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Effect of dietary palm oil on lipoprotein lipases: Lipoprotein levels and tissue lipids in rat
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
4,
207-217 (19%)
Effect of Dietary Palm Oil on Lipoprotein Lipases: Lipoprotein Levels and Tissue Lipids in Rat T. A. PEREIRA,* R. SINNIAH,~ Laboratory
AND N. P. DAS*
*Department of Biochemistry, and TDepartment of Pathology, of Flavonoid Research, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore
Received February 22, 1990; and in revised form June 7, 1990
It is well recognized that plasma concentrations of lipoprotein influence the risk of coronary heart disease (CHD). There is a direct correlation between the levels of low-density lipoprotein (LDL) cholesterol and the incidence of heart disease, whereas high-density lipoprotein (HDL) cholesterol has an inverse correlation and has what has been described as a “protective” effect (1,2). Quantitative determinations of lipids and apolipoproteins may not be sufficient to identify all patients who are at high risk for CHD. Qualitative changes in lipoprotein structure and function, possibly occurring in the vicinity of or within the arterial wall, may enhance the atherogenic potency of the lipoproteins by altering their interactions with other cells and with the arterial wall itself. Atherosclerotic plaque contains foam cells that originate from macrophages saturated with cholesterol and also from smooth muscle cells (3). Dietary lipids are known to cause experimental atherosclerosis in various animal models (4-9). Epidemiological studies clearly indicate that in man the type of dietary fat has a distinct influence on coronary artery disease, which is one of the clinical manifestations of atherosclerosis (10-S). The available data indicate that, in general, long-chain saturated fatty acids promote atherogenesis, whereas polyunsaturated fatty acids, present in vegetable and fish oils, inhibit the formation of atherosclerotic lesions. These dietary fats have been associated with an influence on cholesterol metabolism. It is known that arterial thrombosis is implicated in the formation and complications of atherosclerosis. Consequently, dietary lipids may influence atherosclerosis via the thrombotic pathway as well. In mammals, circulating triacylglycerols cannot cross biological membranes. For these neutral lipids to move from their carrier (chylomicrons, very lowdensity lipoproteins (VLDL) or fat droplets) into the tissues, they must be hydrolyzed with the release of fatty acids. This hydrolysis is performed by two enzymes: lipoprotein lipase (LPL; EC 3.1.1.34), which is located at the endothelium in extrahepatic tissues, and hepatic lipase (HL; EC 3.1.1.3). The hy-
0885-4505/90 $3.00 Copyright All rights of
Q 1990 by Academic Press, Inc. reproductionin my form reserved.
208
PEREIRA,
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AND DAS
TABLE 1 Fatty Acid Profile (wt %) of Dietary Fat Given to Control and Experimental Fatty acid
Rats
Butterfat
RBD palm oil
2.% 11.20 27.80 1.84 12.10 30.30 2.22 1.03 0.52
0.2 1.1 44.0 0.1 4.5 39.2 10.0 0.4 0.4
c:12 c:14 C:l6 C:l6:1 C:l8 C:l8:1 C: 18:2 C: 18:3 c:20: 1
drolytic activity of these two enzymes provides a major pathway by which endogenous or exogenous triacylglycerol-rich particles are cleared from the blood stream. Recently Mattson and Grundy (16) concluded that oleic acid is as effective as linoleic acid in lowering the plasma cholesterol level. We decided to carry out this study using palm oil, which has a monounsaturated/saturated fatty acid ratio of about 1, for our feeding experiments with rats. We were interested in examining whether there was any relationship between the antithrombotic effects of palm oil (15) and some related biochemical parameters. We therefore started a program to investigate the difference in levels of lipoprotein lipase, hepatic lipase, fat distribution in the aorta and liver, total cholesterol, HDL, LDL, and triacylglycerol in young rats (70 g body wt) fed a control or palm oil diet over a period of 10 weeks. The objective of this study was to provide additional information on the nutritional quality of palm oil. This study also tried to establish the fatty acid profile of dietary fats incorporated into the tissues of both the experimental and the control groups of rats, in order to confirm the hypothesis that palm oil, containing 50% saturated fatty acids, does not contribute to risk for coronary heart disease. Butterfat was chosen as the control dietary fat. MATERIALS
AND METHODS
Source of Palm Oil
Refined, bleached, and deodorized (RBD) palm oil was donated by Lam Soon Oil & Soap Manufacturing (S) Rte. Ltd., Singapore. Animals
Male Wistar rats, specific pathogen free and approximately 70 g body wt, were used. Two groups comprising six rats each, were fed a specially formulated powdered diet for 10 weeks. RBD palm oil and butterfat were the fat ingredients in the diets providing energy for basal metabolism and for muscular activity. The fatty acid profiles of RBD palm oil and butterfat are given in Table 1. Table 2 presents the total amount of food mixture consumed in 1 week. Water was
DIETARY
PALM OIL EFFECT
209
ON LIPASES
TABLE 2 The Composition of the Synthetic Powdered Food Mixture Consumed in 1 Week by Rats Diet (g)
Butterfat group
Ingredient Wheat flour Casein Dried yeast powder or-Methionine Sodium chloride RBD palm oil Butterfat Total energy distribution (%) Protein Carbohydrates Fat (Fat difference between two groups as total
520 200 30 0.3 10 95
Palm oil group 520 200 30 0.3 10 80 -
28.4 28.34 51.5 51.41 20.07 20.24 energy percentage is not significant)
Vitamin diet fortification mixture (one capsule)
Mineral mixture (one capsule)
Vitamin A palm&ate Cholecalciferol Thiamine hydrochloride Riboflavin Pyrdioxine hydrochloride Nicotinamide a-Tocopherol acetate Calcium pentothenate Cyanocobalamm (B,,) Ascorbic acid
Ferrous fumarate Cupric oxide Potassium iodide Magnesium sulfate Manganese sulfate Sodium molybdate Potassium sulfate Zinc oxide Calcium carbonate Calcium phosphate dibasic
7000 IU 400 IU 10 mg 10 mg 2 mg 25 mg 101 IU 8 mg 2 Pi? 75 mg
50 mg 1.0 mg 0.15 mg 3.0 mg 1.0 mg 0.2 mg 5.0 mg 1.5 mg 25.0 mg 10.0 mg
Note. Purchased from NYAL Co. of Sydney, Australia.
