lard.8 They also reported significant decreases in the total plasma triglyceride levels in the FO groups. In this experiment, however, the P/S ratio of the control oil (0.2:1) was significantly less than that of the fish oil (1.5:1).
A regression study on monkeys11 in which the same total
polyunsaturates were supplied by the fish oil and control diets found
that fish oil caused no significant changes in the plasma or
lipoprotein fraction cholesterol or
triglyceride levels. However, the daily fish oil and
control oil supplements averaged 168.9 and 76.6 mg/kg body wt,
respectively, which may be too low to influence the
parameters measured. In contrast, the animals in our study
received 435 mg/kg body wt of oil supplements with equal P/S ratios. On
average, the fish oil supplement in our study provided 4.4 g EPA
and 3.2 g DHA daily (143
mg·kg-1·d-1),
which more closely approximates the consumption of Greenland Eskimos
(157
mg·kg-1·d-1
EPA and DHA).30
It is well documented that fish oils cause a decrease in triglycerides (and consequently VLDL, the main triglyceride carrier). This decrease has been suggested to result from the interference of fish oil with the enzymes responsible for triglyceride production and secretion by the liver.31 32 33 34 Fish oil has also been shown to cause an increased conversion of VLDL remnants to LDL particles in pigs35 36 and a downregulation of the LDL receptor.36 37 38 These changes would result in an increased number of LDL particles due to the combination of increased production and decreased removal from the plasma. Consistent with the work of others, in our study fish oil caused an increase in LDL protein concentration, suggesting that there was an increased number of LDL particles in the plasma of these animals.
The saturation levels of the fatty acids in the diet have been shown to
be reflected in the fatty acid composition of the lipoprotein (LDL)
particles.39 40 For example, the enrichment of 18:1 fatty
acid in the diet results in an increased level of 18:1 in the LDL
particle.40 The differences in the fatty acid compositions
of the LDL particles from the FO and CO groups can be explained by
examining the fatty acid composition of the oil supplements. The fatty
acids 18:0, 18:2, and 20:4 are found in very low levels in MaxEPA
(5%) compared with the control oil (52%), whereas the fatty
acids 16:1, 18:3, 20:5, and 22:6 are found in higher proportions in the
fish oil (30.5% in MaxEPA versus 1.3% in the control oil). Similar
differences in the LDL fatty acid profiles from the terminal LDL
particles are seen when the FO and CO groups are compared. Regression
studies in both monkeys and pigs9 11 showed increased
incorporation of the n-3 fatty acids 20:5 and 22:6 into plasma lipids
at the expense of the n-6 fatty acids 18:2 and 20:4.
The increased levels of the highly unsaturated fatty acids in the FO LDL would suggest that these particles would be more susceptible to oxidation15 40 and hence, more atherogenic41 and less likely to allow lesion regression. Results from conjugated diene analysis, showing fish oil LDL particles with a lag time less than half that of the other groups, confirm that the fish oil particles were more susceptible to oxidation. Our laboratory has previously shown that, in addition to decreased lag time during conjugated diene production in LDL from FO pigs, there was an increase in electrophoretic mobility of these particles on agarose gels.12 It has also been shown that LDL from humans eating fish oil–supplemented diets are more susceptible to copper oxidation and macrophage-mediated modification and that both particles are more easily taken up by cultured macrophages,42 suggesting that these particles may be more atherogenic in vivo. In contrast, Nenseter et al43 demonstrated that n-3 fatty acid supplementation did not result in an increased susceptibility of human LDL to copper-induced oxidation.
On the basis of the oxidation profile of the LDL particles as well as the increase in LDL and decrease in HDL cholesterol (all factors that have been shown to be associated with increased atherosclerosis), one would predict that the lesion regression in the FO group would have been retarded by the oil supplement. Our results show that fish oil did not enhance or retard the regression of the atherosclerotic lesions compared with the control oil. Sassen et al8 concluded that in pigs, fish oil enhanced the regression and retarded the progression of coronary atherosclerosis compared with lard-fed animals. In a later experiment,9 however, they showed a significant reduction in the right coronary artery luminal encroachment but no significant reduction in aortic atherosclerosis with fish oil supplementation. It is important to note that in both of the above studies, the P/S ratios of the fish oil and control diets were not matched. In contrast, using monkeys, Fincham et al10 found no enhanced lesion regression with fish oil supplementation compared with a control oil with an equivalent P/S ratio.
In summary, this study examined the effects of a fish oil supplement on lesion regression in the pig model of atherosclerosis. After a switch from a high-fat/high-cholesterol diet to a low-fat/low-cholesterol diet, it was confirmed that lesion regression will occur under these conditions. The addition of a control oil supplement to the regression diet resulted in higher HDL cholesterol levels; however, this favorable change did not affect the extent of lesion regression. In contrast, the addition of a fish oil supplement caused an increase in LDL and a decrease in HDL cholesterol and caused the LDL particle to be more susceptible to oxidation. However, these unfavorable changes were also not reflected in the extent of lesion regression compared with the P/S-matched CO group. The addition of a fish oil supplement to a regression diet did not influence lesion regression in pigs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was funded by the Heart and Stroke Foundation of Ontario (Dr Rogers) and the National Institutes of Health, grant HL-45619 (Dr Guyton).
Received September 21, 1995; accepted July 16, 1996.
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References |
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