Vitamin E Inhibits Fish Oil-Induced Hyperlipidemia and Tissue Lipid Peroxidation in Hamsters

Stan Kubow a'*, Nathalie Goyette a, Selim Kermasha b, Jean Stewart-Phillip c, and Kristine G. Koski a aSchool of Dietetics and Human Nutrition, bDepartment of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, Quebec H9X 3V9, Canada CMontreal General Hospital Research Institute and Department of Medicine,

McGill University, Montreal, Quebec H3G 1A4, Canada

ABSTRACT: Previous research has linked hyperlipidemia with increased serum concentrations of lipid peroxidation products; however, a specific association between diet-induced oxidative stress and hyperlipidemia has not been studied. In the present study, the relationship between tissue lipid peroxidation and hyperlipidemia induced by ingestion of fish oil was examined. In Experiment I, male Golden Syrian hamsters were fed semi- purified diets composed of 1.6 wt% safflower oil plus 15.0 wt% of either butterfat (BF), safflower oil (SAFF), or high-cholesterol menhaden oil [MHO(H-CHOL)] semipurified diets for 27 d. The cholesterol contents of the diets were adjusted to 0.088%. The MHO(H-CHOL)-fed hamsters exhibited higher serum concen- trations of total cholesterol, triglycerides, apolipoprotein B, and lipid peroxides when compared to the BF and SAFF diet groups. In a further study (Experiment 2), hamsters were fed for 27 d three dietary treatments: (i) MHO(H-CHOL) with no vitamin E content; (ii) a low-cholesterol menhaden oil containing high concentrations of vitamin E (2.5 mg tocopherol/g oil or dietary concentrations of 375 mg/kg) [MHO(L-CHOL) + E]; and (iii) the MHO(L-CHOL + E) with added cholesterol (595 mg/kg) [MHO(L-CHOL) + CHOL + E] to match the cholesterol content of the MHO(H-CHOL). The MHO(L-CHOL) + E and MHO(L- CHOL) + CHOL + E diet groups showed lower concentrations of serum cholesterol, triglycerides, and hepatic lipid peroxides than the MHO(H-CHOL)-treated group. Moreover, in contrast to the hypercholesterolemia caused by the MHO(H-CHOL) feeding, the MHO(L-CHOL)+ E and MHO(L-CHOL) + CHOL + E diets did not show a serum cholesterol-elevating action. This study supports the hypothesis that oxidative stress in the Syrian hamster could play a causal role in dietary-induced hyperlipi- demia which can be inhibited by high vitamin E intake. Lipids 31, 839-847 (1996).

Lipid peroxidation and oxidative stress have been associated with the occurrence of hypercholesterolemia and atheroscle-

*To whom correspondence should be addressed at School of Dietetics and Human Nutrition, Macdonald Campus of McGill University, 21,111 Lakeshore, Ste. Anne de Bellevue, Quebec H9X 3V9, Canada. Abbreviations: ANOVA, analysis of variance; BF, butterfat; HDL, high density lipoprotein; LDL, low density lipoprotein; MHO(H-CHOL), high-cholesterol menhaden oil; MHO(L-CHOL) + E, low-cholesterol menhaden oil with a high vitamin E content; MHO(L-CHOL) + CHOL + E, low-cholesterol menhaden oil with a high vitamin E content and added cholesterol; NRC, National Research Council; PUFA, polyunsaturated fatty acids; SAFF, safflower oil; TBARS, thio- barbituric acid reactive substances; USP, United States Pharmacopeia.

rosis both in humans and experimental animals (1-5). Despite associations of oxidative stress with hyperlipidemia, a possi- ble relationship between diet-induced oxidative stress and diet-induced hyperlipidemia has not been closely examined.

Fish-oil feeding generally results in reductions in plasma triglyceride and very low density lipoprotein concentrations (6), but there does exist conflicting research. Investigations have shown elevated serum triglycerides in the New Zealand rabbit (7) and the Golden Syrian hamster (8) following fish- oil feeding. In addition, the fish oil-fed Syrian hamster had an elevated apolipoprotein B/apolipoprotein AI ratio (8), which may be correlated with an increased risk of atherosclerosis and may be a better predictor of coronary heart disease than plasma lipid concentrations p e r se (9). Fish-oil concentrates given to hypercholesterolemic men have caused increased plasma total and low density lipoprotein (LDL) cholesterol concentrations (10).

Fish oils rich in n-3 polyunsaturated fatty acids (PUFA) that are efficiently incorporated into tissue membrane phos- pholipids displacing n-6 PUFA, and membranes enriched in n-3 fatty acids are likely to be more susceptible to oxidative disruption (11). Dietary fish-oil consumption has been shown to increase aortic lesions in rabbits and cause higher plasma levels of lipid peroxidation products as measured by the pro- duction of thiobarbituric acid reactive substances (TBARS) (12). In addition, oxidative stress generated by fish-oil con- sumption is known to increase the requirement of vitamin E (12). As lipid peroxidation (1) and a low body content of to- copherol (5) have been shown to increase tissue cholesterol concentrations, the accompanying increase in oxidative stress could play a causal role. Indeed, the ability of fish oil to de- crease plasma triglyceride concentrations in the normolipi- demic human has been shown to be markedly enhanced by high supplemental intake of vitamin E (13). Haglund et al.

(13) suggested that the antioxidant effect of vitamin E was in- volved through efficient inhibition of fatty acid peroxidation, thereby allowing larger amounts of active n-3 fatty acids in fish oils to exert triglyceride-lowering effects.

In the present study, two experiments were performed with the Syrian hamster. In Experiment 1, the relationship between tissue concentrations of lipid peroxidation products and serum lipid concentrations was investigated among menhaden

Copyright �9 1996 by AOCS Press 839 .Lipids, Vol. 31, no. 8 (1996)

840 S. KUBOW ETAL.

oil-, butterfat (BF), and safflower oil (SAFF)-based diets. In contrast to fish oils, SAFF feeding was not expected to in- crease tissue lipid peroxidation as tissue concentrations of lipid peroxidation products have previously been shown not to be increased by high n-6 PUFA intake as compared to high- saturated fatty acid intake (14). Dietary studies comparing the effects of different fat sources on blood cholesterol metabo- lism have typically not balanced for the vitamin E content nascent in each fat. Hence, in the initial experiment, the ef- fects of the different fat sources on blood cholesterol metabo- lism and lipid peroxidation were assessed without balancing for the vitamin E content as this could inhibit the effects of fish oil on lipid peroxidation and on blood lipid concentra- tions. In a second experiment, we tested whether a high in- take of vitamin E could modify the dietary cholesterol- induced hyperlipidemia and tissue lipid peroxidation associ- ated with fish oil-based diets.

