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Original Research Communications |
1 From the Department of Human Biology and Nutritional Sciences, University of Guelph, Canada.
2 Supported by research grant T-3633 from the Heart and Stroke Foundation of Ontario. KS received a partnered Doctoral Research Award from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada.
3 Address reprint requests to BJ Holub, Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1. E-mail: bholub{at}uoguelph.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We sought to determine the effects of a fish-oil-derived n-3 fatty acid concentrate on serum lipid and lipoprotein risk factors for cardiovascular disease in postmenopausal women receiving and not receiving HRT, with an emphasis on serum triacylglycerol concentrations and the ratio of triacylglycerol to HDL cholesterol.
Design: Postmenopausal women (n = 36) were grouped according to exogenous hormone use and were randomly allocated to receive 8 capsules/d of either placebo oil (control) or n-3 fatty acid–enriched oil (supplement). The supplement provided 2.4 g eicosapentaenoic acid (EPA) plus 1.6 g docosahexaenoic acid (DHA) daily. Serum lipids and the fatty acid composition of serum phospholipids were determined on days 0 and 28.
Results: Supplementation with n-3 fatty acids was associated with 26% lower serum triacylglycerol concentrations (P < 0.0001), a 28% lower overall ratio of serum triacylglycerol to HDL cholesterol (P < 0.01), and markedly greater EPA and DHA concentrations in serum phospholipids (P < 0.05).
Conclusions: These results show that supplementation with a fish-oil–derived concentrate can favorably influence selected cardiovascular disease risk factors, particularly by achieving marked reductions in serum triacylglycerol concentrations and triacylglycerol:HDL cholesterol in postmenopausal women receiving and not receiving HRT. This approach could potentially reduce the risk of coronary heart disease by 27% in postmenopausal women.
Key Words: Fish oil • n-3 fatty acids • omega-3 fatty acids • total cholesterol • LDL cholesterol • HDL cholesterol • triacylglycerol • postmenopausal women • hormone replacement therapy • cardiovascular disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Menopause is associated with a significant increase in CVD risk (8). Observational studies found lower rates of CHD in postmenopausal women receiving exogenous estrogens. It is thought that the decreases in LDL cholesterol and increases in HDL cholesterol that are associated with estrogen use provide protection against CVD (9). The Heart and Estrogen/Progestin Replacement Study showed recently that hormone replacement therapy (HRT) had no beneficial effect on CVD risk despite improvements in LDL- and HDL-cholesterol concentrations (10). Significant increases in triacylglycerol concentrations were reported with HRT in both the Heart and Estrogen/Progestin Replacement Study (baseline mean + 0.15 mmol/L) (10) and the Postmenopausal Estrogen/Progestin Intervention Trial (baseline mean + 0.14 mmol/L) (11). Fish oils containing n-3 fatty acids have successfully reduced triacylglycerol concentrations in humans (12–14).
Fish oils contain the long-chain polyunsaturated n-3 fatty acids eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Studies to date on n-3 fatty acid supplementation were performed primarily in male or mixed-subject groups. Two studies that examined the effects of fish oil in postmenopausal women did not include a placebo (control) group (15, 16). The purpose of the present study was to determine the effects of n-3 fatty acid supplementation on serum lipids in healthy postmenopausal women receiving and not receiving HRT in a placebo-controlled, double-blind trial. In addition, this intervention study was specifically designed to determine the effects of n-3 fatty acid supplementation on the ratio of triacylglycerol to HDL cholesterol.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 y before the study began were eligible. Individuals taking lipid-altering, blood pressure–altering, or antiinflammatory medications were excluded from the study. A total of 36 subjects took part in the study. One subject withdrew from the study because of an unrelated illness. Of the 35 subjects who completed the study, 19 were receiving either estrogen or combined-hormone therapy and 16 were not receiving any form of HRT. In the group receiving HRT, 14 women had undergone surgical menopause and 5 women had experienced natural menopause. Of the subjects in the HRT group, 74% were taking conjugated equine estrogens, 16% were taking conjugate estrone sulfate, and 11% were taking micronized estradiol. All subjects receiving combined-hormone therapy were taking medroxyprogesterone acetate. In the group of women not receiving HRT, 3 women had undergone surgical menopause and 13 had experienced natural menopause. None of the subjects had been diagnosed with diabetes mellitus or CVD. The Human Ethics Committee of the University of Guelph approved this study, and all subjects gave their written, informed consent.
