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Effects of fish oil and vitamin E on the antioxidant defense system in diet-induced hypercholesterolemic rabbits
Prostaglandins & other Lipid Mediators 66 (2001) 99 –108
Effects of fish oil and vitamin E on the antioxidant defense system in diet-induced hypercholesterolemic rabbits Hsiu-Ching Hsu, Yuan-Teh Lee, Ming-Fong Chen* Department of Internal Medicine (Cardiology), National Taiwan University Hospital 7, Chung-Shan South Road Taipei, 10002, Taiwan Received 8 January 2001; received in revised form 8 February 2001; accepted 25 April 2001
Abstract This study was designed to investigate the effects of fish oil and vitamin E on the antioxidant defense system in hypercholesterolemic rabbits. A high fat and cholesterol diet, with or without supplement by fish oil and/or a vitamin E supplement, was fed to rabbits for 6 weeks. Compared to the reference diet of regular laboratory rabbit chow, a high fat and cholesterol-enriched diet increased atheroma formation, plasma lipid and peroxide levels, decreased blood glutathione levels, and reduced plasma glutathione reductase, glutathione peroxidase, and catalase activities. Fish oil supplementation significantly reduced atheroma and increased glutathione reductase and glutathione peroxidase activities and blood glutathione levels, but increased plasma lipid peroxide levels. Vitamin E supplementation of the fish oil diet enhanced the beneficial effects by increasing glutathione reductase activity and decreasing peroxide levels. These results indicate that a high fat and cholesterol diet attenuates blood glutathione levels and plasma antioxidant enzyme activities, which may account for some of its atherogenic properties. Consumption of fish oil enhances antioxidative defenses against the oxidative stress imposed by hypercholesterolemia, and vitamin E further enhances these beneficial effects. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hypercholesterolemia; Fish oil; Vitamin E; Glutathione; Rabbits
* Corresponding author. Tel.: ⫹886-2-23123456; fax: ⫹886-2-2395-9922. E-mail address: [email protected] (M.-F. Chen). 0090-6980/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 1 ) 0 0 1 4 6 - 0
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1. Introduction Hypercholesterolemia is reported to be one of the most important coronary risk factors [1,2] and increased serum cholesterol concentrations result in the development of atherosclerosis [3]. Many studies have shown that the long chain n-3 polyunsaturated fatty acids contained in fish oil may decrease the risk of cardiovascular diseases and several other chronic illnesses [4]. Experimental studies have indicated that fish oil have multifaceted actions such as prevention of arrhythmias, antithrombotic actions, inhibiting cellular growth factors and monocytes migration, hypolipidemic effect and promotion of the synthesis of nitric oxide [5]. However, because of its high concentrations of unsaturation, fish oil can be rapidly oxidized to lipid peroxides. It has been reported that plasma and tissue levels of lipid peroxides increase in fish oil-fed animals [6 – 8] and humans [9,10]. Lipid peroxidation is considered to be a pivotal mechanism of cell membrane destruction and cell damage, and has been suggested to be associated with the initiation and progression of atherosclerosis [11]. Antioxidants and antioxidative enzymes protect cells and tissues from oxidative injury [12]. Vitamin E, with antioxidative properties, is suggested to reduce the peroxidative damage caused by fish oil-related lipid peroxidation and thereby enhance the beneficial effects of fish oil, and fish oil supplementation is therefore usually combined with vitamin E as an antioxidant. However, little is known about other advantages, drawbacks, or mechanisms of vitamin E in addition to its chain-breaking antioxidative properties [13]. The goals of the present study were to examine the effect of cholesterol on the glutathione and antioxidant enzyme activities and to determine how fish oil and vitamin E supplementation alters the antioxidant defense system in rabbits rendered hypercholesterolemic with a high fat and cholesterol diet.
