atherosclerosis in the Syrian Golden hamster

Regular paper

Effect of conjugated linoleic acid isomers on lipoproteins and

atherosclerosis in the Syrian Golden hamster

Patricia L.Mitchell,Morgan https://www.360docs.net/doc/326033842.html,ngille,Deborah L.Currie,Roger S.McLeod *

Department of Biochemistry and Molecular Biology,Room 9C,Sir Charles Tupper Medical Building,Dalhousie University,5850College Street,Halifax,

Nova Scotia,Canada B3H 1X5

Received 9March 2005;received in revised form 22April 2005;accepted 27April 2005

Available online 13May 2005

Abstract

Conjugated linoleic acid (CLA)is a group of positional and geometric isomers of linoleic acid (LA,C18:2cis -9,cis -12)that are reported to have important biological activities,including protection against atherosclerosis.In this study,the potential role of the individual cis -9,trans -11and trans -10,cis -12isomers of CLA in atherogenesis were compared with LA in the Syrian Golden hamster.Supplementation of a high-fat,high-cholesterol diet (HFHC)with 1%(w/w)cis -9,trans -11CLA or trans -10,cis -12CLA did not significantly affect plasma cholesterol levels compared to supplementation with 1%(w/w)LA.Very low density lipoprotein cholesterol (VLDL-C)was lower and plasma triglycerides (TG)were higher in diets where C18:2fatty acid was added to the HFHC diet,but neither the cis -9,trans -11CLA group nor trans -10,cis -12CLA group was significantly different from the LA control group.CLA supplementation did not significantly affect low density lipoprotein cholesterol (LDL-C).Trans -10,cis -12CLA increased high density lipoprotein cholesterol (HDL-C)levels compared to LA or cis -9,trans -11CLA (P <0.02),and although the ratio of non-HDL-C:HDL-C in the cis -9,trans -11CLA group (1.11F 0.54)and the trans -10,cis -12CLA group (1.11F 0.21)was lower than the LA group (1.29F 0.45),the reduction did not reach statistical significance.Atherosclerosis was assessed in the ascending aorta by measuring the number of aortic cross-sections containing Oil Red O-stained intimal https://www.360docs.net/doc/326033842.html,pared to the LA group (60F 11%),both the cis -9,trans -11CLA group (38F 8%)and the trans -10,cis -12CLA group (28F 7%)had fewer sections displaying a fatty streak lesion,although the differences did not reach statistical significance.These results suggest that individual CLA isomers may reduce atherosclerotic lesion development in the hamster,but when compared to LA,the apparent atheroprotective effects do not correlate with beneficial changes in lipoprotein profile.D 2005Elsevier B.V .All rights reserved.

Keywords:Conjugated linoleic acid;Atherosclerosis;Lipoprotein;Hamster

1.Introduction

Conjugated linoleic acid (CLA)is the term used to describe a group of positional and geometric isomers of linoleic acid (LA,C18:2cis -9,cis -12)with unique bio-logical properties (reviewed in [1,2]).In dairy foods and other fatty tissues of ruminant animals,CLA is present predominantly (>90%)as the isomer with conjugated double bonds at positions 9and 11(cis -9,trans -11)of the 18-carbon acyl chain.Conversely,CLA supplements (which are widely

available)are a mixture containing ¨40%cis -9,trans -11CLA,¨40%trans -10,cis -12CLA and ¨20%other iso-mers.Many of the studies reported so far have utilized mixed isomer CLA supplementation,and as a result,it is not completely clear whether the cis -9,trans -11or the trans -10,cis -12isomer is responsible for the biological effects.

One important but controversial observation is that CLA can decrease the extent of atherosclerosis in experimental animals.A potential mechanism of such a reduction in disease is through the ability of CLA to alter hepatic lipid and lipoprotein metabolism.Dietary CLA supplementation in rabbits [3–6]or hamsters [7,8]decreased the develop-ment of the early lesions of atherosclerosis.In these studies,the decrease in lesions was modest,and comparison

1388-1981/$-see front matter D 2005Elsevier B.V .All rights reserved.doi:10.1016/j.bbalip.2005.04.007

*Corresponding author.Tel.:+19024947013;fax:+19024941355.E-mail address:rmcleod2@dal.ca (R.S.McLeod).

