Differential effects of dietary oils on plasma lipids, lipid peroxidation and adipose tissue lipoprotein lipase activity in rats

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ELSEVIER

Nutrition Research, Vol. 20, No. 8, pp 1113-1123, 2000 Copyright © 2000 Elsevier Science Inc Printed m the USA All rights reserved

0271-5317/00IS-see front matter

PII: S0271-5317(00)00196-2

DIFFERENTIAL EFFECTS OF DIETARY OILS ON PLASMA LIPIDS, LIPID PEROXIDATION AND ADIPOSE TISSUE LIPOPROTEIN LIPASE ACTIVITY IN RATS

Nahla Hwalla Baba, Ph.D 1, Zeina Ghossoub, M.Sc 1, Zuheir Habbal, Ph.D 2 Department of Food Technology and Nutrition t, and Department of Pathology and Laboratory

Medicine 2' American University of Beirut,

ABSTRACT

The potential health benefits of various dietary oils in relation to cardiovascular disease and cancer are currently receiving considerable attention. This study investigated the effects of dietary canola, virgin olive, soybean, and sesame oils on body composition, serum lipids, lipid peroxidation and adipose tissue lipoprotein lipase activity in rats. Thirty-six male Sprague-Dawley rats were fed, ad-libitum, four diets each containing 40% of dietary energy in the form of either canola (CO), virgin olive (VO), soybean (BO), or sesame oils (SO) for seven weeks, with free access to water. At the end of the feeding period, the rats were sacrificed by decapitation and blood samples were analyzed for serum triglycerides (TG), total and HDL-Cholesterol (TC and HDL-C), glucose, insulin, malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE). Carcasses were analyzed for water, fat, and protein. Fat cell size and number, and adipose tissue lipoprotein lipase activity were determined from epididymal fat pads. Results showed a lower deposition of fat and lower percent liver fat in CO and BO groups as compared to SO and VO. Serum analysis also revealed a higher HDL-C/TC in CO group as compared to the other three groups. Serum triglyceride levels were highest in the VO group. SO group manifested the lowest levels of serum lipid peroxides. Lipoprotein lipase activity in adipose tissue homogenates (THATLPLA) per cell was significantly lower in VO as compared to BO and CO fed rats. It can be concluded that feeding CO resulted in a more favorable plasma lipid response than the other three vegetable oils whereas lipid oxidation was significantly lower with SO than CO feeding. © 2000 Elsevier Science lnc

KEY WORDS: Dietary oils, Plasma lipids, Lipid peroxidation, Lipoprotein lipase, Rats.

Corresponding author: Nahla Hwalla Baba, 850 3 '~ Ave # 18, New York NY 10022, Tel: (961) 1-351666, Fax: (961) 1-744460, E-mail: [email protected]

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1114 N.H. BABA et al.

