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Fish oil reduces cholesterol and arachidonic acid content more efficiently in rats fed diets containing low linoleic acid to saturated fatty acid ratios
Biochimica et Biophysics Acta, 962 (1988) 331-344 Elsevier
337
BBA 52930
Fish oil reduces cholesterol and arachidonic acid content more efficiently in rats fed diets containing low linoleic acid to saturated fatty acid ratios Manohar
L. Garg, Antoni A. Wierzbicki, Alan B.R. Thomson and M. Thomas Clandinin
Nutrition and Metabolism Research Group, Department of Foods and Nutrition, Faculty of Home Economics and Division of Gastroenterologv, Faculty of Medicine, University of Alberta, Edmonton (Canada) (Received
Key words:
Fish oil; Arachidonic
25 May 1988)
acid: Cholesterol:
Fatty acid composition;
Serum; (Rat liver)
Rats were fed diets containing a high level of saturated fatty acids (hydrogenated beef tallow) versus a high level of linoleic acid (safflower oil) at both low and high levels of fish oil containing 7.5% (w/w) eicosapentaenoic and 2.5% (w/w) docosahexaenoic acids for a period of 28 days. The effect of feeding these diets on the cholesterol content and fatty acid composition of serum and liver lipids was examined. Feeding diets high in fish oil with safflower oil decreased the cholesterol content of rat serum, whereas feeding fish oil had no significant effect on the cholesterol content of serum when fed in combination with saturated fatty acids. The serum cholesterol level was higher in animals fed safflower oil compared to animals fed saturated fat without fish oil. Consumption of fish oil lowered the cholesterol content of liver tissue regardless of the dietary fat fed. Feeding diets containing fish oil reduced the arachidonic acid content of rat serum and liver lipid fractions, the decrease being more pronounced when fish oil was fed in combination with hydrogenated beef tallow than with safflower oil. These results suggest that dietary n - 3 fatty acids of fish oil interact with dietary linoleic acid and saturated fatty acids differently to modulate enzymes of cholesterol and fatty acid metabolism.
Introduction
Consumption of marine oils is apparently associated with a decreased risk of cardiovascular disease [l-4]. The biochemical changes following consumption of dietary fish oil include a reduction in circulating cholesterol and triacylglycerol levels and a decrease in platelet aggregation [l-4]. These effects have been attributed to the presence of large amounts of eicosapentaenoic (20 : 5( n - 3)) and/or docosahexaenoic (22 : 6( n - 3)) acid [l-4].
Correspondence: M.T. Clandinin, Department of Foods and Nutrition, 318f Home Economics Building, University of Alberta, Edmonton, Alberta, Canada, T6G 2M8. 0005-2760/88/$03.50
0 Elsevier Science. Publishers
B.V. (Biomedical
Recent studies have suggested that 22 : 6( n - 3) is responsible for the cholesterol-lowering effect [5], whereas 20 : 5( n - 3) contributes to changes in eicosanoid metabolism [6], leading to decreased platelet aggregation. A side-effect associated with the consumption of megadoses of fish oil is enhanced bleeding time [7]. Attempts have been made to find the efficacious amount of fish oil that may show desirable beneficial effects without undesirable side-effects, such as enhanced bleeding time. A range of dietary 20 : 5(n - 3) doses has been proposed by different authors [4,8]. A recent study has demonstrated that the balance between saturated and unsaturated fatty acids may be an important factor in the biosynthesis of long-chain polyunsaturated fatty acids (20 : 4(n - 6), 20 : 5( n Division)
338
- 3) 22 : 5(n - 6), 22 : 5(n - 3)) that serve as eicosanoid precursors [9]. We have recently shown that feeding linolenic acid affects synthesis of eicosanoid precursors in a different manner when fed along with saturated or n - 6 fatty acids [lo]. Therefore, it seems logical to hypothesize that dietary 20 : 5(n - 3) may also have a different effect on eicosanoid precursor 20 : 4( n - 6) synthesis when the balance of the diet fat is saturated or unsaturated fatty acids. In the present study, the cholesterol content and fatty acid composition of serum and liver lipid fractions of rats fed fish oil was examined in the presence of high amounts of either saturated fatty acids or linoleic acid. The results suggest that the ratio of dietary 18 : 2( n - 6) to saturated fatty acids may be an important determinant to the hypocholesterolemic and platelet antiaggregatory effects of fish oil. Materials and Methods Animals and diets. Male weanling SpragueDawley rats weighing approx. 40 g were fed Wayne rat chow (Allied Mills Inc., Chicago) for 3 days before switching to the experimental diets. Four high fat diets were prepared from the basal diet described previously in detail [ll]. Each diet contained 200 g fat/kg diet (20%, w/w) from one of the following sources: 90% hydrogenated beef tallow plus 10% safflower oil (hydrogenated beef tallow diet); 100% safflower oil (safflower oil diet); 65% hydrogenated beef tallow plus 25% fish oil plus 10% safflower oil (beef tallow and fish oil diet); 75% safflower oil plus 25% fish oil (safflower oil and fish oil diet). The fatty acid composition of the diets is given (Table I). The hydrogenated beef tallow diet contained mainly saturated fatty acids (86.3%) and enough linoleic acid (9.5%) to prevent essential fatty acid deficiency. The safflower oil diet was enriched with linoleic acid (70.5%). Both the hydrogenated beef tallow and the safflower oil diets at high levels of fish oil provided similar amounts of eicosapentaenoic (7.5 and 7.8%, respectively) and docosahexaenoic acids (2.6 and 2.4%, respectively) but differed in the linoleic acid to saturated fatty acid ratio (0.14 and 2.92, respectively) (Table I). Animals were housed individually in hanging stainless steel cages in a well-ventilated room maintained at 22 + 2’C on a 12/12 h light/dark
