![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA 02454
3To whom correspondence should be addressed. E-mail: kchayes{at}brandeis.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • conjugated linoleic acid (CLA) • plasma lipids • liver cholesterol • hamsters • acute phase response
Conjugated linoleic acid (CLA),4 most notably the cis-9, trans-11 (18:2–9c,11t) and trans-10, cis-12 (18:2–10t,12c) isomers, have been considered anticarcinogenic and antiatherosclerotic at concentrations of 0.5–1% in the diet of animals (1
–4
). A possible connection between CLA and atherosclerosis might reflect an anti-inflammatory potential of CLA (5
). It is well established that increasing the polyunsaturated fatty acid (PUFA) to saturated fatty acid (P/S) ratio typically lowers the LDL/HDL cholesterol profile, and a lower LDL/HDL ratio reduces the risk of atherosclerosis, in part by reducing the inflammatory profile (6
). On the other hand, Nicolosi et al. (2
) reported that CLA (0.06–1.11%) fed to hamsters along with high dietary cholesterol (0.12%) reduced plasma total cholesterol (TC), especially atherogenic apolipoprotein (apo)B-rich lipoproteins and triglycerides (TG). The plasma 
-tocopherol/TC ratio was increased in hamsters fed CLA, which the authors suggested might represent a protective effect of tocopherol against the atherogenic process. Lee et al. (3
) fed rabbits a 0.1% cholesterol diet and 0.5 g/d CLA and found significantly lower plasma total and LDL cholesterol and TG after 3 mo. Furthermore, the LDL/HDL and TC/HDL cholesterol ratios were reduced in the CLA-fed rabbits, which was associated with decreased atherosclerosis. Neither of these experiments measured food intake or body composition, which may be relevant because mice fed CLA had less body fat and more active muscle metabolism (7
,8
), suggesting basic changes in energy balance and lipoprotein metabolism.
On the basis the putative antiatherosclerotic (2
) and anti-inflammatory (5
) effects of CLA and our previous study of lipoprotein changes during acute inflammation (9
), we hypothesized that the anti-inflammatory aspects of CLA might reduce atherosclerosis by modifying the -tocopherol/cholesterol ratio (2
) to blunt the acute phase response (APR) and reduce inflammation. On the other hand, if CLA reduced cholesterol absorption by impeding gut acyl coenzyme A:cholesterol acyltransferase (ACAT) activity (10
), it could reduce plasma cholesterol to lower the risk of developing atherosclerosis. Several experiments were conducted in hamsters to pursue these concepts, two of which are reported here.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
All experiments were conducted in male Syrian hamsters (initial weight 73–76 g) obtained from Charles River (Wilmington, MA). The hamsters were housed individually and kept in a temperature-controlled environment with a 12-h light:dark cycle. All hamsters had free access to water, and fresh diet was provided daily. Fats provided 30% of dietary energy. CLA, generously provided by Dr. Michael Pariza at the University of Wisconsin-Madison contained 90% conjugated linoleic acids with >40% (19:2–9c,11t) and >40% (19:2–10t,12c). Body weight was monitored on a weekly basis, and food intake on a daily basis. The Brandeis University Animal Care and Use Committee approved all protocols and procedures. At the end of each test period, the APR was induced by subcutaneous injection of silver nitrate (AgNO3: 0.75 mL of 20 g/L AgNO3/100 g body, Experiments 1 and 2) and food deprivation overnight in suspended cages, as previously described (9
). Blood was collected via cardiac puncture under CO2/O2 anesthesia for plasma lipid and vitamin analyses. Liver, adipose and adrenals were also collected and weighed.
Initially, pilot experiments were designed to establish a hamster model for testing the hypothesis that CLA reduces the inflammatory response associated with an APR because we had previously described APR in this species (9
). Accordingly, graded intakes (0–0.48%) of CLA were fed to mitigate the APR induced by lipopolysaccharide (LPS, 10 µg/100 g body), but no effect occurred in hamsters fed cholesterol-free diets based on a saturated fat–rich blend with a P/S ratio of 0.3 (data not shown).