given ad libitum. The weight of the animals was recorded weekly throughout the experiment (Fig. 1). At the end of the experiment, the animals were killed under light ether anesthesia. Blood Collection Blood was collected from the inferior vena cava into a test tube containing EDTA (1 mg/ml of blood) and then centrifuged to obtain the plasma for cholesterol and triacylglycerol determinations. The carcasses were immediately dissected after blood, liver, and adipose tissue were removed, weighed, and frozen. GLC Analysis Frozen blood plasma and tissue homogenates of adipose tissue and liver were extracted with Folch’s reagent (32), methylated with BF, (33), and analyzed for component fatty acids using a Varian Model 3700 gas chromatograph. Staining of Tissue Sections The aorta and liver were removed immediately and preserved in formalin. Sections of aorta and liver of animals fed either the control or the experimental
210
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diet were prepared and stained with hematoxylin and eosin (H & E) and frozen sections were stained for fat with Oil Red “0” stains. Plasma
Cholesterol
and Triacylglycerol
Analysis
Total cholesterol, HDL cholesterol, LDL cholesterol, and triacylglycerol in the plasma were analyzed using commercially available diagnostic kits supplied by Boehringer-Mannheim Gmbh Co. (Singapore). Analysis for Lipoprotein and Hepatic Lipase (17) (i) Preparation of the samples for lipoprotein lipase (LPL) assay. The subcutaneous adipose tissue was dissected from the cervical, dorsal, and lumbar regions of the rat and stored at -20°C. The tissue was macerated in a meat grinder, defatted in small pieces with acetone, and extracted for 1 h at 0°C with 0.025 M ammonia solution (50 mg/ml). The insoluble part was removed by centrifugation and the supematant solution was lyophilized. The final powder was dissolved in 0.025 M ammonia (10 mg/ml) immediately before use. (ii) Preparation of samples for hepatic lipase (HL) assay. Liver was removed and stored at -20°C. The tissues were ground and the experimental procedure was followed as above (1). LPL and HL Assay From each sample, the respective lipase assays were carried out using the Boehringer-Mannheim Gmbh Diagnostic Kit, Monotest Lipase, Cat. No. 159697, EC 3.1.1.3. Statistical Analysis The correlation coefficients and their significance cording to Snedecor and Cochran (30).
(t test) were calculated
ac-
RESULTS The mean body weight changes of the experimental and control groups of rats are shown in Fig. 1. The mean values of plasma lipoproteins (total, HDL, and LDL) and triacylglycerol are given in Table 3. The mean values of lipoprotein lipase (LPL) and hepatic endothelial lipase (HL) are given in Table 4. The fatty acid profiles of plasma, liver tissue, and adipose tissue of rats fed RBD palm oil and butterfat are given in Tables 5 and 6, respectively. Histopathology (i) Aorta. Fat stain (Oil Red 0) showed an absence of fat in the endothelium of the aorta of both control and palm oil fed animals. No plaques were observed in the H & E-stained slides of the aorta, thereby confirming the absence of atheroma. In some cases, however, fat is seen to leak into the wall of the aorta from periadventitial fat during the process of sectioning and staining. (ii) Liver. Sections of the liver of control animals showed moderate amounts of fats (+2 grade fat) within the hepatocytes, mainly in the centrizonal areas. H & E staining showed no evidence of cell necrosis.
DIETARY
PALM
OIL EFFECT
211
ON LIPASES
250
200
b .c 4 z 150 6 i 3 til 2 : ( 100
50
0
FIG. 1
2
4 No. of Weeks
6
6
Average weight of rats. 0, Control diet; 0, palm oil diet.
In the palm oil-fed experimental group, there was centrizonal to diffuse fatty change (+2.5) within the hepatocytes. Some liver cells showed spotty cell necrosis and lymphomonocytic cellular infiltration. DISCUSSION Atheroma is a disease process in which lipid materials are deposited in the intima of blood vessels predominantly within smooth muscles and macrophages. The lipids are mainly cholesterol and cholesterol esters. Both chemically and biologically modified forms of LDL are preferentially taken up and degraded by these cells through scavenger receptors (18). Although confirmation that such events are an integral part in the development of atherogenesis is lacking, serum lipid peroxides are reported to be elevated in patients suffering from atherosclerosis. Table 5 shows that palmitic acid (C:16, saturated fatty acid) is deposited at twice the amount in adipose and liver tissues compared to the plasma of rats
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DIETARY
PALM
213
OIL EFFECT ON LIPASES
TABLE 4 Effect of Dietary Palm Oil on Lipoprotein Lipase (LPL) and Hepatic Endothelial after 10 Weeks of Feeding in Rats Diet
LPUmU/g)
HLWJ/d
Control Palm oil
36.0 + 1.26 46.7 + 0.16
7.5 + 0.22 12.9 + 0.14
Note. Correlation values for both groups are LPL:LDL,
Lipase (HL) Liver fat grade
r = 0.617; HL:Liver
2.0 2.5 fat, r = 0.682.
