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School of Nutrition, Faculty of Community Services, Ryerson University, Toronto, Ontario, Canada M5B 2K3 and * Food, Nutrition and Health Program, Faculty of Agricultural Sciences, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4
2To whom correspondence should be addressed. E-mail: ddkitts{at}interchange.ubc.ca
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.001) susceptibility to lipid peroxidation in RBC and liver of SHR and WKY rats. Greater amounts of dietary (n-3) fat enhance tissue susceptibility to oxidation, which can be modulated by increased dietary cholesterol in SHR and WKY rats.
KEY WORDS: • (n-3) fat blends • antioxidant enzymes • lipid oxidation • SHR rats • WKY rats
Increased incorporation of fish and thus, long-chain polyunsaturated fatty acids (PUFA)3 into the diet is recommended in the dietary guidelines of the American Heart Association (AHA) to reduce the incidence and risk factors of cardiovascular disease (1 ). However, the AHA has not recommended the use of (n-3) fatty acid supplements because long-term benefits have not been demonstrated. The hypolipidemic effects of dietary fish oil are fairly consistent in lowering plasma triacylglycerols (TG), with variable effects on total and LDL cholesterol concentrations (1 –3 ). On the other hand, the highly polyunsaturated long-chain fatty acids [i.e., eicosapentaenoic acid (EPA), 20:5(n-3) and docosahexaenoic acid (DHA), 22:6(n-3)] of fish oils are extremely susceptible to lipid peroxidation, even in the presence of added dietary antioxidants (4 ). The data evaluating the effect of fish oil PUFA on oxidative stress and measures of lipid peroxidation in vivo vary with both animal and clinical studies reporting increased oxidation (2 ,5 –8 ), no effect on oxidation (9 ,10 ) and even some indication of decreased susceptibility to induced oxidation (3 ,10 ).
Factors that may influence the results of studies of diets consisting of fish oil or EPA and DHA include animal species and strain (11 –13 ), duration of treatment and the relative amount of fish oil in the diet (3 ,14 ,15 ). The method of determination of susceptibility to lipid peroxidation or the presence of oxidative stress in vivo can also influence whether there is a difference in susceptibility to oxidation (10 ,16 ,17 ). Studies evaluating the effects of dietary fat on plasma lipids, LDL oxidation and antioxidant status have been performed by feeding levels ranging from 5 to 20% dietary fat to rat models (2 ,3 ,7 ,11 ,13 ). Diets formulated to contain between 5 and 10% dietary fat are suggested for small rats according to the nutritional recommendations for laboratory rats outlined by the Canadian Council on Animal Care (18 ). Moreover, there is some evidence indicating that the level of dietary fat influences the activity of antioxidant enzymes in target tissues (11 ,19 ,20 ). Part of the effect of a critical level of dietary fat intake on antioxidant enzyme activities may be associated with the expression of genes encoding the drug-metabolizing enzymes, such as the phase I and II enzymes and that of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) (19 –21 ). Rats fed 20% soybean oil diets showed reduced activities of G6PDH and glutathione peroxidase (GSH-Px, EC 1.11.1.9), a key enzyme in the antioxidant defense system, compared to those fed 5% soybean oil (19 ). Therefore, it seems likely that the amount of dietary fat can play a key role in the influence of PUFA on the oxidative status of target tissues through effects on membrane lipid composition, and in the availability of reducing equivalents [i.e., reduced nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH)] for antioxidant enzyme activity.
