Soy lecithin reduces plasma lipoprotein cholesterol and early atherogenesis in hypercholesterolemic monkeys and hamsters: beyond linoleate

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1 Atherosclerosis 140 (1998) Soy lecithin reduces plasma lipoprotein cholesterol and early atherogenesis in hypercholesterolemic monkeys and hamsters: beyond linoleate Thomas A. Wilson, Craig M. Meservey, Robert J. Nicolosi * Center for Chronic Disease Control, Department of Health and Clinical Science, Room 305, Weed Hall, Uni ersity of Massachusetts Lowell, Lowell, MA 01854, USA Received 13 January 1998; received in revised form 13 April 1998; accepted 4 May 1998 Abstract The current study was designed to investigate the hypocholesterolemic and anti-atherogenic properties of soy lecithin beyond its fatty acid content. In experiment 1, 18 cynomolgus monkeys were divided into three groups of six and fed diets which approximated either the average American diet (AAD), the American Heart Association (AHA) Step I diet, or a modified AHA (maha) Step I diet containing 3.4% soy lecithin for 8 weeks. Plasma samples were collected from food-deprived monkeys and analyzed for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), very low- and low-density lipoprotein cholesterol (non-hdl-c), and triglyceride (TG) concentrations. Group comparisons revealed that monkeys fed the maha Step 1 diet had significantly lower plasma TC ( 46%) and non-hdl-c ( 55%) levels compared to the AAD diet, whereas monkeys fed the AHA Step 1 diet had lesser reductions in plasma TC ( 21%) and non-hdl-c ( 18%) levels. The monkeys fed the maha Step I diet had significantly lower plasma TC ( 32%) and non-hdl-c ( 45%) compared to the monkeys fed the AHA step diet. Also, only the maha Step I diet significantly reduced pre-treatment plasma TC and non-hdl-c levels by 39 and 51%, respectively with no significant effect on plasma HDL-C or TG levels. In experiment 2, 45 hamsters were divided into three groups of 15 and fed the following three modified non-purified diets for 8 weeks: a hypercholesterolemic diet (HCD) containing 10% coconut oil and 0.05% cholesterol, HCD plus 3.4% soy lecithin (+SL), or the HCD with added levels of linoleate and choline equivalent to the +SL diet but no lecithin ( SL). Plasma lipids were determined as in experiment 1 and aortas were perfusion-fixed and Oil Red O stained for morphometric analyses of fatty streak area. Relative to the HCD group, the +SL-treated hamsters had significantly lower plasma TC ( 58%), non-hdl-c ( 73%) and aortic fatty streak area ( 90%). Relative to the SL group, hamsters fed the +SL diet had significantly lower plasma TC ( 33%), non-hdl-c ( 50%) and significantly reduced aortic fatty streak area ( 79%). In conclusion, the first experiment suggests that the cholesterol-lowering efficacy of the AHA Step I diet can be enhanced with the addition of soy lecithin without reducing plasma HDL-C levels, whereas the second experiment suggest that the hypocholesterolemic, and in particular, the anti-atherogenic properties of soy lecithin cannot be attributed solely to its linoleate content Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hamsters; Monkeys; Soy lecithin; Linoleate; Early atherosclerosis 1. Introduction A recent report from the American Heart Association (AHA) shows that upwards of 30% of Americans * Corresponding author. Tel.: ; fax: ; Robert Nicolosi@uml.edu in the year age group have blood cholesterol levels equal to or greater than 240 mg/dl (6.2 mmol/l) [1]. The initial therapeutic approach for these individuals at increased risk for premature coronary heart disease (CHD) is the AHA Step I and Step II diets, which focus on reductions in dietary saturated fat and cholesterol intakes [2]. Thus for many individuals, the ex /98/$ Elsevier Science Ireland Ltd. All rights reserved. PII S (98)

