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Lipid profile of rats fed high-fat diets based on flaxseed, peanut, trout, or chicken skin
Nutrition 22 (2006) 197–205
Basic nutritional investigation
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Lipid profile of rats fed high-fat diets based on flaxseed, peanut, trout, or chicken skin Dennys E. C. Cintra, M.Sc., André G. V. Costa, M.Sc., Maria do Carmo G. Peluzio, D.S., Sérgio L. P. Matta, D.S., Marco Túlio C. Silva, D.S., and Neuza M. B. Costa, Ph.D.* Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil Manuscript received May 4, 2005; accepted September 3, 2005.
Abstract
Objective: Dietary saturated fatty acids are associated with coronary disease. Conversely, dietary monounsaturated polyunsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) seem to exert a protective effect. This study evaluated the lipid profile of rats fed high-fat (HF) diets, with fat added as different sources of PUFA (flaxseed and trout), MUFA (peanut), and saturated fatty acid (chicken skin). Methods: Adult male Wistar rats were placed into six dietary groups (n ⫽ 10): control (normal); high fat, with 1% cholesterol, 10% soy oil, and 5% lard; and four groups fed similar HF diets, with 10% lipid as trout, flaxseed, peanut, or chicken skin. After 28 d the animals were killed. Blood, livers, and adipose tissue samples were collected. Results: A higher level (P ⬍ 0.05) of total serum cholesterol was observed in rats fed the normal diet (93.57 ⫾ 14.95 mg/dL) compared with those fed the HF diet (67.57 ⫾ 12.54 mg/dL). Total cholesterol levels in rats fed the flaxseed diet were lower (P ⬍ 0.05) than in rats fed the other fats. No difference was observed in cholesterol levels between groups fed the peanut and chicken skin diets (P ⬎ 0.05). Animals fed the peanut diet showed decreased body weight gain than did animals in the other treatment groups. There were large lipid and cholesterol depositions in livers of rats fed the HF diet. Lipid deposition in adipose tissue followed the same dietary fatty acid profile, i.e., high levels of -3 PUFA in the flaxseed group, high levels of MUFA in the peanut and chicken skin groups and high levels of -6 PUFA in the trout group. Conclusions: These data indicate that flaxseed is promising for dietary manipulation of hyperlipidemia. © 2006 Elsevier Inc. All rights reserved.
Keywords:
Flaxseed; Trout; Peanut; Chicken skin; Lipid profile; Lipidemia
Introduction Studies to determine the effects of lipids on blood cholesterol date from the 1950s [1] and show that different dietary lipids can modulate plasma cholesterol levels, depending on their fatty acid composition. High levels of saturated fatty acids (SFAs) in the Western diet are associated with the formation of atherosclerotic plaque [2–5]. However, intake of monounsaturated fatty acids (MUFAs) -9, especially oleic acid (18:1-9), and long-chain polyunsaturated fatty acids (PUFAs) of the -3 series, especially eicosapentaenoic acid (20:5-3) and docosahexaenoic
acid (22:6-3), is associated with decreased risk of cardiovascular death [3,6 –11]. Many studies have demonstrated that MUFAs and PUFAs show similar effects on lowering blood cholesterol when SFA is substituted in the diet [12]. The presented study evaluated the effects of MUFAcontaining peanut and PUFA-containing flaxseed and trout diets compared with an SFA-containing chicken skin diet on the lipid profile of rats.
Material and Methods Animals and diet
* Corresponding author. Tel./fax: ⫹55-31-3899-2541. E-mail address: [email protected] (N.M.B. Costa). 0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.09.003
Sixty male adult Wistar rats with an initial body weight of 180 g were obtained from the Biology and Health Sci-
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ences Center of Federal University of Viçosa, MG, Brazil. The animals were randomized to six groups (n ⫽ 10). One group received a control AIN-93 diet [13]; another group received a high-fat (HF) diet of 10% soybean oil, 1% cholesterol, and 5% lard. The other groups received diets similar to the HF diet, but with 10% of lipids provided by trout, peanut, flaxseed, or chicken skin to replace the soybean oil. Compositions of the experimental diets are presented in Table 1. Cornstarch and protein contents of the diets were adjusted to maintain isocaloric diets. Animals were kept in individual cages in a temperaturecontrolled room at 22 ⫾ 2°C, with a 12-h dark/12-h light cycle for 28 d. They received water and diet ad libitum. Food intake and body weights were monitored weekly. Ingredients Trout that were about 200 g in body weight and 14 mo of age were eviscerated, scaled, and kept frozen. Before diet preparation, trout were thawed at room temperature, ground in a meat grinder, and dried in an oven at 60°C for 48 h. Peanuts (Nestlé, São Paulo, Brazil) were donated by a food industry as skinned and ground peanut flour, which was further roasted at 168°C for 25 min before being added to the animal diet. Flaxseed (Mãe Terra®)was obtained in the local market and ground in a multiprocessor. Chicken skin (Pif-Paf®) was obtained in the local market, ground in a meat grinder, and dried in oven at 60°C for 48 h. The drained oil was discarded. Casein was purchased from Sigma and Rhoster (Sigma, São Paulo, Brazil, and Rhoster, São Paulo, Brazil). Sucrose (União®), cornstarch (Maizena®), lard (Sadia®), and soybean oil (Liza®) were purchased in the local market. Dextrinated starch, cellulose, mineral mix, vitamin mix, L-cystine, and choline bitartrate
were purchased from Rhoster. Cholesterol was purchased from Sigma. The moisture [14], protein [14], and lipid [15] contents of trout, peanut, flaxseed, and chicken skin were determined before being added to the diets. Blood and tissue sampling At the end of the experimental period and after 12 h of fasting, animals were killed by CO2. Blood was collected by heart puncture and serum was taken after centrifugation at 2400g for 15 min. Liver slices were fixed in 10% formaldehyde buffer for morphologic analysis and the remaining liver samples were kept for cholesterol analysis. Subcutaneous and visceral adipose tissue samples were taken from the abdominal cavity for fatty acid profile analysis. Lipid extraction, saponification, and esterification Lipid extraction of food components, adipose tissues, and livers of the experimental animals was carried out according to the method of Folch et al. [16]. Fecal lipid was extracted by Soxhlet, using petrol ether (Vetec, Minas Gerais, Brazil) [15]. Lipid saponification and esterification, when required, followed the method of Hartman and Lago [17]. Fatty acid profile Fatty acid profiles of food components and adipose tissues were determined by gas chromatography, using a Shimadzu Model 17A, under the following operating conditions: fused silica capillary column (100 m ⫻ 0.25 mm; SP-2560); hydrogen as the carrier gas, with a flow rate of 20
Table 1 Composition of experimental diets (g/100 g) Ingredients
Normal
HF
Trout
Flaxseed
Peanut
Chicken skin
Casein* Sucrose Cornstarch Dextrinated starch Cellulose Lard Cholesterol Mineral mix Vitamin mix L-cystine Choline bitartrate Soybean oil Trout† Flaxseed† Peanut† Chicken skin†
15.30 10.00 45.26 15.5 5.00 — — 3.50 1.00 0.18 0.25 4.00 — — — —
15.30 10.00 33.26 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 10.00 — — — —
0.00 10.00 22.38 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 — 36.19 — — —
11.80 10.00 25.89 15.5 1.00 5.00 1.00 3.50 1.00 0.18 0.25 — — 24.88 — —
9.86 10.00 28.21 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 — —
11.39 10.00 31.37 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 — — — — 15.81
HF, high fat * To provide 12 g of protein/100 g of diet. † To provide 10 g of lipids/100 g of diet.
20.50 —
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cm/s; oven temperature, initially 140°C for 5 min, was increased up to 240°C at 4°C/min and maintained at 240°C for 30 min; injector, split ratio was 1:50, temperature 250°C; vaporization and detector temperatures were 250°C and 260°C, respectively. The injection volume was 1 L of sample solution. The retention times of methyl ester standards (Sigma, São Paulo, Brazil) were used to identify peaks. Blood lipids Total serum cholesterol levels were analyzed by the method of Allain et al. [18], high-density lipoprotein (HDL) cholesterol by the method of Warnick et al. [19], and triacylglycerol by the method of Fossati and Prencipe [20] by using specific enzymatic kits (Katal, Belo Horizonte, MG, Brazil).
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Table 2 Food composition (g/100 g) Content
Dried trout
Flaxseed
Peanut
Dried chicken skin
Moisture Total lipids Protein Fiber*
— 27.63 56.53 —
8.14 40.18 19.35 18.00
1.42 48.76 30.86 6.40
4.13 63.22 32.49 ND
ND, not determined * From the food label (nutrition facts) or table of food composition [32].