TABLE
1
FATTY ACID COMPOSITION MENTED DIETS
OF
THE
FAT-SUPPLE-
Diets were prepared fresh on a weekly basis and - 20 o C. Fatty acids are designated by the number atoms followed by the number of double bonds. The composition is expressed in %w/w and is the mean four random samples of diet. Diet fat
Beef tallow
Safflower
+ fish oil 14:o 16:0 16:l(n 17:o 18:O 18:l(n 18:l(n 18:2(n 18:3(n 20:4(n
-7)
-9) -7) -6) -3) -6)
20:5(n -3) 22:5(n -3) 22:6(n-3) Total Saturated Monounsaturated n-6 n-3 18 : 2( n -6)/saturated
4.2 28.9 0.5 2.1 51.2 3.2 0.2 9.5 0.1 _ _ _
86.3 4.1 9.5 0.1 0.11
stored at of carbon fatty acid of at least
oil + fish oil
5.0 23.8 3.5 0.5 39.3 5.0 0.9 9.4 0.8 0.4 1.5 0.6 2.6
0.3 7.6 0.1 0.1 5.8 14.6 0.2 70.5 0.6
68.6 9.9 9.8 11.5 0.14
13.8 14.9 70.5 0.6 5.11
_ _
2.0 7.6 3.2 0.6 9.6 6.4 0.4 51.6 0.9 0.3 7.x 0.6 2.4
19.8 10.0 57.9 11.7 2.92
cycle. All diets were prepared weekly and stored at -20°C. Food and water were available to rats ad libitum. The body weights of the animals were monitored once a week throughout the feeding period. Lipid analysis. After 4 weeks of feeding experimental diets, the rats were killed by decapitation (between 0800 and 1000 h). The livers were quickly excised and rinsed in ice-cold saline. Blood samples were also collected for separation of serum by centrifugation. Total lipids from serum and liver samples were extracted with chloroform/methanol (2 : 1) [12] containing 0.005% butylated hydroxytoluene. The total, free and esterified cholesterol contents in aliquots of serum and liver lipid extracts were determined enzymatically [13,14]. Total phospholipids, triacylglycerol and cholesterol esters were separated by thin-layer chromatography on silica gel G plates (Analtech, 250 pm, 20 X 20
339
TABLE
II
BODY WEIGHT, LIVER WEIGHT, LIVER WEIGHT/BODY WEIGHT RATIO FED DIETS CONTAINING LOW AND HIGH LEVELS OF EICOSAPENTAENOIC Rats were fed the diets for 4 weeks. Data are presented significantly different (P > 0.05). Diet fat
Body Liver Liver Food
weight (g) weight (g) weight/body consumption
weight (X) (g/day)
AND FOOD ACID
as means + SD. of five animals
CONSUMPTION
in each diet group.
OF RATS
None of the values are
Beef tallow
Safflower
-
+ fish oil
-
+ fish oil
248 +15 10.0 + 1.2 4.01+ 0.46 19.2 + 1.1
265 +23 10.4 + 1.3 3.94* 0.33 20.8 k 1.0
280 *32 12.2 + 1.9 4.34* 0.28 20.4 + 2.0
214 +20 12.1 + 1.1 4.76+ 0.34 19.9 f 1.2
cm) using a solvent system comprised of petroleum ether/diethyl ether/acetic acid (80 : 20 : 1, v/v) [15]. Phospholipids were subjected to methylation with BF,-methanol (1496, w/w) reagent followed by heating at 100°C for 1 h [16]. Triacylglycerol and cholesterol ester fractions were saponified with 0.5 M methanolic potassium hydroxide by heating at 85’C for 90 min followed by methylation with BF,-methanol (14%, w/w) reagent [16]. The fatty acid methyl esters were extracted in hexane and analyzed by automated capillary gas chromatography (Varian Model 6000 equipped with a flame ionization detector) [17]. Authentic standard mixtures of fatty acid methyl esters were injected to identify serum and liver fatty acid methyl esters. Peak areas and weight percent fatty acid composition were computed using a chromatography data system (Varian Model DS 604). Statistical analyses. Data are expressed as the mean f S.D. The effect of diet fat treatment was determined by analysis of variance procedures. The comparison between individual diet treatments was made using Newman-Keuls multiple range test [18]. Results Body weights, liver weights and food consumption
All animals appeared healthy after 4 weeks of feeding the experimental diets. Dietary fat treatment had no significant effect on body weights, liver weights or liver weight/body weight ratios in
oil
these animals. Rats were offered and consumed similar amounts of food (approx. 20 g/day) irrespective of the dietary regimen (Table II).
Cholesterol content of rat serum and liver
Serum and liver cholesterol concentrations are presented (Fig. 1). Animals fed the safflower oil diet had a significantly higher cholesterol content in serum compared to those fed the hydrogenated beef tallow diet (P < 0.05). Feeding the diet high in fish oil with safflower oil reduced the cholesterol content of serum while the hydrogenated beef tallow and fish oil diet had no significant effect. The increase in the cholesterol level following feeding of the safflower oil diet and the reduction in cholesterol level caused by the safflower oil and fish oil diet was associated with a change in level of the unesterified cholesterol fraction of the serum (Fig. 1). In liver tissue, the level of cholesterol was significantly higher in animals fed the safflower oil diet compared to the group fed the hydrogenated beef tallow diet. For rats fed the diet containing fish oil in combination with hydrogenated beef tallow or with safflower oil, the cholesterol content was significantly lower than that observed for rats fed the hydrogenated beef tallow or safflower oil diets, respectively. The increase in liver cholesterol concentration following feeding of the safflower oil diet was detected in both the free and esterified cholesterol content. The decrease in the liver cholesterol content resulting from both diets
340
Free =‘
Ester
Total
loo-
? g
b l80-
z ’ tj a, 0 6
60-
,T_
40-
q Beef Tallow n Beef Tallow 7 E z
240
180
and Fish Oil t3 Safflower
Oil
•I Safflower Oil and Fish Oil
&
Fig. 1. Effect of dietary
5 0
120
’@
60
C
fish oil on cholesterol content of rat serum and liver. Values are the means + SD. of five rats in each group. Values without a common superscript are significantly different (P c 0.05).
