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Journal of the American College of Nutrition, Vol. 17, No. 6, 586-594 (1998)
Published by the American College of Nutrition

Effects of Dietary n-6 and n-3 Lipids on Antioxidant Defense System in Livers of Exercised Rats

Jaya T. Venkatraman, PhD, CNS, Poomchai Angkeow, MS, Neera Satsangi, PhD and Gabriel Fernandes, PhD, CNS,

Nutrition Program (J.T.V., P.A.), Department of Physical Therapy, Exercise and Nutrition Sciences, State University of New York at Buffalo, Buffalo, New York
Department of Medicine (N.S., G.F.), University of Texas Health Science Center, San Antonio, Texas
Department of Microbiology and Physiology (G.F.), University of Texas Health Science Center, San Antonio, Texas

Address reprint requests to: Jaya T. Venkatraman, PhD, Assistant Professor, Nutrition Program, Department of Physical Therapy, Exercise and Nutrition Sciences, State University of New York at Buffalo, 15 Farber Hall, Buffalo, NY 14214


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objective: The present study was designed to investigate the effects of dietary n-6 and n-3 lipids and exercise on the activities of hepatic antioxidant enzymes and microsomal lipid composition and peroxidation in Fischer-344 male rats.

Methods: Weanling male Fischer-344 rats were fed ad libitum semipurified diets containing 10% corn oil (CO) or 10% fish oil (FO), with equal levels of antioxidants. After 2 months on the diets, weight-matched animals in each diet group were divided into sedentary (S) and exercised (Ex) groups, and the diets were continued. The animals in the exercise group were run on a treadmill 30 to 40 minutes to exhaustion 6 days/week for 2 months. At the end of 2 months, the rats were sacrificed and livers were collected; antioxidant enzymes were determined in the cytosol, fatty acid composition was analyzed in the microsomes, and vitamin E levels were analyzed in the sera.

Results: The rats in the FO-S group exhibited significantly higher liver cytosolic catalase activity, while their superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were significantly lower compared to the CO-S group. The GSH-Px activity was significantly higher in the FO-Ex group compared to FO-S group. The source of dietary lipids significantly influenced the fatty acid composition of the total lipids in the microsomes. Feeding the FO-based diet significantly increased 18:0 and n-3 fatty acids incorporation into the microsomes (18:3, 20:5, 22:5, and 22:6), whereas ingestion of CO resulted in a significant increase in 14:0, 14:1, 18:1, and n-6 fatty acids (18:2 and 20:4). The serum vitamin E levels were significantly higher in the CO groups, and exercise had no effect on vitamin E levels. Exercise significantly decreased the generation of thiobarbituric acid reactive substances (TBARS) by liver microsomes. Consumption of FO, which is highly susceptible to oxidation, did not show any significant changes in membrane lipid peroxidation.

Conclusions: The present study suggests that feeding FO increases the activity of liver cytosolic catalase in FO-S rats and GSH-Px in FO-Ex rats. In addition, exercise significantly decreased the generation of TBARS by the liver microsomal lipids. Serum vitamin E levels were higher in the CO group and exercise did not alter vitamin E levels. This suggests that the amount of vitamin E included in the diets was possibly adequate to cope with the oxidative stress induced during exercise.

Abbreviations: BCA=bicinchoninic acid • CO=corn oil • EDTA=ethylene diamine tetra acetic acid • FO=fish oil • GR=glutathione reductase • GSH-Px=glutathione peroxidase • MDA=malondialdehyde • MUFA=monounsaturated fatty acid • ß-NADPH=ß-nicotinamide adenine dinucleotide phosphate (reduced form) • PLSD=protected least significant difference • PUFA=polyunsaturated fatty acid • SDS=sodium dodecyl sulfate • SOD=superoxide dismutase • TBARS=thiobarbituric acid reactive substances.