Wallis test was used to compare three or more independent samples, followed by Dunn’s test (P ⬍ 0,05), when the comparison was statistically significant. Analyses were carried out using Sigma Stat 2.02 (Software for Windows; Jandel Corp., San Rafael, CA, USA).
Liver cholesterol Results Liver cholesterol analysis was carried out by gas chromatography according to the method of Naeemi et al. [21] by using a Shimadzu Model 17A, under the following operating conditions: column, DB-1 (dimethylpolysiloxane, 30 m ⫻ 0.25 mm); hydrogen as the carrier gas at 175°C at a flow rate of 22 cm/s; oven temperature was initially 280°C for 1 min, was increased up to 300°C at 20°C/min, and maintained at 300°C for 10 min; injector, split ratio was 1:50; vaporization and detector temperatures were 290°C and 300°C, respectively. The injection volume was 1 L of sample solution. Liver morphology Liver slices were dehydrated with ethanol, cleared with xylene, and embedded in paraffin wax (Merck, São Paulo, Brazil). After inclusion, material was cut on a microtome (CUT model 445, Olympus) at 4 m. Liver sections were stained with hematoxylin and eosin (Merck, São Paulo, Brazil). Pictures were taken with a photomicroscope (Model AX70, Olympus) using Kodacolor Gold 100 asa film at 40⫻ magnification. Histologic analysis was qualitative. Statistical analysis Collected data were subjected to the KolmogorovSmirnov test to check for symmetry. For variables with normal distribution, Student’s t test was used to compare two independent samples (normal and HF groups). Analysis of variance was used to compare three or more independent samples (trout, flaxseed, peanut, and chicken skin groups), followed by Tukey’s test (P ⬍ 0.05) when the F value was significant. For asymmetric variables, the Mann-Whitney test was used to compare two independent samples, and the Kruskal-
Table 2 lists moisture, total lipid, protein, and fiber contents. Table 3 presents the fatty acid profiles of trout, flaxseed, peanut, chicken skin, and soybean oil. Trout and peanut showed a high and similar composition of MUFAs (C18:1). Flaxseed showed a high content of the -3 fatty acid ␣-linolenic acid (⬃58%) and chicken skin had a high content of SFA, although its content of MUFAs was comparable to those of trout and peanut. Body weight, food intake, and food efficiency ratio are presented in Table 4. No significant differences were observed in body weight and food intake at the beginning of the study. Nevertheless, the peanut group showed lower (P ⬍ 0.05) body weight gain and food efficiency ratio compared with the trout and flaxseed groups. Total serum cholesterol, HDL cholesterol, and triacylglycerol levels are listed in Table 5. Total cholesterol in animals fed the HF diet, with 1% cholesterol in the diet, was unexpectedly lower than that in animals fed the normal diet. Lower levels (P ⬍ 0.05) of total cholesterol were observed in animals fed the flaxseed diet compared with the peanut and chicken skin diets. Animals fed the flaxseed diet also showed lower levels (P ⬍ 0.05) of triacylglycerol compared with animals fed the chicken skin diet. No other differences in triacylglycerol levels were observed. No significant differences in HDL cholesterol were observed between the normal and HF groups; however, animals in the normal group showed a lower (P ⬍ 0.05) HDL cholesterol/total cholesterol ratio than did the HF group. Liver weights of animals in the normal, flaxseed, peanut, and chicken skin groups were significantly lower than those in the HF group (P ⬍ 0.05), as listed in Table 6. This may be related to the deposition of total lipids and cholesterol in the liver because no difference in body weight was observed across animals in different groups. Table 6 also indicates that the types of lipid in the diet exert an influence on liver
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Table 3 Fatty acid profile (g/100 g of total lipid) Fatty acid
Dried trout
Flaxseed
Peanut
Dried chicken skin
Soybean oil
C14:0 C16:0 C18:0 C23:0 Total SFAs
1.17 23.40 5.13 — 29.70
— 6.45 4.38 — 10.83
— 12.98 3.16 — 16.14
0.55 29.69 6.43 0.35 37.02
— 14.65 1.93 — 16.58
C14:1 C16:1 C18:1 C20:1 Total MUFAs
— 6.31 33.41 — 39.72
— — 18.00 — 18.00
— — 38.25 — 38.25
0.30 6.13 38.09 0.69 45.21
— — 31.66 0.70 32.36
C18:2-6 C20:2 C22:2 C18:3-6 C18:3-3 C20:3-6 C20:4-6 C22:6-3 Total PUFAs
17.19 1.20 — 2.67 0.80 1.07 1.44 6.22 30.58
12.71 — — — 58.47 — — — 71.17
39.33 3.53 1.60 1.15 — — — — 45.61
16.60 — — 0.55 0.62 — — — 17.77
51.06 — — — — — — — 51.06
5:1 —
1.5:1 27:1
5.03:1 —
MUFA ⫹ PUFA/SFA -6/-3
2:1 3:1
8:1 1:4.5
MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid
lipid composition because the content of SFAs in the diets corresponded to the larger depositions of cholesterol and triacylglycerol in the liver. Total liver lipid levels were higher in animals fed the HF diet than in animals fed the normal diet, but no difference was observed between the HF and other groups. Nevertheless, animals in the HF group showed a large deposition of cholesterol in the liver than did animals in the other groups (Table 6). A difference was observed in lipid liver deposition based on photomicrographs of liver parenchyma (Fig. 1). Fecal lipid levels (Table 6) were higher in animals fed the flaxseed diet than in animals in the other groups, likely due to its high dietary fiber content, which increases transit time and fecal bulk. Liver steatosis Figure 1 shows lipid deposition in liver slices. The intensity of liver steatosis was classified as more than 30% of hepato-
cytes affected (⫹), more than 50% of hepatocytes affected (⫹⫹), and more than 75% of hepatocytes affected (⫹⫹⫹). Lipid vesicles appeared in livers of all animals. Hepatocytes of the HF group presented a flattened nucleus due to high lipid content. Lipid deposition in the adipose tissue Dietary lipid was incorporated effectively in visceral and subcutaneous adipose tissues of all experimental animals (Tables 7 and 8). Animals fed the HF diet showed lower contents of SFAs and MUFAs in visceral and subcutaneous adipose tissues (P ⬍ 0.05) compared with the trout group. An opposite effect was observed in relation to the PUFA content of these two groups. This seems to be due to the soybean oil used in the HF diet, which is rich in -6 PUFAs. The fatty acid profiles (SFA, MUFA, and PUFA) of
Table 4 Body weight, body weight gain, food intake, and FER of the experimental groups* Treatment
Initial body weight (g)
Body weight gain (g)
Food intake (g)
FER
Normal HF Trout Flaxseed Peanut Chicken skin
185.80 ⫾ 21.29 183.00 ⫾ 22.71a 178.90 ⫾ 19.38a 169.00 ⫾ 27.72a 184.70 ⫾ 7.80a 178.90 ⫾ 11.98a
114.30 ⫾ 24.47 131.10 ⫾ 23.32ab 140.40 ⫾ 19.02a 140.90 ⫾ 33.18a 105.70 ⫾ 19.78b 128.30 ⫾ 16.90ab
528.47 ⫾ 62.00 512.07 ⫾ 60.54a 467.10 ⫾ 36.76a 483.52 ⫾ 41.88a 493.61 ⫾ 29.96a 512.36 ⫾ 21.24a
0.21 ⫾ 0.03 0.25 ⫾ 0.02ab 0.30 ⫾ 0.03a 0.29 ⫾ 0.06a 0.21 ⫾ 0.03b 0.25 ⫾ 0.03ab
FER, food efficiency ratio; HF, high fat * Mean values followed by the same letter in the column are not different by Tukey’s test (P ⬍ 0.05). No difference was observed between normal and HF groups by Student’s t test (P ⬎ 0.05).