Study design
Both the placebo and the n-3 fatty acid supplement were provided as 1-g capsules. The n-3 fatty acid supplement (Natra Sea 6000; Ocean Nutrition Canada Ltd, Bedford, Nova Scotia) was a triacylglycerol concentrate derived from fish oil. Each capsule contained 
30 mg saturated fatty acids (SFAs), 110 mg monounsaturated fatty acids (MUFAs), 610 mg polyunsaturated fatty acids (PUFAs), and 1.1 mg 
-TE RRR--tocopherol. Each capsule provided 0.3 g EPA and 0.2 g DHA (total EPA and DHA = 0.5 g/capsule). The placebo capsules (control) contained evening primrose oil (Bioriginal; PGE Canada, Saskatoon, Saskatchewan) providing 80 mg SFAs, 65 mg MUFAs, and 833 mg PUFAs that consisted mainly of linoleic acid (18:2n-6). The placebo contained 3.5 mg -TE RRR--tocopheryl acetate/capsule. Subjects received 8 capsules/d of either placebo oil or n-3 fatty acid concentrate oil providing 2.4 g EPA and 1.6 g DHA (total EPA and DHA = 4.0 g/d). All capsules were delivered in a double-blind fashion.
For preliminary screening purposes, blood lipid profiles were measured with a compact, instant blood lipid analyzer (Cholestech LDX System; Cholestech, Hayward, CA). Within each HRT-status grouping (ie, receiving or not receiving HRT), subjects were assigned to either the control or the supplemented group for the 28-d study. The group means of the screened blood lipid values were balanced during group assignment.
Subjects were required to provide venous blood samples after fasting overnight (for 12–14 h) on days 0 and 28 of the study. These samples were analyzed in duplicate for total, LDL, and HDL cholesterol and triacylglycerol; the fatty acid composition of serum phospholipid was also analyzed to assess compliance with the treatment.
Duplicate measures of sitting blood pressure and resting heart rate were determined at each visit with an automatic digital blood pressure monitor (Omron, Vernon Hills, IL). Height and weight were also measured at each visit. Each subject completed a 3-d dietary record (on 2 weekdays and 1 weekend day) before starting the study and once during the study. Dietary records were analyzed by using FOOD PROCESSOR, a nutrition analysis system (version 7.11; ESHA Research, Salem, OR). At the end of the study, subjects completed a one-page questionnaire intended to determine adverse effects and the effectiveness of the blinding.
Laboratory analyses
Blood was collected by venipuncture into evacuated tubes (Vacutainer; Becton Dickinson, Rutherford, NJ). After the samples sat for 1 h, they were centrifuged (1000 x g for 15 min at 30°C). The recovered serum was divided into aliquots and then stored at -80°C until analyzed. Triacylglycerol concentrations were quantified enzymatically [procedure no. 339 (Triglycerides), Sigma Diagnostics, St Louis] and normalized to those analyzed at an Ontario Ministry of Health Licensing and Inspection Branch licensed laboratory (Kodak Ektachem; MDS Laboratory Services, Guelph, Canada) that is subject to the provincial Laboratory Proficiency Testing Program. Total cholesterol concentrations were also determined enzymatically [procedure no. 352 (Cholesterol), Sigma Diagnostics]. HDL-cholesterol concentrations were quantified after precipitation of the serum [procedure 352-3 (HDL cholesterol), Sigma Diagnostics], and LDL-cholesterol concentrations were determined by using the Friedewald equation (17). The fatty acid composition of serum phospholipids was determined by using gas chromatography (18).
Statistical analyses
Statistical analyses were performed by using SAS (SAS Institute, Cary, NC). Because triacylglycerol concentrations were not normally distributed, they were logarithmically transformed before the analyses. Baseline subject characteristics across hormone-use status were compared with independent t tests. Analysis of variance was performed with the general linear models procedure to determine the effects of treatment, HRT use, and time. Individual means were compared by using t tests if the interaction effect was significant. Significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The only significant difference was that subjects receiving HRT had a higher mean HDL-cholesterol concentration than did subjects not receiving HRT. Total cholesterol, triacylglycerol, and triacylglycerol:HDL cholesterol tended to be higher in the women receiving HRT than in those not receiving HRT, but not significantly so.