Previous studies with spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats indicate that the SHR rat, as an animal model of human essential hypertension, shows elevations in red blood cell (RBC) and tissue catalase (CAT, EC 1.11.1.6) and hepatic glutathione reductase (GSSG-Red, EC 1.6.4.2) activities (12 ,22 ). Elevated RBC and heart glutathione levels in SHR rats suggest an adaptation against oxidative stress in vivo related to genetic hypertension (22 ). Diets containing an 8% fish oil blend fed to SHR and WKY rats decreased heart and RBC GSH-Px and superoxide dismutase (SOD) activities, reduced heart GSSG-Red activities, but did not affect the development of hypertension in SHR rats (13 ). Hepatic GSH-Px activities in MO-fed rats were increased in SHR but decreased in WKY rats. A high cholesterol intake (5.0 g/kg diet) increased hepatic GSSG-Red activities and reduced lipid oxidation in RBC and liver of SHR and WKY rats in other studies (3 ,13 ). Thus, diet-induced changes to antioxidant status with an 8% fish oil blend or a high intake of cholesterol were largely tissue specific and did not indicate a great change in oxidative stress in vivo at a relatively lower level of dietary fat intake (3 ,13 ). On the other hand, others reported that Wistar rats fed a 20% fish oil blend diet showed reduced heart and liver antioxidant enzyme activities with increased levels of lipid oxidation products in these tissues, thereby indicating increased oxidative stress in these rats (2 ). Therefore, in the present study, we hypothesized that diets containing 160 g fat/kg diet as blends of (n-6) or (n-3) fatty acids, with varying levels of cholesterol supplementation, would alter tissue and RBC antioxidant status and increase susceptibility to lipid peroxidation. We evaluated the combined effects of diets containing 160 g fat/kg diet as blends consisting of butter as a source of saturated fat; soybean or menhaden oils as sources of (n-6) and (n-3) PUFA, respectively, with either 0.5 or 5.0 g cholesterol/kg diet on tissue antioxidant enzymes, GSH levels and GSH depletion; and TBARS production as indicators of susceptibility to oxidative stress in SHR and WKY rats.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Five-wk-old male SHR and WKY rats were obtained from Charles River Laboratories (Montreal, PQ, Canada). After arrival, rats were housed in hanging stainless steel cages and allowed to acclimatize for 1 wk fed on a nonpurified pelleted stock diet (Ralston Purina, St. Louis, MO). Rats had free access to distilled deionized water throughout the experimental period. Rats were then assigned to one of six treatment groups (n = 8 per group), varying in dietary fat source based on butter (B), soybean oil (SBO) or menhaden oil (MO) and cholesterol level (low cholesterol 0.5 g/kg diet and high cholesterol 5.0 g/kg diet). The low level of cholesterol was used to equalize diet cholesterol contents to that of butter (
0.2 g/100 g). A basal diet mix designed to provide 30 g/kg diet canola oil (Neptune Food Services, Richmond, BC, Canada) in the final experimental diets, and thereby an adequate supply of essential fatty acids (EFA), was formulated before addition of the test fats. The basal diet mix contained (g/kg final diet): casein (ICN Biochemicals, Cleveland, OH), 250; alphacel nondigestible fiber (ICN), 50; sucrose (Neptune), 30; Ca-free mineral mix (ICN), 35; CaCO3 (BDH Chemicals, Toronto, ON, Canada), 20; vitamin mix (ICN), 30; DL-methionine (United States Biochemical, Cleveland, OH), 3.0; choline chloride (Van Waters & Rogers, Abbotsford, BC, Canada), 2.0; monofos (Van Waters & Rogers), 30; canola oil (Neptune), 30. The composition of the mineral and vitamin mixes as well as the monofos were as previously reported (13
). The amount of cornstarch (Neptune) in the diets was adjusted to take into account the varying amounts of sterols added to the individual diets (Table 1
). Final diet compositions contained 130 g/kg of the experimental dietary fats, for a total of 160 g fat/kg diet. Concentrations of butter, soybean or menhaden oils, sterols and cornstarch added to the basal diet mix as well as the final gross energy and dietary fatty acid composition of the experimental diets are provided in Table 1
. The amount of nonsalted butter added to diets was calculated to account for the naturally occurring moisture present in processed butter (20 g moisture/100 g butter). A mild heat treatment (45–50°C, 10–15 min) was used to liquefy the butter to allow uniform distribution of fat and sterols into the final diet blends. Sterols (cholesterol and cholic acid, 2:1 ratio) were added to the liquid fats and thoroughly mixed into the final diets using a Hobart mixer with an aluminum bowl and paddle over a period of 20–25 min. Once mixed, diets were stored in doubled, dark plastic bags at -15°C throughout the experimental period.
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A sample of each experimental diet was taken for fatty acid, gross energy by bomb calorimetry and dry matter content determinations. Analysis of diets for cholesterol confirmed the calculated sterol content of the low and high cholesterol diets. Diets were isonitrogenous and contained comparable levels of energy (i.e., 22.12–22.80 MJ/kg, Table 1 ).
