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Conjugated Linoleic Acid


David Kritchevsky

CONTENTS
8.1
8.2

Introduction
CLA and Cancer
8.2.1 Tissue Culture Studies
8.2.2 Human Studies
8.3 CLA and Experimental Atherosclerosis
8.4 Other Effects of CLA
8.4.1 Effects on Body Fat
8.4.2 Diabetes
8.4.3 Immune System
8.5 Mechanisms of Action
Acknowledgment
References

8.1 INTRODUCTION
Conjugated linoleic acid (CLA) is an inclusive designation of a number of naturally
occurring octadecadienoic acids. CLA is a product of the biohydrogenation of
linoleic acid that occurs in ruminant animals. It is found principally in dairy products
and the meat of ruminant animals; the major isomer in these foods is the cis9, trans11
modification. The concentration of CLA in food sources is about 1 to 10 mg/g fat.
Chin et al. (1992) have published a summary of the levels of CLA found in foods
and Fritsche and Steinhard (1998) estimate that daily intake in man ranges from 0.3
to 1.5 g per person.
Parodi (1999) has described the early history of CLA beginning with its discovery in milk fat in 1932 and discusses the chemistry leading to proof of its structure.
The conjugated diene present in milk fat was shown to be an intermediate in the
course of hydrogenation of linoleic acid by the rumen bacterium Butyrivibrio fibrisolvens (Kepler et al. 1966; Kepler and Tove 1967). CLA might have remained an
interesting component of rumen chemistry if it had not been for the discovery by
Pariza et al. (1979) of an antimutagen in cooked hamburger and the demonstration
by Ha et al. (1987) that the active material was a mixture of CLA isomers, namely,
the cis9, trans11; trans9, trans11; trans10, cis12, and trans10, trans12 modifications.
These observations and the commercial availability of CLA isomers led to a decade

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TABLE 8.1
Influence of Mutagenesis Modulator on
Dimethylbenz(a)anthracene-Induced Skin Tumors in Mice
Papillomas/Mouse
Groupa

Control

Test

A) SENCAR mice
B) SENCAR mice
C) CD-1 mice

30.2 3.4
9.6 0.8
10.0 1.7

13.4 2.4
4.5 0.9
3.2 1.1

0.01
0.01
0.01

A) 20-week study; modulator prepared by ion exchange. B) 19-week study;


modulator prepared by extraction. C) 10-week study; modulator prepared on
florisil.
Source: From Pariza, M.W. and Hargraves, W.A., Carcinogenesis, 6, 591593,
1985. With permission.

of intense research into the anticarcinogenic effects of CLA and, predictably, into
its effects on other diseases. At this writing the bulk of CLA literature still relates
to its anticarcinogenic effects.

8.2 CLA AND CANCER


The material that Pariza et al. (1979) called mutagenesis modulator was found to
be present in uncooked as well as in cooked hamburger and shown to inhibit
isoquinoline-induced mutagenicity in the Ames test (Pariza et al. 1983). Pariza and
Hargraves (1985) showed that mutagenesis modulator could inhibit 7,12-dimethylbenz(a)anthracene (DMBA)-induced epidermal tumors in mice (Table 8.1). This was
2 years before Ha et al. (1987) established its structure.
Base-catalyzed isomerization of linoleic acid-rich oils produces a mixture of
CLA forms, principally the c9,t11 and t10,c12 isomers. The commercial preparations
used in most of the studies described next contain 40 to 45% of each of the major
isomers. This isomer mixture was compared with linoleic acid for ability to inhibit
benzo(a)pyrene (BP)-induced forestomach tumors in mice. CLA inhibited tumor
formation by 40 to 50% whereas linoleic acid was without effect (Ha et al. 1990).
The early studies showed that CLA could affect tumorigenicity when applied
directly to the tumor site. Ip and his colleagues showed that dietary CLA also
inhibited chemically induced mammary tumors in rats independently of type or
amount of dietary fat and independently of type of carcinogen. Ip et al. (1991) fed
female Sprague-Dawley rats a basal diet or one augmented with 0.5, 1.0 or
1.5% CLA. The dietary regimen was instituted 2 weeks prior to administration
of DMBA and continued to the end of the experiment (24 weeks); even the
lowest level of CLA exerted some antiumorigenic effect (Table 8.2).
To address the possibility that the CLA effect was due to interference with the
metabolic conversion of DMBA to an active form, Ip et al. (1994) tested CLA effects
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TABLE 8.2
Effect of Different Levels of Dietary CLA on 7,12Dimethylbenz(a)anthracene-Induced Mammary Tumors in
Sprague-Dawley Rats (30/group)
Tumors
Group
1
2
3
4
5
a

CLA (%)

DMBA

Incidence

No.

