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Dietary octacosanol reduces plasma triacylglycerol levels but not atherogenesis in apolipoprotein E–knockout mice
Nutrition Research 27 (2007) 212 – 217 www.elsevier.com/locate/nutres
Dietary octacosanol reduces plasma triacylglycerol levels but not atherogenesis in apolipoprotein E–knockout mice Zuyuan Xu, Evelyn Fitz, Natalie Riediger, Mohammed H. Moghadasian4 Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6 Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6 Received 30 August 2006; revised 25 January 2007; accepted 31 January 2007
Abstract Epidemiological and clinical studies have shown a significant positive correlation between elevated plasma levels of cholesterol and triacylglycerol (TG) and the incidence of coronary artery disease. Several dietary and pharmacologic agents have been used to improve plasma lipoprotein profile and reduce the risk for cardiovascular diseases. Some clinical trials have shown beneficial effects of dietary policosanol on plasma lipids; however, long-term effects have not been documented. Octacosanol is one of the major components of policosanol mixtures. This study investigated the long-term effects of dietary octacosanol on plasma lipids and atherogenesis in apolipoprotein E–knockout (apo E–KO) mice, a model of spontaneous atherosclerosis. Apo E–KO mice were fed a 0.2% (wt/wt) cholesterol-supplemented diet in the presence (treated group, n = 5) or absence (control group, n = 5) of 1% (wt/wt) dietary octacosanol for 12 weeks. Dietary octacosanol significantly reduced the levels of plasma TG by approximately 70% by week 5 of the study, as compared with the control group. However, plasma total cholesterol levels were slightly increased in the treated group compared with the control group. A decrease in the ratio of HDL to total cholesterol was observed in the octacosanol-treated group compared with controls (0.06 vs 0.08). Despite these changes in plasma lipid profile, dietary octacosanol had no significant effects on the extent and severity of aortic atherosclerosis in this model. In conclusion, our data indicate that dietary octacosanol reduces plasma TG levels in apo E–KO mice fed a bWestern-typeQ diet. The potential lipid-lowering and antiatherogenic effects of dietary octacosanol merit further investigation. D 2007 Elsevier Inc. All rights reserved. Keywords:
Apo E–KO mice; Atherosclerosis; Octacosanol; Lipoproteins; Policosanol
1. Introduction Coronary artery disease (CAD) is still one of the leading causes of morbidity and mortality in the Western world. Mixed dyslipidemias with high plasma levels of total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, and triacylglycerol (TG), and low levels of high-density lipoprotein (HDL) cholesterol play an etiologic role in CAD [1]. Currently, the use of functional foods and 4 Corresponding author. Tel.: +1 204 235 3934; fax: +1 204 231 1151. E-mail address: [email protected] (M.H. Moghadasian). 0271-5317/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2007.01.015
nutraceuticals to treat and prevent chronic diseases, specifically CAD, is very attractive. These products are both economical and widely accepted by the general public as an alternative to synthetic drugs. Octacosanol (CH3[CH2]26CH2OH), a high-molecular-weight primary aliphatic alcohol, is one of the policosanol family members commonly found in the natural wax extracted from various plant parts including fruits, leaves, barks, and wax [2,3]. Several studies have reported cardiovascular benefits of policosanols and octacosanol without major adverse effects [4-7]. However, the exact mechanism for their actions has not been established. Thus, available evidence on safety
Z. Xu et al. / Nutrition Research 27 (2007) 212–217
and efficacy of these dietary agents warrants further consideration of their use in the treatment or prevention of chronic diseases. Octacosanol may decrease the risk of atheroma formation by reducing lipid levels, platelet aggregation, endothelial damage, and the development of foam cells [4]. Policosanol and its major component octacosanol may decrease cholesterol synthesis in the liver before the generation of mevalonate [5]. Octacosanol may downregulate the cellular expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. However, octacosanol does not seem to inhibit HMG-CoA reductase activity in the same way that statins work [8]. Treatment with octacosanoic acid isolated and purified from sugar cane wax suppressed HMG-CoA reductase production in cultured fibroblasts, and this finding suggests a possible depression of de novo synthesis of the enzyme and cholesterol [9]. Some research indicates that octacosanol may enhance the breakdown of LDL particles [4]. In another study, dietary octacosanol was thought to increase lipid catabolism to generate more energy for improvement of motor endurance [10]; and this action may contribute to reductions in plasma TG levels. Octacosanol is already recognized as an ergogenic product used to enhance athletic performance [2]. Despite the above-mentioned effects on lipid metabolism, the impact of dietary policosanols or octacosanol on prevention of atherosclerosis has not been established. Our literature search revealed no long-term clinical studies that examined the effects of these agents on atherosclerosis in humans. Furthermore, to our knowledge, the effects of policosanols or octacosanol on the development or prevention of atherosclerotic lesions in animal models are limited. Therefore, the goal of the current study was to determine the effects of dietary octacosanol, the major component of policosanol mixtures, in a well-established animal model of atherosclerosis. Among the animal models of dyslipidemia and atherosclerosis, the apolipoprotein E–knockout (apo E–KO) mouse model has been widely used to investigate the effects of dietary agents or drugs [11]. This mouse model develops hypercholesterolemia and advanced atherosclerosis, which generally resemble those commonly seen in humans. We have extensive experience using the apo E–KO mouse model to establish cholesterol-lowering and antiatherogenic effects of dietary phytosterols alone or in combination with other lipid-lowering drugs [12-16]. In the current study, we used this robust rodent model to address the following research objectives to test: (a) if dietary octacosanol can modify the plasma lipid profile and (b) if changes in plasma lipids induced by dietary octacosanol influence atherosclerotic lesion development in the aorta in a 12-week experiment. Our previous work showed that 12 weeks is adequate to induce or prevent atherosclerotic lesions in cholesterol-fed apo E–KO mice [12-16]. In
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addition to the daily dietary intake of policosanols, several supplements containing these compounds are currently available to the general public. Therefore, the results of this study will advance knowledge of the cardiovascular benefits of octacosanol to better understand their application in the prevention/treatment of chronic diseases using functional foods and nutraceuticals.