Dietary lipid and fatty acid analyses.
The fatty acid compositions of the experimental diets (Table 1) were determined by gas chromatography after extraction of lipids from diets using Folch’s reagent (24 ). Fatty acids were methylated using BF3 and separated using a Varian Model 3700 GC equipped with an SP-2330 silica capillary column (30 m x 0.25 mm i.d.; Supelco, Bellefonte, PA). The temperature program started at 180°C, 10 min and then increased by 5°C/min up to 195°C. The carrier gas was N2 (40 mL/min) and the internal standard used was 17:0 (Supelco).
Animal feeding.
To ensure a similar postprandial time period for all rats before systolic blood pressure (SBP) measurements, rats were trained to meal-feed from 0900 to 1600 h after 1 wk of free access to experimental diets according to Kitts et al. (25 ). The feed efficiency ratio (FER) was calculated from body weight gained divided by feed intake over the 10-wk experimental period. SBP readings were taken using an indirect tail-cuff method (Harvard Apparatus, South Natick, MA) between 1500 and 1700 h in SHR and WKY rats.
Experimental procedures.
At 14 wk of age, rats were killed by exsanguination under halothane anesthesia at 1300 h in a fed state. Rats were not denied feed before killing, to minimize any potential changes to antioxidant status due to possible stresses of food deprivation. Blood was drawn into heparinized tubes via cardiac puncture followed by centrifugation to separate plasma (1000 x g, 5 min, 4°C) from RBC. Heart and liver tissues were dissected and deposited into chilled 50 mmol/L Tris 0.1 mmol/L EDTA, pH 7.6 buffer.
Aliquots of plasma were taken for total cholesterol (26 ), triacylglycerol (27 ) and phospholipid (28 ) analyses (Boehringer Mannheim, Laval, PQ, Canada). RBC were washed twice with isotonic 0.15 mol/L NaCl before lysis of an aliquot of RBC by 1:10 dilution with double-distilled water and exposure to three freeze/thaw cycles in dry ice/acetone. Heart and liver were prepared as 1.0 g tissue/10 mL buffer homogenates in fresh, chilled 50 mmol/L Tris 0.1 mmol/L EDTA (pH 7.6) buffer using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) at 25% maximum speed, for two cycles of 15 s each. An aliquot of these homogenates was taken to prepare tissue cytosolic fractions for use in the enzymatic assays by centrifugation (105,000 x g, 15 min, 4°C) using a Beckman L2-65 ultracentrifuge and sw40Ti rotor. Protein content of cytosols was determined according to Bradford (29 ). Spectrophotometric determination of enzyme activity was carried out using a Perkin–Elmer Lambda 6B spectrophotometer (Perkin Elmer Cetus Instruments, Norwalk, CT) with temperature control set at 25°C.
Apparent fat digestibility.
At 10-wk of age, rats were removed from their cages and placed in individual metabolic cages (Nalgene, Rochester, NY) for a 5-d balance study. Samples of freeze-dried diet and fecal material were extracted with Folch’s solution (24 ) and filtered into a fat-free aluminum dish to determine total crude lipid content. Apparent fat digestibility was calculated from the difference between intake and excretion expressed as a fraction of total lipid ingested during the balance study.
Analytical methods.
Catalase (CAT, EC.1.11.1.6), glutathione peroxidase (GSH-Px, EC.1.11.1.9), glutathione reductase (GSSG-Red, EC.1.6.4.2) and superoxide dismutase (SOD, EC.1.15.1.1) activities of RBC, heart and liver tissues were determined as previously described (12 ,13 ,22 ). Hemoglobin (Hb) content of RBC hemolysates and tissue cytosols was determined by the method of Drabkin and Austin (30 ).
