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American Journal of Clinical Nutrition, Vol. 79, No. 5, 765-773, May 2004
© 2004 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

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 therapy1,2,3

Ken D Stark and Bruce J Holub

1 From the Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada (BJH), and the Section of Nutritional Neuroscience, Laboratory of Membrane Biochemistry and Biophysics, Division of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD (KDS).

2 Supported by research grant T-3633 from the Heart and Stroke Foundation of Ontario. KDS was a recipient of a partnered Doctoral Research Award from the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research. The DHA concentrate and placebo oil capsules were donated by Martek Biosciences Corporation (Columbia, MD).

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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Dietary docosahexaenoic acid (DHA) has triacylglycerol-lowering potential and undergoes in vivo retroconversion to eicosapentaenoic acid (EPA) in humans. Hormone replacement therapy (HRT) influences circulating lipid concentrations and fatty acid metabolism. DHA supplementation has not been studied in postmenopausal women.

Objective: We studied the effects of supplementation with DHA (free of EPA) on the resulting elevation in EPA and on selected cardiovascular disease risk factors in postmenopausal women.

Design: Women receiving (n = 18) and not receiving (n = 14) HRT completed a randomized, double-blind, placebo-controlled crossover trial with a DHA supplement (2.8 g DHA/d). A washout period of >= 6 wk divided the two 28-d intervention periods. Fasting blood samples were collected for analysis.

Results: In all women, DHA supplementation was associated with significant changes (P < 0.05), including 20% lower serum triacylglycerol concentrations, 8% higher HDL-cholesterol concentrations, a 28% lower overall ratio of serum triacylglycerol to HDL cholesterol, and a 7% decrease in resting heart rate. DHA supplementation resulted in a 45% lower net increase (P = 0.02) in EPA and a 42% lower (P = 0.0028) estimated percentage retroconversion of DHA to EPA [{Delta}EPA/({Delta}EPA + {Delta}DHA) x 100] in women receiving than in those not receiving HRT.

Conclusion: With DHA supplementation, the accumulation of EPA in serum phospholipids is significantly attenuated in postmenopausal women receiving HRT compared with that in women not receiving HRT. DHA supplementation can also favorably influence selected cardiovascular disease risk factors in postmenopausal women.

Key Words: Fish oil • n–3 fatty acids • docosahexaenoic acid • DHA • total cholesterol • LDL cholesterol • HDL cholesterol • triacylglycerol • postmenopausal women • hormone replacement therapy • cardiovascular disease • retroconversion • heart rate • C-reactive protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A combination of in vitro studies using liver preparations (1) and in vivo evaluations in animals and humans has confirmed the retroconversion of docosahexaenoic acid (DHA) to eicosapentaenoic acid (EPA) (2, 3). Because the liver is a primary source of serum phospholipid, increases in EPA in serum phospholipid have been used as a biomarker in humans to estimate the in vivo retroconversion of DHA to EPA after EPA-free DHA supplementation (1, 4, 5). Both the biosynthesis and the degradation of DHA in humans are largely dependent on peroxisomal ß-oxidation, and estrogen has been shown to have significant effects on peroxisome proliferation (6) and ß-oxidation (7-9).

Menopause is associated with increases in concentrations of triacylglycerol, total cholesterol, and LDL cholesterol in serum and decreases in concentrations of HDL cholesterol (10). Menopausal women are also at higher risk of cardiovascular disease (CVD) (10); however, it is believed that increases in CVD events in women are log-linear with age (11). The significant reductions in circulating concentrations of estradiol and estrone (12) are believed to influence hepatic lipid and lipoprotein metabolism.

Intervention studies have shown no cardiovascular benefits of hormone replacement therapy (HRT) and that HRT generally increases triacylglycerol concentrations (13, 14). C-reactive protein (CRP), an important predictor of CVD events (15), was shown to be markedly elevated in women receiving HRT (16, 17). The effects of estrogen therapy on various other coronary heart disease risk factors and cardiovascular biomarkers have been reviewed (18). HRT use has also been associated with changes in the fatty acid composition of various circulating lipids (19-21).