TABLE 5 Fatty Acid Profile (wt %) of Liver, Plasma, and Adipose Tissue of Rats Fed a RBD Palm Oil Diet Fatty acid c:12 c:14 C:16 C:16:1 C:18 C:18:1 c: 18:2 c:20 C: 18:3 c:20: 1 C:22:5 C:22:6
Plasma
Liver 0.25 0.33 37.17 0.19 10.40 38.70 10.36 0.00 0.10 1.10 0.20 1.20
I ++-e k k k
0.01 0.03 1.13 0.07 1.16 1.09 1.10
5 + k 4
0.01 0.02 0.01 0.07
0.15 0.35 20.42 0.28 6.38 30.46 14.09 0.50 0.20 0.54 0.15 1.10
f 0.01 + 0.03 + 1.00 + 0.01 i 0.34 f 2.01 2 1.39 + 0.02 -+ 0.03 + 0.03 f 0.02 +- 0.03
Adipose tissue 0.123 1.970 40.300 0.710 3.110 37.470 15.150 0.00 0.470 0.270 0.130 0.190
f + k + 2 f f
0.010 0.035 1.600 0.040 0.708 3.170 2.750
f 2 I? +
0.170 0.090 0.030 0.030
TABLE 6 Fatty Acid Profile (wt %) of Liver, Plasma, and Adipose Tissue of Rats Fed a Butterfat Diet Butterfat Fatty acid c:12 c:14 C:l6 C:16:1 C:l8 C:18:1 C:l8:2 c:20 C: 18:3 c:20:1 C:22:5 C:22:6
Liver 2.50 IO.60 28.60 1.50 17.80 30.10 1.10 0.10 0.60 0.40 0.20 1.10
2 + 2 2 k -c 2 + 2 2 + +
Plasma 0.11 0.10 1.40 0.01 1.09 1.10 0.01 0.01 0.02 0.01 0.03 0.01
1.50 6.37 16.50 2.80 10.40 29.80 2.10 0.30 1.03 0.30 0.15 1.00
-e 2 a k t r 2 * r k -e 2
0.03 0.13 1.20 0.35 0.58 1.60 0.35 0.04 0.28 0.01 0.02 0.03
Adipose tissue 2.10 10.37 24.88 1.70 6.80 30.20 2.00 0.10 0.80 0.20 0.12 0.20
f 2 f f f + 2 + f f + 2
0.11 0.20 1.70 0.50 0.48 2.61 0.30 0.06 0.17 0.04 0.00 0.00
214
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fed RBD palm oil. C:18 (stearic acid, a saturated fatty acid) is also deposited more in the liver than in the plasma. A possible explanation could be chain elongation of palmitic acid in the liver to form stearic acid or oleic acid by desaturation by enzymes in the liver (36). Palm oil, which contains 39% monounsaturated oleic acid and 10% linoleic acid, has been found to be antithrombotic (15) and it has been wrongly associated with palm kernel oil, which contains 81% palmitic, myristic, and lauric acids which are implicated in atherogenesis (34). The fact that there was no fat deposit in the aorta may mean that the saturated fatty acids might have been removed by lipolysis by cyclic AMP and HDL after deposition. The cyclic AMP was reported to inhibit cholesterol biosynthesis (41) and to exert a regulatory function on platelets, which depends on the balance between thromboxane A2, prostagladin endoperoxide, and prostacyclin PGl, (43). It has also been reported to aid in lipolysis of fat attached to the heart muscle, thereby enhancing HDL as a clearing agent for LDL. The large deposit of palmitic acid in the adipose tissue supports the hypothesis that the saturated fatty acids have been preferentially transferred from the plasma and incorporated into liver and adipose tissue; conversion of stearic acid to oleic acid (36) also supports the above hypothesis. The most likely explanation for the increased hepatic level of fat may be the reduced output of lipids from the liver to the plasma (29). A high level of linoleic acid is essential for normal development of the brain and the retina (40) and its deficiency was reported to impair coordinative performance (39). The fatty acid profiles in plasma, liver, and adipose tissue of rats fed palm oil and butterfat demonstrate that the total fatty acid composition of the dietary oil influenced the disposition of fatty acids in tissue lipids (38). In view of an earlier report (21) it may be pertinent to suggest that the faster rate of weight gain of palm oil-fed rats compared to controls after 5 weeks of the diet (Fig. 1) may be due to icreaseed palatability and the essential fatty acids in the diet. We found that the hepatic lipase level in the experimental rat group was higher than that of the control group (Table 4) and there was a moderately greater amount of fats (+ 2.5 grade fat) compared to the control group ( + 2 grade fat). Fat deposit was reported to increase in the liver when hepatic lipase activity increased (22). LPL activity correlated to LDL cholesterol levels (r = +0.617), and the plasma triacylglycerol levels were correlated inversely (r = -0.536) to HDL cholesterol for both group of rats (Table 4). There was a higher LDL level in the experimental group of rats than in the control group. It is possible that the degree of saturation of the fatty acids (palmitic and oleic acids) of membrane phospholipids could have affected the activities of the membrane bound enzymes LPL and HL and the degree of degredation of LPL by fibroblasts-by both receptor-mediated and receptor-independent pathways (23)-and therefore resulted in higher plasma LDL levels. Palm oil-fed rats had lower triacylglycerol and correspondingly higher HDL levels compared to those of the control group, which had lower HDL and higher triacylglycerol levels (Table 3). Palm oil contains 11% o-6 fatty acids (25). Boberg
DIETARY
PALM
OIL EFFECT ON LIPASES
215
et al. (24) reported that when o-6 fatty acid was included in the diet of rats, the level of triacylglyceride decreased while the HDL and LDL cholesterol levels increased. They attributed their observation to the increased conversion of VLDL to LDL (24). In view of this report, it is likely that our present result is brought about by the w-6 fatty acid that is known to be present in palm oil at a 10% level (25). Keys et al. (19) have developed a formula that correlates dietary fat and serum cholesterol; it may be desirable to use twice as much polyunsaturated fat (P), mainly linoleic acid, than saturated fat (S), mainly palmitic acid, to lower serum cholesterol levels. Although palm oil has a monounsaturated/saturated fatty acid ratio of 1, it does not cause deposition of fats in the aorta and brings about triacylglycerol levels lower than those of the control rats, over a IO-week period (Table 3). The HDL cholesterol to total plasma cholesterol ratio (Table 3) of the experimental group was significantly higher than that of the control group. Currently this ratio is found to be a more useful index for determining the quality and effects of fat on health and indicates that the fat present in the diet leads to a lesser tendency toward development of atherosclerosis (31). The channeling of saturated fatty acid, palmitic and stearic, to adipose and liver tissues may account for the lower concentration of these fatty acids in the plasma and lack of their deposition in the arterial wall, and may also explain the antithrombotic tendency of palm oil. Thus, in rat palm oil diet caused some changes in several lipid panels, which may not contribute to the risk for development of coronary heart disease.