Key words: antioxidant enzymes, catalase, corn oil, exercise, fatty acid composition, fish oil, glutathione peroxidase, lipid peroxidation, microsomes, rat liver, superoxide dismutase, vitamin E


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The susceptibility of an organism to oxidative damage is influenced by the antioxidant defense system’s ability to cope with the stress which in turn can be influenced by nutrition intervention with antioxidants [1]. Inherent antioxidant defense systems consisting of enzymes, such as glutathione peroxidase (GSH-Px), catalase, and superoxide dismutase (SOD), and antioxidant nutrients may participate in coping with oxidative stress [2–4]. As antioxidant enzymes have an important role in the protection against free radical damage, a decrease in the activities or expression of these enzymes may predispose tissues to free radical damage [5,6].

The very long-chain n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentanoic acid (20:5n-3) and docosahexanoic acid (22:6n-3), present in certain marine sources, have been shown to have beneficial effects in delaying the development of various diseases [7–10]. In spite of the beneficial therapeutic effects of fish oil (FO), there has always been concern over the instability of the very long-chain fatty acids and their susceptibility to oxidation. Consumption of high levels of n-3 PUFAs leads to enhanced membrane lipid peroxidation by free radicals [11]. Our earlier studies have indicated that feeding FO adequately supplemented with vitamin E significantly increased the activities and mRNA levels of catalase, GSH-Px, and superoxide dismutase in livers of autoimmunity-prone mice [12].

Though the beneficial effects of chronic exercise are well known, considerable evidence suggests that an excessive amount of deleterious free radicals and reactive oxygen species may be generated during strenuous exercise. It has been suggested that exercise training may enhance the antioxidant defense system to cope with the oxidative stress. As it is generally agreed that strenuous exercise exerts stress on the antioxidant defense system, based on our earlier findings, we were interested in investigating whether feeding FO would induce or enhance the antioxidant defense system in normal rats to enable them to cope better with oxidative stress during exercise. The interactive effects of diet and exercise on the activities of hepatic antioxidant enzymes have not been well established.

It was the objective of the present study to investigate the effects of either dietary n-6 or n-3 lipids and exercise on liver cytosolic antioxidant enzyme activities, microsomal lipid peroxidation, and fatty acid composition. Measuring these indices gives an insight into developing appropriate nutritional strategies based on dietary lipids and antioxidants that enhance the antioxidant defense system’s ability to cope with excess free radicals generated during strenuous exercise.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Diets
Weanling male Fischer-344 rats were purchased from Charles River Laboratories (Wilmington, MA). They were maintained in plastic cages under a 12-hour light/dark cycle. National Institute of Health (NIH) guidelines were strictly followed. They were fed ad libitum semipurified diets containing 10% (w/w) corn oil (CO) (ICN, Irvine, CA) or 10% (w/w) fish oil (FO) (US Department of Commerce, National Marine Fisheries Service, Charleston, NC). The CO and FO diets were prepared weekly and stored at 4°C. The semipurified diets were composed of (% by weight) casein—20%, dextrose—30%, starch—31%, oil—10%, cellulose—3.5%, AIN-93G mineral mixture—3.5%, AIN-76 vitamin mixture—1.5%, dl-methionine—0.3%, and choline chloride—0.2%. The fatty acid composition of the two oils is given in Table 1. Both of the oils contained equal levels of antioxidants (1.3 g or 1300 IU of {alpha}-tocopherol/kg oil, 1.2 g or 13.2 IU of {gamma}-tocopherol/kg oil, and 1 g/kg t-butylhydroquinone) as recommended by the NIH to prevent peroxidation during storage. The FO diet also contained 1% CO (9% FO+1% CO) to meet essential fatty acid requirements. In the experimental diets, the vitamin E concentration was 215 IU/kg, whereas typical rodent diets generally contain 75 IU/kg of vitamin E. Fresh diets were provided daily, and precautions were taken to prevent oxidation of the lipids in the diets. After being on the diets for 2 months, the animals in each diet group were divided into two subgroups (sedentary [S] and exercised [Ex]) and the same diets were continued. Each experimental group consisted of four rats.