MATERIALS A N D M E T H O D S

Experimental design. In Experiment 1, hamsters were fed semipurified diets containing 1.6 wt% SAFF and 15 wt% of either BE SAFE or a high cholesterol (5.76 mg cholesterol/g of oil) menhaden oil [MHO(H-CHOL)] (Table 1). In experi- ment 1, the effects of these different fat sources on blood cho- lesterol metabolism and lipid peroxidation were examined without matching for vitamin E content as additional vitamin E might have compromised effects of MHO(H-CHOL) on lipid peroxidation and on blood lipid concentrations. The MHO(H-CHOL) and BF had nondetectable concentrations of vitamin E, whereas SAFF contained 0.42 g vitamin E/g oil (Table 1). Sufficient vitamin E (6.1 mg vitamin E/kg diet) was added to the BF and MHO(H-CHOL) semipurified diets to meet the National Research Council (NRC) requirements for adequate vitamin E intake for the hamster (15). As BF con-

TABLE 1 Composition of Experimental Diets (g/kg) a

Ingredients BF SAFF MHO(H-CHOL) MHO(L-CHOL) + E + CHOL MHO(L-CHOL) + E

Egg white 150 150 150 150 150 Cornstarch 538 538 538 538 538 Butterfat b 178 . . . . Safflower oil c 16 166 16 16 16 Menhaden oil d - - - - 150 150 150 Cholesterol e

Naturally occurring 0.44 - - 0.88 0.285 0.285 Added 0.44 0.88 - - 0.595 - - Total 0.88 0.88 0.88 0.88 0.285

Cellulose 50 50 50 50 50 Vitamin mix r ~4 14 14 14 14 Mineral mixg 80 80 80 80 80

aBF = butterfat; SAFF = safflower oil, MHO(H-CHOL) = high cholesterol (5.76 mg cholesterol/g oil) menhaden oil; MHO(L- CHOL) + CHOL + E = low cholesterol (1.9 mg cholesterol/g oil) MHO with high vitamin E content (2.5 mg vitamin E/g off) and added cholesterol (595 mg/kg); MHO(L-CHOL) + E = low cholesterol (1.9 mg choIesterol/g oil) MHO with high vita- min E content (2.5 mg vitamin E/g oil). bNondetectable concentrations of vitamin E in BF. Fatty acid composition of BF is as follows (mg%): 4:0, 3.3%; 6:0, 2.3%; 8:0, 1.6%; 10:0, 3.9%; 12:0, 4.0%; 14:0,12.5%; 14:1, 1.2%; 16:0, 33.1%; 16:1n-9, 1.7%; 18:0, 11.6%; 18:1n-9, 21.0%; 18:2n-6, 3.8%. tin Experiments 1 and 2, all diets contained a minimum of 1.6 wt% SAFF to prevent essential fatty acid deficiency. Vitamin E concentration of SAFF is 0.42 g/g oil. Fatty acid composition of SAFF is as follows (mg%): 14:0, 0.3%; 16:0, 11.9%; 18:0, 2.3%; 18:1 n-9, 14.1%; 18:2n-6, 70.9%; 18:3 n-3, 0.5%. aNondetectable concentrations of vitamin E in MHO(H-CHOL) and 2.5 mg vitamin E/g oil in MHO(L-CHOL). Fatty acid composition of H-CHOL MHO is as follows (rag%): 14:0, 15.2%; 16:0, 22.3%; 16:1 n-9, 12.7%; 18:0, 3.9%; 18:1 n-9, 10:0%; 18:2n-6, 2.8%; 18:3n-3, 1.1%; 18:4n-3, 4.5%; 20:1n-9, 1.4%; 20:4n-6, 0.5%; 20:5n-3, 12.7%; 22:1n-9, 1.3%; 22:4n-6, 0.5%; 22:5n-3, 1.5%; 22:6n-3, 9.6%. Fatty acid composition of MHO(L-CHOL) is as follows (rag%): 14:0, 7.8%; 16:0, 14.7%; 16:1n-9, 0.2%; 18:0, 2.7%; 18:1n-9, 5.2%; 18:2n-6, 1.4%; 18:3n-3, 0.9%; 18:4n-3, 2.5%; 20:1n-9, 0.8%; 20:4n-6, 0.8%; 20:5n-3, 13.6%; 22:1 n-9, 0.2%; 22:4n-6, 0.2%; 22:5n-3, 2.5%; 22:6n-3, 10.2%. eCholesterol United States Pharmacopeia (USP) was added to the oil, BF, and MHO(L-CHOL) diets at 880, 440, and 595 (mg/kg diet), respectively. No cholesterol was added to the MHO(H-CHOL) diet because it naturally contains 5.76 mg cho- lesterol/g of oil. BF naturally contains 2.39 mg cholesterol/g BF, MHO(L-CHOL) contains 1.9 mg cholesterol/g oil, and SAFF contains no cholesterol. ~'itamin mix supplied the following (mg/kg diet): niacin, 135; calcium pantothenate, 60; riboflavin, 22.5; pyridoxine, 9; thiamin, 30; folic acid, 3; biotin, 0.9; cyanocobalamine, 0.015; menaquinone, 6; choline, 6000; @-tocopheryl acetate (1350 IU/g), 6.1 ; cholecalciferol (400,000 IU/g), 9.32; vitamin A palmitate (250,000 IU/g), 40.2. Only 18.5 mg/kg of vita- min A palmitate (250,000 IU/g) was provided to the BF diets since vitamin A occurs naturally in BF at a level of 30.07 IU/g. gMineral mix supplied the following (mg/kg diet): calcium carbonate, 22.48; potassium phosphate, 25.16; sodium chloride, 5.72; magnesium sulfate, 4.46; cupric carbonate, 0.0045; cobalt chloride, 0.0666; sodium fluoride, 0.00008; potassium io- date, 0.0040; ferric sulfate, 1.045; manganous carbonate, 0.0115; sodium selenite, 0.00033; zinc carbonate, 0.0265.