0.5
1.0
1.5
1.5

+
+
+
+
0

80.0
66.7
46.7a
40.0a
0

81
55a
36a
32a
0

Multiplicity
2.7
1.8
1.2
1.1
0

0.3
0.2a
0.2a
0.1a

p < 0.05 compared to control.

Source: After Ip, C. et al., Cancer Res., 51, 61186124, 1991.

TABLE 8.3
Effect of CLA on Mammary Tumors in Sprague-Dawley Rats
Induced by 7,12-Dimethylbenz(a)anthracene (DMBA) or
Methylnitrosourea (MNU)
Tumors
Carcinogen
DMBA
DMBA
MNU
MNU
a
b

CLAa (%)

Incidence

Number

80
52b
88
60b

62
38b
76
50b

CLA fed from weaning to 1 week postcarcinogen administration (5 weeks).


p < 0.05 compared to control.

Source: After Ip, C. et al., Nutr. Cancer, 24, 241247, 1995.

on the carcinogenicity of methylnitrosourea (MNU; a direct acting carcinogen). In


a 36-week study they found that dietary levels of CLA as low as 0.1% decreased
significantly the carcinogenicity of DMBA. They also found that 1% dietary CLA
reduced the incidence of DMBA-induced mammary tumors by 35% and the incidence of MNU-induced mammary tumors by 32% (Table 8.3). CLA was shown to
reduce the proliferative activity of ductal and lobuloalveolar mammary epithelial
cells by 15 and 23%, respectively. This observation offered a suggestion that CLA
might affect mammary tumorigenesis by a direct effect on the target organ. CLA
feeding for only a short period in the rats life corresponding to postweaning and
puberty was enough to reduce tumorigenesis caused by subsequent administration
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of MNU by about 25 to 30%, depending on time of MNU treatment (Ip et al. 1995).
The same study showed that feeding 1% CLA as a triglyceride or as the free fatty
acid had virtually the same effect on MNU-induced tumorigenesis.
The amount and type of dietary fat can influence the course of experimental
carcinogenesis; linoleic acid enhances experimental carcinogenesis. The possibility
that elevated levels of linoleic acid might swamp the CLA effect was tested in rats
fed a fat blended to reflect the fatty acid composition of the American diet. The ratio
of saturated to monounsaturated to polyunsaturated fatty acids in the blended fat
was 1:1:1. Inhibition of DMBA-induced mammary tumorigenesis was virtually the
same in rats fed 1% CLA together with 10, 13.3, 16.7 or 20% of the blended fat
(Ip et al. 1996). The amount of linoleic acid in the diet (2 or 12%) did not affect
inhibition of DMBA-induced mammary cancer by CLA.
Liew et al. (1995) studied the effect of CLA on colon carcinogenesis induced
by 2-amino-3-methylimidazo[4,5f]quinoline (IQ) in F344 rats. IQ alone yielded
100% incidence of aberrant crypt foci (4.3 2.4 ACF per rat); when CLA (0.5%)
was added to the diet, ACF incidence was reduced by 40% and ACF per rat was
reduced to 1.1 1.3 (p < 0.05). When the diet contained safflower oil, ACF incidence
was 100% but ACF per rat was reduced by 26% (3.2 1.7). Dietary CLA reduced
IQDNA adducts in the liver by 27% and in the colon by 41%.
Belury et al. (1996) showed that increasing levels of dietary CLA reduced
promotion of skin tumors in mice promoted by phorbol ester (12-0-tetradecanoyl
phorbol-13-acetate). Papilloma yield after 24 weeks in mice fed 0, 0.5, 1.0 or 1.5%
CLA was 6.71, 5.92, 4.83, and 4.67, respectively. Level of inhibition by the two
higher concentrations of CLA was statistically significant (p < 0.05).
The SCID (severe combined immunodeficient) mouse provides a vehicle for
examining effects of human tumor cells in an experimental animal model (Cesano
et al. 1992). Subcutaneous injection of tumor cells into this model results in tumor
growth at the site of injection as well as metastatic proliferation of the tumors. SCID
mice were fed 1% CLA for 2 weeks prior to subcutaneous inoculation of 107 MDAMB468 cells (human breast adenocarcinoma). The mice were followed for 14 weeks.
After 9 weeks, the weight of tumors in the treated mice was 74% (p < 0.01) lower
than in the controls and tumor area (mm3) was reduced by 87% (p < 0.01). At 14
weeks, tumor weight in treated mice was reduced by 30% (p < 0.02) and tumor
weight by 62% (p < 0.05). No breast cancer cells spread to lungs, peripheral blood
or bone marrow (Visonneau et al. 1997).
SCID mice were also fed a semipurified diet containing 1% CLA for 2 weeks
prior to injection with 5 106 DU-145 cells (human prostatic carcinoma) and
followed for 12 weeks. In addition to a control group maintained on laboratory
ration, a third was fed a semipurified diet containing 1% linoleic acid. Food intake
(1 g/day) at week 12 was similar in the control and linoleic acid-fed groups (about
4 g) and significantly lower in the CLA group (about 3 g). Tumor volume measured
at 4 weeks was similar in all three groups; by 8 weeks tumor volume in the linoleic
acid-fed mice was slightly larger than in the controls and significantly larger
(p < 0.05) than in CLA-fed group. By 12 weeks, tumor growth in the CLA-fed group
was only slightly greater than it was at 4 weeks (about 400 mm3), whereas in the
linoleic-acid and control groups it had risen several fold. Tumor mass was lowest
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in the CLA-fed group (p < 0.001) against both controls and highest in the mice fed
linoleic acid (p < 0.05) against commercial diet). CLA-fed mice exhibited a dramatically reduced number of lung metastases compared to the other two groups; they
showed no metastases to any other tissue (Cesano et al. 1998).