INTRODUCTION

There is considerable interest in determining the potential health benefits of vegetable oils in relation to risk factors for atherosclerosis, heart disease and cancer. Whether different dietary oils rich in monounsaturated fatty acids have similar effects on serum lipid levels still needs to be elucidated. Olive oil, has been recommended as superior for both its ability to lower serum cholesterol levels similar to other vegetable oils rich in monounsaturated fatty acids and its potential to inhibit serum lipid peroxidation in rats as compared to oils rich in polyunsaturated fatty acids (1, 2). More recently canola oil emerged as another recommendable dietary oil, which is rich in oleic acid similar to olive oil and with low levels of saturated fatty acids as compared to other vegetable oils. Moreover, compared to olive oil, canola oil contains higher levels of linoleic acid, which has a hypocholesterolemic potential (3,4), and diets enriched with canola oil reduced total cholesterol, LDL cholesterol and apoB 100 in humans as compared to safflower oil (5). In addition canola oil has been recommended because it contains high levels of ct-linolenic acid; marine sources of linolenic acid as well as vegetable sources, have been shown to decrease cytokines which act as cellular mediators in the pathology of atherosclerosis in human (6) and in mice (7). Moreover n-3 ct-linolenic acid in vegetable oils have been shown to cause less serum lipid oxidation in rats than does fish oils (8,9,10). More recently olive oil enriched with ct- linolenic acid was shown to mimick the effects of canola oil resulting in reduced triglycerides, total cholesterol, and increasing HDL-C/TC ratio as compared to unenriched olive oil (1) Canola oil was also found to be antithrombic (11,12), and had the ability to decrease platelet aggregation (6). It contains a lower ratio of n-6 to n-3 fatty acids which may allow for better conversion of linolenic acid to eicosapentenasic acid (EPA) known to decrease serum cholesterol in rats (13, 14, 15). Other benefits for canola oil include its ability to reduce the likelihood of a transient ischemic event that may lead to life threatening cardiac arrhythmia (16). In the present study canola oil and olive oil rich in monounsaturated fatty acid are compared to two other seed oils namely sesame and soybean oils. Soybean oil is rich in linolenic acid and is similar to sesame oil in its linoleic acid content. Linolenic acid in sesame oil and olive oil constitutes a minor portion of their total fatty acids. Sesame oil, also, contains phenols - sesamol and sesamolin - which makes it unique oil because of its superior oxidative stability (6, 17, 18, 19, and 20). Another important aspect of dietary oils is their tocopherol content and antioxidative ability, as lipid peroxidation appears to make a significant contribution to the development of atherosclerosis or to the sequalae of ischemic injury to the brain (21). The oxidative modification of LDL has been implicated in the pathogenesis of atherosclerosis (22, 23). The present study attempted to compare these commonly consumed oils in relation to their effects on body composition, serum lipids, lipid peroxidation, and adipose tissue lipoprotein lipase activity.

MATERIALS & METHODS

Animal and Experimental Diets. Thirty-six male Sprague Dawley rats were housed individually in metallic cages in an animal room with twelve-hour light and dark cycles. The room was maintained at a temperature of 21°C. The rats were fed an adaptation diet ad-libitum for one week. At the end of this period, the rats were divided into four groups and fed diets containing canola oil (CO), soybean oil (BO), virgin olive oil (VO), or sesame oil (SO) as 40% of dietary energy. The rats had free access to food and water. The composition of the four experimental diets is shown in Table 1. The fatty acid profile of the experimental oils is shown in Table 2. Fatty acid composition of the four experimental oils was determined by gas chromatography (24). Caloric content of the fed diets was determined by bomb calorimetry (Parr 1341 Plain Oxygen Bomb Calorimeter, Illinois, USA). Food intake and body weights were recorded three times a week. At the end of the feeding period (seven weeks), the rats were fasted overnight, weighed,

FATTY ACIDS & PLASMA LIPIDS 1115

killed by decapitation, and blood samples were immediately collected and serum was separated by centrifugation at 2000 rpm for later determination of glucose, insulin, and blood lipids. Serum lipid peroxidation was determined by measuring lipid peroxidation products MDA and 4-HNE in serum. For determination of MDA and 4-HNE, test tubes containing EDTA were used to prevent blood coagulation. The protocol was approved by the University Research Board.

Lipoorotein Lioase Assay and Fat Cell Number. Individual fat pads from the epididymal, perirenal, and omental fat depots were dissected and weighed. Total dissectable fat was later obtained by adding the weights of individual pads. Two samples of approximately 200 mg and two samples of 50 mg were excised from the right epididymal fat depot, rapidly weighed and immediately frozen to -80°C for later assay of tissue homogenate adipose tissue lipoprotein lipase (TH-LPLA) and heparin releasable adipose tissue LPL (HR-ATLPLA), respectively as described by Nilson-Ehle et al. (25).