containing fish oil was found to be associated with the esterified and not with the unesterified cholesterol content (Fig. 1). Fatty acid composition of serum and liver lipids The fatty acid composition of rat serum and liver phospholipid, triacylglycerol and cholesterol ester fractions is illustrated (Figs. 2-4). Feeding the diet rich in safflower oil to rats increased the 18 : 2(n - 6) and 20 : 4(n - 6) contents in serum and liver phospholipids with an accompanied decrease in saturated (16 : 0 and/or 18 : 0) and (16 : 1, 18 : l(n - 9), 18 : l(n monounsaturated 7)) fatty acids and docosahexaenoic acid (22 : 6( n - 3)). Feeding fish oil with hydrogenated beef tallow reduced the 20 : 4(n - 6) content of both serum (61%) and liver (66%) phospholipids and was followed by a concomitant increase in the 20 : 5(n - 3) and 22 : 6(n - 3) content compared with that of the hydrogenated beef tallow diet.
Animals fed the diet containing fish oil in combination with safflower oil also exhibited a reduced 20 : 4(n - 6) content in phospholipids, but to a lesser extent (33% in serum and 31% in liver). Similarly, feeding animals the safflower oil and fish oil diet resulted in an accumulation of smaller amounts of 20 : 5( n - 3) and 22 : 6( n - 3) than observed for animals fed the hydrogenated beef tallow and fish oil diet. The fatty acid composition found in the serum triacylglycerol fraction resembled that of the dietary fat fed. For example, animals fed the diet containing hydrogenated beef tallow exhibited high levels of saturated (52.8%) and monounsaturated fatty acids (28.7%), those fed the safflower oil diet exhibited elevated levels of 18 : 2( n - 6) (60.7%) and rats fed diets containing fish oil had higher levels of 20 : 5( n - 3) and 22 : 6( n - 3) (16.1% and 9.7%) in serum triacylglycerols. In this lipid fraction, fish oil supplementation also de-
341
Liver
Serum b
PL EI Beef Tallow
TG
CE
n Beef Tallow
PL I3 Safflower
and Fish Oil
TG Oil
CE EI Safflower Oil and Fish Oil
Fig. 2. Effect of dietary fish oil on saturated and monousaturated fatty acid content of rat serum and liver lipid fractions: PL, phospholipid; TG, triacylglycerol; CE, cholesterol ester. Values are the meansfS.D. of five rats in each group. Values without a common superscript are significantly different (P < 0.05).
creased the 20 : 4(n - 6) levels both in the serum and in liver tissue. Feeding diets high in fish oil produced changes in the fatty acid composition of liver triacylglycerols similar to those in the serum. The cholesterol ester fraction of serum lipids exhibited the largest changes in 20 : 4(n - 6) (71% decrease) and 20 : 5( n - 3) (39.4-fold increase) content following consumption of the diet containing fish oil in association with hydrogenated beef tallow. Fish oil intake in combination with safflower oil could lower 20 : 4(n - 6) levels only by 8% and could accumulate 20 : 5(n - 3) to a much smaller extent. In the liver tissue, the 20 : 4(n - 6) content was lowered only when fish oil was fed in association with hydrogenated beef tallow and not with safflower oil. Accumulation of
20 : 5(n - 3) in the liver cholesterol ester fraction was greater when fish oil was fed along with hydrogenated beef tallow than when it was fed with safflower oil. Discussion Previous studies have demonstrated that consumption of fish oil reduces circulating lipid levels in animals as well as in human subjects [l-4,19-21] and is associated with decreased thrombus formation [22,23]. It has been suggested that 20 : 5( n - 3) present in fish oil competes with 20 : 4(n - 6) at the level of the cyclooxygenase, leading to decreased formation of thromboxane A, and increased synthesis of thromboxane A,, which, unlike thromboxane A 2, is not pro-aggregatory. Some
342
Serum
Liver
60 ‘;; k -0 .0 ; .P) 0 .f 1
48
36
24
Q Beef Tallow n Beef Tallow and Fish Oil c
q Safflower
Oil
LI Safflower Oil and Fish Oil
0
PL
TG
CE
PL
TG
CE
Fig. 3. Effect of dietary fish oil on linoleic and arachidonic acid content of rat serum and liver lipid fractions: PL, phospholipid; triacylglycerol; CE, cholesterol ester. Values are the means i S.D. of five rats in each group. Values without a common superscript significantly different (P i 0.05).
