Micronutrient-enriched rapeseed oils reduce cardiovascular disease risk factors in rats fed a high-fat diet

Atherosclerosis 213 (2010) 422–428

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Micronutrient-enriched rapeseed oils reduce cardiovascular disease risk factors in rats fed a high-fat diet夽 Lucilla Attorri a , Antonella Di Biase a , Rita Di Benedetto a , Patrizia Rigato b , Antonio Di Virgilio a , Serafina Salvati a,∗ a b

Department of Public Veterinary Health and Food Safety, Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome, Italy Department of Pathology, San Giuseppe Hospital, V.le XXIV Maggio snc, 00040, Marino, Rome, Italy

a r t i c l e

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Article history: Received 13 January 2010 Received in revised form 3 June 2010 Accepted 6 July 2010 Available online 15 July 2010 Keywords: Rapeseed oil Micronutrients Cardiovascular diseases Plasma lipids Antioxidant enzymatic activities High-fat diet Liver

a b s t r a c t Many epidemiological studies have demonstrated that vegetable food consumption is associated with a reduced risk of cardiovascular diseases. The beneficial effects have been attributed to the content of bioactive molecules present in large quantities in plant food. The main proposal of this study was to evaluate in vivo whether micronutrient-enriched rapeseed oils (optimised oils) obtained using different crushing and refining procedures and characterised by different quantities and qualities of micronutrients, could have any beneficial effect on lipid profile and antioxidant status of plasma and liver. Sprague–Dawley rats were fed a high-fat diet for 4 weeks. The lipid source consisted of 20% optimised rapeseed oils with different quantities and qualities of micronutrients. The control group received traditional refined rapeseed oil. The experimental optimised oils all had a hypolipidaemic effect. In the group fed the highest levels of micronutrients, the reduction in plasma and hepatic triglycerides reached 25% and 17%, respectively, that of cholesterol 20% and 14%, respectively. In plasma, the ferric antioxidant capacity, superoxide dismutase, glutathione peroxidase and reduced glutathione significantly increased and lipid peroxidation decreased in parallel with the enhancement of micronutrients. The same trend was observed in the liver, except for glutathione peroxidase which was not affected by optimised oils. These results indicate that a regular intake of optimised rapeseed oils can help to improve lipid status and prevent oxidative stress, providing evidence that optimised oils could be a functional food with potentially important cardioprotective properties. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Cardiovascular diseases (CVDs) are the leading causes of disability and death in industrialized nations and much of the developing world. Over the past three decades it has become clear that the onset and progression of atherosclerosis, the pathological basis of CVD, result from a combination of abnormalities in lipoprotein metabolism, oxidative stress and chronic inflammation [1]. Many controlled intervention studies [2–5] have reported that rapeseed oil reduces serum total cholesterol (TC) and/or low-

夽 OPTIM’OILS “Valorisation of healthy lipidic micronutrients by optimising food processing of edible oils and fats” is a Specific Targeted Research Project supported by the thematic priority “Food Quality and Safety” of the European Commission 6th Framework Program – Contract no FOOD – CT-2006-36318 – www.optimoils.com – Contact: [email protected]. ∗ Corresponding author. Tel.: +39 06 49902574; fax: +39 06 49387149. E-mail address: [email protected] (S. Salvati). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.07.003

density lipoprotein cholesterol (LDL-C) when consumed in place of fats containing saturated fatty acids (SFA). The beneficial effects have been attributed to its low content of SFA and high level of n-3 polyunsaturated fatty acids (PUFAs). Rapeseeds also contain minor bioactive compounds such as tocopherol present in all isomeric structures: ␣, ␤, ␦ and ␥; Coenzyme Q (CoQ), phytosterols and phenols [6–10]. These bioactive compounds have a potent antioxidant activity, characterised by their ability to scavenge or neutralise reactive oxidant species directly. The best protection for animal cells may be obtained by a combination of antioxidants. The various bioavailable antioxidants may work in concert to upgrade the complex antioxidant network necessary to sustain cellular function [11]. Previous studies have shown that polyphenols cooperate with vitamins C and E [12] and ␤-sitosterol [13], and that ␣-tocopherol synergises with ␥-tocopherol [14] to raise antioxidant capacity higher than that provided by each separate compound. However, the industrial processes currently used in the production of edible oils (extraction and refining) are not optimally suited to the satisfactory preservation of these minor nutritional compounds. The present investigation is part of the European Union Project OPTIM’OILS (Valorisation of healthy lipidic micronutrients by opti-

