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Influence of graded levels of conjugated linoleic acid (CLA) on experimental atherosclerosis in rabbits
Nutrition Research 22 (2002) 1275–1279 www.elsevier.com/locate/nutres
Influence of graded levels of conjugated linoleic acid (CLA) on experimental atherosclerosis in rabbits David Kritchevskya,*, Shirley A. Teppera, Scott Wrighta, Susanne K. Czarneckib a
b
The Wistar Institute, Philadelphia, PA 19104, USA Department of Chemistry, Chestnut Hill College, Philadelphia, PA 19118, USA
Received 31 January 2002; received in revised form 26 June 2002; accepted 29 June 2002
Abstract Dietary CLA inhibits experimental atherosclerosis when fed at a level of 0.1%. In this study rabbits were fed 0.2% cholesterol as part of a semi-purified diet which contained 0, 0.05, 0.075, 0.10 or 0.5% CLA. At levels as low as 0.05% of the diet CLA reduced severity of atherosclerosis in the aortic arch by 20% and in the thoracic aorta by 8%. As dietary concentrations of CLA rose severity of atherosclerosis fell. When the diet contained 0.10% CLA severity of atherosclerosis in the aortic arch and thoracic aorta fell by 40 (p ⬍ 0.05) and 33%, respectively. CLA fed at 0.5% of the diet lowered severity of atherosclerosis in the aortic arch and thoracic aorta by 60% (p ⬍ 0.05) and 56% (p ⬍ 0.05), respectively. The effects observed at lower levels of CLA suggest effective levels of dietary CLA may be achieved in a normal human diet. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Atherosclerosis; Cholesterol; Conjugated linoleic acid (CLA); Diet; Rabbits
1. Introduction Conjugated linoleic acid (CLA) is a collective term covering a mixture of positional and geometric isomers of octadecadienoic acid (18:2) in which the double bonds are contiguous (conjugated) rather than being separated by a methylene group as they are in linoleic acid. The principal isomers of CLA present in commercially available material are the cis9, trans11 and the trans10, cis12 modifications. The former is the principal form of CLA found * Corresponding author. Tel.: ⫹1-215-898-3713; fax: ⫹1-215-898-3995. E-mail address: [email protected] (D. Kritchevsky). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 4 3 2 - 3
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D. Kritchevsky et al. / Nutrition Research 22 (2002) 1275–1279
Table 1 Atherogenic diet Ingredient Casein DL-methionine Sucrose Corn starch Coconut oil Corn oil Cellulose Mineral mix Vitamin mix Choline bitartrate Cholesterol
%
% Calories
25.00 0.20 20.48 20.00 13.00 1.00 15.00 4.00 1.00 0.12 0.20
25.9
100.00
100.0
20.9 20.7 30.2 2.3
in the meat and milk of ruminant animals [3]. In our earlier studies [1,2] we have shown that dietary levels of CLA as low as 0.5% could significantly lower the severity of atherosclerosis in cholesterol-fed rabbits. On average CLA is present in milk and milk products at a level of about 5 mg/g fat, the level in beef is somewhat lower [3]. McGuire et al. [4] have discussed dietary sources and intake of CLA in humans. In Germany the intake of c9,t11-CLA is 430 mg/day in men and 350 mg/day in women. In Finland, people ingesting a diet high in dairy foods eat about 310 mg of the c9,t11 isomer daily. In the State of Washington intake of the c9,t11 isomer is about 110-140 mg/day. Thus it would be very difficult to achieve 0.5% CLA in a normal human diet. Our aim was to determine the lowest dietary concentration at which CLA would inhibit experimental atherogenesis.