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276

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between studies was difficult,as a standard methodology for lesion induction and assessment was not employed.In rabbits[3–6],CLA supplementation decreased lesion development.The total aorta area covered by lesions was decreased,measured morphometrically after Sudan IV staining of neutral lipids[5]or using a0–4scale for lesion severity[3,4,6].In the earlier studies[3–5],rabbits were supplemented with a mixed isomer CLA preparation, whereas in the most recent work[6],mixed and individual isomers were compared.In the latter study,all CLA supplemented groups had reduced lesions.

A similar trend was observed in hamsters[7,8]using mixed CLA isomer supplementation.Both LA and CLA reduced atherosclerotic lesions,although the CLA mixture was more effective.However,the effectiveness of individual CLA isomers in comparison to LA has not previously been reported.

In the mouse,CLA may increase[9]or decrease[10] lesion development.Additionally,however,the mouse may be particularly sensitive to abnormalities of liver metabolism when the diets contain the trans-10,cis-12isomer[11,12].

The hamster may be particularly appropriate for studies that can have translational application to humans because the hamster has a similar lipoprotein system.While most rodents carry plasma cholesterol primarily in the high density lipoprotein(HDL)fraction,the hamster can trans-port substantial amounts of cholesterol in the low density (LDL)and very low density(VLDL)lipoproteins when the diet contains cholesterol[13,14].In addition,hamster and human liver both produce VLDL containing only apoB100 [15],while the rat and mouse produce both apoB100-and apoB48-containing VLDL particles.As in the human system,the hamster has plasma cholesteryl ester transfer activity[16],whereas the rat and mouse do not.Therefore, the hamster is a convenient and relevant small animal model for studies of lipoprotein metabolism and atherosclerosis.

The role of specific CLA isomers,especially comparing the cis-9,trans-11and trans-10,cis-12isomers for effects on lipoprotein metabolism and atherosclerosis,has not been studied extensively.In the rabbit,each CLA isomer had minimal effects on plasma cholesterol and triglycerides[6]. It has been suggested[17],based on a comparison of mixed and purified isomer supplementation in the hamster,that decreases in LDL cholesterol(LDL-C)and HDL cholesterol (HDL-C)and increases in VLDL triglycerides(VLDL-TG) were mediated by the trans-10,cis-12CLA isomer.CLA isomers may have different effects on human plasma lipoproteins as well[18],as the cis-9,trans-11isomer may decrease and the trans-10,cis-12isomer may increase levels of atherogenic lipoproteins.In contrast,studies in the human hepatoma cell line,HepG2,have suggested that the trans-10,cis-12CLA isomer is less effective than other fatty acids as a stimulus for VLDL and TG secretion [19,20].CLA isomers may also enhance hepatic clearance of lipoproteins[12,21].Thus,the effects of CLA isomers on plasma lipoproteins and atherosclerosis are not clear.

The current investigation examined the effect of isomer-specific CLA supplementation on plasma lipids,lipopro-teins and atherosclerosis in the Syrian Golden hamster.Our data indicate that,when compared to LA,both CLA isomers may reduce the extent of early atherosclerotic lesions,even though the changes in lipoprotein profile are modest.

2.Materials and methods

2.1.Animals and diets

All procedures involving animals were approved by the Dalhousie University Committee on Laboratory Animals in accordance with the guidelines of the Canadian Council on Animal Care.Outbred male Syrian Golden hamsters[CRL: LVG(SYR),7–8weeks old]were obtained from Charles River Laboratories(Montreal,Quebec)and housed on a12-h light/dark cycle.After1–2weeks of acclimation to the animal unit on chow diet,the animals were separated into three groups of6animals each and housed in individual cages to allow for the monitoring of body weight and food intake.The experimental(HFHC)diet contained20%(w/w) hydrogenated coconut oil,2%(w/w)safflower oil and 0.12%cholesterol and were prepared by Research Diets, Inc.(New Brunswick,NJ)on a base of standard chow (Purina5001).Linoleic acid or purified CLA isomer(Nu-Chek-Prep,Inc.,Elysian,MN;>90%pure as assessed by gas chromatography)was added to the experimental diet to 1%(w/w)as an ethanol solution and the ethanol was then removed by lyophilization.All diets were stored atà208C until use and pellets were replenished twice weekly.Food and water were available ad libitum and the hamsters were weighed each week.Intake of the high-fat diet was lower than the intake of the chow diet,but the caloric intake was the same and animals maintained constant body weight for 12weeks on either diet.The fatty acid analysis of each of the test diets(Table1)revealed that the supplemental Table1