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Table 5 Total serum cholesterol, HDL-C, triacylglycerol, and HDL-C/total cholesterol ratio of the animals* Treatment
Total cholesterol (mg/dL) Mean ⫾ SD
Normal HF Trout Flaxseed Peanut Chicken skin
93.57 ⫾ 14.95 67.57 ⫾ 12.54 83.87 ⫾ 14.41 60.87 ⫾ 7.02 101.40 ⫾ 17.31 107.84 ⫾ 9.52
HDL-C (mg/dL)
Median †
96.53 63.00a 81.79ab 61.56a 105.49b 106.79b
Mean ⫾ SD 55.28 ⫾ 7.03 58.77 ⫾ 9.34 51.94 ⫾ 13.97 41.57 ⫾ 6.96 46.18 ⫾ 6.46 64.38 ⫾ 16.93
Triacylglycerol (mg/dL) Median
Mean ⫾ SD
53.87 62.66a 45.48ab 38.54b 45.76ab 62.82a
76.85 ⫾ 18.07 53.36 ⫾ 11.36 62.60 ⫾ 23.25 49.54 ⫾ 6.57 55.98 ⫾ 12.95 77.21 ⫾ 16.99
HDL-C/total cholesterol ratio
Median †
72.50 53.38ab 57.61ab 51.99a 56.45ab 83.99b
Mean ⫾ SD
Median
0.59 ⫾ 0.07 0.90 ⫾ 0.19 0.62 ⫾ 0.09 0.68 ⫾ 0.14 0.46 ⫾ 0.08 0.59 ⫾ 0.14
0.59† 0.87a 0.65ab 0.64ab 0.47b 0.63b
HDL-C, high-density lipoprotein cholesterol; HF, high fat; SD, standard deviation. * Medians followed by the same letter in the column are not different by Kruskal-Wallis test, complemented with Dunn’s multiple comparisons test (P ⬎ 0.05). † A significant difference was found between normal and HF groups by Mann-Whitney test (P ⬍ 0.05).
animals fed the flaxseed and peanut diets were similar with respect to visceral adipose tissue but not to subcutaneous adipose tissue. This may be due to high lipid mobility in subcutaneous adipose tissue.
Discussion Levels of fiber, lipid, and fatty acid in flaxseed in this study were similar to those found by Prasad [22] and Walisundera et al. [23]. The high content of ␣-linolenic acid (58%) may play an important role in decreasing the risk of coronary heart disease (CHD) by lowering levels of lowdensity lipoprotein cholesterol, as reported in the literature [22,24,25]. Low levels of SFA, especially palmitic acid (C16:0), may be another protective factor against CHD [26]. Trout is reported to have a higher content of -3 fatty acids eicosapentaenoic acid and docosahexaenoic acid in the liver than in the filet [27,28]. This may explain the low levels of these fatty acids found in this study for trout, which showed a high content of MUFAs (C18:1 and C16:1). The high content of MUFAs is associated with a low incidence of CHD [29] because it decreases total cholesterol (10%) and low-density lipoprotein cholesterol (14%) [30]. This effect, however, depends on levels of SFA in the diet [31]. Chicken skin, which has a high content of MUFAs
(C18:1 and C16:1), presented the highest content of SFAs (C16:0) among the foods evaluated in this study and, hence, low PUFA/SFA ratio. Palmitic acid (C16:0) is associated with the risk of CHD because of its effect in increasing blood cholesterol levels and platelet aggregation [26]. The lipid and protein contents of peanut found in this study were similar to those reported in the literature [30,32]. The peanut diet, however, resulted in a low food efficiency ratio, perhaps due to the presence of limiting amino acid and antinutrient factors. Although these factors may be present in other plant proteins such as flaxseed, the addition of casein to the flaxseed diet improved its protein quality and enhanced its food efficiency ratio. Animals fed the peanut diet showed lower body weight gain over the study period (P ⬍ 0.05). This trend has also been reported for humans [29,33]. The effect of nuts in lowering body mass index is not completely understood, but it is hypothesized that the content of PUFAs compared with that of SFAs may decrease body weight [34] and that the lipid intake may stimulate satiety [33]. Animals fed the HF diet showed lower levels of blood cholesterol compared with animals fed the normal diet. This unexpected result may be due to an inhibition of 3-hydroxy3-methylglutaryl coenzyme A reductase by a feedback mechanism caused by the high intake of dietary cholesterol (1%). Moreover, cholesterol deposition in the liver was high
Table 6 Liver weight, total liver lipids, liver cholesterol, and fecal lipids of the animals* Treatment
Liver weight (g) (n ⫽ 8)
Total liver lipids (mg/g) (n ⫽ 8)
Liver cholesterol (mg/g) (n ⫽ 8)
Total fecal lipids (mg/g) (n ⫽ 10)
Normal HF Trout Flaxseed Peanut Chicken skin
10.87 ⫾ 1.51† 15.80 ⫾ 2.61a 13.77 ⫾ 1.72ab 13.31 ⫾ 1.23b 12.82 ⫾ 0.93b 13.25 ⫾ 1.75b
33.40 ⫾ 11.50† 135.20 ⫾ 73.00a 68.40 ⫾ 41.10a 57.00 ⫾ 31.00a 99.80 ⫾ 53.60a 83.00 ⫾ 37.20a
3.35 ⫾ 1.58† 34.50 ⫾ 17.43a 15.30 ⫾ 9.82b 12.46 ⫾ 8.15b 12.97 ⫾ 6.39b 13.06 ⫾ 6.41b
29.69 ⫾ 12.63† 106.33 ⫾ 15.51a 123.51 ⫾ 35.41a 201.60 ⫾ 20.05b 123.35 ⫾ 23.63a 113.54 ⫾ 19.02a
HF, high fat * Means followed by the same letter in the column are not different by Tukey’s test (P ⬍ 0.05). † A significant difference was observed between normal and HF groups by Student’s t test (P ⬍ 0.05).
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Fig. 1. Photomicrographs of liver parenchyma of animals fed the control (normal), HF, trout, flaxseed, peanut, or chicken diet (hematoxylin and eosin, original magnification 440⫻). Arrows indicate lipid vesicles. (⫹), greater than 30% steatosis; (⫹⫹), greater than 50% steatosis; (⫹⫹⫹), greater than 75% steatosis; HF, high fat.
in the HF group, which may explain why cholesterol was low in the bloodstreams of these animals. Animals fed the flaxseed diet showed the lowest levels of serum cholesterol. The content of fatty acid may be associated with this effect because unsaturation has been reported to be inversely related to serum cholesterol levels [35]. The ␣-linolenic fatty acid (C18:3) and the fiber contents of
flaxseed may also be associated to its cholesterol-lowering property. According to Hadley and Mitchell-Fetch [36], the ratio of soluble to insoluble dietary fiber may vary from 20:80 to 40:60 in flaxseed. The soluble fiber found in oat bran can decrease cholesterol levels by 12% to 26% when associated with a diet high in saturated fat. Flaxseed also contains lignans, which show important biological activi-
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Table 7 Fatty acid profile of visceral adipose tissue (%) of the animals* Fatty acid
Normal
HF
Trout
Flaxseed
Peanut
Chicken skin
C14:0 C16:0 C18:0 Total SFAs
1.66 ⫾ 0.09 30.59 ⫾ 4.69 3.97 ⫾ 0.89 36.23 ⫾ 4.54
1.12 ⫾ 0.11a 24.1 ⫾ 3.91a 3.63 ⫾ 0.46a 28.92 ⫾ 3.97a
1.53 ⫾ 0.09b 31.48 ⫾ 5.69b 5.37 ⫾ 1.55b 39.22 ⫾ 7.6b
1.37 ⫾ 0.12bc 26.11 ⫾ 3.19ab 4.82 ⫾ 0.72ab 32.50 ⫾ 3.4ab
1.33 ⫾ 0.11c 27.54 ⫾ 4.31ab 3.72 ⫾ 0.48a 32.68 ⫾ 4.28ab
1.43 ⫾ 0.6ac 29.59 ⫾ 3.06ab 4.49 ⫾ 0.75ab 35.56 ⫾ 3.74ab
C16:1-7 C18:1-9 Total MUFAs
8.65 ⫾ 0.78† 37.01 ⫾ 3.56 45.93 ⫾ 4.25
4.00 ⫾ 0.55a 35.68 ⫾ 2.42a 40.13 ⫾ 3.65a
5.56 ⫾ 0.92b 41.46 ⫾ 4.37bd 48.18 ⫾ 5.21bc
5.24 ⫾ 0.69b 38.17 ⫾ 2.88ad 44.49 ⫾ 3.55ab
5.55 ⫾ 0.39b 43.13 ⫾ 2.83bc 49.38 ⫾ 3.22bc
6.12 ⫾ 0.85b 46.95 ⫾ 2.07c 53.08 ⫾ 2.35c
C18:2-6 C18:3-3 Total PUFAs
16.78 ⫾ 4.13† 1.12 ⫾ 0.37 17.84 ⫾ 4.35†
28.90 ⫾ 4.08a 1.65 ⫾ 0.43a 30.94 ⫾ 3.95a
12.21 ⫾ 2.79b — 12.60 ⫾ 3.03bd
10.48 ⫾ 1.95b 11.11 ⫾ 4.7b 23.01 ⫾ 5.24ce
17.74 ⫾ 2.76c — 17.94 ⫾ 2.48de
11.36 ⫾ 1.87b — 11.36 ⫾ 1.87b
Total -6 Total -3 MUFA ⫹ PUFA/SFA -6/-3
17.21 1.41 1.5:1 12:1
29.50 2.41 2.5:1 12:1
13.18 0.26 1.5:1 50:1
11.8 11.81 2:1 1:1
18.28 0.47 2:1 39:1
11.36 — 2:1 ‡
HF, high fat; MUFA, monounsaturated fatty acid; PUFA,polyunsaturated fatty acid; SFA, saturated fatty acid * Mean values followed by the same letter in the line are not different by Tukey’s test (P ⬍ 0.05). † A significant difference was observed between normal and HF groups by Student’s t-test (P ⬍ 0.05). ‡ No -3 was found in the chicken skin group. Total fatty acids may include other fatty acids not listed in Table 7.