20 : 5(n - 3) is also converted to prostacyclin of the 3+eries, which has anti-aggregatory properties similar to those of PGI 2 generated from 20 : 4( n 6) [24,25]. Thus, the net result of fish oil consumption is to alter the eicosanoid balance, which in turn inhibits the platelet response to clot formation. We have recently shown that dietary fish oil inhibits A6- and A5-desaturase activities and reduces 20 : 4( n - 6) biosynthesis from 18 : 2( n - 6) [26,27]. Eicosanoids are derived from this common precursor, 20 : 4(n - 6). Therefore, it is conceivable that limitation in 20 : 4(n - 6) biosynthesis will impair thromboxane A, and prostacyclin I, formation. The present study demonstrates that fish oil lowers 20 : 4(n - 6) more efficiently when fed in combination with a high level of dietary saturated fatty acids than when it is fed with a high level of dietary 18 : 2(n - 6). However,
TG. are
20 : 5( n - 3) and/or 22 : 6( n - 3) accumulate in phospholipid, triacylglycerol and cholesterol ester fractions of rat serum and liver lipids to a greater extent when fish oil and saturated fat are fed together. Although we did not measure levels of thromboxane and prostacyclin in these animals, the 20 : 4(n - 6) content is known to reflect the pattern of thromboxane A, and prostacyclin I, produced [1,4] and the presence of 20 : 5( n - 3) indicates competitive inhibition [1,4] of the cyclooxygenase and formation of thromboxane A3 and prostacyclin I,. Thus, it is apparent that consumption of fish oil is likely to impair platelet responsiveness more efficiently when it is combined with hydrogenated beef tallow rather than with safflower oil. The smaller change in the 20 : 4( n - 6) content when rats are fed the diet containing fish oil and
343
Liver
Serum -
50r 0 Beef Tallow n Beef Tallow and Fish Oil 0 Safflower
Oil
q Safflower Oil and Fish Oil
b
!i z
b
6
Jz
z
s
3
0” 0
PL
TG
CE
PL
TG
CE
Fig. 4. Effect of dietary fish oil on eicosapentaenoic and docosahexaenoic acid content of rat serum and liver lipid fractions: PL, phospholipid; TG, triacylglycerol; CE, cholesterol ester. Values are the means+S.D. of five rats in each group. Values without a common superscript are significantly different (P < 0.05).
safflower oil together may be due to the high dietary 18 : 2(n - 6) content, resulting in a tendency to increase 20 : 4(n - 6) synthesis, an effect opposite to that known to be caused by the 20 : 5(n - 3) in fish oil [26,27]. When fish oil and hydrogenated beef tallow are fed, the impairment of 20 : 4(n - 6) biosynthesis by 20 : 5(n - 3) at the level of cyclooxygenase is maximal and is not opposed by substrate competition, thus resulting in maximum inhibition of platelet aggregation. The only known side-effect associated with consumption of megadoses of n - 3 fatty acids, as in the case of Greenland Eskimos, is an enhanced bleeding tendency [l-4]. Attempts have been made to define an appropriate proportion of dietary 20 : 5(n - 3) that may show beneficial effects without having side-effects, such as increase in clotting time [4,8]. Seafoods or fish oils are not a major part of the North American or European diet. Based upon extrapolation of the present study
to man, it is conceivable that a small amount of fish oil may inhibit the response of platelets to thrombus formation, provided that the major source of dietary fat is saturated fats. In liver tissue, the cholesterol content was lowered by dietary fish oil, irrespective of the nature of the dietary fat. Consumption of fish oil rich in 20 : 5(n - 3) and/or 22 : 6(n - 3) has recently been shown to accelerate the flow of cholesterol towards bile formation and secretion [28]. Thus, the decrease in the cholesterol content following the feeding of fish oil may be due to increased utilization of cholesterol for bile synthesis in the liver. Alternatively, the reduction in the liver cholesterol concentration after fish oil feeding may be due to the inhibition of hydroxy-’ methylglutarylcoenzyme A reductase activity [29], a rate-limiting enzyme in the cholesterol biosynthetic pathway. This latter alternative merits further investigation.
344
In summary, feeding fish oil in association with saturated fatty acids produces a greater reduction in the 20 : 4(n - 6) content and accumulation of more 20 : 5( n - 3) and/or 22 : 6( n - 3) in serum and liver lipid fractions than when fish oil is fed with a diet high in 18 : 2(n - 6). Dietary fish oil lowers the cholesterol content in liver regardless of the dietary fat fed. Althought it is inappropriate to interpret the animal data in humans, the present study suggests that fish oil may have greater antithrombotic effects in populations consuming fats from animal sources (beef tallow, lard, etc.) than those eating mainly vegetable oils (safflower oil, corn oil, etc.). Therefore, the data presented in this manuscript could provide a useful basis for formulating human clinical trials to examine the efficacy of fish oil supplementation on platelet aggregation, cholesterol and triacylglycerol metabolism in subjects consuming diets with low versus high linoleic acid to saturated fatty acid ratios. Acknowledgements This ces and M.T.C. Fellow Medical Wharton
work was supported by the Natural ScienEngineering Research Council of Canada. is a Scholar and M.L.G. is a Postdoctoral of the Alberta Heritage Foundation for Research. The technical assistance of T. is gratefully acknowledged.
References Kinsella, J.E. (1986) Food Technology 40, 89-97. Von Schacky, C. (1987) Ann. Int. Med. 107, 890-899. Dyerberg, J. (1986) Nutr. Rev. 44, 125-134. Herold, P.M. and Kinsella, J.E. (1986) Amer. J. Clin Nutr. 43, 566-598. Kobatake, Y. Kuroda, K., Jinnouchi, H., Nishide, E. and Innami, S. (1984) J. Nutr. Sci. Vitaminol. 30, 357-372.