Fatty acid composition of test diets(mg/g)

Fatty acid Diet

LA cis-9,

trans-11

CLA

trans-10,

cis-12

CLA

C8:012.48.711.5

C10:010.37.49.9

C12:083.462.282.2

C14:032.825.131.3

C16:025.417.320.4

C18:020.113.613.6

C18:110.77.07.0

C18:2(LA)27.814.215.9

cis-9,trans-11CLA ND9.1ND trans-10,cis-12CLA ND ND 6.8 ND,not detected.

P.L.Mitchell et al./Biochimica et Biophysica Acta1734(2005)269–276 270

conjugated fatty acid was present at approximately3–6%of the total fatty acids.

2.2.Measurement of lipids and lipoproteins

Hamsters were anesthetized with Somnitol(50mg/kg) and exanguinated by ventricular puncture.Blood was collected into tubes containing1A L of500mM EDTA solution,as anticoagulant,and the final concentration was adjusted to1mM upon the completion of the blood collection.Following centrifugation to remove the blood cells,the plasma was recovered and used immediately for density gradient ultracentrifugation(see below),or stored at à808C for plasma lipid analysis.

For density gradient ultracentrifugation analysis of plasma lipoproteins,sucrose was dissolved in freshly isolated plasma(800–1000A L)to a final concentration of 12.5%sucrose(w/v).The plasma was then diluted to1.6 mL with12.5%sucrose solution(in phosphate-buffered saline,PBS).A4mL polyallomer ultracentrifuge tube (Beckman-Coulter)was used to construct a discontinuous sucrose gradient by overlayering1.2mL PBS on top of the 12.5%sucrose plasma sample and underlayering the plasma sample with670A L of25%sucrose and670A L of47% sucrose solution(both in PBS).The plasma lipoproteins were resolved by ultracentrifugation at55,000rpm for20h in an SW60Ti rotor.Upon completion,aliquots(200A L for 20fraction gradients or330A L for12fraction gradients) were collected from the top of the gradient.Cholesterol and triglyceride concentrations in plasma samples and gradient fractions were determined by enzymatic assays from Roche Diagnostics or Sigma-Aldrich,respectively,adapted to microtiter plate format.For plasma lipid analyses,5–10A L aliquots were assayed;for density gradient ultracentrifuga-tion fractions,10–25A L aliquots were assayed.Lipoprotein cholesterol and triglyceride content was calculated as the area under the curve of the density gradient profile.

2.3.Quantification of atherosclerosis in the hamster aorta

Following exanguination of the hamster,the abdominal aorta was cut and the vasculature was perfused with20–25 mL of saline by injection into the ventricle puncture site. Once the effluent was clear of blood,the vasculature was perfused with20–25mL of2%paraformaldehyde in saline. Heart and aorta were then excised,placed in2%parafor-maldehyde and stored at48C.The ascending aorta (approximately4mm in length)was removed and mounted in embedding medium(Tissue-Tek O. C.T.,Sakura Finetech USA,Inc.,Torrance,CA)on the stage of a freezing microtome(atà35toà408C).Approximately80 to100sections of10A m each were prepared from each tissue sample and mounted on gelatin-coated microscope slides.The slides were allowed to dry overnight at room temperature and then stained with Oil Red O,to identify neutral lipid accumulations,and counterstained with hema-toxylin[22].Images were captured using a Zeiss Axiovert microscope and7–15sections from each animal were randomized and evaluated for intimal lipid accumulation by an investigator who was blinded to the origin of the section. The raw images were processed with Adobe Photoshop R software to optimize the detection of the lipid stain and quantified using Scion Image n software.A standard color representing the lipid-stained area was selected for each series of sections.Threshold for color detection was set at 60for the lesion area and40for the total cross-sectional area.A similar approach to lesion quantitation has been previously reported[23].Preliminary sectioning had estab-lished that,as previously described[13,14],the majority of the lipid infiltration occurred in the proximal ascending aorta and that the aortic root and the descending aorta of the hamster were usually free of lesions.The lesion area was defined as the percent of the cross-sectional area stained with Oil Red O.A significant lesion was defined as one in which more than0.1%of the total cross-sectional area was stained with Oil Red O.