ties, such as decreasing platelet aggregation, serum cholesterol, and the risk of cancer and act as an antioxidant [22,37]. Therefore, the effect of flaxseed in decreasing serum cholesterol does not seem to be due only to its C18:3 content, but rather to the synergistic action of its constituents. This was also reported by Jenkins et al. [25] who found lower serum lipid levels in normocholesterolemic and hypercholesterolemic subjects who were fed flaxseed lipid. No difference (P ⬍ 0.05) was observed in total serum cholesterol between animals fed the peanut and chicken skin diets. Chicken skin presented high levels of SFAs (C16:0 and C18:0). It has been shown that the atherogenic effect of
these SFAs may be enhanced by a cholesterol-rich diet [38], which leads to high levels of low-density lipoprotein and very low-density lipoprotein cholesterol and a small liver lipid deposition. Conversely, chicken skin also showed a high content of MUFAs (C18:1), which has been reported to play an important role in decreasing serum total cholesterol and increasing HDL cholesterol. HDL cholesterol was not significantly different (P ⬎ 0.05) between the normal and HF groups. However, animals fed the HF and chicken skin diets showed higher HDL cholesterol levels (P ⬍ 0.05) than did animals in the highPUFA flaxseed group. Although PUFAs are associated with
Table 8 Fatty acid profile of subcutaneous adipose tissue (%) of the animals* Fatty acid
Normal
HF
Trout
Flaxseed
Peanut
Chicken skin
C14:0 C16:0 C18:0 Total SFAs
1.7 ⫾ 0.16† 33.13 ⫾ 2.8† 4.08 ⫾ 0.47 39.07 ⫾ 2.99†
1.28 ⫾ 0.13a 24.62 ⫾ 0.83a 3.65 ⫾ 0.36a 29.66 ⫾ 0.83a
1.53 ⫾ 0.15b 29.56 ⫾ 1.06b 4.71 ⫾ 0.43b 35.85 ⫾ 1.04b
1.44 ⫾ 0.11ab 25.96 ⫾ 1.75a 4.67 ⫾ 0.58b 32.13 ⫾ 2.17ab
1.41 ⫾ 0.11ab 25.08 ⫾ 4.29a 3.97 ⫾ 0.14ab 30.64 ⫾ 4.27a
1.44 ⫾ 0.14ab 30.31 ⫾ 1.05b 4.28 ⫾ 0.68ab 36.04 ⫾ 1.59bc
C16:1-7 C18:1-9 Total MUFAs
7.74 ⫾ 0.62† 36.04 ⫾ 0.84† 43.84 ⫾ 0.87†
3.15 ⫾ 0.46a 33.06 ⫾ 1.12a 36.22 ⫾ 1.13a
5.08 ⫾ 0.62b 42.41 ⫾ 1.13b 48.11 ⫾ 1.14b
3.51 ⫾ 0.66a 35.31 ⫾ 1.87c 38.83 ⫾ 1.87c
4.71 ⫾ 0.59b 44.76 ⫾ 1.56d 50.34 ⫾ 2.36bd
5.36 ⫾ 0.7b 45.26 ⫾ 1.28d 50.7 ⫾ 0.82d
C18:2 -6 C 18:3 -3 Total PUFAs
16.39 ⫾ 3.18† 1.02 ⫾ 0.17 17.07 ⫾ 3.68†
32.11 ⫾ 1.55a 1.92 ⫾ 0.22a 34.10 ⫾ 1.61a
15.59 ⫾ 1.67bc — 16.03 ⫾ 1.59bde
15.19 ⫾ 1.5bc 13.83 ⫾ 2.49b 29.03 ⫾ 3.61c
18.72 ⫾ 3.35b — 19.01 ⫾ 3.31c
13.23 ⫾ 2.35c — 13.23 ⫾ 2.35bd
Total -6 Total -3 MUFA ⫹ PUFA/SFA -6/-3
16.39 1.02 1.5:1 16:1
32.5 1.92 2:1 17:1
17.37 — 1.5:1
15.19 13.83 2:1 1:1
19.04 0.38 2:1 50:1
13.23 — 1.5:1
‡
HF, high fat; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid * Mean values followed by the same letter in the line are not different by Tukey’s test (P ⬍ 0.05). † A significant difference was observed between normal and HF groups by Student’s t test (P ⬍ 0.05). ‡ No -3 was found in the trout and chicken skin groups. Total fatty acids may include other fatty acids not listed in Table 8.
‡
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decreasing cholesterol and risk of CHD, they may also decrease HDL cholesterol, probably due to inhibition of apolipoprotein A-1 synthesis. This apolipoprotein activates lecithin-cholesterol acyl transferase, which esterifies cholesterol bound in the circulating HDLs [39]. Animals fed the normal diet had higher levels of triacylglycerol than did animals fed the HF diet, which may be due to the higher content of carbohydrate in the former diet as a consequence of its lower fat content, as shown in Table 1. Animals fed the chicken skin diet had higher (P ⬍ 0.05) triacylglycerol levels than did animals fed the flaxseed diet. This difference may be attributed to the fatty acid profile of these diets because it has been demonstrated that the -3 fatty acids decrease triacylglycerol levels in hyperlipidemic subjects and in Eskimos from Greenland who consume a diet low in saturated fat [40,41]. The diet high in saturated fat increased blood cholesterol up to 25%. This seems to be the result of liver lipid deposition, which provides acetyl coenzyme A to liver cells for cholesterol synthesis [42]. The excessive liver lipid deposition leads to steatosis [43], which represents an imbalance between triacylglycerol synthesis in the liver and its secretion [44]. Among the fatty acids, MUFA C18:1 was found to be more abundant in both adipose tissues analyzed, followed by C16:0 and C18:2 -6. The same profile was observed by Garaulet et al. [45] in subcutaneous and visceral adipose tissues in humans. Animals in the flaxseed group showed the most distinctive behavior, characterized by a high -3 deposition in adipose tissue, which reflects its content of -3. Similarly, in a study carried out by Walisundera et al. [23] in pigs fed 5% flaxseed for 8 wk, a deposition of 18:3-3 was found in animal organs and tissues. However, docosahexaenoic acid (C22:6) provided by trout was not deposited in both tissues analyzed. This may be due to the fact that this is a fatty acid essential for membrane formation and the function of nervous tissue and vision, where it may represents about 40% of the phospholipids of these tissues [45,46] and play an important role in the immune system [47].
Conclusion Analysis of trout showed a higher content of -6 than of -3 fatty acids, although it is a cold-water fish. The same lipid profile was observed in adipose tissues of animals fed a trout diet. This diet did not decrease blood cholesterol and showed no protective effect on the liver parenchyma of these animals. The flaxseed diet was the most efficient diet in decreasing total serum cholesterol and triacylglycerol levels and for protecting the liver parenchyma. This seems to be due to its content of -3 fatty acids and the presence of lignans and soluble fiber of flaxseed. Moreover, this diet produced higher fecal lipid output and lower liver lipid deposition compared with the other treatments. The peanut
diet showed the lowest food efficiency ratio, which may represent a positive effect that should be investigated in future studies for body weight control in obese subjects. The peanut diet also proved to a good source of MUFA C18:1 and showed a potential for decreasing triacylglycerol levels in rats. It seems that the negative effects of the high SFA content in the chicken skin diet were counterbalanced by the positive effects of its MUFA content. Other animal models should be considered in future studies to evaluate the effects of different lipid sources in the context of a high cholesterol diet and to elucidate the mechanisms by which the diet may modulate lipid profile.
Acknowledgements The authors thank Dr. Yara Tabata and Dr. Marcos Rigolino for the donation of the trout, Katal for the donation of the analytical kits, Dr. Sylvia Franceschini for statistical advice, Dr. Antônio Marcos dos Santos for technical assistance with the histologic analysis, and Dr. April Mason for comments.