2. Methods and materials 2.1. Animals and diets Male 4-week-old apo E–KO mice were purchased from the Jackson Laboratory (Bar Harbor, Me) and acclimated to the animal facility for 1 week, during which time they were fed standard mouse chow. Thereafter, one group of mice (control group, n = 5) was fed with Pico Lab Mouse diet (Ren’s Feed and Supply Ltd, Oakville, Ontario) supplemented with 0.2% (wt/wt) cholesterol (Sigma-Aldrich, Oakville, Ontario). The other group (treated group, n = 5) was fed with the same diet supplemented with 1% (wt/wt) of a polycosanol mixture from rice wax containing N96% octacosanol. This mixture is referred to as octacosanol in this study. The ingredients of the Pico Lab Mouse Diet are presented in Table 1. Octacosanol was generously provided by Sino Pharmaceuticals Co, Richmond, British Columbia. A certificate of analysis provided by the company indicates that the policosanol mixture used in this study was composed of 96.3% 1-octacosanol, 1.2% 1-hexacosanol, 0.4% 1-heptacosanol, 0.2% 1-nonacosanol, and 1.3% 1-tiacosanol. The certificate also indicates that ethanol was used as the solvent for gas chromatography analysis of the mixture. The dose of the octacosanol was estimated based on previous studies [6,17,18]. Both groups of mice (control and treated) had similar average body weights and plasma cholesterol levels at baseline. Mice were housed in groups during the study. One mouse from the octacosanol-treated group was euthanized by an overdose of pentobarbital (RO Burrell Lab, St Boniface Hospital Research Centre, Winnipeg, Manitoba) because of fighting injuries on the second week of the study. The effects of octacosanol on the development of atherosclerotic lesions in the aortic roots of apo E–KO mice were conducted through 12 weeks. Body weight was recorded Table 1 Proximate contents of the Pico Lab Mouse Diet Amount (g/100 g) Protein Fat Carbohydrate (starch) Minerals and vitamins Fiber Moisture
20.5 9.0 53.0 4.8 2.7 10.0
This basal diet contained cholesterol (0.2% wt/wt) for the control group (n = 5) and the treated group (n = 5). The treated group was fed the basal diet supplemented with 1% octacosanol (wt/wt).
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weekly, and nonfasting blood samples were taken at baseline and after 5 weeks of treatment from the jugular vein of lightly anesthetized mice (halothane inhalation); because of human errors, lipid measurements in final plasma samples were not possible. Similarly, because of a lack of adequate amounts of plasma sample measurements, the plasma levels of octacosanol in the treated animals were not determined. It should be noted that numerous studies from our laboratory and others have shown that 5 weeks is long enough to observe the effects of intervention on plasma lipid levels in rodents and human subjects [12-16,19-22]. Therefore, we believe that the effects of octacosanol on lipid values at week 12 would be similar to those at week 5. At week 12, the mice were euthanized by an injection (intraperitoneal) of pentobarbital. Final blood samples were collected through heart puncture, and the hearts and aortas were fixed in 10% buffered formalin (Fisher Scientific, Ottawa, Ontario) for histologic examination [13-15]. The experimental protocol and animal use were approved by the Animal Care Committee at the University of Manitoba, Winnipeg, Manitoba. 2.2. Lipid analysis Plasma was prepared from blood samples and used for determination of TC, TG, and HDL cholesterol levels using appropriate enzymatic kits from Diagnostic Chemicals Limited (Charlottetown, Prince Edward Island) as previously described [12-15]. Standard precipitation method was used to prepare the HDL fraction [23]. The mean of HDL cholesterol levels was divided by the mean of TC levels to calculate the ratio of HDL/TC as previously described [24]. All lipid measurements were performed using internal standard solutions provided by the manufacturer (Thermo DMA, Arlington, Tex). 2.3. Histology and morphometry evaluations of atherosclerotic lesions Hearts from mice were placed in 10% buffered formalin and cut transversely at the level of atria; the atrial portions were embedded in optimal cutting temperature compound
Fig. 2. Plasma TC concentrations (mean F SD, mmol/L) at baseline and at week 5 of the experiment for the control and octacosanol-treated apo E–KO mice.
(Fisher Scientific) and sectioned at the level of aortic valve cusps. Once the aortic valves were apparent, 10-lm sections were mounted on glass slides and stained with hematoxylin and eosin (H&E, Fisher Scientific) and oil red O (ORO, Sigma-Aldrich) for histologic and morphometric examinations [13-15]. The ORO-stained sections were used to estimate atherosclerotic lesion size and lesion-lumen ratio in the aortic roots of both treated and control mice using Image Pro-Plus software (Media Cybernetics Inc, Silver Spring, Md) [13-15]. 2.4. Statistical analysis Data are presented as mean and standard deviation. A 2-tailed Student t test using Microsoft Office Excel (Microsoft, Redmond, Wash) was used to identify statistically significant differences between the treated and control mice at the level of P b .05 as previously described [12,14,23]. 3. Results 3.1. Body weight Both of the experimental groups gained weight at comparable rates during the study period as illustrated in Fig. 1. The data indicate that octacosanol was well tolerated by the mice and no apparent adverse effect was observed.
Table 2 The effects of octacosanol treatment at week 5 on plasma TG levels and HDL cholesterol concentrations in apo E–KO mice
Fig. 1. Body weights (mean F SD) at baseline and during the experimental period for the control and octacosanol-treated apo E–KO mice.
Measurements
Control (n = 5)
Octacosanol (n = 4)
TG (mmol/L) HDL cholesterol (mmol/L) HDL/TC ratio
1.26 F 0.32 0.79 F 0.27 0.08
0.41 F 0.294 0.78 F 0.15 0.06
Values are mean F standard deviation. The control group was fed cholesterol (0.2% wt/wt), and the octacosanol-treated mice was fed the same diet but supplemented with 1% octacosanol (wt/wt) for 12 weeks. 4 P b .05 as compared with the control group.
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Fig. 3. Representative photomicrographs of aortic roots of control (A and C) and octacosanol-treated (B and D) apo E–KO mice. A and B, Cellular and morphological features of the atherosclerotic lesions. C and D, Extent of lipid deposits in the atherosclerotic lesions. A and B, H&E staining; C and D, ORO staining.
3.2. Plasma lipid levels The levels of plasma TC at baseline and after 5 weeks of treatment are shown in Fig. 2. By week 5 of the study, the control group had a significantly ( P b .05) higher TC (increase of 47%) compared with the baseline value, indicating the effect of dietary cholesterol on plasma cholesterol levels. The addition of octacosanol to the diet further augmented plasma cholesterol levels; however, this effect was not statistically significant ( P = .34) (Fig. 2). The HDL cholesterol levels were comparable between the octacosanol-treated mice and the control group (Table 2). The mean ratio of HDL cholesterol to TC in the control group and octacosanol group were 0.08 and 0.06, respectively. The reduction in the ratio of HDL to TC was most likely due to increases in TC levels in the octacosanol-treated mice. Table 2 also presents the TG levels in both groups of mice at week 5 of the study. Octacosanol treatment was associated with a significant ( P b .05) reduction of up to 70% in plasma TG level compared with those in the control group.
sections (Fig. 3C and D) illustrate the presence of lipid-rich atherosclerotic lesions in both groups of control and octacosanol-treated mice. The H&E staining revealed no noticeable difference in the quality and morphological characteristics of the atherosclerotic lesions between the control group and octacosanol-treated group (Fig. 3A and B). Table 3 summarizes the morphometric analysis of the atherosclerotic lesions in the aortic roots of the mice. No statistically significant difference was observed in lesion size between the octacosanol-treated and control groups (0.48 vs 0.42 mm2).