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Table 1 Micronutrients in different rapeseed oils. mg/kg oil or ppm

RAP REF

RAP 1 DH-COOKP

RAP 2 EXT-HEX

RAP 3 TSE-BETH

Sterols Tocopherols Phospholipids Phenols (in eq., caffeic acid) CoQ9 + CoQ10

7913 614 0 81 85.4

7629 708 105 386 57.6

9003 750 428 352 269.5

13,511 1677 0 495 262.9

mising food processing of edible oils and fat), which aims to improve the processes currently used for seed oil production in order to increase the levels of micronutrients and to develop new healthy oils to be marketed in the European Union. The aim within the EU project of the present study was to evaluate in vivo whether rapeseed oils enriched in micronutrients on account of the different crushing and soft-refining procedures adopted in their preparation would favourably affect plasma lipid metabolism and the antioxidant defences. Diets containing 20% rapeseed oils as lipid source were administered to rats for 4 weeks. In rats, a high-fat diet (HFD) induces obesity, dyslipidaemia and hypertension and decreases antioxidant capacity [15,16]. The interaction among these factors plays an important role in the pathophysiology of CVD. Despite the consumption of an HFD, the micronutrient-enriched rapeseed oils as fat source have favourable effects on lipid levels and oxidative stress in plasma and liver. 2. Materials and methods 2.1. Optimised oils Rapeseed oils obtained by different technical procedures (optimised oils) were supplied by CREOL and ITERG (PessacFrance). The three main crushing procedures used to obtain higher levels of micronutrients were: (1) cooking and pressing after dehulling or flaking (DH-COOKP), (2) hexane extraction after cold pressing (EXT-HEX) and (3) ethanol extraction after twin-screw extrusion (TSE-BETH). A fraction of the rapeseed oil obtained by TSE-BETH was enriched with gum phospholipids (PL). The micronutrient composition of the different oils is shown in Table 1. Tocopherols and sterols were determined by ANIA (Valencia-Spain); the former were quantified by high-performance liquid chromatography coupled to photodiode array detection (HPLC/DAD), using the official NF ISO method 9936 while the latter were quantified by gas chromatography using the methodology based on the EN ISO 12228:1999 standard. Phospholipids were determined by HPLC according to Rombaut et al. [17] using FUSAGx (Gembloux-Belgium). Phenols, analysed by AgroParisTech (Massay-France), were quantified following NF ISO 9936. CoEQ was determined by reverse-phase high-performance liquid chromatog˜ et al. raphy with a mass detector as described by Rodriguez-Acuna [18]. 2.2. Animals and diets Sprague–Dawley rats weighing 150–170 g were obtained from Harlan-Nossan (Milano-Italy). The animals were housed in stainless steel cages (two rats per cage) at a controlled room temperature of 24 ◦ C, under a 12:12 light:dark cycle. After 1 week of acclimatisation, they were divided into 5 groups (n = 10 rats per group). They were allowed ad libitum access to food and tap water. All groups received the same basal synthetic diet containing 18% casein, 0.3% dl methionine, 39% rice starch, 15% sucrose, 3% fibre, 3.5% salt mixture (AIN-76), 1% vitamin mixture (AIN-76), 0.2% choline chloride, 20% fat. The lipid fraction was provided by different rapeseed oils. Experimental diets were administered for 4 weeks. Group 1