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Basic nutritional investigation
www.elsevier.com/locate/nut
Lipid profile of rats fed high-fat diets based on flaxseed, peanut, trout, or chicken skin Dennys E. C. Cintra, M.Sc., André G. V. Costa, M.Sc., Maria do Carmo G. Peluzio, D.S., Sérgio L. P. Matta, D.S., Marco Túlio C. Silva, D.S., and Neuza M. B. Costa, Ph.D.* Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil Manuscript received May 4, 2005; accepted September 3, 2005.
Abstract
Objective: Dietary saturated fatty acids are associated with coronary disease. Conversely, dietary monounsaturated polyunsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) seem to exert a protective effect. This study evaluated the lipid profile of rats fed high-fat (HF) diets, with fat added as different sources of PUFA (flaxseed and trout), MUFA (peanut), and saturated fatty acid (chicken skin). Methods: Adult male Wistar rats were placed into six dietary groups (n ⫽ 10): control (normal); high fat, with 1% cholesterol, 10% soy oil, and 5% lard; and four groups fed similar HF diets, with 10% lipid as trout, flaxseed, peanut, or chicken skin. After 28 d the animals were killed. Blood, livers, and adipose tissue samples were collected. Results: A higher level (P ⬍ 0.05) of total serum cholesterol was observed in rats fed the normal diet (93.57 ⫾ 14.95 mg/dL) compared with those fed the HF diet (67.57 ⫾ 12.54 mg/dL). Total cholesterol levels in rats fed the flaxseed diet were lower (P ⬍ 0.05) than in rats fed the other fats. No difference was observed in cholesterol levels between groups fed the peanut and chicken skin diets (P ⬎ 0.05). Animals fed the peanut diet showed decreased body weight gain than did animals in the other treatment groups. There were large lipid and cholesterol depositions in livers of rats fed the HF diet. Lipid deposition in adipose tissue followed the same dietary fatty acid profile, i.e., high levels of -3 PUFA in the flaxseed group, high levels of MUFA in the peanut and chicken skin groups and high levels of -6 PUFA in the trout group. Conclusions: These data indicate that flaxseed is promising for dietary manipulation of hyperlipidemia. © 2006 Elsevier Inc. All rights reserved.
Keywords:
Flaxseed; Trout; Peanut; Chicken skin; Lipid profile; Lipidemia
Introduction Studies to determine the effects of lipids on blood cholesterol date from the 1950s [1] and show that different dietary lipids can modulate plasma cholesterol levels, depending on their fatty acid composition. High levels of saturated fatty acids (SFAs) in the Western diet are associated with the formation of atherosclerotic plaque [2–5]. However, intake of monounsaturated fatty acids (MUFAs) -9, especially oleic acid (18:1-9), and long-chain polyunsaturated fatty acids (PUFAs) of the -3 series, especially eicosapentaenoic acid (20:5-3) and docosahexaenoic
acid (22:6-3), is associated with decreased risk of cardiovascular death [3,6 –11]. Many studies have demonstrated that MUFAs and PUFAs show similar effects on lowering blood cholesterol when SFA is substituted in the diet [12]. The presented study evaluated the effects of MUFAcontaining peanut and PUFA-containing flaxseed and trout diets compared with an SFA-containing chicken skin diet on the lipid profile of rats.
Material and Methods Animals and diet
* Corresponding author. Tel./fax: ⫹55-31-3899-2541. E-mail address: [email protected] (N.M.B. Costa). 0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2005.09.003
Sixty male adult Wistar rats with an initial body weight of 180 g were obtained from the Biology and Health Sci-
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ences Center of Federal University of Viçosa, MG, Brazil. The animals were randomized to six groups (n ⫽ 10). One group received a control AIN-93 diet [13]; another group received a high-fat (HF) diet of 10% soybean oil, 1% cholesterol, and 5% lard. The other groups received diets similar to the HF diet, but with 10% of lipids provided by trout, peanut, flaxseed, or chicken skin to replace the soybean oil. Compositions of the experimental diets are presented in Table 1. Cornstarch and protein contents of the diets were adjusted to maintain isocaloric diets. Animals were kept in individual cages in a temperaturecontrolled room at 22 ⫾ 2°C, with a 12-h dark/12-h light cycle for 28 d. They received water and diet ad libitum. Food intake and body weights were monitored weekly. Ingredients Trout that were about 200 g in body weight and 14 mo of age were eviscerated, scaled, and kept frozen. Before diet preparation, trout were thawed at room temperature, ground in a meat grinder, and dried in an oven at 60°C for 48 h. Peanuts (Nestlé, São Paulo, Brazil) were donated by a food industry as skinned and ground peanut flour, which was further roasted at 168°C for 25 min before being added to the animal diet. Flaxseed (Mãe Terra®)was obtained in the local market and ground in a multiprocessor. Chicken skin (Pif-Paf®) was obtained in the local market, ground in a meat grinder, and dried in oven at 60°C for 48 h. The drained oil was discarded. Casein was purchased from Sigma and Rhoster (Sigma, São Paulo, Brazil, and Rhoster, São Paulo, Brazil). Sucrose (União®), cornstarch (Maizena®), lard (Sadia®), and soybean oil (Liza®) were purchased in the local market. Dextrinated starch, cellulose, mineral mix, vitamin mix, L-cystine, and choline bitartrate
were purchased from Rhoster. Cholesterol was purchased from Sigma. The moisture [14], protein [14], and lipid [15] contents of trout, peanut, flaxseed, and chicken skin were determined before being added to the diets. Blood and tissue sampling At the end of the experimental period and after 12 h of fasting, animals were killed by CO2. Blood was collected by heart puncture and serum was taken after centrifugation at 2400g for 15 min. Liver slices were fixed in 10% formaldehyde buffer for morphologic analysis and the remaining liver samples were kept for cholesterol analysis. Subcutaneous and visceral adipose tissue samples were taken from the abdominal cavity for fatty acid profile analysis. Lipid extraction, saponification, and esterification Lipid extraction of food components, adipose tissues, and livers of the experimental animals was carried out according to the method of Folch et al. [16]. Fecal lipid was extracted by Soxhlet, using petrol ether (Vetec, Minas Gerais, Brazil) [15]. Lipid saponification and esterification, when required, followed the method of Hartman and Lago [17]. Fatty acid profile Fatty acid profiles of food components and adipose tissues were determined by gas chromatography, using a Shimadzu Model 17A, under the following operating conditions: fused silica capillary column (100 m ⫻ 0.25 mm; SP-2560); hydrogen as the carrier gas, with a flow rate of 20
Table 1 Composition of experimental diets (g/100 g) Ingredients
Normal
HF
Trout
Flaxseed
Peanut
Chicken skin
Casein* Sucrose Cornstarch Dextrinated starch Cellulose Lard Cholesterol Mineral mix Vitamin mix L-cystine Choline bitartrate Soybean oil Trout† Flaxseed† Peanut† Chicken skin†
15.30 10.00 45.26 15.5 5.00 — — 3.50 1.00 0.18 0.25 4.00 — — — —
15.30 10.00 33.26 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 10.00 — — — —
0.00 10.00 22.38 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 — 36.19 — — —
11.80 10.00 25.89 15.5 1.00 5.00 1.00 3.50 1.00 0.18 0.25 — — 24.88 — —
9.86 10.00 28.21 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 — —
11.39 10.00 31.37 15.5 5.00 5.00 1.00 3.50 1.00 0.18 0.25 — — — — 15.81
HF, high fat * To provide 12 g of protein/100 g of diet. † To provide 10 g of lipids/100 g of diet.
20.50 —
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cm/s; oven temperature, initially 140°C for 5 min, was increased up to 240°C at 4°C/min and maintained at 240°C for 30 min; injector, split ratio was 1:50, temperature 250°C; vaporization and detector temperatures were 250°C and 260°C, respectively. The injection volume was 1 L of sample solution. The retention times of methyl ester standards (Sigma, São Paulo, Brazil) were used to identify peaks. Blood lipids Total serum cholesterol levels were analyzed by the method of Allain et al. [18], high-density lipoprotein (HDL) cholesterol by the method of Warnick et al. [19], and triacylglycerol by the method of Fossati and Prencipe [20] by using specific enzymatic kits (Katal, Belo Horizonte, MG, Brazil).
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Table 2 Food composition (g/100 g) Content
Dried trout
Flaxseed
Peanut
Dried chicken skin
Moisture Total lipids Protein Fiber*
— 27.63 56.53 —
8.14 40.18 19.35 18.00
1.42 48.76 30.86 6.40
4.13 63.22 32.49 ND
ND, not determined * From the food label (nutrition facts) or table of food composition [32].