2. Materials and methods The atherogenic diets used (Table 1) were prepared and pelletted by Dyets, Inc., Bethlehem, PA. This mixture of CLA isomers was obtained from Natural ASA, Hovdebygda, Norway and contained 42.8% of the c9,t11 isomer and 44.8% of the t10,c12 isomer. The diets were isocaloric. CLA was added at the expense of sucrose. Forty male, New Zealand White rabbits, starting weight 2550 ⫾ 35 gm, were obtained from Covance, Denver, PA and segregated into five groups of 8 having the same average weight. The animals were housed in individual stainless steel cages in a temperature-controlled, humidified room maintained on a 12-hr light/dark cycle. The rabbits were allowed free access to food and water. The rabbits were fed the atherogenic diet plus 0, 0.05, 0.075, 0.10 or 0.50 CLA. After 90 days on the diet the rabbits were sedated deeply by barbiturate injection and exsanguinated. Serum total and HDL cholesterol and triglycerides were determined using appropriate commercially available kits (Sigma, St. Louis, MO). Aortas were removed, cleaned and the severity of lesions graded visually using a 0-4 scale [5]. Statistical analysis of the data was carried out
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Table 2 Necropsy data: rabbits fed atherogenic diet ⫾ different levels of CLA for 90 days Group
Number % CLA Weight gain, g Liver wt. g Liver % body wt Serum lipids, mg/dl Cholesterol %HDL-cholesterol Triglycerides Atherosclerosis Aortic arch Thoracic aorta % area
A
B
C
D
E
8/8 — ⫺28 ⫾ 98 60.1 ⫾ 3.7 2.32 ⫾ 0.13
8/8 0.05 ⫺181 ⫾ 101 60.1 ⫾ 6.3 2.59 ⫾ 0.24
7/8 0.075 43 ⫾ 52 65.6 ⫾ 2.9 2.62 ⫾ 0.07a
8/8 0.10 268 ⫾ 103 60.2 ⫾ 3.0 2.15 ⫾ 0.11 ab
6/8 0.5 ⫺67 ⫾ 97 69.4 ⫾ 3.8 2.83 ⫾ 0.20b
411 ⫾ 28 6.0 ⫾ 0.6 144 ⫾ 22a
476 ⫾ 23 4.7 ⫾ 0.3 255 ⫾ 35a
416 ⫾ 39 9.1 ⫾ 2.2 172 ⫾ 47
532 ⫾ 47 7.0 ⫾ 1.6 171 ⫾ 27
419 ⫾ 44 9.0 ⫾ 3.5 224 ⫾ 43
1.25 ⫾ 0.16 ab 0.75 ⫾ 0.13a 13.5 ⫾ 1.8a
1.00 ⫾ 0.21 0.69 ⫾ 0.21 11.8 ⫾ 1.9
0.86 ⫾ 0.14 0.57 ⫾ 0.07 9.7 ⫾ 1.1
0.75 ⫾ 0.13a 0.50 ⫾ 0.13 9.4 ⫾ 1.7
0.50 ⫾ 0.18b 0.33 ⫾ 0.11a 6.7 ⫾ 2.4a
All data ⫾ SEM. Values in horizontal row bearing same letter are significantly different, p 0.05.
using software programs designed by Cricket Software, Malvern PA. StatWorks version 1.2 was used for numerical data and Cricket Graph version 1.3.2 was used for the bar graph. The experimental protocol was approved by the institutional IUCAC.
3. Results The results are presented in Table 2. Serum cholesterol levels were in the range of 400-500 mg/dl (average 451 ⫾ 23 mg/dl). Our earlier studies showed that CLA had no significant effect on plasma cholesterol levels [1,2]. HDL-cholesterol levels were elevated in the animals fed 0.075– 0.50% CLA. Triglyceride levels were elevated in the CLA-fed rabbits, consistent with our earlier observations. The degree of elevation of triglyceride levels did not appear to be dose-related. The severity of atherosclerosis in both the aortic arch and thoracic aorta fell with increasing levels of dietary CLA. In the aortic arch severity of lesions was reduced by 20, 31, 40, and 60% in rabbits fed 0.05, 0.075, 0.10, and 0.50% CLA. Reduction of severity of lesions in the thoracic aorta was 8, 24, 33, and 56% with increasing levels of dietary CLA. In an earlier study [2] we showed that 0.5% dietary CLA lowered severity of aortic arch and thoracic aorta lesions by 28% (p ⬍ 0.05) and 41%, respectively. We had shown previously [2] that 0.1% dietary CLA lowered severity of atherosclerosis (aortic arch plus thoracic aorta) by 34%. In the current study severity was reduced by 37.5%. Thus, even though severity of atherosclerosis seen in this study was lower than that seen in the earlier experiment the relative reduction of atherosclerosis was similar. When 0.5% CLA was present in the diet the lowering of severity was 59% in this study compared to 64% in the previous one. The effect of increasing levels of CLA on severity of atherosclerosis is presented graphically in Fig. 1.