Because these pilot studies did not show an effect of CLA on APR, a slight modification was introduced. In Experiment 1, eight hamsters were divided into two groups and fed a cholesterol-free diet (Table 1
) with or without CLA (0 and 0.48% CLA) for 12 wk. The dietary fat was changed to corn oil (P/S 4.0) on the chance that a high PUFA diet might be more effective in eliciting an APR challenge for CLA because PUFA arguably have more peroxidative capacity (12
) (proinflammatory) than the stable saturated fat fed in the pilot studies. Vitamin E, which can be considered anti-inflammatory (13
), was removed from the vitamin mix to prevent the rise in plasma vitamin E associated with purified diets in hamsters and encountered in the pilot experiments (45–70 µmol/L). The atypically high plasma vitamin E in hamsters fed purified diets might mask the effects of CLA supplements, especially if CLA functioned by enhancing vitamin E antioxidant efficacy.
|
,8
). Additionally, a high level of cholesterol (0.3%) was added to a diet containing a relatively unsaturated fat blend (P/S = 2.0, Table 3
). The cholesterol supplement was intended to create a more stressful dietary model, similar to studies in which CLA was protective against atherosclerosis in hamsters and rabbits (2
,3
). Hamsters (n = 24) were allotted to four groups and fed these diets (± 0.3% cholesterol, 0 or 1% CLA) for 7 wk. Again, diets lacked a vitamin E supplement to reduce its potential influence on the APR.
|
Total plasma cholesterol and triglycerides were determined by enzymatic assays (Sigma Diagnostics, kit #352 for cholesterol and #336 for triglycerides, Sigma Chemical, St. Louis, MO). Plasma lipoprotein fractions were isolated from hamsters in the first experiment by discontinuous density gradient ultracentrifugation according to the method of Goulinet and Chapman (14
). Ultracentrifugation was performed using a SW 41-Ti rotor (Beckman) in a Beckman L8–55 ultracentrifuge at 54 x 107 g/min at 15°C for 48 h. After ultracentrifugation, the triglyceride meniscus was collected in the first 0.5 mL. Successive fractions of 0.4 mL were then collected and assayed for cholesterol.
Hepatic esterified cholesterol (EC).
Liver free and EC concentrations were determined by HPLC based on the method of Kim and Chung (15
). Liver (100 mg) was ground with anhydrous sodium sulfate and extracted two times with chloroform. The combined extract was evaporated under nitrogen and redissolved in the mobile phase solvent (50:50, isopropanol/acetonitrile). A 100-µL sample was injected into the HPLC and eluted isocratically with acetonitrile/isopropanol (50:50, v/v) at 2.0 mL/min using a Waters Radial-Pak Resolve C18 cartridge column (8 mm x 10 cm, 10-µm particles; Waters, Milford, MA). The absorbance of the eluate was measured at a wavelength of 210 nm using a UV detector. Free cholesterol and individual cholesterol ester concentrations were calculated by comparing the peak areas of samples with those obtained for standards (Sigma Chemical)
Plasma retinol and -tocopherol.
Plasma retinol and -tocopherol were assayed using HPLC according to the method of Bieri et al. (16
). All standards (retinol, retinyl acetate, d--tocopherol and d--tocopheryl acetate) were obtained from Hoffman-La Roche (Nutley, NJ). In brief, plasma was mixed with an ethanol solution containing internal standards (retinyl acetate and d--tocopheryl acetate) and extracted with hexane. A portion of the hexane extract was evaporated under nitrogen, redissolved in methanol and injected into a C18 reverse-phase, 150 mm x 4.6 mm HPLC column (Supelcosil, LC-18, 5-µm particles; Supelco, Bellefonte, PA). The mobile phase (methanol/water, 96:4) was delivered at a flow rate of 2.0 mL/min (model 110A pump; Beckman Instruments, Berkeley, CA). Retinol and -tocopherol were detected at 290 nm with a Beckman UV detector and their concentrations were quantified in relation to the internal standards.
Statistics.
Statistical analysis was performed on a Macintosh iMAC (Apple Computer, Cupertino, CA) using StatView 5.0.1 (SAS Institute, Cary, NC). The effect of CLA in Experiment 1 was assessed by Student’s t test, and APR was assessed by two-way repeated-measures ANOVA. In Experiment 2, two-way ANOVA was used to determine CLA and cholesterol effects before APR. When ANOVA indicated a significant effect (P < 0.05) between groups, differences were determined by the Tukey-Kramer post-hoc test. The effect of CLA and dietary cholesterol on plasma variables during APR was assessed by analysis of covariance, using CLA as the independent variable and dietary cholesterol as the covariate. In this case, variance between treatments was heterogeneous; therefore, a log transformation of the data was applied before the analysis. Differences were considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Hamsters fed CLA in the first experiment gained 28% less weight and had less perirenal adipose tissue than those fed the control diet (P < 0.05, Table 2
). Their relative liver weights were 20% greater (P < 0.05, data not shown), but their absolute liver weights did not differ from controls. Plasma TC and TG did not differ between groups when measured in hamsters deprived of food overnight. Removing dietary -tocopherol normalized the plasma tocopherol concentration to <30 µmol/L, or half that recorded in pilot experiments in which tocopherol was added at 100 µg/g (data not shown).