The independence of fasting triacylglycerol concentrations (serum or plasma) as a risk factor for coronary heart disease has been debated (22, 23), but more and more evidence suggests that triacylglycerol concentrations are definitely an independent risk factor in women (22, 24, 25). In addition, the ratio of triacylglycerol to HDL cholesterol has been identified as a stronger predictor of myocardial infarction than either total:HDL cholesterol or LDL:HDL cholesterol (24). DHA supplementation has also been shown to significantly decrease triacylglycerol concentrations in humans (4, 26). Supplementation with n–3 polyunsaturated fatty acids (PUFAs) derived from fish oil was previously shown to reduce serum triacylglycerol concentrations in postmenopausal women (27-29), although no studies to date have specifically examined DHA supplementation in postmenopausal women while controlling for the use of HRT. The present placebo-controlled, double-blind crossover trial studied the effects of DHA supplementation on serum lipids and lipoproteins and other CVD risk factors in postmenopausal women receiving and not receiving HRT. We also examined possible interactive effects between HRT status and the apparent retroconversion of DHA to EPA.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Thirty-eight postmenopausal women aged 45–70 y, who had their last menses >= 1 y before the start of the studies, were recruited from the Guelph, Ontario, vicinity. Excluded were women diagnosed with diabetes mellitus or cardiovascular disease, women who consumed fish more than once per week, and women who consumed supplements containing either n–3 PUFAs or phytoestrogens. Eighteen women receiving HRT and 14 women not receiving HRT completed the study requirements. Four subjects withdrew from the study: 2 subjects complained of fatigue and nausea, 1 subject was unable to swallow the required number of capsules each day, and 1 subject was unable to meet the scheduled sample collection dates because of unforeseen travel complications. In addition, 2 subjects were removed from the study because they failed to comply with the supplementation regimen (as determined by a count of returned capsules and the low concentrations of DHA in serum phospholipid during the DHA supplementation phase of the experiment).

All of the postmenopausal women not receiving HRT had experienced a natural onset of menopause. In the group receiving HRT, 10 women had undergone surgical menopause, and 8 women had experienced a natural onset of menopause. The specific estrogen replacement regimens of those receiving HRT were as follows: 22% were using conjugated equine estrogens, 61% were using conjugate estrone sulfate, 11% were using micronized estradiol, and 6% were using estropipate; all women receiving combined-hormone therapy (44% of the women receiving HRT) took medroxyprogesterone acetate. The Human Ethics Committee of the University of Guelph approved the studies, and all of the subjects gave their written informed consent.

Study design
The subjects reported to the Human Nutraceutical Research Unit at the University of Guelph for data and sample collection and for treatment allocation. The subjects were required to refrain from consuming fish for 2 wk before commencing and during the study. At the first visit, the subjects were also required to have a completed general health questionnaire, which included a survey on medications, dietary supplements, and monthly fish and soy consumptions, and a 3-d dietary record (2 weekdays and 1 weekend day).

Martek Biosciences Corporation (Columbia, MD) provided both the DHA concentrate (Neuromins) and the placebo oil as opaque 500-mg capsules in coded containers to ensure subject and investigator blinding. Each capsule of the algal source of DHA contained 180 mg saturated fatty acids, 94 mg monounsaturated fatty acids, 234 mg PUFAs, and 0.13 mg {alpha}-tocopherol equivalents ({alpha}-TE) RRR-{alpha}-tocopheryl acetate and 0.13 mg ascorbyl palmitate as antioxidants. Each capsule provided 230 mg DHA and no EPA. Treated subjects received 2.8 g DHA from 12 capsules per day. Each placebo oil capsule contained a mixture of corn and soy oil that provided {approx}81 mg saturated fatty acids, 132 mg monounsaturated fatty acids, 300 mg PUFAs that consisted mainly of linoleic acid (18:2n–6), 0.13 mg {alpha}-TE RRR-{alpha}-tocopheryl acetate, and 0.13 mg ascorbyl palmitate. All subjects were required to consume 12 capsules/d for the 28 d of each phase of the study.