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HYPERLIPIDEMIA AND LIPID PEROXlDATION 841

tained 2.39 mg cholesterol/g and SAFF contained no choles- terol, United States Pharmacopeia (USP) cholesterol was added to the SAFF and BF diets at 880 and 440 mg (mg/kg diet), respectively, to match levels in the MHO(H-CHOL). In Experiment 2, hamsters were fed one of three semipurified diets which consisted of: (i) the high-cholesterol MHO(H- CHOL); (ii) a low-cholesterol menhaden oil which also con- tained high amounts of vitamin E (2.5 mg vitamin E/g oil) equivalent to a dietary concentration of 375 mg/kg [MHO(L- CHOL) + E]; and (iii) the low-cholesterol MHO(L-CHOL) + E oil (2.5 mg vitamin E/g oil) which was supplemented with USP cholesterol (595 mg/kg diet) [MHO(L-CHOL) + CHOL + E] to match the high-cholesterol content found in MHO(H- CHOL).

Animals and diets. Male Syrian hamsters, weighing 110-120 g, were purchased at seven weeks of age from Charles River Inc. (St-Constant, Quebec, Canada). The ani- mals were fed a commercial nonpurified diet (Mouse Chow; Purina, Montreal, Quebec, Canada) upon arrival. After an adaptation period of three weeks, the hamsters received semi- purified diets for 27 d in two separate experiments. Thirty-six hamsters were weighed and randomly assigned to three semi- purified diet groups in Experiment 1, whereas in Experiment 2, twenty-one hamsters were weighed and allocated randomly to three semipurified diet groups. In Experiments 1 and 2, all diets contained minimally sufficient vitamin E (6.1 mg vita- min E/kg diet) to meet the NRC requirements of vitamin E for the hamster (15). In all diets, butylated hydroxytoluene was added to dietary fat sources at a level of 0.1% of lipid weight to protect fats from oxidation. The MHO(H-CHOL) was purchased from U.S. Biochemical Corporation (Cleve- land, OH), and the MHO(L-CHOL) was a gift from the Fish Oil Biomedical Test Material Program of the National Ma- rine Fisheries Service (Washington, D.C.). The differences in fatty acid content between the MHO(L-CHOL) and MHO(H- CHOL) oils as indicated in Table 1 were minimal, i.e., total n-3 content (mg %) = 29.4 [MHO(L-CHOL)] and 29.7 [MHO(H-CHOL)]; n-6/n-3 ratio = 0.09 [MHO(L-CHOL)] and 0.12 [MHO(H-CHOL)]; unsaturation indices = 167 [MHO(L-CHOL)] and 184 [MHO(H-CHOL)]; eicosapen- taenoic acid (20:5n-3)/arachidonic acid (20:4n-6) ratio = 17 [MHO(L-CHOL)] and 25.4 [MHO(H-CHOL)]; 20:5n-3/do- cosahexaenoic acid (22:6n-3) ratio = 1.3 [MHO(H-CHOL)] and 1.3 [MHO(L-CHOL)]. Based on previous literature, the magnitude of the differences in fatty acid composition would not be expected to impact upon serum lipid concentrations (16,17). Unused fresh diets were kept in plastic bags at 4~ and bags were flushed with nitrogen after each opening dur- ing the trial. Samples of stored semipurified diet were mea- sured for TBARS on a weekly basis throughout the experi- ment to obtain an index of dietary lipid peroxidation by a modification (18) of the method of Dahle et al. (19). Analysis of all semipurified diets yielded nondetectable values of TBARS, e.g., below 20 nmol of reference standard (malondi- aldehyde bisdiethyl acetal). Suitable vitamin and mineral mixes for hamster needs were developed following the NRC

requirements (15) and were given at 1.5 times the require- ments to ensure an adequate intake of those nutrients. BF (Lactancia, Victoriaville, Quebec, Canada)contained 18.4% water (manufacturer's specifications). This factor was taken into account in the formulation of the BF diet (e.g., 177.6 g/kg of BF was added into the diet instead of 150.0 g/kg of lipid that was added in the other diets) in order to make the diets isoenergetic.

The animals were housed individually in plastic bottom wired top cages and kept in a temperature-controlled room (23-25~ with a 12-h automatically maintained light-dark cycle (light cycle 0700 to 1900). They were allowed access to food and water ad libitum. Fresh diets were fed to the ani- mals daily, and uneaten food was discarded. Body weight was measured every second day, and food intake was recorded daily. Care and handling of the animals conformed to the guidelines of the McGill University Animal Care Committee and Canadian Council of Animal Care (20).

Tissue sampling and preparation. On day 1 and day 27, blood was collected using heparinized capillary tubes by retro-orbital sinus puncture, from 14-h fasted hamsters anes- thetized with CO 2. On day 27, the hamsters were killed by de- capitation under CO 2 anesthesia. Hearts and livers were ex- cised and cleaned of blood with ice-cold saline, and the tis- sues were weighed. The hearts were fixed on corks for histological examination. Livers were freeze-clamped in liq- uid nitrogen and stored at -80~ until analyses. Aliquots of blood samples were allowed to clot at room temperature, cen- trifuged at 6000 x g for 10 rain, and stored at 4~ until they were analyzed within two days for total cholesterol, total triglyceride, and high density lipoprotein cholesterol (HDL- C) content. The remaining serum was stored at -20~ for de- termination of serum apolipoprotein A-I, apolipoprotein B, alanine aminotransferase, and lipid peroxides at a later date.