2. Materials and methods 2.1. Animals and diets Male New Zealand white rabbits weighing 2.5–3 Kg were studied. They were randomly divided into 5 groups of 8, each of which was fed a different diet. The reference rabbits (Ref) were fed regular laboratory rabbit chow containing protein 16.2%, fat 6.5%, crude fiber 14.0%, ash 7.3%, nitrogen-free extract 50%, salt and vitamin mix (Purina Inc., St. Louis, MO, USA) for the entire study period. To the diets of the other 4 study groups were added 1% (wt %) cholesterol (Wako Co. Osaka, Japan); two of these groups (C and CE) received 10% (wt %) coconut oil (Yeali Co., Taipei, Taiwan), while the other two (CF and CFE) supplemented with 10% (wt %) fish oil (99.8% refined edible fish oil, Tama Co. Tokyo, Japan). Fatty acid composition of the diets was the same as a previous study [14]. The CE and CFE groups were also given ␣-tocopherol (synthetic form containing equimolar stereoisomers; Merck Ltd. Darmstadt, Germany) at a dose of 450 mg/kg diet. Salt and vitamin mix (ICN Biomedicals, INC. Aurora, Ohio, USA) were added to the diets to meet the American Institute of Nutrition standards [15]. All rabbits were housed in individual cages with raised screen bottoms in a room maintained at a constant temperature and kept on a 12:12 h
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light-dark cycle. Food and water were given daily and any unused food discarded. The study conformed to the guidelines for the care and use of animals approved by the National Taiwan University Medical Center. 2.2. Study protocol Eight rabbits in each group were fed the study diets for 6 weeks. At the end of the study period, 10 ml of venous blood from an ear vein of each animal was drawn into a tube containing 3.4 mM ethylenediaminetetraacetic acid (EDTA) for the analysis of plasma triglyceride, cholesterol, vitamin E, and lipid peroxides concentrations and antioxidative enzyme activities. The reduced and oxidized glutathione contents were also measured in whole blood. Then, the rabbits were anesthetized and killed for delineation of atherosclerotic lesions in the ascending aorta. 2.3. Biochemical determinations 2.3.1. Plasma concentrations of cholesterol, triglyceride, vitamin E and thiobarbituric acid reactive substances (TBARS) For analysis of cholesterol, triglyceride, vitamin E and TBARS, plasma was recovered by centrifugation at 4°C, 1500 g for 15 min. Butylated hydroxytoluene (2 mM) was added to the plasma to prevent further auto-oxidation for TBARS determination. Plasma cholesterol and triglyceride concentrations were determined by automated enzymatic methods [16,17]. Plasma vitamin E concentrations were measured by high performance liquid chromatography with fluorescence detection (excitation at 205 nm, emission at 340 nm) [18]. The plasma TBARS content was used to represent lipid peroxides levels and measured by fluorometric assay (excitation at 515 nm, emission at 552 nm) using 1,1,3,3-tetraethoxypropane as a standard, as described by Wasowicz et al. [19,20]. 2.3.2. Determination of reduced (GSH) and oxidized (GSSG) glutathione concentrations in blood Blood total glutathione (GSH⫹GSSG) concentrations were determined as described by Murphy et al. [21]. Briefly, the blood cells were lysised by 0.01 M phosphate buffer, and subsequently the GSSG was reduced by glutathione reductase, then the GSH reacted with 5,5⬘-dithiobis-(2-nitrobenzoic acid) to form chromophoric products. The absorbance was measured at 410 nm and the concentration calculated from the standard curve using a known concentration of GSSG or GSH. For the GSSG assay, a final concentration of 0.02 N N-ethylmaleimide was added to the blood lysate to remove GSH by forming a stable complex to prevent its participation in assay. Then, the lysate was extracted by at least 10 volumes of diethyl ether to ensure complete removal of the non-reacted sulfhydryl reagent, which can inhibit glutathione reductase reactivity in the assay. After the extraction process, the solution was added to the assay mixture for GSSG determination. GSH concentrations were calculated by subtracting the GSSG concentrations from the GSSG⫹GSH concentrations.
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2.3.3. Assessment of glutathione reductase, glutathione peroxidase and catalase enzyme activities The measurements of glutathione peroxidase and glutathione reductase activities were based on the consumption of nicotinamide adenine dinucleotide phosphate following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively [22]. The assay for catalase is based on it’s peroxidative activity as described by Johansson and Borg [23]. These procedures were performed in an Epose 5060 aotoanalyzer (Eppendorf Corp, Hamburg, Germany). All biochemical measurements were performed in triplicate and the mean values were recorded. 2.3.4. Tissue harvest for the delineation of atherosclerotic lesions The rabbits were anesthetized and killed for delineation of atherosclerotic lesions [24]. A midline thoracotomy was performed, and the ascending aorta was quickly removed from the aortic valve cups to the Arifice of the left subclavian artery. The excised aorta was fixed in neutral 10% formaldehyde for 24 h, rinsed in 70% ethanol, and immersed in Herxeheimer’s solution (5% Sudan IV in ethanol and acetate) at room temperature 15 min. The tissues were transferred to 80% ethanol for 20 min and washed in running water for 1 h. The percentage of aortic intimal infiltrated by lipids was delineated by planimetry from the distribution of sudanophilia. 2.4. Statistics All data are expressed as the means ⫾ SDs. Differences between groups were assessed by analysis of variance followed by a Tukey’s test. Statistical analyses were performed with SAS (version 6.011; SAS Institute Inc, Cary, NC). P ⬍ 0.05 was considered statistically significant.
3. Results 3.1. Plasma lipid and antioxidant concentrations The body weight gains of all 5 groups were not significant differences. Table 1 shows that plasma triglyceride and cholesterol concentrations increased markedly in all four study groups treated with diets containing high fat and cholesterol, and that supplementation with fish oil, vitamin E, or fish oil plus vitamin E did not affect this result. Compared to the reference group, rabbits treated with high fat and cholesterol not only had increased plasma lipid concentrations, but also had increased plasma vitamin E concentrations and smaller vitamin E/cholesterol ⫹ triglyceride ratios. Compared to the C group, plasma vitamin E concentrations were markedly increased in the CE group and slightly decreased in the CF group; the ratios were also increased or decreased in the CE or CF groups, respectively. However, in the CFE group, these values were higher than those in the CF group. As shown in Table 2, rabbits fed a diet containing high fat and cholesterol had decreased
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Table 1 Plasma cholesterol (CHOL), triglyceride (TG) and vitamin (VIT) E concentrations in rabbits Group
CHOL (mg/dL)
TG (mg/dL)
Vit E (mol/L)
Vit E/CHOL⫹TG (nmole/mg)
Ref C CE CF CFE
57 ⫾ 10a 1423 ⫾ 300b 1323 ⫾ 405b 1291 ⫾ 353b 1221 ⫾ 362b
54 ⫾ 9.2a 193 ⫾ 89b 186 ⫾ 84b 201 ⫾ 60b 221 ⫾ 76b
8.4 ⫾ 2.0a 22.8 ⫾ 7.7b 106.4 ⫾ 28.3c 16.4 ⫾ 3.8b,d 39.5 ⫾ 8.0b,e
7.5 ⫾ 3.2a 4.3 ⫾ 1.6b 8.6 ⫾ 3.2b 2.1 ⫾ 0.4c 3.2 ⫾ 0.4d
All data are the means ⫾ SDs, n ⫽ 8. Within a column, values with the same superscript letters are not significantly different from each other. Ref: reference group fed a regular laboratory chow; C: high fat and cholesterol-fed; CE: high fat and cholesterol ⫹ vitamin E-fed; CF: high cholesterol ⫹ fish oil-fed, CFE: high cholesterol ⫹ fish oil ⫹ vitamin E-fed group.