Table 3 Morphometric characteristics of atherosclerotic lesions in the control and octacosanol-treated apo E–KO mice at week 12 Measurement 2
Control (n = 5)
Octacosanol (n = 4)
0.42 F 0.14 1.26 F 0.22 0.32 F 0.078
0.48 F 0.11 1.27 F 0.22 0.38 F 0.07
3.3. Atherosclerotic lesion development
Lesion size (mm ) Lumen area (mm2) Lesion-lumen ratio
Representative photomicrographs of aortic roots from the 2 groups of mice are shown in Fig. 3. The ORO-stained
Values are mean F standard deviation. The control group was fed cholesterol (0.2% wt/wt), and the octacosanol-treated mice was fed the same diet but supplemented with 1% octacosanol (wt/wt) for 12 weeks.
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4. Discussion The results from animal studies are not consistent for the effects of octacosanol on lipoproteins. Arruzazabala et al [25] showed that 5 to 200 mg/kg of policosanols for 4 weeks significantly reduced TC and TG levels without significant changes in HDL cholesterol levels in New Zealand rabbits. A dose-response relationship was reported for the effects on plasma cholesterol but not for TG levels. However, further studies by the same group of investigators [26] were unable to reproduce these beneficial effects of policosanols in hypercholesterolemic (exogenously induced) New Zealand rabbits over 60 days of study. Several clinical studies have shown that policosanols may reduce TC levels by 8% to 21% in patients with hypercholesterolemia [27-32]. However, wheat germ policosanol failed to lower plasma cholesterol in subjects with normal or mildly elevated cholesterol concentrations [33]. In agreement with the latter clinical study, we observed a lack of cholesterol-lowering effects of octacosanol in apo E–KO mice. Clinical trials have shown some benefits of policosanol treatment on reducing TG levels. One study reported a significant decrease (33%) in TG levels in healthy volunteers [27]. However, in rats, the addition of octacosanol (10 g/kg diet) to a high-fat diet significantly decreased serum TG concentrations [17]. Our study supports previous research showing a significant decrease in plasma TG levels. Although the mechanisms of TG-lowering effects of policosanols are not fully understood, it has been suggested that octacosanol may increase lipid catabolism [10]. It is possible that octacosanol increases the production and activities of lipoprotein and hepatic lipases and thereby reduces the levels of circulating TG. Another possibility is that octacosanol may increase the uptake of TG-rich particles by hepatocytes. One may expect that reductions in TG concentrations are accompanied by increases in HDL cholesterol levels. The study of Arruzazabala et al [25] showed that treatment with 5 to 200 mg/kg of policosanols for 4 weeks did not alter HDL cholesterol levels in New Zealand rabbits. In agreement with these observations, there were no significant alterations in HDL cholesterol levels in octacosanol-treated apo E–KO mice in the present study. In contrast, clinical studies have reported significant increases in HDL cholesterol levels with the ingestion of these compounds [27,32]. Arruzazabala et al [25] showed that New Zealand rabbits receiving 25 or 200 mg/kg policosanols for 60 days were mostly free of atherosclerotic lesions, and this was associated with a reduction in the levels of thromboxane A2 and an increase in prostacyclin levels. Similarly, Wistar rats treated with 0.5, 2.5, 5, and 25 mg/kg of policosanols developed less atherosclerotic lesions as compared with the controls [34]. In an effort to identify the potential mechanism for the antiatherogenic effects of policosanol, Ng et al [35] investigated the effects of these natural compounds on LDL oxidation and bile acid excretion.
Policosanol showed no antioxidant activity in human LDL particles but increased bile acid secretion in hamsters [35]. We observed no noticeable differences in the quality and morphological characteristics of the atherosclerotic lesions between the control group and octacosanol-treated apo E–KO mice in the present study. Both groups of mice had comparable lesion size (0.48 vs 0.42 mm2) and lesionlumen ratio (0.38 vs 0.32), indicating a lack of antiatherogenic properties of octacosanol over 12 weeks in this mouse model. In summary, several studies reported potential cholesterol-lowering and antiatherogenic properties of policosanols. In our experiment using a small number of mice, we observed a TG-lowering effect of octacosanol in cholesterol-fed apo E–KO mice. Despite these effects, octacosanoltreatment did not change atherogenesis in this mouse model. One reason for this could be the nonsignificant increases in plasma TC levels observed in octacosanol-treated mice and the limited number of animals used. It remains unclear if dietary octacosanol can affect dietary cholesterol absorption. Furthermore, a longer treatment protocol (more than 12 weeks) may be needed to observe the antiatherogenic properties of octacosanol in this animal model of atherosclerosis. Future studies are needed to characterize the potential antiatherogenic activities of octacosanol. Acknowledgment Technical support of Behzad Yeganeh is greatly appreciated. Support from Sino Pharmaceuticals Co is also greatly appreciated. References [1] Singh BK, Mehta JL. Management of dyslipidemia in the primary prevention of coronary heart disease. Curr Opin Cardiol 2002; 17:503 - 11. [2] Taylor JC, Rapport L, Lockwood GB. Octacosanol in human health. Nutrition 2003;19:192 - 5. [3] Hargrove JL, Greenspan P, Hartle DK. Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes. Exp Biol Med 2004;229:215 - 26. [4] Varady KA, Wang Y, Jones PJ. Role of policosanols in the prevention and treatment of cardiovascular disease. Nutr Rev 2003;61:376 - 83. [5] Menendez R, Amor AM, Gonzalez RM, Fraga V, Mas R. Effect of policosanol on the hepatic cholesterol biosynthesis of normocholesterolemic rats. Biol Res 1996;29:253 - 7. [6] Kabir Y, Kimura S. Biodistribution and metabolism of orally administered octacosanol in rats. Ann Nutr Metab 1993;37:33 - 8. [7] Aleman CL, Mas R, Hernandez C, Rodeiro I, Cerejido E, Noa M, et al. A 12-month study of policosanol oral toxicity in Sprague Dawley rats. Toxicol Lett 1994;70:77 - 87. [8] McCarty MF. Policosanol safely down-regulates HMG-CoA reductase—potential as a component of the Esselstyn regimen. Med Hypotheses 2002;59:268 - 79. [9] Menendez R, Mas R, Amor AM, Rodeiros I, Gonzalez RM, Alfonso JL. Inhibition of cholesterol biosynthesis in cultured fibroblasts by D003, a mixture of very long chain saturated fatty acids. Pharmacol Res 2001;44:299 - 304.
