Effect of a fish-oil concentrate on serum lipids in postmenopausal

women receiving and not receiving hormone replacement therapy

in a placebo-controlled, double-blind trial13
Ken D Stark, Eek J Park, Valerie A Maines, and Bruce J Holub
ABSTRACT
Background: n3 Fatty acid supplementation lowered serum
triacylglycerol concentrations in studies in which most of the
subjects were male. The effects of n3 fatty acid supplementation in postmenopausal women receiving and not receiving hormone replacement therapy (HRT) have received little attention.
Objective: We sought to determine the effects of a fish-oil-derived
n3 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
n3 fatty acidenriched 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 n3 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-oilderived 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.
Am J Clin Nutr 2000;72:38994.
KEY WORDS
Fish oil, n3 fatty acids, omega-3 fatty acids,
total cholesterol, LDL cholesterol, HDL cholesterol, triacylglycerol,
postmenopausal women, hormone replacement therapy,
cardiovascular disease

INTRODUCTION
Elevated serum lipid concentrations have been identified
clearly as major risk factors for coronary heart disease (CHD)

(1). High total cholesterol and LDL-cholesterol concentrations

and low HDL-cholesterol concentrations are well-established
risk factors for CHD. Until recently, the role of triacylglycerol
concentration (serum or plasma) as an independent risk factor
was debated (25). A recent meta-analysis suggested that a 0.1mmol/L increase in triacylglycerol is associated with a 3.2%
increase in cardiovascular disease (CVD) in men and a 7.6%
increase in women (5). After adjustment for HDL-cholesterol
concentration and other risk factors, these increases in risk
dropped to 1.4% and 3.7%, respectively; however, triacylglycerol remained an independent risk factor for CVD (5). The possibility that triacylglycerol is a more important risk factor for
myocardial infarction in women than in men was reported previously (6). A recent study showed that the relative risk of CHD
was 3.17 in women and 1.83 in men with triacylglycerol concentrations in the highest tertile, as compared with the lowest
tertile (7). The ratio of triacylglycerol to HDL cholesterol was
shown to be a stronger predictor of myocardial infarction than
was either total:HDL cholesterol or LDL:HDL cholesterol (6).
The recently completed Atherosclerosis Risk In Communities
Study concluded that the pattern of high triacylglycerol concentrations, together with low HDL-cholesterol concentrations, is
involved in the transition from atheroma to atherothrombosis (7).
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

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@uoguelph.ca.
Received July 2, 1999.
Accepted for publication January 31, 2000.

Am J Clin Nutr 2000;72:38994. Printed in USA. 2000 American Society for Clinical Nutrition

389

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STARK ET AL

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 n3 fatty acids have successfully
reduced triacylglycerol concentrations in humans (1214).
Fish oils contain the long-chain polyunsaturated n3 fatty
acids eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n3). Studies to date on n3 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 n3 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 n3 fatty acid supplementation on the
ratio of triacylglycerol to HDL cholesterol.

SUBJECTS AND METHODS

Subjects
Postmenopausal women between the ages of 43 and 60 y were
recruited from the University of Guelph area. Only women who
had had their last menses 1 y before the study began were eligible. Individuals taking lipid-altering, blood pressurealtering,
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 n3 fatty acid supplement were provided as 1-g capsules. The n3 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 (18:2n6). The placebo contained 3.5 mg
-TE RRR--tocopheryl acetate/capsule. Subjects received 8 cap-

sules/d of either placebo oil or n3 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 1214 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  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.

RESULTS
The baseline characteristics of the subjects are shown according to HRT status in Table 1. The only significant difference was

EFFECT OF n3 FATTY ACIDS IN POSTMENOPAUSAL WOMEN

TABLE 1
Baseline characteristics of postmenopausal women receiving or not
receiving hormone replacement therapy (HRT)1
Women not
receiving HRT
(n = 16)

Women
receiving HRT
(n = 19)

Age (y)
52.1 1.0
51.1 0.8
Height (m)
1.63 0.02
1.65 0.01
Weight (kg)
70.7 4.2
74.2 3.3
Systolic BP (mm Hg)
124.7 4.5
129.4 4.5
Diastolic BP (mm Hg)
86.1 4.2
86.4 2.4
Mean arterial pressure (mm Hg)
99.0 4.2
100.8 3.0
Heart rate (beats/min)
69.3 3.0
72.1 2.4
BMI (kg/m2)
26.6 1.6
27.3 1.3
Total cholesterol (mmol/L)
5.22 0.27
5.81 0.16
HDL cholesterol (mmol/L)
1.57 0.09
1.91 0.142
LDL cholesterol (mmol/L)
3.13 0.23
3.17 0.12
Triacylglycerols (mmol/L)
1.14 0.15
1.57 0.17
Total:HDL cholesterol
3.48 0.25
3.29 0.21
LDL:HDL cholesterol
2.12 0.21
1.85 0.16
Triacylglycerols:HDL cholesterol
0.79 0.12
0.95 0.13
1
x SEM. BP, blood pressure. For serum cholesterol, 1 mmol/L =
38.66 mg/dL; for triacylglycerols, 1 mmol/L = 88.57 mg/dL.
2
Significantly different from women not receiving HRT, P < 0.05 (independent t test).

