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Biochemical and Molecular Actions of Nutrients
Research Communication

Fish Oil Containing Phytosterol Esters Alters Blood Lipid Profiles and Left Ventricle Generation of Thromboxane A₂ in Adult Guinea Pigs^{1 ,2}

H. Stephen Ewart^*3, Laura K. Cole, Jaroslav Kralovec^*, Heather Layton, Jonathan M. Curtis^*, Jeffrey L. C. Wright^*,4 and Mary G. Murphy

^* Ocean Nutrition Canada, Limited, Halifax, NS, Canada and the Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada

³To whom correspondence should be addressed. E-mail: sewart{at}ocean-nutrition.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study was designed to investigate the lipid-lowering ability of a novel dietary ingredient composed of phytosterols esterified to (n-3) polyunsaturated fatty acids (PUFA) [PS(n-3)]. Adult guinea pigs were fed a test diet supplemented with PS(n-3) (25 g/kg) and corn oil (CO, 5g/kg), whereas the diet fed to control guinea pigs was supplemented with CO only (30 g/kg). Cholesterol was added to both diets (0.8 g/kg). After 3–4 wk of consuming the diets, serum total cholesterol (TC) and triacylglyercol (TAG) in the PS(n-3) group were 36 and 29% lower, respectively, than levels in controls (P < 0.05). The lower TC levels in the PS(n-3) group reflected a 38% reduction in non-HDL cholesterol (non-HDL-C), whereas the HDL-C concentration was unaffected. Analysis of cardiac left ventricle indicated that generation of the proaggregatory, arrhythmic eicosanoid, thromboxane A₂, was >60% lower in the PS(n-3)-supplemented guinea pigs than in CO controls (P < 0.001). This study demonstrates that the TAG-lowering and eicosanoid-modifying properties of the fish oil (n-3) PUFA are retained when they are provided in the diet in ester linkage with hypocholesterolemic phytosterols.

KEY WORDS: • phytosterol esters • fish oil • serum cholesterol • thromboxane A₂ • guinea pigs

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Dyslipidemia is widely recognized as a major risk factor in the development of cardiovascular disease (1 –7 ). Hallmark features are elevations in circulating levels of total cholesterol (TC),⁵ particularly LDL cholesterol (LDL-C), as well as decreases in HDL cholesterol (HDL-C) and hypertriglyceridemia. The risk with each parameter is compounded when they are present in combination. For example, hypertriglyceridemia combined with elevated LDL-C and a LDL-C:HDL-C ratio >5 increases the risk of a coronary event sixfold relative to hypertriglyceridemia alone (2 ). As such, maximum reduction of lipid-related risks of cardiovascular disease requires an agent that promotes collective decreases in triacylglycerols (TAG) and LDL-C and, at the same time, increases HDL-C levels.

Substances that hold considerable promise as hypolipidemic agents are the long-chain (n-3) polyunsaturated fatty acids (PUFA), which are concentrated in fish and other marine oils. Studies examining the effects of fish-oil (n-3) PUFA on blood lipids have consistently demonstrated an 30% reduction in TAG (8 ). In addition, dietary fish oil PUFA have antithrombotic properties related in part to their ability to reduce formation of thromboxane A₂ (TXA₂), a proaggregatory eicosanoid (9 ). Increasingly, the (n-3) PUFA are being shown also to have direct antiarrhythmic properties, due to their abilities to regulate cardiac ion channel activities and contractility (10 ).

Fish-oil PUFA do not appear to reduce blood total cholesterol levels, and may even mildly elevate LDL-C (8 ). One family of dietary agents that do lower cholesterol is the plant sterols (11 ,12 ). Phytosterols and phytostanols, or their fatty acyl esters, interfere with intestinal absorption of dietary and biliary cholesterol (11 ). The esterification of plant sterols with fatty acids greatly improves their solubility, which facilitates their addition to fat-based foods. Both phytosterol and phytostanol esters are added as functional food ingredients to margarine and appear to be equally effective in reducing total cholesterol and LDL-C in normocholesterolemic and mildly hypercholesterolemic individuals (13 ).

In a new approach to maximizing lipid-lowering effects, we have combined the TAG-reducing properties of (n-3) PUFA with the hypocholesterolemic actions of phytosterols to form a dietary supplement of phytosterols esterified to fish oil enriched in (n-3) PUFA [PS(n-3)]. This is a unique formulation because other currently available phytosterols are esterified primarily with vegetable oil fatty acids, including monounsaturates and the (n-6) PUFA precursor, linoleic acid, contained in canola oil. The primary purpose of the present study was to determine whether supplementing diets of adult guinea pigs with PS(n-3) would affect blood lipid levels in a predictable manner. Guinea pigs are a particularly good model to use for such studies because unlike the more commonly used rats and mice, they have blood lipid profiles and responses to dietary fatty acids that resemble those of humans (14 ). We showed previously that (n-3) PUFA supplementation reduces production of TXA₂ by guinea pig left ventricle (15 ); here, we also determined whether this property was retained when the PUFA were presented in the diet as phytosterol esters.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials.