Z. Xu et al. / Nutrition Research 27 (2007) 212–217 [10] Shimura S, Hasegawa T, Takano S, Suzuki T. Studies on the effect of octacosanol on motor endurance in mice. Nutr Rep Int 1987; 36:1029 - 38. [11] Moghadasian MH, Frohlich JJ, McManus BM. Advances in experimental dyslipidemia and atherosclerosis. Lab Invest 2001;81: 1173 - 83. [12] Nashed B, Yeganeh B, HayGlass KT, Moghadasian MH. Antiatherogenic effects of dietary plant sterols are associated with inhibition of proinflammatory cytokine production in Apo E–KO mice. J Nutr 2005;135:2438 - 44. [13] Yeganeh B, Moshtaghi-Kashanian GR, Declercq V, Moghadasian MH. Combination of dietary phytosterols plus niacin or fenofibrate: effects on lipid profile and atherosclerosis in apo E–KO mice. J Nutr Biochem 2005;16:222 - 8. [14] Moghadasian MH, McManus BM, Pritchard PH, Frohlich JJ. bTall oilQ–derived phytosterols reduce atherosclerosis in Apo E–deficient mice. Arterioscler Thromb Vasc Biol 1997;17:119 - 26. [15] Moghadasian MH, McManus BM, Godin DV, Rodrigues B, Frohlich JJ. Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E–deficient mice: possible mechanisms of action. Circulation 1999;99:1733 - 9. [16] Moghadasian MH, Nguyen LB, Shefer S, McManus BM, Frohlich JJ. Histologic, hematologic, and biochemical characteristics of apo E–deficient mice: effects of dietary cholesterol and phytosterols. Lab Invest 1999;79:355 - 64. [17] Kato S, Karino K, Hasegawa S, Nagasawa J, Nagasaki A, Eguchi M, et al. Octacosanol affects lipid metabolism in rats fed on a high-fat diet. Br J Nutr 1995;73:433 - 41. [18] Kim H, Park S, Han DS, Park T. Octacosanol supplementation increases running endurance time and improves biochemical parameters after exhaustion in trained rats. J Med Food 2003;6:345 - 51. [19] Pritchard PH, Li M, Zamfir C, Lukic T, Novak E, Moghadasian MH. Comparison of cholesterol-lowering efficacy and anti-atherogenic properties of hydrogenated versus non-hydrogenated (Phytrol) tall oilderived phytosterols in apo E–deficient mice. Cardiovasc Drugs Ther 2003;17:443 - 9. [20] Lukic T, Wasan KM, Zamfir D, Moghadasian MH, Pritchard PH. Disodium ascorbyl phytostanyl phosphate reduces plasma cholesterol concentrations and atherosclerotic lesion formation in apolipoprotein E–deficient mice. Metabolism 2003;52:425 - 31. [21] Elam MB, Hunninghake DB, Davis KB, Garg R, Johnson C, Egan D, et al. Effect of niacin on lipid and lipoprotein levels and glycemic control in patients with diabetes and peripheral arterial disease: the ADMIT study: a randomized trial. Arterial Disease Multiple Intervention Trial. JAMA 2000;284:1263 - 70. [22] Kuwabara K, Murakami K, Todo M, Aoki T, Asaki T, Murai M, et al. A novel selective peroxisome proliferator-activated receptor alpha
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
217
agonist, 2-methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220), potently decreases plasma triglyceride and glucose levels and modifies lipoprotein profiles in KK-Ay mice. J Pharmacol Exp Ther 2004;309:970 - 7. Moghadasian MH, McManus BM, Nguyen LB, Shefer S, Nadji M, Godin DV, et al. Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans. FASEB J 2001; 15:2623 - 30. Moghadasian MH. Dietary phytosterols reduce probucol-induced atherogenesis in apo E–KO mice. Atherosclerosis 2006;188:28 - 34. Arruzazabala ML, Carbajal D, Mas R, Molina V, Valdes S, Laguna A. Cholesterol-lowering effects of policosanol in rabbits. Biol Res 1994;27:205 - 8. Arruzazabala ML, Noa M, Menendez R, Mas R, Carbajal D, Valdes S, et al. Protective effect of policosanol on atherosclerotic lesions in rabbits with exogenous hypercholesterolemia. Braz J Med Biol Res 2000;33:835 - 40. Hernandez F, Illnait J, Mas R, Castano G, Fernandez L, Gonzalez M, et al. Effect of policosanol on serum lipids and lipoproteins in healthy volunteers. Curr Ther Res 1992;51:568 - 75. Pons P, Ma´s R, Illnait J, Ferna´ndez L, Rodriguez M, Robaina C, Ferna´ndez JC. Efficacy and safety of policosanol in patients with primary hypercholesterolemia. Curr Ther Res 1992;52:507 - 13. Pons P, Rodrı´guez M, Ma´s R, Illnait J, Ferna´ndez L, Robaina C, et al. One-year efficacy and safety of policosanol in patients with type II hypercholesterolemia. Curr Ther Res 1994;55:1084 - 92. Aneiros E, Calderon B, Ma´s R, Illnait J, Castan˜o G, Ferna´ndez L, et al. Effect of successive dose increases of policosanol on the lipid profile and tolerability of treatment. Curr Ther Res 1993;54:304 - 12. Aneiros E, Ma´s R, Calderon B, Illnait J, Ferna´ndez L, Castan˜o G, et al. Effect of policosanol in lowering cholesterol levels in patients with type II hypercholesterolemia. Curr Ther Res 1995;56:176 - 82. Canetti M, Moreira M, Mas R, Illnait J, Fernandez L, Fernandez J, et al. A two-year study on the efficacy and tolerability of policosanol in patients with type II hyperlipoproteinaemia. Int J Clin Pharmacol Res 1995;15:159 - 65. Lin Y, Rudrum M, van der Wielen RP, Trautwein EA, McNeill G, Sierksma A, et al. Wheat germ policosanol failed to lower plasma cholesterol in subjects with normal to mildly elevated cholesterol concentrations. Metabolism 2004;53:1309 - 14. Noa M, Mas R, de la Rosa MC, Magraner J. Effect of policosanol on lipofundin-induced atherosclerotic lesions in rats. J Pharm Pharmacol 1995;47:289 - 91. Ng CH, Leung KY, Huang Y, Chen ZY. Policosanol has no antioxidant activity in human low-density lipoprotein but increases excretion of bile acids in hamsters. J Agric Food Chem 2005; 53:6289 - 93.
