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ELSEVIER Atherosclerosis 108 (1994) 19-25

Conjugated linoleic acid and atherosclerosis in rabbits

Kisun N. Lee”, David Kritchevskyb, Michael W. Pariza”*

“FoodResearch Institute, Depurtmenr of Food Mkrohiology und Toxicology.
University of Wisconsin-Madison, 1925 Willow Drive. Madison. WI 53706, USA
bThe Wisrur Institute. Philudelphiu. PA, USA

(Received 30 August 1993: revision received 7 February 1994; accepted 2 March 1994)


Conjugated linoleic acid (CLA) consists of a series of positional and geometric dienoic isomers of hnoleic acid that
occur naturally in foods. CLA exhibits antioxidant activity in vitro and in vivo. To assess the effect of CLA on athero-
sclerosis, 12 rabbits were fed a semi-synthetic diet containing 14% fat and 0.1% cholesterol for 22 weeks. For 6 of these
rabbits, the diet was augmented with CLA (0.5 g CLAirabbit per day). Blood samples were taken monthly for lipid
analysis. By 12 weeks total and LDL cholesterol and triglycerides were markedly lower in the CLA-fed group. In-
terestingly, the LDL cholesterol to HDL cholesterol ratio and total cholesterol to HDL cholesterol ratio were
significantly reduced in CLA-fed rabbits. Examination of the aortas of CLA-fed rabbits showed less atherosclerosis.

Ke~worris: Antioxidant; Conjugated linoleic acid (CLA); Atherosclerosis; Lipoproteins; Semi-purified diet; Rabbit

1. Introduction Conjugated dienoic derivatives of linoleic acid

(CLA) are a series of positional and geometric iso-
Clinical studies have shown that increased levels mers of linoleic acid which occur naturally in
of low density lipoprotein (LDL) in plasma con- foods derived from ruminant animals [5]. Since
tribute significantly to the pathogenesis of athero- CLA exhibits antioxidant activity both in vivo [6]
sclerosis [l). Oxidative modification of LDL may and in vitro [7], we undertook an investigation of
enhance its atherogenicity [2]. It has also been the effect of CLA on experimental atherogenesis in
shown that treatment of spontaneously athero- rabbits.
sclerotic Watanabe heritable hyperlipidemic rab-
bits with probucol, a potent antioxidant, inhibits 2. Materials and methods
the progression of atherosclerosis [3]. It has been
reported that butylated hydroxytoluene (BHT) has 2.1. Experimental design
an anti-atherogenic effect in normal cholesterol- Six male and six female New Zealand White rab-
fed rabbits [4]. bits weighing between 3.3 kg and 4.2 kg were ran-
domly assigned to one of two groups of six (three
* Corresponding author. Tel.: 608 263 6955; Fax: 608 263 of each sex per group). The rabbits were fed a
i 114. semi-synthetic high-fat diet for a 4 week adapt-

0021-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved

SSDI 0021-9150(94)05240-J
20 K. N. Lee et al. /Atherosclerosis 108 (1994) 19-25

Table I bituric acid-reactive substances (TBARS), an indi-

Composition of experimental diet (g/kg) cator of lipid peroxidation level, were determined
in the plasma [12].
Casein, ‘vitamin-free’ test 250.0
t_-Arginine HCI 6.0
rn-Methionine 2.0 2.3. Tissue cholesterol analysis
sucrose 194.6 Livers were excised and weighed. Aliquots of liver
Corn starch 193.0 or aorta were homogenized in chloroformimetha-
Coconut oil, hydrogenated 120.0
no1 2: 1 [ 131and analyzed for free and total choles-
Corn oil 20.0
Cellulose 150.0
terol [14].
Vitamin mix, Teklad (40060) 10.0
Mineral mix 53.4 2.4. Morphological and histological evaluation of
Cholesterol 1.0 atherosclerosis
See Materials and methods for experimental detail. The aortas were excised from the rabbits. Excess
fat was carefully removed from the adventitial side
of the aorta, which was then washed under running
ation period. Then the animals received the same water. Aortas were opened longitudinally, pinned
semi-synthetic high fat diet containing 0. lo/ocholes- to a corkboard, fixed in 10% buffered formalin, and
terol for a 22 week experimental period (Table 1). stained by Sudan IV to reveal sudanophilic plaques
In addition, during the experimental period, rab- [ 151.Morphometric assessment of lipid depots was
bits in the CLA-treatment group were given 0.5 g graded in double blind fashion by two independent
CLA per day per rabbit: this amount was adopted observers before and after staining. For light
based on previous studies in our lab. We used the microscopy, segments of the thoracic and abdom-
same amount of coconut oil as our control because inal aortas were embedded in paraffin and stained
it was the major fat in our basal semi-synthetic diet. with hematoxylin and eosin. The thoracic and ab-
Throughout the experimental period, the rabbits dominal aortas were subjected to histological exam-
were restricted to 100 g food per day. Food con-
sumption and body weights were recorded daily and
weekly, respectively. At 4 week intervals, 17 h fast-
ing blood samples were drawn from central ear veins
--O-- Control
and used for plasma lipid analysis. At the end of
22 weeks the rabbits were sacrificed using an over-
dose of sodium phenobarbital.
2.2. Plasma lipid analysis
Blood samples were collected in tubes contain-
ing EDTA (1.5 mg/ml). Plasma was obtained im-
mediately by low speed centrifugation at 4°C. Very
low density lipoprotein (VLDL) was isolated from
fresh plasma by preparative ultracentrifugation [8].
High density lipoprotein (HDL) was isolated by
precipitation of the apo B containing lipoproteins
with sodium phosphotungstate-magnesium chlo-
ride [9]. Total cholesterol, VLDL cholesterol, and 0 4 8 12 16 20 24
HDL cholesterol in the plasma were measured en-
zymatically [lo]. LDL cholesterol was determined
by subtraction of VLDL and HDL cholesterol from Fig, I. Total plasma cholesterol * S.E. in rabbits fed experi-
total cholesterol. Triglyceride in the plasma was mental diet with or without CLA. The f-value for the 12-22
measured by an enzymatic method [l 11. Thiobar- week period was 0.124.
K. N. Lee et al. / Atherosclero.sis 108 (1994) 19-25 21