8.2.1 TISSUE CULTURE STUDIES


A number of in vitro studies of CLA have reported on the growth of cells in culture.
Visonneau et al. (1996) examined the comparative effects of different concentrations
of CLA or linoleic acid (104, 105, and 106 M) on a number of cell lines. Five
breast carcinoma cell lines were tested: MBA-MB468, MCF7, MDA-MB 231, HS578T, and BT-474. Except for the BT-474 line, CLA inhibited growth at every
concentration. Linoleic acid at a concentration of 104 M stimulated cell growth. At
the lowest concentration, linoleic acid was generally inhibitory. CLA inhibited
growth at all concentrations and inhibited growth of a prostatic carcinoma cell line
(DU145) as well as a melanoma cell line (WM451) at all three concentrations. CLA
at 104 M inhibited growth of a colon carcinoma cell line (HT-29) and a glioblastoma
cell line (U87-MG).
Shultz and his co-workers (1992a, b) and Cunningham et al. (1997) studied three
human cell lines. When CLA was incubated with human malignant melanoma (M21HPB), colorectal (HT-29), or breast cancer (MCF-7) cells, reduction in growth,
depending on time and dose, was observed. All three cell lines, when incubated with
CLA, incorporated less tritium-labeled leucine than did controls, suggesting cytotoxicity. Linoleic acid and CLA were used at concentrations of 1.78 to 7.14 105.
Linoleic acid stimulated MCF-7 cell growth initially but became inhibitory at 8 to
12 days. CLA, on the other hand, was inhibitory at all concentrations; inhibition
reached 100% after 12 days. These researchers investigated the possibility that the
CLA effect involved inhibition of eicosanoid synthesis. Their findings suggested that
CLA activity was mediated through inhibition of lipoxygenase activity. Linoleic acid,
but not CLA, increased peroxide concentrations in normal human breast cells (HMEC)
and MCF-7 cells. Treatment of MCF-7 cells with CLA and a cyclooxygenase inhibitor
(indomethacin) stimulated cell growth, but when CLA was used together with a
lipoxygenase inhibitor (nordihydroguaiaretic acid, NGDA) growth was inhibited.
Durgam and Fernandes (1997) attributed the inhibitory effect of CLA on growth
of human breast cancer cells to interference with the hormone-regulated mitogenic
pathway. They compared CLA effects on estrogen responsive (ER) MCF-7 cells and
ER negative MDA-MB-231 cells. They found that CLA (3.5 105 M) decreased
MCF-7 cell growth significantly but had no effect on the growth of the MDAMB-231 cells.
Schonberg and Krokau (1995) suggested that the CLA effect involved lipid
peroxidation. In contrast, using a human hepatoma cell line (HepG2), Igarashi and
Miyazawa (2001) attributed growth inhibition by CLA to alterations in fatty acid
metabolism but not lipid peroxidation. Their indication of peroxidation was formation of thiobarbituric acid-reactive substances (TBARS), which was 6% higher in
cells incubated with linoleic acid than in those incubated with CLA. The level of
TBARS production was about the same as that seen in control cells and in every
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case was strongly inhibited by addition of a-tocopherol. Total fatty acid content of
HepG2 cells incubated with CLA was 32% greater than controls and 53% greater
than cells grown with linoleic acid. The major differences between the fatty acid
composition of control cells and those grown with CLA was in the higher content
of palmitic acid (+35%), palmitoleic acid (+83%) and stearic acid (+37%). Cellular
lipids were increased significantly in cells supplemented with CLA. The data suggest
stimulation of de novo lipid synthesis.
Studies of the inhibitory effects of CLA on DNA adduct formation with IQ or
PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) show that, in general,
CLA reduces adduct formation so it may act as a blocking agent under certain
conditions. The mechanism by which CLA blocks DNA adduct formation has not
been elucidated (Liew et al. 1995; Schut et al. 1997; Josyula and Schut 1998).
An active area of research and speculation concerns effects of CLA on eicosanoid
metabolism. Many investigators have shown that CLA is readily incorporated into
cell membrane phospholipids; thus, CLA may displace arachidonic acid in phospholipids. In a study of effects of CLA in MCF-7 and SW480 cancer cells, Miller et al.
(2001) found that the c9, t11 isomer significantly decreased conversion of arachidonic acid to PGE2. The underlying finding was that CLA reduced incorporation of
arachidonic acid into the phosphatidyl choline (lecithin) that is the preferential
substrate for phospholipase A2, which, in turn, releases arachidonic acid for
eicosanoid synthesis. Arachidonic acid is released from phospholipids by the action
of phospholipase A2 and converted to eicosanoids along the cyclooxygenase pathway
(yielding prostaglandins and thromboxanes) as well as the lipoxygenase pathway
(yielding leukotrienes). Eicosanoids affect cell proliferation, inflammation and
immunity, all of which may influence carcinogenesis.