Biochemical analysis. Serum measurements of cholesterol, triglyceride, and HDL-C were obtained by automated enzy- matic methodology with the Abbott VP Super System (Ab- bott, Irving, TX) using Abbott enzymatic reagent kits (Abbott kit 6095 for cholesterol, 6097 for triglycerides, and 6039 for HDL-C). Serum alanine aminotransferase was measured via an enzymatic assay (Sigma kit 59 for aminotransferase; Sigma Chemical Co., St. Louis, MO) as an index of possible liver damage. Apolipoproteins A-I and B were measured using enzymatic assays (356 for apolipoprotein A-I and 357 for apolipoprotein B; Sigma Chemical Co.). The serum ala- nine aminotransferase, apolipoproteins A-I and B enzymatic assays were analyzed using the Abbott VP Super System. Serum and liver were thawed and analyzed for lipid hydroper- oxides using an adaptation of a lipid hydroperoxide kit assay (Kamiya Biomedical, Thousand Oaks, CA) based on the method of Oshishi et al. (21). The lipid was extracted by the method of Folch et al. (22) and reconstituted in different quantities of isopropranol (different dilutions) due to the vari- able amounts of lipids in each sample. The absorbance of the sample was determined at 620 nm using a microplate reader (Titertek Multiskan Plus MKII; ICN Biomedicals, Cleveland,

Lipids, Vol. 31, no. 8 (1996)

842 S. KUBOW ETAL.

OH) using a 620 nm filter. The concentration of TBARS in livers was assayed as described previously (18). Concurrent measurements of lipid hydroperoxides and TBARS were made, as previous research has indicated that concentrations of these indices appear to peak at different time points in he- patic tissues in relation to the induction of the oxidative stress (23). The cholesterol content of the dietary fats was deter- mined by gas-liquid chromatography by the method of Heuck et al. (24). The fatty acid composition of the dietary fats was determined by gas-liquid chromatography following fatty acid methyl esters formation and has been described previ- ously (25).

Histopathology. The heart was sectioned midway through the atria, and the aorta was cut distally to the sinus valves and fixed on a cork. Cryostat frozen sections, 10 Bm thick, were cut at three different levels and stained with oil red O and counter- stained with haematoxylin. The sections were examined mi- croscopically, and a similar section of each aorta was scored from 0 to 10 according to a modification of the method of Roberts and Thompson (26). Aorta were examined for the pres- ence of foam cells and lesions in the aortic sinus wall. The his- tology was done on 50% of hearts, taken via random alloca- tion, in each diet group (i.e., six hearts per dietary treatment).

Statistical analysis. Statistical analysis of the data was per- formed using the Statistical Analysis System for personal com- puters (Version 6.04, 1992; SAS Institute, Cary, NC). Two-way analysis of variance was used to determine the influence of time and dietary treatment on parameters measured at the beginning and end of the experiment (body weight, triglycerides, choles- terol, HDL-C). In Experiment !, for serum lipid measures, body weight change was used as a covariate in the statistical model of two-way analysis of variance (ANOVA). One-way ANOVA was used to determine whether there was a main effect of diet on cumulative food intake, liver weight, serum alanine trans- ferase, apolipoproteins A-I and B, apolipoprotein A-I/B, serum liver lipid peroxides, and liver TBARS. In Experiments 1 and 2, when a significant difference occurred among the main effects of diet and time in two-way ANOVA analyses or when a signif- icant main effect of diet was observed via one-way ANOVA, mean differences among treatment groups were identified by least significant means for a priori comparisons. In Experiment 1, the following a priori comparisons were done to determine the effect of dietary source of fat on serum lipoprotein and tis- sue lipid peroxidation concentrations: (i) BF vs. SAFF, (ii) SAFF vs. MHO(H-CHOL), and (iii) BF vs. MHO(H-CHOL). Pearson's correlations between serum lipids and apolipoproteins and serum measures of oxidative stress (lipid peroxides and TBARS) were performed. In Experiment 2, the specified com- parisons were: (i) MHO(H-CHOL) vs. MHO(L-CHOL) + E, (ii) MHO(H-CHOL) vs. MHO(L-CHOL) + CHOL + E , and (iii) MHO(L-CHOL) + E vs. MHO(L-CHOL) + CHOL + E. Cumulative food intake in Experiment 2 was put as a covariate in the statistical model of two-way ANOVA for serum lipid measures. The values were expressed as means _+ SE for all pa- rameters measured. In all comparisons, P < 0.05 was consid- ered as the minimum level of significance.

RESULTS

Experiment 1. Food intake, weight gain, and tissue weight. There were no significant differences in cumulative food in- takes among the BF (170.9 _+ 3.7 g), SAFF (163.0 +_ 3.7 g), and MHO(H-CHOL) (155.1 _+ 3.2 g) diets. Body weight change from day 0 to day 27 was significantly higher (P < 0.05) in the BF (+4.4 _+ 2.1 g) and the SAFF (+5.3 _+ 1.8 g) groups as compared to the MHO(H-CHOL) group (-1.9 _+ 1.4 g). No differences in the magnitude of body weight change were found between the BF group and the SAFF group. Liver weight as a percentage of body weight, however, was lower (P < 0.05) in the BF (6.35 _+ 0. l 1%) and the SAFF (6.08 _ 0.07%) groups as compared to fish oil-fed (6.70 _+ 0.12%) group suggesting an association between fish-oil feeding and hepatomegaly. The BF and SAFF groups were not different from each other in terms of liver weight as a percentage body weight. Liver weights were not different among the dietary treatments (data not shown).

Serum cholesterol, triglycerides, and apolipoproteins. Be- cause significant differences were observed in terms of body weight change, body weight change was included as a covari- ate in the two-way ANOVA statistical analyses for serum lipid analyses. Body weight as a covariate, however, did not statisti- cally influence serum lipid concentrations. In general, for total serum cholesterol concentrations, significant main effects of diet (P < 0.0001), time (P < 0.0001), and a significant diet x time (P < 0.000 l) interaction were observed (Table 2).

In terms of the changes in total serum cholesterol, a signif- icant main effect of diet (P < 0.0001) was observed. As would be expected from the high-saturated fat content of BE the BF group showed a significantly higher final concentration of serum cholesterol and a larger increase in total serum choles- terol than did hamsters fed the polyunsaturated SAFF diet (Table 2). The MHO(H-CHOL) hamsters had significantly higher final levels of serum cholesterol than did the hamsters fed either the SAFF and BF diets. In addition, hamsters fed the MHO(H-CHOL) diet had greater change in total serum cholesterol concentrations over the 27-d feeding period than the BF and the SAFF groups (Table 2), indicating a hyper- cholesterolemic action of MHO feeding relative to the other lipid-based diets.