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. A significant time effect was detected for dietary PUFA intake, which increased between days 0 and 28 because of the supplements taken. The subjects received either 4.9 g PUFA/d from the n-3 fatty acid supplements or 6.7 g PUFA/d from the placebo. A treatment x HRT use x time interaction was detected (P < 0.05, not shown) for dietary fiber, which may be a spurious finding; there were no significant differences in fiber intake between individual subject groups by t test. No other treatment x HRT use x time interactions were detected in this study; therefore, the data were collapsed for women receiving and not receiving HRT when analyses for such interactions were performed (Table 2 and Tables 3 and 4).
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. There was an effect of HRT on HDL-cholesterol concentration and triacylglycerol:HDL cholesterol and a time effect on LDL-cholesterol concentration, triacylglycerol concentration, and triacylglycerol:HDL cholesterol. Both triacylglycerol concentration and triacylglycerol:HDL cholesterol had highly significant treatment x time interactions (P < 0.0005); there was a trend (P = 0.061) for treatment x HRT use x time interaction for triacylglycerol:HDL cholesterol. Treatment with n-3 fatty acids was associated with a highly significant decrease (26%) in triacylglycerol concentrations. The response of triacylglycerol concentrations to n-3 fatty acid supplementation was somewhat variable: 7 subjects had reductions of 5–15%, 5 subjects had reductions of 20–40%, and 4 subjects had reductions of 40–60%. The 2 subjects (of the 18 assigned to n-3 fatty acid supplementation) who had no reductions in triacylglycerol concentrations were receiving HRT. Triacylglycerol:HDL cholesterol in postmenopausal women decreased significantly by 28% with n-3 fatty acid supplementation. There were no significant changes in total:HDL cholesterol, LDL:HDL cholesterol, total cholesterol concentration, heart rate, systolic blood pressure, diastolic blood pressure, or mean arterial pressure during the 28-d study.
The fatty acid compositions of serum phospholipids are shown in Table 4. No treatment x HRT use x time interactions were detected because the serum phospholipid fatty acids responded similarly to n-3 fatty acid supplementation in the women receiving and not receiving HRT. EPA and DHA concentrations rose by 446% (from 1.18% to 6.44% by weight) and 64% (from 3.9% to 6.4% by weight), respectively, with n-3 fatty acid supplementation. The ratio of n-3 to n-6 fatty acids was approximately tripled in response to n-3 fatty acid supplementation (P < 0.0001). Concentrations of all 3 n-6 PUFAs (18:2n-6, 20:3n-6, and 20:4n-6) decreased significantly (P < 0.0005) with n-3 fatty acid supplementation.
On the basis of the results of the questionnaire, 56% of the subjects who took the n-3 fatty acid supplement and 47% of the subjects who took the placebo identified their treatment correctly. Two subjects who took the n-3 fatty acid supplements complained of occasional nausea. No other adverse effects were reported.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A review of human and animal studies showed that treatment with n-3 fatty acids decreases triacylglycerol concentrations by 25–30% in normolipidemic subjects (14). In the present study, n-3 fatty acid supplementation in postmenopausal women decreased triacylglycerol concentrations by 26% (mean decrease of 0.36 mmol/L). This reduction could decrease CVD risk by a predicted 27% in postmenopausal women on the basis of a recent meta-analysis of triacylglycerol concentration (serum or plasma) as an independent risk factor for CVD (5). Two previous studies of fish oil in postmenopausal women showed triacylglycerol reductions of 30% in women not receiving HRT (15) and decreases of 28% (15) and 8% (16) in women receiving HRT; these studies were not placebo controlled, however. Although there were no significant HRT-use effects or interactions with regard to triacylglycerol concentrations in this study, n-3 fatty acid supplementation decreased triacylglycerol concentration by 35% overall (mean decrease of 0.45 mmol/L) in postmenopausal women not receiving HRT and by 19% overall (mean decrease of 0.26 mmol/L) in women receiving HRT.