The subjects were randomly allocated to a coded supplement (as provided by the manufacturer) in the first 28-d period, completed a washout period of >=6 wk, and then received the opposing supplement in the final 28-d period. Blood samples and anthropometric data were collected on days 0 and 28 of each of the intervention periods. The subjects were required to complete 3-d dietary records during each intervention phase. Blood was analyzed for serum concentrations of triacylglycerol, total cholesterol, HDL cholesterol, glucose, insulin, high-sensitivity CRP, and fatty acid composition of serum phospholipid.

At the conclusion of each phase of the trial, the subjects completed a one-page questionnaire to determine the effectiveness of the blinding and to allow reporting of side effects. The correct treatment was identified in 37.5% of the instances, whereas the wrong treatment and "no opinion" were selected in 31.3% of the instances each. Therefore, blinding in this study was assumed to be successful. The subjects completing the trial reported no other side effects.

Laboratory analyses
Blood samples were collected from the subjects by venipuncture into evacuated tubes (Vacutainer; Becton Dickinson, Rutherford, NJ) after the subjects had fasted overnight (for 12–14 h). After being allowed to sit for 1 h, the samples were centrifuged (1000 x g for 15 min at 30 °C), and the recovered serum was divided into aliquots. The serum was stored at –80 °C until analyzed. Triacylglycerol, total cholesterol, HDL cholesterol, and glucose were analyzed on a Synchron CX DELTA automated sample processor (Beckman Coulter Inc, Fullerton, CA) with the appropriate reagent systems. LDL cholesterol was determined by using the equation developed by Friedwald et al (30), except for one subject receiving HRT whose LDL-cholesterol data for day 0 of each intervention phase were not included in the statistical analyses because the corresponding triacylglycerol concentrations were > 5.0 mmol/L. Insulin concentrations were measured by radioimmunassay (Pharmacia Insulin RIA 100; Pharmacia & Upjohn Diagnostics Inc, Mississauga, Ontario), and high-sensitivity CRP was measured on a Bering Nephelometer with appropriate reagents (Dade Behring Inc, Deerfield, IL).

Fatty acid compositions of serum phospholipids were determined by gas-liquid chromatography. Lipids were extracted from serum samples according to the method of Folch et al (31) as modified by Holub and Skeaff (32) in the presence of the internal standard phosphatidyl choline diheptadecanoyl (NuCheck Prep, Elysian, MN). The serum phospholipids were separated from the neutral lipids by thin-layer chromatography. The fatty acid methyl esters were prepared from the phospholipid fraction by the method of Morrison and Smith (33) and were analyzed on a Varian 3800 gas-liquid chromatograph (Palo Alto, CA) with a 60-m DB-23 capillary column (0.32 mm internal diameter).

Duplicate measures of sitting blood pressures and resting heart rate were taken at each visit with an Omron HEM-712C automatic digital blood pressure monitor (Vernon Hills, IL). Height and weight measures were also taken at each visit. Dietary records were analyzed with FOOD PROCESSOR NUTRITION ANALYSIS software, version 7.11 (ESHA Research, Salem, OR).