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  		Table 1. Fatty Acid Composition of the Oils1

 
Exercise Protocol
Exercise was performed on a rodent treadmill (Quinton Instruments, Seattle) with the following protocol as described by Fernandes et al [13]. After training for several days on a treadmill containing 10 running compartments, each rat thereafter ran daily (between 3 to 4 p.m.) for 30 to 50 minutes, 6 days/week at 0.6 to 1.0 miles/hour and at 10° angle to provide resistance for running. The treadmill consists of rubber belts which are driven at a controlled speed. Each rat was trained to recognize three signals: breaking an electric beam (created noise); mild, continuous air flow; and a slight, variable, electric shock, which was administered when rats, choosing not to run, slid down into a wireholder contact. The rats learned to avoid the shock by keeping a regular (slightly elevated) pace with the belt. At 4 months, the rats were sacrificed humanely (between 9 to 10 a.m.), and livers were collected from the rats and stored at -80°C for enzyme and lipid analyses.

Chemicals
Xanthine oxidase, ferricytochrome C, glutathione reductase (GR), glutathione, ß-nicotinamide adenine dinucleotide phosphate (reduced form) (ß-NADPH), 30% hydrogen peroxide, and t-butylhydroperoxide were purchased from Sigma Chemical Co. (St. Louis, MO). Organic solvents were purchased from J. T. Baker Inc. (Phillipsburg, NJ) and fatty acid methyl ester standards were purchased from Nu-Chek (Elysian, MN) and Supelco (Belfonte, PA).

Preparation of Rat Liver Cytosol and Microsomes
Rat liver cytosol and microsomes were prepared by differential centrifugation. Approximately 2.0 g of liver was homogenized in 20 mL of ice-cold 50 mM phosphate buffer (pH 7.0) with a Tekmar Tissuemizer (Tekmar Company, Cincinnati, OH). The liver homogenate was centrifuged at 10,000xg for 20 minutes at 4°C, and the pellet was discarded. The supernatant was further centrifuged at 105,000xg for 90 minutes at 4°C. The microsomal pellet was resuspended in phosphate buffer (50 mM, pH 7.0). The microsomes and the supernatant (cytosolic fraction) were frozen in liquid nitrogen and stored at -70°C until analyzed.

Determination of Protein in the Rat Liver Cytosol and Microsomes
The protein content of the rat liver cytosolic fractions were determined [14]. The protein content in the rat liver microsomes was determined by the microplate procedure described by the supplier of the bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Company, Rockford, IL).

Determination of Antioxidant Enzymes in Rat Liver Cytosol
The enzyme kinetics were measured in a Beckman DU-65 UV/visible recording spectrophotometer (Beckman Instruments, Fullerton, CA).

Catalase Activity (EC 1.11.1.6):
Catalase activity was measured at 22°C by monitoring the decomposition of hydrogen peroxide as described by Aebi [15]. The reaction mixture consisted of 2.0 mL of the liver cytosol suspended in phosphate buffer (50 mM, pH 7.0), and 1.0 mL of hydrogen peroxide solution (30 mM). The absorbance was recorded for 2 minutes at 240 nm immediately after adding hydrogen peroxide solution. A concentration of 100 µg of protein was selected for the catalase assay. Catalase activity was expressed as moles of hydrogen peroxide reduced/min/mg protein.

Superoxide Dismutase Activity (EC 1.15.1.1):
Superoxide dismutase activity was determined at 22°C by using the xanthine oxidase/cytochrome C system [16]. The reaction mixture consisted of 2.1 mL potassium phosphate buffer (50 mM containing 0.1 mM EDTA, pH 7.8), 0.3 mL xanthine (1.0 mM), 0.1 mL deoxycholate (1%), 0.1 mL potassium cyanide (1.5 mM), 0.3 mL ferricytochrome C (0.1 mM), 20 µl xanthine oxidase (diluted), and 80 µl liver cytosol suspended in distilled water. The absorbance was recorded at 550 nm for 4 minutes. The conditions for SOD assay were optimized by determining the dose response of SOD to increasing concentrations of cytosolic protein (12.5 to 400 µg) and a concentration of 100 µg of protein was chosen for the SOD assay. Superoxide dismutase activity was expressed as units of SOD/minute/mg protein. One unit of SOD was defined as the amount of SOD required to inhibit the rate of cytochrome C reduction by 50%.