Previous studies showed that the triacylglycerol-lowering effect of fish oil is greater (as both percentage and absolute decreases) in subjects with higher initial triacylglycerol concentrations (13, 19). Interestingly, in the present study, the women receiving HRT tended to have higher serum triacylglycerol concentrations at study entry (P = 0.092) than did the women not receiving HRT (Table 1), but the triacylglycerol-lowering effect was nonsignificantly smaller in the former group with the n-3 fatty acid intervention (Table 3). Previous studies showed that the use of estrogen or combined HRT can increase serum or plasma triacylglycerol concentrations (10, 11). Oral estrogen may increase triacylglycerol concentrations by raising the production of VLDL (20). Estrogen administration also increases the triacylglycerol content of HDL and LDL particles (21). EPA and DHA in fish oil are thought to lower serum triacylglycerol concentrations by several mechanisms, including inhibition of fatty acid and triacylglycerol biosynthesis in the liver and reduction of the assembly and secretion rate of VLDL-triacylglycerol (12, 22).
Little information is available about the direct effects of n-3 fatty acid supplementation on triacylglycerol:HDL cholesterol. Recently, this ratio was shown to be strongly associated with the risk of myocardial infarction (6) and to be a possible marker for the progression of atherosclerosis (7). The marked suppression of triacylglycerol:HDL cholesterol reported here (28% overall in postmenopausal women) provides a basis for the use of this specific ratio in future clinical trials of fish-oil–based nutraceutical therapy with respect to CVD-related morbidity and mortality. The trend (P = 0.061) toward a treatment x HRT use x time interaction for triacylglycerol:HDL cholesterol suggests that HRT may compromise the extent of the beneficial effects of EPA and DHA on this risk factor; triacylglycerol:HDL cholesterol decreased by 39% in women not receiving HRT and by 20% in women receiving HRT. Total:HDL cholesterol and LDL:HDL cholesterol, considered to be predictors of CHD risk (6, 23), did not change significantly in this study. This further emphasizes the importance of routinely monitoring the effects of intervention strategies on triacylglycerol:HDL cholesterol.
As reviewed extensively (12–14), individuals (mostly male subjects) with elevated triacylglycerol concentrations tend to exhibit a moderate rise in LDL-cholesterol concentrations with fish-oil therapy providing 3 g EPA and DHA/d. This pattern tended to occur in some of our subjects who were receiving HRT. (All 4 subjects who had initial fasting triacylglycerol concentrations >2.0 mmol/L had an overall increase in LDL-cholesterol concentration of 10%.) This pattern was not evident in the women not receiving HRT. Fish-oil (EPA and DHA) therapy taken with pharmaceutical (24) or nutraceutical (25) agents for combined lowering of triacylglycerol and LDL-cholesterol concentrations may be advisable, along with blood lipid monitoring, for individual management of CVD risk.
There is evidence that LDL particle size increases with fish-oil supplementation; it was suggested that this may reduce the atherogenic potential of LDL (26), although further study is needed to confirm this. In addition, n-3 fatty acids can reduce CHD risk via other mechanisms, such as antiatherogenic, antithrombotic, and antiarrhythmic actions (19, 27). This could be of interest because of the tendency of HRT to increase the rate of venous thromboembolism (9, 28). In 1999, evidence supporting the efficacy of fish oil for preventing CVD was reported by von Schacky et al (29). These authors found that a relatively low dose of fish oil (3.3 g EPA and DHA for 3 mo followed by 1.65 g EPA and DHA for 21 mo) resulted in decreased progression and increased regression of CHD, as determined by coronary angiography. In addition, the GISSI-Prevenzione Trial (Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico) reported significant decreases over 3.5 y in the rates of death, nonfatal myocardial infarction, and stroke with low-dose EPA and DHA treatment (0.85–0.88 g/d) in post–myocardial infarction patients (30).
In our study, only 5 of 35 subjects could be classified as clinically hypertriacylglycerolemic according to current definitions (serum triacylglycerol >2.3 mmol/L) (1). However, lowering of serum or plasma triacylglycerol concentrations may provide significant CVD risk reduction in individuals with baseline concentrations <2.3 mmol/L. Individuals with plasma triacylglycerol concentrations of 1.22 mmol/L were shown to have a relative risk (adjusted for multiple risk factors, including HDL cholesterol) of 2.2 for myocardial infarction, compared with individuals with plasma triacylglycerol concentrations of 0.79 mmol/L (6). In women specifically, it was shown that plasma triacylglycerol concentrations >0.94 mmol/L are associated with increased risk of death from CHD (3). Note that 17 of 35 subjects (50%) in our study had serum triacylglycerol concentrations reflecting increased CHD risk (0.94–2.3 mmol/L) but did not have overt hypertriacylglycerolemia.