ination. Measurement of plaque thickness was made LJ” ,

at the point of maximal thickness of each section.

The sections were projected onto paper using a light
image projector. The outline of each arterial wall
and plaques on that wall were traced, and then the
plaque to wall volume ratio was measured. Two
other characteristics were also assessed: deposition
of lipids and the development of nontibrous and
fibrous connective tissues. The severity of these
characteristics was categorized as mild (if less than
25%) or severe (if more than 25%).

2.5. Statistical analysis

Results are expressed in mean f standard error
unless otherwise stated. Data were analyzed by the 0 4 8 12 16 20 24
Student’s t-test or analysis of variance (ANOVA).
Differences for semi-quantified histological data
were tested using the contingency table method for
Fig. 2. Total plasma triglyceride f SE. in rabbits fed experi-
a nonparametric statistical analysis. The rela- mental diet with or without CLA. The P-value for the O-22
tionships between variables were tested with the week period was 0.157.
Pearson correlation coefficient. Differences were
considered significant if the P-value was less than
0.05. However, some P-values greater than 0.05 groups over the 22 week experimental period.
are reported in the text and figure legends. There was no significant effect of CLA feeding on
plasma LDL cholesterol until 8 weeks. However,
3. Results and discussion by 12 weeks the LDL cholesterol concentration for

There was no difference in food intake between

the groups throughout the study (58.4 f 2.2 g/day 800

for control vs. 60.9 f 6.9 g/day for the CLA-fed + Control

group). Interestingly, during the first few weeks of

the experimental period the CLA-fed rabbits gain-
ed more weight than the control rabbits, indicating
an effect on feed efficiency which will be the sub-
ject of a report to be published elsewhere. How-
ever, at the completion of the experimental period,
body weights for the two groups were similar
(3.87 f 0.12 kg vs. 3.91 f 0.26 kg for control and
CLA-fed animals, respectively).
Throughout the experiment, all the rabbits
responded to the atherogenic diet similarly, with
marked increases in plasma total cholesterol com-
pared with values obtained prior to cholesterol 06
0 4 8 12 16 20 24
feeding (Fig. 1). The same observation was made
for plasma triglycerides (Fig. 2). As might be ex-
pected, the VLDL and LDL cholesterol levels in- Fig. 3. LDL cholesterol f SE. in rabbits fed experimental diet
creased as total plasma cholesterol increased. Fig. with or without CLA. The P-value for the O-22 week period
3 shows the LDL cholesterol profiles for both was 0.090 and for the 12-22 week period was 0.017.
22 K. N. Lee et al. /Atherosclerosis 108 (1994) 19-2.5

bits. However, Fig. 4 shows that the ratio of LDL

-O- Control cholesterol to HDL cholesterol in the plasma of
CLA-fed rabbits also was significantly (P < 0.05)
20 -
+ CLA lower than that for control rabbits. The ratio of
total cholesterol to HDL cholesterol between the
groups is of borderline significance (Fig. 5).
There was no difference in hepatic cholesterol
content between CLA-treated and control groups.
On the other hand, the CLA group displayed 30%
less cholesterol deposition in the aorta (18.8 f 4.6
mg/g and 13.2 f 2.5 mg/g for control and CLA
group, respectively.) The difference is not statistic-
ally significant but is nonetheless consistant with
the results of histological examination.
0 4 8 12 16 20 24 All rabbits exhibited atherosclerotic lesion dis-
tributions that are characteristic of this experi-
mental model. Lesions consisted of fatty streaks,
Fig. 4. LDL cholesterol/HDL cholesterol ratio f SE. in rab- mainly affecting the aortic arch and the descending
bits fed experimental diet with or without CLA. The P-value thoracic aorta. The effect on the abdominal aorta
for the O-22 week period was 0.060 and for the 12-22 week was variable. Aortic atherosclerotic involvement
period was 0.040. was assessed by determining the percentage of
total aortic surface covered by fatty lesions. The
the CLA-fed group was significantly (P < 0.02) involved aortic surface of the control group was
lower than that of the control group. We did not 55 f 14% whereas for the CLA group the value
find a significant difference in HDL cholesterol was 43 f 11%. This difference is not statistically
levels between the CLA-fed and the control rab- significant but is nonetheless consistant with data
on cholesterol deposition cited above.
The histological examination of the aortas of
both groups of animals showed the same type of