2.4.Statistical analyses

The large variation of the data within groups necessitated a non-parametric statistical analysis.Significance of the differences between diet treatment groups was determined using the Mann–Whitney U-test or the Kruskal–Wallis one-way analysis using rank sums(Statistical Package for the Social Sciences,SPSS).

3.Results

3.1.Plasma lipid and lipoprotein analysis

In an initial study,we established the effect of the HFHC diet on plasma lipids and lipoprotein profile in the hamster. The HFHC diet increased plasma lipid levels within2 weeks,and values for HFHC and chow-fed animals were calculated as the average value for4animals sacrificed between weeks4and10(Table2).The plasma cholesterol was elevated nearly ten-fold in the HFHC diet group (3951F692A g/mL)(all values are mean F S.E.)compared to the chow control group(400F47A g/mL)(P<0.05). Plasma triglycerides were only modestly elevated in the HFHC group(1087F193A g/mL)compared to the chow control(741F51A g/mL)(P=0.25).Density gradient ultracentrifugation analysis(Fig.1)indicated that the HFHC diet increased the levels of all plasma lipoprotein classes.In particular,the cholesterol associated with the VLDL and LDL fractions was increased more than ten-fold,although the cholesterol associated with HDL was also increased more than two-fold(Table2).Overall,this raised the ratio of atherogenic(non-HDL)to anti-atherogenic(HDL)lipopro-tein cholesterol from0.23F0.04in the chow group to 1.54F0.63in the HFHC group.These lipid and lipoprotein

P.L.Mitchell et al./Biochimica et Biophysica Acta1734(2005)269–276271

changes were associated with the appearance of Oil Red O-stained lesions(fatty streaks)in the proximal region of the ascending aorta.

It has been shown previously that the addition of linoleic acid(LA)to a high-fat,high-cholesterol diet decreases plasma cholesterol and triglycerides in hamsters[8].There-fore,in order to examine the changes associated specifically with the isomers of CLA,we used an LA supplemented diet for the control group.The plasma cholesterol levels for

the Fig.1.Density gradient ultracentrifugation analysis of hamster lipoproteins

after10weeks on chow or high-fat,high-cholesterol(HFHC)diet.Syrian

Golden hamsters were fed the indicated diet ad libitum,and after4–10

weeks,the animals were sacrificed and blood samples were collected.

Plasma lipoproteins were separated by sucrose density gradient ultra-

centrifugation and separated into12equal fractions from the top(fraction1)

of the gradient.Cholesterol and triglyceride in each fraction were

determined enzymatically and are expressed as mean F S.E.for4animals

from each diet group.

Table3

Effect of1%CLA diets on lipid and lipoprotein profiles of the HFHC-fed

Syrian Golden hamster

LA cis-9,trans-11

CLA

trans-10,cis-12

CLA

VLDL-TG(A g/mL)973F2121136F2901416F240

VLDL-C(A g/mL)583F262431F198502F132

LDL-C(A g/mL)687F229520F213885F213

HDL-C(A g/mL)952F41865F861219F88*

Non-HDL-C:HDL-C 1.29F0.45 1.11F0.54 1.11F0.21

Plasma TC(A g/mL)3105F5402806F6403422F436

Plasma TG(A g/mL)1578F2251994F4802586F191

Plasma lipoproteins from individual animals were separated by density

gradient ultracentrifugation,as indicated in Fig.2.For each animal,

lipoprotein cholesterol(C)and triglycerides(TG)were determined

enzymatically in each gradient fraction and assigned to VLDL(fractions

1–4),LDL(fractions5–10)or HDL(fractions12–20).Data represent

mean F S.E.for6animals per group.