Glutathione Peroxidase Activity (EC 1.11.1.9):
Glutathione peroxidase activity was determined at 22°C by using a coupled-enzyme system [17]. The final volume of the reaction mixture was 1.0 mL. It consisted of 0.5 mL potassium phosphate buffer (0.1 M, pH 7.0/1 mM EDTA), 0.1 mL glutathione (10 mM), 0.1 mL glutathione reductase (2.4 U/mL), and 0.1 mL liver cytosol suspended in potassium phosphate buffer (0.1 M containing 1 mM EDTA, pH 7.0). The mixture was preincubated for 4 minutes at 22°C, and 0.1 mL of ß-NADPH (1.5 mM) was added. The absorbance was read at 340 nm for 4 minutes. Thereafter, 0.1 mL of t-butyl hydroperoxide (12 mM) was added, and the absorbance was recorded under the same conditions. The linearity of GSH-Px activity as a function of protein was examined using 25 to 500 µg of cytosolic proteins (data not presented). A concentration of 100 µg of protein was selected for the assay. Glutathione peroxidase activity was expressed as µmoles ß-NADPH oxidized/minute/g protein.

Fatty Acid Analysis of Microsomal Lipids
Microsomal lipids were extracted [18] and methyl esters were prepared [19]. The fatty acid methyl esters were analyzed using a gas chromatograph (Shimadzu, model 9A, Columbia, MD) equipped with a column packed with 10% SP-2330 on 100/120 chromosorb WAW (Supelco, Belfonte, PA). Nitrogen was used as the carrier gas. The injection port temperature was 250°C and the oven temperature was maintained at 200°C. At the time of injection, the fatty acid methyl esters were reconstituted in 25 µl of carbon disulfide and 0.5 µl of each sample was injected into the gas chromatograph. The running time of each sample was approximately 35 minutes. The peaks were identified using fatty acid methyl ester standard #68A (Nu Chek, Elysian, MN) and PUFA-1 (Supelco, Belfonte, PA). The areas under the peaks were measured using an integrator (Shimadzu, model C-RIA Chromatopac). The peroxidation index was calculated based on the following formula:=(monoenesx1)+(dienesx2)+(trienesx3)+(tetraenesx4)+ (pentaenesx5)+(hexaenesx6) [20].

Determination of Lipid Peroxidation in the Microsomes
Thiobarbituric acid reactive substances (TBARS) were measured by the modified spectrophotometric assay [21]. The reaction mixture consisted of liver microsomes suspended in 0.4 mL of distilled water, 0.1 mL of sodium dodecyl sulfate (SDS, 8.1%), 0.75 mL of acetic acid (20%), and 0.75 mL of thiobarbituric acid aqueous solution (0.8%). The glass tubes containing reaction mixture were heated in a boiling water bath for 30 min. Tubes were cooled, 2.0 mL of n-butanol was added to each tube, and tubes were vortexed vigorously and centrifuged at 3000 rpm for 10 minutes at 22°C. The clear upper layer was taken for an absorbance reading at 535 nm. Microsomal lipid peroxidation increased linearly with increase in the concentration of microsomal protein. A concentration of 0.5 mg of protein was selected for the TBARS assay. TBARS generation was calculated based on extinction coefficient (1.56x105M-1 cm-1) and expressed as nM malondialdehyde (MDA)/mg protein.

Serum Vitamin E Analysis
Vitamin E content of the sera was analyzed by high-pressure liquid chromatography (Waters HPLC .510, Milford, MA) using a C-18 microBondapak 300x8 mm stainless steel column [22]. The analysis was carried out isocratically using methanol-water (95:5 v/v) as mobile phase using UV detection at 280 nm. Sera were deproteinized with ethanol containing 0.01% butylated hydroxytoluene. After centrifuging, the supernatant was extracted with five volumes of hexane, evaporated under nitrogen, and redissolved in ethanol. Tocopheryl acetate was used as an internal standard.