Another independent sample of 200 mg was also excised from the same site for immediate determination of percent fat and fat cell size and number using the photomicroscopic method of Lavau et al. (26). The liver was also dissected out, weighed, and approximately 1 GM samples from liver were obtained and frozen at -20°C for later determination of percent liver fat. Body_ Composition. Carcasses were weighed and then dried in the oven at a temperature of 80°C. Total body water was calculated from the difference between carcass weight before and after drying. Percent body fat, percent liver fat and percent epididymal fat were determined according to Folsch et al. (27). Percent body protein was analyzed using Macro-Kjeldahl method (28). Feces from each rat were collected during the last three days of feeding, weighed, and dried in the oven at 100°C for five days and ground to powder form. Percent fecal fat was determined by Folsch et al. (27).

Serum Lioids and Lipid Peroxidation. Serum cholesterol, HDL-C and triglycerides were analyzed by enzymatic colorimetric tests ( Boehringer Mannheim, Catalogue No. 1121952, 3524, 1121952 respectively). Glucose was analyzed using the hexokinase/G6P-DH colorimetric test ( Boehringer Mannheim catalogue No. 1447521). Insulin was determined using radioimmunoassay kit (lnsulin-CT CIS Bio International). Malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4- HNE) were assayed using a kit provided by Calbiochem (Calbiochem's Lipid Peroxidation Assay Kit, catalogue No. 437634).

Statistical Analysis. Data were analyzed by ANOVA and means were compared by Duncan's Multiple Range test using MSTAT package (29).

RESULTS

Nutritional Parameters. Body weight, body composition and feed intake were similar in the four groups of rats fed whether CO, BO, VO, or SO.

1 116 N.H. BABA et al.

TABLE 1

The Composition of Four Experimental Diets

Ingredients CO BO VO SO (g/100g)

Canola Oil 22.71 Soybean Oil 22.71 Olive Oil 22.71 Sesame Oil 22.71 Casein 16.44 16.44 16.44 16.44 Alphacel I 12.71 12.71 12.71 12.71 Sucrose 45.18 45.18 45.18 45.18 Salt Mix (USPXIV) 2 4.00 4.00 4.00 4.00 Vitamin Mix 3 0.6008 0.6008 0.6008 0.6008 Kcal/g 6.0 6.0 6.0 6.0 Composition of the diet from ICN Nutritional Biochemicals. 1. Commercial name for cellulose. 2. USPXIV salt mixture from ICN nutritional biochemicals contains (g/100g mixture); calcium

carbonate 6.86; calcium citrate 30.83; calcium biphosphate 11.28; manganese carbonate 3.52; magnesium sulfate 3.83; potassium chloride 12.47; potassium phosphate 21.88; sodium chloride 7.71; copper sulfate 0.0078; ferric citrate 1.53; manganese sulfate 0.02; potassium aluminum sulfate 0.009; potassium iodide 0.004; sodium fluoride 0.051.

3. Vitamins were added as Vitamin Diet Fortification Mixture from ICN nutritional biochemicals which contains (g/kg mixture); vitamin A acetate 1.8; vitamin D concentrate 0.125; ct- Tocopherol 22.0; ascorbic acid 45.0; Inositol 5.0; choline chloride 75.0; Menadione 2.25; P- amino-benzoic acid 5.0; Niacin 4.25; Riboflavin 1.0; Pyridoxine hydrochloride 1.0; Thiamin- hydrochloride 1.0; calcium pantothenate 3.0; biotin 0.020, folic acid 0.090; vitamin B12 0.00135.

TABLE2

Fatty Acid Composition* of Canola, Soybean, Virgin Olive, and Sesame Oils.

CO BO VO SO %

Palmitic (16:0) 4.4 10.4 9.1 9.3 Palmitoleic (16:1) - 0.8 Stearic (18:0) 1.9 4.0 2.5 5.0 Oleic (18:1) 62.4 23.2 81.6 43.6 Linoleic (18:2) 21.3 54.7 5.4 42.1 Linolenic (18:3) 9.9 7.6 0.7 <0.5 *Trace acids (less than 0.1%) were excluded.

Carcass Analysis. The proximate analysis of carcasses showed that percent body fat was significantly lower in the CO and BO than VO and SO groups. Liver fat was highest in the SO followed by VO, CO and BO fed rats. CO and BO fed rats had similar liver weight, which was less than that of VO and SO fed groups (Table 3). Percent body protein did not differ among the four groups.