Tissue and RBC susceptibility to in vitro forced peroxidation was determined by two methods as previously described (12 ,13 ,22 ). Briefly, depletion of sulfhydryl groups (as a measure of GSH) was assessed by incubation of RBC with 0.30 mmol/L added H2O2; heart homogenates with 0.20 mmol/L added H2O2 and liver homogenates with 0.20 mmol/L added H2O2 for 30 min at 37°C, followed by reaction of the supernatant with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB; Sigma, St. Louis, MO) and the absorbance read at 412 nm. Second, production of 2-thiobarbituric acid reactive substances (TBARS) in heart and liver and malondialdehyde (MDA) in RBC was also determined after incubation with H2O2 (5.0 mmol/L added H2O2 for both RBC and heart; 40.0 mmol/L added H2O2 for liver) and reaction of the supernatant with 35 mmol/L 2-thiobarbituric acid (Sigma) in 25 mmol/L NaOH with heating in a boiling water bath for 15 min. Tissue TBARS were expressed as absorbance units at 532 nm.
Statistics.
All data are expressed as means ± SEM. Three-way MANOVA (SPSS 8.0 for Windows; SPSS, Chicago, IL) was used to test for differences between experimental treatments and any interactions between rat strain, dietary cholesterol level and dietary fat source. Where treatment differences and corresponding interactions did exist, the source of the differences at a P 0.05 significance level was identified by the Student–Newman–Keuls multiple-range test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Short-chain fatty acids (SCFA) (4:0, 6:0, 8:0 and 10:0), unique to the butter blend diets, totaled 36 g/kg total fatty acids (Table 1) . Medium-chain fatty acids (12:0 and 14:0) in B and MO blend diets totaled 120 and 56 g/kg total fatty acids, respectively. Diet content of monounsaturated 18:1(n-9) was similar for B and MO diets, and lower than that of SBO diets. PUFA content of diets as 18:2(n-6) was SBO > MO > B and for 18:3(n-3) was MO > SBO > B, as expected. MO diets were unique in their content of the very long chain (n-3) PUFA including EPA and DHA. The calculated polyunsaturated to saturated fatty acid ratios (P:S) for the B, SBO and MO diets were 0.22, 1.64 and 1.34, respectively. The ratios of (n-6) to (n-3) PUFA in the B, SBO and MO diets were 3.8, 4.2 and 0.96, respectively.
Rat growth, apparent fat digestibility and systolic blood pressure.
Body weight gained was not affected by rat strain, dietary fat source or cholesterol level (Table 2 ). Moreover, the FER of rats over the 10-wk experimental period was not different between rat strains or dietary treatment groups (SHR, 0.185 ± 0.008 and WKY, 0.202 ± 0.011). Apparent digestibility of dietary fats was not different between rat strains at 10 wk of age. However, apparent fat digestibility was reduced (P < 0.001) in rats fed high cholesterol diets and in rats fed MO diets than in rats fed B or SBO diets (P = 0.048). Systolic blood pressure at 13 wk of age confirmed that SHR rats were hypertensive (P < 0.001) compared to WKY rats (Table 2) . However, the SBP of rats was not influenced by either dietary fat source or cholesterol level.
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Plasma total cholesterol (TC) concentrations were lower (P < 0.001) in SHR than in WKY rats (Table 3 ). Groups fed high cholesterol diets showed increased (P < 0.001) plasma TC levels compared to those fed low cholesterol diets. This difference was particularly evident in WKY rats, as demonstrated by the rat strain and dietary cholesterol level interaction (P < 0.001). Rats fed MO diets, compared to rats fed B or SBO, showed reduced (P < 0.001) plasmaTC concentrations, particularly in those fed high cholesterol diets, as indicated by the dietary cholesterol level and fat source interaction (P = 0.001). Plasma triacylglycerol (TG) levels did not differ between SHR and WKY rats (Table 3) . However, plasma TG concentrations were greatest in B-fed groups and lowest (P < 0.001) in MO-fed groups. The influence of dietary cholesterol level on plasma TG concentrations was greatest in B-fed groups compared to SBO- and MO-fed groups, as indicated by the dietary cholesterol and fat source interaction (P = 0.005). Plasma TG (P < 0.001) and phospholipid (PL) levels (P = 0.005) in rats were both decreased in high compared to low cholesterol diet groups (Table 3) . Plasma PL concentrations were greater (P < 0.001) in WKY than in SHR rats.