|
. Analysis of plasma cholesterol distribution among lipoproteins revealed that CLA always expanded the VLDL fraction and that during the APR, a shift into LDL and out of HDL occurred in both the control and CLA-fed hamsters, with the shift exaggerated in hamsters fed CLA (Fig. 1
).
|
In the second experiment, absolute liver weights did not differ in CLA-supplemented hamsters in the absence of cholesterol (Table 3
), although they were significantly heavier when adjusted for the slightly lower body weight (not shown). Feeding 0.3% cholesterol greatly increased liver weights in both groups of hamsters and significantly depressed adipose weight, whereas CLA had no effect on adipose mass over this shorter experimental period. Surprisingly, in hamsters fed an excess load of 0.3% dietary cholesterol, 1% CLA significantly reduced liver EC by 40% (Table 3)
and plasma TC by 27% (Table 4
) compared with the response by control hamsters not receiving CLA. No difference was noted in plasma TG across diet groups.
|
. TG also rose during APR in hamsters fed no dietary cholesterol, whereas TG declined in hamsters fed cholesterol. Clinically this group was the most lethargic during APR. As with the pilot data, retinol decreased in all groups during APR and was unaffected by CLA. -Tocopherol increased along with TC in all groups during APR, with CLA supplementation almost doubling the increase. However, the -tocopherol/cholesterol molar ratio was not significantly altered (Table 4)
. |
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
These studies examined CLA from two perspectives, acute inflammation and dietary cholesterol loading, each designed to explore different aspects of the CLA effect on cholesterol metabolism. On the basis of our hypothesis that the putative antiatherosclerotic potential of CLA might reflect an anti-inflammatory action, the first approach tested the capacity of CLA to modulate inflammatory changes in lipoproteins during APR. The APR in hamsters can be elicited as an immediate, short-term reaction to an inflammatory stressor, such as silver nitrate (9
) or endotoxin (LPS) (17
), initiating reactions that include depressed appetite, lethargy and mobilization of adipose reserves. In conjunction with increased cytokine activity, hypercholesterolemia and hypertriglyceridemia develop (9
,17
), and the decline in plasma retinol serves as a convenient marker of APR (18
). CLA did not improve either the lethargy or the LDL/HDL lipoprotein profile during APR in the current experiments, suggesting that CLA exerted no detectable anti-inflammatory effect either by reducing the LDL/HDL cholesterol ratio (6
) or by increasing the -tocopherol/cholesterol ratio (13
). In addition, although -tocopherol increased with APR, which might be considered anti-inflammatory and beneficial (13
), this was unrelated to CLA and may reflect increased adipose turnover and the simultaneous mobilization of tocopherol and cholesterol during APR because the -tocopherol/cholesterol ratio was unchanged (see below).
An APR was effectively induced in all challenged hamsters, as evidenced by the sharp decline in plasma retinol. As expected (9
), plasma TC increased during APR compared with baseline. This rise in TC 24 h into the APR was independent of dietary cholesterol intake and is consistent with the observed increase in LDL in hamsters (9
) and rats (19
), which was postulated (9
) and demonstrated (19
) to reflect an APR-induced decrease in hepatic LDL receptor activity. Surprisingly, the percentage increase in TC induced by APR (apoB-rich lipoproteins) was amplified significantly by 1% CLA supplementation, in the absence of dietary cholesterol. This additional rise in TC, attributed to CLA only during APR, could reflect acute cholesterol influx into the plasma pool, decreased clearance or a combination of the two superimposed on a system already constrained by the depressed LDL receptor activity attributed to APR itself. Acute cholesterol influx would imply either cholesterol secretion by the liver or cholesterol release from adipose tissue because food intake and cholesterol absorption essentially cease during the APR in hamsters. Because a previous study in hamsters found that hepatic cholesterol and TG secretion are negligible during the APR (9
), adipose mobilization of cholesterol (and tocopherol) plus further impairment of lipoprotein clearance were the likely explanation for the CLA-induced TC increase during APR. If CLA can impair cholesterol transport across gut mucosa (see below), it may similarly impair cholesterol transport into bile to increase plasma cholesterol.
CLA and cholesterol transport.