RAP 4 TSE-BETH + PL 16,059 1677 752 469 272.6

received the reference refined rapeseed oil (RAP REF) and was the control group; Group 2: oil obtained by cooking and pressing (DH-COOKP); Group 3: oil obtained by hexane extraction after extrusion (EXT-HEX); Group 4: oil obtained by ethanol extraction after extrusion (TSE-BETH); Group 5 oil obtained by TSE-BETH but enriched in gum phospholipids. The diet was prepared under vacuum and stored in the dark at 4 ◦ C. Animals were weighed twice each week and food intake was recorded weekly to monitor the growth rate of rats and the potential effect of diet on food consumption. 2.3. Tissue processing At the end of the experiment, fasting blood samples were collected in 0.2% EDTA from the left atrium of the heart of rats anaesthetised by a mixture of medetomidine and ketamine (1:1, v/v). Blood was centrifuged at 1500 × g for 10 min at 4 ◦ C and plasma was separated, aliquoted and stored at −80 ◦ C until analysis. Livers were immediately excised, weighed and divided into smaller pieces and stored at −80 ◦ C for lipid and enzymatic analysis. For histological examination, the right lobe was fixed in a buffer solution of 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) for routine histology and with Masson’s trichrome (MT) for collagen. Stained areas were viewed using an optical microscope at 40×. All animal experiments were performed according to European Community Council Directive 86/609/ECC and Italian legislation (DL 116/92) on animal experimentation. 2.4. Plasma and liver lipids Concentrations of TC, triglycerides (TG), LDL-C and high-density lipoprotein cholesterol (HDL-C) in plasma were determined by enzymatic colorimetric methods using commercial kits (BPCBiosed, Italy). The hepatic lipids were extracted using Folch’s method [19] and TC and TG concentrations were analysed by the same enzymatic kits used for the plasma analysis. 2.5. Lipid peroxidation Aliquots of liver were mixed with a solution of phosphate buffer (10 mM, pH 7.4) and potassium chloride (11.5 g/L), homogenised and centrifuged at 12,000 × g for 20 min. Supernatant fractions (liver homogenates) were collected to determine lipid peroxidation. The assay of thiobarbituric acid reactive substance (TBARS) was based on the method of Yagi [20]. Plasma or liver homogenates were mixed with trichloroacetic acid and centrifuged at 1500 × g for 10 min. Supernatant fractions were incubated with thiobarbituric acid and butylated hydroxytoluene solution at 80 ◦ C for 40 min. After cooling, the samples were centrifuged for 10 min (1500 × g) at 4 ◦ C. The supernatant absorbance was read at 535 nm. The concentration was calculated using an extinction coefficient of 1.56 × 105 M−1 cm−1 and expressed as ␮mol TBARS/mL plasma and ␮mol TBARS/g liver.

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Table 2 Effect of different optimised rapeseed oils on various plasma and hepatic lipid parameters. Group 1 refined oil Plasma Triglyceride (mg/dl) Total cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) HDL/LDL

17.6 46.9 39.7 7.2 5.5

Liver Triglyceride (mg/100 g) Total cholesterol (mg/100 g)

928 ± 12 54.9 ± 1.4

± ± ± ± ±

0.4 0.2 0.2 0.2 0.2

Group 2 DH-COOKP 16.1 42.0 35.1 6.0 5.8

± ± ± ± ±

Group 3 EXT-HEX

0.3a 0.4a 0.1a 0.1a 0.1

14.5 39.3 31.9 6.0 5.4

845 ± 102 51.9 ± 2.3

± ± ± ± ±

0.4a,b 0.8a 0.1a,b 0.0a 0.3

858 ± 25 52.9 ± 2.5

Group 4 TSE-BETH 13.6 37.9 31.9 5.2 6.1

± ± ± ± ±

0.1a,b,c 0.4a,b,c 0.0a,b 0.2a,b,c 0.3a,b,c

810 ± 16 49.5 ± 1.6

Group 5 TSE-BETH + PL 13.3 37.5 31.9 4.0 7.9

± ± ± ± ±

0.5a,b,c 0.3a,b,c 0.1a,b 0.1a,b,c,d 0.3a,b,c,d

769 ± 19e 47.4 ± 2.2e

Group 1: totally refined rapeseed oil; Group 2: cooking and pressing after dehulling or flaking (DH-COOKP); Group 3: hexane extraction after cold pressing (EXT-HEX); Group 4: ethanol extraction after twin-screw extrusion (TSE-BETH); Group 5: fraction of the rapeseed oil obtained by TSE-BETH enriched with gum phospholipids (PL). Values represent the mean ± SD of seven samples for plasma and at least three samples for liver. a p < 0.001 vs. Group 1. b p < 0.001 vs. Group 2. c p < 0.001 vs. Group 3. d p < 0.001 vs. Group 4. e p < 0.05 vs. Group 5.

2.6. Total plasma antioxidant activity

2.9. Statistical analysis

Plasma antioxidant activity was analysed by the ferric-reducing antioxidant potential (FRAP) method according to Benzie and Strain [21] using a DU 800 spectrophotometer (Beckman, CA, USA) equipped with a thermostatically controlled cell-holder. The method is based on the reduction of the Fe3+ -2,4,6-tripyridyls-triazine complex to ferrous Fe2+ at low pH. Briefly, 800 ␮L of FRAP reagent prepared daily was mixed with 50 ␮L of diluted sample and the absorbance was recorded at 595 nm after 0 and 30 min of incubation at 37 ◦ C. The change in absorbance was calculated for each sample and compared with a FeSO4 ·7H2 O standard solution tested in parallel. Values were expressed as ␮mol/L of Fe2+ .