Statistical analyses
Statistical analyses were performed with SAS for WINDOWS, version 8.02 (SAS Institute Inc, Cary, NC). Triacylglycerol and CRP concentrations were log transformed to allow parametric statistical analyses. Simple comparisons between women receiving and those not receiving HRT at study entry were completed by use of independent t tests. The general linear models procedure was used for analysis of variance (ANOVA). No significant effects of either order of treatment (DHA-placebo versus placebo-DHA) or intervention phase (first versus second) were detected in this crossover trial, which indicated that the washout period was successful. Dietary intakes were compared by two-factor repeated-measures ANOVA (HRT status and diet period), and CVD risk factors and fatty acid compositions were compared by three-factor repeated-measures ANOVA (HRT status, supplementation status, and time). HRT status x supplementation status x time interactions were detected for EPA, 22:0, docosapentaenoic acid (DPA), and the ratio of DHA to EPA only. To facilitate interpretation, the means for supplementation status x time interactions are shown in the tables, because this was the most common interaction detected. Changes in EPA, 22:0, DPA, and DHA:EPA were analyzed by two-way ANOVA with interaction for HRT status and supplementation status and are shown separately. Post hoc analyses of individual means were determined by Tukey’s procedure. Changes in selected CVD risk factors in postmenopausal women were compared by paired t tests. Parsimonious modeling starting with interactions for HRT status, supplementation status, and time with BMI as a covariate was completed for CRP by analysis of covariance (ANCOVA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The characteristics of the subjects entering the study are shown according to HRT status in Table 1Go. The women receiving HRT had significantly (by independent t tests) higher serum triacylglycerol concentrations (by 90% overall), triacylglycerol:HDL cholesterol (by 104%), and CRP concentrations (by 165%). Although the LDL-cholesterol concentrations of the women not receiving HRT were higher than those of the women receiving HRT, this difference was not significant (P = 0.057).


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  		TABLE 1 Entry characteristics of postmenopausal women receiving or not receiving hormone replacement therapy (HRT)1

 
The dietary intakes of the women at entry to the study and during the control and supplementation periods are shown according to HRT status in Table 2Go. After two-factor repeated-measures ANOVA for HRT status and diet period, a moderate effect of HRT use was detected. The women receiving HRT had a lower intake of carbohydrates (and a lower percentage of energy from carbohydrates) and higher percentages of energy from fat and alcohol. The women receiving HRT also had significantly higher intakes of monounsaturated and polyunsaturated fats. The increase in PUFA intakes during the placebo and DHA supplementations (diet period effect) was not significant (P = 0.10).


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  		TABLE 2 Daily dietary intakes (including supplements for control and supplemented groups), body weight, and BMI of postmenopausal women not receiving or receiving hormone replacement therapy (HRT) at study entry (baseline) and after placebo (control) and docosahexaenoic acid supplementation1

 
The results of the trial were analyzed with three-factor repeated-measures ANOVA with interaction for HRT status, supplementation status, and time. No HRT status x supplementation status x time interactions were detected for any of the selected CVD risk factors. The results are shown before and after supplementation in Table 3Go. Supplementation status x time interactions were detected for HDL cholesterol, triacylglycerol, triacylglycerol:HDL cholesterol, and heart rate. The main effects of HRT status reflected the baseline HRT status comparisons (Table 1Go), with the addition of LDL cholesterol reaching significance (P = 0.014). In the most parsimonious ANCOVA model, both HRT status (ß = 0.71) and BMI (ß = 0.09) were significantly associated with CRP (P < 0.0001). CRP concentrations were unaffected by DHA supplementation.


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  		TABLE 3 Effects of docosahexaenoic acid supplementation on selected risk factors for cardiovascular disease in postmenopausal women1

 
Changes in CVD risk factors after DHA supplementation (and their significance) for all women combined are illustrated in Figure 1Go. Briefly, the DHA treatment was associated with a 20% (0.32 ± 0.09 mmol/L) decrease in triacylglycerol concentrations, an 8% (0.13 ± 0.03 mmol/L) increase in HDL-cholesterol concentrations, a 28% (0.30 ± 0.08 mmol/L) decrease in triacylglycerol:HDL cholesterol, and a 7% (4.8 ± 1.3 beats/min) decrease in heart rate.



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  		FIGURE 1. Mean (± SEM) changes in serum lipids and heart rate in postmenopausal women regardless of hormone replacement therapy status after supplementation with docosahexaenoic acid (n = 32) and a placebo control (n = 32) for 28 d. The ratio of triacylglycerol (TG) to HDL cholesterol (HDL-C) was derived from mmol/L values of TG and HDL-C.