The CLA-induced increment in TC elevation during the APR in both experiments suggests that CLA may have exacerbated the problem of LDL clearance, already depressed by the APR (9
,19
). Plasma cholesterol removal may have been further compromised by impaired disposition of hepatic cholesterol during 1% CLA intake. Because the hepatic CE pool was not reduced by CLA in the absence of dietary cholesterol, conversion to esters did not appear to be impaired. If direct secretion as biliary cholesterol was depressed, a process already slowed by APR because eating and gut function all but cease, the result would eventually increase LDL, and possibly HDL. Note that the decline in HDL during APR was somewhat abated by CLA, which would contribute to the observed rise in TC.
On the other hand, CLA significantly reduced TC (especially the apoB-rich lipoprotein fractions) and liver CE in hamsters fed cholesterol. Because TC reduction was already apparent at baseline (before APR), CLA appeared to depress gut cholesterol transport (absorption), which would support an earlier report of impaired gut ACAT activity in CLA-fed hamsters (10
). Thus, impaired cholesterol transport across the gut during high cholesterol intake or from the liver into bile during excretion could account for certain aspects of altered cholesterol metabolism in our hamsters.
In our study, CLA did not affect plasma lipids before APR induction in the absence of dietary cholesterol, presumably because the putative impairment in cholesterol transport by CLA was below detectable limits when examined without a dietary cholesterol stressor. These observations may explain why CLA appeared to reduce atherogenesis in hamsters and rabbits fed CLA in conjunction with high cholesterol diets (2
,3
), whereas it may be ineffective (20
) or even deleterious (21
,22
) when not associated with reduced cholesterol absorption.
CLA and adipose tissue.
The reduced adipose stores in hamsters in Experiment 1 confirm and extend CLA involvement in adipose tissue metabolism in other species. Reduced adipose tissue (and weight gain) supports previous observations by Park et al. (7
) and Azain et al. (23
) in which CLA supplementation decreased body fat and enhanced muscle mass.
Finally, it should be emphasized that to detect any benefit from CLA in hamsters, it was necessary to supplement 1% CLA (2% of energy), which is an amount much greater than can be realized from any natural food source. Furthermore, the potential benefit from CLA as a TC-lowering agent required the simultaneous intake of cholesterol at an extremely high level. Thus, the posited attributes of CLA against experimental atherosclerosis may, in fact, depend on an abnormally high intake of cholesterol.
|
FOOTNOTES |
|---|
2 Present address: Department of Physiology and Biophysics, State University of New York at Stony Brook, New York, NY 11794.
4 Abbreviations used: ACAT, Acyl-CoA; apo, apolipoprotein; APR, acute phase response; CLA, conjugated linoleic acid; EC, esterified cholesterol; LPS, lipopolysaccharide; PUFA, polyunsaturated fatty acids; P/S, polyunsaturated fatty acids/saturated fatty acids; TC, total cholesterol; TG, triglycerides.
Manuscript received 25 February 2002. Initial review completed 13 March 2002. Revision accepted 30 October 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Pariza, M. W., Park, Y. & Cook, M. E. (1999) Conjugated linoleic acid and the control of cancer and obesity. Toxicol. Sci. 52:107-110.
2. Nicolosi, R. J., Rogers, E. J., Kritchevsky, D., Scimeca, J. A. & Huth, P. J. (1997) Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 22:266-277.[Medline]
3. Lee, K. N., Kritchevsky, D. & Pariza, M. W. (1994) Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19-25.[Medline]
4. Ip, C., Chin, S. F., Scimeca, J. A. & Pariza, M. W. (1991) Mammary cancer prevention by conjugated dienoic derivatives of linoleic acid. Cancer Res. 51:6118-6124.
5. Miller, C. C., Park, Y., Pariza, M. W. & Cook, M. E. (1994) Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochem. Biophys. Res. Commun. 198:1107-1112.[Medline]
6. Navab, M., Berliner, J. A., Subbanagounder, G., Hama, S., Lusis, A. J., Castellani, L. W., Reddy, S., Shih, D., Shi, W., Watson, A. D., Van Lenten, B. J., Vora, D. & Fogelman, A. M. (2001) HDL and the inflammatory response induced by LDL-derived oxidized phospholipids. Arterioscler. Thromb. Vasc. Biol. 21:481-488.
7. Park, Y., Albright, K. J., Liu, W., Storkson, J. M., Cook, M. E. & Pariza, M. W. (1997) Effect of conjugated linoleic acid on body composition in mice. Lipids 32:853-858.[Medline]
8. West, D. B., Delany, J. P., Camet, P. M., Blohm, F., Truett, A. A. & Scimeca, J. (1998) Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse. Am. J. Physiol. 275:R667-R672.
9. Gonnerman, W. A., Lim, M., Sipe, J. D., Hayes, K. C. & Cathcart, E. S. (1996) The acute phase response in Syrian hamsters elevates apolipoprotein serum amyloid A (apoSAA) and disrupts lipoprotein metabolism. Amyloid Int. J. Exp. Clin. Invest. 3:261-269.