All data are presented as the mean ± SD. The data were evaluated by one-way ANOVA. Post hoc Tukey’s test was performed to evaluate differences between groups. Correlations (r) between different CVD risk factors were performed using Pearson’s product-moment correlation coefficient. SPSS was used for the statistical analyses. Statistical significance was considered at p < 0.05.

2.7. Enzymatic antioxidant activity Superoxide dismutase (SOD-EC 1.15.1.1) and glutathione peroxidase (GPX-EC. 1.11.1.9) activities were evaluated in plasma and liver by commercial kits (Cayman, MI, USA) following the manufacturer’s instructions.

3. Results 3.1. Food intake and body weight No significant differences were observed in food intake or in body weight gain among all groups. The average body weight was 176.0 ± 4.1, 170.4 ± 2.3, 175.8 ± 5.1, 174.0 ± 3.7, 171.0 ± 3.4 g at the beginning of the experiment and 306.6 ± 14.4, 308.8 ± 17.4, 323.2 ± 16.0, 323.8 ± 22.5, 317.0 ± 7.5 g after 4 weeks of dietary treatment for Groups 1–5, respectively. There were no significant differences in rat liver weight among groups (12.5 ± 1.8, 12.4 ± 1.9, 11.4 ± 2.3, 13.5 ± 1.7, 12.5 ± 0.4 g for Groups 1–5, respectively).

2.8. Glutathione assay

3.2. Plasma and hepatic lipids

Plasma and hepatic reduced glutathione (GSH) contents were measured using a commercial kit (Cayman, MI, USA). This method is based on a chemical reaction that involves the formation of a chromophoric thione, which has maximum absorbance at 405 nm.

Values of lipid parameters obtained after feeding rats the experimental diets for 4 weeks are summarised in Table 2. Plasma TG and TC levels were significantly lower in the optimised oil groups than in the refined rapeseed oil group and the reduction was in parallel with the increase in micronutrient levels in the oils. PL enrichment

Table 3 Effect of different optimised rapeseed oils on plasma FRAP and plasma and hepatic TBARS.

Plasma FRAP (␮mol/L) TBARS (␮mol/mL) Liver TBARS (␮mol/g)

Group 1 refined oil

Group 2 DH-COOKP

Group 3 EXT-HEX

Group 4 TSE-BETH

Group 5 TSE-BETH + PL

66.41 ± 3.2 0.97 ± 0.00

75.90 ± 3.6a 0.86 ± 0.00a

81.20 ± 6.3a 0.76 ± 0.00a,b

84.32 ± 3.0a,b 0.64 ± 0.00a,b,c

86.30 ± 2.5a,b 0.63 ± 0.00a,b,c

5.97 ± 0.10a,b

5.03 ± 0.08a,b,c

7.04 ± 0.06

6.91 ± 0.06

4.23 ± 0.05a,b,c,d

Group1: totally refined rapeseed oil; Group 2: cooking and pressing after dehulling or flaking (DH-COOKP); Group 3: hexane extraction after cold pressing (EXT-HEX); Group 4: ethanol extraction after twin-screw extrusion (TSE-BETH); Group 5: fraction of the rapeseed oil obtained by TSE-BETH enriched with gum phospholipids (PL). Values represent the mean ± SD of seven samples for plasma and at least three samples for liver. a p < 0.001 vs. Group 1. b p < 0.001 vs. Group 2. c p < 0.001 vs. Group 3. d p < 0.001 vs. Group 4.

