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Departamento de Nutrición y Bromatología I (Nutrición), Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain and * Instituto de Ciencia y Tecnología de los Alimentos, Universidad de León, León, Spain
2To whom correspondence should be addressed. E-mail: frasan{at}farm.ucm.es.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • liver lipids • olive oil-fried sardines • (n-3) polyunsaturated fatty acids • serum lipoproteins
According to Varela and Ruiz Roso (1), fat is possibly the compound of the Mediterranean diet that is of greatest importance in the diet-health relationship. Moreover, differences in fat intake between Mediterranean and northern European countries are not due only to the composition of the fat consumed but also to how the fat is consumed. One particularly interesting feature of fat intake in Spain, and in the Mediterranean countries in general, is the high percentage of total fat provided by cooking fat (e.g., oils in the frying process). In the Mediterranean countries, 
50% of total fat intake comes from cooking (2).
Several models have been used for studying cholesterol and atherogenesis, but no single one is considered perfect for extrapolating results to humans (3). Rats are the animals most used in cholesterol metabolism studies and one of the most often used to study the cholesterolemic effect of proteins (3–7). In many studies, bovine bile or colic acid has been used extensively in animal studies to increase dietary cholesterol absorption and thus the hypercholesterolemic effect of this sterol (3–8).
(n-3) PUFA have hypolipemic properties (9). The inclusion of sardines fried in olive oil (7) or those fried in sunflower oil (9) in the diet of rat markedly decreased the hypercholesterolemic induction of dietary cholesterol. Although other studies in young animals have demonstrated that the effect of fish protein on serum cholesterol is average compared with that of protein from land animals (e.g., casein) and plants (e.g., soybean) (10), data concerning the effect of fish protein on the different lipoproteins are still rather scarce. Several amino acids may exert significant effects on serum cholesterol levels (11). In addition, more recent studies suggest that dietary taurine reduces serum cholesterol and liver cholesterol levels in rats fed a high cholesterol diet (6).
We tested the hypothesis that the use of fried sardines as the only source of dietary fat and protein in growing hypercholesterolemic rats that had undergone cholesterol loading would have a greater effect in normalizing serum lipid and lipoprotein values, liver fat and the fatty acid profile than a combination of casein and fat extracted from the fried sardines. This has relevance for humans for two reasons. First, many studies have analyzed the effect of (n-3) PUFA in the composition of fresh fish, but fried fish has not been similarly considered. Second, few previously published works have studied the effect on all lipoproteins. The effect of consuming (n-3) PUFA from fried, rather than raw fish is very important because of the exchange of lipids and minor compounds that occurs between the oil used for frying and the fish during frying (12–14). Thus, the effect of consuming fried fish might be rather different from the effect of fresh fish. Fried fish is commonly consumed in Mediterranean countries, in which the total and coronary heart disease mortality and morbidity rates are still rather low (15). Because of the lack of studies on the effect of lipoproteins, such information acquired from animal models increases the probability of success of dietary prophylaxis and therapy in human hypercholesterolemia and atherogenesis. Recognizing the increasing consumption of oily fish, and thus of fried fish, this study in hypercholesterolemic growing rats assessed 1) the acceptability of diets containing fried sardines as the only source of fat and protein; 2) the effect of a fried-sardine cholesterol-free diet on the lipoprotein profile; 3) the respective effects of fried-fish fat and fried-fish protein on the lipoprotein profile; and 4) the dietary effects of these two fried-fish components on liver composition.