Statistical Analysis
The data shown are mean±SEM. Data were statistically analyzed using the Statview 4.0/Super ANOVA package software (Abacus Concepts, Berkeley, CA). Where a significant F ratio was found (p<0.05), Fisher’s protected least significant difference (PLSD) test was used to test the differences between groups for significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body Weights
The effects of dietary lipids and exercise on the body weights of rats are shown in Table 2. The rats fed the FO diet had significantly lower (p<0.05) body weights than rats fed the CO diet. In addition, exercise slightly decreased the body weights in the CO-fed group by 4%, but this decrease was not statistically significant due to variation in the body weight.


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  		Table 2. Effects of Dietary Lipids and Exercise on the Body Weights in Rats1

 
Activities of Hepatic Cytosolic Antioxidant Enzymes
Catalase Activity:
In the sedentary groups of rats, the FO-diet-fed rats exhibited significantly higher (p<0.05) catalase activity compared to the CO-diet-fed rats (Fig. 1). In the exercised group, although the catalase activity was higher, the difference was not statistically significant.



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  		Fig. 1. Effects of lipids on cytosolic catalase activity in sedentary and exercised rats. Values (mean±SEM, n=4) with different superscripts are significantly different at p<0.05 as revealed by Fisher’s PLSD test; CO=corn oil; FO=fish oil.

 
Superoxide Dismutase (SOD) Activity:
The SOD activity was significantly (p<0.0001) altered by the types of dietary lipids and exercise (Fig. 2), and there was significant interaction between types of dietary lipids and exercise. In the CO-diet-fed groups, SOD activity in exercised rats decreased significantly (p<0.01) compared to the sedentary rats. In the FO-diet-fed group, the cytosolic SOD activity in exercised rats and sedentary rats was similar. SOD activity was significantly higher in the CO-S group compared to all the other groups.



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  		Fig. 2. Effects of lipids and exercise on cytosolic superoxide dismutase activity in rats. One unit of SOD was defined as the amount of SOD required to inhibit the rate of cytochrome c by 50%. Values (mean±SEM, n=4) with different superscripts are significantly different at p<0.05 as revealed by Fisher’s PLSD test; CO=corn oil; FO=fish oil.

 
Glutathione Peroxidase (GSH-Px) Activity:
In the sedentary groups, the liver cytosols from FO-diet-fed rats showed significantly lower (p<0.05) GSH-Px activity compared to the CO-diet-fed rats (Fig. 3). However, no significant differences between the FO- and CO-diet-fed rats were observed in the exercised group. GSH-Px activity was significantly higher in the FO-Ex group compared to FO-S group.



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  		Fig. 3. Effects of lipids and exercise on cytosolic glutathione peroxidase activity in rats. Values (mean±SEM, n=4) with different superscripts are significantly different at p<0.05 as revealed by Fisher’s PLSD test; CO=corn oil; FO=fish oil.

 
Fatty Acid Composition of Liver Microsomal Total Lipids
The microsomal fatty acid composition was significantly affected (p<0.0001) by the types of dietary lipids, while exercise did not alter the fatty acid composition (Table 3). The liver microsomes of CO-diet-fed rats exhibited higher levels of n-6 PUFAs (18:2 and 20:4) compared to the FO-diet-fed rats. On the other hand, feeding the FO diet (rich in n-3 PUFAs) resulted in a significant increase (p<0.01) in 18:0, and n-3 fatty acids (18:3, 20:5, 22:5, and 22:6) in the liver microsomes. Exercise exhibited no significant effect on n-6 or n-3 fatty acids incorporated into hepatic microsomal lipids. CO intake resulted in high levels of 14:0, 14:1, 18:1, 18:2n-6 and 20:4n-6 in the liver microsomes.


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  		Table 3. Effect of Dietary Lipids and Exercise on Fatty Acid Composition of Hepatic Microsomal Total Lipids in Rats1

 
The levels of saturates, n-3 fatty acids, and peroxidizability index were significantly higher in the FO-diet-fed groups while n-6 and P/S and n-6/n-3 ratios were lower (Table 4). Total n-6 fatty acids were significantly higher (p<0.05) in CO fed groups while total n-3 fatty acids were significantly higher (p<0.05) in the liver microsomes from FO-diet-fed groups. The n-6/n-3 ratios were significant lower (p<0.05) in the liver microsomes of FO-diet-fed rats than in the CO-diet-fed rats.