2 148 T.A. Wilson et al. / Atherosclerosis 140 (1998) pected degree of cholesterol-lowering (up to 10%) [2] may not be sufficient to prevent the need for drug intervention. Moreover, the high-density lipoprotein cholesterol (HDL-C) reductions seen with AHA Step I and Step II diets [3,4] may not improve the lipoprotein profile, i.e., total cholesterol (TC)/HDL-C or low density lipoprotein cholesterol (LDL-C)/HDL-C ratios. Several studies have indicated that lecithin, a phosphatidyl choline-containing phospholipid, has hypocholesterolemic properties. Data on its hypocholesterolemic properties however have not always concurred possibly as a result of differences in the degree of initial hypercholesterolemia of the population studied, and the type and level of lecithin implemented. For example, the cholesterol-lowering effects of lecithin have mainly been observed in animals [5 8] and in humans [9 11] when hyperlipidemia exists and not in the normolipemic state [10,12]. The underlying mechanism(s) for the hypocholesterolemic properties of lecithin during hypercholesterolemia have not been elucidated. Whether the cholesterol-lowering effects are independent of its linoleate content or some other component has not been sufficiently addressed. A few studies have been undertaken in the past several years to evaluate the hypocholesterolemic effect of unsaturated lecithin (soy lecithin) compared with saturated lecithin (egg lecithin). Those studies conducted in humans have generally failed to show an effect for soy lecithin beyond its well-known unsaturated fatty acid effect [10,13 16]. Recently it was suggested that most studies involving lecithin supplementation have lacked a proper control group to take into account the effect of lecithin s two unsaturated fatty acid moieties [17]. Therefore, the current study was designed to: (i) examine the effect of soy lecithin on plasma lipid concentrations and (ii) balance the fatty acid profile of the lecithin experimental group against that of a lecithin control group by adding soy oil, glycerol, H 2 PO 4, choline chloride, ethanolamine, and inositol to the latter diet and comparing that to a third group consuming a hypercholesterolemic diet to assess the effects of lecithin itself with a group having a comparable fatty acid profile and phospholipid composition. 2. Materials and methods 2.1. Experimental animals and diets In experiment 1, 18 male cynomolgus monkeys (Macaca fascicularis), approximately 5 7 years of age were individually housed in stainless steel cages with free access to food and water. The monkeys were fed diets approximating the average American diet (AAD) Table 1 Composition of diets of monkeys in experiment 1 (% energy) Dietary component AAD AHA Step I Casein Soy protein Dextrin/sucrose Coconut oil Olive oil Corn oil Cholesterol a Cellulose b Soy lecithin c maha Step I a Cholesterol intake in mg/day. b Cellulose intake in g/day. c Soy lecithin intake in g/day. Equivalent to 0.78 mg of pure lecithin per kj day 1. (36% energy as fat, 15% energy as saturated fat (SFA), 15% energy as monounsaturated fat (MUFA) and 6% energy as polyunsaturated fat (PUFA) and 0.08% cholesterol) for 6 weeks. After 6 weeks of feeding the AAD, the monkeys were bled and fasting plasma total cholesterol (TC) concentrations were measured. Plasma TC values were arranged in ascending order and sorted into three groups of six monkeys each based on lowest to highest pretreatment plasma TC concentrations. One group continued on the AAD while the other monkeys were fed either the AHA Step I diet (30% kcal as fat; 9% SFA, 14% MUFA, and 7% PUFA and 0.04% cholesterol), or a modified AHA Step I diet plus 3.4% soy lecithin (maha) for 8 weeks (Tables 1 and 2). In experiment 2, 45 8-week-old male F1B hamsters (Biobreeders, Fitchburg, MA) were individually housed in stainless steel suspended rodent cages with free access to food and water. Hamsters were randomly assigned to three experimental groups of 15 per group and were fed one of three experimental diets for 8 weeks (Table Table 2 Composition of diets of monkeys in experiment 1 (g/kg of diet) Dietary component AAD AHA Step I maha Step I Casein Soy protein Cystine Dextrin Sucrose Coconut oil Olive oil Corn oil Cholesterol Salt mix a Vitamin mix a Choline Banana flavor Cellulose Soy lecithin 34.1 a Composition reported in Brousseau et al. [18].