Moreover, the use of a cholesterol-free diet had two goals: 1) to test the rate of recovery after cholesterol loading, and 2) to imitate abrupt dietary changes in persons diagnosed with dyslipemia who have been advised to markedly decrease consumption of cholesterol-raising agents by sharply limiting their intake of saturated fatty acids (SFA) and cholesterol and to increase fiber, monounsaturated (MUFA) and PUFA in their diet. Oily fish are rich in (n-3) fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the American Heart Association (9) recommends at least 2 servings of fish/wk to benefit from their cardioprotective effects.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Olive oil with an acid value of 0.4 mg KOH/g (Carbonell, Cordoba, Spain) and sardines (Sardina pilchardus, Walb.) were purchased at a local store. Acid casein, wheat starch and microcrystalline cellulose were obtained from Central Ibérica de Drogas, S.A. (Madrid, Spain); DL-methionine, sucrose, all minerals and vitamins, BHT, butylated hydroxyanisole, sodium hydroxide, boron trifluoride/methanol complex (solution 200 g/L in methanol), hexane, chloroform, methanol, sodium chloride, minerals and vitamins, and rhodamine B from Merck, Darmstadt, Germany; chromatography standards (lecithin, cholesterol, cholesterol oleate and tripalmitin) from Sigma, St. Louis, MO; cholesterol and bovine bile from Farmitalia-Carlo Erba, Madrid, Spain.
Frying.
Sardines (600–700 g) with head, scales, guts and backbone removed were opened out into a fan-shape and fried in a domestic fryer with a 3 L capacity in olive oil for 4 min at 180°C. Once fried, the sardines were freeze-dried and kept in a nitrogen atmosphere at -20°C until analyses were carried out and diets prepared.
Animals and maintenance.
Growing male Wistar rats (Instituto de Nutrición y Bromatología, Facultad de Farmacia, Universidad Complutense, Madrid, Spain) were individually housed in metabolic cells kept in a temperature-controlled room (22.3 ± 1.8°C) with a 12-h light:dark cycle. The study protocol was approved by a Committee for Animal Studies of the School of Pharmacy of the Universidad Complutense of Madrid and was performed according to NIH guidelines (16).
Experimental design.
Rats (n = 28) were fed commercial rat pellets (Sanders, Madrid, Spain) from weaning to the onset of the study. At 6 wk of age, when they weighed 65 g, and after a 3-d adaptive period, they were switched to a hypercholesterolemic diet containing casein plus olive oil plus a cholesterol-raising agent (COC diet) for a 3-wk hypercholesterolemia induction period. Then, rats (n = 21) were tested for serum cholesterol, divided into three groups of 7 rats each with a similar mean cholesterol value and SD, and fed the experimental diets. Rats consumed water and food ad libitum. The COC diet contained 14 g/100g protein, 10 g/100g fat and 2 g/100g cholesterol plus 0.5 g/100g bovine bile as a cholesterol-raising agent. The mineral and vitamin mixtures were prepared as indicated by the NRC (17). (Table 1). During the experimental period, one group of 7 rats was fed a cholesterol-free diet containing casein and olive oil (CO diet); another 7 rats were fed a diet containing sardines fried in olive oil as the only source of protein and fat (S diet), and a third group consumed a diet containing casein as the protein source and fat extracted from sardines fried in olive oil as the fat source (CSF diet) (Table 1). The analyzed composition of the experimental diets is shown in Table 2.
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Food intake was checked daily and body weight was measured on alternate days. Blood samples were collected by tail puncture after 16–18 h of food deprivation at the end of the hypercholesterolemia induction period from rats that were going to be assigned to groups S, CSF and CO. At the end of the hypercholesterolemia induction period, blood samples were also collected by cardiac puncture from rats from the COC group that had been deprived of food for 16–18 h and from the S, CSF and CO groups at the end of the experimental period. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg body) and killed by blood extraction via carotid puncture.
Serum lipid and lipoprotein analyses.
Serum was separated by low speed centrifugation at 1500 x g at 4°C for 30 min. Lipoproteins were isolated from serum by 7 h of density gradient ultracentrifugation at 232,000 x g and 8°C (18). Total cholesterol (TC), free cholesterol, triacylglycerides and phospholipids were analyzed according to standard enzymatic procedures of Boehringer, Mannheim, Germany (kits 172626, 310328, 701904, 691844,respectively). All intra-assay and interassay CV were <5.5%.
Proximate composition.
Moisture was determined by drying at 100°C to a constant weight (16). Protein was determined by the Kjeldahl procedure (19). Sardine, diet and liver fats were extracted (20) and purified (21).
Liver lipid fractions.