Dietary octacosanol reduces plasma triacylglycerol levels but not atherogenesis in apolipoprotein E–knockout mice Zuyuan Xu, Evelyn Fitz, Natalie Riediger, Mohammed H. Moghadasian4 Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6 Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada R2H 2A6 Received 30 August 2006; revised 25 January 2007; accepted 31 January 2007
Abstract Epidemiological and clinical studies have shown a significant positive correlation between elevated plasma levels of cholesterol and triacylglycerol (TG) and the incidence of coronary artery disease. Several dietary and pharmacologic agents have been used to improve plasma lipoprotein profile and reduce the risk for cardiovascular diseases. Some clinical trials have shown beneficial effects of dietary policosanol on plasma lipids; however, long-term effects have not been documented. Octacosanol is one of the major components of policosanol mixtures. This study investigated the long-term effects of dietary octacosanol on plasma lipids and atherogenesis in apolipoprotein E–knockout (apo E–KO) mice, a model of spontaneous atherosclerosis. Apo E–KO mice were fed a 0.2% (wt/wt) cholesterol-supplemented diet in the presence (treated group, n = 5) or absence (control group, n = 5) of 1% (wt/wt) dietary octacosanol for 12 weeks. Dietary octacosanol significantly reduced the levels of plasma TG by approximately 70% by week 5 of the study, as compared with the control group. However, plasma total cholesterol levels were slightly increased in the treated group compared with the control group. A decrease in the ratio of HDL to total cholesterol was observed in the octacosanol-treated group compared with controls (0.06 vs 0.08). Despite these changes in plasma lipid profile, dietary octacosanol had no significant effects on the extent and severity of aortic atherosclerosis in this model. In conclusion, our data indicate that dietary octacosanol reduces plasma TG levels in apo E–KO mice fed a bWestern-typeQ diet. The potential lipid-lowering and antiatherogenic effects of dietary octacosanol merit further investigation. D 2007 Elsevier Inc. All rights reserved. Keywords:
Apo E–KO mice; Atherosclerosis; Octacosanol; Lipoproteins; Policosanol
1. Introduction Coronary artery disease (CAD) is still one of the leading causes of morbidity and mortality in the Western world. Mixed dyslipidemias with high plasma levels of total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, and triacylglycerol (TG), and low levels of high-density lipoprotein (HDL) cholesterol play an etiologic role in CAD [1]. Currently, the use of functional foods and 4 Corresponding author. Tel.: +1 204 235 3934; fax: +1 204 231 1151. E-mail address: [email protected] (M.H. Moghadasian). 0271-5317/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2007.01.015
nutraceuticals to treat and prevent chronic diseases, specifically CAD, is very attractive. These products are both economical and widely accepted by the general public as an alternative to synthetic drugs. Octacosanol (CH3[CH2]26CH2OH), a high-molecular-weight primary aliphatic alcohol, is one of the policosanol family members commonly found in the natural wax extracted from various plant parts including fruits, leaves, barks, and wax [2,3]. Several studies have reported cardiovascular benefits of policosanols and octacosanol without major adverse effects [4-7]. However, the exact mechanism for their actions has not been established. Thus, available evidence on safety
Z. Xu et al. / Nutrition Research 27 (2007) 212–217
and efficacy of these dietary agents warrants further consideration of their use in the treatment or prevention of chronic diseases. Octacosanol may decrease the risk of atheroma formation by reducing lipid levels, platelet aggregation, endothelial damage, and the development of foam cells [4]. Policosanol and its major component octacosanol may decrease cholesterol synthesis in the liver before the generation of mevalonate [5]. Octacosanol may downregulate the cellular expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. However, octacosanol does not seem to inhibit HMG-CoA reductase activity in the same way that statins work [8]. Treatment with octacosanoic acid isolated and purified from sugar cane wax suppressed HMG-CoA reductase production in cultured fibroblasts, and this finding suggests a possible depression of de novo synthesis of the enzyme and cholesterol [9]. Some research indicates that octacosanol may enhance the breakdown of LDL particles [4]. In another study, dietary octacosanol was thought to increase lipid catabolism to generate more energy for improvement of motor endurance [10]; and this action may contribute to reductions in plasma TG levels. Octacosanol is already recognized as an ergogenic product used to enhance athletic performance [2]. Despite the above-mentioned effects on lipid metabolism, the impact of dietary policosanols or octacosanol on prevention of atherosclerosis has not been established. Our literature search revealed no long-term clinical studies that examined the effects of these agents on atherosclerosis in humans. Furthermore, to our knowledge, the effects of policosanols or octacosanol on the development or prevention of atherosclerotic lesions in animal models are limited. Therefore, the goal of the current study was to determine the effects of dietary octacosanol, the major component of policosanol mixtures, in a well-established animal model of atherosclerosis. Among the animal models of dyslipidemia and atherosclerosis, the apolipoprotein E–knockout (apo E–KO) mouse model has been widely used to investigate the effects of dietary agents or drugs [11]. This mouse model develops hypercholesterolemia and advanced atherosclerosis, which generally resemble those commonly seen in humans. We have extensive experience using the apo E–KO mouse model to establish cholesterol-lowering and antiatherogenic effects of dietary phytosterols alone or in combination with other lipid-lowering drugs [12-16]. In the current study, we used this robust rodent model to address the following research objectives to test: (a) if dietary octacosanol can modify the plasma lipid profile and (b) if changes in plasma lipids induced by dietary octacosanol influence atherosclerotic lesion development in the aorta in a 12-week experiment. Our previous work showed that 12 weeks is adequate to induce or prevent atherosclerotic lesions in cholesterol-fed apo E–KO mice [12-16]. In
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addition to the daily dietary intake of policosanols, several supplements containing these compounds are currently available to the general public. Therefore, the results of this study will advance knowledge of the cardiovascular benefits of octacosanol to better understand their application in the prevention/treatment of chronic diseases using functional foods and nutraceuticals.
