- Email: [email protected]
The comparative reductions of the plasma lipids and lipoproteins by dietary polyunsaturated fats: Salmon oil versus vegetable oils
The Comparative Reductions of the Plasma Lipids and Lipoproteins by Dietary Polyunsaturated Fats: Salmon Oil Versus Vegetable Oils William
S. Harris, William
The lower plasma lipid levels and lower incidence unusual fatty acids present (C20:5)
acid composition. mg/dl
acids and are of the omega-3
All diets contained
in Greenland
In 4 wk the salmon oil diet reduced respectively.
lipid class after
(p < 0.005).
HDL cholesterol
fatty acid family. We have compared
the salmon
plasma cholesterol
HE RELATIVE rarity of coronary heart disease and thrombosis in an Eskimo popuiation has raised the possibility that their unusual diet might be Most Eskimos and Northwest Coast protective.‘A Indians traditionally have derived the bulk of their food from the sea. Eskimos consumed seal, whale and fish, whereas the Northwest Coast Indians ate fish, especially salmon. Such diets were high in fat, protein and cholesterol.’ The fat of cold water marine animals, unlike land animals, is unique in that it contains large quantities of o(omega)-3 fatty acids (Fig. 1). The principal w-3 fatty acids in fish oil are eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6). These acids are uncommon in Western diets which typically contain mostly saturated and monounsaturated fatty acids. The predominant polyunsaturated fatty acid of the Western diet is linoleic acid (Cl8:2w6). More than 20 yr ago, polyunsaturated fat was found to have a hypocholesterolemic effect when substituted for saturated fat in the diet.’ ’ Since polyunsaturated fish oils seemed no more effective than vegetable oils in lowering plasma cholesterol levels,8 further explorations of the hypolipidemic effects of fish oils were not carried out. The recent discovery that the prostaglandins derived from eicosapentaenoic acid have biological effects different than those derived from arachidonic acid (C20:4w6)” has generated further interest in tish oils. The goal of the present metabolic study was to determine the effects of a diet rich in w-3 fatty acids from salmon oil upon the plasma lipids and lipoproteins in normal subjects under controlled conditions. This diet was compared to two control diets, one containing levels of saturated fat typical of the American diet and the other rich in the w-6 polyunsaturated fatty acid, linoleic acid. We found that, although both of the polyunsaturated fat diets reduced the plasma
Merabohsm,Vol.
32, No. 2 (Februarvl.
1983
fatty
(p < 0.001)
and
oil diet caused similar decreases
fatty acids comprised
diet. Fish oils contain
oil diet high in
they differed only in fatty
levels changed from 128 to 108 and 13 to 8
levels did not change. The vegetable
levels. The omega-3
that the
a salmon oil diet
fat and to a vegetable
levels from 188 to 162 mg/dl
LDL and VLDL cholesterol
useful in the control of both hypercholesterolemia
T
Eskimos suggested
These fatty acids are eicosapentaenoic
40% of the total calories as fat and 500 mg of cholesterol;
levels but did not lower triglyceride
in each plasma potentially
(C22:6)
levels from 77 to 48 mg/dl
(p < 0.005).
cholesterol
diseases
high levels of these unique fatty acids to a control diet high in saturated
linoleic acid ((X8:2). triglyceride
of atherosclerotic
P. McMurry
in their diet of seal and fish may be anti-atherogenic.
and docosahexaenoic
containing
E. Connor, and Martha
acids which
in
up to 30% of the total fatty acids may be metabolically
unique
and
and hypertriglyceridemia.
cholesterol levels, only the salmon oil diet caused a significant decrease in triglyceride levels. Neither decreased HDL concentrations. MATERIALS
AND
METHODS
Subjects Twelve healthy adults (6 men and 6 women) participated in the study. They ranged in age from 21 to 56 yr (mean 40 -r 13.1) and weighed from 53 to 97 kg (mean 72 f 13). The mean percent of ideal body weight was I 14 i- 15%.Plasma cholesterol levels at entry to the study ranged from 132 to 259 mg/dl (mean 192 + 41) and triglyceride levels from 41 to 162 mg/dl (mean 85 * 36). All subjects were either students or staff at the Oregon Health Sciences University. Physical activity levels remained constant throughout the study. Informed consent was given by each subject prior to entry into the study in accordance with the requirements of our Committee on Human Research.
Study
Design
The study was carried out in the Clinical Research Center (CRC) and all meals were prepared by the CRC metabolic kitchen. Subjects were free to carry on their normal daily activities during the study although all meals were eaten at the CRC (except for an occasional weekend meal which was taken home). All I2 subjects consumed the salmon oil and control diets; 7 subjects took all three diets. This
From the Division of Endocrinology~Metabolism/Clinical Nutrition, Department of ‘Medicine, Oregon Health Sciences C~nivrrsitv. Portland. Oregon. Receivedfor publication July 20. 1982 Supported in part b_v a research grant lHL-75687) from the National Heart, Lung, and Blood Institute and by the Clinical Research Center grant IRR334j from the Division of Research Resources of the National Institutes of Health. William S. Harris. Ph.D.. is a research fellow and trainee, National Heart, Lun,g and BIood Institute (Training grant HL07295) Address reprint requests should be sent to William E. Connor, Ph.D. L465-Lipid Research Lab, Oregon Health Sciences University Portland, Oregon 97201. 8 1983 by Grune & Stratton. Inc. 00260495/83/3202~0016%01.00/0
179
180
HARRIS,
FATTY ACID NOMENCLATURE FAMILY FATTY ACID w3
Eicosopentaenoic Acid (C20:5 ~3)
w6
Linoleic ACid (Cl8:2w6)
w9
Oleic Acid (Cl8:lw9)
DIETARY SOURCES
mRCOOH 3
H3Cw
6
Marine Oils,
RkOOH R'iOOH
n/vvvv H3C
9’
Vegetable
Three diets which differed only in fatty acid composition were fed in random order for 4 wk each: a “saturated” control diet, a salmon oil diet containing considerable amounts of w-3 fatty acids, and a vegetable oil diet high in w-6 fatty acids. Each diet provided 40% of the calories as fat, 15% as protein and 45% as carbohydrate. The fatty acid composition of the three dietary fats is given in Table I. Each diet provided about 500 mg/day of cholesterol. The control diet was designed to simulate the composition of a typical American diet’s and was made up of whole foods (fruits. vegetables and grains), plus a liquid formula which contained most of the fat and protein. Cholesterol was supplied as egg yolk, and fat as cocoa butter and peanut oil. The vegetable oil diet was identical to the control diet except that a mixture of safflower and corn oil (stripped to reduce the quantity of plant sterols from 1,000 to 300 mg/dl) provided the dietary fat. The use of a formula was necessary in order to balance the fat. cholesterol and protein content of the salmon oil diet. The latter contained the same carbohydrate sources as the control diets, but the formula component was replaced by salmon fillets and salmon oil. Salmon was chosen as the source of w-3 fatty acids because of its ready accessibility in the Pacific Northwest, its exceptionally good taste, and its high fat content (ca. 15%).It also contains relatively high proportions of w-3 fatty acids. A 3-wk “washout period” was inserted after the salmon diet for most subjects during which they consumed their usual home diets. This period was inserted to allow the w-3 fatty acid levels in the plasma to return to normal. This occurred in IO-14 days. Vitamin and mineral supplements for all three diets were provided to meet the Recommended Dietary Allowances of the Food and Nutrition Board of the National Research Council.”