blood GSH concentrations, but the GSSG concentrations were not different significantly from the reference group. However, the GSSG/GSH ratios were markedly increased. The CE group had higher blood GSH and GSSG concentrations but had lower GSSG/GSH ratios than the C group. Compared to the high fat and cholesterol-treated group, fish oil supplementation increased the GSH and GSSG concentrations, but the GSSG/GSH ratios were not significantly different. The CFE group did not show any synergistic effect of vitamin E and fish oil on the increase in glutathione concentrations. 3.2. Plasma antioxidative enzyme activities Table 3 shows that glutathione reductase, glutathione peroxidase, and catalase activities decreased significantly in rabbits fed high fat and cholesterol. The same effect was seen in the CE group. The CF group had higher glutathione reductase and glutathione peroxidase activities than the C group; no difference was seen in catalase activity. The CFE group showed a synergistic effect of fish oil and vitamin E on glutathione reductase activity, but not on glutathione peroxidase or catalase activity.
Table 2 Blood reduced (GSH) and oxidized (GSSG) glutathione concentrations in rabbits Group
GSH (mg/L)
Ref C CE CF CFE
313.8 ⫾ 52.1 67.4 ⫾ 28.2b 221.2 ⫾ 64.5c 147.4 ⫾ 50.7d 186.5 ⫾ 40.7c,d a
GSSG (mg/L)
GSSG/GSH (%)
5.0 ⫾ 2.9 5.1 ⫾ 3.5a 10.8 ⫾ 3.2b 16.1 ⫾ 5.3b 15.1 ⫾ 4.6b
1.6 ⫾ 0.5a 7.5 ⫾ 3.3b 4.9 ⫾ 2.4c 9.2 ⫾ 4.2b 8.1 ⫾ 3.1b
a
All data are the means ⫾ SDs, n ⫽ 8. Within a column, values with the same superscript letters are not significantly different from each other. Ref: reference group fed a regular laboratory chow; C: high fat and cholesterol-fed; CE: high fat and cholesterol ⫹ vitamin E-fed; CF: high cholesterol ⫹ fish oil-fed, CFE: high cholesterol ⫹ fish oil ⫹ vitamin E-fed group.
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Table 3 Plasma enzyme activities of glutathione reductase (GR), glutathione peroxidase (GP) and catalase (CA) in rabbits Group
GR (U/L)
Ref C CE CF CFE
1007 ⫾ 128 341 ⫾ 168b 320 ⫾ 153b 972 ⫾ 207a,c 1363 ⫾ 388a,d a
GP (kU/L)
CA (kU/L)
20.6 ⫾ 3.3 2.8 ⫾ 1.0b 3.1 ⫾ 1.5b 5.5 ⫾ 1.3c 4.3 ⫾ 1.9b,c
29.6 ⫾ 4.2a 4.3 ⫾ 2.6b 3.9 ⫾ 2.6b 5.1 ⫾ 3.6b 4.7 ⫾ 2.6b
a
All data are the means ⫾ SDs, n ⫽ 8. Within a column, values with the same superscript letter are not significantly different from each other. Ref: reference group fed a regular laboratory chow; C: high fat and cholesterol-fed; CE: high fat and cholesterol ⫹ vitamin E-fed; CF: high cholesterol ⫹ fish oil-fed, CFE: high cholesterol ⫹ fish oil ⫹ vitamin E-fed group.
3.3. Plasma lipid peroxides As shown in Fig. 1, high fat and cholesterol-fed rabbits had elevated plasma TBARS concentrations. Vitamin E added to the high fat and cholesterol diet did not significantly change this effect. The CF group had increased plasma TBARS concentrations; this effect was significantly reduced in the CFE group. 3.4. Atherosclerotic lesions in ascending aorta Fig. 2 shows that high fat and cholesterol-fed rabbits had marked atheroma formation compared to the reference group. Addition of vitamin E alone to the high fat and cholesterol
Fig. 1. Plasma thiobarbituric acid-reactive substances (TBARS) concentrations in reference (Ref) rabbits or in rabbits treated with high fat and cholesterol diet (C), with high fat, high cholesterol and vitamin E diet (CE), with high cholesterol and fish oil diet (CF), or with high cholesterol, fish oil and vitamin E (CFE). The results are presented as the means ⫾ SDs (n ⫽ 8 for each group). Values with the superscript letters are: a ⬍ b ⬍ c ⬍ d.