For serum triglycerides measured on day 0 and day 27, sig- nificant main effects of diet (P < 0.05), time (P < 0.005), and a significant interaction of diet x time (P < 0.05) were ob- served. The change from day 0 to day 27 in serum triglyceride concentrations also demonstrated a significant main effect of diet (P < 0.001). The MHO(H-CHOL) group demonstrated higher final concentrations of triglycerides than either the BF or SAFF groups. The change (day 0-27) in triglyceride con- centrations in the MHO(H-CHOL) hamsters was also signifi- cantly greater than in the BF- and SAFF-fed animals, indicat- ing a hypertriglyceridemic effect of fish-oil intake. No differ- ences were observed in either final serum triglyceride concentrations or change (day 0-27) in serum triglyceride concentrations between the BF and SAFF diets.

Lipids, Vol. 31, no. 8 (1996)

HYPERLIPIDEMIA AND LIPID PEROXIDATION

TABLE 2 Effects of Dietary Fats on Serum Concentrations of Total Cholesterol, Triglycerides, HDbC, and Apolipoprotelns A-I and B in Hamsters (Experiment 1)a

843

BF SAFF MHO(H-CHOL)

Total cholestero~ (mg/dl_) Day 0 Day 27 Change

Total triglycerides (mgZdL)

Day 0 Day 27 Change

HDUC (mg/dL) Day 0 Day 27 Change

Apo A-I (mg/dL) Apo B (mg/dL) Apo A-I/Apo B

(12) 108.87 • 5.73 (12) 154.67 • 7.54

(12) 45.81 • 8.81

(12) 97.29 • 6.58 (12) 101.62 • 5.81 a

(12) 4.33 + 8.72 a

(12) 100.58 • 4.49 (12) 183.86 • 6.97 b'c

(12) 83.28 + 7.68 b'c

(12) 230.33 • 10.19 (12) 205.80 + 11.61 (12) 200.03 + 14.15 (12) 233.96 + 21.35 (12) 222.35 • 15.90 (12) 303.95 + 26.65 b'c

(12) -6.37 • 22.25 (12) 16.56 • 10.36 (12) 103.92 • 20.06 b,c

(10) 59.12 + 2.33 (10) 54.52 + 2.68 (10) 54.29 • 2.00 (12) 7 i .38 • 3.86 (10) 76.17 • 8.18 (12) 83.04 + 8.80

(10) 6.05 • 2.65 (10) 6.62 • 3.18 (10) 10.87 • 5.52 (5) 64.50 + 2.04 (5) 62.80 -4- 1.53 (5) 61.50 + 2.32

(12) 16.35 • 1.57 (12) 15.21 • 1.20 (12) 37.85 • 2.33 b,c (5) 5.32 • 0.43 (5) 5.10 • 0.88 (5) 1.77 • 0.17 b'c

aValues are means • SE. Number in parentheses equals number of hamsters. Hamsters were fed BF, SAFF, and MHO(H- CHOL)-based semipurified diets for 27 d. See Table 1 for other abbreviations. Means within each row with superscript let- ters indicate the following comparisons are significantly different (P< 0.05) by least significant means: aBF vs. SAFF; bSAFF vs. MHO(H-CHOL); CSF vs. MHO(H-CHOL). Body weight change (day 0-27) was used as a covariate in the statistical model of two-way analysis of varriance (ANOVA). Significance of main effects of two-way ANOVA for total serum cholesterol (diet: P< 0.05; time: P< 0.005; diet x time: P< 0.05), total serum triglycerides (diet, P< 0.0001; time: P< 0.0001; diet x time: P< 0.0001), serum high density lipoprotein-choleserol (HDL-C), (diet: nonsignificant; time: P < 0.0001; diet x time: non- significant). Significant main effects of one-way ANOVA for serum cholesterol change (P < 0.0001 ), serum triglyceride change (P< 0.001), serum apo B (P <0.0001 ), serum apolipoprotein (Apo) A-I/Apo-B (P < 0.001 ).

ANOVA showed no significant diet or time effects on serum HDL-C concentrations. On the other hand, the MHO(H-CHOL) animals had concentrations of apolipopro- tein B that were greater than hamsters fed the BF and SAFF diets. In addition, hamsters fed the MHO(H-CHOL) diet had significantly lower ratios of apolipoprotein A-I/apolipopro- tein B than the other two diets.

Serum and liver lipid oxidation products. The source of fat influenced the levels of serum lipid peroxides as animals fed the MHO(H-CHOL) diet had significantly higher serum lev- els of lipid peroxides than the hamsters fed the SAFF and BF diets (Table 3). Liver lipid peroxide and TBARS levels were not affected by the source of fat. When all dietary treatments were grouped together, correlations were observed between serum lipid peroxides and a number of serum indices includ- ing total serum cholesterol (change) (r = 0.42; P < 0.004), total serum triglycerides (change) (r = 0.47; P < 0.0009), serum apolipoprotein B (r = 0.69; P < 0.0001), and the serum apolipoprotein A-I to apolipoprotein B ratio (r = -0.72; P < 0.0004).

Serum alanine aminotransferase and histopathology. There were no significant differences observed among the treatment groups in terms of serum alanine aminotransferase levels (data not shown). Morphometric evaluation of the his- tologic sections showed the absence of foam cells. No athero- sclerotic lesions in the aortic sinus wall and no significant dif- ferences in intimal thickness were observed among dietary treatments (data not shown).

Experiment 2. Food intake, weight gain, and tissue weight. There were no differences in cumulative food intake between the MHO(H-CHOL) (161 _+ 6 g) group and either the MHO(L-CHOL) + CHOL + E (148 _+ 5 g) or the MHO(L- CHOL) + E (167 -e 5 g) groups; however, food intake was sig- nificantly lower (P < 0.05) in the MHO(L-CHOL) + CHOL + E group when compared with the MHO(L-CHOL) + E group. Although total weight gain did not differ among the dietary treatments (data not shown), liver weight at day 27 was sig- nificantly higher (P < 0.05) in the MHO(H-CHOL)-fed group (6.14 _+ 0.37 g) as compared with the MHO(L-CHOL) + E (4.82 _+ 0.21 g) and MHO(L-CHOL) + CHOL + E (5.14 _+ 0.21 g) groups. Similarly, liver weight as a percentage of body weight was significantly higher in the MHO(H-CHOL)-fed animals (4.50 _+ 0.20%) relative to the MHO(L-CHOL) + E (3.60 _+ 0.07%) and MHO(L-CHOL) + CHOL + E (4.00 _+ 0.20%) groups, indicating that the hepatomegaly associated with MHO feeding in Experiment 1 could be alleviated by high vitamin E intake. The MHO(L-CHOL) + E and MHO(L- CHOL) + CHOL + E diet groups were not significantly dif- ferent from each other in terms of liver weight or liver weight as a percentage of body weight.