FATTY ACIDS & PLASMA LIPIDS 1117

TABLE 3

Proximate Analysis of Carcasses, Liver Weight, and Percent Liver Fat of Four Groups of Rats Fed Diets Containing 40% of Dietary Calories as Canola, Soybean, Virgin Olive, or Sesame Oil

CO BO VO SO Mean _+ SE

Fat (%) 9 . 1 + 0 . 2 6 c 9.6_+0.39 c 1 5 . 3 + 0 . 3 4 ~ 1 1 . 4 + 0 . 2 2 b

Protein (%) 1 8 . 7 + 0 . 2 2 1 8 . 0 + 0 . 2 7 1 7 . 7 + 0 . 3 3 18.1 + 0 . 1 9 Water (%) 62.7 + 1.01" 60.2 _+ 0.91 ab 57.9 + 1.17 b 60.3 + 1.09 "b Liver Weight (g) 13.5 + 0.47 b 12.8 + 0.60 b 16.1 + 1.17 a 16.0 + 0.98" Liver Fat (%) 8 . 3 + 0 . 3 8 ¢ 4.7_+0.38 d 1 1 . 1 + 0 . 1 9 b 1 2 . 5 + 0 . 3 4 a

a,b,c values in the same row with different superscripts are significantly different at p < 0.05.

Serum Total Cholesterol, HDL-C. Trielvcerides, MDA, 4-HNE, Glucose, and Insulin. Total plasma cholesterol, HDL-C and insulin levels were similar among the four groups of rats. Triglyceride levels were significantly lower in CO and BO as compared to the other two groups. HDL-C/TC was significantly higher in the CO group as compared to the other three groups. CO rats were shown to have the highest and SO the lowest levels of MDA and 4-HNE and the differences were statistically significant for MDA and M D A + 4 - H N E (Table 4).

T A B L E 4

Serum Glucose, Total Cholesterol, HDL-C, Triglycerides, Insulin, and Lipid Peroxides Levels in Rats Fed Diets Containing 40% of Dietary Calories as Canola, Soybean, Virgin Olive, and

Sesame Oils

CO BO VO SO Mean + SE

Glucose (mg/dl) 96.9_+ 6.19 c 109.0_ + 6.08 bc 127.9_+ 6.97 a 120.9_+ 5.00 ~ Total Cholesterol (mg/dl) 97.6 + 5.84 102.0_+ 6.28 104.9_+ 9.11 103.1_+ 5.92 Triglycerides (mg/dl) 70.6 + 10.18 b 65.6-+ 8.40 b 142.6-+ 26.96 a 106.0-+ 14.73 ab HDL-C (mg/dl) 64.8-+ 2.99 65.1+ 2.70 56.4-+ 4.47 62.1+ 4.30 HDL-C/Total Cholesterol 0.7 + 0.02 ~ 0.6-+ 0.20 b 0.5_+ 0.02 ~ 0.6+ 0.01 b Insulin (~tlU/ml) 8.4_+ 1.62 6.8-+0.78 9.1-+ 2.37 7.6-+ 1.40 MDA(p.mole/lit) 107.9_+ 7.18 a 91.6-+11.39 ~b 89.3-+ 6.19 ~b 72.2-+ 7.07 b 4-HNE (Ixmole/lit) 181.7 -+ 1.55 165.5+ 10.79 179.0+ 1.94 155.4+ 12.64 MDA + 4-HNE (Ixmole/lit) 289.5_+ 7.68 ~ 257.1_+ 19.7& b 264.2+ 10.07 ab 226.7+ 16.7 b

a,b,c values in the same row with different superscripts are significantly different at p < 0.05.

Adipose Tissue Lipoprotein Lipase Activity (ATLPLA), Fat Cell Size, Fat Cell Number, and Fat Cell Volume. Tissue homogenate (TH) and heparin releasable (HR) /~TLPLA/fat cell are shown in Table 5. There was no significant difference in the heparin releasable ATLPLA/cel l between the four groups. However, tissue homogenate ATLPLA/fat cell showed that VO fed rats had a significantly lower ATLPLA/fa t cell than BO group. There was also no significant difference between the four experimental groups in fat cell number per gram epididymal fat (Table 5). Fat cell size was smallest in CO and highest in VO fed rats.