Laboratory Methods weekly fasting blood samples were collected into standard disodium EDTA (I gm/dl) tubes. The fresh plasma was analyzed for cholesterol and triglycerides with an Auto Analyzer II (Technican Instruments, Tarrytown, N.Y.).” The last three values for each 4-wk dietary period were averaged for the purposes of statistical comparisons. Lipid levels had stabilized after 2 wk. The plasma cholesterol levels of high density. low density, and very low density lipoproteins (HDL, LDL and VLDL, respectively) were measured before and after precipitation with heparin and manganese chloride.12 Plasma and dietary fatty acids were measured by a combination of thin layer and gas chromatography according to previously described methods.13 Twice
Fish
Oils
Vegetable Oils; Animal Fats
occurred because the vegetable oil phase was added to the study after we began to see the positive effects of the salmon oil diet. Thus. the first 5 subjects did not eat the vegetable oil diet. Compliance was estimated by the stability of body weights, daily personal contact with the subjects and the presence of w-3 fatty acids in the plasma lipids.
AND
McMURRY
Fig. 1. Fatty acid families and dietary sources. Fatty acid families are distinguished by the position of the first double bond counting from the terminal (~1 methyl group of the molecule (w is the last letter of the Greek alphabet). Members of any particular w family may be metabolically interconverted but no conversions from one w family to another occur in mammals. The fatty acids listed are common members of their respective w families. An alternative system of nomenclature in common use replaces the w with an n (e.g., 18:2w6 = 18:2(n-6)).
STRUCTURE
H,C
CONNOR,
Statistical Analysis Since each subject served as his own control, statistical analysis was made using paired methodologies. When the effects of only two diets were compared, the Student’s paired t-test was used. When comparisons were made among all three diets, a one-way analysis of variance with repeated measures was used followed by the NewmanKeuls test.14
RESULTS
All subjects found little difficulty in eating their daily meals, some of which contained up to 1 lb of salmon steak. The salmon oil (3-6 tablespoonsful daily) was well tolerated by all subjects. No gastrointestinal discomfort was reported and bowel habits remained regular. There were no signficant weight changes throughout the study.
The Composition of the Dietary Fatty Acids The control diet had a fatty acid pattern similar to the typical Western diet, whereas the salmon oil diet was greatly enriched with the two major w-3 fatty acids (Table 1). The w-3 family provided about 20% of Table 1. The Major Fatty Acids of the Three Experimental Diets Percent Vegetable Fatty Acid
Control’
16:0
19
16:lw7
Salmon
Oil*
011
12
15 6
1
0
18:0
17
3
18:lw9
40
26
18:206
18
54
3
0
0
10
20: lw9
6 23
20:5w3
0
0
9
22:lwll
0
0
9
22:5w3 Total
0
0
11
saturated
+
22:6w3
36
16
25 48
Total
monounsaturated
41
27
Total
w-6
18
54
Total
w-3
Iodine P/S
#t
*Dietary 011, safflower tlodine
0.5 fat sources:
control;
cocoa
20
121
69
ratio
3
butter
168
3.4 and peanut
1.3 011; vegetable
and corn oil. numbers
calculated
from
the fatty
acid compositions.
HYPOLIPIDEMIC EFFECTS OF FISH OIL
181
the total fatty acids in the salmon diet, which in an intake of 20-29 g of o-3 fatty acids per control and vegetable oil diets had virtually acids of this class. The content of linoleic lower in the salmon oil diet than in the control still supplied an adequate quantity of this fatty acid.
resulted day. The no fatty acid was diet but essential
The Fatty Acid Composition of the Plasma Lipids
Plasma Lipid and Lipoprotein Levels
The plasma levels of cholesterol fell from 188 mg/dl during the control diet to 162 mg/dl during the salmon oil (p < 0.001). In similar fashion VLDL cholesterol levels fell from I3 to 8 mg/dl (p < 0.001) while LDL decreased from 128 to 108 mg/dl (p < 0.005). HDL cholesterol levels were not affected by the salmon oil diet. Plasma triglyceride levels fell from 77 to 48 mg/dl (p < 0.005). In order to study the potential differences between o-3 and o-6 polyunsaturated fatty acids, the effects of the salmon and vegetable oil diets were compared to the control diet in 7 subjects (Tables 2 and 3). Cholesterol levels were reduced to a similar extent (- 11%) by both polyunsaturated fat diets. However, the salmon oil had an additional hypotriglyceridemic effect. In these 7 subjects the plasma triglyceride levels were reduced by 33% (p < 0.01) whereas the vegetable oil diet did not decrease triglyceride values. These findings were also reflected in the changes in VLDL cholesterol levels which were unchanged by the vegetaTable 2.
The Plasma Cholesterol
and Triglyceride
ble oil diet but were reduced by 50% on the salmon oil diet (p < 0.01). LDL cholesterol levels fell by about 11% or both of the polyunsaturated fat diets, whereas HDL cholesterol remained unchanged from either test diet.
Concentrations*
Total w-3 fatty acids (C20:5, C22:5, C22:6) increased greatly in the plasma lipids after the ingestion of salmon oil. Cholesteryl esters, triglycerides and phospholipids all contained between 25% and 30% w-3 fatty acids. Changes were detectable within 1 day and peak concentrations were reached by 10 days. The salmon oil diet also caused a rapid fall in linoleic acid levels to about 50% of control values in most lipid fractions. In data not portrayed in Fig. 2, the arachidonic acid (C20:4w6) content was reduced by 23% in the phospholipid fraction. The total amounts of saturated and monounsaturated fatty acids in the plasma lipids were only mildly affected by the w-3 diet. DISCUSSION
The most striking finding of this study was the ability of salmon oil to lower plasma triglyceride and VLDL levels as well as plasma cholesterol and LDL levels in normolipidemic subjects. Although the hypocholesterolemic effect of marine oils was a consistent finding in the previous studies of the 195Os, its hypotriglyceridemic action was not appreciated.‘-* In more of Subjects Fed Control, Salmon Oil and Vegetable
Cholesterol(mg/dll Subpcts
Salmon Oil
C0ntKll
Oil Diets
Trlglycende(mg/dl) Vegetable Oil
Salmon 011
COlWOl
Vegetable 011
219
179
216
96
57
91
2
194
146
157
93
36
78
3
122
108
110
49
35
52
4
211
191
185
104
70
98
5
204
198
193
57
43
58
6
156
147
138
57
37
53
7
231
222
220
79
75
98
191 k 38
170 + 39
174 + 41
Sub-mean + SD.
I
1
76 i 22
50+
p < 0.01
p <
L
17
0.01
p
I p < 0.05
8
164
141
53
9
194
166
158
64
10
164
127
36
36 46
11
IL Overall Mean -+ S.D (Twelve subjects)
152
136
75
248
186
64
188 + 37
162 + 34
I
1 p < 0.001
*Mean of last 3 determinations of each dietary period
75 :’ 21
-1
39
35
77 k 33
1
48-r
15
I p < 0.005
c 0.01
182
HARRIS, CONNOR, AND McMURRY
Table 3. The Cholesterol
Concentrations
(mg/dl)*
of Different
Plasma Lipoprotein
Vegetable
Fractions of Subjects Fed Control, Salmon Oil and
Oil Diets LDL
VLDLt SAllOfl
Vegetable
Salmon
HDL Vegetable
Salmon
011
011
Control
011
Oil
COlWOl
10
20
151
143
149
Vegetable
011
011
59
48
48
1
23
2
13
3
14
119
80
104
60
61
61
3
7
4
7
69
61
64
41
36
43
4
9
6
12
163
139
140
39
52
47
5
10
6
6
130
132
131
64
63
64
6
9
6
7
76
60
67
55
69
56
184
165
150
56
46
57
127 i- 43
111 i 43
115 + 37
53 + 9.6
54 k 11.4
54 k 7.8
39
7
- 11 12 + 5.3
Sub-mean ? S.D.