8.2.2 HUMAN STUDIES


A basic interest lies in relating experimental findings to possible CLA effects on
human cancers. Knekt et al. (1996) reported that a high intake of milk is correlated
with a reduced risk of breast cancer and Knekt and Jrvinen (1999) have reviewed
the field. Analysis of epidemiological evidence relating to dairy products and risk
of breast cancer is confusing. High consumption of dairy products has been linked
to both reduced and elevated risks. Many modifying factors are in these studies,
such as age, body weight, menopause, components of the specific dairy products
under consideration, other components of the diet and caloric intake.
Lavillonniere and Bougnoux (1999) report preliminary data from a study of the
CLA content of breast adipose tissue from women with breast cancer and controls.
CLA content of control tissue was elevated compared to that in afflicted tissue. Aro
et al. (2000) studied serum and dietary CLA in Finnish women with breast cancer.
They examined 195 cases (35% premenopausal) and 208 controls (36% premenopausal). In postmenopausal women, dietary CLA, serum CLA, myristic acid and
trans-vaccenic acid were significantly lower in cases than in controls.
Animal data are almost unanimous in finding CLA to inhibit experimental
tumorigenesis and tumor cell proliferation in vitro. There are few data relating CLA
in the human diet to anticarcinogenesis. CLA is a component of a class of dietary
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foods (animal products) that have borne the brunt of the epidemiological blame for
human carcinogenesis. Continuing experimental evidence from CLA research may
lead to reconsideration of dietary effects on carcinogenesis.