Serum cholesterol and triglycerides. Since significantly different amounts of diets were consumed, food intake was included as a covariate for the two-way ANOVA for assess- ing the statistical comparisons of serum lipid values in Ex- periment 2. However, differences in cumulative food intake did not contribute to significant changes in plasma lipid con-

Lipids, Vol. 31, no. 8 (1996)

844 S. KUBOW ETAL.

TABLE 3 Effects of Dietary Fats on Serum and Liver Lipid Peroxides (LPO) and Liver Thiobarbituric Acid Reactive Sub- stances (TBARS) in Hamsters (Experiment 1) a

BF SAFF M H O ( H - C H O L )

LPO Serum (nmol/mL) (12) 13.56 + 3.88 (12) 20.23 + 5.90 (12) 62.00 • 7.73 b'c Liver (nmol/mg fat) (12) 2.50 • 0.53 (12) 1.36 • 0.23 (12) 2.11 • 0.44

TBARS Liver (nmol/mg fat) (12) 8.98 + 2.02 (11 9.71 • 3.38 (11 ) 10.78 + 1.82

aValues are means + SE. Number in parentheses equals number of hamsters. See Table 1 for other abbreviations. Hamsters were fed BF, SAFF, and MHO(H-CHOL)-based semipurified diets for 27 ft. Means within each row with superscript letters indicate the fol lowing comparisons are significantly different (P < 0.05) by least significant means: aBF vs. SAFF; bSAFF vs. MHO(H-CHOL); CBF vs. MHO(H-CHOL). Significance of main effect of one-way ANOVA for serum LPO (P < 0.0001 ).

centrations. In general, for total serum cholesterol concentra- tions, significant diet (P < 0.001), time (P < 0.01), and diet x time (P < 0.005) interactions were observed (Table 4). A sig- nificant diet (P < 0.05) effect was observed for change (day 0-27) in serum cholesterol concentrations. For the MHO(H- CHOL) group, serum cholesterol increased significantly (P <0.05) from baseline after 27 d of feeding. On the other hand, no significant differences were observed for the MHO(L- CHOL) + CHOL + E and the MHO(L-CHOL) + E groups with respect to the comparison of cholesterol concentrations on day 27 with baseline values. The MHO(H-CHOL)-fed hamsters also had significantly higher final concentrations of total serum cholesterol than did either the MHO(L-CHOL) + E or the MHO(L-CHOL) + CHOL + E groups (Table 4). Vit-

amin E, however, did not completely overcome the hyper- cholesterolemic effect of cholesterol as no significant differ- ences were observed between the MHO(H-CHOL) and the MHO (L-CHOL) + E + CHOL groups when the change in serum cholesterol between day 0 and day 27 was measured. The change in serum cholesterol over the 27-d feeding period was significantly lower in the MHO(L-CHOL) + E animals as compared with the other groups (Table 4). Overall, these data suggest that the hypercholesterolemic action of MHO was partially inhibited by high vitamin E intake.

A significant diet (P < 0.001) effect was observed in terms of total serum triglycerides, and a significant diet (P < 0.05) effect was observed for change (day 0-27) in serum triglyc- eride concentrations. After the 27-d feeding, the serum

TABLE 4 Effects of Vitamin E and Cholesterol in MHO-Based Diets on Serum Concentrations of Total Cholesterol, Triglycerides, and HDL-C in Hamsters (Experiment 2) a

M H O ( H - C H O L ) MHO(L -CHOL) + CHOL + E MHO(L -CHOL) + E

Total cholesterol (mg/dL) Day 0 (7) 150.00 + 11.94 d (7) 125.52 + 4.85 (7) 130.74 + 9.49 Day 27 (6) 214.83 + 32.48 a'b (7) 140.23 + 25.54 (7) 85.74 + 7.43 Change (6) 64.83 + 35.42 b (7) 27.48 + 29.05 c (7) -51 .53 + 6.09

Total tr iglycerides (mg/dL) Day 0 (7) 227.32 + 25.99 (7) 196.73 • 12.92 (7) 181.69 • 20.13 Day 27 (6) 332.03 • 21.98 a,b (7) 230.75 + 16.69 c (7) 165.76 + 19.12 Change (6) 156.72 • 44.07 a'b (7) 34.02 + 19.91 (7) -6 .51 + 22.90

HDL-C (mg/dL) Day 0 (7) 103.63 + 7.77 d (7) 83.81 + 4.51 d (7) 87.90 • 10.70 d Day 27 (6) 65.47 • 6.04 a'b (7) 50.69 + 3.17 (7) 49.32 • 2.56 Change (6) -38 .16 • 10.00 (7) -31.1 8 + .63 (7) -41.01 + 8.60

aValues are means + SE. Number in parentheses equals number of hamsters. See Tables 1 and 2 for abbreviations. Ham- sters were fed for 27 d one of three semipurified diets which consisted of: (i) a high-cholesterol MHO(H-CHOL); (ii) a low- cholesterol MHO which also contained high amounts of vitamin E (2.5 mg vitamin E/g oil) equivalent to a dietary concen- tration of 375 mg/kg [MHO(L-CHOL) + El; and (iii) the low-cholesterol MHO(L-CHOL) + E oil (2.5 mg vitamin E/g oil) which was supplemented with United States Pharmacopeia cholesterol (595 mg/kg diet) [MHO(L-CHOL) + CHOL + El to match the high-cholesterol content found in MHO(H-CHOL). Means within each row with superscript letters indicate the fol lowing comparisons are significantly different (P < 0.05) by least significant Means: aMHO(H-CHOL) vs. MHO(L-CHOL) + CHOL + E; b MHO(H-CHOL) vs. MHO(L-CHOL) + E; CMHO(L-CHOL) + E vs. MHO(L-CHOL) + CHOL + E. Means within each column with the supercript letter dindicate that the comparison of day 0 vs. day 27 is significantly different (P < 0.05) by least significant means. Cumulative food intake (day 0-27) was used as a covariate in the statistical model of two-way ANOVA. Significance of main effects of two-way ANOVA for total serum cholesterol (diet: P <0.001 ; time: P < 0.01 ; diet x time: P < 0.005), total serum triglycerides (diet, P < 0.001; time, nonsignificant; diet x time: nonsignificant), serum HDL-C (diet: P < 0.0001 ; time: P < 0.0001; diet x time: P < 0.0001 ). Significance of main effect of one-way ANOVA for serum choles- terol change (P< 0.05) and serum triglyceride change (P< 0.05).