7 6 I 2.2
___16 12 + 5.3
I
--
1 p < 0.05
p < 0.01
p < 0.01
p < 0.05
Overall
8
11
8
123
94
31
9
32
18
134
132
37
32
10
7
6
109
89
47
35
11
14
9
90
65
57
57
12
12
7
188
131
59
52
128 f 39
108 ? 37
50 t 10.9
49 t 12.0
mean
13 27.3
* so.
8 f 3.8
(Twelve subjects) I
1 p
<
0.001
p < 0.005
of last 3 determtinatlons of each dietary period.
‘Mean
tVLDL: very low density lipoprotein, LDL: low density lipoprotein, HDL: high density lipoprotein.
recent studies fish oil feeding has invariably led to lower triglyceride levels.‘s,‘6 No other reported study to date, however, has compared fish oil to both polyunsaturated vegetable oils and saturated fats in the same individuals using diets with controlled fatty acid compositions. Our finding that, of the three fats tested, only salmon oil was hypotriglyceridemic, indicates that its polyunsaturated w-3 fatty acids may have different metabolic effects than w-6 fatty acids as will be discussed later. 40 35 B 2 T * B k . *
30 25 20
;
I5
8
IO 5
TRIGLYCERIDES
Fig. 2. This family
The w-3 fatty acid content
is expressed (C20:5
table
oil diet
open
columns;
+
as the C22:5
were
the
salmon
percent
+
C22:6).
same oil diet
PHOSPHOLIPIDS
cR~::~R~yL
of the plasma
of total The
as the
w-3
fatty
levels
control
= striped
lipid fractions.
acids diet.
columns
from
during
Control
(mean
the
w-3
the vegediet
and S.D.).
=
Although this study was primarily designed to compare the hypolipidemic effects of two polyunsaturated oils: salmon oil vs. vegetable oil, we were also able to make comparisons between the two major classes of polyunsaturated fatty acids: w-6 vs. w-3. This comparison was possible because the salmon oil diet contained some 8% of calories as w-3 fatty acids and the control diet contained 7.2% of calories as w-6 fatty acids. Since these two diets did not differ greatly in amounts of saturated and monounsaturated fatty acids, their effects upon the plasma lipids may be primarily ascribed to their respective contents of the w-6 or o-3 polyunsaturates. Viewed from this perspective, the w-3 fatty acids were not only more hypotriglyceridemic, but were also more hypocholesterolemic than the w-6 fatty acids, on a gram-for-gram basis. Thus, it is not unreasonable to conclude that the w-3 fatty acids were more hypolipidemic than the major w-6 fatty acid, linoleic. The reasons for the greater potency of the w-3 fatty acids are not completely clear. Certainly, their amount of unsaturation is much greater. The primary o-6 fatty acid, linoleic acid, contains two double bonds per molecule. The principal w-3 fatty acids in salmon oil (20:5 and 22:6) have an average of 5.5 double bonds per molecule. Thus, the o-3 fatty acids provided about 2.75 times as much “unsaturation” as the w-6 fatty acids, gram-for-gram. If the hypolipidemic effect resulted from the total amount of unsaturation in the
HYPOLIPIDEMIC EFFECTS OF FISH OIL
dietary fat, then an equivalent amount of “unsaturation” provided as w-6 fatty acids should lower lipids to the same extent as the fish oil diet did. This hypothesis was tested in our study. The vegetable oil diet provided a ratio of 2.7 to 1.0 of w-6 fatty acids to the w-3 fatty acids in salmon oil. Both oils had the same hypocholesterolemic effect. Thus, the cholesterol-lowering effect of these oils seemed to be a function of their total unsaturation. an observation which has been made frequently over the years.” The hypotriglyceridemic effect, however, did not appear to be linked to the degree of unsaturation per se and perhaps was related to the w-3 structure. Other dietary variables which may have been responsible for the hypolipidemic effects of the salmon oil diet should also be considered. Besides the major differences in w-3 fatty acid content, fish oils also contain relatively high levels of the C20 and C22 monounsaturated fatty acids (Table 1). Very low levels of these fatty acids were found in the plasmas of our subjects, corroborating the work of other researchers.lL.IX It is not known whether these fatty acids are simply poorly absorbed or are rapidly metabolized. Since the Cl 6 and Cl 8 monounsaturates have been found to have neutral effects upon the plasma lipid levels,‘” WC have no reason to believe that these longer chain monounsaturates should be hypolipidemic. A second consideration is the differences in the dietary protein sources. The principal protein source during the salmon oil phase was salmon flesh; during the other two dietary phases it was casein. There is data in rabbits which indicate that fish protein is no different than casein in terms of its effect upon the plasma cholesterol levels.2” Hence, the difference in dietary protein were probably not responsible for the lipid changes. The effects of polyunsaturated vegetable oils upon plasma triglyceride levels have been studied by several investigators who have reached divergent conclusions. Some have noted that vegetable oils decreased triglyceride levels” ” while others have found no effect.25m’R Among those reporting a hypotriglyceridemic effect of vegetable oils was one study in which normal and hypertriglyceridemic subjects were examined for only a IO-day dietary period.14 In the study employing the longest dietary periods (7-9 wk per period) the vegetable oil had no effect upon plasma triglyceride levels.‘6 Thus it seems likely that 10 days may have been too short to allow the plasma lipids to stabilize. In two other studies2’.‘3 plasma triglyceride levels were depressed by only 14%-l 5% during vegetable oil feeding. In our study, the feeding of polyunsaturated vegetable oils had no effect upon the plasma triglyceride levels. Polyunsaturated vegetable oils have also been found
183
to lower HDL cholesterol levels in some studies.‘3.‘6.2’ However, the results of this study support the findings by others that vegetable oil feeding does not affect plasma HDL levels.24.27 On the other hand, fish oil feeding has been found to have a slightly elevating effect’5,2’ or no effect upon HDL cholesterol levels.‘6 Our findings, conducted under controlled dietary conditions, confirm the latter finding. The total incorporation of w-3 fatty acids into the plasma lipid classes in this study was higher than has been reported previously.R.‘5.‘b.” This undoubtedly occurred because of the higher intake w-3 fatty acids in this study (21-29 g) compared to earlier studies (4-8 g). The finding of higher levels of w-3 fatty acids in the plasma lipids than in the diet implies that there may be preferential incorporation of the w-3 fatty acids over the other dietary fatty acids. The ultimate metabolic consequences of such high levels of w-3 fatty acids in the plasma lipids are not known. The exact mechanism(s) by which w-3 fatty acids exert their influence on plasma lipids cannot be determined from this study. The similar reduction of plasma triglyceride and VLDL cholesterol levels (- 38%) after the salmon oil diet suggested that the fall in triglyceride levels occurred from a reduction of the total amount of VLDL in the blood rather than from a change in the VLDL lipid composition. Thus. the hypotriglyceridemic effect probably resulted from either a more rapid hydrolysis and removal of VLDL particles enriched with w-3 fatty acids or an inhibition of VLDL synthesis or secretion. Other potential mechanisms which should also be considered include: ( I) structural alterations in lipoproteins leading to changes in their interactions with lipolytic enzymes (2) changes in the composition of plasma membrane lipids altering the affinity of membrane-bound enzymes for lipoproteins,” and (3) an inhibitory influence of w-3 fatty acids on the hepatic biosynthesis of either fatty acids,j’ triglyceride or apoprotein B3’ or the assembly or secretion of VLDL. A reduction in free fatty acids released from adipose tissue could lead to lower hepatic triglyceride synthesis.” Prostaglandins derived from eicosapentaenoic or arachidonic acids might inhibit the release of free fatty acids from adipose tissue, although the role of prostaglandins in lipolysis is still unclear.34 The effects of the salmon oil diet upon platelet lipid composition and function have recently been reported from our laboratory.3” Platelets became enriched in o-3 fatty acids with a resultant prolongation of bleeding time and reduced adhesiveness. Because of their dual antithrombotic and hypolipidemic effects, marine oils rich in o-3 fatty acids may have important nutritional applications in the prevention and treatment of atherosclerotic disease.