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RBC CAT activity was greater (P < 0.001) in SHR (0.062 ± 0.003 k/mg Hb) than in WKY rats (0.056 ± 0.002 k/mg Hb), but was not influenced by dietary treatment. RBC SOD activity was not different between rat strains (SHR, 3.46 ± 0.22 and WKY, 3.39 ± 0.21 U/mg Hb) or dietary treatment groups. Similarly, RBC GSH-Px activities were not different between rat strains (SHR, 49.3 ± 2.6 and WKY, 49.9 ± 2.5 nmol NADPH min-1 · mg Hb-1) or dietary treatment groups. RBC GSSG-Red activities were not different between rat strains (SHR, 2.44 ± 0.22 and WKY, 2.34 ± 0.19 nmol NADPH min-1 · mg Hb-1), although an interaction between rat strain and dietary fat source (P = 0.014) was identified in groups fed MO diets (SHR, 2.61 ± 0.26 vs. WKY, 2.24 ± 0.28 nmol NADPH min-1 · mg Hb-1) compared to groups fed B and SBO diets (SHR, 2.35 ± 0.20 vs. WKY, 2.40 ± 0.14 nmol NADPH min-1 · mg Hb-1).
CAT activity in heart tissue was negligible once correction for enzyme activity attributed to contaminating RBC was taken into consideration (data not shown). Heart SOD activity was not different between rat strains (SHR, 26.4 ± 1.9 and WKY, 25.8 ± 1.5 U/mg protein) or dietary treatment groups. Similarly, heart GSH-Px activities were not different between rat strains (SHR, 95.5 ± 5.1 and WKY, 101.9 ± 6.9 nmol NADPH min-1 · mg protein-1) or dietary treatments. Heart GSSG-Red activities did not differ between rat strains (SHR, 32.9 ± 3.0 and WKY, 30.4 ± 2.4 nmol NADPH min-1 · mg protein-1) or dietary treatment groups.
Liver CAT activity was greater (P = 0.010) in SHR than in WKY rats and lower (P < 0.001) in SBO-fed rats than in B- and MO-fed rats (Fig. 1A, B ). Liver SOD activity was reduced (P = 0.032) in rats fed high rather than low cholesterol diets; the difference was greater for WKY than for SHR rats, as demonstrated by the significant rat strain by dietary cholesterol level interaction (P < 0.001) (Fig. 1 C, D). Liver GSSG-Red activities were greater (P = 0.004) in SHR than in WKY rats (Fig. 2A, B ). Liver GSSG-Red activities also increased (P = 0.028) in groups fed high rather than low cholesterol diets; this was particularly evident in SHR rats, as demonstrated by the interaction between rat strain and dietary cholesterol level (P = 0.003). SHR rats fed SBO blend diets tended to have decreased liver GSSG-Red activities compared to WKY rats, resulting in an interaction (P < 0.001) between rat strain and dietary fat source. Liver GSH-Px activities were lower (P < 0.001) in SHR than in WKY rats (Fig. 2 C, D); moreover, there was a tendency for GSH-Px activities to be reduced in groups fed high rather than low cholesterol diets (P = 0.065).
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Depletion of sulfhydryl groups (as GSH) in RBC was greater (P = 0.014) in MO-fed rats than in B- or SBO-fed rats (Fig. 3A, B ). Rat strain and dietary cholesterol level did not influence RBC GSH depletion. Rats fed SBO diets had greater (P < 0.001) MDA production in RBC than B and MO groups. This finding was particularly evident with WKY rats, as demonstrated by the interaction (P = 0.005) between rat strain and dietary fat (Fig. 3 C, D). RBC MDA production was reduced (P < 0.001) in rats fed high compared to low cholesterol diets, regardless of dietary fat blend.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the present study, SHR and WKY rats fed 160 g fat blends/kg diet did not show notable differences in body wt gained or FER between rat strains, dietary fat or cholesterol treatments, despite the fact that the apparent digestibility of fat at 10 wk of age was slightly reduced by feeding diets containing high cholesterol or MO. These results were in contrast to previous reports, which identified rat strain differences for FER (SHR < WKY) and dietary fat source effects on body wt gained (menhaden oil diets < butter, beef tallow and soybean oil) in studies that fed lower fat diets (i.e., 5–8% dietary fat) to young (5 to 14 wk of age) SHR and WKY rats (13 ,36 ,37 ) and to SHR and WKY rats fed varying levels of fat up to 22 wk (38 ). The greater amount of dietary fat and thus energy, provided by diets to young, growing rats in the present study likely contributed to the lack of differences noted between treatment groups.