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  		Table 4. Effects of Dietary Lipids and Exercise on Saturates, MUFAs, PUFAs, P/S Ratio and Peroxidizability Index of Liver Microsomal Lipids in Rats1

 
Lipid Peroxidation of Liver Microsomes
The FO diet had no significant effect on microsomal lipid peroxidation when compared to rats fed the CO diets, as measured by TBARS generation (Fig. 4). It was interesting to note that exercise significantly decreased the generation of TBARS by microsomal lipids in the present study.



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  		Fig. 4. Effect of dietary lipids and exercise on TBARS generation in the liver microsomes of rats. Values (mean±SEM, n=4) with different superscripts are significantly different at p<0.05; CO=corn oil; FO=fish oil; MDA=malondialdehyde.

 
Vitamin E Levels in Sera
The serum vitamin E levels are presented in Fig. 5. Serum vitamin E levels were significantly higher (p<0.0001) in the CO-diet-fed groups compared to the FO-diet-fed groups. Exercise had no effect on serum vitamin E levels.



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  		Fig. 5. Effects of dietary lipids and exercise on serum vitamin E levels in rats. Values (mean±SEM, n=4) with different superscripts are significantly different at p<0.05; CO=corn oil; FO=fish oil. 

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is evident from our earlier work that aging and/or different dietary lipids have differential effects on the activities of antioxidant enzymes [12,23–26]. The results from the present study show that under sedentary conditions, ad libitum feeding of FO significantly increased catalase activity, whereas the activities of SOD and GSH-Px were generally decreased as compared to the CO fed group. A number of studies have indicated that consumption of FO not only decreases plasma triglycerides but also significantly influences the activities of antioxidant enzymes and their mRNA expression. Increases in both activities and mRNA expression of catalase, SOD, and GSH-Px enzymes after feeding FO supplemented with adequate vitamin E has been reported by us in our earlier study [12]. In addition, n-3 fatty acids at hypotriglyceridemic doses enhanced the activities of hepatic antioxidant defense enzymes, particularly catalase, GSH-Px, and glutathione reductase activity [27]. In contrast, Iritani and Ikeda [28] found that the activities of the three key antioxidant enzymes, catalase, SOD, and GSH-Px, in liver cytosols increased with CO intake. Decreases in CuZn-SOD and Mn-SOD activity in liver homogenates of rats fed FO has been reported [6].

Data from the present study showed increase in catalase activity and decrease in both SOD and GSH-Px in sedentary rats after feeding FO. Although the level of H2O2 produced was not measured in this study, it has been reported that increased peroxisomal ß-oxidation associated with a high rate of H2O2 production was observed with feeding n-3 fatty acids [29,30]. The increased H2O2 has to be removed by enhanced catalase activity which is located mainly in the peroxisomes which may explain the increases in the catalase activity in the FO-fed groups observed in the present study.

The activities of both GSH-Px and SOD were lower in the FO fed groups. With regards to the effect of exercise, it generally did not decrease the activities of the three key antioxidant enzymes in the present study. Significant increases in the activities of GSH-Px, GR, SOD, and catalase in skeletal muscle after exercise in rats have been reported [31] suggesting exercise may enhance the antioxidant defense system. Liver has a high metabolic rate and plays an important role in detoxification in the body [32] hence, it is equipped with abundant antioxidant enzymes and other scavenging systems.

Previous studies show that elevation in the activities of catalase and SOD in the muscle occur after endurance exercise training [33,34]. Highly trained runners have the highest levels of erythrocyte vitamin E, glutathione reductases, and catalase compared to sedentary people [35]. Training is also known to have differential effects on antioxidant enzymes [36].