HARRIS, CONNOR. AND McMURRY
184
ciated. We wish to thank the dietetic and nursing staff of the Clinical
ACKNOWLEDGMENT The authors are grateful to Oregon Aqua Foods, Inc., of Astoria, Oregon, for providing the salmon oil and fillets for this study. The valuable discussions with Dr. Roger Illingworth are also appre-
Research Center for their skillful care of the subjects. Excellent clerical assistance was provided by Joanne Skirving, Patricia Schade, and Paula Bisaccio. Statistical analysis was conducted with the help of Dr. Gary Sexton.
REFERENCES 1. Bang HO, Dyerberg J, Hydrne N: The composition of food consumed by Greenlandic Eskimos. Acta Med Stand 220:69-73, 1976 2. Bang HO, Dyerberg J: Plasma lipids and lipoproteins in Greenlandic West Coast Eskimos. Acta Med Stand 192:85-94. 1972 3. Dyerberg, J, Bang HO, Stofferson E, et al: Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis. Lancet ii:] 17-l 19, 1978 4. Dyerberg J, Bang HO: Hemostatic function and platelet polyunsaturated fatty acids in Eskimos. Lancet ii:433-435, 1979 5. Bronte-Stewart B, Antonis A, Eales L, et al: Effects of feeding different fats on serum cholesterol levels. Lancet i:521-526, 1956 6. Malmros H, Wigand G: The effect on serum cholesterol of diets containing different fats. Lancet ii:]-8, 1957 7. Keys A, Anderson JT, Grande F: “Essential” fatty acids, degree of unsaturation and effects of corn (maize) oil on the serum cholesterol level in man. Lancet i:66-68, 1957 8. Ahrens EH. lnsull W. Hirsch J, et al: The effect on human serum iipidsof a dietary fat, highly unsaturated, but poor in essential fatty acids. Lancet i:ll5-119. 1959 9. Needleman P, Raz A, Minkes MS, Ferrendelli JA, Sprecher H: Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. Proc Nat1 Acad Sci USA 76:9444948, 1979 IO. Ahrens EH, Boucher CA: The composition of a simulated American diet. J Am Diet Ass 73613-620, 1978 Il. Recommended Dietary Allowances, 8th Ed.. Food and Nutrition Board, National Research Council of the National Academy of Sciences, 1974 12. Manual of Laboratory Operations, Lipid Research Clinics Program, Vol. 1, Lipid and Lipoprotein Analysis, DHEW Publication No. (NIH)
75-628,
1974
13. Wene JD, Connor WE, Den Besten L: The development of essential fatty acid deficiency in healthy men fed a fat-free diet intravenously and orally. J Clin Invest 56: 127-l 34, 1975 14. Winer BJ: Siatisticalprinciples of experimental edition, McGraw-Hill, New York, 197 1, pp 261-273.
design, 2nd
15. von Lossonczy TO, Ruiter A, Bronsgeest-Schoute HC, et al: The effect of a fish diet on serum lipids in healthy human subjects. Am J Clin Nutr 31:1340-1346, 1978 16. Bronsgeest-Schoute HC, van Gent CM, Luten JB, et al: The effects of various intakes of w-3 fatty acids on the blood lipid composition in healthy human subjects. Am J Clin Nutr 34:17521757, 1981 17. Goodnight SH, Harris WS, Connor WE, et al: Polyunsaturated fatty acids, hyperlipidemia and thrombosis. Arteriosclerosis 2:87-l 13, 1982 18. Ruiter A, Jongbloed AN, VanGent CM, et al: The influence of dietary mackerel oil on the condition of organs and on blood lipid composition in the young, growing pig. Am J Clin Nutr 31:21592166, 1978 19. Hegsted DM, McGandy RB, Myers ML, et al: Quantitative effects of dietary fat on serum cholesterol in man. Am J Clin Nutri 17:28ll295, 1965
20. Carroll KK: The role of dietary protein in hypercholesterolemia and atherosclerosis. Lipids 13:360-365, 1978 21. Sanders TAB, Vickers M, Haines AP: Effect of blood lipids and haemostasis of a supplement of cod-liver oil, rich in eicosapentaeonic and docosahexaenoic acids in healthy young men. Clin Sci 61:317-324, 1981 22. Nestel PJ, Havenstein N, Scott TW. et al: Polyunsaturated ruminant fats and cholesterol metabolism in man. Aust NZ J Med 4:497-501,1974 23. Shepherd J, Packard CJ, Grundy SM. et al: Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man. J Lipid Res 21:9lI99. 1980 24. Chait A, Onitiri A, Nicoll A, et al: Reduction of serum triglyceride levels by polyunsaturated fat. Studies on the mode of action and on very low density lipoprotein composition. Atherosclerosis 20:347-364, 1974 25. Nestel PJ, Havenstein N, Whyte HM, et al: Lowering of plasma cholesterol and enhanced sterol excretion with the comsumption of polyunsaturated ruminant fats. N Eng J Med 228:379-382, 1973 26. Turner JD, Le N-A, Brown WV: Effect of changing dietary fat saturation on low density lipoprotein metabolism in man. Am J Physiol 241:E57-E63, 1981 27. Nestel PJ, Havenstein N, Homma Y, et al: Increased sterol excretion with polyunsaturated-fat, high-cholesterol diets. Metabolism 24:189-198. 1975 28. Connor WE. Witiak DT, Stone OB, et al: Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of different fatty acid composition. J Clin Invest 48:1363-1375. 1969 29. Shepherd J, Packard CJ, Patsch JR, et al: Effects of dietary polyunsaturated and saturated fat on the properties of high density lipoproteins and metabolism of apolipoprotein A-l. J Clin Invest 61:1582-1592, 1978 30. Gavigan SJP. Knight BL: Catabolism of low density lipoprotein by fibroblasts cultured in medium supplemented with saturated or unsaturated free fatty acids. Biochem Biophys Acta 665:632-635, 1981 31. lritani N, lnoguchi K, Endo M, et al: Identification of shellfish fatty acids and their effects on lipogenic enzymes. Biochem Biophys Acta 618:378-382, 1980 32. Shepherd J, Packard CJ, Taunton D, et al: Effects of dietary fat saturation on the composition of very low density lipoproteins and on the metabolism of their major apoprotein, apolipoprotein B. Biochem Sot Tram 6:779-78 I, 1978 33. Have1 RJ, Kane JP, Balasse ED, et al: Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest 49:2017-2035, 1970 34. Lipinski BA, Mathias MM: Prostaglandin production and lipolysis in isolated rat adipocytes as effected by dietary fat. Prostaglandins 16:957-963, 1978 35. Goodnight SH, Harris WS. Connor WE: The effects of dietary w-3 fatty acids upon platelet composition and function in man: a prospective, controlled study. Blood 58:880-885, 1981
S. Harris, William
The lower plasma lipid levels and lower incidence unusual fatty acids present (C20:5)
acid composition. mg/dl
acids and are of the omega-3
All diets contained
in Greenland
In 4 wk the salmon oil diet reduced respectively.
lipid class after
(p < 0.005).