2. Methods and materials 2.1. Animals and diets Male 4-week-old apo E–KO mice were purchased from the Jackson Laboratory (Bar Harbor, Me) and acclimated to the animal facility for 1 week, during which time they were fed standard mouse chow. Thereafter, one group of mice (control group, n = 5) was fed with Pico Lab Mouse diet (Ren’s Feed and Supply Ltd, Oakville, Ontario) supplemented with 0.2% (wt/wt) cholesterol (Sigma-Aldrich, Oakville, Ontario). The other group (treated group, n = 5) was fed with the same diet supplemented with 1% (wt/wt) of a polycosanol mixture from rice wax containing N96% octacosanol. This mixture is referred to as octacosanol in this study. The ingredients of the Pico Lab Mouse Diet are presented in Table 1. Octacosanol was generously provided by Sino Pharmaceuticals Co, Richmond, British Columbia. A certificate of analysis provided by the company indicates that the policosanol mixture used in this study was composed of 96.3% 1-octacosanol, 1.2% 1-hexacosanol, 0.4% 1-heptacosanol, 0.2% 1-nonacosanol, and 1.3% 1-tiacosanol. The certificate also indicates that ethanol was used as the solvent for gas chromatography analysis of the mixture. The dose of the octacosanol was estimated based on previous studies [6,17,18]. Both groups of mice (control and treated) had similar average body weights and plasma cholesterol levels at baseline. Mice were housed in groups during the study. One mouse from the octacosanol-treated group was euthanized by an overdose of pentobarbital (RO Burrell Lab, St Boniface Hospital Research Centre, Winnipeg, Manitoba) because of fighting injuries on the second week of the study. The effects of octacosanol on the development of atherosclerotic lesions in the aortic roots of apo E–KO mice were conducted through 12 weeks. Body weight was recorded Table 1 Proximate contents of the Pico Lab Mouse Diet Amount (g/100 g) Protein Fat Carbohydrate (starch) Minerals and vitamins Fiber Moisture
20.5 9.0 53.0 4.8 2.7 10.0
This basal diet contained cholesterol (0.2% wt/wt) for the control group (n = 5) and the treated group (n = 5). The treated group was fed the basal diet supplemented with 1% octacosanol (wt/wt).
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weekly, and nonfasting blood samples were taken at baseline and after 5 weeks of treatment from the jugular vein of lightly anesthetized mice (halothane inhalation); because of human errors, lipid measurements in final plasma samples were not possible. Similarly, because of a lack of adequate amounts of plasma sample measurements, the plasma levels of octacosanol in the treated animals were not determined. It should be noted that numerous studies from our laboratory and others have shown that 5 weeks is long enough to observe the effects of intervention on plasma lipid levels in rodents and human subjects [12-16,19-22]. Therefore, we believe that the effects of octacosanol on lipid values at week 12 would be similar to those at week 5. At week 12, the mice were euthanized by an injection (intraperitoneal) of pentobarbital. Final blood samples were collected through heart puncture, and the hearts and aortas were fixed in 10% buffered formalin (Fisher Scientific, Ottawa, Ontario) for histologic examination [13-15]. The experimental protocol and animal use were approved by the Animal Care Committee at the University of Manitoba, Winnipeg, Manitoba. 2.2. Lipid analysis Plasma was prepared from blood samples and used for determination of TC, TG, and HDL cholesterol levels using appropriate enzymatic kits from Diagnostic Chemicals Limited (Charlottetown, Prince Edward Island) as previously described [12-15]. Standard precipitation method was used to prepare the HDL fraction [23]. The mean of HDL cholesterol levels was divided by the mean of TC levels to calculate the ratio of HDL/TC as previously described [24]. All lipid measurements were performed using internal standard solutions provided by the manufacturer (Thermo DMA, Arlington, Tex). 2.3. Histology and morphometry evaluations of atherosclerotic lesions Hearts from mice were placed in 10% buffered formalin and cut transversely at the level of atria; the atrial portions were embedded in optimal cutting temperature compound
Fig. 2. Plasma TC concentrations (mean F SD, mmol/L) at baseline and at week 5 of the experiment for the control and octacosanol-treated apo E–KO mice.
(Fisher Scientific) and sectioned at the level of aortic valve cusps. Once the aortic valves were apparent, 10-lm sections were mounted on glass slides and stained with hematoxylin and eosin (H&E, Fisher Scientific) and oil red O (ORO, Sigma-Aldrich) for histologic and morphometric examinations [13-15]. The ORO-stained sections were used to estimate atherosclerotic lesion size and lesion-lumen ratio in the aortic roots of both treated and control mice using Image Pro-Plus software (Media Cybernetics Inc, Silver Spring, Md) [13-15]. 2.4. Statistical analysis Data are presented as mean and standard deviation. A 2-tailed Student t test using Microsoft Office Excel (Microsoft, Redmond, Wash) was used to identify statistically significant differences between the treated and control mice at the level of P b .05 as previously described [12,14,23]. 3. Results 3.1. Body weight Both of the experimental groups gained weight at comparable rates during the study period as illustrated in Fig. 1. The data indicate that octacosanol was well tolerated by the mice and no apparent adverse effect was observed.
Table 2 The effects of octacosanol treatment at week 5 on plasma TG levels and HDL cholesterol concentrations in apo E–KO mice
Fig. 1. Body weights (mean F SD) at baseline and during the experimental period for the control and octacosanol-treated apo E–KO mice.
Measurements
Control (n = 5)
Octacosanol (n = 4)
TG (mmol/L) HDL cholesterol (mmol/L) HDL/TC ratio
1.26 F 0.32 0.79 F 0.27 0.08
0.41 F 0.294 0.78 F 0.15 0.06
Values are mean F standard deviation. The control group was fed cholesterol (0.2% wt/wt), and the octacosanol-treated mice was fed the same diet but supplemented with 1% octacosanol (wt/wt) for 12 weeks. 4 P b .05 as compared with the control group.
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Fig. 3. Representative photomicrographs of aortic roots of control (A and C) and octacosanol-treated (B and D) apo E–KO mice. A and B, Cellular and morphological features of the atherosclerotic lesions. C and D, Extent of lipid deposits in the atherosclerotic lesions. A and B, H&E staining; C and D, ORO staining.
3.2. Plasma lipid levels The levels of plasma TC at baseline and after 5 weeks of treatment are shown in Fig. 2. By week 5 of the study, the control group had a significantly ( P b .05) higher TC (increase of 47%) compared with the baseline value, indicating the effect of dietary cholesterol on plasma cholesterol levels. The addition of octacosanol to the diet further augmented plasma cholesterol levels; however, this effect was not statistically significant ( P = .34) (Fig. 2). The HDL cholesterol levels were comparable between the octacosanol-treated mice and the control group (Table 2). The mean ratio of HDL cholesterol to TC in the control group and octacosanol group were 0.08 and 0.06, respectively. The reduction in the ratio of HDL to TC was most likely due to increases in TC levels in the octacosanol-treated mice. Table 2 also presents the TG levels in both groups of mice at week 5 of the study. Octacosanol treatment was associated with a significant ( P b .05) reduction of up to 70% in plasma TG level compared with those in the control group.
sections (Fig. 3C and D) illustrate the presence of lipid-rich atherosclerotic lesions in both groups of control and octacosanol-treated mice. The H&E staining revealed no noticeable difference in the quality and morphological characteristics of the atherosclerotic lesions between the control group and octacosanol-treated group (Fig. 3A and B). Table 3 summarizes the morphometric analysis of the atherosclerotic lesions in the aortic roots of the mice. No statistically significant difference was observed in lesion size between the octacosanol-treated and control groups (0.48 vs 0.42 mm2).