Interestingly, other workers identified abnormalities in the hypothalamic–pituitary– adrenal–gonadal axis in SHR compared to WKY rats as responsible for growth and SBP differences between strains when low or high fat diets are fed to rats (39
,40
). Wexler (39
) reported that SHR rats fed a high fat diet were relatively cachetic, with reduced pituitary gland weights, compared to SHR controls fed a low fat nonpurified diet, as well as WKY counterparts. In this same study, the high fat diet (20% suet, 5% cholesterol, 2% cholic acid) inhibited the development of hypertension in SHR, thought to be related to the hypopituitarism associated with the high fat diet. In the present study, total body wt gained (144 g over 10 wk) by rats fed a 160 g fat/kg diet was lower than that reported previously when SHR and WKY rats were fed 80 g fat/kg diet [154 g over 10 wk (13
)]. This difference likely indicates a dietary fat level effect on energy balance in these rats. The link between a high fat diet and hypopituitarism in SHR rats, and thus related effects on growth and SBP, may involve both the type and amount of fat in the diets as well as the treatment period. For example, other workers reported reductions in SBP of SHR after feeding fish oil diets or diets enriched with EPA and DHA or 
-linolenic acid (GLA) (41
–43
). The greatest hypotensive effects were observed in SHR rats fed EPA + DHA + GLA at 1.7% of the diet (41
), purified EPA + DHA as ethyl esters at 3.31% of the diet (43
) or 9.2% GLA in the diet (42
). In comparison, the MO blend diets used in this study contained 17.6 g EPA + DHA/kg diet. Thus, the amounts of long-chain PUFA (EPA + DHA) consumed in diets by SHR rats in this study were likely insufficient over the 10-wk feeding period to result in a hypotensive effect, particularly in the absence of dietary GLA, which shows potent hypotensive effects related to increased biosynthesis of the vasodilatory prostanoid PGI2 (41
,42
).
In the present study, dietary fat source had a profound effect on not only plasma TC concentrations but also TG levels. These changes have been attributed to modulation of the lipogenic enzymes of the pentose phosphate pathway (21 ,34 ). Previous studies also indicated that the hypocholesterolemic effect of dietary fish oil can be attributed to decreases in LDL-cholesterol and the VLDL- + LDL-cholesterol/HDL-cholesterol ratio (3 ,9 ), reflecting the inhibitory effect of dietary fish oil on the hepatic synthesis and secretion of TG-rich VLDL (44 ). Other mechanisms underlying this change in plasma cholesterol include the inhibition of HMG-coA reductase activity (45 ) as well as enhanced bile acid and cholesterol secretion in fish oil–fed rats (46 ). These effects of dietary fish oil on biliary cholesterol secretion could likely contribute to the reductions in plasma TC in SHR and WKY rats fed high cholesterol MO diets. The dietary cholesterol level and fat source effects observed in the present study were more pronounced in WKY than in SHR rats because of the well-recognized hypocholesterolemia of the SHR (12 ,22 ,41 ). Iritani and co-workers (47 ) demonstrated in vitro that incorporation of acetate into cholesterol was reduced in liver tissue slices from SHR compared to WKY rats. Thus, rat strain differences in plasma TC response to dietary treatments in this study were indicative of the genetic differences in cholesterol metabolism between SHR rats and their related WKY counterparts.
Dietary fat sources rich in (n-6) and (n-3) PUFA, particularly 18:2(n-6), reduced hepatic protein levels as well as mRNA and thereby, synthesis and activity of G6PDH (19 ,21 ,34 ). These effects in turn reduce hepatic lipogenesis and plasma TG levels, as seen in SBO groups in this study. In addition, the hypotriacylglycerolemic effects of dietary fish oil are well documented and may involve a combination of metabolic effects, including membrane alterations associated with (n-3) PUFA enrichment of membrane PL, inhibition of hepatic fatty acid synthase, G6PDH and acetyl-coA carboxylase activities at the mRNA level, as well as induction of peroxisomal ß-oxidation (34 ,48 ).