HDL cholesterol
fatty acid family. We have compared
the salmon
plasma cholesterol
HE RELATIVE rarity of coronary heart disease and thrombosis in an Eskimo popuiation has raised the possibility that their unusual diet might be Most Eskimos and Northwest Coast protective.‘A Indians traditionally have derived the bulk of their food from the sea. Eskimos consumed seal, whale and fish, whereas the Northwest Coast Indians ate fish, especially salmon. Such diets were high in fat, protein and cholesterol.’ The fat of cold water marine animals, unlike land animals, is unique in that it contains large quantities of o(omega)-3 fatty acids (Fig. 1). The principal w-3 fatty acids in fish oil are eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6). These acids are uncommon in Western diets which typically contain mostly saturated and monounsaturated fatty acids. The predominant polyunsaturated fatty acid of the Western diet is linoleic acid (Cl8:2w6). More than 20 yr ago, polyunsaturated fat was found to have a hypocholesterolemic effect when substituted for saturated fat in the diet.’ ’ Since polyunsaturated fish oils seemed no more effective than vegetable oils in lowering plasma cholesterol levels,8 further explorations of the hypolipidemic effects of fish oils were not carried out. The recent discovery that the prostaglandins derived from eicosapentaenoic acid have biological effects different than those derived from arachidonic acid (C20:4w6)” has generated further interest in tish oils. The goal of the present metabolic study was to determine the effects of a diet rich in w-3 fatty acids from salmon oil upon the plasma lipids and lipoproteins in normal subjects under controlled conditions. This diet was compared to two control diets, one containing levels of saturated fat typical of the American diet and the other rich in the w-6 polyunsaturated fatty acid, linoleic acid. We found that, although both of the polyunsaturated fat diets reduced the plasma
Merabohsm,Vol.
32, No. 2 (Februarvl.
1983
fatty
(p < 0.001)
and
oil diet caused similar decreases
fatty acids comprised
diet. Fish oils contain
oil diet high in
they differed only in fatty
levels changed from 128 to 108 and 13 to 8
levels did not change. The vegetable
levels. The omega-3
that the
a salmon oil diet
fat and to a vegetable
levels from 188 to 162 mg/dl
LDL and VLDL cholesterol
useful in the control of both hypercholesterolemia
T
Eskimos suggested
These fatty acids are eicosapentaenoic
40% of the total calories as fat and 500 mg of cholesterol;
levels but did not lower triglyceride
in each plasma potentially
(C22:6)
levels from 77 to 48 mg/dl
(p < 0.005).
cholesterol
diseases
high levels of these unique fatty acids to a control diet high in saturated
linoleic acid ((X8:2). triglyceride
of atherosclerotic
P. McMurry
in their diet of seal and fish may be anti-atherogenic.
and docosahexaenoic
containing
E. Connor, and Martha
acids which
in
up to 30% of the total fatty acids may be metabolically
unique
and
and hypertriglyceridemia.
cholesterol levels, only the salmon oil diet caused a significant decrease in triglyceride levels. Neither decreased HDL concentrations. MATERIALS
AND
METHODS
Subjects Twelve healthy adults (6 men and 6 women) participated in the study. They ranged in age from 21 to 56 yr (mean 40 -r 13.1) and weighed from 53 to 97 kg (mean 72 f 13). The mean percent of ideal body weight was I 14 i- 15%.Plasma cholesterol levels at entry to the study ranged from 132 to 259 mg/dl (mean 192 + 41) and triglyceride levels from 41 to 162 mg/dl (mean 85 * 36). All subjects were either students or staff at the Oregon Health Sciences University. Physical activity levels remained constant throughout the study. Informed consent was given by each subject prior to entry into the study in accordance with the requirements of our Committee on Human Research.
Study
Design
The study was carried out in the Clinical Research Center (CRC) and all meals were prepared by the CRC metabolic kitchen. Subjects were free to carry on their normal daily activities during the study although all meals were eaten at the CRC (except for an occasional weekend meal which was taken home). All I2 subjects consumed the salmon oil and control diets; 7 subjects took all three diets. This
From the Division of Endocrinology~Metabolism/Clinical Nutrition, Department of ‘Medicine, Oregon Health Sciences C~nivrrsitv. Portland. Oregon. Receivedfor publication July 20. 1982 Supported in part b_v a research grant lHL-75687) from the National Heart, Lung, and Blood Institute and by the Clinical Research Center grant IRR334j from the Division of Research Resources of the National Institutes of Health. William S. Harris. Ph.D.. is a research fellow and trainee, National Heart, Lun,g and BIood Institute (Training grant HL07295) Address reprint requests should be sent to William E. Connor, Ph.D. L465-Lipid Research Lab, Oregon Health Sciences University Portland, Oregon 97201. 8 1983 by Grune & Stratton. Inc. 00260495/83/3202~0016%01.00/0
179
180
HARRIS,
FATTY ACID NOMENCLATURE FAMILY FATTY ACID w3
Eicosopentaenoic Acid (C20:5 ~3)
w6
Linoleic ACid (Cl8:2w6)
w9
Oleic Acid (Cl8:lw9)
DIETARY SOURCES
mRCOOH 3
H3Cw
6
Marine Oils,
RkOOH R'iOOH
n/vvvv H3C
9’
Vegetable
Three diets which differed only in fatty acid composition were fed in random order for 4 wk each: a “saturated” control diet, a salmon oil diet containing considerable amounts of w-3 fatty acids, and a vegetable oil diet high in w-6 fatty acids. Each diet provided 40% of the calories as fat, 15% as protein and 45% as carbohydrate. The fatty acid composition of the three dietary fats is given in Table I. Each diet provided about 500 mg/day of cholesterol. The control diet was designed to simulate the composition of a typical American diet’s and was made up of whole foods (fruits. vegetables and grains), plus a liquid formula which contained most of the fat and protein. Cholesterol was supplied as egg yolk, and fat as cocoa butter and peanut oil. The vegetable oil diet was identical to the control diet except that a mixture of safflower and corn oil (stripped to reduce the quantity of plant sterols from 1,000 to 300 mg/dl) provided the dietary fat. The use of a formula was necessary in order to balance the fat. cholesterol and protein content of the salmon oil diet. The latter contained the same carbohydrate sources as the control diets, but the formula component was replaced by salmon fillets and salmon oil. Salmon was chosen as the source of w-3 fatty acids because of its ready accessibility in the Pacific Northwest, its exceptionally good taste, and its high fat content (ca. 15%).It also contains relatively high proportions of w-3 fatty acids. A 3-wk “washout period” was inserted after the salmon diet for most subjects during which they consumed their usual home diets. This period was inserted to allow the w-3 fatty acid levels in the plasma to return to normal. This occurred in IO-14 days. Vitamin and mineral supplements for all three diets were provided to meet the Recommended Dietary Allowances of the Food and Nutrition Board of the National Research Council.”