The triacylglycerol, phospholipid and cholesterol fractions were separated by TLC on a 60 F254S plate (Merck) using a petroleum ether/diethyl ether/acetic acid mixture (85:15:1) as eluent. Lecithin, cholesterol oleate, pentadecanoic acid and tripalmitin were used as reference standards. Spots were visualized by 1 g/L rhodamine B.
Fatty acid analyses.
The fatty acid methyl esters of olive oil, fish fat, diet fat and liver lipid fractions were analyzed by GC as previously indicated (22).
Statistical analyses.
The experimental groups were compared with the COC group by the Mann Whitney U test. Experimental groups were compared by the Kruskal-Wallis test. When a significant variation was identified, a pairwise comparison was performed using the Mann Whitney U test. Differences were considered significant at P < 0.05. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Rats later assigned to the different experimental groups consumed similar amounts of foods during the hypercholesterolemic induction period (data not shown). Food intake was not affected during the experimental period by the different diets (Table 3). Groups fed the S and CSF diets ate significantly more SFA, palmitic acid and (n-3) PUFA, less MUFA and less oleic acid than the CO group (data not shown). Body weight gain, the feed efficiency ratio (FER: g weight gain/g food intake) and the protein efficiency ratio (PER: g weight gain/g protein intake) did not differ among the groups after the induction period (data not shown) and experimental periods (Table 3).
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The hypercholesterolemic induction period led to moderate hypercholesterolemia (4.79 ± 0.39 mmol/L). The consumption of the experimental diets decreased the serum total cholesterol levels. The S diet generally had the greatest hypocholesterolemic effect. This diet also induced the highest serum phospholipid levels, whereas CO-fed rats had the lowest triacylglyceride concentrations. The cholesterol/phospholipid ratio was lower in the S group and higher in the CO group than in the CSF group (Table 4).
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The VLDL fraction in the CSF groups was rich in triacylglycerides, whereas that of the CO group was rich in cholesterol esters. The LDL fraction of the S group contained lower total, free and esterified cholesterol concentrations than that of the CSF group. The HDL fraction of the S group contained more TC, free and esterified cholesterol, and phospholipid than the CO and CSF groups. On the basis of the results presented in Table 4, the main lipoprotein carrier of TC and free and esterified cholesterol was HDL in the S group, whereas it was VLDL in the CO group. Phospholipids were transported mainly by the HDL fraction and in a significantly higher proportion in the S group than in the other groups. Triacylglycerides were carried mainly by VLDL in the CSF group but by HDL in the CO and S groups.
Liver weight and proximate composition.
The CSF and S groups had greater liver weights than the CO group. S-fed rats had greater relative liver weight than the CO group. The liver of the CO and CSF groups had more fat and less moisture than the S group (Table 5).
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CSF- and CO-fed rats had higher TC, free and esterified liver cholesterol than the S counterparts (Table 5). The S group had significantly more (n-3) PUFA and SFA, and less (n-6) PUFA and MUFA in whole liver (data not shown) than the CO group. The CSF group generally had intermediate values. The oleic acid/stearic acid ratio, used as an index of 
9-desaturase activity (23,24), was significantly higher in the CO group (9.26 ± 0.46) than in the S and CSF groups (4.56 ± 0.43 and 6.68 ± 0.13, respectively). The triacylglyceride fraction of the CO group contained significantly more MUFA and SFA, and less (n-6) and (n-3) PUFA than that of S and CSF groups (Table 6). The phospholipid fraction contained more (n-3) PUFA and less (n-6) PUFA in the S and CSF groups than in the CO group (Table 6). (n-3) PUFA and SFA were significantly higher and MUFA were lower in the cholesterol ester fraction of the S and SCF groups than in that of the CO group (Table 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Consistent with other studies (4,5,7,9,26) the cholesterol/casein/oleic acid diet induced moderate hypercholesterolemia in rats during the hypercholesterolemic induction period, with the presence of large amounts of ß-VLDL, high amounts of LDL and low amounts of HDL. On the other hand, the withdrawal of dietary cholesterol had a remarkable hypocholesterolemic effect. Nonetheless, most modifications in the lipoprotein profile were significantly different among the three experimental groups. Viejo (27) found that in rats fed cholesterol-enriched casein diets, the withdrawal of dietary cholesterol significantly lowered the level of serum cholesterol. However, the presence of (n-3) PUFA helped to decrease the serum cholesterol level. This effect was even greater when rats consumed whole fried sardines. Thus, discussion of the results should take into account the fat and protein composition of the diets.