10. Yeung, C.H.T., Yang, L., Huang, Y., Wang, J. & Chen, Z. Y. (2000) Dietary conjugated linoleic acid mixture affects the activity of intestinal acyl coenzyme A:cholesterol acyltransferase in hamsters. Br. J. Nutr. 84:935-941.[Medline]
11. Hayes, K. C., Stephan, Z. F., Pronczuk, A., Lindsey, S. & Verdon, C. (1989) Lactose protects against estrogen-induced pigment gallstones in hamsters fed nutritionally adequate purified diets. J Nutr. 119:1726-1736.
12. Reaven, P. D., Grasse, B. J. & Tribble, D. L. (1994) Effects of linoleate-enriched and oleate-enriched diets in combination with alpha-tocopherol on the susceptibility of LDL and LDL subfractions to oxidative modification in humans. Arterioscler. Thromb. 14:557-566.
13. Devaraj, S., Harris, A. & Jialal, I. (2002) Modulation of monocyte-macrophage function with alpha-tocopherol: implications for atherosclerosis. Nutr. Rev. 60:8-14.[Medline]
14. Goulinet, S. & Chapman, M. J. (1993) Plasma lipoproteins in the golden Syrian hamster (Mesocricetus auratus): heterogeneity of apoB- and apoA-I-containing particles. J. Lipid Res. 34:943-959.[Abstract]
15. Kim, J. C. & Chung, T. H. (1984) Direct determination of the free cholesterol and individual cholesteryl esters in serum by high pressure liquid chromatography. Korean J. Biochem. 16:69-77.
16. Bieri, J. G., Tollier, T. J. & Catigliani, G. L. (1979) Simultaneous determination of retinol and alpha-tocopherol in plasma and red blood cells by high pressure liquid chromatography. Am. J. Clin. Nutr. 32:2143-2149.
17. Feingold, K. R, Hardardottir, I., Memon, R., Krul, E. J. T., Moser, A., Taylor, J. M. & Grunfeld, C. (1993) Effect of endotoxin on cholesterol biosynthesis and distribution in serum lipoprotein in Syrian hamsters. J. Lipid Res. 34:2147-2158.[Abstract]
18. Stephensen, C. B. & Gildengorin, G. (2000) Serum retinol, the acute phase response, and the apparent misclassification of vitamin A status in the third National Health and Nutrition Examination Survey. Am. J. Clin. Nutr. 72:1170-1178.
19. Liao, W., Rudling, M. & Angelin, B. (1996) Endotoxin suppresses rat hepatic low-density lipoprotein receptor expression. Biochem. J. 313:873-878.
20. Benito, P., Nelson, G. J., Kelley, D. S., Bartolini, G., Schmidt, P. C. & Simon, V. (2001) The effect of conjugated linoleic acid on plasma lipoproteins and tissue fatty acid composition in humans. Lipids 36:229-23.[Medline]
21. Munday, J. S., Thompson, K. G. & James, K. A. (1999) Dietary conjugated linoleic acids promote fatty streak formation in the C57BL/6 mouse atherosclerosis model. Br. J. Nutr. 81:251-255.[Medline]
22. Stangl, G. I., Muller, H. & Kirchgessner, M. (1999) Conjugated linoleic acid effects on circulating hormones, metabolites and lipoproteins, and its proportion in fasting serum and erythrocyte membranes of swine. Eur. J. Nutr. 38:271-277.[Medline]
23. Azain, M. J., Hausman, D. B., Sisk, M. B., Flatt, W. P. & Jewell, D. E. (2000) Dietary conjugated linoleic acid reduces rat adipose tissue cell size rather than cell number. J. Nutr. 130:1548-1554.
This article has been cited by other articles:
|
|
 |
|
  L. D Whigham, A. C Watras, and D. A Schoeller Efficacy of conjugated linoleic acid for reducing fat mass: a meta-analysis in humans Am. J. Clinical Nutrition, May 1, 2007; 85(5): 1203 - 1211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. H. Terpstra Effect of conjugated linoleic acid on body composition and plasma lipids in humans: an overview of the literature Am. J. Clinical Nutrition, March 1, 2004; 79(3): 352 - 361. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yu-Poth, D. Yin, G. Zhao, P. M. Kris-Etherton, and T. D. Etherton Conjugated Linoleic Acid Upregulates LDL Receptor Gene Expression in HepG2 Cells J. Nutr., January 1, 2004; 134(1): 68 - 71. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