Laboratory Methods weekly fasting blood samples were collected into standard disodium EDTA (I gm/dl) tubes. The fresh plasma was analyzed for cholesterol and triglycerides with an Auto Analyzer II (Technican Instruments, Tarrytown, N.Y.).” The last three values for each 4-wk dietary period were averaged for the purposes of statistical comparisons. Lipid levels had stabilized after 2 wk. The plasma cholesterol levels of high density. low density, and very low density lipoproteins (HDL, LDL and VLDL, respectively) were measured before and after precipitation with heparin and manganese chloride.12 Plasma and dietary fatty acids were measured by a combination of thin layer and gas chromatography according to previously described methods.13 Twice
Fish
Oils
Vegetable Oils; Animal Fats
occurred because the vegetable oil phase was added to the study after we began to see the positive effects of the salmon oil diet. Thus. the first 5 subjects did not eat the vegetable oil diet. Compliance was estimated by the stability of body weights, daily personal contact with the subjects and the presence of w-3 fatty acids in the plasma lipids.
AND
McMURRY
Fig. 1. Fatty acid families and dietary sources. Fatty acid families are distinguished by the position of the first double bond counting from the terminal (~1 methyl group of the molecule (w is the last letter of the Greek alphabet). Members of any particular w family may be metabolically interconverted but no conversions from one w family to another occur in mammals. The fatty acids listed are common members of their respective w families. An alternative system of nomenclature in common use replaces the w with an n (e.g., 18:2w6 = 18:2(n-6)).
STRUCTURE
H,C
CONNOR,
Statistical Analysis Since each subject served as his own control, statistical analysis was made using paired methodologies. When the effects of only two diets were compared, the Student’s paired t-test was used. When comparisons were made among all three diets, a one-way analysis of variance with repeated measures was used followed by the NewmanKeuls test.14
RESULTS
All subjects found little difficulty in eating their daily meals, some of which contained up to 1 lb of salmon steak. The salmon oil (3-6 tablespoonsful daily) was well tolerated by all subjects. No gastrointestinal discomfort was reported and bowel habits remained regular. There were no signficant weight changes throughout the study.
The Composition of the Dietary Fatty Acids The control diet had a fatty acid pattern similar to the typical Western diet, whereas the salmon oil diet was greatly enriched with the two major w-3 fatty acids (Table 1). The w-3 family provided about 20% of Table 1. The Major Fatty Acids of the Three Experimental Diets Percent Vegetable Fatty Acid
Control’
16:0
19
16:lw7
Salmon
Oil*
011
12
15 6
1
0
18:0
17
3
18:lw9
40
26
18:206
18
54
3
0
0
10
20: lw9
6 23
20:5w3
0
0
9
22:lwll
0
0
9
22:5w3 Total
0
0
11
saturated
+
22:6w3
36
16
25 48
Total
monounsaturated
41
27
Total
w-6
18
54
Total
w-3
Iodine P/S
#t
*Dietary 011, safflower tlodine
0.5 fat sources:
control;
cocoa
20
121
69
ratio
3
butter
168
3.4 and peanut
1.3 011; vegetable
and corn oil. numbers
calculated
from
the fatty
acid compositions.
HYPOLIPIDEMIC EFFECTS OF FISH OIL
181
the total fatty acids in the salmon diet, which in an intake of 20-29 g of o-3 fatty acids per control and vegetable oil diets had virtually acids of this class. The content of linoleic lower in the salmon oil diet than in the control still supplied an adequate quantity of this fatty acid.
resulted day. The no fatty acid was diet but essential
The Fatty Acid Composition of the Plasma Lipids
Plasma Lipid and Lipoprotein Levels
The plasma levels of cholesterol fell from 188 mg/dl during the control diet to 162 mg/dl during the salmon oil (p < 0.001). In similar fashion VLDL cholesterol levels fell from I3 to 8 mg/dl (p < 0.001) while LDL decreased from 128 to 108 mg/dl (p < 0.005). HDL cholesterol levels were not affected by the salmon oil diet. Plasma triglyceride levels fell from 77 to 48 mg/dl (p < 0.005). In order to study the potential differences between o-3 and o-6 polyunsaturated fatty acids, the effects of the salmon and vegetable oil diets were compared to the control diet in 7 subjects (Tables 2 and 3). Cholesterol levels were reduced to a similar extent (- 11%) by both polyunsaturated fat diets. However, the salmon oil had an additional hypotriglyceridemic effect. In these 7 subjects the plasma triglyceride levels were reduced by 33% (p < 0.01) whereas the vegetable oil diet did not decrease triglyceride values. These findings were also reflected in the changes in VLDL cholesterol levels which were unchanged by the vegetaTable 2.
The Plasma Cholesterol
and Triglyceride
ble oil diet but were reduced by 50% on the salmon oil diet (p < 0.01). LDL cholesterol levels fell by about 11% or both of the polyunsaturated fat diets, whereas HDL cholesterol remained unchanged from either test diet.
Concentrations*
Total w-3 fatty acids (C20:5, C22:5, C22:6) increased greatly in the plasma lipids after the ingestion of salmon oil. Cholesteryl esters, triglycerides and phospholipids all contained between 25% and 30% w-3 fatty acids. Changes were detectable within 1 day and peak concentrations were reached by 10 days. The salmon oil diet also caused a rapid fall in linoleic acid levels to about 50% of control values in most lipid fractions. In data not portrayed in Fig. 2, the arachidonic acid (C20:4w6) content was reduced by 23% in the phospholipid fraction. The total amounts of saturated and monounsaturated fatty acids in the plasma lipids were only mildly affected by the w-3 diet. DISCUSSION
The most striking finding of this study was the ability of salmon oil to lower plasma triglyceride and VLDL levels as well as plasma cholesterol and LDL levels in normolipidemic subjects. Although the hypocholesterolemic effect of marine oils was a consistent finding in the previous studies of the 195Os, its hypotriglyceridemic action was not appreciated.‘-* In more of Subjects Fed Control, Salmon Oil and Vegetable
Cholesterol(mg/dll Subpcts
Salmon Oil
C0ntKll
Oil Diets
Trlglycende(mg/dl) Vegetable Oil
Salmon 011
COlWOl
Vegetable 011
219
179
216
96
57
91
2
194
146
157
93
36
78
3
122
108
110
49
35
52
4
211
191
185
104
70
98
5
204
198
193
57
43
58
6
156
147
138
57
37
53
7
231
222
220
79
75
98
191 k 38
170 + 39
174 + 41
Sub-mean + SD.
I
1
76 i 22
50+
p < 0.01
p <
L
17
0.01
p
I p < 0.05
8
164
141
53
9
194
166
158
64
10
164
127
36
36 46
11
IL Overall Mean -+ S.D (Twelve subjects)
152
136
75
248
186
64
188 + 37
162 + 34
I
1 p < 0.001
*Mean of last 3 determinations of each dietary period
75 :’ 21
-1
39
35
77 k 33
1
48-r
15
I p < 0.005
c 0.01
182
HARRIS, CONNOR, AND McMURRY
Table 3. The Cholesterol
Concentrations
(mg/dl)*
of Different
Plasma Lipoprotein
Vegetable
Fractions of Subjects Fed Control, Salmon Oil and
Oil Diets LDL
VLDLt SAllOfl
Vegetable
Salmon
HDL Vegetable
Salmon
011
011
Control
011
Oil
COlWOl
10
20
151
143
149
Vegetable
011
011
59
48
48
1
23
2
13
3
14
119
80
104
60
61
61
3
7
4
7
69
61
64
41
36
43
4
9
6
12
163
139
140
39
52
47
5
10
6
6
130
132
131
64
63
64
6
9
6
7
76
60
67
55
69
56
184
165
150
56
46
57
127 i- 43
111 i 43
115 + 37
53 + 9.6
54 k 11.4
54 k 7.8
39
7
- 11 12 + 5.3
Sub-mean ? S.D.