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did not affect TG and TC content since no significant differences were observed between Groups 4 and 5. The plasma TC reduction affected lipoprotein levels. In optimised oil groups both LDL-C and HDL-C levels were lower than controls. LDL-C reduction was combined with the increase in micronutrients in the oils and the largest reduction (∼45%) was in Group 5. Conversely, HDL-C levels were less affected by micronutrient levels and PL addition since no significant differences were observed in HDL-C levels among Groups 3–5, in which the levels of bioactive compounds were different. However, the HDL-C/LDL-C ratio, calculated to evaluate the atherosclerosis risk, was increased in groups with the highest micronutrient levels, namely 4 and 5, indicating a reduced risk of atherosclerosis. The hepatic TC and TG levels were also lower in rats fed optimised oils than in controls, although the reduction was significant (p < 0.01 and <0.05, respectively) only in the group that received PL-enriched oil, namely Group 5, as shown in Table 2. 3.3. Biomarkers of oxidative stress The FRAP values, an indicator of total antioxidant defence, and plasma TBARS levels, biomarkers of lipid peroxidation, were significantly influenced by feeding with micronutrient-enriched oils. The experimental diets caused an increase in plasma ferric antioxidant capacity with a concomitant decrease in plasma TBARS values as shown in Table 3. The plasma TBARS and FRAP values reached the highest reduction (∼35%) and increase (∼30%), respectively, in Group 5 and the regression coefficient test indicated a significant correlation between them (r = −0.97; p < 0.001). The hepatic TBARS content decreased with a trend similar to plasma TBARS and the greatest reduction (∼40%) was again observed in Group 5. 3.4. Plasma and hepatic antioxidant status To study the effect of optimised oils on plasma and hepatic antioxidant status of rats fed high-fat diets antioxidant defence system capabilities were evaluated. As shown in Fig. 1(A–C) both plasma enzymatic activity and GSH levels were significantly higher in all groups fed optimised rapeseed oils than in controls. Similar to the plasma lipid profile, the SOD activity (Fig. 1A) and GSH levels (Fig. 1C) increased with the level of micronutrient enhancement in oils and the highest activity was detected in optimised rapeseed oils with the highest content of bioactive compounds. However, the increase induced by PL addition (Group 5 vs. Group 4) was statistically significant (p < 0.01) only in GSH content. No significant differences in plasma GPX activity were observed among different optimised oil groups (Fig. 1B). Hepatic enzymatic and non-enzymatic antioxidant activities showed the same trend as plasma antioxidant defences, although the differences in optimised groups were less marked than in controls. SOD activity and GSH levels increased in all optimised groups, but the increase was significantly higher (p < 0.01) in Groups 3–5 than in controls (Fig. 1D and F). Conversely, no differences were observed in the GPX activity among experimental groups as presented in Fig. 1E. 3.5. Liver histology As shown in Fig. 2(A and B), the livers of rats fed an HFD for 4 weeks showed presence of a large number of circular lipid droplets. These lipid inclusions were reduced in both size and number in the liver of the optimised groups (Fig. 2C and D). No evident differences in hepatic morphology were observed among optimised groups. These results seem to correspond to the hepatic TG and TC profile shown in Table 2. No signs of inflammation or collagen deposits

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were observed in any experimental groups as shown in Fig. 2(B and D).

4. Discussion Over the past two decades there has been an expansion in the production of low-erucic acid rapeseed oil, which has the potential to improve consumers’ health on account of its high content of oleic acid, and to be an important source of n-3 fatty acids (␣linolenic acid). Other authors have shown that rapeseed oil reduces serum TC and/or LDL-C when fed in place of higher SFA-containing fats in controlled intervention studies [2,22,5]. Our results were in agreement with these findings, since totally refined rapeseed oil induced hypolipidaemic effects in rats fed an HFD. In general, high-fat diets significantly increase TC levels in the serum as compared with the normal control diet in rats [23]. In the present study when traditional refined rapeseed oils were administered as lipid source TG and TC levels were significantly reduced, by 70% and 35%, respectively, compared with rats fed a chow diet (data not shown). These hypolipidaemic effects are enhanced in rats fed rapeseed oils obtained by crushing and refining procedures that prevent the loss of micronutrients. In effect, micronutrient-enriched rapeseed oils further reduced plasma lipid content and the decrease was accompanied by a reduction in both LDL-C and HDL-C. The LDL-C levels were affected by the content of micronutrients and PL addition in the oils while HDL-C levels were less influenced by micronutrient levels, no differences being observed among Groups 3–5. The molecular mechanisms responsible for the observed lipidlowering effects of optimised rapeseed oils could be due to one or more effects of its active components on potential sites of actions leading to decreased lipid biosynthesis and/or enhanced cholesterol elimination. The observed hypolipidaemic effect of a diet containing refined rapeseed oil could be due to a high n-3 PUFA content, whose effects in decreasing lipid markers of CVD risks are well known [24]. Rats fed a high-fat diet rich in linolenic acid showed a decrease in plasma TG levels and a suppression of hepatic FAS activity [25]. On the other hand, rapeseed oil rich in n-3 unsaturated fatty acids increased hepatic LDL receptor activity, decreased LDL production and increased LDL clearance [26]. The further reduction in TC and LDL-C levels in optimised groups could be explained by the higher levels of phytosterols and phenols in the oils. Recently, Micallef and Garg [27] have shown the efficacy on plasma lipid profile of concomitant supplementation of phytosterols and n-3 PUFAs in subjects with hyperlipidaemia. The authors provided evidence of a synergistic reduction in total and LDL-C and complementary effects on TG and HDL-C concentration by concomitant dietary supplementation of n-3 fatty acids and phytosterols. Phytosterols can exert their effect of decreasing TC in plasma either by the down-regulation of acyl CoA: cholesterol acyltransferase activity (ACAT) or hydroxyl-3-methyl-glutaryl-CoA reductase activity (HMGR) or by increasing CH excretion [28]. It has also been shown that the consumption of phytosterols reduces LDL-C concentration by increasing LDL receptor expression [29]. The hypolipidaemic effect of phytosterols could be enhanced by the phenol content in optimised oils. Woo et al. [30] showed a hypolipidaemic effect in rats fed a high-fat diet supplemented with phenols: hyperlipidaemia was reduced via a decrease in HMG-CoA reductase and ACAT activities and an increase in lipoprotein lipase (LPL) activity. The high content of antioxidant compounds such as tocopherols, CoQ and phenols present in optimised oils could beneficially influence CVD markers, increasing the total antioxidant activity in plasma. Some studies (2–5) support the oxidative theory of