6 Croft. K.D., Beilin. L.J., Legge, F.M. and Vandongen. R. (1987) Lipids 22. 6477650. 7 Dyerberg, J.. Bang. H.O., Stoffersen. E.. Moncada, S. and Vane. J.R. (1978) Lancet ii, 117-119. 8 Nestel, P.J. (1987) Amer. J. Clin. Nutr. 45. 1161-1167. 9 Cook. H.W. and Spence. M.W. (1987) Lipids 22. 613-619. 10 Garg, M.L.. Wierzbicki, A.A., Thomson, A.B.R. and Clandinin, M.T. (1988) Biochim. Biophys. Acta 962, 330-336. 71 Clandinin, M.T. and Yamashiro. S. (1982) J. Nutr. 112, 825-828. 12 Folch. J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226. 497-509. 13 Trinder, P. (1969). Ann. Clin. Biochem. 6, 24427. 14 Siedel. J.. Schlumberger. H.. KIose. S.. Ziegenhorn. J. and Wahlefeld. A.W. (1981) J. Chn. Chem. Clin. Biochem. 19. 8388839. 15 Garg, M.L.. Snoswell, A.M. and Sabine, J.R. (1985) Nutr. Rep. Intl. 32, 17-26. 16 Metcalfe, L.D. and Schmitz, A.A. (1961) Anal. Chem. 33. 363-364. 17 Hargreaves, K.M. and Clandinin. M.T. (1987) Biochim. Biophys. Acta 918, 97-105. 18 Steel, R.G.D. and Torrie. J.H. (1980) Principles and Procedures of Statisitics: A Biometrical Approach. McGraw-Hill, New York. 19 Harris. W.S., Connor, W.E. and McMurry, M.P. (1983) Metabolism 32, 1799184. 20 Phillipson. B.E.. Rothrock, D.W.. Connor. W.E., Harris. W.S. and Illingworth. D.R. (1985) New Engl. J. Med. 312. 1210-1216. 21 Nestel, P.J.. Connor, W.E.. Reardon, M.F.. Connor. S.. Wong, S. and Boston, R. (1984) J. Clin. Invest. 74. 82-89. 22 Ahmed. A.A. and Holub. B.J. (1984) Lipids 19, 617-624. 23 Dyerberg, J. and Jorgensen. K.A. (1982) Prog. Lipid Res. 21, 255-269. 24 Dyerberg, J. and Bang, H.O. (1979) Lancet ii. 433-435. 25 Salmon, J.A. (1987) Biochem. Sot. Trans. 15, 324-326. 26 Garg. M.L., Sebokova, E.. Thomson, A.B.R. and Clandinin. M.T. (1988) Biochem. J. 249, 351-356. 27 Garg, M.L., Thomson, A.B.R. and Clandinin. M.T. (1988) J. Nutr. 118, 661-668. 28 Balasubramaniam, S., Simons, L.A., Chang. S. and Hickie. J.B. (1985) J. Lipid Res. 26, 684-689. 29 Field. F.J., Albright, E.J. and Mathur. S.N. (1987) J. Lipid Res. 28. 50-58.
337
BBA 52930
Fish oil reduces cholesterol and arachidonic acid content more efficiently in rats fed diets containing low linoleic acid to saturated fatty acid ratios Manohar
L. Garg, Antoni A. Wierzbicki, Alan B.R. Thomson and M. Thomas Clandinin
Nutrition and Metabolism Research Group, Department of Foods and Nutrition, Faculty of Home Economics and Division of Gastroenterologv, Faculty of Medicine, University of Alberta, Edmonton (Canada) (Received
Key words:
Fish oil; Arachidonic
25 May 1988)
acid: Cholesterol:
Fatty acid composition;
Serum; (Rat liver)
Rats were fed diets containing a high level of saturated fatty acids (hydrogenated beef tallow) versus a high level of linoleic acid (safflower oil) at both low and high levels of fish oil containing 7.5% (w/w) eicosapentaenoic and 2.5% (w/w) docosahexaenoic acids for a period of 28 days. The effect of feeding these diets on the cholesterol content and fatty acid composition of serum and liver lipids was examined. Feeding diets high in fish oil with safflower oil decreased the cholesterol content of rat serum, whereas feeding fish oil had no significant effect on the cholesterol content of serum when fed in combination with saturated fatty acids. The serum cholesterol level was higher in animals fed safflower oil compared to animals fed saturated fat without fish oil. Consumption of fish oil lowered the cholesterol content of liver tissue regardless of the dietary fat fed. Feeding diets containing fish oil reduced the arachidonic acid content of rat serum and liver lipid fractions, the decrease being more pronounced when fish oil was fed in combination with hydrogenated beef tallow than with safflower oil. These results suggest that dietary n - 3 fatty acids of fish oil interact with dietary linoleic acid and saturated fatty acids differently to modulate enzymes of cholesterol and fatty acid metabolism.
Introduction
Consumption of marine oils is apparently associated with a decreased risk of cardiovascular disease [l-4]. The biochemical changes following consumption of dietary fish oil include a reduction in circulating cholesterol and triacylglycerol levels and a decrease in platelet aggregation [l-4]. These effects have been attributed to the presence of large amounts of eicosapentaenoic (20 : 5( n - 3)) and/or docosahexaenoic (22 : 6( n - 3)) acid [l-4].