11 18 N.H. BABA et al.

TABLE 5

ATLPLA Per Fat Cell, Fat Cell Size, and Fat Cell Number in Rats Fed Diets Containing 40% of Dietary energy as Canola, Soybean, Virgin Olive, and Sesame Oils

CO BO VO SO Mean + SE

Fat Cell Diameter (txm) 51.3 + 2.59 53.4 + 2.42 60.4 + 2.42 55.7 _+ 2.09 Fat Cell Volume (~m 3) xl03 109.8 +14.23 b 119.7 + 12.06 b 160.9 +14.4 ~ 131.6 -+11.55 ~b Fat Cell Number (xl06) 69.5 + 3.70 71.9 + 5.23 81.0 + 2.75 70.4+ 5.09 TH ATLPLA/cell (p~mole FFA/hr)(xl0 -1~) 5.3 + 0.56 ~b 6.1 + 0.99 a 2.3 + 0.17 ~ 3.6 +-- 0.39 bc HR ATLPLA/cell (~tmole FFA/hr)(xl0 13) 1.3 + 0.25 2.2 + 0.49 1.3 + 0.32 1.6 _+ 0.19

a,b values in the same row with different superscripts are significantly different at p < 0.05.

DI~SCUSSION

Fatty acids in dietary lipids have been implicated in the response of serum lipids to feeding different oils and fats. This study examined whether commonly consumed dietary oils differing in their degree of unsaturation and type of fatty acids vary in their effects on plasma lipids and lipid peroxidation on rats. Although the results cannot be extrapolated to humans, they may suggest that further exploratory experiments on humans be worth conducting.

The oils compared in the present study had distinguishing characteristics. CO and VO are both high in oleic acid but differ in their content of the polyunsaturated fatty acids; linoleic and linolenic. BO is the richest in linoleic acid and SO has the bulk of its fatty acids divided equally between oleic and linoleic acid. Feeding rats diets containing the above oils resulted in differential effects on body composition, and plasma lipid profile. CO and BO fed rats deposited lower percentages of body fat than did VO and SO fed rats. There is little information in the literature about whether all types of dietary fats have the same ability to promote adipose tissue deposition. This study shows that in a high fat diet the type of fat can affect the degree of deposition of adipose tissue. This is in agreement with the study of Yaqoub et al. (30) which showed that high fat diets that differed in the type of fat did not lead to similar adipose tissue deposition in the epididymal fat pad in rats, and rats fed evening primrose oil and menhaden fish oil deposited less fat than olive oil and safflower oil fed rats. Studies on humans also showed that high fat diets differing in fatty acid composition had differential effects on fat tissue deposition during weight recovery after low food intake and olive oil rich diets resulted in increased body fat as compared to lard, coconut, safflower or fish oils (31).

In the present study BO feeding resulted in lower percentage of liver fat than feeding the other three oils. This could be related to the finding that oleic acid is a far more suitable substrate than linoleic acid for triglyceride biosynthesis and release by the liver (32). Kouhout et al. (32) reported that the rate of fat oxidation by the liver mitochondria is directly related to the fatty acid composition of the oil, and is inversely related to the chain length of the saturated fatty acids and to the number of double bonds.

In this study serum triglyceride levels were found to be different after feeding the four oils. CO and BO fed rats had lower serum triglyceride levels than VO and SO fed rats. Several studies in rats and humans reported similar results. Yaqoub et al. (30) showed that rats fed high fat diets containing olive oil and hydrogenated coconut oil had increased serum triacylglycerol concentrations compared with rats fed low fat diet, or high fat diets containing evening primrose

FATTY ACIDS & PLASMA LIPIDS 1119

oil, Menhaden fish oil, or safflower oils. In Humans, Dullo et al. (31) showed that ingesting high levels of olive oil leads to a significant increase in serum triglycerides and cholesterol levels as compared to safflower oil.