SUMMARY
The aims of our study were to investigate the effect of dietary palm oil on the levels of lipoprotein lipase, hepatic lipase, fat distribution (in the aorta and liver), and total cholesterol, HDL, LDL, and triacylglycerol levels in young rats (70 g body wt) over a period of 10 weeks. Palm oil-fed rats showed higher growth rate and lower triacylglycerol levels than the control group. Hepatic lipase activity was correlated to the liver fat distribution (correlation coefficient, r = +0.682) as seen by histopathological sections and was similar for both the palm oil and the control diets. Palm oil-fed rats exhibited a significantly higher HDL cholesterol to total plasma cholesterol ratio when compared to animals fed the control diet. The triacylglycerol levels correlated inversely to the HDL cholesterol levels (r = -0.536) while the lipoprotein lipase (LPL) activity correlated directly to the LDL level (r = +0.617) for both groups of animals. The fatty acid profiles of adipose and liver tissues and plasma revealed that saturated fatty acidspalmitic and stearic-were preferentially incorporated in liver and adipose tissues and less in the plasma. This accounts for lack of deposition in the arterial wall and for the antithrombotic tendency of palm oil. Thus, our present findings suggest that dietary palm oil may not contribute to the risk for coronary heart disease.
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ACKNOWLEDGMENTS We thank Dr. L. Retnam of the Animal House, Dr. G. S. Shahi of the Department of Physiology, Dr. T. N. Khan of the Department of Pathology, and Mr. J. Lau of the Department of Biochemistry for technical assistance and Messrs. T. C. Ow and T. C. Tan of the Department of Pathology for the preparation of tissue sections. We are grateful to the National University of Singapore for Research Grant RP 890339.
REFERENCES 1. Castelli, W. P., Abbott, R. D., and McNamara, P. M., Circulation 67, 730-738 (1983). 2. Wilson, P. W., Garrison, R. J., Castelli, W. P., and Feinlib, M., Amer. J. Curdiol. 46, 649-655 (1980). 3. Ross, R., N. 4. Vies, R. O.,
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22. 23. 24. 25. 26. 27.
Engl. J. Med. 314, 488-500 (1986). Buller, J., Gottenbos, J. J., and Thomasson, H. J., J. Atheroscler.
Res. 4, 170183 (1964). Kritchevsky, D., Ann. N.Y. Acud. Sci. 162, 80-88 (1%9). Malmros, H., Lancet 2, 479-484 (1%9). Wissler, R. W., and Vesselinovitch, D., Adv. Exp. Med. Biol. 60, 65-76 (1975). Weber, G., in “Regression of Arterial Lesions: Facts and Problems” (L. A. Carlson, R. Paoletti, C. Sirtori, and G. Weber, Eds.), pp. l-13, International Conference on Atherosclerosis. Raven Press, New York, 1978. Howard, C. F., Jr., Adv. Exp. Med. Biol. 60, 13-31 (1975). Kannel, W. B., in “Results of the Epidemiologic Investigation of Ischaemic Heart Disease: Illustrated by the Framingham Study” (des Haas, Hemker, and Snellen, Eds.), pp. 272-310. University Press, Leiden, 1970. Stamler, J., Med. Clin. North Amer. 57, 5-46 (1973). Heyden, S., in “The Role of Fats in Human Nutrition” (A. J. Vergroesen, Ed.), pp. 43-113, Academic Press, London/New York, 1975. Renaud, S., Morazain, R., McGregor, L., and Baudier, F., Huemostusis 8, 234-251 (1979). Kronmann, N., and Green, A., Actu Med. Scund. 208, 401-406 (1980). Honstra, G., Hennisen, A. A. H. M., Tan, D. T. S., and Kalafuzs, R., “Beneficial Effects of Palm Oil on Arterial Thrombosis (Rat) and Atherosclerosis (Rabbit).” Palm oil Research Institute of Malaysia, Publication No. ISBN 967-961-013-6, 1986. Mattson, F. H., and Grundy, S. M., J. Lipid Res. 26, 194-202 (1985). Nilsson-EhIe, P., and Ekman, R., Artery 3, 194-209 (1977). Goldstein, J. L., Ho, Y. K., and Brown, M. S., Annu. Rev. Biochem. 46, 897-930 (1977). Keys, A., Anderson, J. T., and Grande, F., Metablism 14, 747-775 (1965). Sturdevant, R. A. L., Pearce, M. L., and Dayton, S., N. Engl. J. Med. 288, 24-27 (1973). Umoh, I. B., Ayalogu, E. O., and Oke, 0. L., Food Chem. 10, 83-95 (1983). Haug, A., Hostmark, A. T., and Spydevold, O., Actu Physiof. Scund. 125, 609-617 (1985). Dietsky, J. M., Klin. Wochenschr. 62, 338-345 (1984). Boberg, M., Vessby, B., and Selinus, I., Actu Med. &and. 228, 153-160 (1986). Chong, Y. H., Palm Oil Research Institute of Malaysia. Ministry of Primary Industries, MaIaysiaBulletin (1988). Horrobin, D. F., and Manku, M. S., Lipids 18, 558-562 (1983). Wood, D. A., Butler, S., Riemersma, R. A., Thomson, M., and Oliver, M. F., Lancer 2, 117122 (1984).