3 T.A. Wilson et al. / Atherosclerosis 140 (1998) Table 3 Composition of non-purified hamster diets in experiment 2 (g/kg diet) Dietary component HCD +SL SL Chow a Coconut oil Cholesterol Soy lecithin b 34.1 Soy oil Glycerol 0.75 H 2 PO Choline chloride 1.35 Ethanolamine 0.57 Inositol 1.10 a Chow is Purina rodent chow c5001. Equivalent to 1.0 mg pure lecithin per kj day 1. 3). Group 1 (HCD) was fed a hypercholesterolemic non-purified diet (PMI rodent chow c5001, PMI Feeds, St. Louis, MO) containing 10% coconut oil and 0.05% cholesterol. Group 2 (+SL) was fed the same diet supplemented with 3.4% soy lecithin (Central Soya, Fort Wayne, IN). Group 3 ( SL) was fed the same diet with equivalent levels of fatty acids, choline, ethanolamine, inositol, and glycerol as in the +SL diet, but no lecithin. Ethanolamine, inositol, and glycerol were added to the SL diet to balance the level of phospholipids that are present in the +SL diet. The lipid and fatty acid compositions of the soy lecithin used in the monkey and hamster diets of experiments 1 and 2 and the soy oil used in the hamster diets of experiment 2 are shown in Table 4. The 3.4% wt./wt. level of lecithin in the monkey study is equivalent to 10 g/day in a human consuming 2000 kcal/day and was chosen from our previous study in monkeys [8] and is in keeping with the recent study of Wojcickl Table 4 Lipid compositions of soy lecithin used in experiments 1 and 2, and soy oil used in experiment 2 (%) Lipid component Soy lecithin Soy oil Triglycerides Phospholipids Phosphatidylcholine 40.4 Phosphatidylethanolamine 35.1 Phosphatidylinositol 24.5 Fatty acids Palmitate (16:0) Stearate (18:0) Oleate (18:1) Linoleate (18:2) Linolenate (18:3) Other a a Other fatty acids include: myristate (14:0), palmitoleate (16:1), margarate (17:0), arachidate (20:0), gadoleate (20:1), and behenate (22:0). et al. [11]. This level was also chosen for the hamster study and is equivalent to 36 g/day in a human consuming 2000 kcal/day and is within the amount also fed to humans [17]. A non-purified diet, rather than a semipurified diet, was used because published data from our laboratory [19] and those from another [20], have demonstrated that the non-purified diet produced a lipoprotein profile (predominantly non-hdl-c) more similar to humans. All animals were maintained in AAALAC (American Association for the Accreditation of Laboratory Animal Care) accredited facilities, in an environmentally controlled atmosphere (20 C) on a 12/12-h light/dark cycle. The animals were maintained in accordance with the guidelines of the Committee on Animals of the University of Massachusetts at Lowell Research Foundation and the guidelines prepared by the Committee on Care in Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (DHEW publication no , revised 1985) Lipid measurements In experiment 1, blood from food-deprived monkeys was collected via the saphenous vein prior to and after 4, 6, and 8 weeks of dietary treatment into heparincontaining tubes and plasma harvested after low-speed centrifugation at 2500 g for 15 min at 4 C. Plasma total cholesterol (TC) [21] and triglycerides (TG) [22] were measured enzymatically and, after the apo B-containing lipoproteins VLDL and LDL were precipitated with phosphotungstate reagent [23], the supernatant was assayed for HDL-C (Sigma, St. Louis, MO). The non-hdl-c fraction (very low-density lipoprotein cholesterol (VLDL-C) +LDL-C) in monkeys was determined by subtracting the HDL-C from the TC. In experiment 2, at 4, 6, and 8 weeks of treatment, blood from food-deprived hamsters was collected via the retro-orbital sinus into heparinized tubes and plasma lipids and lipoprotein cholesterols were analyzed as above Quantification of aortic fatty streak area For experiment 2, at the time of euthanasia, hamsters were anesthetized with an i.p. injection of sodium pentobarbital and aortic tissue was obtained for fatty streak analysis as previously described [24,25]. After blood was collected through a cardiac puncture of the left ventricle, the perfusion needle was inserted through the puncture site and a 10% buffered formalin solution was perfused through the animal s cardiovascular system with physiological pressure. The right atrium was punctured to provide an outlet for the fixative. After 2 min, the flow of fixative was slowed,