Wallis test was used to compare three or more independent samples, followed by Dunn’s test (P ⬍ 0,05), when the comparison was statistically significant. Analyses were carried out using Sigma Stat 2.02 (Software for Windows; Jandel Corp., San Rafael, CA, USA).
Liver cholesterol Results Liver cholesterol analysis was carried out by gas chromatography according to the method of Naeemi et al. [21] by using a Shimadzu Model 17A, under the following operating conditions: column, DB-1 (dimethylpolysiloxane, 30 m ⫻ 0.25 mm); hydrogen as the carrier gas at 175°C at a flow rate of 22 cm/s; oven temperature was initially 280°C for 1 min, was increased up to 300°C at 20°C/min, and maintained at 300°C for 10 min; injector, split ratio was 1:50; vaporization and detector temperatures were 290°C and 300°C, respectively. The injection volume was 1 L of sample solution. Liver morphology Liver slices were dehydrated with ethanol, cleared with xylene, and embedded in paraffin wax (Merck, São Paulo, Brazil). After inclusion, material was cut on a microtome (CUT model 445, Olympus) at 4 m. Liver sections were stained with hematoxylin and eosin (Merck, São Paulo, Brazil). Pictures were taken with a photomicroscope (Model AX70, Olympus) using Kodacolor Gold 100 asa film at 40⫻ magnification. Histologic analysis was qualitative. Statistical analysis Collected data were subjected to the KolmogorovSmirnov test to check for symmetry. For variables with normal distribution, Student’s t test was used to compare two independent samples (normal and HF groups). Analysis of variance was used to compare three or more independent samples (trout, flaxseed, peanut, and chicken skin groups), followed by Tukey’s test (P ⬍ 0.05) when the F value was significant. For asymmetric variables, the Mann-Whitney test was used to compare two independent samples, and the Kruskal-
Table 2 lists moisture, total lipid, protein, and fiber contents. Table 3 presents the fatty acid profiles of trout, flaxseed, peanut, chicken skin, and soybean oil. Trout and peanut showed a high and similar composition of MUFAs (C18:1). Flaxseed showed a high content of the -3 fatty acid ␣-linolenic acid (⬃58%) and chicken skin had a high content of SFA, although its content of MUFAs was comparable to those of trout and peanut. Body weight, food intake, and food efficiency ratio are presented in Table 4. No significant differences were observed in body weight and food intake at the beginning of the study. Nevertheless, the peanut group showed lower (P ⬍ 0.05) body weight gain and food efficiency ratio compared with the trout and flaxseed groups. Total serum cholesterol, HDL cholesterol, and triacylglycerol levels are listed in Table 5. Total cholesterol in animals fed the HF diet, with 1% cholesterol in the diet, was unexpectedly lower than that in animals fed the normal diet. Lower levels (P ⬍ 0.05) of total cholesterol were observed in animals fed the flaxseed diet compared with the peanut and chicken skin diets. Animals fed the flaxseed diet also showed lower levels (P ⬍ 0.05) of triacylglycerol compared with animals fed the chicken skin diet. No other differences in triacylglycerol levels were observed. No significant differences in HDL cholesterol were observed between the normal and HF groups; however, animals in the normal group showed a lower (P ⬍ 0.05) HDL cholesterol/total cholesterol ratio than did the HF group. Liver weights of animals in the normal, flaxseed, peanut, and chicken skin groups were significantly lower than those in the HF group (P ⬍ 0.05), as listed in Table 6. This may be related to the deposition of total lipids and cholesterol in the liver because no difference in body weight was observed across animals in different groups. Table 6 also indicates that the types of lipid in the diet exert an influence on liver
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Table 3 Fatty acid profile (g/100 g of total lipid) Fatty acid
Dried trout
Flaxseed
Peanut
Dried chicken skin
Soybean oil
C14:0 C16:0 C18:0 C23:0 Total SFAs
1.17 23.40 5.13 — 29.70
— 6.45 4.38 — 10.83
— 12.98 3.16 — 16.14
0.55 29.69 6.43 0.35 37.02
— 14.65 1.93 — 16.58
C14:1 C16:1 C18:1 C20:1 Total MUFAs
— 6.31 33.41 — 39.72
— — 18.00 — 18.00
— — 38.25 — 38.25
0.30 6.13 38.09 0.69 45.21
— — 31.66 0.70 32.36
C18:2-6 C20:2 C22:2 C18:3-6 C18:3-3 C20:3-6 C20:4-6 C22:6-3 Total PUFAs
17.19 1.20 — 2.67 0.80 1.07 1.44 6.22 30.58
12.71 — — — 58.47 — — — 71.17
39.33 3.53 1.60 1.15 — — — — 45.61
16.60 — — 0.55 0.62 — — — 17.77
51.06 — — — — — — — 51.06
5:1 —
1.5:1 27:1
5.03:1 —
MUFA ⫹ PUFA/SFA -6/-3
2:1 3:1
8:1 1:4.5
MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid
lipid composition because the content of SFAs in the diets corresponded to the larger depositions of cholesterol and triacylglycerol in the liver. Total liver lipid levels were higher in animals fed the HF diet than in animals fed the normal diet, but no difference was observed between the HF and other groups. Nevertheless, animals in the HF group showed a large deposition of cholesterol in the liver than did animals in the other groups (Table 6). A difference was observed in lipid liver deposition based on photomicrographs of liver parenchyma (Fig. 1). Fecal lipid levels (Table 6) were higher in animals fed the flaxseed diet than in animals in the other groups, likely due to its high dietary fiber content, which increases transit time and fecal bulk. Liver steatosis Figure 1 shows lipid deposition in liver slices. The intensity of liver steatosis was classified as more than 30% of hepato-
cytes affected (⫹), more than 50% of hepatocytes affected (⫹⫹), and more than 75% of hepatocytes affected (⫹⫹⫹). Lipid vesicles appeared in livers of all animals. Hepatocytes of the HF group presented a flattened nucleus due to high lipid content. Lipid deposition in the adipose tissue Dietary lipid was incorporated effectively in visceral and subcutaneous adipose tissues of all experimental animals (Tables 7 and 8). Animals fed the HF diet showed lower contents of SFAs and MUFAs in visceral and subcutaneous adipose tissues (P ⬍ 0.05) compared with the trout group. An opposite effect was observed in relation to the PUFA content of these two groups. This seems to be due to the soybean oil used in the HF diet, which is rich in -6 PUFAs. The fatty acid profiles (SFA, MUFA, and PUFA) of
Table 4 Body weight, body weight gain, food intake, and FER of the experimental groups* Treatment
Initial body weight (g)
Body weight gain (g)
Food intake (g)
FER
Normal HF Trout Flaxseed Peanut Chicken skin
185.80 ⫾ 21.29 183.00 ⫾ 22.71a 178.90 ⫾ 19.38a 169.00 ⫾ 27.72a 184.70 ⫾ 7.80a 178.90 ⫾ 11.98a
114.30 ⫾ 24.47 131.10 ⫾ 23.32ab 140.40 ⫾ 19.02a 140.90 ⫾ 33.18a 105.70 ⫾ 19.78b 128.30 ⫾ 16.90ab
528.47 ⫾ 62.00 512.07 ⫾ 60.54a 467.10 ⫾ 36.76a 483.52 ⫾ 41.88a 493.61 ⫾ 29.96a 512.36 ⫾ 21.24a
0.21 ⫾ 0.03 0.25 ⫾ 0.02ab 0.30 ⫾ 0.03a 0.29 ⫾ 0.06a 0.21 ⫾ 0.03b 0.25 ⫾ 0.03ab
FER, food efficiency ratio; HF, high fat * Mean values followed by the same letter in the column are not different by Tukey’s test (P ⬍ 0.05). No difference was observed between normal and HF groups by Student’s t test (P ⬎ 0.05).