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Fig. 1. The effect of feeding optimised oils on superoxide dismutase (A plasma SOD; D liver SOD); glutathione peroxidase (B plasma GPX; E liver GPX) and glutathione (C plasma GSH; E liver GSH) Group1: total refined rapeseed oil; Group 2: cooking and pressing after dehulling or flaking (DH-COOKP); Group 3: hexane extraction after cold pressing (EXT-HEX); Group 4: ethanol extraction after twin-screw extrusion (TSE-BETH); Group 5: a fraction of the rapeseed oil obtained by TSE-BETH was enriched with gum phospholipids (PL). Values represent the mean ± SD of seven samples for plasma and at least three samples for liver. a p < 0.05 vs. Group 1; b p < 0.001 vs. Group 2; c p < 0.001 vs. Group 3 and d p < 0.001 vs. Group 4.

atherosclerosis, which is why dietary antioxidants have attracted considerable attention as agents for the prevention and treatment of [31] CVD progression [32]. Our study shows that optimised rapeseed oils strongly reduce plasma TBARS levels and the decrease was negatively correlated with ferric plasma antioxidant activity. Lipid peroxidation is regarded as one of the basic mechanisms of cellular damage caused by reactive oxygen species. Free radical-mediated lipid peroxidation leads to an accumulation of lipid peroxidation products, which in turn propagate lipid peroxidation and cause serious damage to the membrane and changes in intracellular enzymes, resulting in loss of cell function. Dietary antioxidants offset the oxidative stress indirectly by enhancing the natural defences of cells and/or directly by scavenging the free radical species. The endogenous antioxidant defence has both enzymatic and non-enzymatic components that prevent radical formation, remove radicals before damage can occur, repair oxidative damage and eliminate damaged molecules. This endogenous antioxidant defence consists of components such as GSH and various antioxidant enzymes such as SOD and GPX. GSH is one of the most common

biological antioxidants and its function includes the removal of free radicals such as superoxide anions and alkoxy radicals and acting as a substrate for GPX and glutathione reductase. SOD is one of the major enzymes of the endogenous antioxidant defence system that catalyses the dismutation of superoxide anions, while GPX catalyses the reduction of H2 O2 and hydroperoxides to nontoxic products. It is known that an HFD significantly depletes the antioxidant system on account of increased utilisation to combat the oxidative stress in hyperlipidaemic rats [33]. The significant increase in GSH levels and SOD activity observed in plasma when the micronutrient content of oils is enhanced probably indicates that optimised oils can either increase the biosynthesis of GSH and the expression and/or activity of SOD, or reduce the extent of oxidative stress, leading to less GSH degradation and SOD depletion, or both effects may be present. GPX activity was higher in optimised groups compared with controls, though no significant differences were observed among the former groups. In spite of the low plasma TG and TC levels, the histological examination showed a fat deposition in the rat livers in all experimental groups. However, lipid metabolism is regulated mainly by