Table 3 Morphometric characteristics of atherosclerotic lesions in the control and octacosanol-treated apo E–KO mice at week 12 Measurement 2
Control (n = 5)
Octacosanol (n = 4)
0.42 F 0.14 1.26 F 0.22 0.32 F 0.078
0.48 F 0.11 1.27 F 0.22 0.38 F 0.07
3.3. Atherosclerotic lesion development
Lesion size (mm ) Lumen area (mm2) Lesion-lumen ratio
Representative photomicrographs of aortic roots from the 2 groups of mice are shown in Fig. 3. The ORO-stained
Values are mean F standard deviation. The control group was fed cholesterol (0.2% wt/wt), and the octacosanol-treated mice was fed the same diet but supplemented with 1% octacosanol (wt/wt) for 12 weeks.
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4. Discussion The results from animal studies are not consistent for the effects of octacosanol on lipoproteins. Arruzazabala et al [25] showed that 5 to 200 mg/kg of policosanols for 4 weeks significantly reduced TC and TG levels without significant changes in HDL cholesterol levels in New Zealand rabbits. A dose-response relationship was reported for the effects on plasma cholesterol but not for TG levels. However, further studies by the same group of investigators [26] were unable to reproduce these beneficial effects of policosanols in hypercholesterolemic (exogenously induced) New Zealand rabbits over 60 days of study. Several clinical studies have shown that policosanols may reduce TC levels by 8% to 21% in patients with hypercholesterolemia [27-32]. However, wheat germ policosanol failed to lower plasma cholesterol in subjects with normal or mildly elevated cholesterol concentrations [33]. In agreement with the latter clinical study, we observed a lack of cholesterol-lowering effects of octacosanol in apo E–KO mice. Clinical trials have shown some benefits of policosanol treatment on reducing TG levels. One study reported a significant decrease (33%) in TG levels in healthy volunteers [27]. However, in rats, the addition of octacosanol (10 g/kg diet) to a high-fat diet significantly decreased serum TG concentrations [17]. Our study supports previous research showing a significant decrease in plasma TG levels. Although the mechanisms of TG-lowering effects of policosanols are not fully understood, it has been suggested that octacosanol may increase lipid catabolism [10]. It is possible that octacosanol increases the production and activities of lipoprotein and hepatic lipases and thereby reduces the levels of circulating TG. Another possibility is that octacosanol may increase the uptake of TG-rich particles by hepatocytes. One may expect that reductions in TG concentrations are accompanied by increases in HDL cholesterol levels. The study of Arruzazabala et al [25] showed that treatment with 5 to 200 mg/kg of policosanols for 4 weeks did not alter HDL cholesterol levels in New Zealand rabbits. In agreement with these observations, there were no significant alterations in HDL cholesterol levels in octacosanol-treated apo E–KO mice in the present study. In contrast, clinical studies have reported significant increases in HDL cholesterol levels with the ingestion of these compounds [27,32]. Arruzazabala et al [25] showed that New Zealand rabbits receiving 25 or 200 mg/kg policosanols for 60 days were mostly free of atherosclerotic lesions, and this was associated with a reduction in the levels of thromboxane A2 and an increase in prostacyclin levels. Similarly, Wistar rats treated with 0.5, 2.5, 5, and 25 mg/kg of policosanols developed less atherosclerotic lesions as compared with the controls [34]. In an effort to identify the potential mechanism for the antiatherogenic effects of policosanol, Ng et al [35] investigated the effects of these natural compounds on LDL oxidation and bile acid excretion.
Policosanol showed no antioxidant activity in human LDL particles but increased bile acid secretion in hamsters [35]. We observed no noticeable differences in the quality and morphological characteristics of the atherosclerotic lesions between the control group and octacosanol-treated apo E–KO mice in the present study. Both groups of mice had comparable lesion size (0.48 vs 0.42 mm2) and lesionlumen ratio (0.38 vs 0.32), indicating a lack of antiatherogenic properties of octacosanol over 12 weeks in this mouse model. In summary, several studies reported potential cholesterol-lowering and antiatherogenic properties of policosanols. In our experiment using a small number of mice, we observed a TG-lowering effect of octacosanol in cholesterol-fed apo E–KO mice. Despite these effects, octacosanoltreatment did not change atherogenesis in this mouse model. One reason for this could be the nonsignificant increases in plasma TC levels observed in octacosanol-treated mice and the limited number of animals used. It remains unclear if dietary octacosanol can affect dietary cholesterol absorption. Furthermore, a longer treatment protocol (more than 12 weeks) may be needed to observe the antiatherogenic properties of octacosanol in this animal model of atherosclerosis. Future studies are needed to characterize the potential antiatherogenic activities of octacosanol. Acknowledgment Technical support of Behzad Yeganeh is greatly appreciated. Support from Sino Pharmaceuticals Co is also greatly appreciated. References [1] Singh BK, Mehta JL. Management of dyslipidemia in the primary prevention of coronary heart disease. Curr Opin Cardiol 2002; 17:503 - 11. [2] Taylor JC, Rapport L, Lockwood GB. Octacosanol in human health. Nutrition 2003;19:192 - 5. [3] Hargrove JL, Greenspan P, Hartle DK. Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes. Exp Biol Med 2004;229:215 - 26. [4] Varady KA, Wang Y, Jones PJ. Role of policosanols in the prevention and treatment of cardiovascular disease. Nutr Rev 2003;61:376 - 83. [5] Menendez R, Amor AM, Gonzalez RM, Fraga V, Mas R. Effect of policosanol on the hepatic cholesterol biosynthesis of normocholesterolemic rats. Biol Res 1996;29:253 - 7. [6] Kabir Y, Kimura S. Biodistribution and metabolism of orally administered octacosanol in rats. Ann Nutr Metab 1993;37:33 - 8. [7] Aleman CL, Mas R, Hernandez C, Rodeiro I, Cerejido E, Noa M, et al. A 12-month study of policosanol oral toxicity in Sprague Dawley rats. Toxicol Lett 1994;70:77 - 87. [8] McCarty MF. Policosanol safely down-regulates HMG-CoA reductase—potential as a component of the Esselstyn regimen. Med Hypotheses 2002;59:268 - 79. [9] Menendez R, Mas R, Amor AM, Rodeiros I, Gonzalez RM, Alfonso JL. Inhibition of cholesterol biosynthesis in cultured fibroblasts by D003, a mixture of very long chain saturated fatty acids. Pharmacol Res 2001;44:299 - 304.