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Fig. 2. Atherosclerotic lesion in the ascending aorta of rabbits. Rabbits were fed a regular laboratory chow (Ref) or a high fat and cholesterol diet (C), a high fat, high cholesterol and vitamin E diet (CE), a high cholesterol and fish oil diet (CF), or a high cholesterol, fish oil and vitamin E (CFE). The results are presented as the means ⫾ SDs (n ⫽ 8 for each group). Values with the superscript letters are: a ⬍ b ⬍ c ⬍ d.
diet did not significantly decrease the atherosclerotic lesions. Fish oil supplementation attenuated this effect, and addition of vitamin E had synergistic effect in decreasing atheroma formation.
4. Discussion It has been reported that hypercholesterolemia imposes free radical-mediated peroxidation, which initiates and potentiates atherosclerosis [3,25–28]. Many studies have indicated that elevated plasma cholesterol concentrations may modify the biochemical properties of blood components and the arterial intima, thus enhancing atherogenesis [29,30]. The present study shows that high cholesterol and fat feeding increased plasma TBARS concentration and paradoxically increased plasma vitamin E concentrations by almost three-fold. We speculated that the increased vitamin E was released from tissue in order to protect the increased plasma lipid against oxidation. However, the vitamin E/triglyceride⫹cholesterol ratios, which are indicator of lipid protection [31], were decreased. These observations are consistent with the atherogenic property of cholesterol. The well known biochemical functions of glutathione are in maintaining protein sulfhydryl groups and in detoxifying substances, including xenobiotics, metals, hydrogen peroxide, and oxygen radicals [32–34]. The antioxidant defense system counteracts the toxic activity of free radicals and peroxides [35]. We speculated that cholesterol attenuates blood glutathione concentrations and plasma antioxidant enzyme activities and that this may contribute to its atherogenic property. The linkage between free radical production and the consumption of antioxidative enzyme activity has not yet been established. However, it has been suggested that increasing the antioxidant capacity might be useful in preventing peroxidation and cellular damage [36]. The present study showed beneficial effects of fish oil supplementation on blood glutathione
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concentrations and plasma glutathione reductase and glutathione peroxidase activities in hypercholesterolemic rabbits. Moreover, the ascending aorta atheroma was markedly reduced by fish oil supplementation. These results may indicate that fish oil supplementation attenuated the oxidative stress produced by hypercholesterolemia [37]. The fish oil/cholesterol and the fish oil/cholesterol/vitamin E-treated groups showed similar responses of antioxidant enzyme to oxidative stress. This effect was not seen in rabbits fed high fat and cholesterol alone or plus vitamin E. Based on these results, it was excluded that the increases in antioxidant enzyme activities were responses to the high plasma lipid peroxides. The fact that the hypotriglyceridemic effect of fish oil was not seen in the present study indicates that lowering of triglyceride concentrations is not always effective in all hyperlipidemias; however, the enhancement of antioxidant defense activities contributes to its anti-atherogenic property. Vitamin E supplementation of fish oil-treated rabbits increased plasma glutathione reductase activity and decreased plasma TBARS significantly. These results indicate that the effects of vitamin E are partly mediated through changes in antioxidant enzyme activity and partly through its chain-breaking antioxidant activity. The dose of vitamin E supplemented was controversial. However, it has been reported that, even at the high dose of 1000 IU/kg chow (671 mg/kg chow), vitamin E supplementation was beneficial to endothelial functions of cholesterol-fed rabbits [38]. Plasma TBARS concentrations in rabbits fed high cholesterol and fish oil and given vitamin E supplementation were still higher than in rabbits not given fish oil; it is not known whether a higher vitamin E dose then 450 mg/kg diet is required to achieve a more beneficial effect. Previous studies [24] have shown that, despite the higher plasma TBARS concentrations, the vitamin E, same dose as used in this study, can augment the anti-atherosclerotic effect of fish oil, and that the combination of vitamin E and fish oil is more effective than vitamin E alone in terms of anti-atherogenesis. Although TBARS are worldwide used to present lipid peroxide, it measures only malondialdehyde, which is one of a variety of aldehydes produced during lipid peroxidation. Further studies are necessary to find a sensitive plasma parameter as an index for accurately monitoring the anti-atherogenic properties and to clarify whether a higher vitamin E dose is more effective. A fish oil supplement given to high cholesterol-fed rabbits reduced atheroma formation, hampered blood glutathione concentrations decreases, even the GSSH/GSH ratios were not significant change, and increased plasma glutathione reductase and peroxidase activities. This may suggest that an overall increase in redox activity at the site of formation of the atheroma may hamper its formation. A vitamin E supplement given to fish oil-treated rabbits augments these beneficial effects not only because of its chain-breaking antioxidant activity, but also by enhancement of glutathione reductase activity. These results indicate that the combination of vitamin E and fish oil enhances antioxidative effects in the body.
Acknowledgments The study was supported partially by the NSC grant 89-2314-B002-031 from the National Science Council of the Republic of China. The authors are grateful to miss Lin-Lin Chu and Zu-Ohr Hsiau for technical assistances.