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D. Kritchevsky et al. / Nutrition Research 22 (2002) 1275–1279
Fig. 1. Rabbits fed 0.2% cholesterol and varying levels of CLA for 90 days. Trend for significance of decreased severity of lesions with increasing level of dietary CLA: arch, p ⫽ 0.011; thoracic, p ⫽ 0.057.
Linear regression analysis shows that the trend for decreased severity of lesions with increased dose of CLA is highly significant for the aortic arch (p ⫽ 0.011) and barely misses significance for the thoracic aorta (p ⫽ 0.057). The current study shows that a dietary concentration of CLA as low as 0.05% leads to reduction in severity of lesions.
4. Discussion CLA fed at levels of 0.5–1.0% lowers severity of atherosclerosis in cholesterol-fed rabbits [1,2] and severity of aortic sudanophilia in cholesterol-fed hamsters [6,7]. The effective dietary levels of CLA are more than one could expect to achieve in a normal human diet. Hence we examined CLA effects at lower levels of intake. The results show that levels of CLA as low as 0.05% exert some inhibitory effect on atherosclerosis. These observations suggest that examining effects of even lower levels of CLA fed for longer periods of time might provide helpful data. One other aspect of CLA research is important here. As noted above the material used in this and earlier studies, by us and by others, is a mixture of about equal proportions (about 40% each) of the two major CLA isomers, c9,t11 and t10,c12. It is now becoming apparent that the two isomers are not biologically equivalent. A CLA mixture (0.5%) fed to weanling
D. Kritchevsky et al. / Nutrition Research 22 (2002) 1275–1279
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mice for 20 days resulted in accretion of lean body mass and deposition of less body fat [8]. It was shown later that the t10,c12 isomer was solely responsible for this effect [9]. With availability of the major individual CLA isomers in pure form and at reasonable price it should now be possible to ascertain if the effect on atherosclerosis is due to one specific isomer or if both major isomers exerts the anti-atherogenic effect. Studies to determine the effects of the individual isomers are in progress.
Acknowledgments This work was supported by a grant from the Eugene Garfield Foundation and by a Research Career Award (HL-00734) from the National Institutes of Health (USA).
References [1] Lee KN, Kritchevsky D, Pariza MW. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 1994;108:19 –25. [2] Kritchevsky D, Tepper SA, Wright S, Tso P, Czarnecki SK. Influence of conjugated linoleic acid (CLA) on establishment and progression of atherosclerosis in rabbits. J Am Cell Nutr 2000;19:472S–7S. [3] Chin SF, Liu W, Storkson JM, Ha YL, Pariza MW. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogenes. J Food Comp Anal 1992;5:185–97. [4] McGuire MK, McGuire MA, Ritzenthaler K, Shultz TD. Dietary sources and intakes of conjugated linoleic acid intake in humans. In: Yurawecz MP, Mossoba MM, Kramer JKG, Pariza MW, Nelson GJ, editors. Advances in conjugated linoleic acid research, Vol. 1. Champaign, IL: AOCS Press, 1999. p. 369 –77. [5] Duff GL, McMillan GC. The effect of alloxan diabetes on experimental atherosclerosis in the rabbit. J Exp Med 1949;89:611–30. [6] Nicolosi RJ, Rogers EJ, Kritchevsky D, Scimeca JA, Huth PJ. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 1997;22:266 – 77. [7] Wilson TA, Nicolosi RJ, Chrysam M, Kritchevsky D. Conjugated linoleic acid reduces early aortic atherosclerosis greater than linoleic acid in hypercholesterolemic hamsters. Nutr Res 2000;20:1795– 805. [8] Park Y, Albright KJ, Liu W, Storkson JM, Cook ME, Pariza MW. Effect of conjugated linoleic acid on body composition in mice. Lipids 1997;32:853– 8. [9] Park Y, Storkson JM, Albright KJ, Liu W, Pariza MW. Evidence that the trans-10, cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids 1999;34:235– 41.