7 6 I 2.2
___16 12 + 5.3
I
--
1 p < 0.05
p < 0.01
p < 0.01
p < 0.05
Overall
8
11
8
123
94
31
9
32
18
134
132
37
32
10
7
6
109
89
47
35
11
14
9
90
65
57
57
12
12
7
188
131
59
52
128 f 39
108 ? 37
50 t 10.9
49 t 12.0
mean
13 27.3
* so.
8 f 3.8
(Twelve subjects) I
1 p
<
0.001
p < 0.005
of last 3 determtinatlons of each dietary period.
‘Mean
tVLDL: very low density lipoprotein, LDL: low density lipoprotein, HDL: high density lipoprotein.
recent studies fish oil feeding has invariably led to lower triglyceride levels.‘s,‘6 No other reported study to date, however, has compared fish oil to both polyunsaturated vegetable oils and saturated fats in the same individuals using diets with controlled fatty acid compositions. Our finding that, of the three fats tested, only salmon oil was hypotriglyceridemic, indicates that its polyunsaturated w-3 fatty acids may have different metabolic effects than w-6 fatty acids as will be discussed later. 40 35 B 2 T * B k . *
30 25 20
;
I5
8
IO 5
TRIGLYCERIDES
Fig. 2. This family
The w-3 fatty acid content
is expressed (C20:5
table
oil diet
open
columns;
+
as the C22:5
were
the
salmon
percent
+
C22:6).
same oil diet
PHOSPHOLIPIDS
cR~::~R~yL
of the plasma
of total The
as the
w-3
fatty
levels
control
= striped
lipid fractions.
acids diet.
columns
from
during
Control
(mean
the
w-3
the vegediet
and S.D.).
=
Although this study was primarily designed to compare the hypolipidemic effects of two polyunsaturated oils: salmon oil vs. vegetable oil, we were also able to make comparisons between the two major classes of polyunsaturated fatty acids: w-6 vs. w-3. This comparison was possible because the salmon oil diet contained some 8% of calories as w-3 fatty acids and the control diet contained 7.2% of calories as w-6 fatty acids. Since these two diets did not differ greatly in amounts of saturated and monounsaturated fatty acids, their effects upon the plasma lipids may be primarily ascribed to their respective contents of the w-6 or o-3 polyunsaturates. Viewed from this perspective, the w-3 fatty acids were not only more hypotriglyceridemic, but were also more hypocholesterolemic than the w-6 fatty acids, on a gram-for-gram basis. Thus, it is not unreasonable to conclude that the w-3 fatty acids were more hypolipidemic than the major w-6 fatty acid, linoleic. The reasons for the greater potency of the w-3 fatty acids are not completely clear. Certainly, their amount of unsaturation is much greater. The primary o-6 fatty acid, linoleic acid, contains two double bonds per molecule. The principal w-3 fatty acids in salmon oil (20:5 and 22:6) have an average of 5.5 double bonds per molecule. Thus, the o-3 fatty acids provided about 2.75 times as much “unsaturation” as the w-6 fatty acids, gram-for-gram. If the hypolipidemic effect resulted from the total amount of unsaturation in the
HYPOLIPIDEMIC EFFECTS OF FISH OIL
dietary fat, then an equivalent amount of “unsaturation” provided as w-6 fatty acids should lower lipids to the same extent as the fish oil diet did. This hypothesis was tested in our study. The vegetable oil diet provided a ratio of 2.7 to 1.0 of w-6 fatty acids to the w-3 fatty acids in salmon oil. Both oils had the same hypocholesterolemic effect. Thus, the cholesterol-lowering effect of these oils seemed to be a function of their total unsaturation. an observation which has been made frequently over the years.” The hypotriglyceridemic effect, however, did not appear to be linked to the degree of unsaturation per se and perhaps was related to the w-3 structure. Other dietary variables which may have been responsible for the hypolipidemic effects of the salmon oil diet should also be considered. Besides the major differences in w-3 fatty acid content, fish oils also contain relatively high levels of the C20 and C22 monounsaturated fatty acids (Table 1). Very low levels of these fatty acids were found in the plasmas of our subjects, corroborating the work of other researchers.lL.IX It is not known whether these fatty acids are simply poorly absorbed or are rapidly metabolized. Since the Cl 6 and Cl 8 monounsaturates have been found to have neutral effects upon the plasma lipid levels,‘” WC have no reason to believe that these longer chain monounsaturates should be hypolipidemic. A second consideration is the differences in the dietary protein sources. The principal protein source during the salmon oil phase was salmon flesh; during the other two dietary phases it was casein. There is data in rabbits which indicate that fish protein is no different than casein in terms of its effect upon the plasma cholesterol levels.2” Hence, the difference in dietary protein were probably not responsible for the lipid changes. The effects of polyunsaturated vegetable oils upon plasma triglyceride levels have been studied by several investigators who have reached divergent conclusions. Some have noted that vegetable oils decreased triglyceride levels” ” while others have found no effect.25m’R Among those reporting a hypotriglyceridemic effect of vegetable oils was one study in which normal and hypertriglyceridemic subjects were examined for only a IO-day dietary period.14 In the study employing the longest dietary periods (7-9 wk per period) the vegetable oil had no effect upon plasma triglyceride levels.‘6 Thus it seems likely that 10 days may have been too short to allow the plasma lipids to stabilize. In two other studies2’.‘3 plasma triglyceride levels were depressed by only 14%-l 5% during vegetable oil feeding. In our study, the feeding of polyunsaturated vegetable oils had no effect upon the plasma triglyceride levels. Polyunsaturated vegetable oils have also been found
183
to lower HDL cholesterol levels in some studies.‘3.‘6.2’ However, the results of this study support the findings by others that vegetable oil feeding does not affect plasma HDL levels.24.27 On the other hand, fish oil feeding has been found to have a slightly elevating effect’5,2’ or no effect upon HDL cholesterol levels.‘6 Our findings, conducted under controlled dietary conditions, confirm the latter finding. The total incorporation of w-3 fatty acids into the plasma lipid classes in this study was higher than has been reported previously.R.‘5.‘b.” This undoubtedly occurred because of the higher intake w-3 fatty acids in this study (21-29 g) compared to earlier studies (4-8 g). The finding of higher levels of w-3 fatty acids in the plasma lipids than in the diet implies that there may be preferential incorporation of the w-3 fatty acids over the other dietary fatty acids. The ultimate metabolic consequences of such high levels of w-3 fatty acids in the plasma lipids are not known. The exact mechanism(s) by which w-3 fatty acids exert their influence on plasma lipids cannot be determined from this study. The similar reduction of plasma triglyceride and VLDL cholesterol levels (- 38%) after the salmon oil diet suggested that the fall in triglyceride levels occurred from a reduction of the total amount of VLDL in the blood rather than from a change in the VLDL lipid composition. Thus. the hypotriglyceridemic effect probably resulted from either a more rapid hydrolysis and removal of VLDL particles enriched with w-3 fatty acids or an inhibition of VLDL synthesis or secretion. Other potential mechanisms which should also be considered include: ( I) structural alterations in lipoproteins leading to changes in their interactions with lipolytic enzymes (2) changes in the composition of plasma membrane lipids altering the affinity of membrane-bound enzymes for lipoproteins,” and (3) an inhibitory influence of w-3 fatty acids on the hepatic biosynthesis of either fatty acids,j’ triglyceride or apoprotein B3’ or the assembly or secretion of VLDL. A reduction in free fatty acids released from adipose tissue could lead to lower hepatic triglyceride synthesis.” Prostaglandins derived from eicosapentaenoic or arachidonic acids might inhibit the release of free fatty acids from adipose tissue, although the role of prostaglandins in lipolysis is still unclear.34 The effects of the salmon oil diet upon platelet lipid composition and function have recently been reported from our laboratory.3” Platelets became enriched in o-3 fatty acids with a resultant prolongation of bleeding time and reduced adhesiveness. Because of their dual antithrombotic and hypolipidemic effects, marine oils rich in o-3 fatty acids may have important nutritional applications in the prevention and treatment of atherosclerotic disease.