The S and CSF groups consumed diets containing 20 g/100 g (n-3) PUFA. In these groups, the main decrease in the serum TC levels was in cholesterol ester. These two groups had a normalized lipoprotein profile because VLDL and LDL transported less cholesterol and HDL more cholesterol compared with the CO group. However, the percentage contribution of lipids to the VLDL lipid mass in CO-fed rats was similar to that of the VLDL composition of hypercholesterolemic rats (27).
The hypocholesterolemic effect of fish consumption is still not clear, and several hypotheses have been proposed. According to Nestel (28), (n-3) PUFA decrease the hypercholesterolemic induction of dietary cholesterol. The same author (29) suggested that the usefulness of fish oils in treating hypercholesterolemia is doubtful except when the excess cholesterol is in the serum VLDL fraction. Our results suggest that sardine oil may reduce the outflow of VLDL from the liver. It has been suggested that (n-3) PUFA decrease synthesis of VLDL by affecting the synthesis of apolipoprotein (apo) B100 and triacylglyceride (30). Moreover, the oleic acid-(n-3) PUFA interaction positively affects the lipoprotein profile (29,31).
S-fed rats had a more normal lipoprotein profile than CSF-fed rats, probably due to the protein amino acid composition (3,10,32) or to the way peptides were delivered during digestion (33). According to Shirai et al. (34), sardines are a much richer source of taurine than soy or whey proteins. Moreover, the amino acid profile of sardines is between that of casein and soybean protein (10). These findings suggest that the cholesterolemic effects of fish protein are between those of casein and soybean protein, and thus explain the lower serum cholesterol levels of the S group compared with those of the CO and CSF groups in the present study. Significant effects of amino acid dietary supplementation on serum cholesterol levels have been reported (35). However, only the sulfur-containing amino acids had significant effects when added to the diet at levels of 0.5 and 1 g/100 g; the addition of methionine increased the cholesterol level, whereas the addition of cystine decreased it (35). Years ago, Kritchevsky (36) suggested that the dietary lysine/arginine ratio influenced plasma cholesterol levels. Other authors have suggested that tyrosine, arginine and glycine affect these values (11). More recently, Yokogoshi et al. (6) reported the hypocholesterolemic effect of a diet containing taurine on hypercholesterolemic rats. Nonetheless, although proteins and their amino acid constituents affect serum cholesterol levels, the mode of the cholesterolemic action of dietary protein is not yet clear. Yokogoshi et al. (6) suggested that the hypocholesterolemic effect of taurine is due mainly to enhanced cholesterol degradation and bile acid excretion.
The serum cholesterol/phospholipid ratio has been used as a hypercholesterolemic and atherogenic marker (37). The S group had the lowest cholesterol/phospholipid ratio, indicating that the effect of whole sardines on lipoprotein metabolism went further than the mere decrease of serum cholesterol. In fact, the S group had lower lipid concentrations of VLDL and LDL and greater HDL lipid levels than the CSF group. Although it was not possible to measure the different apo concentrations in the lipoprotein fractions, the lipid changes observed indicated that the total mass of VLDL and LDL decreased, whereas that of HDL increased in the S and CSF groups compared with the CO group. Moreover, the S diet normalized the lipoprotein profile to a greater degree than did the CSF diet because the HDL fraction was the main transporter of all serum lipids. These data appear to be at least partially related to the higher taurine content of the S diet because, according to Yokogoshi et al. (6), taurine supplementation raised the low HDL levels in hypercholesterolemic rats.