4 150 T.A. Wilson et al. / Atherosclerosis 140 (1998) Table 5 Plasma lipoprotein cholesterol and triglyceride concentrations before and after dietary treatment in monkeys (mmol/l) Diet TC Non-HDL-C HDL-C TG Before After Before After Before After Before After AAD a a AHA Step I a a maha Step I a b a b Values represent means S.E.M.; n=6. Values in a column not sharing a superscript are different at P Values in a row not sharing a superscript are significantly different at P and fixation continued for an additional 25 min. The heart and thoracic aorta were removed and fixation completed by pinning the tissue down overnight in its natural shape in 10% formalin. Specimens were stored in vials containing phosphate buffer solution (PBS) at 4 C until being analyzed for fatty streak area. To measure the extent of the fatty streak formation in the aortic arch, a piece of aortic tissue extending from as close to the heart as possible to the branch of the left subclavian artery was used. The tissue was cleaned, rinsed quickly with isopropanol, and placed in a vial containing 3% Oil Red O stain (0-0625, Sigma, St. Louis, MO) in 60% isopropanol for 15 min. Subsequently, the Oil Red O stained tissue was carefully and gently removed from the outside surface of the arch. The arch was dissected vertically in two, then cut open longitudinally along the inferior border and mounted on a glass slide, endothelial side up. Specimens were covered with Apathy s mounting media and cover slips, and analyzed under 145 magnification. A computerized image analysis system (Image Technology, Cresskill, NJ), attached to a compound light microscope, was used to measure the total Oil Red O stained area of each aortic arch, and size of the arch was measured (SigmaScan, Jandel Scientific, San Rafael, CA) to calculate the fatty streak area ( m 2 ) per aortic arch area (mm 2 ) Statistical analyses SigmaStat software (Jandel Scientific, San Rafael, CA) was used for all statistical evaluations. A repeated measures one-way analysis of variance (RM ANOVA) was used to analyze the monkey data, while an ANOVA was used to analyze the hamster data. When statistical significance was found by ANOVA, the Student Newman Keuls separation of means was used to determine group differences. The Pearson Product Moment method was used for determination of correlations between variables for which values from individual animals were used. All other values are expressed as mean S.E.M. and statistical significance was set at P 0.05 [26]. 3. Results 3.1. Experiment 1 The mean body weights for the 18 monkeys on each diet were as follows: AAD, kg; AHA Step I diet, kg, and maha Step I diet, kg (mean S.E.M.). The monkeys being fed the AHA Step I diet plus soy lecithin consumed approximately 0.78 mg of pure lecithin per kj day 1. Plasma lipid concentrations after being fed the AAD for the initial 6 weeks are presented in Table 5. There were no significant differences observed between the three groups of monkeys for any plasma lipid variable after 6 weeks of consuming the AAD (before treatment) (Table 5). Plasma concentrations at 4, 6, and 8 weeks of dietary treatment were not significantly different so were averaged for statistical analysis. After 8 weeks, compared to the AAD and AHA Step I diet, the monkeys consuming the maha Step I diet had significantly lower plasma TC ( 46%, P and 32%, P 0.04) and non-hdl-c ( 55%, P 0.03 and 45%, P 0.04), respectively. No other significant differences were observed between diets for plasma HDL-C and TG (Table 5) although the maha Step I diet reduced plasma TG approximately 50% relative to the AAD but only by 25% relative to pretreatment concentrations. Also, monkeys on the maha Step I diet had lower plasma TC and non-hdl-c levels ( 39%, P 0.06 and 51%, P 0.06, respectively) after 8 weeks of dietary treatment compared to pretreatment levels (Table 5). No other diets produced different plasma lipid levels between pre- and post-treatments (Table 5) Experiment 2 After 8 weeks of consuming the HCD, +SL and SL diets, final body weights were not significantly different among groups; 131 2, 126 3, and g, respectively (mean S.E.M.). The hamsters fed the +SL diet consumed approximately 1.0 mg of pure lecithin per kj day 1 and 57% of fat as linoleate while the hamsters fed the SL diet consumed 55% of fat as linoleate.

5 T.A. Wilson et al. / Atherosclerosis 140 (1998) The +SL group had significantly lower plasma TC ( 58%, P ) and non-hdl-c ( 73%, P ) levels than hamsters consuming the HCD. Compared to the HCD group, hamsters fed the SL diets had significantly lower plasma TC ( 37%, P 0.002) and non-hdl-c ( 47%, P 0.002) levels. Relative to SL fed hamsters, the +SL group had significantly lower plasma TC ( 33%, P ), and non-hdl-c ( 50%, P ). No differences in plasma HDL-C levels were seen between groups, therefore, the non-hdl-c:hdl-c ratio reflected the differences in non-hdl-c and was also significantly different between all groups. Plasma TG were significantly lower in the +SL and SL groups compared to the HCD group (54%, P and 43%, P 0.03; respectively) (Table 6). Aortic fatty streak area, a measure of early atherosclerosis, was significantly lower in the +SL group ( 90%, P ) and the SL group ( 49%, P=0.0001) compared to the HCD fed animals (Table 6). The +SL group had 79% less fatty streak involvement than the SL group (P ) (Table 6). There was a strong positive correlation between plasma LDL-C level and early atherosclerosis when all three groups were analyzed together (r=0.70, P ), but no significant correlations were found within any individual diet group (data not shown). 