Corn oil was obtained commercially from Best Foods Canada (Etobicoke, ON). The Vitros kits for separating HDL from total cholesterol and for quantitating blood cholesterol were purchased from Ortho-Clinical Diagnostics, (Rochester, NY). Phytosterol (85% stigmasterol) was obtained from Fabrichem, (Fairfield, CT). Other chemicals were from Sigma-Aldrich Canada, (Oakville, ON). The starting marine oil was produced by Ocean Nutrition Canada, (Mulgrave, NS) as a concentrate of eicosapentaenoic acid (EPA; 299 mg/g oil) and docosahexaenoic acid (DHA; 214 mg/g oil).

Phytosterol ester synthesis.

The EPA/DHA concentrate (18.2 mmol) was stirred at 150–155°C for 2 h under vacuum (5 mm Hg). A dispersed mixture of dry phytosterol (12.1 mmol) and sodium methoxide (3.0 mmol) was then added within 1 h and the reaction mixture stirred at 150–155°C for an additional 22 h. During this period the reaction was monitored by TLC and HPLC. The reaction product was separated from the unreacted starting material by hexane precipitation of the phytosterol, followed by repeated extraction with methanol. Typical yields of pure phytosterol fish oil conjugates were in the range of 70%.

Sterol esters were characterized by combined size exclusion chromatography with refractive index detection and positive ion atmospheric pressure chemical ionization mass spectrometry. Intact sterol ester MH⁺ ions were observed and their structures confirmed by tandem mass spectrometry that showed fragment ions corresponding to both the fatty acyl and sterol moieties.

Animals and diets.

The compositions of the control and the test diets are provided in Table 1 . In brief, diets were prepared with commercial nonpurified guinea pig pellets (Pro Lab Guinea Pig Formula, PMI Feeds, St. Louis, MO) that were ground and mixed with cholesterol (0.8 g/kg diet). The test diet was supplemented with PS(n-3) (25 g/kg diet) and corn oil (CO, 5 g/kg diet). The control diet was supplemented entirely with CO (30 g/kg diet). Each diet was reconstituted into pellets using a Hobart pelletizer at the Sandy Cove Facility of the National Research Council (Halifax, NS). The diets were stored at -20°C under N₂ in 150-g portions. Male albino guinea pigs (10–14 d old; Harlan, Indianapolis, IN) weighing between 120 and 240 g were fed the diets (9/dietary group) for 23–28 d. They were given free access to food and to water supplemented with ascorbic acid (200 mg/L) and kept in the animal care quarters of the Sir Charles Tupper Medical School, Dalhousie University under a 12-h light:dark cycle. They were fed at the same time each day; uneaten food was weighed and discarded, and the cups were washed thoroughly. The guinea pigs were weighed every 2–3 d; at the end of the treatment period, they were deprived of food overnight and killed the next day with an overdose of sodium pentobarbital (Somnitol, 340 g/L). The hearts were quickly removed into preweighed tubes, flash frozen and stored at -86°C. These studies were carried out in full compliance with regulations of the Canadian Council on Animal Care.

Blood was taken after overnight food deprivation from the thoracic vein 4 d before (baseline lipid levels) and 9 d after diet supplementation began. Blood collection at the end of the feeding period was by cardiac puncture after the guinea pigs were anesthetized by intraperitoneal injection with sodium pentobarbital (Somnitol, 65 mg/mL). Serum was separated from RBC by centrifugation (5,000 x g, 10 min), transferred to cryovials and stored at -80°C before analysis. Lipid analysis was performed using Vitros diagnostic products (Ortho-Clinical Diagnostics, Rochester, NY). The methodologies for TAG (Vitros TRIG Slides) and TC (Vitros CHOL Slides) are based on enzymatic methods as described by Spayd et al. (16 ) and Allain et al. (17 ), respectively. HDL-C was separated from TC using the Vitros Magnetic HDL-Cholesterol Reagent as described by Warnick et al. (18 ).

Thromboxane analysis.