8.3 CLA AND EXPERIMENTAL ATHEROSCLEROSIS


Reports that CLA might exhibit antioxidant activity in vivo and in vitro and
knowledge that oxidized cholesterol (Imai et al. 1976) and oxidized low density
lipoprotein (LDL) (Steinberg et al. 1989) significantly promoted atherogenesis
prompted investigation of the effects of CLA on experimental atherosclerosis.
In the first study, control and test groups of six rabbits each (three male, three
female) were fed a semipurified diet containing 0.1% cholesterol for 22 weeks. The
diet of the test rabbits was augmented with 0.5 g/d of CLA. At necropsy, plasma
total cholesterol, LDL-cholesterol, and triglyceride levels were lower than those of
controls but not significantly so. The plasma LDL/high-density lipoprotein (HDL)
cholesterol ratios were 16.5 for the control group and 10.9 for the CLA-fed group.
Histological examination of the aortas showed maximal plaque thickness (mm) in
the thoracic aorta to be the same in both groups; however, maximal plaque thickness
in the abdominal aorta of CLA-fed rabbits was 27% smaller. Lipid deposition and
connective tissue development were less severe in the aortas of the CLA-fed
rabbits (Lee et al. 1994). On the basis of the thiobarbituric acid-reactive substances assay, levels of plasma peroxides were the same for both groups.
A second experiment was designed to confirm the apparent antiatherogenicity
of CLA and also to examine its effects on progression or regression of established
atherosclerotic lesions. Once established in rabbits, atheromata do not regress after
removal of the atherogenic stimulus and may become slightly more severe. Some
hypolipidemic drugs added to a cholesterol-free diet after lesions have been established may cause a slight reduction in severity of atherosclerosis. A group of 30
rabbits was fed a semipurified atherogenic diet containing 0.2% cholesterol (control);
10 rabbits were fed the same diet augmented with 1% CLA. After 90 days, the rabbits
receiving the control diet were bled and randomized into three groups of 10 rabbits
each of equal serum cholesterol levels. One group of control rabbits and the CLAfed rabbits were necropsied. The two remaining groups were placed on a cholesterolfree diet with or without 1% CLA. These rabbits were necropsied 90 days later.
No significant differences were found in weight gain or serum lipids. However,
the rabbits fed CLA exhibited 31% less severe atheromata in the aortic arch and a
40% lower severity in the thoracic aorta (Table 8.4). After rabbits were on the
regression regimen for 90 days, serum cholesterol had fallen by 83% on the control
regimen and 67% on the CLA diet. The percentage of HDL-cholesterol had risen
by 121 and 74%, respectively. Serum triglycerides were unchanged in the control
group but were 58% lower in the CLA-fed animals. Examination of the level of
atherosclerosis revealed practically no change in the control rabbits; however, severity of atherosclerosis in the aortic arch and thoracic aorta of the CLA-fed rabbits
had been reduced by 31 and 30%, respectively. The involved area had been reduced
by 31% (Table 8.5).
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TABLE 8.4
Necropsy Data for Rabbits Fed 0.2% Cholesterol with
(Control) or without 1% CLA for 90 Days
Group
(n = 10)

Weight gain (g)


Liver weight (g)
Liver (% body weight)
Serum (mg/mL)
Cholesterol
% HDL-Ca
Triglyceride
Aorta (04 scale)
Arch
Thoracic
Area (%)
a

Control

CLA

822 1.22
109 5
3.13 0.14

742 87
111 7
3.24 0.18

430 40
6.1 0.44
77 6

559 53
3.6 0.51
135 36

2.39 0.47
2.35 0.36
49 1

1.65 0.37
1.40 0.40
30 10

HDL-C = high-density lipoprotein cholesterol.

Source: After Kritchevsky, D. et al., J. Am. Coll. Nutr., 19, 474S477S, 2000.

TABLE 8.5
Necropsy Data for Rabbits with Established Atherosclerosis Fed
Corn Oil with (Control) or without 1% CLA for 90 Days
Group
(n = 10)

Weight gain (g)


Liver weight (g)
Liver (% body weight)
Serum (mg/mL)
Cholesterol
% HDL-Ca
Triglyceride
Aorta (04 scale)
Arch
Thoracic
Area (%)
a

Control

CLA

461 65
99 9
2.33 0.20

246 37
83 6
2.31 0.13

73 10
13.5 1.02
77 13

140 24
10.6 0.81
57 5

2.35 0.35
2.30 0.40
51 11

1.65 0.26
1.65 0.22
34 6

HDL-C = high-density lipoprotein cholesterol.

Source: After Kritchevsky, D. et al., J. Am. Coll. Nutr., 19, 474S477S, 2000.