Correspondence: M.T. Clandinin, Department of Foods and Nutrition, 318f Home Economics Building, University of Alberta, Edmonton, Alberta, Canada, T6G 2M8. 0005-2760/88/$03.50
0 Elsevier Science. Publishers
B.V. (Biomedical
Recent studies have suggested that 22 : 6( n - 3) is responsible for the cholesterol-lowering effect [5], whereas 20 : 5( n - 3) contributes to changes in eicosanoid metabolism [6], leading to decreased platelet aggregation. A side-effect associated with the consumption of megadoses of fish oil is enhanced bleeding time [7]. Attempts have been made to find the efficacious amount of fish oil that may show desirable beneficial effects without undesirable side-effects, such as enhanced bleeding time. A range of dietary 20 : 5(n - 3) doses has been proposed by different authors [4,8]. A recent study has demonstrated that the balance between saturated and unsaturated fatty acids may be an important factor in the biosynthesis of long-chain polyunsaturated fatty acids (20 : 4(n - 6), 20 : 5( n Division)
338
- 3) 22 : 5(n - 6), 22 : 5(n - 3)) that serve as eicosanoid precursors [9]. We have recently shown that feeding linolenic acid affects synthesis of eicosanoid precursors in a different manner when fed along with saturated or n - 6 fatty acids [lo]. Therefore, it seems logical to hypothesize that dietary 20 : 5(n - 3) may also have a different effect on eicosanoid precursor 20 : 4( n - 6) synthesis when the balance of the diet fat is saturated or unsaturated fatty acids. In the present study, the cholesterol content and fatty acid composition of serum and liver lipid fractions of rats fed fish oil was examined in the presence of high amounts of either saturated fatty acids or linoleic acid. The results suggest that the ratio of dietary 18 : 2( n - 6) to saturated fatty acids may be an important determinant to the hypocholesterolemic and platelet antiaggregatory effects of fish oil. Materials and Methods Animals and diets. Male weanling SpragueDawley rats weighing approx. 40 g were fed Wayne rat chow (Allied Mills Inc., Chicago) for 3 days before switching to the experimental diets. Four high fat diets were prepared from the basal diet described previously in detail [ll]. Each diet contained 200 g fat/kg diet (20%, w/w) from one of the following sources: 90% hydrogenated beef tallow plus 10% safflower oil (hydrogenated beef tallow diet); 100% safflower oil (safflower oil diet); 65% hydrogenated beef tallow plus 25% fish oil plus 10% safflower oil (beef tallow and fish oil diet); 75% safflower oil plus 25% fish oil (safflower oil and fish oil diet). The fatty acid composition of the diets is given (Table I). The hydrogenated beef tallow diet contained mainly saturated fatty acids (86.3%) and enough linoleic acid (9.5%) to prevent essential fatty acid deficiency. The safflower oil diet was enriched with linoleic acid (70.5%). Both the hydrogenated beef tallow and the safflower oil diets at high levels of fish oil provided similar amounts of eicosapentaenoic (7.5 and 7.8%, respectively) and docosahexaenoic acids (2.6 and 2.4%, respectively) but differed in the linoleic acid to saturated fatty acid ratio (0.14 and 2.92, respectively) (Table I). Animals were housed individually in hanging stainless steel cages in a well-ventilated room maintained at 22 + 2’C on a 12/12 h light/dark
TABLE
1
FATTY ACID COMPOSITION MENTED DIETS
OF
THE
FAT-SUPPLE-
Diets were prepared fresh on a weekly basis and - 20 o C. Fatty acids are designated by the number atoms followed by the number of double bonds. The composition is expressed in %w/w and is the mean four random samples of diet. Diet fat
Beef tallow
Safflower
+ fish oil 14:o 16:0 16:l(n 17:o 18:O 18:l(n 18:l(n 18:2(n 18:3(n 20:4(n
-7)
-9) -7) -6) -3) -6)
20:5(n -3) 22:5(n -3) 22:6(n-3) Total Saturated Monounsaturated n-6 n-3 18 : 2( n -6)/saturated
4.2 28.9 0.5 2.1 51.2 3.2 0.2 9.5 0.1 _ _ _
86.3 4.1 9.5 0.1 0.11
stored at of carbon fatty acid of at least
oil + fish oil
5.0 23.8 3.5 0.5 39.3 5.0 0.9 9.4 0.8 0.4 1.5 0.6 2.6
0.3 7.6 0.1 0.1 5.8 14.6 0.2 70.5 0.6
68.6 9.9 9.8 11.5 0.14
13.8 14.9 70.5 0.6 5.11
_ _
2.0 7.6 3.2 0.6 9.6 6.4 0.4 51.6 0.9 0.3 7.x 0.6 2.4
19.8 10.0 57.9 11.7 2.92
cycle. All diets were prepared weekly and stored at -20°C. Food and water were available to rats ad libitum. The body weights of the animals were monitored once a week throughout the feeding period. Lipid analysis. After 4 weeks of feeding experimental diets, the rats were killed by decapitation (between 0800 and 1000 h). The livers were quickly excised and rinsed in ice-cold saline. Blood samples were also collected for separation of serum by centrifugation. Total lipids from serum and liver samples were extracted with chloroform/methanol (2 : 1) [12] containing 0.005% butylated hydroxytoluene. The total, free and esterified cholesterol contents in aliquots of serum and liver lipid extracts were determined enzymatically [13,14]. Total phospholipids, triacylglycerol and cholesterol esters were separated by thin-layer chromatography on silica gel G plates (Analtech, 250 pm, 20 X 20
339
TABLE
II
BODY WEIGHT, LIVER WEIGHT, LIVER WEIGHT/BODY WEIGHT RATIO FED DIETS CONTAINING LOW AND HIGH LEVELS OF EICOSAPENTAENOIC Rats were fed the diets for 4 weeks. Data are presented significantly different (P > 0.05). Diet fat
Body Liver Liver Food
weight (g) weight (g) weight/body consumption
weight (X) (g/day)
AND FOOD ACID
as means + SD. of five animals
CONSUMPTION
in each diet group.
OF RATS
None of the values are
Beef tallow
Safflower
-
+ fish oil
-
+ fish oil
248 +15 10.0 + 1.2 4.01+ 0.46 19.2 + 1.1
265 +23 10.4 + 1.3 3.94* 0.33 20.8 k 1.0
280 *32 12.2 + 1.9 4.34* 0.28 20.4 + 2.0
214 +20 12.1 + 1.1 4.76+ 0.34 19.9 f 1.2
cm) using a solvent system comprised of petroleum ether/diethyl ether/acetic acid (80 : 20 : 1, v/v) [15]. Phospholipids were subjected to methylation with BF,-methanol (1496, w/w) reagent followed by heating at 100°C for 1 h [16]. Triacylglycerol and cholesterol ester fractions were saponified with 0.5 M methanolic potassium hydroxide by heating at 85’C for 90 min followed by methylation with BF,-methanol (14%, w/w) reagent [16]. The fatty acid methyl esters were extracted in hexane and analyzed by automated capillary gas chromatography (Varian Model 6000 equipped with a flame ionization detector) [17]. Authentic standard mixtures of fatty acid methyl esters were injected to identify serum and liver fatty acid methyl esters. Peak areas and weight percent fatty acid composition were computed using a chromatography data system (Varian Model DS 604). Statistical analyses. Data are expressed as the mean f S.D. The effect of diet fat treatment was determined by analysis of variance procedures. The comparison between individual diet treatments was made using Newman-Keuls multiple range test [18]. Results Body weights, liver weights and food consumption
All animals appeared healthy after 4 weeks of feeding the experimental diets. Dietary fat treatment had no significant effect on body weights, liver weights or liver weight/body weight ratios in
oil
these animals. Rats were offered and consumed similar amounts of food (approx. 20 g/day) irrespective of the dietary regimen (Table II).