28. Honstra, G., Nutr. Rev. 45, 205-207 (1987). 29. Haug, A., and Hostmark, A. T., J. Nutr. 117, 1011-1017 (1987). 30. Snedecor, G. W., and Cochran, W. G., “Statistical Methods,” p. 172. Iowa State University Press, Ames, IA, 1971. 31. Kris-Etherton, P. M., Chih Ying Ho, and Fosmire, M. A., J. Nutr. 114, 1675-1682 (1984). 32. Folch, J., Lees, M., and Sloane-Stanley, G. H. S., J. Biol. Chem. 226, 497-509 (1957).
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33. Metcalfe, L. D., Smitz, A. A., and Pelka, J. B., Anal. Chem. 33, 363-364 (l%l). 34. Kritchevsky, D., Tepper, S. A., Bises, G., and Klurfeld, D. M., Atherosclerosis
41, 279-284
(1982). 35. 36.
37. 38. 39. 40.
41. 42. 43.
Beare, J. L., Canad. J. Biochem. 39, 1855-1863 (1961). Elovson, J., Biochim. Biophys. Acta 106, 480-494 (1%5). Pugh, E. L., and Kates, M., Lipids 19, 48-55 (1984). Nwokolo, E. N., and Kitts, D. D., Food Chem. 30, 219-229 (1988). Lumpty, M. S., and Walker, B. L., .I. Nutr. 106, 86-93 (1976). Neuringer, M., and Connor, W. E., Nutr. Rev. 44, 285-294 (1986). Horrobin, D. F., Med. Hypotheses 6, 785-800 (1980). Trevke, and Meyer, Nature (London) 200, 363-364 (1963). Gorman, R. R., Bunting, S., and Miller, 0. V., Adv. Cyclic Nucleotide
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MEDICINE
AND
METABOLIC
BIOLOGY
4,
207-217 (19%)
Effect of Dietary Palm Oil on Lipoprotein Lipases: Lipoprotein Levels and Tissue Lipids in Rat T. A. PEREIRA,* R. SINNIAH,~ Laboratory
AND N. P. DAS*
*Department of Biochemistry, and TDepartment of Pathology, of Flavonoid Research, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Republic of Singapore
Received February 22, 1990; and in revised form June 7, 1990
It is well recognized that plasma concentrations of lipoprotein influence the risk of coronary heart disease (CHD). There is a direct correlation between the levels of low-density lipoprotein (LDL) cholesterol and the incidence of heart disease, whereas high-density lipoprotein (HDL) cholesterol has an inverse correlation and has what has been described as a “protective” effect (1,2). Quantitative determinations of lipids and apolipoproteins may not be sufficient to identify all patients who are at high risk for CHD. Qualitative changes in lipoprotein structure and function, possibly occurring in the vicinity of or within the arterial wall, may enhance the atherogenic potency of the lipoproteins by altering their interactions with other cells and with the arterial wall itself. Atherosclerotic plaque contains foam cells that originate from macrophages saturated with cholesterol and also from smooth muscle cells (3). Dietary lipids are known to cause experimental atherosclerosis in various animal models (4-9). Epidemiological studies clearly indicate that in man the type of dietary fat has a distinct influence on coronary artery disease, which is one of the clinical manifestations of atherosclerosis (10-S). The available data indicate that, in general, long-chain saturated fatty acids promote atherogenesis, whereas polyunsaturated fatty acids, present in vegetable and fish oils, inhibit the formation of atherosclerotic lesions. These dietary fats have been associated with an influence on cholesterol metabolism. It is known that arterial thrombosis is implicated in the formation and complications of atherosclerosis. Consequently, dietary lipids may influence atherosclerosis via the thrombotic pathway as well. In mammals, circulating triacylglycerols cannot cross biological membranes. For these neutral lipids to move from their carrier (chylomicrons, very lowdensity lipoproteins (VLDL) or fat droplets) into the tissues, they must be hydrolyzed with the release of fatty acids. This hydrolysis is performed by two enzymes: lipoprotein lipase (LPL; EC 3.1.1.34), which is located at the endothelium in extrahepatic tissues, and hepatic lipase (HL; EC 3.1.1.3). The hy-
0885-4505/90 $3.00 Copyright All rights of
Q 1990 by Academic Press, Inc. reproductionin my form reserved.