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.
Dietary intakes and body weight are compared between the
supplemented and control groups in Table 2. 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 n3 fatty acid supplements or 6.7 g PUFA/d from the placebo. A treatment  HRT
use  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  HRT use  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).
Serum lipid concentrations for all subjects are shown in Table
3. 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  time interactions (P < 0.0005); there
was a trend (P = 0.061) for treatment  HRT use  time interaction for triacylglycerol:HDL cholesterol.
Treatment with n3 fatty acids was associated with a highly
significant decrease (26%) in triacylglycerol concentrations. The
response of triacylglycerol concentrations to n3 fatty acid supplementation was somewhat variable: 7 subjects had reductions of
515%, 5 subjects had reductions of 2040%, and 4 subjects had
reductions of 4060%. The 2 subjects (of the 18 assigned to n3
fatty acid supplementation) who had no reductions in triacylglycerol concentrations were receiving HRT. Triacylglycerol:HDL cho-

391

lesterol in postmenopausal women decreased significantly by 28%

with n3 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  HRT use  time interactions
were detected because the serum phospholipid fatty acids
responded similarly to n3 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 n3
fatty acid supplementation. The ratio of n3 to n6 fatty acids
was approximately tripled in response to n3 fatty acid supplementation (P < 0.0001). Concentrations of all 3 n6 PUFAs
(18:2n6, 20:3n6, and 20:4n6) decreased significantly
(P < 0.0005) with n3 fatty acid supplementation.
On the basis of the results of the questionnaire, 56% of the subjects who took the n3 fatty acid supplement and 47% of the subjects who took the placebo identified their treatment correctly.
Two subjects who took the n3 fatty acid supplements complained of occasional nausea. No other adverse effects were reported.

DISCUSSION
This study evaluated the effects of n3 fatty acid supplementation on serum lipids in postmenopausal women who were
either receiving or not receiving HRT. The n3 fatty acid supplement (fish-oilderived EPA and DHA concentrate) significantly reduced serum triacylglycerol concentrations, similarly in
both HRT-status groups, without affecting other lipid variables
that we measured. Triacylglycerol:HDL cholesterol also decreased
in the subjects who took the n3 fatty acid supplement, particularly in the women not receiving HRT.
A review of human and animal studies showed that treatment
with n3 fatty acids decreases triacylglycerol concentrations by
2530% in normolipidemic subjects (14). In the present study,
n3 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, n3 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 n3

392

STARK ET AL

TABLE 2
Daily dietary intakes (including supplements on day 28), body weight, and BMI of postmenopausal women at study entry (day 0) and after n3 fatty acid
supplementation (day 28)1
Control group (n = 17)
Weight (kg)
BMI (kg/m2)
Protein
(% of energy)
(g)
Carbohydrate
(% of energy)
(g)
Fat
(% of energy)
(g)
Saturated fat (g)
Monounsaturated fat (g)
Polyunsaturated fat (g)2
Dietary fiber (g)
Cholesterol (mg)
Alcohol (% of energy)
Total energy (kJ)
1
x SEM.
2

Supplemented group (n = 18)

Day 0
Day 28

Day 0

Day 28

73.5 4.8
27.6 1.7

74.2 4.9
27.9 1.8

71.7 2.4
26.4 1.1

71.4 2.3
26.3 1.0

16.6 1.6
75.9 8.8

16.7 1.4
77.4 9.4

17.2 1.0
79.5 4.5

15.5 1.0
69.2 6.3

51.7 2.1
236 14

48.5 1.6
224 19

50.3 1.6
240 15

48.1 1.8
211 14

31.2 1.9
63.7 5.5
21.7 1.8
18.4 2.1
9.9 1.1
18.4 2.0
198 19
0.76 0.35
7578 361

33.7 1.8
66.5 4.8
20.7 1.9
19.0 1.6
15.8 1.0
19.0 2.4
201 30
1.70 0.61
7478 508

30.2 1.4
64.3 4.9
22.0 2.2
20.3 2.1
11.8 1.0
20.3 1.1
241 35
2.33 0.90
7837 439

32.6 1.4
63.5 4.4
19.5 1.4
19.8 2.0
16.4 1.0
19.8 1.5
235 26
3.57 1.29
7197 405

Significant time effect, P < 0.0005 (ANOVA).