*Significantly different from cis-9,trans-11CLA(P<

0.02).

Fig.2.Density gradient ultracentrifugation analysis of hamster lipoproteins

after10weeks on high-fat,high-cholesterol(HFHC)diet supplemented

with1%(w/v)linoleic acid,cis-9,trans-11CLA or trans-10,cis-12CLA.

Syrian Golden hamsters were fed the indicated diet ad libitum,and after10

weeks,blood samples were collected at sacrifice.Plasma lipoproteins were

separated by sucrose density gradient ultracentrifugation and separated into

20equal fractions from the top(fraction1)of the gradient.Cholesterol and

triglyceride in each fraction were determined enzymatically and are

expressed as mean F S.E.for6animals per diet group.

Table2

Effect of HFHC diet on lipid and lipoprotein profiles of the Syrian Golden

hamster

CHOW HFHC

VLDL-TG(A g/mL)414F93993F231*

VLDL-C(A g/mL)63F191012F383*

LDL-C(A g/mL)73F21793F248*

HDL-C(A g/mL)564F811228F82*

Non-HDL-C:HDL-C0.23F0.04 1.54F0.63*

Plasma TC(A g/mL)400F433951F810*

Plasma TG(A g/mL)741F511087F193

Plasma lipoproteins from individual animals were separated by density

gradient ultracentrifugation,as indicated in Fig.1.For each animal,

lipoprotein cholesterol(C)and triglycerides(TG)were determined

enzymatically in each gradient fraction and assigned to VLDL(fractions

1–3),LDL(fractions4–6)or HDL(fractions7–12).Data represent

mean F S.E.for4animals per group.

*Significantly different from chow(P<0.05).

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272

animals fed LA,cis-9,trans-11CLA or trans-10,cis-12 CLA were not significantly different(P=0.50)(Table3). Nevertheless,cis-9,trans-11CLA tended to decrease and trans-10,cis-12CLA tended to increase plasma cholesterol compared to LA.Although plasma triglycerides were higher in both the cis-9,trans-11CLA(1994F480A g/mL)and trans-10,cis-12CLA(2586F191A g/mL)groups than in the LA group(1578F225A g/mL),the large variation within each group precluded the assignment of statistical significance to the differences(P=0.11).As shown in Table 3and Fig.2,VLDL-C levels were similar in all three groups,and while LDL-C was higher in the trans-10,cis-12 CLA group than in the other two groups,this difference was not statistically significant(P=0.59).Supplementation with trans-10,cis-12CLA significantly increased the HDL-C (P<0.02)compared to either LA or cis-9,trans-11CLA (Fig.2and Table3)but did not significantly reduce the ratio of non-HDL-C to HDL-C.

3.2.Analysis of atherosclerotic lesions

Fig.3shows representative examples of the Oil Red O-stained fatty streak lesions in each group of hamsters.An objective,quantitative assessment of lesion size and frequency was developed,enlisting a blinded observer and image analysis,and this technique was then applied to aortic lesions in the animals receiving a fatty acid sup-plemented diet.The total cross-sectional area of the aortic sections did not differ in the three groups(Fig.4A). Although there was a tendency for the lesion area in

the Fig.3.Atherosclerosis in the Syrian Golden hamster.Hamsters were fed a high-fat,high-cholesterol(HFHC)diet supplemented with1%(w/v)LA(A),cis-9, trans-11CLA(B)or trans-10,cis-12CLA(C)for10weeks.Aortae were fixed in2%paraformaldehyde and10-A m frozen sections of the ascending aorta were prepared.After mounting,slides were stained with Oil Red O.Arrowhead represents a fatty streak lesion in representative samples from each group.

P.L.Mitchell et al./Biochimica et Biophysica Acta1734(2005)269–276273

cis -9,trans -11CLA group to be larger than that of either the LA or the trans -10,cis -12CLA group,this was not statistically significant,if the lesion area was expressed as either absolute area (Fig.4B)or as percent of total vessel cross-sectional area (Fig.4C).