208
PEREIRA,
SINNIAH,
AND DAS
TABLE 1 Fatty Acid Profile (wt %) of Dietary Fat Given to Control and Experimental Fatty acid
Rats
Butterfat
RBD palm oil
2.% 11.20 27.80 1.84 12.10 30.30 2.22 1.03 0.52
0.2 1.1 44.0 0.1 4.5 39.2 10.0 0.4 0.4
c:12 c:14 C:l6 C:l6:1 C:l8 C:l8:1 C: 18:2 C: 18:3 c:20: 1
drolytic activity of these two enzymes provides a major pathway by which endogenous or exogenous triacylglycerol-rich particles are cleared from the blood stream. Recently Mattson and Grundy (16) concluded that oleic acid is as effective as linoleic acid in lowering the plasma cholesterol level. We decided to carry out this study using palm oil, which has a monounsaturated/saturated fatty acid ratio of about 1, for our feeding experiments with rats. We were interested in examining whether there was any relationship between the antithrombotic effects of palm oil (15) and some related biochemical parameters. We therefore started a program to investigate the difference in levels of lipoprotein lipase, hepatic lipase, fat distribution in the aorta and liver, total cholesterol, HDL, LDL, and triacylglycerol in young rats (70 g body wt) fed a control or palm oil diet over a period of 10 weeks. The objective of this study was to provide additional information on the nutritional quality of palm oil. This study also tried to establish the fatty acid profile of dietary fats incorporated into the tissues of both the experimental and the control groups of rats, in order to confirm the hypothesis that palm oil, containing 50% saturated fatty acids, does not contribute to risk for coronary heart disease. Butterfat was chosen as the control dietary fat. MATERIALS
AND METHODS
Source of Palm Oil
Refined, bleached, and deodorized (RBD) palm oil was donated by Lam Soon Oil & Soap Manufacturing (S) Rte. Ltd., Singapore. Animals
Male Wistar rats, specific pathogen free and approximately 70 g body wt, were used. Two groups comprising six rats each, were fed a specially formulated powdered diet for 10 weeks. RBD palm oil and butterfat were the fat ingredients in the diets providing energy for basal metabolism and for muscular activity. The fatty acid profiles of RBD palm oil and butterfat are given in Table 1. Table 2 presents the total amount of food mixture consumed in 1 week. Water was
DIETARY
PALM OIL EFFECT
209
ON LIPASES
TABLE 2 The Composition of the Synthetic Powdered Food Mixture Consumed in 1 Week by Rats Diet (g)
Butterfat group
Ingredient Wheat flour Casein Dried yeast powder or-Methionine Sodium chloride RBD palm oil Butterfat Total energy distribution (%) Protein Carbohydrates Fat (Fat difference between two groups as total
520 200 30 0.3 10 95
Palm oil group 520 200 30 0.3 10 80 -
28.4 28.34 51.5 51.41 20.07 20.24 energy percentage is not significant)
Vitamin diet fortification mixture (one capsule)
Mineral mixture (one capsule)
Vitamin A palm&ate Cholecalciferol Thiamine hydrochloride Riboflavin Pyrdioxine hydrochloride Nicotinamide a-Tocopherol acetate Calcium pentothenate Cyanocobalamm (B,,) Ascorbic acid
Ferrous fumarate Cupric oxide Potassium iodide Magnesium sulfate Manganese sulfate Sodium molybdate Potassium sulfate Zinc oxide Calcium carbonate Calcium phosphate dibasic
7000 IU 400 IU 10 mg 10 mg 2 mg 25 mg 101 IU 8 mg 2 Pi? 75 mg
50 mg 1.0 mg 0.15 mg 3.0 mg 1.0 mg 0.2 mg 5.0 mg 1.5 mg 25.0 mg 10.0 mg
Note. Purchased from NYAL Co. of Sydney, Australia.
given ad libitum. The weight of the animals was recorded weekly throughout the experiment (Fig. 1). At the end of the experiment, the animals were killed under light ether anesthesia. Blood Collection Blood was collected from the inferior vena cava into a test tube containing EDTA (1 mg/ml of blood) and then centrifuged to obtain the plasma for cholesterol and triacylglycerol determinations. The carcasses were immediately dissected after blood, liver, and adipose tissue were removed, weighed, and frozen. GLC Analysis Frozen blood plasma and tissue homogenates of adipose tissue and liver were extracted with Folch’s reagent (32), methylated with BF, (33), and analyzed for component fatty acids using a Varian Model 3700 gas chromatograph. Staining of Tissue Sections The aorta and liver were removed immediately and preserved in formalin. Sections of aorta and liver of animals fed either the control or the experimental
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diet were prepared and stained with hematoxylin and eosin (H & E) and frozen sections were stained for fat with Oil Red “0” stains. Plasma
Cholesterol
and Triacylglycerol
Analysis
Total cholesterol, HDL cholesterol, LDL cholesterol, and triacylglycerol in the plasma were analyzed using commercially available diagnostic kits supplied by Boehringer-Mannheim Gmbh Co. (Singapore). Analysis for Lipoprotein and Hepatic Lipase (17) (i) Preparation of the samples for lipoprotein lipase (LPL) assay. The subcutaneous adipose tissue was dissected from the cervical, dorsal, and lumbar regions of the rat and stored at -20°C. The tissue was macerated in a meat grinder, defatted in small pieces with acetone, and extracted for 1 h at 0°C with 0.025 M ammonia solution (50 mg/ml). The insoluble part was removed by centrifugation and the supematant solution was lyophilized. The final powder was dissolved in 0.025 M ammonia (10 mg/ml) immediately before use. (ii) Preparation of samples for hepatic lipase (HL) assay. Liver was removed and stored at -20°C. The tissues were ground and the experimental procedure was followed as above (1). LPL and HL Assay From each sample, the respective lipase assays were carried out using the Boehringer-Mannheim Gmbh Diagnostic Kit, Monotest Lipase, Cat. No. 159697, EC 3.1.1.3. Statistical Analysis The correlation coefficients and their significance cording to Snedecor and Cochran (30).