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 n3
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-oilbased nutraceutical
therapy with respect to CVD-related morbidity and mortality.
The trend (P = 0.061) toward a treatment  HRT use  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

TABLE 3
Effects of n3 fatty acid supplementation on blood pressure and serum lipid concentrations in postmenopausal women1
Day 0
Total cholesterol (mmol/L)
HDL cholesterol (mmol/L)2
LDL cholesterol (mmol/L)3
Triacylglycerols (mmol/L)3,4
Total:HDL cholesterol
LDL:HDL cholesterol
Triacylglycerol:HDL cholesterol24
Systolic BP (mm Hg)
Diastolic BP (mm Hg)
Mean arterial pressure (mm Hg)
Heart rate (beats/min)
1
x SEM. BP, blood pressure.
2

Control group (n = 17)

Day 28

5.57 0.25
1.86 0.16
3.10 0.20
1.33 0.18
3.26 0.26
1.88 0.21
0.83 0.14
123.6 4.0
82.8 3.3
96.4 3.4
73.3 2.3

6.02 0.19
1.88 0.16
3.48 0.12
1.42 0.18
3.45 0.20
2.05 0.17
0.85 0.12
117.4 5.8
81.3 3.7
93.3 4.3
70.0 2.1

Supplemented group (n = 18)

Day 0
Day 28
5.51 0.20
1.66 0.10
3.20 0.16
1.41 0.16
3.48 0.20
2.05 0.15
0.93 0.12
130.4 4.7
89.1 3.0
102.9 3.5
68.8 2.9

Significant HRT-use effect, P < 0.05 (ANOVA).

Significant time effect, P < 0.05 (ANOVA).
4
Significant treatment  time interaction, P < 0.0005 (ANOVA).
5,7
Significantly different from control group on day 28 (t test of difference scores): 5 P < 0.0001, 7 P < 0.01.
6,8
Significantly different from baseline of supplemented group (paired t test): 6 P < 0.0005, 8 P < 0.05.
3

5.54 0.25
1.69 0.10
3.37 0.20
1.05 0.125,6
3.37 0.15
2.06 0.13
0.67 0.097,8
127.9 4.8
86.4 3.2
100.3 3.7
66.0 2.2

EFFECT OF n3 FATTY ACIDS IN POSTMENOPAUSAL WOMEN

393

TABLE 4
Effects of n3 fatty acid supplementation on fatty acid composition of serum phospholipids in postmenopausal women1
Control group (n = 17)
Fatty acid

Day 0

Day 28

Day 0

Supplemented group (n = 18)

Day 28

% by wt of total fatty acids

16:0
26.6 0.7
26.9 0.6
28.0 0.6
28.5 0.7
18:0
13.3 0.3
13.4 0.3
12.6 0.3
13.8 0.32,3
18:1
12.2 0.4
11.1 0.34
12.2 0.2
10.3 0.22,3
18:2n6
17.7 0.4
17.6 0.5
17.4 0.5
12.8 0.42,3
20:3n6
3.1 0.2
3.9 0.24
2.7 0.1
1.4 0.12,3
20:4n6 (AA)
10.1 0.3
11.0 0.44
9.9 0.3
8.4 0.32,3
20:5n3 (EPA)
1.06 0.07
0.76 0.08
1.18 0.05
6.44 0.102,3
22:5n3
0.85 0.10
0.78 0.09
0.84 0.06
1.56 0.172,3
22:6n3 (DHA)
3.5 0.3
3.3 0.3
3.9 0.2
6.4 0.22,3
n6
33.0 0.4
34.5 0.64
32.0 0.5
23.9 0.42,3
n3
5.6 0.5
5.0 0.4
6.1 0.3
14.5 0.52,3
n3:n6
0.17 0.02
0.14 0.01
0.19 0.01
0.61 0.032,3
4
EPA:AA
0.10 0.02
0.07 0.01
0.12 0.01
0.78 0.032,3
1
x SEM. AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. There was a significant treatment  time effect for all the
fatty acids listed, P < 0.0005 (ANOVA).
2
Significantly different from supplemented group on day 0, P < 0.05 (paired t test).
3
Significantly different from control group on day 28, P < 0.05 (t test of difference scores).
4
Significantly different from control group on day 0, P < 0.05 (paired t test).

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 (1214), 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, n3 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 nellInfarto 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
(0.850.88 g/d) in postmyocardial 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 (0.942.3 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 n3 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
n3 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-oilderived 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, n3 fatty acid supplementation was effective in reducing triacylglycerol:HDL cholesterol by 28%. Further
studies are needed to elucidate the interactions of n3 fatty acid
supplementation with specific HRT regimens and to determine
the long-term effects of n3 fatty acid supplementation on cardiovascular events and mortality in postmenopausal women.

394

STARK ET AL

We thank Margaret Berry and Heather Roelfsma for their technical assistance in this investigation and our subjects for their commitment to this study.

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