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INTRODUCTION
Elevated serum lipid concentrations have been identified
clearly as major risk factors for coronary heart disease (CHD)
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
390
STARK ET AL
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
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).
391
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
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
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
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
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
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
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
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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