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Effects of fish oil and vitamin E on the antioxidant defense system in diet-induced hypercholesterolemic rabbits Hsiu-Ching Hsu, Yuan-Teh Lee, Ming-Fong Chen* Department of Internal Medicine (Cardiology), National Taiwan University Hospital 7, Chung-Shan South Road Taipei, 10002, Taiwan Received 8 January 2001; received in revised form 8 February 2001; accepted 25 April 2001
Abstract This study was designed to investigate the effects of fish oil and vitamin E on the antioxidant defense system in hypercholesterolemic rabbits. A high fat and cholesterol diet, with or without supplement by fish oil and/or a vitamin E supplement, was fed to rabbits for 6 weeks. Compared to the reference diet of regular laboratory rabbit chow, a high fat and cholesterol-enriched diet increased atheroma formation, plasma lipid and peroxide levels, decreased blood glutathione levels, and reduced plasma glutathione reductase, glutathione peroxidase, and catalase activities. Fish oil supplementation significantly reduced atheroma and increased glutathione reductase and glutathione peroxidase activities and blood glutathione levels, but increased plasma lipid peroxide levels. Vitamin E supplementation of the fish oil diet enhanced the beneficial effects by increasing glutathione reductase activity and decreasing peroxide levels. These results indicate that a high fat and cholesterol diet attenuates blood glutathione levels and plasma antioxidant enzyme activities, which may account for some of its atherogenic properties. Consumption of fish oil enhances antioxidative defenses against the oxidative stress imposed by hypercholesterolemia, and vitamin E further enhances these beneficial effects. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hypercholesterolemia; Fish oil; Vitamin E; Glutathione; Rabbits
* Corresponding author. Tel.: ⫹886-2-23123456; fax: ⫹886-2-2395-9922. E-mail address: [email protected] (M.-F. Chen). 0090-6980/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 1 ) 0 0 1 4 6 - 0
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1. Introduction Hypercholesterolemia is reported to be one of the most important coronary risk factors [1,2] and increased serum cholesterol concentrations result in the development of atherosclerosis [3]. Many studies have shown that the long chain n-3 polyunsaturated fatty acids contained in fish oil may decrease the risk of cardiovascular diseases and several other chronic illnesses [4]. Experimental studies have indicated that fish oil have multifaceted actions such as prevention of arrhythmias, antithrombotic actions, inhibiting cellular growth factors and monocytes migration, hypolipidemic effect and promotion of the synthesis of nitric oxide [5]. However, because of its high concentrations of unsaturation, fish oil can be rapidly oxidized to lipid peroxides. It has been reported that plasma and tissue levels of lipid peroxides increase in fish oil-fed animals [6 – 8] and humans [9,10]. Lipid peroxidation is considered to be a pivotal mechanism of cell membrane destruction and cell damage, and has been suggested to be associated with the initiation and progression of atherosclerosis [11]. Antioxidants and antioxidative enzymes protect cells and tissues from oxidative injury [12]. Vitamin E, with antioxidative properties, is suggested to reduce the peroxidative damage caused by fish oil-related lipid peroxidation and thereby enhance the beneficial effects of fish oil, and fish oil supplementation is therefore usually combined with vitamin E as an antioxidant. However, little is known about other advantages, drawbacks, or mechanisms of vitamin E in addition to its chain-breaking antioxidative properties [13]. The goals of the present study were to examine the effect of cholesterol on the glutathione and antioxidant enzyme activities and to determine how fish oil and vitamin E supplementation alters the antioxidant defense system in rabbits rendered hypercholesterolemic with a high fat and cholesterol diet.
2. Materials and methods 2.1. Animals and diets Male New Zealand white rabbits weighing 2.5–3 Kg were studied. They were randomly divided into 5 groups of 8, each of which was fed a different diet. The reference rabbits (Ref) were fed regular laboratory rabbit chow containing protein 16.2%, fat 6.5%, crude fiber 14.0%, ash 7.3%, nitrogen-free extract 50%, salt and vitamin mix (Purina Inc., St. Louis, MO, USA) for the entire study period. To the diets of the other 4 study groups were added 1% (wt %) cholesterol (Wako Co. Osaka, Japan); two of these groups (C and CE) received 10% (wt %) coconut oil (Yeali Co., Taipei, Taiwan), while the other two (CF and CFE) supplemented with 10% (wt %) fish oil (99.8% refined edible fish oil, Tama Co. Tokyo, Japan). Fatty acid composition of the diets was the same as a previous study [14]. The CE and CFE groups were also given ␣-tocopherol (synthetic form containing equimolar stereoisomers; Merck Ltd. Darmstadt, Germany) at a dose of 450 mg/kg diet. Salt and vitamin mix (ICN Biomedicals, INC. Aurora, Ohio, USA) were added to the diets to meet the American Institute of Nutrition standards [15]. All rabbits were housed in individual cages with raised screen bottoms in a room maintained at a constant temperature and kept on a 12:12 h