 
The three-factor ANOVA of the fatty acid compositions of serum phospholipid showed significant HRT status x supplementation status x time interactions for EPA, 22:0, DPA, and DHA:EPA. The changes in concentrations and in the ratio are shown in Figure 2Go. DHA supplementation raised EPA concentrations significantly more in the women not receiving HRT (from 1.12 ± 0.09% by wt to 1.78 ± 0.11% by wt) than in the women receiving HRT (from 0.91 ± 0.06% by wt to 1.27 ± 0.06% by wt). DHA supplementation resulted in increases in 22:0 in the women not receiving HRT and decreases in 22:0 in the women receiving HRT, but the individual means of the changes in 22:0 were not significantly different by Tukey’s procedure. Concentrations of DPA at day 0 were significantly higher in the women not receiving HRT than in the women receiving HRT for both intervention phases (1.13 ± 0.04% by wt compared with 0.85 ± 0.04% by wt, respectively, for the supplement phase). The DHA intervention decreased DPA concentrations to similar concentrations in both the women not receiving HRT and those receiving HRT (0.44 ± 0.04% by wt compared with 0.43 ± 0.02% by wt, respectively). The women not receiving HRT also had a significantly lower DHA:EPA after supplementation (from 3.49 ± 0.26% by wt to 6.13 ± 0.46% by wt) than did the women receiving HRT (from 3.99 ± 0.34% by wt to 7.54 ± 0.28% by wt).



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  		FIGURE 2. Mean (± SEM) changes in selected fatty acids in serum phospholipid before and after supplementation with a placebo control or docosahexaenoic acid (DHA) in postmenopausal women not receiving (n = 14) and receiving (n = 18) hormone replacement therapy (HRT). The ratio of DHA to eicosapentaenoic acid (EPA) was derived from the percentage by weight of total fatty acids. DPA, docosapentaenoic acid. Columns headed by different letters within a specific fatty acid are significantly different, P < 0.05 (Tukey’s post hoc analysis). An HRT status x supplementation status interaction was detected for each of the changes by two-factor repeated-measures ANOVA.

 
Numerous supplementation status x time interactions were determined by ANOVA for the remaining fatty acids studied; however, no significant differences in the individual means of 16:0, 18:0, and 24:1n–9 were found by Tukey’s procedure. The effects of DHA compared with placebo on the serum phospholipid fatty acids of postmenopausal women are shown in Table 4Go. Briefly, DHA supplementation significantly increased the percentage of n–3 PUFAs, largely by resulting in a deficit of n–6 PUFAs, although the percentages of some individual monounsaturated fatty acids were also significantly decreased. Supplementation with DHA was associated with a 179% increase in DHA concentrations and a 26% decrease in arachidonic acid concentrations in serum phospholipid. Various HRT status main effects were detected and reflect previous findings in the literature (21), and a single HRT status x time interaction was detected for DHA. These results are difficult to interpret with concurrent DHA and placebo control supplementation.


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  		TABLE 4 Effects of docosahexaenoic acid (DHA) supplementation on the fatty acid composition of serum phospholipid in postmenopausal women1