+ ContTol lesions, characterized by an accumulation of lipids
I and matrix fibers in the hyperplasic areas of the
--&-...- aA
inner layer of the intima as shown in Fig. 6. Table
2 gives the maximal thickness of plaque and pla-
que to wall volume ratio of each representative
section. The lesions in the thoracic aorta of all ani-
.S 20 mals were larger and more severe than abdominal
lesions. However, CLA-fed rabbits appeared to
have less developed lesions in their abdominal aor-
10 tas than the control rabbits. The plaque to wall
volume ratio of abdominal aortas between the
groups approached significance (P < 0.09). The
0 data (Table 3) also show that CLA-fed rabbits ex-
0 4 8 12 16 20 24 hibited less histological evidence of atherogenesis
in lipid deposition and in connective tissue de-
Fig. 5. Total cholesterol/HDL cholesterol ratio f SE. in rab-
There were many correlations between vari-
bits fed experimental diet with or without CLA. The P-value ables, including the end markers of atherosclerosis
for the 12-22 week period was 0.078. and plasma lipids. In both groups, a correlation
K. N. Lee et al. /Atherosclerosis I08 (1994) 19-25 23

Fig. 6. Light micrographs of representative aortic sections from the control rabbits (A) and CLA-treated rabbits (B). Sections were
taken from similar areas of the thoracic aorta. Hematoxylin and eosin stain. x 100.

between the aortic atherosclerosis involvement (%)

and final LDL cholesterol (r = 0.7626, P < 0.05)
was found. The aortic atherosclerosis involvement
Table 2
(“/I) in the control group also correlated with aver-
Aortic maximal plaque thickness and plaque to wall volume
ratio of rabbits fed experimental diet with or without CLA for
age LDL cholesterol (Y = 0.9357, P < 0.05) and
22 weeks with average plasma total triglycerides (r = 0.9577,
P < 0.05). Aortic cholesterol content was correl-
Control CLA ated with final plasma total cholesterol (v =
Mosimal thickness of pluque (mm) 0.8071, P < O.Ol), and with average plasma total
Thoracic 0.33 f 0.03 0.33 ?? 0.09 cholesterol (r = 0.7115, P ~0.05). There was a
Abdominal 0.22 f 0.05 0.16 zt 0.03
correlation between the abdominal plaque to wall
Pluque lo M.uII volume rtllio
volume ratio and average plasma total choleste-
Thoracic 0.547 f 0. I55 0.483 ?? 0. I70 rol/HDL cholesterol ratio (r = 0.9938, P < 0.01)
Abdominal 0.345 f 0. IO7 0. I I3 f 0.030 in the CLA group. Average LDL cholesterol/HDL
See Materials and methods for experimental detail. There were
cholesterol ratio and hepatic cholesterol content
no statistically significant differences between control and was negatively correlated (r = -0.8854, P < 0.05)
CLA-treated animals. in this CLA group. It is believed that the retarda-
24 K. N. Lee et al. /Atherosclerosis 108 (1994) 19-25

Table 3
The histological evaluation of the lipid deposition and the connective tissue development in aortas of rabbits fed experimental diet
with or without CLA for 22 weeks

Thoracic aorta Abdominal aorta

Control n CLA n P Control n CLA n P

Lipid deposition
Mild I 3 2 4
Severe 5 3 4 2
0.07 0.10

Connective tissue development

Mild 2 5 3 5
Severe 4 I 3 I
0.01 0.07

See Materials and methods for experimental detail.

tion of atherosclerosis by CLA is at least in part that CLA is clearly established as an anticar-
due to the changes in lipoprotein metabolism. cinogen [6,7].
Our observation is supported by Nicolosi et al.
[ 161. They very recently reported that CLA-fed Acknowledgements
hamsters had significantly lower plasma total and
LDL cholesterols as well as triglycerides levels. This work was supported in part by the College
They also found that CLA-fed animals had a signi- of Agricultual and Life Sciences, University of
ficant decrease in aortic streak formation. Wisconsin-Madison, research grants provided by
Based on the TBARS assay, lipid peroxidation the Wisconsin Milk Marketing Board and Kraft-
in the plasma appeared similar for the two groups General Foods, unrestricted gift funds adminis-
(1.67 f 0.11 nmol/ml for control, 1.64 f 0.12 tered by the Food Research Institute, University
nmol/ml for the CLA-treated group). However, of Wisconsin-Madison, and Research Career
the TBARS assay does not detect all lipid peroxi- Award HL00734 from the National Institute of
dation products and is also subject to interference Health to D. Kritchevsky. We thank Dr. Stanley
by other substances [17]. In addition, the TBARS Goldfarb for histological analysis.
assay does not always correlate with the progres-
sion of atherosclerosis [ 181.
Using the TBARS assay as an index of lipid
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