HARRIS, CONNOR. AND McMURRY
184
ciated. We wish to thank the dietetic and nursing staff of the Clinical
ACKNOWLEDGMENT The authors are grateful to Oregon Aqua Foods, Inc., of Astoria, Oregon, for providing the salmon oil and fillets for this study. The valuable discussions with Dr. Roger Illingworth are also appre-
Research Center for their skillful care of the subjects. Excellent clerical assistance was provided by Joanne Skirving, Patricia Schade, and Paula Bisaccio. Statistical analysis was conducted with the help of Dr. Gary Sexton.
REFERENCES 1. Bang HO, Dyerberg J, Hydrne N: The composition of food consumed by Greenlandic Eskimos. Acta Med Stand 220:69-73, 1976 2. Bang HO, Dyerberg J: Plasma lipids and lipoproteins in Greenlandic West Coast Eskimos. Acta Med Stand 192:85-94. 1972 3. Dyerberg, J, Bang HO, Stofferson E, et al: Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis. Lancet ii:] 17-l 19, 1978 4. Dyerberg J, Bang HO: Hemostatic function and platelet polyunsaturated fatty acids in Eskimos. Lancet ii:433-435, 1979 5. Bronte-Stewart B, Antonis A, Eales L, et al: Effects of feeding different fats on serum cholesterol levels. Lancet i:521-526, 1956 6. Malmros H, Wigand G: The effect on serum cholesterol of diets containing different fats. Lancet ii:]-8, 1957 7. Keys A, Anderson JT, Grande F: “Essential” fatty acids, degree of unsaturation and effects of corn (maize) oil on the serum cholesterol level in man. Lancet i:66-68, 1957 8. Ahrens EH. lnsull W. Hirsch J, et al: The effect on human serum iipidsof a dietary fat, highly unsaturated, but poor in essential fatty acids. Lancet i:ll5-119. 1959 9. Needleman P, Raz A, Minkes MS, Ferrendelli JA, Sprecher H: Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. Proc Nat1 Acad Sci USA 76:9444948, 1979 IO. Ahrens EH, Boucher CA: The composition of a simulated American diet. J Am Diet Ass 73613-620, 1978 Il. Recommended Dietary Allowances, 8th Ed.. Food and Nutrition Board, National Research Council of the National Academy of Sciences, 1974 12. Manual of Laboratory Operations, Lipid Research Clinics Program, Vol. 1, Lipid and Lipoprotein Analysis, DHEW Publication No. (NIH)
75-628,
1974
13. Wene JD, Connor WE, Den Besten L: The development of essential fatty acid deficiency in healthy men fed a fat-free diet intravenously and orally. J Clin Invest 56: 127-l 34, 1975 14. Winer BJ: Siatisticalprinciples of experimental edition, McGraw-Hill, New York, 197 1, pp 261-273.
design, 2nd
15. von Lossonczy TO, Ruiter A, Bronsgeest-Schoute HC, et al: The effect of a fish diet on serum lipids in healthy human subjects. Am J Clin Nutr 31:1340-1346, 1978 16. Bronsgeest-Schoute HC, van Gent CM, Luten JB, et al: The effects of various intakes of w-3 fatty acids on the blood lipid composition in healthy human subjects. Am J Clin Nutr 34:17521757, 1981 17. Goodnight SH, Harris WS, Connor WE, et al: Polyunsaturated fatty acids, hyperlipidemia and thrombosis. Arteriosclerosis 2:87-l 13, 1982 18. Ruiter A, Jongbloed AN, VanGent CM, et al: The influence of dietary mackerel oil on the condition of organs and on blood lipid composition in the young, growing pig. Am J Clin Nutr 31:21592166, 1978 19. Hegsted DM, McGandy RB, Myers ML, et al: Quantitative effects of dietary fat on serum cholesterol in man. Am J Clin Nutri 17:28ll295, 1965
20. Carroll KK: The role of dietary protein in hypercholesterolemia and atherosclerosis. Lipids 13:360-365, 1978 21. Sanders TAB, Vickers M, Haines AP: Effect of blood lipids and haemostasis of a supplement of cod-liver oil, rich in eicosapentaeonic and docosahexaenoic acids in healthy young men. Clin Sci 61:317-324, 1981 22. Nestel PJ, Havenstein N, Scott TW. et al: Polyunsaturated ruminant fats and cholesterol metabolism in man. Aust NZ J Med 4:497-501,1974 23. Shepherd J, Packard CJ, Grundy SM. et al: Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man. J Lipid Res 21:9lI99. 1980 24. Chait A, Onitiri A, Nicoll A, et al: Reduction of serum triglyceride levels by polyunsaturated fat. Studies on the mode of action and on very low density lipoprotein composition. Atherosclerosis 20:347-364, 1974 25. Nestel PJ, Havenstein N, Whyte HM, et al: Lowering of plasma cholesterol and enhanced sterol excretion with the comsumption of polyunsaturated ruminant fats. N Eng J Med 228:379-382, 1973 26. Turner JD, Le N-A, Brown WV: Effect of changing dietary fat saturation on low density lipoprotein metabolism in man. Am J Physiol 241:E57-E63, 1981 27. Nestel PJ, Havenstein N, Homma Y, et al: Increased sterol excretion with polyunsaturated-fat, high-cholesterol diets. Metabolism 24:189-198. 1975 28. Connor WE. Witiak DT, Stone OB, et al: Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of different fatty acid composition. J Clin Invest 48:1363-1375. 1969 29. Shepherd J, Packard CJ, Patsch JR, et al: Effects of dietary polyunsaturated and saturated fat on the properties of high density lipoproteins and metabolism of apolipoprotein A-l. J Clin Invest 61:1582-1592, 1978 30. Gavigan SJP. Knight BL: Catabolism of low density lipoprotein by fibroblasts cultured in medium supplemented with saturated or unsaturated free fatty acids. Biochem Biophys Acta 665:632-635, 1981 31. lritani N, lnoguchi K, Endo M, et al: Identification of shellfish fatty acids and their effects on lipogenic enzymes. Biochem Biophys Acta 618:378-382, 1980 32. Shepherd J, Packard CJ, Taunton D, et al: Effects of dietary fat saturation on the composition of very low density lipoproteins and on the metabolism of their major apoprotein, apolipoprotein B. Biochem Sot Tram 6:779-78 I, 1978 33. Have1 RJ, Kane JP, Balasse ED, et al: Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest 49:2017-2035, 1970 34. Lipinski BA, Mathias MM: Prostaglandin production and lipolysis in isolated rat adipocytes as effected by dietary fat. Prostaglandins 16:957-963, 1978 35. Goodnight SH, Harris WS. Connor WE: The effects of dietary w-3 fatty acids upon platelet composition and function in man: a prospective, controlled study. Blood 58:880-885, 1981