Sections (80–100 mg) of cardiac left ventricle were minced on ice and incubated for TXA₂ generation as described by Murphy et al. (15 ). Briefly, the minced sections were washed and incubated at 37°C in oxygenated buffer to allow tissue production of TXA₂. The samples were then centrifuged (10,000 x g, 5 min), and the supernatants removed, acidified with 1 mol/L HCl and extracted with ethyl acetate. The ethyl acetate was dried under N₂, after which the residue was reconstituted in chloroform and stored at -80°C. All samples were then quantified for TXB₂ (the stable metabolite of TXA₂) by enzyme immunoassay using a kit from Amersham (Oakville, ON). The color intensities were read using a Bio-Rad Model 3550 Microplate Reader (Bio-Rad, Mississauga, ON). It was noted that there could be limited cross-reactivity of the antibodies with the trienoic prostanoids derived from EPA.

Statistical analysis.

Data are expressed as means ± SD. Comparisons were made using Student’s t test, with significant differences defined as P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Effects of supplement on food consumption and weight gain.

During the study, the guinea pigs in both the CO- and PS(n-3)-supplemented groups consumed 28 g of food/d during wk 1 and 33–36 g of food/d by wk 4. The one exception was the day after the blood sampling (d 10) when consumption dropped to 8 g for both groups. For this reason, a second blood sampling planned for wk 3 of the study was canceled. With the exception of the 2 d after low food consumption, rates of weight gain did not differ between the CO and PS(n-3) groups (7.5 ± 1.2 and 7.9 ± 0.9 g/d, respectively, n = 9). Based on the average food consumption during wk 4, the guinea pigs ingested an average of 0.9 g of fatty acids and 0.03 g of cholesterol each day.

Blood lipid profiles.

After 2 wk of consuming the diets, TAG levels did not differ between the CO- and the PS(n-3)-fed groups (Fig. 1A ). After 4 wk, however, serum TAG was 28% lower in the PS(n-3)-fed group than in the CO group (P < 0.05).

View larger version (20K): [in this window] [in a new window]   FIGURE 1 Serum triacylglycerol (TAG, panel A) and total cholesterol (TC, panel B) concentrations in guinea pigs fed diets supplemented with corn oil (CO, controls) or with phytosterols esterified to (n-3) polyunsaturated fatty acids (PUFA) [PS(n-3)]. Values are means ± SD, n = 8 (baseline and wk 2) or 9 (wk 4). *Significantly different from the control value at that time, P < 0.05.

TXB₂ generation by ventricular tissue from PS(n-3)-supplemented guinea pigs (n = 8) was > 60% lower than in ventricular tissue from CO-fed (n = 9) guinea pigs (4.98 ± 2.17 pg/mg ventricle vs. 13.17 ± 3.71 pg/mg ventricle, respectively, P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A significant reduction in risk of various forms of cardiac disease, including arrhythmias, angina, myocardial infarction and heart failure, is achieved at least in part by lowering blood total and LDL cholesterol levels, lowering TAG and increasing HDL-C (8 ). Dietary phytosterols incorporated into margarines or butter have been shown to be effective in lowering TC and LDL-C, but do not affect TAG or HDL-C (13 ). By comparison, intake of moderate amounts of (n-3) PUFA consistently lowers TAG but has little if any effect on blood cholesterol levels (8 ). Nevertheless, ingestion of moderate amounts of oily fish or fish-oil supplements several times a week reduces the risk of death due to acute cardiac events by up to 50%, an outcome that is better than that achieved by conventional drug therapy (8 ,19 ,20 ). It would be beneficial, therefore, to produce a dietary supplement that combines the cholesterol-lowering properties of phytosterols with the TAG-lowering effects of (n-3) PUFA. The PS(n-3) used in this study is the first to do so because most phytosterol esters have as their constituents fatty acids derived from vegetable oils.

The major fish oil (n-3) PUFA, DHA and EPA, like most dietary fatty acids, are ingested primarily as TAG. Several recent studies have shown that the bioavailability of (n-3) PUFA is retained when they are provided as ethyl esters (21 ); however, there is no information available regarding the effectiveness of (n-3) PUFA when they are in the form of phytosterol esters. The present study demonstrated that adult guinea pigs fed the PS(n-3)-supplemented diet had lower blood levels of TAG, TC and non-HDL-C than did CO-fed guinea pigs. This outcome would be predicted if the effects of diets containing (n-3) PUFA alone (15 ) or phytosterol alone (22 ) were combined. It was beyond the scope of the present study to investigate the mechanisms underlying the lipid-lowering effects of the PS(n-3); however, it is presumed that they are similar to the mechanisms proposed for the individual components. Mechanisms that have been proposed for (n-3) PUFA-mediated reductions in circulating TAG levels include inhibition of hepatic TAG synthesis and/or secretion, reduced intestinal and hepatic apolipoprotein B species (B48, B100), which are responsible for TAG clearance, and increased lipoprotein lipase activity (23 ). The lower production of TXA₂ (measured as TXB₂) in ventricles of the PS(n-3)-fed guinea pigs (relative to CO controls) is a functional consequence of ingestion of (n-3) PUFA as TAG, in part due to the ability of these PUFA to reduce availability of the eicosanoid precursor, arachidonic acid (15 ). With respect to the phytosterols, their hypocholesterolemic actions have been attributed to several mechanisms, with blockade of cholesterol absorption as the primary candidate. Reduced absorption could be related to displacement of cholesterol from bile salt micelles, reduction of cholesterol esterification and/or increased bile salt excretion (22 ,24 ). If we assume that the PS(n-3) are substrates for sterol esterase(s) in the intestinal lumen of guinea pigs, it follows that the released and absorbed (n-3) PUFA, and the nonabsorbed phytosterols, exert their effects on blood lipids through one or more of the mechanisms cited.