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Table 5 Total serum cholesterol, HDL-C, triacylglycerol, and HDL-C/total cholesterol ratio of the animals* Treatment
Total cholesterol (mg/dL) Mean ⫾ SD
Normal HF Trout Flaxseed Peanut Chicken skin
93.57 ⫾ 14.95 67.57 ⫾ 12.54 83.87 ⫾ 14.41 60.87 ⫾ 7.02 101.40 ⫾ 17.31 107.84 ⫾ 9.52
HDL-C (mg/dL)
Median †
96.53 63.00a 81.79ab 61.56a 105.49b 106.79b
Mean ⫾ SD 55.28 ⫾ 7.03 58.77 ⫾ 9.34 51.94 ⫾ 13.97 41.57 ⫾ 6.96 46.18 ⫾ 6.46 64.38 ⫾ 16.93
Triacylglycerol (mg/dL) Median
Mean ⫾ SD
53.87 62.66a 45.48ab 38.54b 45.76ab 62.82a
76.85 ⫾ 18.07 53.36 ⫾ 11.36 62.60 ⫾ 23.25 49.54 ⫾ 6.57 55.98 ⫾ 12.95 77.21 ⫾ 16.99
HDL-C/total cholesterol ratio
Median †
72.50 53.38ab 57.61ab 51.99a 56.45ab 83.99b
Mean ⫾ SD
Median
0.59 ⫾ 0.07 0.90 ⫾ 0.19 0.62 ⫾ 0.09 0.68 ⫾ 0.14 0.46 ⫾ 0.08 0.59 ⫾ 0.14
0.59† 0.87a 0.65ab 0.64ab 0.47b 0.63b
HDL-C, high-density lipoprotein cholesterol; HF, high fat; SD, standard deviation. * Medians followed by the same letter in the column are not different by Kruskal-Wallis test, complemented with Dunn’s multiple comparisons test (P ⬎ 0.05). † A significant difference was found between normal and HF groups by Mann-Whitney test (P ⬍ 0.05).
animals fed the flaxseed and peanut diets were similar with respect to visceral adipose tissue but not to subcutaneous adipose tissue. This may be due to high lipid mobility in subcutaneous adipose tissue.
Discussion Levels of fiber, lipid, and fatty acid in flaxseed in this study were similar to those found by Prasad [22] and Walisundera et al. [23]. The high content of ␣-linolenic acid (58%) may play an important role in decreasing the risk of coronary heart disease (CHD) by lowering levels of lowdensity lipoprotein cholesterol, as reported in the literature [22,24,25]. Low levels of SFA, especially palmitic acid (C16:0), may be another protective factor against CHD [26]. Trout is reported to have a higher content of -3 fatty acids eicosapentaenoic acid and docosahexaenoic acid in the liver than in the filet [27,28]. This may explain the low levels of these fatty acids found in this study for trout, which showed a high content of MUFAs (C18:1 and C16:1). The high content of MUFAs is associated with a low incidence of CHD [29] because it decreases total cholesterol (10%) and low-density lipoprotein cholesterol (14%) [30]. This effect, however, depends on levels of SFA in the diet [31]. Chicken skin, which has a high content of MUFAs
(C18:1 and C16:1), presented the highest content of SFAs (C16:0) among the foods evaluated in this study and, hence, low PUFA/SFA ratio. Palmitic acid (C16:0) is associated with the risk of CHD because of its effect in increasing blood cholesterol levels and platelet aggregation [26]. The lipid and protein contents of peanut found in this study were similar to those reported in the literature [30,32]. The peanut diet, however, resulted in a low food efficiency ratio, perhaps due to the presence of limiting amino acid and antinutrient factors. Although these factors may be present in other plant proteins such as flaxseed, the addition of casein to the flaxseed diet improved its protein quality and enhanced its food efficiency ratio. Animals fed the peanut diet showed lower body weight gain over the study period (P ⬍ 0.05). This trend has also been reported for humans [29,33]. The effect of nuts in lowering body mass index is not completely understood, but it is hypothesized that the content of PUFAs compared with that of SFAs may decrease body weight [34] and that the lipid intake may stimulate satiety [33]. Animals fed the HF diet showed lower levels of blood cholesterol compared with animals fed the normal diet. This unexpected result may be due to an inhibition of 3-hydroxy3-methylglutaryl coenzyme A reductase by a feedback mechanism caused by the high intake of dietary cholesterol (1%). Moreover, cholesterol deposition in the liver was high
Table 6 Liver weight, total liver lipids, liver cholesterol, and fecal lipids of the animals* Treatment
Liver weight (g) (n ⫽ 8)
Total liver lipids (mg/g) (n ⫽ 8)
Liver cholesterol (mg/g) (n ⫽ 8)
Total fecal lipids (mg/g) (n ⫽ 10)
Normal HF Trout Flaxseed Peanut Chicken skin
10.87 ⫾ 1.51† 15.80 ⫾ 2.61a 13.77 ⫾ 1.72ab 13.31 ⫾ 1.23b 12.82 ⫾ 0.93b 13.25 ⫾ 1.75b
33.40 ⫾ 11.50† 135.20 ⫾ 73.00a 68.40 ⫾ 41.10a 57.00 ⫾ 31.00a 99.80 ⫾ 53.60a 83.00 ⫾ 37.20a
3.35 ⫾ 1.58† 34.50 ⫾ 17.43a 15.30 ⫾ 9.82b 12.46 ⫾ 8.15b 12.97 ⫾ 6.39b 13.06 ⫾ 6.41b
29.69 ⫾ 12.63† 106.33 ⫾ 15.51a 123.51 ⫾ 35.41a 201.60 ⫾ 20.05b 123.35 ⫾ 23.63a 113.54 ⫾ 19.02a
HF, high fat * Means followed by the same letter in the column are not different by Tukey’s test (P ⬍ 0.05). † A significant difference was observed between normal and HF groups by Student’s t test (P ⬍ 0.05).
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Fig. 1. Photomicrographs of liver parenchyma of animals fed the control (normal), HF, trout, flaxseed, peanut, or chicken diet (hematoxylin and eosin, original magnification 440⫻). Arrows indicate lipid vesicles. (⫹), greater than 30% steatosis; (⫹⫹), greater than 50% steatosis; (⫹⫹⫹), greater than 75% steatosis; HF, high fat.
in the HF group, which may explain why cholesterol was low in the bloodstreams of these animals. Animals fed the flaxseed diet showed the lowest levels of serum cholesterol. The content of fatty acid may be associated with this effect because unsaturation has been reported to be inversely related to serum cholesterol levels [35]. The ␣-linolenic fatty acid (C18:3) and the fiber contents of
flaxseed may also be associated to its cholesterol-lowering property. According to Hadley and Mitchell-Fetch [36], the ratio of soluble to insoluble dietary fiber may vary from 20:80 to 40:60 in flaxseed. The soluble fiber found in oat bran can decrease cholesterol levels by 12% to 26% when associated with a diet high in saturated fat. Flaxseed also contains lignans, which show important biological activi-
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203
Table 7 Fatty acid profile of visceral adipose tissue (%) of the animals* Fatty acid
Normal
HF
Trout
Flaxseed
Peanut
Chicken skin
C14:0 C16:0 C18:0 Total SFAs
1.66 ⫾ 0.09 30.59 ⫾ 4.69 3.97 ⫾ 0.89 36.23 ⫾ 4.54
1.12 ⫾ 0.11a 24.1 ⫾ 3.91a 3.63 ⫾ 0.46a 28.92 ⫾ 3.97a
1.53 ⫾ 0.09b 31.48 ⫾ 5.69b 5.37 ⫾ 1.55b 39.22 ⫾ 7.6b
1.37 ⫾ 0.12bc 26.11 ⫾ 3.19ab 4.82 ⫾ 0.72ab 32.50 ⫾ 3.4ab
1.33 ⫾ 0.11c 27.54 ⫾ 4.31ab 3.72 ⫾ 0.48a 32.68 ⫾ 4.28ab
1.43 ⫾ 0.6ac 29.59 ⫾ 3.06ab 4.49 ⫾ 0.75ab 35.56 ⫾ 3.74ab
C16:1-7 C18:1-9 Total MUFAs
8.65 ⫾ 0.78† 37.01 ⫾ 3.56 45.93 ⫾ 4.25
4.00 ⫾ 0.55a 35.68 ⫾ 2.42a 40.13 ⫾ 3.65a
5.56 ⫾ 0.92b 41.46 ⫾ 4.37bd 48.18 ⫾ 5.21bc
5.24 ⫾ 0.69b 38.17 ⫾ 2.88ad 44.49 ⫾ 3.55ab
5.55 ⫾ 0.39b 43.13 ⫾ 2.83bc 49.38 ⫾ 3.22bc
6.12 ⫾ 0.85b 46.95 ⫾ 2.07c 53.08 ⫾ 2.35c
C18:2-6 C18:3-3 Total PUFAs
16.78 ⫾ 4.13† 1.12 ⫾ 0.37 17.84 ⫾ 4.35†
28.90 ⫾ 4.08a 1.65 ⫾ 0.43a 30.94 ⫾ 3.95a
12.21 ⫾ 2.79b — 12.60 ⫾ 3.03bd
10.48 ⫾ 1.95b 11.11 ⫾ 4.7b 23.01 ⫾ 5.24ce
17.74 ⫾ 2.76c — 17.94 ⫾ 2.48de
11.36 ⫾ 1.87b — 11.36 ⫾ 1.87b
Total -6 Total -3 MUFA ⫹ PUFA/SFA -6/-3
17.21 1.41 1.5:1 12:1
29.50 2.41 2.5:1 12:1
13.18 0.26 1.5:1 50:1
11.8 11.81 2:1 1:1
18.28 0.47 2:1 39:1
11.36 — 2:1 ‡
HF, high fat; MUFA, monounsaturated fatty acid; PUFA,polyunsaturated fatty acid; SFA, saturated fatty acid * Mean values followed by the same letter in the line are not different by Tukey’s test (P ⬍ 0.05). † A significant difference was observed between normal and HF groups by Student’s t-test (P ⬍ 0.05). ‡ No -3 was found in the chicken skin group. Total fatty acids may include other fatty acids not listed in Table 7.