Liver microsomes from rats fed the FO diet exhibited higher levels of n-3 fatty acids, while microsomes from the CO fed groups showed higher levels of n-6 fatty acids. The increase in n-3 fatty acids in liver microsomes from FO fed rats is attributed to the high levels of n-3 fatty acids in fish oil. Arachidonic acid (20:4n-6) is a precursor for proinflammatory prostaglandins and leukotrienes. Our observations were similar to those reported by other investigators in both normal rats and autoimmune mice [12,37,38].

Lipid peroxidation is frequently used as an indication of tissue oxidative stress as a result of free radical attack on the cell membrane [39]. However, several factors may influence lipid peroxidation in the microsomal membranes after consumption of highly unsaturated fatty acids, including the cellular antioxidant enzyme activities, and the cellular concentrations of vitamin E, a chain-breaking antioxidant. Feeding FO supplemented with up to 150 mg vitamin E/kg diet is reported to reduce the susceptibility of the rat liver and kidney to lipid peroxidation [40]. Also, a reduction in the lipid peroxidation of rat liver and plasma after feeding FO supplemented with vitamin E (209 IU/kg diet) has been reported [22]. In the present study, each diet was supplemented with vitamin E at the level of 215 IU/kg diet. Though the vitamin E level of hepatic microsomes was not determined in the present study, this issue was addressed earlier in our laboratory [41] when we conducted experiments on B/W mice and studied the effect of 75 and 500 I.U. of vitamin E in FO- and CO-based diets on the vitamin E level of plasma and microsomal and mitrochondrial membranes prepared from liver and other tissues. The plasma and membrane levels of vitamin E increased when the vitamin E level in the diet was increased. The level of lipid peroxidation in the sedentary groups of FO-diet-fed rats was lower than in the CO-diet-fed group. It is possible that the level of vitamin E supplemented in this study may be adequate to protect the liver tissue from free radical damage. Several studies have demonstrated increases in the generation of free radicals during strenuous exercise leading to tissue damage [42–44]. In addition, exercise has been shown to increase the need for vitamin E [45] to provide adequate protection to the tissues from oxidative stress.

Studies have demonstrated elevated levels of lipid peroxide by-products following acute physical activities especially in ultra marathon runners and trained and untrained subjects [46,47] and animals [43] following physical activity. However, not all studies demonstrate evidence of oxidant stress following exercise [36,47,48]. An exhaustive exercise bout in male college students, who ingested 300 mg/day of vitamin E for 4 weeks, had significantly lower serum MDA levels after exercise than did the control subjects who were given placebos [49]. Recent research [50] has suggested that exercise-induced free radical generation is abolished in the myocardium of rats supplemented with vitamin E for a period of 60 days. Both the ineffectiveness of vitamin E supplementation in ameliorating muscle injury in rats [51] and humans [52], and improved muscular endurance in mice supplemented with vitamin E [53] have been reported. Increases in plasma tocopherol during cycling to exhaustion due to mobilization of vitamin E from other tissues to prevent lipid peroxidation has been reported [54].

In summary, the findings from the present study reveal that feeding FO increases the activities of cytosolic catalase and exercise increased the activity of GSH-Px in rat liver cytosol. In addition, exercise decreased generation of TBARS in microsomal lipids. It can be speculated that the level of vitamin E supplemented in these diets may be adequate to cope with additional oxidative stress exerted by exercise in the Fisher-344 rat model. As serum vitamin E levels were lower in the FO-diet-fed groups, it is possible that vitamin E from tissues may be getting mobilized to plasma in order to prevent lipid peroxidation to cope with oxidative stress during exercise. It is evident from literature that vitamin E from tissues, mainly liver, may be getting mobilized to maintain serum vitamin E levels to reduce oxidation.


   ACKNOWLEDGMENTS
 
This research was funded by NIH grants IR15AR/AI43517, AG13693, DE10863, AG14541 and University of Buffalo Startup Funds. The authors wish to acknowledge Dr. Atif B. Awad, Dr. Carol Fink-Delbalso, and Dr. Diane Bofinger for their valuable suggestions, Dr. David Pendergast for critically reviewing the manuscript and Paula Kelly for expert editorial assistance.

Received November 1, 1997. Accepted April 1, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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