(t test) were calculated
ac-
RESULTS The mean body weight changes of the experimental and control groups of rats are shown in Fig. 1. The mean values of plasma lipoproteins (total, HDL, and LDL) and triacylglycerol are given in Table 3. The mean values of lipoprotein lipase (LPL) and hepatic endothelial lipase (HL) are given in Table 4. The fatty acid profiles of plasma, liver tissue, and adipose tissue of rats fed RBD palm oil and butterfat are given in Tables 5 and 6, respectively. Histopathology (i) Aorta. Fat stain (Oil Red 0) showed an absence of fat in the endothelium of the aorta of both control and palm oil fed animals. No plaques were observed in the H & E-stained slides of the aorta, thereby confirming the absence of atheroma. In some cases, however, fat is seen to leak into the wall of the aorta from periadventitial fat during the process of sectioning and staining. (ii) Liver. Sections of the liver of control animals showed moderate amounts of fats (+2 grade fat) within the hepatocytes, mainly in the centrizonal areas. H & E staining showed no evidence of cell necrosis.
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OIL EFFECT
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ON LIPASES
250
200
b .c 4 z 150 6 i 3 til 2 : ( 100
50
0
FIG. 1
2
4 No. of Weeks
6
6
Average weight of rats. 0, Control diet; 0, palm oil diet.
In the palm oil-fed experimental group, there was centrizonal to diffuse fatty change (+2.5) within the hepatocytes. Some liver cells showed spotty cell necrosis and lymphomonocytic cellular infiltration. DISCUSSION Atheroma is a disease process in which lipid materials are deposited in the intima of blood vessels predominantly within smooth muscles and macrophages. The lipids are mainly cholesterol and cholesterol esters. Both chemically and biologically modified forms of LDL are preferentially taken up and degraded by these cells through scavenger receptors (18). Although confirmation that such events are an integral part in the development of atherogenesis is lacking, serum lipid peroxides are reported to be elevated in patients suffering from atherosclerosis. Table 5 shows that palmitic acid (C:16, saturated fatty acid) is deposited at twice the amount in adipose and liver tissues compared to the plasma of rats
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OIL EFFECT ON LIPASES
TABLE 4 Effect of Dietary Palm Oil on Lipoprotein Lipase (LPL) and Hepatic Endothelial after 10 Weeks of Feeding in Rats Diet
LPUmU/g)
HLWJ/d
Control Palm oil
36.0 + 1.26 46.7 + 0.16
7.5 + 0.22 12.9 + 0.14
Note. Correlation values for both groups are LPL:LDL,
Lipase (HL) Liver fat grade
r = 0.617; HL:Liver
2.0 2.5 fat, r = 0.682.
TABLE 5 Fatty Acid Profile (wt %) of Liver, Plasma, and Adipose Tissue of Rats Fed a RBD Palm Oil Diet Fatty acid c:12 c:14 C:16 C:16:1 C:18 C:18:1 c: 18:2 c:20 C: 18:3 c:20: 1 C:22:5 C:22:6
Plasma
Liver 0.25 0.33 37.17 0.19 10.40 38.70 10.36 0.00 0.10 1.10 0.20 1.20
I ++-e k k k
0.01 0.03 1.13 0.07 1.16 1.09 1.10
5 + k 4
0.01 0.02 0.01 0.07
0.15 0.35 20.42 0.28 6.38 30.46 14.09 0.50 0.20 0.54 0.15 1.10
f 0.01 + 0.03 + 1.00 + 0.01 i 0.34 f 2.01 2 1.39 + 0.02 -+ 0.03 + 0.03 f 0.02 +- 0.03
Adipose tissue 0.123 1.970 40.300 0.710 3.110 37.470 15.150 0.00 0.470 0.270 0.130 0.190
f + k + 2 f f
0.010 0.035 1.600 0.040 0.708 3.170 2.750
f 2 I? +
0.170 0.090 0.030 0.030
TABLE 6 Fatty Acid Profile (wt %) of Liver, Plasma, and Adipose Tissue of Rats Fed a Butterfat Diet Butterfat Fatty acid c:12 c:14 C:l6 C:16:1 C:l8 C:18:1 C:l8:2 c:20 C: 18:3 c:20:1 C:22:5 C:22:6
Liver 2.50 IO.60 28.60 1.50 17.80 30.10 1.10 0.10 0.60 0.40 0.20 1.10
2 + 2 2 k -c 2 + 2 2 + +
Plasma 0.11 0.10 1.40 0.01 1.09 1.10 0.01 0.01 0.02 0.01 0.03 0.01
1.50 6.37 16.50 2.80 10.40 29.80 2.10 0.30 1.03 0.30 0.15 1.00
-e 2 a k t r 2 * r k -e 2
0.03 0.13 1.20 0.35 0.58 1.60 0.35 0.04 0.28 0.01 0.02 0.03
Adipose tissue 2.10 10.37 24.88 1.70 6.80 30.20 2.00 0.10 0.80 0.20 0.12 0.20
f 2 f f f + 2 + f f + 2
0.11 0.20 1.70 0.50 0.48 2.61 0.30 0.06 0.17 0.04 0.00 0.00
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fed RBD palm oil. C:18 (stearic acid, a saturated fatty acid) is also deposited more in the liver than in the plasma. A possible explanation could be chain elongation of palmitic acid in the liver to form stearic acid or oleic acid by desaturation by enzymes in the liver (36). Palm oil, which contains 39% monounsaturated oleic acid and 10% linoleic acid, has been found to be antithrombotic (15) and it has been wrongly associated with palm kernel oil, which contains 81% palmitic, myristic, and lauric acids which are implicated in atherogenesis (34). The fact that there was no fat deposit in the aorta may mean that the saturated fatty acids might have been removed by lipolysis by cyclic AMP and HDL after deposition. The cyclic AMP was reported to inhibit cholesterol biosynthesis (41) and to exert a regulatory function on platelets, which depends on the balance between thromboxane A2, prostagladin endoperoxide, and prostacyclin PGl, (43). It has also been reported to aid in lipolysis of fat attached to the heart muscle, thereby enhancing HDL as a clearing agent for LDL. The large deposit of palmitic acid in the adipose tissue supports the hypothesis that the saturated fatty acids have been preferentially transferred from the plasma and incorporated into liver and adipose tissue; conversion of stearic acid to oleic acid (36) also supports the above hypothesis. The most likely explanation for the increased hepatic level of fat may be the reduced output of lipids from the liver to