This study confirms a recent report that dietary phytosterols decrease total and non-HDL-C in adult guinea pigs (22 ). The majority (>90%) of circulating cholesterol in guinea pigs is found in the non-HDL fraction, which mirrors the human situation qualitatively. Nevertheless, quantitative differences may arise. For example, the 36–38% lower values for total and non-HDL-C in guinea pigs fed the PS(n-3) supplement (relative to CO controls) are considerably greater than the 9–14% reductions reported for humans ingesting 2 g of phytosterols daily (25 ). This may reflect the relatively high amounts of phytosterol given to the guinea pigs on a weight basis. However it was recently reported that in humans, ingestion of 1.6 g/d of phytosterols is sufficient to produce a reduction in plasma cholesterol to the degree that additional amounts of dietary phytosterol do not elicit further clinical reductions (26 ). The exaggerated response in guinea pigs could be related to several variables including diet, baseline cholesterol status and age (25 ). Thus, differences in cholesterol handling and clearance by the intestinal tract among guinea pigs and humans could result in differences in the overall effect of phytosterols on plasma cholesterol levels.

In conclusion, phytosterol esters enriched with (n-3) fatty acids from fish oil represent a novel nutritional approach to simultaneously lowering plasma TAG and cholesterol levels. Esterification of phytosterols with fatty acids is necessary to increase phytosterol solubility so that cholesterol-lowering levels are achieved when they are added to foods. Replacement of the commonly used vegetable oil fatty acids with DHA and EPA from fish oils fulfils the solubilizing role. The PS(n-3) has the added benefit of having TAG-lowering properties, without the need for additional dietary fat. Both phytosterols and (n-3)-PUFA are normal dietary components, and as such, their use would be expected to present minimal side effects and safety concerns. The principal concern regarding the safety of (n-3)-PUFA is the potential to increase bleeding time (27 ), whereas concern regarding safety of phytosterols relate to their potential to reduce absorption of fat-soluble (pro-) vitamins such as ß-carotene (24 ). The recent FDA qualified health claim on (n-3) PUFA supports the use of 3 g/d total EPA plus DHA from all dietary sources (28 ). An effective intake of phytosterol is 1.6 g/d (29 ), an amount that does not appear to adversely affect plasma carotenoid levels. An intake of PS(n-3) of 2.5–3 g/d would provide 1.6–1.9 g/d phytosterol and 0.5–0.7 g/d EPA plus DHA. Thus, the use of PS(n-3) may be a safe and effective alternative to conventional lipid-lowering drugs.

	ACKNOWLEDGMENTS

 
The authors thank Sylvia Craig of the Sir Charles Tupper Animal Care Facility for taking the blood samples from the guinea pigs during the feeding study. The authors also wish to acknowledge the help of Santosh Lall and Sean Tibbetts and the use of equipment at the National Research Council-Institute for Marine Biosciences in preparing the diets.

	FOOTNOTES

 
¹ A portion of the data and findings reported in this manuscript are included in a patent application submitted to the World Intellectual Property Organization (WIPO) and published by the WIPO as part of the application process.

² Supported by Ocean Nutrition Canada, Ltd., Industrial Research Assistance Program of the National Research Council of Canada and the Heart and Stroke Foundation of New Brunswick (Canada).

⁴ Present address: UNC-Wilmington, Center for Marine Science, Wilmington, NC.

⁵ Abbreviations: CO, corn oil; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; PS(n-3), phytosterol esterified to fish oil enriched in (n-3) polyunsaturated fatty acids; PUFA, polyunsaturated fatty acids; TAG, triacylglycerol; TC, total cholesterol; TXA₂ (B₂), thromboxane A₂ (B₂).

Manuscript received 2 October 2001. Revision accepted 12 March 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ewart, H. S. Articles by Murphy, M. G. Search for Related Content PubMed PubMed Citation Articles by Ewart, H. S. Articles by Murphy, M. G.