In the present study, blinding was assumed to be successful because only 56% of subjects in the n-3 fatty acid group and only 47% of subjects in the placebo group identified their treatment correctly. Random guessing would result in 50% of subjects in each group identifying their treatment correctly. Subject compliance with the study protocol was determined to be high on the basis of counts of returned capsules and analysis of the fatty acid composition of serum phospholipids. The latter was shown to be a suitable biological marker for dietary n-3 fatty acid (EPA and DHA) intake (31). Another study found that DHA concentrations in serum phospholipids are inversely associated with risk of CHD (32).
In conclusion, supplementation with fish-oil–derived EPA and DHA concentrate reduced triacylglycerol concentrations significantly, by 26%, in postmenopausal women. This effect was estimated to decrease the risk of CHD by 27% in postmenopausal women. In addition, n-3 fatty acid supplementation was effective in reducing triacylglycerol:HDL cholesterol by 28%. Further studies are needed to elucidate the interactions of n-3 fatty acid supplementation with specific HRT regimens and to determine the long-term effects of n-3 fatty acid supplementation on cardiovascular events and mortality in postmenopausal women.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopheryl acetate and fish oil on low-density-lipoprotein oxidation in postmenopausal women with and without hormone-replacement therapy. Am J Clin Nutr 1996;63:184–93.
-3 fatty acids on plasma lipids and lipoproteins in patients with combined hyperlipidemia. Arterioscler Thromb 1993;13:1755–62.–3 fatty acids on coronary atherosclerosis. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 1999;130:554–62.
-3 fatty acids: essential fatty acids with important biological effects, and serum phospholipid fatty acids with important biological effects, and serum phospholipid fatty acids as markers of dietary –3 fatty acid intake. Am J Clin Nutr 1993;57(suppl):801S–6S.This article has been cited by other articles:
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  I. Rudkowska Functional foods for cardiovascular disease in women Menopause Int, June 1, 2008; 14(2): 63 - 69. [Abstract] [Full Text] [PDF]
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 C. E. Lok, M. Allon, S. Donnelly, M. Dorval, B. Hemmelgarn, L. Moist, M. J. Oliver, M. Tonelli, and K. Stanley Design of the fish oil inhibition of stenosis in hemodialysis grafts (FISH) study Clinical Trials, August 1, 2007; 4(4): 357 - 367. [Abstract] [PDF]
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 D. Faeh, K. Minehira, J.-M. Schwarz, R. Periasamy, S. Park, and L. Tappy Effect of Fructose Overfeeding and Fish Oil Administration on Hepatic De Novo Lipogenesis and Insulin Sensitivity in Healthy Men Diabetes, July 1, 2005; 54(7): 1907 - 1913. [Abstract] [Full Text] [PDF]
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 K. D Stark and B. J Holub Differential eicosapentaenoic acid elevations and altered cardiovascular disease risk factor responses after supplementation with docosahexaenoic acid in postmenopausal women receiving and not receiving hormone replacement therapy Am. J. Clinical Nutrition, May 1, 2004; 79(5): 765 - 773. [Abstract] [Full Text] [PDF]
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M. Laidlaw and B. J Holub Effects of supplementation with fish oil-derived n-3 fatty acids and {gamma}-linolenic acid on circulating plasma lipids and fatty acid profiles in women Am. J. Clinical Nutrition, January 1, 2003; 77(1): 37 - 42. [Abstract] [Full Text] [PDF]
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 B. J. Holub Clinical nutrition: 4. Omega-3 fatty acids in cardiovascular care Can. Med. Assoc. J., March 1, 2002; 166(5): 608 - 615. [Full Text] [PDF]
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 C. Crone, G. Gabriel, and T. N. Wise Non-Herbal Nutritional Supplements--The Next Wave: A Comprehensive Review of Risks and Benefits for the C-L Psychiatrist Psychosomatics, August 1, 2001; 42(4): 285 - 299. [Abstract] [Full Text] [PDF]
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