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light-dark cycle. Food and water were given daily and any unused food discarded. The study conformed to the guidelines for the care and use of animals approved by the National Taiwan University Medical Center. 2.2. Study protocol Eight rabbits in each group were fed the study diets for 6 weeks. At the end of the study period, 10 ml of venous blood from an ear vein of each animal was drawn into a tube containing 3.4 mM ethylenediaminetetraacetic acid (EDTA) for the analysis of plasma triglyceride, cholesterol, vitamin E, and lipid peroxides concentrations and antioxidative enzyme activities. The reduced and oxidized glutathione contents were also measured in whole blood. Then, the rabbits were anesthetized and killed for delineation of atherosclerotic lesions in the ascending aorta. 2.3. Biochemical determinations 2.3.1. Plasma concentrations of cholesterol, triglyceride, vitamin E and thiobarbituric acid reactive substances (TBARS) For analysis of cholesterol, triglyceride, vitamin E and TBARS, plasma was recovered by centrifugation at 4°C, 1500 g for 15 min. Butylated hydroxytoluene (2 mM) was added to the plasma to prevent further auto-oxidation for TBARS determination. Plasma cholesterol and triglyceride concentrations were determined by automated enzymatic methods [16,17]. Plasma vitamin E concentrations were measured by high performance liquid chromatography with fluorescence detection (excitation at 205 nm, emission at 340 nm) [18]. The plasma TBARS content was used to represent lipid peroxides levels and measured by fluorometric assay (excitation at 515 nm, emission at 552 nm) using 1,1,3,3-tetraethoxypropane as a standard, as described by Wasowicz et al. [19,20]. 2.3.2. Determination of reduced (GSH) and oxidized (GSSG) glutathione concentrations in blood Blood total glutathione (GSH⫹GSSG) concentrations were determined as described by Murphy et al. [21]. Briefly, the blood cells were lysised by 0.01 M phosphate buffer, and subsequently the GSSG was reduced by glutathione reductase, then the GSH reacted with 5,5⬘-dithiobis-(2-nitrobenzoic acid) to form chromophoric products. The absorbance was measured at 410 nm and the concentration calculated from the standard curve using a known concentration of GSSG or GSH. For the GSSG assay, a final concentration of 0.02 N N-ethylmaleimide was added to the blood lysate to remove GSH by forming a stable complex to prevent its participation in assay. Then, the lysate was extracted by at least 10 volumes of diethyl ether to ensure complete removal of the non-reacted sulfhydryl reagent, which can inhibit glutathione reductase reactivity in the assay. After the extraction process, the solution was added to the assay mixture for GSSG determination. GSH concentrations were calculated by subtracting the GSSG concentrations from the GSSG⫹GSH concentrations.
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2.3.3. Assessment of glutathione reductase, glutathione peroxidase and catalase enzyme activities The measurements of glutathione peroxidase and glutathione reductase activities were based on the consumption of nicotinamide adenine dinucleotide phosphate following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively [22]. The assay for catalase is based on it’s peroxidative activity as described by Johansson and Borg [23]. These procedures were performed in an Epose 5060 aotoanalyzer (Eppendorf Corp, Hamburg, Germany). All biochemical measurements were performed in triplicate and the mean values were recorded. 2.3.4. Tissue harvest for the delineation of atherosclerotic lesions The rabbits were anesthetized and killed for delineation of atherosclerotic lesions [24]. A midline thoracotomy was performed, and the ascending aorta was quickly removed from the aortic valve cups to the Arifice of the left subclavian artery. The excised aorta was fixed in neutral 10% formaldehyde for 24 h, rinsed in 70% ethanol, and immersed in Herxeheimer’s solution (5% Sudan IV in ethanol and acetate) at room temperature 15 min. The tissues were transferred to 80% ethanol for 20 min and washed in running water for 1 h. The percentage of aortic intimal infiltrated by lipids was delineated by planimetry from the distribution of sudanophilia. 2.4. Statistics All data are expressed as the means ⫾ SDs. Differences between groups were assessed by analysis of variance followed by a Tukey’s test. Statistical analyses were performed with SAS (version 6.011; SAS Institute Inc, Cary, NC). P ⬍ 0.05 was considered statistically significant.
3. Results 3.1. Plasma lipid and antioxidant concentrations The body weight gains of all 5 groups were not significant differences. Table 1 shows that plasma triglyceride and cholesterol concentrations increased markedly in all four study groups treated with diets containing high fat and cholesterol, and that supplementation with fish oil, vitamin E, or fish oil plus vitamin E did not affect this result. Compared to the reference group, rabbits treated with high fat and cholesterol not only had increased plasma lipid concentrations, but also had increased plasma vitamin E concentrations and smaller vitamin E/cholesterol ⫹ triglyceride ratios. Compared to the C group, plasma vitamin E concentrations were markedly increased in the CE group and slightly decreased in the CF group; the ratios were also increased or decreased in the CE or CF groups, respectively. However, in the CFE group, these values were higher than those in the CF group. As shown in Table 2, rabbits fed a diet containing high fat and cholesterol had decreased
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Table 1 Plasma cholesterol (CHOL), triglyceride (TG) and vitamin (VIT) E concentrations in rabbits Group
CHOL (mg/dL)
TG (mg/dL)
Vit E (mol/L)
Vit E/CHOL⫹TG (nmole/mg)
Ref C CE CF CFE
57 ⫾ 10a 1423 ⫾ 300b 1323 ⫾ 405b 1291 ⫾ 353b 1221 ⫾ 362b
54 ⫾ 9.2a 193 ⫾ 89b 186 ⫾ 84b 201 ⫾ 60b 221 ⫾ 76b
8.4 ⫾ 2.0a 22.8 ⫾ 7.7b 106.4 ⫾ 28.3c 16.4 ⫾ 3.8b,d 39.5 ⫾ 8.0b,e
7.5 ⫾ 3.2a 4.3 ⫾ 1.6b 8.6 ⫾ 3.2b 2.1 ⫾ 0.4c 3.2 ⫾ 0.4d
All data are the means ⫾ SDs, n ⫽ 8. Within a column, values with the same superscript letters are not significantly different from each other. Ref: reference group fed a regular laboratory chow; C: high fat and cholesterol-fed; CE: high fat and cholesterol ⫹ vitamin E-fed; CF: high cholesterol ⫹ fish oil-fed, CFE: high cholesterol ⫹ fish oil ⫹ vitamin E-fed group.