Lipids, Vol. 31, no. 8 (1996)

HYPERLIPIDEMIA AND LIPID PEROXIDATION 845

triglyceride concentrations were higher in the MHO(H- CHOL)-fed animals relative to the other diet groups, suggest- ing that the hypertriglyceridemia produced by MHO feeding was inhibited by the supplemental vitamin E. In addition, a hypertriglyceridemic effect of dietary cholesterol occurred as total serum triglyceride concentrations were significantly higher in the MHO(L-CHOL) + CHOL + E group as com- pared with the MHO(L-CHOL) + E animals. Also, when the change between initial and final triglyceride concentrations was compared, the MHO(H-CHOL) group had a significantly greater change in triglyceride concentrations as compared with both the MHO(L-CHOL) + E and the MHO(L-CHOL) + CHOL + E groups.

For serum HDL-C, significant diet (P < 0.0001), time (P < 0.0001), and diet x time interaction (P < 0.0001) effects were observed. All three diet groups showed a significant (P < 0.0001) drop in serum HDL-C values from baseline after the 27-d feeding. Serum HDL-C at day 27 was significantly higher in the MHO(H-CHOL) group than in either the MHO(L-CHOL) + E group or the MHO(L-CHOL) + CHOL + E group, but no significant differences were observed among all treatments when changes in HDL-C concentrations were analyzed.

Liver lipid peroxides. Livers from hamsters fed the MHO(H-CHOL) diet had significantly higher (P < 0.05) lipid peroxide concentrations (0.29 _+ 0.05 nM/mg protein) as com- pared with either the MHO(L-CHOL) + E (0.15 _+ 0.02 nM/mg protein) group or the MHO(L-CHOL) + CHOL + E (0.15 _+ 0.03 nM/mg protein) group supporting the idea that supplemental vitamin E produced an inhibitory effect on MHO-mediated hepatic lipid peroxidation. The MHO(L- CHOL) + E animals did not differ significantly in terms of liver peroxide concentrations from the MHO(L-CHOL) + CHOL + E hamsters.

DISCUSSION

A possible etiological role of oxidative stress in hyperlipi- demia has been suggested from animal studies showing that a deficiency of vitamin E, an important antioxidant that pro- tects cell membranes against oxidative damage and lipid per- oxidation, increases tissue cholesterol (5). The administration of fish oils to animals has previously been indicated to cause a low body content of vitamin E due to enhanced in vivo lipid peroxidation of n-3 fatty acids (27), and supplementation of vitamin E well above normal physiological requirements was needed in both mice (28) and primates (29) to normalize tis- sue vitamin E concentrations. Moreover, induction of oxida- tive stress in rats by administration of xenobiotic compounds, such as phenobarbital, has been demonstrated to increase total cholesterol and LDL-cholesterol (30). Conversely, supple- mentation of vitamin E at a level of 2000 mg vitamin E/kg diet has been shown to decrease plasma cholesterol concen- trations in the Watanabe heritable hyperlipidemic rabbit (31). The findings of the present study lend further support for the concept that oxidative stress could play a significant role in

disrupting cholesterol metabolism which can be ameliorated via high intakes of vitamin E.

Our findings show that the hyperlipidemic response in the hamster to a high intake of fish oil is related to the oxidative challenge induced by this diet. Experiment 1 showed that in- take of MHO was associated with increased serum lipid per- oxidation relative to hamsters fed either BF or SAFF-based diets. This observation is consistent with findings of higher serum concentrations of lipid peroxidation products observed in humans (13) and experimental animals such as rabbits (10) and rats (12) following fish oil supplementation. It is well es- tablished that polyunsaturated fats increase the requirement for vitamin E by increasing the susceptibility of cell mem- branes to oxidative degradation (32). As observed previously with n-6 PUFA diets (14), levels of tissue lipid peroxidation products were not increased by high n-6 fatty acid intake as compared with the high saturated fatty acid intake via the BF diet.

The high concentrations of serum lipid peroxides in the MHO(H-CHOL)-fed animals suggest that the body content of vitamin E was not adequate to protect highly oxidizable n-3 PUFA from oxygen-free radical attack. In support of this concept, the fish oil diets with a high vitamin E content in Ex- periment 2 were associated with lowered tissue lipid peroxi- dation. In the second experiment, fish oils with a high toco- pherol content were associated with a serum cholesterol-low- ering action even when matched for the higher cholesterol content of the hyperlipidemic MHO. The highly significant correlations between serum levels of lipid peroxidation and various apolipoprotein and lipoprotein fractions also suggest that the increased levels of oxidative stress could contribute to the adverse MHO(H-CHOL)-induced apolipoprotein and lipoprotein profiles. The results thus present the interesting possibility that depletion of tissue vitamin E and lipid peroxi- dation could be involved in the hyperlipidemic response of the hamster to fish oils. Human studies support the concept that supplemental vitamin E could affect the serum lipid-low- ering effects of fish oil as subjects receiving vitamin E demonstrated a more marked fish oil-induced decrease in blood triglyceride concentrations than those receiving fish oil alone (13). As blood cholesterol concentrations were unaf- fected by vitamin E supplementation in the presence of fish oil intake, Haglund et al. (13) suggested that triglyceride syn- thesis might be more disrupted by peroxidation of fatty acids than cholesterol synthesis. The present study does not sup- port this hypothesis in the hamster as both blood components were adversely affected by the MHO(H-CHOL) treatment. Moreover, both the cholesterol and triglyceride serum frac- tions showed lower concentrations (Table 4) in the presence of high intake of vitamin E when fish oil was given in Exper- iment 2. Differences in fish oil metabolism between the human and the hamster could account for the differential re- sults observed in terms of serum cholesterol concentrations between the two studies.