4. Discussion The aims of this study were to evaluate the response of plasma lipids and lipoprotein cholesterol, and the development of early atherosclerosis to dietary soy lecithin and also to examine whether the responses were due to linoleate composition of soy lecithin or to some other constituent. In experiment 1, the addition of soy lecithin to the AHA Step I diet, resulted in a significant reduction in plasma TC ( 46%) and non-hdl-c ( 55%) levels compared only to a 21% reduction in Table 6 Plasma lipoprotein cholesterol concentrations and aortic fatty streak area (AFSA) of hamsters fed the HCD, +SL and SL diets HCD +SL SL TC (mmol/l) a b c Non-HDL-C (mmol/l) a b c HDL-C (mmol/l) Non-HDL-C:HDL-C a b c (mol/mol) TG (mmol/l) a b c AFSA ( m 2 /mm a b c 100) Values are means S.E.M., n=15. Values in a row not sharing a superscript are different at P plasma TC and a 18% reduction in non-hdl-c levels in monkeys fed the AHA Step I diet, relative to the AAD. While these reductions with lecithin-treatment are striking, they are in the range reported in a previous study in which decreases in plasma TC and LDL-C of 33 and 34%, respectively, were observed in hyperlipemic patients [11]. The lack of a significant cholesterol-lowering effect in experiment 1 with the AHA Step I diet relative to the AAD, is consistent with the variable responses noted in the adult treatment panel report of NCEP2 [2] and with observations we have seen in our laboratory with monkeys [27,28]. In both of these studies, monkeys fed similar diets to the AAD and AHA Step I, plasma TC and non-hdl-c levels were lowered approximately 17 and 16%, respectively, by the AHA Step I diet compared to the AAD [27,28]. In experiment 2, both the +SL and SL diets in hypercholesterolemic hamsters significantly reduced plasma TC and non-hdl-c without significantly affecting HDL-C thus decreasing the non-hdl-c:hdl- C. Both the +SL and SL diets relative to the HCD-fed animals significantly reduced the development of early atherosclerosis, although the fatty streak area in +SL fed animals was further reduced by 79% relative to the SL group. While it is possible that the changes induced by lecithin were due to the PUFA present in the lecithin, several studies have shown in both animals and humans that the PUFA components of lecithin do not have a hypocholesterolemic effect [5,8,10]. Since the lipid responses in the present study were different for the two treatment diets, we conclude that hypocholesterolemic properties of soy lecithin cannot be completely explained by its linoleate content. Contrary to previous work [10] which showed that soy lecithin increased serum HDL-C and had no effect on TC, we observed in both experiments a significant decrease in plasma TC and non-hdl-c with no significant effect on HDL-C. Also, the present study showed a significant decrease in plasma TG concentrations (experiment 2), which is in agreement with another study [11]. Data examining the development of early atherosclerosis in the present study are also in agreement with previous work showing a decrease in the amount of cholesterol in the aorta of guinea pigs following treatment with soybean phospholipid [29]. It is not clear through which mechanism(s) that soy lecithin induces its hypocholesterolemic or anti-atherogenic effects. An anti-atherogenic effect of dietary lecithin could result from the relationship between phospholipids and HDL-C. Previous studies have shown that lecithin can be partially absorbed intact by the intestine [30] and incorporated preferentially into HDL [31]. Also, soy lecithin is known to act as a good substrate for lecithin-cholesterol acyltransferase (LCAT) activity [32]. This enzyme is associated with the

6 152 T.A. Wilson et al. / Atherosclerosis 140 (1998) irreversible formation of HDL 2 from HDL 3 and its capacity to carry cholesterol from peripheral tissues, such as the aorta, back to the liver, where cholesterol can be converted to bile acids [32]. A previous study has also shown that the feeding of lecithin to rats increased LCAT activity, which increased cholesterol removal from the blood via fecal bile acid excretion [7]. The data presented in this study clearly show that soy lecithin is effective in reducing plasma cholesterol and triglyceride concentrations in the hypercholesterolemic hamster (experiment 2) and increases the reduction in plasma TC and non-hdl-c observed in monkeys fed the AHA Step I diet (experiment 1). However, in experiment 2, the diet containing soy lecithin ( +SL) produced greater hypocholesterolemic effects than the diet containing equivalent amounts of linoleate and choline ( SL), suggesting some other possible component of soy lecithin also exerts hypocholesterolemic effects. Acknowledgements We would like to thank Leena Laitinen, Donato Vespa, and Lorraine Misner for their technical support. References [1] Chait A, Brunzell JD, Denke MA, Eisenberg D, Ernst ND, Franklin FA, Ginsberg H, Kotchen TA, Kuller L, Mullis RM, Nichaman MZ, Nicolosi RJ, Schaefer EJ, Stone NJ, Weidman WH. Rational of the Diet-Heart Statement of the American Heart Association: report of the Nutrition Committee. Circulation 1993;88: [2] NCEP Expert Panel. Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel II). 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