Influence of graded levels of conjugated linoleic acid (CLA) on experimental atherosclerosis in rabbits David Kritchevskya,*, Shirley A. Teppera, Scott Wrighta, Susanne K. Czarneckib a
b
The Wistar Institute, Philadelphia, PA 19104, USA Department of Chemistry, Chestnut Hill College, Philadelphia, PA 19118, USA
Received 31 January 2002; received in revised form 26 June 2002; accepted 29 June 2002
Abstract Dietary CLA inhibits experimental atherosclerosis when fed at a level of 0.1%. In this study rabbits were fed 0.2% cholesterol as part of a semi-purified diet which contained 0, 0.05, 0.075, 0.10 or 0.5% CLA. At levels as low as 0.05% of the diet CLA reduced severity of atherosclerosis in the aortic arch by 20% and in the thoracic aorta by 8%. As dietary concentrations of CLA rose severity of atherosclerosis fell. When the diet contained 0.10% CLA severity of atherosclerosis in the aortic arch and thoracic aorta fell by 40 (p ⬍ 0.05) and 33%, respectively. CLA fed at 0.5% of the diet lowered severity of atherosclerosis in the aortic arch and thoracic aorta by 60% (p ⬍ 0.05) and 56% (p ⬍ 0.05), respectively. The effects observed at lower levels of CLA suggest effective levels of dietary CLA may be achieved in a normal human diet. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Atherosclerosis; Cholesterol; Conjugated linoleic acid (CLA); Diet; Rabbits
1. Introduction Conjugated linoleic acid (CLA) is a collective term covering a mixture of positional and geometric isomers of octadecadienoic acid (18:2) in which the double bonds are contiguous (conjugated) rather than being separated by a methylene group as they are in linoleic acid. The principal isomers of CLA present in commercially available material are the cis9, trans11 and the trans10, cis12 modifications. The former is the principal form of CLA found * Corresponding author. Tel.: ⫹1-215-898-3713; fax: ⫹1-215-898-3995. E-mail address: [email protected] (D. Kritchevsky). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 4 3 2 - 3
1276
D. Kritchevsky et al. / Nutrition Research 22 (2002) 1275–1279
Table 1 Atherogenic diet Ingredient Casein DL-methionine Sucrose Corn starch Coconut oil Corn oil Cellulose Mineral mix Vitamin mix Choline bitartrate Cholesterol
%
% Calories
25.00 0.20 20.48 20.00 13.00 1.00 15.00 4.00 1.00 0.12 0.20
25.9
100.00
100.0
20.9 20.7 30.2 2.3
in the meat and milk of ruminant animals [3]. In our earlier studies [1,2] we have shown that dietary levels of CLA as low as 0.5% could significantly lower the severity of atherosclerosis in cholesterol-fed rabbits. On average CLA is present in milk and milk products at a level of about 5 mg/g fat, the level in beef is somewhat lower [3]. McGuire et al. [4] have discussed dietary sources and intake of CLA in humans. In Germany the intake of c9,t11-CLA is 430 mg/day in men and 350 mg/day in women. In Finland, people ingesting a diet high in dairy foods eat about 310 mg of the c9,t11 isomer daily. In the State of Washington intake of the c9,t11 isomer is about 110-140 mg/day. Thus it would be very difficult to achieve 0.5% CLA in a normal human diet. Our aim was to determine the lowest dietary concentration at which CLA would inhibit experimental atherogenesis.