Z. Xu et al. / Nutrition Research 27 (2007) 212–217 [10] Shimura S, Hasegawa T, Takano S, Suzuki T. Studies on the effect of octacosanol on motor endurance in mice. Nutr Rep Int 1987; 36:1029 - 38. [11] Moghadasian MH, Frohlich JJ, McManus BM. Advances in experimental dyslipidemia and atherosclerosis. Lab Invest 2001;81: 1173 - 83. [12] Nashed B, Yeganeh B, HayGlass KT, Moghadasian MH. Antiatherogenic effects of dietary plant sterols are associated with inhibition of proinflammatory cytokine production in Apo E–KO mice. J Nutr 2005;135:2438 - 44. [13] Yeganeh B, Moshtaghi-Kashanian GR, Declercq V, Moghadasian MH. Combination of dietary phytosterols plus niacin or fenofibrate: effects on lipid profile and atherosclerosis in apo E–KO mice. J Nutr Biochem 2005;16:222 - 8. [14] Moghadasian MH, McManus BM, Pritchard PH, Frohlich JJ. bTall oilQ–derived phytosterols reduce atherosclerosis in Apo E–deficient mice. Arterioscler Thromb Vasc Biol 1997;17:119 - 26. [15] Moghadasian MH, McManus BM, Godin DV, Rodrigues B, Frohlich JJ. Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E–deficient mice: possible mechanisms of action. Circulation 1999;99:1733 - 9. [16] Moghadasian MH, Nguyen LB, Shefer S, McManus BM, Frohlich JJ. Histologic, hematologic, and biochemical characteristics of apo E–deficient mice: effects of dietary cholesterol and phytosterols. Lab Invest 1999;79:355 - 64. [17] Kato S, Karino K, Hasegawa S, Nagasawa J, Nagasaki A, Eguchi M, et al. Octacosanol affects lipid metabolism in rats fed on a high-fat diet. Br J Nutr 1995;73:433 - 41. [18] Kim H, Park S, Han DS, Park T. Octacosanol supplementation increases running endurance time and improves biochemical parameters after exhaustion in trained rats. J Med Food 2003;6:345 - 51. [19] Pritchard PH, Li M, Zamfir C, Lukic T, Novak E, Moghadasian MH. Comparison of cholesterol-lowering efficacy and anti-atherogenic properties of hydrogenated versus non-hydrogenated (Phytrol) tall oilderived phytosterols in apo E–deficient mice. Cardiovasc Drugs Ther 2003;17:443 - 9. [20] Lukic T, Wasan KM, Zamfir D, Moghadasian MH, Pritchard PH. Disodium ascorbyl phytostanyl phosphate reduces plasma cholesterol concentrations and atherosclerotic lesion formation in apolipoprotein E–deficient mice. Metabolism 2003;52:425 - 31. [21] Elam MB, Hunninghake DB, Davis KB, Garg R, Johnson C, Egan D, et al. Effect of niacin on lipid and lipoprotein levels and glycemic control in patients with diabetes and peripheral arterial disease: the ADMIT study: a randomized trial. Arterial Disease Multiple Intervention Trial. JAMA 2000;284:1263 - 70. [22] Kuwabara K, Murakami K, Todo M, Aoki T, Asaki T, Murai M, et al. A novel selective peroxisome proliferator-activated receptor alpha
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
217
agonist, 2-methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220), potently decreases plasma triglyceride and glucose levels and modifies lipoprotein profiles in KK-Ay mice. J Pharmacol Exp Ther 2004;309:970 - 7. Moghadasian MH, McManus BM, Nguyen LB, Shefer S, Nadji M, Godin DV, et al. Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans. FASEB J 2001; 15:2623 - 30. Moghadasian MH. Dietary phytosterols reduce probucol-induced atherogenesis in apo E–KO mice. Atherosclerosis 2006;188:28 - 34. Arruzazabala ML, Carbajal D, Mas R, Molina V, Valdes S, Laguna A. Cholesterol-lowering effects of policosanol in rabbits. Biol Res 1994;27:205 - 8. Arruzazabala ML, Noa M, Menendez R, Mas R, Carbajal D, Valdes S, et al. Protective effect of policosanol on atherosclerotic lesions in rabbits with exogenous hypercholesterolemia. Braz J Med Biol Res 2000;33:835 - 40. Hernandez F, Illnait J, Mas R, Castano G, Fernandez L, Gonzalez M, et al. Effect of policosanol on serum lipids and lipoproteins in healthy volunteers. Curr Ther Res 1992;51:568 - 75. Pons P, Ma´s R, Illnait J, Ferna´ndez L, Rodriguez M, Robaina C, Ferna´ndez JC. Efficacy and safety of policosanol in patients with primary hypercholesterolemia. Curr Ther Res 1992;52:507 - 13. Pons P, Rodrı´guez M, Ma´s R, Illnait J, Ferna´ndez L, Robaina C, et al. One-year efficacy and safety of policosanol in patients with type II hypercholesterolemia. Curr Ther Res 1994;55:1084 - 92. Aneiros E, Calderon B, Ma´s R, Illnait J, Castan˜o G, Ferna´ndez L, et al. Effect of successive dose increases of policosanol on the lipid profile and tolerability of treatment. Curr Ther Res 1993;54:304 - 12. Aneiros E, Ma´s R, Calderon B, Illnait J, Ferna´ndez L, Castan˜o G, et al. Effect of policosanol in lowering cholesterol levels in patients with type II hypercholesterolemia. Curr Ther Res 1995;56:176 - 82. Canetti M, Moreira M, Mas R, Illnait J, Fernandez L, Fernandez J, et al. A two-year study on the efficacy and tolerability of policosanol in patients with type II hyperlipoproteinaemia. Int J Clin Pharmacol Res 1995;15:159 - 65. Lin Y, Rudrum M, van der Wielen RP, Trautwein EA, McNeill G, Sierksma A, et al. Wheat germ policosanol failed to lower plasma cholesterol in subjects with normal to mildly elevated cholesterol concentrations. Metabolism 2004;53:1309 - 14. Noa M, Mas R, de la Rosa MC, Magraner J. Effect of policosanol on lipofundin-induced atherosclerotic lesions in rats. J Pharm Pharmacol 1995;47:289 - 91. Ng CH, Leung KY, Huang Y, Chen ZY. Policosanol has no antioxidant activity in human low-density lipoprotein but increases excretion of bile acids in hamsters. J Agric Food Chem 2005; 53:6289 - 93.