In this study, the LPL activity data paralleled that of triglycerides. BO fed rats had the highest LPL activity and lowest triglyceride level, whereas VO fed rats showed lowest LPL activity with concomitant high triglyceride levels. Inverse relation between serum triglyceride levels and lipoprotein lipase activity was reported by Vanheek et al. (33, 34) in rabbits and by Lottenberg et al. (35) in rats. It has also been shown that rats fed oleic acid rich oils had lower activities of lipoprotein lipase relative to rats fed linoleic acid (36). Furthermore, Kirkland et al. (37) through their work on cultured rat adipocytes, showed that oleic acid enriched cultures manifested a decrease in lipoprotein lipase activity. Levy et al. (38, 39) found that rats fed olive oil demonstrated a decreased activity of lipoprotein lipase and an increased triglyceride biosynthesis and release by the liver, which resulted in triglyceride enrichment of VLDL. In the same study, the researchers showed that a diet rich in PUFAs caused an increase in ATLPLA activity with a concomitant decrease in serum triglycerides.

A high HDL-C/TC ratio was shown in this study in response to CO feeding. Similar observations were reported by Fernandez et al. (40) in their study on guinea pigs. Moreover, Lichtenstein et al. (41) showed the highest, although not a significant, TC/HDL-C ratio in olive oil as compared to corn and canola oil fed humans, and in a study on normolipidemic men, Perez-Jimenez et al. (42) reported high concentrations of total cholesterol, LDL-cholesterol, and apo B levels in response to feeding an olive oil rich diet as compared to NCEP-1 diet. They also showed that, squalene a direct precursor to cholesterol synthesis was 17 times higher in olive oil than in oleic acid-rich sunflower oil.

Lipid oxidation, a process mediated by free radicals, is considered to be important in the development of atherosclerosis (43). One way of estimating free radical activity is to determine the concentration in plasma of malondialdehyde (MDA) and 4-hydroxy-2 (E)-nonenal (4-HNE) which are byproducts of lipid peroxidation. In this study, feeding canola oil resulted in higher concentrations of MDA and 4-HNE in CO fed rats whereas feeding SO resulted in lowest oxidation products. Halliwel et al. (21), demonstrated higher stability of saturated and monosaturated oils in lipid peroxidation as compared to polyunsaturated oils. The fatty acid composition of sesame oil being high in saturates (15% in sesame compared to 6% in canola), and having around 43 % oleic acid may account for the low content of lipid peroxides in SO fed rats. However, sesame has high amounts of linoleic acid (~ 42%), and Ohrvall et al. (44) states that MDA concentrations in plasma are inversely correlated to the proportion of linoleic acid in serum lipoprotein lipids. His findings suggested that other factors, such as, the availability of antioxidants, polyphenols and others, may be of greater importance for intravascular lipid peroxidation. White (8), showed that sesame oil contains small amounts of tocopherols but has good amounts of other phenols which makes it a unique oil because of its superior oxidative stability. Olive oil has the biophenol which was recently shown to improve in vivo an antioxidant defenses (45). On the other hand, canola oil, although rich in oleic acid (~62.5 %), has low content of saturates and of tocopherols (46) which are removed during the refining processes, and its high content of ~x-linolenic acid (=10 %) may render it susceptible to lipid peroxidation.

The present study, therefore, showed that CO feeding had beneficial effects resulting in lower deposition of body fat, increased HDL-C/total cholesterol ratio, low total cholesterol and low fasting blood glucose levels. These results may be attributed to the fact that canola oil has low levels of saturated fats and high levels of oleic and linoleic acid. Sesame oil feeding resulted in the lowest level of lipid peroxides. It can be concluded that feeding CO resulted in a more

1120 N.H. BABA et al.

favorable plasma lipid response than the other three vegetable oils whereas lipid oxidation was significantly lower with SO than CO feeding.

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Accepted for publication February 10, 2000.