ties, such as decreasing platelet aggregation, serum cholesterol, and the risk of cancer and act as an antioxidant [22,37]. Therefore, the effect of flaxseed in decreasing serum cholesterol does not seem to be due only to its C18:3 content, but rather to the synergistic action of its constituents. This was also reported by Jenkins et al. [25] who found lower serum lipid levels in normocholesterolemic and hypercholesterolemic subjects who were fed flaxseed lipid. No difference (P ⬍ 0.05) was observed in total serum cholesterol between animals fed the peanut and chicken skin diets. Chicken skin presented high levels of SFAs (C16:0 and C18:0). It has been shown that the atherogenic effect of
these SFAs may be enhanced by a cholesterol-rich diet [38], which leads to high levels of low-density lipoprotein and very low-density lipoprotein cholesterol and a small liver lipid deposition. Conversely, chicken skin also showed a high content of MUFAs (C18:1), which has been reported to play an important role in decreasing serum total cholesterol and increasing HDL cholesterol. HDL cholesterol was not significantly different (P ⬎ 0.05) between the normal and HF groups. However, animals fed the HF and chicken skin diets showed higher HDL cholesterol levels (P ⬍ 0.05) than did animals in the highPUFA flaxseed group. Although PUFAs are associated with
Table 8 Fatty acid profile of subcutaneous adipose tissue (%) of the animals* Fatty acid
Normal
HF
Trout
Flaxseed
Peanut
Chicken skin
C14:0 C16:0 C18:0 Total SFAs
1.7 ⫾ 0.16† 33.13 ⫾ 2.8† 4.08 ⫾ 0.47 39.07 ⫾ 2.99†
1.28 ⫾ 0.13a 24.62 ⫾ 0.83a 3.65 ⫾ 0.36a 29.66 ⫾ 0.83a
1.53 ⫾ 0.15b 29.56 ⫾ 1.06b 4.71 ⫾ 0.43b 35.85 ⫾ 1.04b
1.44 ⫾ 0.11ab 25.96 ⫾ 1.75a 4.67 ⫾ 0.58b 32.13 ⫾ 2.17ab
1.41 ⫾ 0.11ab 25.08 ⫾ 4.29a 3.97 ⫾ 0.14ab 30.64 ⫾ 4.27a
1.44 ⫾ 0.14ab 30.31 ⫾ 1.05b 4.28 ⫾ 0.68ab 36.04 ⫾ 1.59bc
C16:1-7 C18:1-9 Total MUFAs
7.74 ⫾ 0.62† 36.04 ⫾ 0.84† 43.84 ⫾ 0.87†
3.15 ⫾ 0.46a 33.06 ⫾ 1.12a 36.22 ⫾ 1.13a
5.08 ⫾ 0.62b 42.41 ⫾ 1.13b 48.11 ⫾ 1.14b
3.51 ⫾ 0.66a 35.31 ⫾ 1.87c 38.83 ⫾ 1.87c
4.71 ⫾ 0.59b 44.76 ⫾ 1.56d 50.34 ⫾ 2.36bd
5.36 ⫾ 0.7b 45.26 ⫾ 1.28d 50.7 ⫾ 0.82d
C18:2 -6 C 18:3 -3 Total PUFAs
16.39 ⫾ 3.18† 1.02 ⫾ 0.17 17.07 ⫾ 3.68†
32.11 ⫾ 1.55a 1.92 ⫾ 0.22a 34.10 ⫾ 1.61a
15.59 ⫾ 1.67bc — 16.03 ⫾ 1.59bde
15.19 ⫾ 1.5bc 13.83 ⫾ 2.49b 29.03 ⫾ 3.61c
18.72 ⫾ 3.35b — 19.01 ⫾ 3.31c
13.23 ⫾ 2.35c — 13.23 ⫾ 2.35bd
Total -6 Total -3 MUFA ⫹ PUFA/SFA -6/-3
16.39 1.02 1.5:1 16:1
32.5 1.92 2:1 17:1
17.37 — 1.5:1
15.19 13.83 2:1 1:1
19.04 0.38 2:1 50:1
13.23 — 1.5:1
‡
HF, high fat; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid * Mean values followed by the same letter in the line are not different by Tukey’s test (P ⬍ 0.05). † A significant difference was observed between normal and HF groups by Student’s t test (P ⬍ 0.05). ‡ No -3 was found in the trout and chicken skin groups. Total fatty acids may include other fatty acids not listed in Table 8.
‡
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decreasing cholesterol and risk of CHD, they may also decrease HDL cholesterol, probably due to inhibition of apolipoprotein A-1 synthesis. This apolipoprotein activates lecithin-cholesterol acyl transferase, which esterifies cholesterol bound in the circulating HDLs [39]. Animals fed the normal diet had higher levels of triacylglycerol than did animals fed the HF diet, which may be due to the higher content of carbohydrate in the former diet as a consequence of its lower fat content, as shown in Table 1. Animals fed the chicken skin diet had higher (P ⬍ 0.05) triacylglycerol levels than did animals fed the flaxseed diet. This difference may be attributed to the fatty acid profile of these diets because it has been demonstrated that the -3 fatty acids decrease triacylglycerol levels in hyperlipidemic subjects and in Eskimos from Greenland who consume a diet low in saturated fat [40,41]. The diet high in saturated fat increased blood cholesterol up to 25%. This seems to be the result of liver lipid deposition, which provides acetyl coenzyme A to liver cells for cholesterol synthesis [42]. The excessive liver lipid deposition leads to steatosis [43], which represents an imbalance between triacylglycerol synthesis in the liver and its secretion [44]. Among the fatty acids, MUFA C18:1 was found to be more abundant in both adipose tissues analyzed, followed by C16:0 and C18:2 -6. The same profile was observed by Garaulet et al. [45] in subcutaneous and visceral adipose tissues in humans. Animals in the flaxseed group showed the most distinctive behavior, characterized by a high -3 deposition in adipose tissue, which reflects its content of -3. Similarly, in a study carried out by Walisundera et al. [23] in pigs fed 5% flaxseed for 8 wk, a deposition of 18:3-3 was found in animal organs and tissues. However, docosahexaenoic acid (C22:6) provided by trout was not deposited in both tissues analyzed. This may be due to the fact that this is a fatty acid essential for membrane formation and the function of nervous tissue and vision, where it may represents about 40% of the phospholipids of these tissues [45,46] and play an important role in the immune system [47].
Conclusion Analysis of trout showed a higher content of -6 than of -3 fatty acids, although it is a cold-water fish. The same lipid profile was observed in adipose tissues of animals fed a trout diet. This diet did not decrease blood cholesterol and showed no protective effect on the liver parenchyma of these animals. The flaxseed diet was the most efficient diet in decreasing total serum cholesterol and triacylglycerol levels and for protecting the liver parenchyma. This seems to be due to its content of -3 fatty acids and the presence of lignans and soluble fiber of flaxseed. Moreover, this diet produced higher fecal lipid output and lower liver lipid deposition compared with the other treatments. The peanut
diet showed the lowest food efficiency ratio, which may represent a positive effect that should be investigated in future studies for body weight control in obese subjects. The peanut diet also proved to a good source of MUFA C18:1 and showed a potential for decreasing triacylglycerol levels in rats. It seems that the negative effects of the high SFA content in the chicken skin diet were counterbalanced by the positive effects of its MUFA content. Other animal models should be considered in future studies to evaluate the effects of different lipid sources in the context of a high cholesterol diet and to elucidate the mechanisms by which the diet may modulate lipid profile.
Acknowledgements The authors thank Dr. Yara Tabata and Dr. Marcos Rigolino for the donation of the trout, Katal for the donation of the analytical kits, Dr. Sylvia Franceschini for statistical advice, Dr. Antônio Marcos dos Santos for technical assistance with the histologic analysis, and Dr. April Mason for comments.
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