blood GSH concentrations, but the GSSG concentrations were not different significantly from the reference group. However, the GSSG/GSH ratios were markedly increased. The CE group had higher blood GSH and GSSG concentrations but had lower GSSG/GSH ratios than the C group. Compared to the high fat and cholesterol-treated group, fish oil supplementation increased the GSH and GSSG concentrations, but the GSSG/GSH ratios were not significantly different. The CFE group did not show any synergistic effect of vitamin E and fish oil on the increase in glutathione concentrations. 3.2. Plasma antioxidative enzyme activities Table 3 shows that glutathione reductase, glutathione peroxidase, and catalase activities decreased significantly in rabbits fed high fat and cholesterol. The same effect was seen in the CE group. The CF group had higher glutathione reductase and glutathione peroxidase activities than the C group; no difference was seen in catalase activity. The CFE group showed a synergistic effect of fish oil and vitamin E on glutathione reductase activity, but not on glutathione peroxidase or catalase activity.
Table 2 Blood reduced (GSH) and oxidized (GSSG) glutathione concentrations in rabbits Group
GSH (mg/L)
Ref C CE CF CFE
313.8 ⫾ 52.1 67.4 ⫾ 28.2b 221.2 ⫾ 64.5c 147.4 ⫾ 50.7d 186.5 ⫾ 40.7c,d a
GSSG (mg/L)
GSSG/GSH (%)
5.0 ⫾ 2.9 5.1 ⫾ 3.5a 10.8 ⫾ 3.2b 16.1 ⫾ 5.3b 15.1 ⫾ 4.6b
1.6 ⫾ 0.5a 7.5 ⫾ 3.3b 4.9 ⫾ 2.4c 9.2 ⫾ 4.2b 8.1 ⫾ 3.1b
a
All data are the means ⫾ SDs, n ⫽ 8. Within a column, values with the same superscript letters are not significantly different from each other. Ref: reference group fed a regular laboratory chow; C: high fat and cholesterol-fed; CE: high fat and cholesterol ⫹ vitamin E-fed; CF: high cholesterol ⫹ fish oil-fed, CFE: high cholesterol ⫹ fish oil ⫹ vitamin E-fed group.
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Table 3 Plasma enzyme activities of glutathione reductase (GR), glutathione peroxidase (GP) and catalase (CA) in rabbits Group
GR (U/L)
Ref C CE CF CFE
1007 ⫾ 128 341 ⫾ 168b 320 ⫾ 153b 972 ⫾ 207a,c 1363 ⫾ 388a,d a
GP (kU/L)
CA (kU/L)
20.6 ⫾ 3.3 2.8 ⫾ 1.0b 3.1 ⫾ 1.5b 5.5 ⫾ 1.3c 4.3 ⫾ 1.9b,c
29.6 ⫾ 4.2a 4.3 ⫾ 2.6b 3.9 ⫾ 2.6b 5.1 ⫾ 3.6b 4.7 ⫾ 2.6b
a
All data are the means ⫾ SDs, n ⫽ 8. Within a column, values with the same superscript letter are not significantly different from each other. Ref: reference group fed a regular laboratory chow; C: high fat and cholesterol-fed; CE: high fat and cholesterol ⫹ vitamin E-fed; CF: high cholesterol ⫹ fish oil-fed, CFE: high cholesterol ⫹ fish oil ⫹ vitamin E-fed group.
3.3. Plasma lipid peroxides As shown in Fig. 1, high fat and cholesterol-fed rabbits had elevated plasma TBARS concentrations. Vitamin E added to the high fat and cholesterol diet did not significantly change this effect. The CF group had increased plasma TBARS concentrations; this effect was significantly reduced in the CFE group. 3.4. Atherosclerotic lesions in ascending aorta Fig. 2 shows that high fat and cholesterol-fed rabbits had marked atheroma formation compared to the reference group. Addition of vitamin E alone to the high fat and cholesterol
Fig. 1. Plasma thiobarbituric acid-reactive substances (TBARS) concentrations in reference (Ref) rabbits or in rabbits treated with high fat and cholesterol diet (C), with high fat, high cholesterol and vitamin E diet (CE), with high cholesterol and fish oil diet (CF), or with high cholesterol, fish oil and vitamin E (CFE). The results are presented as the means ⫾ SDs (n ⫽ 8 for each group). Values with the superscript letters are: a ⬍ b ⬍ c ⬍ d.