To gain insight into the frequency of lesions in the aortic sections,we quantified the number of sections which contained a lesion of significant size,defined as >0.1%of the total cross-sectional area.This parameter may be envisioned as a surrogate for the lesion area revealed by en face staining for fatty streak lesions.A scatter diagram of the number of lesion sections in each animal (Fig.5)

revealed that most of the animals in the LA supplemented group had more lesion sections than did either of the CLA supplemented groups.The mean number of lesion sections in the cis -9,trans -11CLA supplemented animals was reduced by one-third (38F 8%)compared to LA (60F 11%),and the number in the trans -10,cis -12CLA was reduced by one-half (28F 7%).Nevertheless,the differences did not reach statistical significance by non-parametric analysis (P =0.09).

Thus,although CLA supplementation reduced the frequency of fatty streak lesions in the hamster aorta substantially,neither isomer had a significant effect on the hamster lipoprotein profile.

4.Discussion

Our results indicate that CLA supplementation of a HFHC diet of the outbred Syrian Golden hamster tends to reduce the development of atherosclerotic lesions in the aorta.Furthermore,the cis -9,trans -11and the trans -10,cis -12CLA isomers were similar in their ability to reduce lesions.Nevertheless,and consistent with the results that have been published previously using mixed isomer preparations,the variability among animals within a group was large and we could not demonstrate statistically sig-nificant changes in lesion size or frequency.Importantly,however,we did find that the effects of individual isomers are evident when LA is used as the control comparator group.

Several methodologies have been employed in earlier work with CLA to evaluate lesion development,including visual lesion grading [5]and en face lipid staining [7],and this has made comparison of lesions between studies difficult.To eliminate observer bias and to specifically examine the effects of CLA isomers,we have employed

an

Fig.4.Quantification of atherosclerosis in sections from the aorta of the Syrian Golden hamster.Microscopic images from the aorta of hamsters fed HFHC diet supplemented with 1%(w/v)LA,cis -9,trans -11CLA or trans -10,cis -12CLA for 10weeks were processed using Adobe Photoshop and Scion Image software to identify the total cross-sectional area of the vessel section (A)or the Oil Red O-stained area (B).In Panel (C),the lesion area is expressed as percent of total cross-sectional area of the vessel.Bars represent mean F S.E.of 7–15sections per animal,6animals per

group.

Fig.5.Quantification of atherosclerosis in sections from the aorta of the Syrian Golden hamster.Microscopic images from the aorta of hamsters fed HFHC diet supplemented with LA,cis -9,trans -11or trans -10,cis -12CLA for 10weeks were processed using Adobe Photoshop and Scion Image software as described in Fig.4.The number of sections with lesions greater than 0.1%of total cross-sectional area was determined for each animal (7–15sections per animal)and calculated as a percent of total sections analysed.

P .L.Mitchell et al./Biochimica et Biophysica Acta 1734(2005)269–276

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LA supplemented control group and objectively quantified lesions in blinded sections by image analysis.As observed by others[8],we found that the supplementation of the HFHC diet with a diene fatty acid(LA)decreased lesion size(data not shown),but that in comparison to the LA control group,individual CLA isomers seemed to have additional beneficial effects.These observations should be verified in larger studies and,perhaps,by using an animal model with greater susceptibility to rapid atherosclerotic lesion development.Although the advantages of the hamster are important,including the similarity with human lipo-protein metabolism,the atherosclerotic lesions in this model develop slowly.

We found no difference in food consumption or final body weight of the hamsters on the three different diets during the10-week study period,which is consistent with previous work in rabbits and hamsters[3,5,24].However, since hamsters fed a mixed isomer diet had significantly lower weight gain than did either the cis-9,trans-11or LA supplemented groups[25],and mixed isomer diet decreased fat depot weights compared to cis-9,trans-11alone[17], there may be synergistic effects on body weight or com-position when both cis-9,trans-11and trans-10,cis-12 isomers are supplemented simultaneously.