Differences in RBC and tissue antioxidant enzyme activities and glutathione reducing equivalents between rat strains were largely consistent between SHR and WKY rats fed semipurified diets in this study and those fed a nonpurified diet in previous studies (12 ,22 ). Hypertension in SHR rats has been associated with elevations in RBC and tissue CAT to metabolize excess H2O2 (12 ,22 ). This protection against oxidative stress in vivo would be particularly important for SHR compared to WKY rats because of the presence of reduced hepatic GSH-Px (12 ,22 ). Elevated RBC and heart GSH levels in SHR rats may serve as a reservoir of reducing equivalents against oxidation when required. In comparison, the much more metabolically active liver was characterized by enhanced GSSG-Red activities in SHR rats to regenerate GSH levels to the same concentration as that in WKY rats. Taken together, rat strain differences in antioxidant enzyme activities indicate a balance between enzymatic and nonenzymatic radical scavenging and detoxification in response to oxidative stress in these tissues.
The in vivo response to a higher fat diet, particularly one rich in long-chain PUFA, is regarded as a form of oxidative stress (19 ,49 ). The effects of varying dietary fat source in the 160 g fat blends/kg diet on RBC and tissue antioxidant enzyme activities in SHR and WKY rats were observed mainly in the hepatic tissue, with the exception of RBC GSSG-Red activities in the present study. MO blend diets, compared to SBO and B blend diets, enhanced RBC GSSG-Red activities in SHR rats, with the opposite effect in WKY rats. These observations coincided with reductions in RBC GSH levels in MO-fed rats, indicating an enhanced requirement for GSH-reducing equivalents in the presence of enhanced oxidative stress associated with the long-chain (n-3) PUFA in fish oil. Dietary fat sources from SBO and MO blend diets influenced hepatic antioxidant enzyme activities and GSH levels, respectively. The (n-6) PUFA of SBO are elongated and desaturated during the synthesis of arachidonic acid [20:4(n-6)], one of the eicosanoid precursors, which undergoes conversion via cyclooxygenase to release prostanoid products and the superoxide radical, O2-·. Excess O2-· inhibits CAT activity (31 ,50 ), as observed in hepatic tissue of SBO blend–fed rats in this study. In these same rats, hepatic SOD activity was not different between dietary fat treatments, which supports the suggestion that excess O2-· levels generated in hepatic tissues had occurred. Similarly, hepatic GSSG-Red activities in SBO blend– fed rats were reduced in the present study, compared to MO- and B blend–fed counterparts. The reactivity of O2-· is generally considered to be rather low compared to that of OH·, but the protonated form of O2-·, HO2-·, is somewhat more reactive (50 ) and may interact with membrane-bound systems in vivo such as GSSG-Red. Increased oxidative stress occurring in vivo was suspected with enhanced hepatic GSH levels in MO-fed rats compared to SBO- and B-blend–fed rats. This result can be explained by the need to accommodate the greater levels of lipid peroxidation and reactive oxygen species generated by the higher levels of long-chain (n-3) PUFA in membrane PL molecules.
The impact of dietary fat source on RBC and tissue susceptibility to oxidation and oxidative stress may also reflect the incorporation of dietary PUFA into membrane bilayer PL molecules. Indeed, greater amounts of membrane PL PUFA are associated with lipid peroxidation–mediated cell membrane damage, in that tissues from rats fed a 16–20% corn or soybean oil diet showed enhanced basal levels of tissue TBARS, conjugated dienes and induced TBARS (19 ,49 ). Preformed long-chain PUFA from the diet such as EPA and DHA are readily incorporated into the sn-2 position of bilayer PL molecules (32 ). Moreover, 20:4(n-6) is the most abundant long-chain PUFA in bilayer PL and a precursor in eicosanoid metabolism. Therefore, the fatty acid composition of tissue and RBC membrane PL can influence the generation of ROS and tissue susceptibility to oxidative stress in vitro. The present study is the first to show that RBC, heart and hepatic tissue from SHR and WKY rats fed a 160 g fat blends/kg diet showed enhanced susceptibility to in vitro lipid peroxidation, in contrast to those fed an 80 g fat blends/kg diet in our previous study (3 ). Dietary 18:2(n-6), as a precursor to 20:4(n-6), would be expected to enhance both membrane PL content of the latter PUFA and susceptibility of RBC and tissue lipids to peroxidation in vitro (19 ,49 ). Moreover, the long-chain (n-3) PUFA in fish oil are noted to be particularly susceptible to lipid peroxidation (4 ). The levels of TBARS generated from the heart and hepatic tissue from MO-fed groups in this study confirmed the increased susceptibility of tissues to lipid peroxidation with fish oil diets previously reported by others (2 ). Consumption of a higher fat diet (160–200 g fat/kg diet) was critical to these results, given that previous studies have shown that 80 g MO fat blend/kg diet in fact reduced tissue peroxidizability related to improved membrane stability attributed to the close packing of EPA and DHA in plasma membrane PL molecules (3 ). Treatment differences of GSH depletion could also reflect tissue levels of antioxidant substrate. For example, RBC and hepatic GSH depletion in MO-fed rats were enhanced and decreased, respectively, in response to the reduced and enhanced basal levels of GSH in these rats compared to B- and SBO-fed rats.