2. Materials and methods The atherogenic diets used (Table 1) were prepared and pelletted by Dyets, Inc., Bethlehem, PA. This mixture of CLA isomers was obtained from Natural ASA, Hovdebygda, Norway and contained 42.8% of the c9,t11 isomer and 44.8% of the t10,c12 isomer. The diets were isocaloric. CLA was added at the expense of sucrose. Forty male, New Zealand White rabbits, starting weight 2550 ⫾ 35 gm, were obtained from Covance, Denver, PA and segregated into five groups of 8 having the same average weight. The animals were housed in individual stainless steel cages in a temperature-controlled, humidified room maintained on a 12-hr light/dark cycle. The rabbits were allowed free access to food and water. The rabbits were fed the atherogenic diet plus 0, 0.05, 0.075, 0.10 or 0.50 CLA. After 90 days on the diet the rabbits were sedated deeply by barbiturate injection and exsanguinated. Serum total and HDL cholesterol and triglycerides were determined using appropriate commercially available kits (Sigma, St. Louis, MO). Aortas were removed, cleaned and the severity of lesions graded visually using a 0-4 scale [5]. Statistical analysis of the data was carried out
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Table 2 Necropsy data: rabbits fed atherogenic diet ⫾ different levels of CLA for 90 days Group
Number % CLA Weight gain, g Liver wt. g Liver % body wt Serum lipids, mg/dl Cholesterol %HDL-cholesterol Triglycerides Atherosclerosis Aortic arch Thoracic aorta % area
A
B
C
D
E
8/8 — ⫺28 ⫾ 98 60.1 ⫾ 3.7 2.32 ⫾ 0.13
8/8 0.05 ⫺181 ⫾ 101 60.1 ⫾ 6.3 2.59 ⫾ 0.24
7/8 0.075 43 ⫾ 52 65.6 ⫾ 2.9 2.62 ⫾ 0.07a
8/8 0.10 268 ⫾ 103 60.2 ⫾ 3.0 2.15 ⫾ 0.11 ab
6/8 0.5 ⫺67 ⫾ 97 69.4 ⫾ 3.8 2.83 ⫾ 0.20b
411 ⫾ 28 6.0 ⫾ 0.6 144 ⫾ 22a
476 ⫾ 23 4.7 ⫾ 0.3 255 ⫾ 35a
416 ⫾ 39 9.1 ⫾ 2.2 172 ⫾ 47
532 ⫾ 47 7.0 ⫾ 1.6 171 ⫾ 27
419 ⫾ 44 9.0 ⫾ 3.5 224 ⫾ 43
1.25 ⫾ 0.16 ab 0.75 ⫾ 0.13a 13.5 ⫾ 1.8a
1.00 ⫾ 0.21 0.69 ⫾ 0.21 11.8 ⫾ 1.9
0.86 ⫾ 0.14 0.57 ⫾ 0.07 9.7 ⫾ 1.1
0.75 ⫾ 0.13a 0.50 ⫾ 0.13 9.4 ⫾ 1.7
0.50 ⫾ 0.18b 0.33 ⫾ 0.11a 6.7 ⫾ 2.4a
All data ⫾ SEM. Values in horizontal row bearing same letter are significantly different, p 0.05.
using software programs designed by Cricket Software, Malvern PA. StatWorks version 1.2 was used for numerical data and Cricket Graph version 1.3.2 was used for the bar graph. The experimental protocol was approved by the institutional IUCAC.
3. Results The results are presented in Table 2. Serum cholesterol levels were in the range of 400-500 mg/dl (average 451 ⫾ 23 mg/dl). Our earlier studies showed that CLA had no significant effect on plasma cholesterol levels [1,2]. HDL-cholesterol levels were elevated in the animals fed 0.075– 0.50% CLA. Triglyceride levels were elevated in the CLA-fed rabbits, consistent with our earlier observations. The degree of elevation of triglyceride levels did not appear to be dose-related. The severity of atherosclerosis in both the aortic arch and thoracic aorta fell with increasing levels of dietary CLA. In the aortic arch severity of lesions was reduced by 20, 31, 40, and 60% in rabbits fed 0.05, 0.075, 0.10, and 0.50% CLA. Reduction of severity of lesions in the thoracic aorta was 8, 24, 33, and 56% with increasing levels of dietary CLA. In an earlier study [2] we showed that 0.5% dietary CLA lowered severity of aortic arch and thoracic aorta lesions by 28% (p ⬍ 0.05) and 41%, respectively. We had shown previously [2] that 0.1% dietary CLA lowered severity of atherosclerosis (aortic arch plus thoracic aorta) by 34%. In the current study severity was reduced by 37.5%. Thus, even though severity of atherosclerosis seen in this study was lower than that seen in the earlier experiment the relative reduction of atherosclerosis was similar. When 0.5% CLA was present in the diet the lowering of severity was 59% in this study compared to 64% in the previous one. The effect of increasing levels of CLA on severity of atherosclerosis is presented graphically in Fig. 1.