In spite of increases in lipid peroxidation induced by the fish oil supplement, lipid peroxidation did not exert athero-

Lipids, Vol. 31, no. 8 (1996)

846 S. KUBOW ETAL.

genic effects over the 27-d study period. This result does not discount the possiblity that atherogenic lesions may have oc- curred at a later period in the feeding study as aortic lesions were visible in fish oil-fed rabbits after five months of feed- ing (11). Although fatty streaks and foam cells have previ- ously been reported in the Golden Syrian hamster after a four-week feeding (33), the atherogenic effects were caused by feeding 15% BF in the presence of much higher levels of cholesterol (3%) than was used in the present study (0.088%).

A major confounding effect between the two types of MHO oils was the higher cholesterol content of the MHO(H-CHOL). Despite the addition of matching levels of cholesterol to the MHO(L-CHOL), however, significant dif- ferences existed in terms of concentrations of serum choles- terol and triglycerides between the two oils. Moreover, only the MHO(H-CHOL) group experienced a significant rise in serum cholesterol concentrations from baseline values (Table 4). In Experiment 2, the serum lipid concentrations of MHO(L-CHOL) + CHOL + E were intermediary between those o f tile other two groups, thereby indicating that the high cholesterol content of MHO(H-CHOL) was partially responsible for the hyperlipidemic effect of the MHO(H- CHOL) oil. The minimal differences in fatty acid content between the MHO(H-CHOL) and the MHO(L-CHOL) oils were not likely responsible for the observed differences in serum lipid concentrations from the feeding of these two oils. Data from animal feeding studies that have compared fish oils with different fatty acid compositions with dietary levels of cholesterol adjusted to be the same. These studies have shown that the dietary ratios of 20:5n-3 to 20:4n-6 (16) and 20:5n-3 to 22:6n-3 (17) were the variables that caused differences in the hypolipidemic action among fish oils from different species. In particular, a hypolipidemic action was associated with fish oils with more than threefold higher di- etary 20:5n-3t20:4n-6 ratios in contrast to fish oils that showed no hypolipidemic effect (16). In the present study, however, the differences in these dietary ratios were small between the two oils (Table 1). In fact, the trends in tlaese ratios observed in the two MHO, deviated in the opposite direction. Hence, in terms of hypolipidemic action the hy- perlipidemic M H O without added vitamin E had a higher 20:5n-3/20:4n-6 ratio (25.4) as compared to the hypolipi- demic MHO with a high vitamin E content which had a ratio of 17. Four- to tenfold differences in the 20:5n-3 to 22:6n-3 ratio among fish oils have been indicated to result in differ- ential decreases in plasma concentrations of total choles- terol, LDL, and HDL (17). The dietary 20:5n-3 to 22:6n-3 ratio, however, was identical in the two MHO, 1.3. These data support the claim that difference in the vitamin E con- tent of the oils rather than the fatty acid composition was the major factor involved in the observed differences in lipid- lowering action between the two oils.

An increase in the serum triglyceride concentrations and a decrease in the apolipoprotein A-I/apolipoprotein B ratio have all been previously observed in the Golden Syrian ham-

ster following feeding of a fish oil-based semipurified diet (8). In contrast to the present experiments, however, Hayes et al.

(8) did not observe the hypercholesterolemic response to fish- oil feeding. The contrasting results in terms of serum choles- terol observed between our two studies could be accounted by differences in strain of Syrian hamster. Although the Source of Syrian hamster was not indicated in the Hayes et al.

study, a variability has been noted among Syrian hamsters from different commercial sources with respect to their lipi- demic responses to dietary parameters (34).

Fish-oil intake in our study resulted in an increase in the liver weight/body weight ratio as in other studies (35). How- ever, unlike other work (36), no increase in serum alanine aminotransferase was observed with fish-oil intake. Some in- vestigators have suggested that the hepatomegaly could be re- lated to the peroxisomal proliferation induced by fish-oil feeding and that the peroxisomal proliferation was a cause of the reduced synthesis and secretion of very low density lipoprotein triglyceride (37). Although peroxisomal prolifera- tion was not measured, the fish oil-induced hepatomegaly in both Experiments 1 and 2 was associated with serum triglyc- eride-raising rather than triglyceride-iowering effects of fish oil, suggesting that the hepatomegaly may be dissociated from triglyceride-lowering effects of fish oil. As the he- patomegaly and hepatic lipid peroxidation were decreased in the presence of vitamin E supplementation, it is possible that fish oil-induced hepatomegaly could be related to oxidative stress at the hepatic level.

The present study shows that dietary vitamin E can modu- late fish oil-induced changes in blood lipid metabolism and that a diet containing high amounts of fish oil induces an ox- idative challenge. Lipid peroxidation and tocopherol deple- tion could be involved in the hyperlipidemic response of the hamster to fish oils since high vitamin E intake was associ- ated with the production of hypolipidemic effects when MHO was fed. The highly significant correlations between serum levels of lipid peroxidation and various apolipoprotein and lipoprotein fractions lend further support to a role of lipid per- oxidation in the diet-induced alterations in serum lipoprotein metabolism. Additional studies are needed in the hamster to investigate the extent to which fish oil consumption increase the requirement for antioxidant nutrients. Overall, the present studies indicate that the relationship between diet-induced ox- idative stress and hyperlipidemia has been largely overlooked and is deserving of further study to fully evaluate its impor- tance.

ACKNOWLEDGMENTS

Grant support from the National Sciences and Engineering Research Council of Canada (to S. Kubow) and the Dairy Farmers of Canada (to S. Kubow, S. Kermasha, K.G. Koski, and J.-S. Phillips) is grate- fully acknowledged. The authors thank the excellent technical assis- tance of Susan Smith and Aghdas Zamani. The gift of MHO(L- CHOL) from the Fish Oil Biomedical Test Material Program of the National Marine Fisheries Service in Washington, D.C., is appreci- ated.

Lipids, Vol. 31, no. 8 (1996)

HYPERLIPIDEMIA AND LIPID PEROXIDATION 847

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[Received September 19, 1995; and in final revised form April 19, 1995; Revision accepted May 20, 1996]

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