Cholesterol content of rat serum and liver
Serum and liver cholesterol concentrations are presented (Fig. 1). Animals fed the safflower oil diet had a significantly higher cholesterol content in serum compared to those fed the hydrogenated beef tallow diet (P < 0.05). Feeding the diet high in fish oil with safflower oil reduced the cholesterol content of serum while the hydrogenated beef tallow and fish oil diet had no significant effect. The increase in the cholesterol level following feeding of the safflower oil diet and the reduction in cholesterol level caused by the safflower oil and fish oil diet was associated with a change in level of the unesterified cholesterol fraction of the serum (Fig. 1). In liver tissue, the level of cholesterol was significantly higher in animals fed the safflower oil diet compared to the group fed the hydrogenated beef tallow diet. For rats fed the diet containing fish oil in combination with hydrogenated beef tallow or with safflower oil, the cholesterol content was significantly lower than that observed for rats fed the hydrogenated beef tallow or safflower oil diets, respectively. The increase in liver cholesterol concentration following feeding of the safflower oil diet was detected in both the free and esterified cholesterol content. The decrease in the liver cholesterol content resulting from both diets
340
Free =‘
Ester
Total
loo-
? g
b l80-
z ’ tj a, 0 6
60-
,T_
40-
q Beef Tallow n Beef Tallow 7 E z
240
180
and Fish Oil t3 Safflower
Oil
•I Safflower Oil and Fish Oil
&
Fig. 1. Effect of dietary
5 0
120
’@
60
C
fish oil on cholesterol content of rat serum and liver. Values are the means + SD. of five rats in each group. Values without a common superscript are significantly different (P c 0.05).
containing fish oil was found to be associated with the esterified and not with the unesterified cholesterol content (Fig. 1). Fatty acid composition of serum and liver lipids The fatty acid composition of rat serum and liver phospholipid, triacylglycerol and cholesterol ester fractions is illustrated (Figs. 2-4). Feeding the diet rich in safflower oil to rats increased the 18 : 2(n - 6) and 20 : 4(n - 6) contents in serum and liver phospholipids with an accompanied decrease in saturated (16 : 0 and/or 18 : 0) and (16 : 1, 18 : l(n - 9), 18 : l(n monounsaturated 7)) fatty acids and docosahexaenoic acid (22 : 6( n - 3)). Feeding fish oil with hydrogenated beef tallow reduced the 20 : 4(n - 6) content of both serum (61%) and liver (66%) phospholipids and was followed by a concomitant increase in the 20 : 5(n - 3) and 22 : 6(n - 3) content compared with that of the hydrogenated beef tallow diet.
Animals fed the diet containing fish oil in combination with safflower oil also exhibited a reduced 20 : 4(n - 6) content in phospholipids, but to a lesser extent (33% in serum and 31% in liver). Similarly, feeding animals the safflower oil and fish oil diet resulted in an accumulation of smaller amounts of 20 : 5( n - 3) and 22 : 6( n - 3) than observed for animals fed the hydrogenated beef tallow and fish oil diet. The fatty acid composition found in the serum triacylglycerol fraction resembled that of the dietary fat fed. For example, animals fed the diet containing hydrogenated beef tallow exhibited high levels of saturated (52.8%) and monounsaturated fatty acids (28.7%), those fed the safflower oil diet exhibited elevated levels of 18 : 2( n - 6) (60.7%) and rats fed diets containing fish oil had higher levels of 20 : 5( n - 3) and 22 : 6( n - 3) (16.1% and 9.7%) in serum triacylglycerols. In this lipid fraction, fish oil supplementation also de-
341
Liver
Serum b
PL EI Beef Tallow
TG
CE
n Beef Tallow
PL I3 Safflower
and Fish Oil
TG Oil
CE EI Safflower Oil and Fish Oil
Fig. 2. Effect of dietary fish oil on saturated and monousaturated fatty acid content of rat serum and liver lipid fractions: PL, phospholipid; TG, triacylglycerol; CE, cholesterol ester. Values are the meansfS.D. of five rats in each group. Values without a common superscript are significantly different (P < 0.05).
creased the 20 : 4(n - 6) levels both in the serum and in liver tissue. Feeding diets high in fish oil produced changes in the fatty acid composition of liver triacylglycerols similar to those in the serum. The cholesterol ester fraction of serum lipids exhibited the largest changes in 20 : 4(n - 6) (71% decrease) and 20 : 5( n - 3) (39.4-fold increase) content following consumption of the diet containing fish oil in association with hydrogenated beef tallow. Fish oil intake in combination with safflower oil could lower 20 : 4(n - 6) levels only by 8% and could accumulate 20 : 5(n - 3) to a much smaller extent. In the liver tissue, the 20 : 4(n - 6) content was lowered only when fish oil was fed in association with hydrogenated beef tallow and not with safflower oil. Accumulation of
20 : 5(n - 3) in the liver cholesterol ester fraction was greater when fish oil was fed along with hydrogenated beef tallow than when it was fed with safflower oil. Discussion Previous studies have demonstrated that consumption of fish oil reduces circulating lipid levels in animals as well as in human subjects [l-4,19-21] and is associated with decreased thrombus formation [22,23]. It has been suggested that 20 : 5( n - 3) present in fish oil competes with 20 : 4(n - 6) at the level of the cyclooxygenase, leading to decreased formation of thromboxane A, and increased synthesis of thromboxane A,, which, unlike thromboxane A 2, is not pro-aggregatory. Some
342
Serum
Liver
60 ‘;; k -0 .0 ; .P) 0 .f 1
48
36
24
Q Beef Tallow n Beef Tallow and Fish Oil c
q Safflower
Oil
LI Safflower Oil and Fish Oil
0
PL
TG
CE
PL
TG
CE
Fig. 3. Effect of dietary fish oil on linoleic and arachidonic acid content of rat serum and liver lipid fractions: PL, phospholipid; triacylglycerol; CE, cholesterol ester. Values are the means i S.D. of five rats in each group. Values without a common superscript significantly different (P i 0.05).