1278
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Fig. 1. Rabbits fed 0.2% cholesterol and varying levels of CLA for 90 days. Trend for significance of decreased severity of lesions with increasing level of dietary CLA: arch, p ⫽ 0.011; thoracic, p ⫽ 0.057.
Linear regression analysis shows that the trend for decreased severity of lesions with increased dose of CLA is highly significant for the aortic arch (p ⫽ 0.011) and barely misses significance for the thoracic aorta (p ⫽ 0.057). The current study shows that a dietary concentration of CLA as low as 0.05% leads to reduction in severity of lesions.
4. Discussion CLA fed at levels of 0.5–1.0% lowers severity of atherosclerosis in cholesterol-fed rabbits [1,2] and severity of aortic sudanophilia in cholesterol-fed hamsters [6,7]. The effective dietary levels of CLA are more than one could expect to achieve in a normal human diet. Hence we examined CLA effects at lower levels of intake. The results show that levels of CLA as low as 0.05% exert some inhibitory effect on atherosclerosis. These observations suggest that examining effects of even lower levels of CLA fed for longer periods of time might provide helpful data. One other aspect of CLA research is important here. As noted above the material used in this and earlier studies, by us and by others, is a mixture of about equal proportions (about 40% each) of the two major CLA isomers, c9,t11 and t10,c12. It is now becoming apparent that the two isomers are not biologically equivalent. A CLA mixture (0.5%) fed to weanling
D. Kritchevsky et al. / Nutrition Research 22 (2002) 1275–1279
1279
mice for 20 days resulted in accretion of lean body mass and deposition of less body fat [8]. It was shown later that the t10,c12 isomer was solely responsible for this effect [9]. With availability of the major individual CLA isomers in pure form and at reasonable price it should now be possible to ascertain if the effect on atherosclerosis is due to one specific isomer or if both major isomers exerts the anti-atherogenic effect. Studies to determine the effects of the individual isomers are in progress.
Acknowledgments This work was supported by a grant from the Eugene Garfield Foundation and by a Research Career Award (HL-00734) from the National Institutes of Health (USA).
References [1] Lee KN, Kritchevsky D, Pariza MW. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 1994;108:19 –25. [2] Kritchevsky D, Tepper SA, Wright S, Tso P, Czarnecki SK. Influence of conjugated linoleic acid (CLA) on establishment and progression of atherosclerosis in rabbits. J Am Cell Nutr 2000;19:472S–7S. [3] Chin SF, Liu W, Storkson JM, Ha YL, Pariza MW. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogenes. J Food Comp Anal 1992;5:185–97. [4] McGuire MK, McGuire MA, Ritzenthaler K, Shultz TD. Dietary sources and intakes of conjugated linoleic acid intake in humans. In: Yurawecz MP, Mossoba MM, Kramer JKG, Pariza MW, Nelson GJ, editors. Advances in conjugated linoleic acid research, Vol. 1. Champaign, IL: AOCS Press, 1999. p. 369 –77. [5] Duff GL, McMillan GC. The effect of alloxan diabetes on experimental atherosclerosis in the rabbit. J Exp Med 1949;89:611–30. [6] Nicolosi RJ, Rogers EJ, Kritchevsky D, Scimeca JA, Huth PJ. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 1997;22:266 – 77. [7] Wilson TA, Nicolosi RJ, Chrysam M, Kritchevsky D. Conjugated linoleic acid reduces early aortic atherosclerosis greater than linoleic acid in hypercholesterolemic hamsters. Nutr Res 2000;20:1795– 805. [8] Park Y, Albright KJ, Liu W, Storkson JM, Cook ME, Pariza MW. Effect of conjugated linoleic acid on body composition in mice. Lipids 1997;32:853– 8. [9] Park Y, Storkson JM, Albright KJ, Liu W, Pariza MW. Evidence that the trans-10, cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids 1999;34:235– 41.