the plasma (29). A high level of linoleic acid is essential for normal development of the brain and the retina (40) and its deficiency was reported to impair coordinative performance (39). The fatty acid profiles in plasma, liver, and adipose tissue of rats fed palm oil and butterfat demonstrate that the total fatty acid composition of the dietary oil influenced the disposition of fatty acids in tissue lipids (38). In view of an earlier report (21) it may be pertinent to suggest that the faster rate of weight gain of palm oil-fed rats compared to controls after 5 weeks of the diet (Fig. 1) may be due to icreaseed palatability and the essential fatty acids in the diet. We found that the hepatic lipase level in the experimental rat group was higher than that of the control group (Table 4) and there was a moderately greater amount of fats (+ 2.5 grade fat) compared to the control group ( + 2 grade fat). Fat deposit was reported to increase in the liver when hepatic lipase activity increased (22). LPL activity correlated to LDL cholesterol levels (r = +0.617), and the plasma triacylglycerol levels were correlated inversely (r = -0.536) to HDL cholesterol for both group of rats (Table 4). There was a higher LDL level in the experimental group of rats than in the control group. It is possible that the degree of saturation of the fatty acids (palmitic and oleic acids) of membrane phospholipids could have affected the activities of the membrane bound enzymes LPL and HL and the degree of degredation of LPL by fibroblasts-by both receptor-mediated and receptor-independent pathways (23)-and therefore resulted in higher plasma LDL levels. Palm oil-fed rats had lower triacylglycerol and correspondingly higher HDL levels compared to those of the control group, which had lower HDL and higher triacylglycerol levels (Table 3). Palm oil contains 11% o-6 fatty acids (25). Boberg
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215
et al. (24) reported that when o-6 fatty acid was included in the diet of rats, the level of triacylglyceride decreased while the HDL and LDL cholesterol levels increased. They attributed their observation to the increased conversion of VLDL to LDL (24). In view of this report, it is likely that our present result is brought about by the w-6 fatty acid that is known to be present in palm oil at a 10% level (25). Keys et al. (19) have developed a formula that correlates dietary fat and serum cholesterol; it may be desirable to use twice as much polyunsaturated fat (P), mainly linoleic acid, than saturated fat (S), mainly palmitic acid, to lower serum cholesterol levels. Although palm oil has a monounsaturated/saturated fatty acid ratio of 1, it does not cause deposition of fats in the aorta and brings about triacylglycerol levels lower than those of the control rats, over a IO-week period (Table 3). The HDL cholesterol to total plasma cholesterol ratio (Table 3) of the experimental group was significantly higher than that of the control group. Currently this ratio is found to be a more useful index for determining the quality and effects of fat on health and indicates that the fat present in the diet leads to a lesser tendency toward development of atherosclerosis (31). The channeling of saturated fatty acid, palmitic and stearic, to adipose and liver tissues may account for the lower concentration of these fatty acids in the plasma and lack of their deposition in the arterial wall, and may also explain the antithrombotic tendency of palm oil. Thus, in rat palm oil diet caused some changes in several lipid panels, which may not contribute to the risk for development of coronary heart disease.
SUMMARY
The aims of our study were to investigate the effect of dietary palm oil on the levels of lipoprotein lipase, hepatic lipase, fat distribution (in the aorta and liver), and total cholesterol, HDL, LDL, and triacylglycerol levels in young rats (70 g body wt) over a period of 10 weeks. Palm oil-fed rats showed higher growth rate and lower triacylglycerol levels than the control group. Hepatic lipase activity was correlated to the liver fat distribution (correlation coefficient, r = +0.682) as seen by histopathological sections and was similar for both the palm oil and the control diets. Palm oil-fed rats exhibited a significantly higher HDL cholesterol to total plasma cholesterol ratio when compared to animals fed the control diet. The triacylglycerol levels correlated inversely to the HDL cholesterol levels (r = -0.536) while the lipoprotein lipase (LPL) activity correlated directly to the LDL level (r = +0.617) for both groups of animals. The fatty acid profiles of adipose and liver tissues and plasma revealed that saturated fatty acidspalmitic and stearic-were preferentially incorporated in liver and adipose tissues and less in the plasma. This accounts for lack of deposition in the arterial wall and for the antithrombotic tendency of palm oil. Thus, our present findings suggest that dietary palm oil may not contribute to the risk for coronary heart disease.
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ACKNOWLEDGMENTS We thank Dr. L. Retnam of the Animal House, Dr. G. S. Shahi of the Department of Physiology, Dr. T. N. Khan of the Department of Pathology, and Mr. J. Lau of the Department of Biochemistry for technical assistance and Messrs. T. C. Ow and T. C. Tan of the Department of Pathology for the preparation of tissue sections. We are grateful to the National University of Singapore for Research Grant RP 890339.
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