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Fig. 2. Histological appearance of liver sections from total refined rapeseed oil (A and B) and from optimised oil (C and D). Left-hand panels show sections stained with haematoxylin and eosin. Right-hand panels show sections stained with trichrome.

the liver and when dietary lipids exceed the capacity of hepatic cells to oxidise fatty acids, as in animals fed an HFD, the result is large deposits of TG in vacuoles, leading to steatosis. Micronutrientenriched rapeseed oils improve both the biochemical markers and histological evidence of hepatic lipid accumulation, indicating that the optimised oils attenuate the steatosis induced by an HFD. Although the steatotic liver is more vulnerable to oxidative stress, TBARS and GSH levels, and SOD and GPX activities in groups fed refined rapeseed oil showed values within the range of rats fed a standard chow diet (data not shown). A consistent decrease in TBARS and an increase in antioxidant markers were induced by feeding with optimised oils (Groups 3–5). These results are similar to those observed in plasma, but the variations in hepatic endogenous antioxidants were less pronounced and the increase in GPX activity was not statistically significant. The minor histopathological damage observed in livers from rats fed optimised oils could be due to the high content of antioxidants that protect against fibrosis by scavenging free radicals [34]. Poliphenols can also act with an alternative mechanism as they decrease the formation of inflammatory and fibrogenic mediators by preventing the activation of Kupffer cells [35]. The increase in endogenous antioxidant defences could be due not only to the quantity of each antioxidant present, but also to the combination of different antioxidants in optimised oils. Antioxidants do not work in isolation; rather they are part of an interlinking set of redox antioxidant cycles [36], which has been termed the “antioxidant network”. Antioxidants with different chemical properties may recharge each other in an integrated manner, and may be needed for the proper protection of all compartments in a cell or an organism. Such interactions have been demonstrated in vitro for ␣-tocopherol, ␣-tocotrienol, vitamin C, lipoic acid and thiols by Packer et al. [37]. Flavonoids have also been shown to act in synergy with vitamin C and ␣-tocopherol and other bioactive plant compounds to enhance LDL resistance to oxidation [12].

In conclusion, the present results suggest that micronutrientenriched oils favourably affect cardiovascular risk factors in rats fed an HFD. Clinical studies in humans will be required to confirm the beneficial effects of optimised rapeseed oils in subjects at increased risk of CVD. Acknowledgments The authors wish to thank Mr. Agostino Eusepi for his assistance in animal care and management. References [1] Hansson GK. Inflammation, atherosclerosis, coronary artery disease. N Engl J Med 2005;352:1685–95. [2] Vega-López S, Ausman LM, Jalbert SM, Erkkilä AT, Lichtenstein AH. Palm and partially hydrogenated soybean oils adversely alter lipoprotein profiles compared with soybean and canola oils in moderately hyperlipidemic subjects. Am J Clin Nutr 2006;84:54–62. [3] McDonald BE, Gerrard JM, Bruce VM, Corner EJ. Comparison of the effect of canola oil and sunflower oil on plasma lipids and lipoproteins and on in vivo thromboxane A2 and prostacyclin production in healthy young men. Am J Clin Nutr 1989;50:1382–8. [4] Seppänen-Laakso T, Vanhannen H, Laakso I, Kohtamaki H, Viikari J. Replacement of margarine on bread by rapeseed and olive oils: effects on plasma fatty acid composition and serum cholesterol. Ann Nutr Metab 1993;37:161– 74. [5] Kratz M, Cullen P, Kannenberg F, et al. Effects of dietary fatty acids on the composition and oxidability of low-density lipoprotein. Eur J Clin Nutr 2002;56:72–81. ˜ RR, Brenne E, Lacoste F. Determination of coenzyme Q10 and Q9 in [6] Acuna vegetable oils. J Agric Food Chem 2008;56:6241–5. [7] Marwede V, Shierholt A, Moellers C, Becker HC. Genotype environment interactions and heritability of tocopherol contents in canola. Crop Sci 2004;44:728–31. [8] Goffman FD, Velasco L, Becker HC. Tocopherols accumulation in developing seeds and pods of rapeseed (Brassica napus L.). Fett/Lipid 1999;101:400– 3. [9] Hamama AA, Bhardwaj HL, Starner DE. Genotype and growing location effects on phytosterols in canola oil. J Am Oil Chem Soc 2003;80:1121–6.

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