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TABLE 8.6
Necropsy Data for Rabbits Fed 0.2% Cholesterol with 0.0 (Control), 0.1, 0.5,
or 1.0% CLA for 90 Days
Group
(n = 8)
Control
Weight gain (g)
Liver weight (g)
Liver (% body weight)
Serum (mg/dL)
Cholesterol (%)
% HDL-Ca
Triglyceride
Aorta (04 scale)
Arch
Thoracic
Area (%)
% Ester cholesterol
a

0.1% CLA

0.5% CLA

1.0% CLA

104 46
68 5
2.73 0.16

3 108
77 6
3.22 0.35

67 43
66 4
2.63 0.12

50 79
78 6
3.35 0.32

983 118
5.0 0.9
190 32

1281 116
3.3 0.54
246 47

1263 104
3.3 0.58
205 48

1103 134
5.0 1.14
216 38

2.36 0.39b,c
2.21 0.42e,f
44 12g
74.7

1.69 0.23d
1.31 0.28
32 7h
52.0

0.88 0.20b,d
0.75 0.21e
11 4g,h
34.1

1.00 0.28c
0.94 0.27f
18 6
44.3

HDL-C = high-density lipoprotein cholesterol.


Values in a horizontal row bearing the same letter are significantly different.

bh

Source: After Kritchevsky, D. et al., J. Am. Coll. Nutr., 19, 474S477S, 2000.

The next study was designed to investigate different concentrations of CLA for
their antiatherogenic potential as well as their effects on pre-established lesions.
Accordingly, one large group of 40 rabbits was fed the same diet augmented with
0.1, 0.5, or 1.0% CLA. The postcholesterol-feeding phase of the study involved
feeding the cholesterol-free diet or the same diet containing 0.1, 0.5 or 1.0% CLA
to rabbits with established lesions. The results of the first phase of the study are
summarized in Table 8.6. As in the earlier study, no differences in serum lipids were
present. The effects of CLA on atherogenesis were striking. Even at 0.1% of the diet,
CLA reduced severity of atherosclerosis in the aortic arch and thoracic aorta by 28
and 41%, respectively. When fed as 0.5 or 1% of the diet, CLA significantly reduced
atherosclerotic involvement in the aortic arch and thoracic aorta. The percentage of
esterified cholesterol present in the aortic tissue is a value that can also be used as
a measure of severity of atherosclerosis. This parameter paralleled the other findings.
Rabbits with pre-established atherosclerosis were placed into four groups of
seven rabbits each. One group was fed the control diet and the others were fed the
same diet plus 0.1, 0.5 or 1.0% cholesterol. As in the earlier study, after 90 days of
consuming a cholesterol diet, the severity of lesions in the aortic arch and thoracic
aorta was increased (by 12 and 4%, respectively). Addition of 0.1 or 0.5% CLA to
the diet had virtually no effect on the severity of atherosclerosis (Table 8.7). However,
consistent with the earlier study, at 1% of the diet, CLA reduced the severity of
aortic arch atheromata by 27% and that in the thoracic aorta by 45% (p < 0.01)
(Kritchevsky et al. 2000).
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TABLE 8.7
Necropsy Data for Rabbits with Established Atherosclerosis Fed Corn Oil
with 0.0 (Control), 0.1, 0.5, or 1% CLA for 90 Days
Group
Control
(n = 7)
Weight gain (g)
Liver weight (g)
Liver (% body weight)
Aorta (04 scale)
Arch
Thoracic
Area (%)
ae

0.1% CLA
(n = 6)

0.5% CLA
(n = 7)

1.0% CLA
(n = 6)

312 99
52 3
1.82 0.07b

265 104
58 6
2.05 0.16

298 84
64 5a
2.28 0.20b,c

242 73
47 3a
1.75 0.13a

2.64 0.28
2.29 0.36d
53 7

2.25 0.28
2.33 0.44
53 10

2.50 0.29
2.00 0.15e
49 5

1.95 0.40
1.25 0.17d,e
30 10

Values in a horizontal row bearing the same letter are significantly different.

Source: After Kritchevsky, D. et al., J. Am. Coll. Nutr., 19, 474S477S, 2000.