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Fig. 2. Atherosclerotic lesion in the ascending aorta of rabbits. Rabbits were fed a regular laboratory chow (Ref) or a high fat and cholesterol diet (C), a high fat, high cholesterol and vitamin E diet (CE), a high cholesterol and fish oil diet (CF), or a high cholesterol, fish oil and vitamin E (CFE). The results are presented as the means ⫾ SDs (n ⫽ 8 for each group). Values with the superscript letters are: a ⬍ b ⬍ c ⬍ d.
diet did not significantly decrease the atherosclerotic lesions. Fish oil supplementation attenuated this effect, and addition of vitamin E had synergistic effect in decreasing atheroma formation.
4. Discussion It has been reported that hypercholesterolemia imposes free radical-mediated peroxidation, which initiates and potentiates atherosclerosis [3,25–28]. Many studies have indicated that elevated plasma cholesterol concentrations may modify the biochemical properties of blood components and the arterial intima, thus enhancing atherogenesis [29,30]. The present study shows that high cholesterol and fat feeding increased plasma TBARS concentration and paradoxically increased plasma vitamin E concentrations by almost three-fold. We speculated that the increased vitamin E was released from tissue in order to protect the increased plasma lipid against oxidation. However, the vitamin E/triglyceride⫹cholesterol ratios, which are indicator of lipid protection [31], were decreased. These observations are consistent with the atherogenic property of cholesterol. The well known biochemical functions of glutathione are in maintaining protein sulfhydryl groups and in detoxifying substances, including xenobiotics, metals, hydrogen peroxide, and oxygen radicals [32–34]. The antioxidant defense system counteracts the toxic activity of free radicals and peroxides [35]. We speculated that cholesterol attenuates blood glutathione concentrations and plasma antioxidant enzyme activities and that this may contribute to its atherogenic property. The linkage between free radical production and the consumption of antioxidative enzyme activity has not yet been established. However, it has been suggested that increasing the antioxidant capacity might be useful in preventing peroxidation and cellular damage [36]. The present study showed beneficial effects of fish oil supplementation on blood glutathione
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concentrations and plasma glutathione reductase and glutathione peroxidase activities in hypercholesterolemic rabbits. Moreover, the ascending aorta atheroma was markedly reduced by fish oil supplementation. These results may indicate that fish oil supplementation attenuated the oxidative stress produced by hypercholesterolemia [37]. The fish oil/cholesterol and the fish oil/cholesterol/vitamin E-treated groups showed similar responses of antioxidant enzyme to oxidative stress. This effect was not seen in rabbits fed high fat and cholesterol alone or plus vitamin E. Based on these results, it was excluded that the increases in antioxidant enzyme activities were responses to the high plasma lipid peroxides. The fact that the hypotriglyceridemic effect of fish oil was not seen in the present study indicates that lowering of triglyceride concentrations is not always effective in all hyperlipidemias; however, the enhancement of antioxidant defense activities contributes to its anti-atherogenic property. Vitamin E supplementation of fish oil-treated rabbits increased plasma glutathione reductase activity and decreased plasma TBARS significantly. These results indicate that the effects of vitamin E are partly mediated through changes in antioxidant enzyme activity and partly through its chain-breaking antioxidant activity. The dose of vitamin E supplemented was controversial. However, it has been reported that, even at the high dose of 1000 IU/kg chow (671 mg/kg chow), vitamin E supplementation was beneficial to endothelial functions of cholesterol-fed rabbits [38]. Plasma TBARS concentrations in rabbits fed high cholesterol and fish oil and given vitamin E supplementation were still higher than in rabbits not given fish oil; it is not known whether a higher vitamin E dose then 450 mg/kg diet is required to achieve a more beneficial effect. Previous studies [24] have shown that, despite the higher plasma TBARS concentrations, the vitamin E, same dose as used in this study, can augment the anti-atherosclerotic effect of fish oil, and that the combination of vitamin E and fish oil is more effective than vitamin E alone in terms of anti-atherogenesis. Although TBARS are worldwide used to present lipid peroxide, it measures only malondialdehyde, which is one of a variety of aldehydes produced during lipid peroxidation. Further studies are necessary to find a sensitive plasma parameter as an index for accurately monitoring the anti-atherogenic properties and to clarify whether a higher vitamin E dose is more effective. A fish oil supplement given to high cholesterol-fed rabbits reduced atheroma formation, hampered blood glutathione concentrations decreases, even the GSSH/GSH ratios were not significant change, and increased plasma glutathione reductase and peroxidase activities. This may suggest that an overall increase in redox activity at the site of formation of the atheroma may hamper its formation. A vitamin E supplement given to fish oil-treated rabbits augments these beneficial effects not only because of its chain-breaking antioxidant activity, but also by enhancement of glutathione reductase activity. These results indicate that the combination of vitamin E and fish oil enhances antioxidative effects in the body.
Acknowledgments The study was supported partially by the NSC grant 89-2314-B002-031 from the National Science Council of the Republic of China. The authors are grateful to miss Lin-Lin Chu and Zu-Ohr Hsiau for technical assistances.
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