Intake of (n-3) PUFA, mainly by the S group, induced greater liver weight and relative liver weight. These results agree with those of other authors who found a liver weight increase in rats fed (n-3) PUFA (25). However, liver fat concentration decreased significantly in the S group. Moreover, macroscopic observation clearly showed lower steatosis in the S group than in the other groups (data not shown). Hepatic triacylglyceride and cholesterol (free, esterified and total) levels were affected by the fat and protein composition of the diets; the S group had the lowest hepatic cholesterol concentration. These findings concur with those of other authors who found that the protein source (38) and (n-3) PUFA affected liver cholesterol content (39), and suggest that fish protein increased the effect of (n-3) PUFA on liver cholesterol. Yokogoshi et al. (6) found that liver cholesterol levels decrease in hypercholesterolemic rats, due to increased bile acid excretion as a result of cholesterol 7-
-hydroxylase enhancement, when taurine was added to the casein diet. According to Viejo (27) bile acid excretion was greater in rats fed fried sardines than in casein-fed rats. Moreover, the cholesterol absorption rate was greater in casein-fed rats than in those fed sardines (27). These data explain the greater drop in hepatic cholesterol concentrations in the S-fed rats and partially explain the mode of action of this dietary component.
The composition of plasma and many tissue lipids in humans and animals reflects the type and amount of dietary lipids consumed (40–42). In fact, the total liver and triacylglyceride fractions of CO-fed rats had lower (n-3) PUFA percentages than those of their S and CSF counterparts. These results agree with those of many authors who found that inclusion of fish fat in the diet increases hepatic (n-3) PUFA contents and decreases arachidonic acid levels (43). Nevertheless, the fatty acid composition of the liver also depends on cholesterol ester pool formation. The livers of all groups had a high percentage of MUFA in the cholesterol ester faction. This finding agrees with studies in which rats consume or had consumed cholesterol in their diets and confirms the strong tendency of cholesterol to esterify with oleic acid, which occurs in the liver of these rats (27). The hepatic free cholesterol/esterified cholesterol ratio data suggest that the enzyme acyl-CoA:cholesterol acyltransferase was highly stimulated in all experimental groups. Oleic acid is a preferential substrate for free cholesterol esterification in the liver and helps keep the synthesis of hepatic apo B100-E receptors as high as possible (44) and the serum TC levels as low as possible in hypercholesterolemic animals.
The greater hypocholesterolemic effect of the S diet, already discussed, seems to be confirmed by the liver fatty acid profile of the S group, which had a lower percentage of oleic acid and lower -9 desaturase activity (given by the palmitoleic acid/palmitic acid and oleic acid/stearic acid ratios).
Liver phospholipid concentration did not vary among the experimental groups, but protein and the fat sources of the diet affected the fatty acid composition of this fraction. Consistent with others, the inclusion of (n-3) PUFA in diets increased the content of EPA and DHA and decreased that of arachidonic acid in organ phospholipids (45).
These changes are relevant, due to the role of (n-3) PUFA in reducing myocardial cell injury and liver disease by inhibiting both prostanoid and leukotriene pathways and growth factors (46,47). Previous data (5,7) suggested that the inclusion of sunflower oil or sardines fried in olive oil in diets enriched in cholesterol and bovine bile decreased several markers of liver damage.
In conclusion, diets containing sardines fried in olive oil or the fat extracted from those fried sardines were well accepted by growing Wistar rats. The withdrawal of dietary cholesterol markedly reduced serum cholesterol levels in the three rat groups but this decrease, after cholesterol loading, was greater in the S-fed rats than in the CSF-fed rats, and greater in the CSF-fed rats than in the CO-fed rats. The lipoprotein profile of S-fed rats was more normal than that of rats fed casein plus the fat extracted from fried sardines. CO-fed rats still had ß-VLDL. The (n-3) PUFA content in hepatic triacylglyceride, cholesterol ester and phospholipid fractions increased due to the consumption of sardines fried in olive oil or of the fat extracted from those fried sardines. The present findings suggest that the consumption of whole sardines fried in olive oil is preferable to that of their extracted fat for the treatment of hypercholesterolemia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; CO, casein and olive oil; CSF, casein plus the fat extracted from sardines fried in olive oil; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FER, feed efficiency ratio; MUFA, monounsaturated fatty acids; PER, protein efficiency ratio; S, sardines fried in olive oil; SFA, saturated fatty acids; TC, total cholesterol.
Manuscript received 7 January 2003. Initial review completed 28 January 2003. Revision accepted 22 April 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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