Conversely, greater levels of cell membrane and LDL cholesterol, as previously reported in hypercholesterolemic SHR and WKY rats, have been associated with decreased in vitro forced peroxidation (3 ). The decreases in MDA or TBARS in RBC and hepatic cell membranes, respectively, are attributed to the protective antioxidant and stabilizing effects of enhanced membrane cholesterol against oxidative stress (3 ,51 ). However, these effects, observed in rats fed high cholesterol diets (5.0 g cholesterol/kg diet), are combined with other undesirable effects, such as a fatty liver with an abnormal macroscopic appearance and the enhanced susceptibility to GSH depletion seen in hepatic tissue in this study and in previous studies (3 ).
In the present study, rats fed high cholesterol diets showed a complex pattern of alterations in antioxidant enzyme activities and GSH levels in hepatic tissue that could not be solely attributed to a reduction in hepatic G6PDH activity previously reported in high cholesterol–fed rats (52 ). In fact, hepatic GSSG-Red activities were enhanced in high cholesterol–fed rats, particularly SHR compared to WKY rats. This observation was associated with decreased levels of GSH in hepatic tissue of high cholesterol–fed rats, a possible indicator of a response to enhanced oxidative stress in these rats. Similarly, hepatic SOD activities were reduced in high cholesterol–fed rats, which is indicative of inhibition of SOD by elevated levels of H2O2 attributed to the slight reduction in hepatic GSH-Px observed in this study (13 ,52 ).
In summary, in the present study, oxidative stress in vivo from long-chain (n-6) and (n-3) PUFA contained in SBO or MO diet blends increased RBC and tissue susceptibility to lipid peroxidation. Diets containing 160 g MO-based fat blend/kg diet enhanced heart and hepatic tissue lipid peroxidation in vitro. SBO blend diets also enhanced lipid peroxidation in RBC of SHR and WKY rats. Alterations in antioxidant enzyme activities were tissue specific and likely reflected dietary fat source and intake level effects on energy metabolism and oxidative status. High cholesterol diets reduced hepatic GSH levels and exerted a protective effect against in vitro oxidation of RBC and hepatic tissue of SHR and WKY rats. The SHR rats provided a unique model for studying genetic essential hypertension, and also revealed disturbances in cholesterol metabolism and dietary fat–mediated alterations to plasma lipid and antioxidant metabolism.
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FOOTNOTES |
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3 Abbreviations used: B, butter; CAT, catalase; DHA, docosahexanoic acid; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); EPA, eicosapentaenoic acid; FER, feed efficiency ratio; G6PDH, glucose-6-phosphate-dehydrogenase; GLA, -linolenic acid; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSG-Red, glutathione reductase; GST, glutathione-S-transferase; Hb, hemoglobin; HMPS, hexose monophosphate shunt; MDA, malondialdehyde; MO, menhaden oil; PL, phospholipid; PUFA, polyunsaturated fatty acids; SBO, soybean oil; SBP, systolic blood pressure; SHR, spontaneously hypertensive rats; SOD, superoxide dismutase; TBARS, 2-thiobarbituric acid reactive substances; TC, total cholesterol; TG, triacylglycerol; WKY, Wistar Kyoto rats.
Manuscript received 16 September 2002. Initial review completed 21 October 2002. Revision accepted 6 December 2002.
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