 
The changes ({Delta}) in serum phospholipid concentrations of EPA, DHA, and a previously defined marker for retroconversion [{Delta}EPA/({Delta}EPA + {Delta}DHA) x 100] (5) for the women receiving and not receiving HRT after DHA supplementation were also examined by use of independent t tests. The increases in EPA with DHA supplementation were significantly greater in the women not receiving HRT than in the women receiving HRT (0.66 ± 0.11% by wt compared with 0.36 ± 0.07% by wt; P = 0.02), whereas there were no significant differences in the changes in DHA. The apparent estimated retroconversion of DHA to EPA was significantly different, being 8.96 ± 1.31% in the women not receiving HRT and 5.22 ± 1.00% in the women receiving HRT (P = 0.028).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study evaluated the effects of an algal DHA supplement (free of EPA) on serum lipids and lipoproteins, the fatty acid composition (including of EPA) of serum phospholipid, and other CVD risk factors in postmenopausal women receiving and not receiving HRT. Our results indicate that elevations in EPA after DHA supplementation are lower in women receiving HRT than in women not receiving HRT. For all women combined, DHA supplementation was also associated with significant decreases in serum concentrations of triacylglycerol and the ratio of triacylglycerol to HDL cholesterol, a significant increase in HDL cholesterol, and an unexpected decrease in resting heart rate. The effects on these CVD risk factors did not differ significantly between the women receiving and those not receiving HRT.

Conquer and Holub (5) estimated the retroconversion of DHA to EPA in young adult omnivores and vegetarians to be 7.4% and 11.4%, respectively. In comparison, the estimate of retroconversion in the present study for the women not receiving HRT was similar (9.0%), but that for the women receiving HRT was significantly lower (5.2%). One could speculate that the use of oral HRT may affect hepatic lipid and fatty acid metabolism; however, definite conclusions will require the use of isotopically labeled molecules (34).

Many biochemical reactions could result in changes in the fatty acid composition of serum phospholipid, a recognized biomarker of n–3 fatty acid status (35). Peroxisomal-mediated metabolism is involved in DHA retroconversion to EPA (3, 36-38), and it has been shown that estradiol can induce peroxisome proliferation (6, 39, 40). Treatment with 17ß-estradiol has been shown to prevent death from hepatic and cardiac lipid accumulation in male mice lacking peroxisome proliferator-activated receptor {alpha} (7), to restore gene expression and enzyme activities of fatty acid ß-oxidation, and to prevent hepatic steatosis in aromatase-deficient mice that lack intrinsic estrogen production (8). An increase in peroxisomal ß-oxidation, as suggested by previous research (6-8, 39, 40), would result in greater peroxisomal retroconversion of DHA to EPA. In the present study, the estimated percentage retroconversion of DHA to EPA was significantly lower in the women receiving HRT than in those not receiving HRT. However, Gronn et al (1) showed that clofibrate, a potent inducer of peroxisomal ß-oxidation, did not cause an increase in concentrations of EPA, but that a greater percentage of labeled DHA was completely oxidized, indicating stimulated mitochondrial ß-oxidation as well. Previously, estrogen was shown to support the maximal capacity of mitochondrial lipid oxidation in rat skeletal muscle (9). Therefore, it is possible that both mitochondrial and peroxisomal fatty acid ß-oxidation are higher in women receiving oral HRT. The pattern of women receiving HRT having consistently depressed (although not always significantly different) concentrations of EPA and DHA compared with concentrations in women not receiving HRT supports a hypothesis of increased ß-oxidation with HRT use.

Note that a higher concentration of combined DHA plus EPA in plasma phospholipid was reported in the Cardiovascular Health Study to markedly lower the risk of fatal ischemic heart disease after adjustment for risk factors (41). In the present study, these combined concentrations rose from 4.5 ± 1.0% of fatty acids to 11.3 ± 1.5% in all subjects with DHA supplementation (data not shown). It has also been shown that increased intake of EPA and DHA is associated with a lower risk of coronary heart disease, particularly coronary heart disease death (42).

The decreases in DPA concentrations in serum phospholipid in the present study were shown previously with DHA supplementation (5). This is likely a result of DHA competition for esterification at the sn-2 position of glycerophospholipids by either de novo synthesis or acyl transferase reactions. The significantly smaller decrease in DPA concentrations in the women receiving HRT is difficult to interpret, but may be a result of lower baseline concentrations of DPA in these women. Lower DPA concentrations with HRT use have been shown previously (43).