The commercial availability of the two major CLA isomers has made it possible
to test them individually for their effects in experimental atherogenesis. Rabbits were
fed an atherogenic diet containing 0.2% cholesterol and the same diet augmented
with 0.5% of the isomer mix, c9,t11-CLA or t-10,c12-CLA. All three test diets
inhibited atherogenesis to the same extent (about 55 4%).
Administration of cholesterol and saturated fat to hamsters leads to aortic
sudanophilia (Kowala et al. 1991). Nicolosi et al. (1997) fed male hamsters (10 per
group) an atherogenic diet consisting of 88.9% commercial ration, 10% coconut oil,
1% safflower oil, and 0.12% cholesterol or the same diet augmented with CLA (0.25
to 5.0%) or linoleic acid (5%). Total cholesterol levels were reduced in all of the
test groups. The fatty streak area was reduced by 19, 26, and 30%, respectively, in
hamsters fed 0.25, 0.50, or 1.0% CLA. Linoleic acid feeding reduced fatty streak
area by 25%. It was determined in a subsequent study (Wilson et al. 2000) in which
hamsters (12 per group) were fed the atherogenic diet alone, or the diet plus 1%
CLA or 1% linoleic acid, that CLA and linoleic acid significantly lowered total
plasma cholesterol levels (control, 327 16; CLA, 285 11; LA, 264 8 mg/dL).
The aortic fatty streak area (mm2/mm2) was 19.4 2.25 in the controls, 10.3
1.55 in the hamsters fed CLA and 17.1 2.62 in those fed linoleic acid. The
difference between the CLA and control groups was significant. LDL oxidation was
reduced significantly in the CLA-fed hamsters.

8.4 OTHER EFFECTS OF CLA


8.4.1 EFFECTS

ON

BODY FAT

When CLA is fed to growing animals it leads to reduced fat deposition and increased
body mass. Park et al. (1997, 1999a) showed that when CLA (0.5 to 1.0%) was fed
to growing mice, body fat was severely reduced and weight gain could be accounted
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for by increased body protein. The effect was seen in mice fed a CLA mix but it
has been shown that the t10,c12 isomer is the active entity vis--vis fat loss and
protein accretion (Park et al. 1999b). The effect on fat loss and protein accretion has
also been seen in growing pigs (Dugan et al. 1997). Studies in tissue culture have
shown CLA to enhance lipolysis (Park et al. 1999b), suppress triglyceride accumulation and enhance apoptosis (Evans et al. 2000).

8.4.2 DIABETES
CLA (1.5%) fed to the Zucker diabetic fatty (fa/fa; ZDF) rat results in significant
reductions in fasting glucose (24%), insulin (47%), triglycerides (51%) and free
fatty acids (49%). In contrast, a diet containing thiazolidinedione reduces the same
parameters by 24, 82, 86, and 60%, respectively. The study was suggested by the
observation that CLA was a PPARg activator (Belury and Vanden Heuvel 1999).

8.4.3 IMMUNE SYSTEM


Sugano et al. (1999) have reviewed the effects of CLA on immune function. CLA
(0.5 or 1.0% in the diet) lowers the levels of prostaglandin E2 in the serum of rats
significantly (by 14 and 24%, respectively). Dietary CLA (1%) will lower leukotriene
(LT) B4 levels in spleen and lung by 19 and 35% and LTC4 levels in these organs
by 8 and 68%. Levels of immunoglobulins A, G, and M in rat spleen lymphocytes
are increased significantly by dietary CLA even when fed at levels as low as 0.05%.
Immunoglobulin production in mesenteric lymph node lymphocytes is reduced significantly by dietary CLA. CLA may exert its antiallergic effects through its regulation of eicosanoid production and enhancement of immunoglobulin production.

8.5 MECHANISMS OF ACTION


The mechanisms of CLA action are moot. CLA blocks DNA adduct formation,
which may be one way in which it affects carcinogenesis (Liew et al. 1995). CLA
is incorporated into cell membrane phospholipids (Sugano et al. 1997). CLA may
displace arachidonic acid, reducing its incorporation into lecithin, which is the
preferential substrate for phospholipase A2, which releases arachidonic acid for
eicosanoid synthesis. Thus, the cyclooxygenase pathway to prostaglandins and
thromboxanes and the lipoxygenase pathway to leukotrienes are inhibited. The
interactions of CLA with the PPAR family may also be a key to its action. At present
we have a wide array of potential mechanisms that may be different for every
different type of CLA activity. The relatively easy work of cataloging CLA effects
is almost finished. We now are entering the more complex and difficult phase of
CLA research how does it do what it does?

ACKNOWLEDGMENT
The author was supported, in part, by a Research Career Award (HL-00734) from
the National Institutes of Health (DK).
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