20 : 5(n - 3) is also converted to prostacyclin of the 3+eries, which has anti-aggregatory properties similar to those of PGI 2 generated from 20 : 4( n 6) [24,25]. Thus, the net result of fish oil consumption is to alter the eicosanoid balance, which in turn inhibits the platelet response to clot formation. We have recently shown that dietary fish oil inhibits A6- and A5-desaturase activities and reduces 20 : 4( n - 6) biosynthesis from 18 : 2( n - 6) [26,27]. Eicosanoids are derived from this common precursor, 20 : 4(n - 6). Therefore, it is conceivable that limitation in 20 : 4(n - 6) biosynthesis will impair thromboxane A, and prostacyclin I, formation. The present study demonstrates that fish oil lowers 20 : 4(n - 6) more efficiently when fed in combination with a high level of dietary saturated fatty acids than when it is fed with a high level of dietary 18 : 2(n - 6). However,
TG. are
20 : 5( n - 3) and/or 22 : 6( n - 3) accumulate in phospholipid, triacylglycerol and cholesterol ester fractions of rat serum and liver lipids to a greater extent when fish oil and saturated fat are fed together. Although we did not measure levels of thromboxane and prostacyclin in these animals, the 20 : 4(n - 6) content is known to reflect the pattern of thromboxane A, and prostacyclin I, produced [1,4] and the presence of 20 : 5( n - 3) indicates competitive inhibition [1,4] of the cyclooxygenase and formation of thromboxane A3 and prostacyclin I,. Thus, it is apparent that consumption of fish oil is likely to impair platelet responsiveness more efficiently when it is combined with hydrogenated beef tallow rather than with safflower oil. The smaller change in the 20 : 4( n - 6) content when rats are fed the diet containing fish oil and
343
Liver
Serum -
50r 0 Beef Tallow n Beef Tallow and Fish Oil 0 Safflower
Oil
q Safflower Oil and Fish Oil
b
!i z
b
6
Jz
z
s
3
0” 0
PL
TG
CE
PL
TG
CE
Fig. 4. Effect of dietary fish oil on eicosapentaenoic and docosahexaenoic acid content of rat serum and liver lipid fractions: PL, phospholipid; TG, triacylglycerol; CE, cholesterol ester. Values are the means+S.D. of five rats in each group. Values without a common superscript are significantly different (P < 0.05).
safflower oil together may be due to the high dietary 18 : 2(n - 6) content, resulting in a tendency to increase 20 : 4(n - 6) synthesis, an effect opposite to that known to be caused by the 20 : 5(n - 3) in fish oil [26,27]. When fish oil and hydrogenated beef tallow are fed, the impairment of 20 : 4(n - 6) biosynthesis by 20 : 5(n - 3) at the level of cyclooxygenase is maximal and is not opposed by substrate competition, thus resulting in maximum inhibition of platelet aggregation. The only known side-effect associated with consumption of megadoses of n - 3 fatty acids, as in the case of Greenland Eskimos, is an enhanced bleeding tendency [l-4]. Attempts have been made to define an appropriate proportion of dietary 20 : 5(n - 3) that may show beneficial effects without having side-effects, such as increase in clotting time [4,8]. Seafoods or fish oils are not a major part of the North American or European diet. Based upon extrapolation of the present study
to man, it is conceivable that a small amount of fish oil may inhibit the response of platelets to thrombus formation, provided that the major source of dietary fat is saturated fats. In liver tissue, the cholesterol content was lowered by dietary fish oil, irrespective of the nature of the dietary fat. Consumption of fish oil rich in 20 : 5(n - 3) and/or 22 : 6(n - 3) has recently been shown to accelerate the flow of cholesterol towards bile formation and secretion [28]. Thus, the decrease in the cholesterol content following the feeding of fish oil may be due to increased utilization of cholesterol for bile synthesis in the liver. Alternatively, the reduction in the liver cholesterol concentration after fish oil feeding may be due to the inhibition of hydroxy-’ methylglutarylcoenzyme A reductase activity [29], a rate-limiting enzyme in the cholesterol biosynthetic pathway. This latter alternative merits further investigation.
344
In summary, feeding fish oil in association with saturated fatty acids produces a greater reduction in the 20 : 4(n - 6) content and accumulation of more 20 : 5( n - 3) and/or 22 : 6( n - 3) in serum and liver lipid fractions than when fish oil is fed with a diet high in 18 : 2(n - 6). Dietary fish oil lowers the cholesterol content in liver regardless of the dietary fat fed. Althought it is inappropriate to interpret the animal data in humans, the present study suggests that fish oil may have greater antithrombotic effects in populations consuming fats from animal sources (beef tallow, lard, etc.) than those eating mainly vegetable oils (safflower oil, corn oil, etc.). Therefore, the data presented in this manuscript could provide a useful basis for formulating human clinical trials to examine the efficacy of fish oil supplementation on platelet aggregation, cholesterol and triacylglycerol metabolism in subjects consuming diets with low versus high linoleic acid to saturated fatty acid ratios. Acknowledgements This ces and M.T.C. Fellow Medical Wharton
work was supported by the Natural ScienEngineering Research Council of Canada. is a Scholar and M.L.G. is a Postdoctoral of the Alberta Heritage Foundation for Research. The technical assistance of T. is gratefully acknowledged.
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