In the present study, algal DHA (free of EPA) supplementation decreased serum triacylglycerol concentrations by 20% and increased HDL-cholesterol concentrations by 8%. The results of previous studies with algal DHA oil in humans were similar (4, 26). However, the present study is the first to show these changes in postmenopausal women either receiving or not receiving HRT. Ours is also the first study to show that DHA supplementation can significantly decrease triacylglycerol:HDL cholesterol. The potential cardiovascular benefits of reducing triacylglycerol:HDL cholesterol and triacylglycerol concentrations in women without overt hypertriacylglycerolemia has been discussed previously (28). Studies have shown that purified EPA and DHA reduce plasma triacylglycerol concentrations similarly, but that HDL-cholesterol concentrations tend to be increased only with DHA supplementation (44, 45). The hypotriglyceridemic effect of n–3 fatty acids is mediated by several mechanisms such as inhibition of fatty acid and triacylglycerol synthesis and depressed assembly and secretion of VLDL triacylglycerol (46, 47). The mechanism by which DHA supplementation increases HDL cholesterol is unknown but may be associated with decreases in lipid transfer protein activity (48).

Heart rate was 5 beats/min lower after supplementation with DHA. Similar results were shown previously with purified DHA but not with purified EPA supplementation (49, 50). The effects on heart rate are possibly related to the antiarrhythmic effects of n–3 fatty acids, which were reviewed recently (51, 52). Briefly, it has been suggested that the antiarrhthymic effects of n–3 fatty acids depend on DHA accumulation in myocardial cell membranes and that the effect is related to stabilization against Ca2+ overload, thromboxane production, ischemic acidosis, and ischemic K+ loss. It has also been suggested that increased DHA content in cardiac phospholipids may regulate the ß-adrenergic transduction mechanism (53). Reductions in heart rate were shown to be associated with decreased all-cause mortality (54) and sudden cardiac death (55).

An association between dietary fat intake and increased estrogen concentrations during the menstrual cycle of premenopausal women was shown previously (56). In the present study, an HRT status effect was detected for increased percentage of energy from dietary fat and monounsaturated and polyunsaturated dietary fat intake, and decreased percentage of energy from dietary carbohydrate and decreased total carbohydrate intake. In this study, CRP concentrations were 2.5 times higher in the women receiving HRT than in the women not receiving HRT. Elevated CRP with HRT use was reported previously (16, 17). No effect of DHA on the elevated CRP concentrations found in the women receiving HRT was noted.

In conclusion, supplementation with DHA resulted in a significantly greater increase in EPA concentrations and a significantly greater estimated percentage retroconversion of DHA to EPA in postmenopausal women not receiving HRT than in postmenopausal women receiving HRT. DHA supplementation in postmenopausal women was also associated with changes in serum triacylglycerol concentrations (20% decrease), HDL-cholesterol concentrations (8% increase), the ratio of serum triacylglycerol to HDL cholesterol (28% decrease), and heart rate (7% decrease). Further research on the effects of estrogen on mitochondrial and peroxisomal ß-oxidation is required to determine the mechanism of the apparent decrease in in vivo DHA retroconversion to EPA in women receiving HRT. These results also show that DHA supplementation can favorably influence selected cardiovascular disease risk factors and potentially reduce the risk of coronary heart disease in postmenopausal women.


   ACKNOWLEDGMENTS
 
We thank Linda Arterburn of Martek Biosciences Corporation (Columbia, MD) for donating the DHA concentrate and placebo oil capsules. We also thank Cheryl Anderson and Patricia Swidinsky for venipuncture assistance in this investigation and our subjects for their commitment to this study.

KDS recruited the subjects and collected, analyzed, and interpreted the data. He was also involved in the design of the study and was the primary writer of the manuscript. BJH obtained funding for the study and was involved in the design of the study, data interpretation, and writing of the manuscript. Neither of the authors had a conflict of interest with the funding agencies or with Martek Biosciences Corporation.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication May 1, 2003. Accepted for publication October 8, 2003.




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