Although not a specific goal of the present work,we found that both LA and CLA supplementation of the HFHC diet reduced plasma cholesterol in the hamster(compare Tables2and3).Previous work[8]in the hamster has indicated that the dienoic fatty acids decreased plasma cholesterol and increased plasma triglycerides compared to the unsupplemented HFHC diet.The authors found that mixed isomer CLA increased triglycerides more than did LA.However,in three other hamster studies[7,24,25],CLA decreased or had no effect on triglyceride levels.Thus, although CLA may reduce plasma cholesterol and not affect plasma triglycerides,these changes are not different from the effects of LA.

Unlike some of the other studies of the effects of CLA, we can find no correlation between an atheroprotective lipoprotein profile and decreased atherosclerotic plaque formation in the current study.Lee et al.[5]reported a correlation between aortic involvement and LDL-C con-centration and suggested that this change may be a marker of atherosclerotic development in rabbits.However,this effect may be found with any dienoic fatty acid supple-ment,since a significant decrease in plasma cholesterol and plasma non-HDL-C was found in hamsters fed a high-cholesterol diet supplemented with LA compared to the high-cholesterol diet alone[8].Similarly,the decrease in plasma triglycerides and cholesterol found with cis-9, trans-11CLA supplementation was similar to that found using LA.In rats,plasma cholesterol and TG were not affected by mixed CLA isomers,although there was a decrease in the total lipids in VLDL[26].In the current study,cis-9,trans-11and trans-10,cis-12supplemented diets did not significantly change any of the tested lipoprotein parameters compared to the LA supplemented control group.

There are several potential reasons for the failure to achieve statistical significance for the beneficial effects of CLA.First,our sample size was quite small(n=6per group),and the variability in the response of each group was quite large.One of the animals in the control LA group clearly did not respond as anticipated,as the lesions were much less frequent than in the other animals in this group(see Fig.5).Second,the variability within the groups could be a consequence of using an animal model that is not clearly defined genetically,and is also consistent with our observation that the variation in cholesterol levels in response to the dietary modifications was also quite large.Third,the lesions that we observed in the treatment groups were not extensive and must be considered very early fatty streak lesions.The value of CLA supplementa-tion may be more readily established using other animal models in which atherosclerosis develops more rapidly, such as the apoE-deficient mouse[27]or the LDL receptor-deficient mouse[28].However,in these models, lipoprotein metabolism may be substantially different from humans.Additional information may be obtained by examining the effects in the hamster over a longer treatment period,during which more complicated lesions can develop[13].

Our observations of the effects of CLA on hamster lipoprotein metabolism are similar to those reported in human studies.So far,no significant changes in plasma cholesterol[29–31]have been detected and there is a tendency for CLA to decrease TG.CLA mixtures contain-ing50%or80%of the cis-9,trans-11CLA isomer were found to decrease plasma TG and VLDL levels[31].These effects did not usually coincide with improvements in LDL-C or in HDL-C[30,31];in fact,HDL-C was decreased in one study[29].Thus,the modest effects of CLA that we have described in the hamster model may be similar to those found in human studies.Nevertheless,we should not rule out a potential impact on atherogenesis. Further studies are warranted to assess the effects of CLA and its isomeric forms on other parameters that may impact on the atherogenic process,including macrophage function [2,32].

In summary,we have found that individual CLA isomers can substantially reduce the frequency of atherosclerotic lesions in the hamster.Although the degree of protection did not reach statistical significance,this is the first study to indicate that each individual CLA isomer has protective effects when compared to the non-conjugated diene LA. Since we found no substantive changes in plasma lip-oprotein metabolism,we suggest that CLA may affect atherogenesis without altering plasma lipid or lipoprotein levels in this animal model.Further studies will be required, using more susceptible animal models or in larger studies over a longer term,in order to clarify the atheroprotective role and mechanism of CLA action.

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Acknowledgements

The authors acknowledge the expert guidance of Dr.W. R.Currie,Ms.Brenda Ross and Mr.Steven Whitefield of the Department of Anatomy and Cell Biology during the assessment of atherosclerosis.We also thank Jessica Miller for a critical reading of the manuscript.This work was supported by grants(to R.S.M.)from Dairy Farmers of Canada and the Canadian Institutes of Health Research. References

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