Effects of Nutritional and Excessive Levels of Selenium on Red Blood Cells of Rats Fed a High Cholesterol Diet Gamaleldin I. Harisa, Osama M. AboSalem, El-sayed M. El-sayed & Gamal Shazly Biological Trace Element Research ISSN 0163-4984 Volume 152 Number 1 Biol Trace Elem Res (2013) 152:41-49 DOI 10.1007/s12011-012-9588-1
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Author's personal copy Biol Trace Elem Res (2013) 152:41–49 DOI 10.1007/s12011-012-9588-1
Effects of Nutritional and Excessive Levels of Selenium on Red Blood Cells of Rats Fed a High Cholesterol Diet Gamaleldin I. Harisa & Osama M. Abo-Salem & El-sayed M. El-sayed & Gamal Shazly
Received: 28 November 2012 / Accepted: 20 December 2012 / Published online: 6 January 2013 # Springer Science+Business Media New York 2013
Abstract In this study, we investigated the effects of selenium (Se) on the properties of erythrocytes and atherogenic index in the presence and absence of high cholesterol diet (HCD). The effect of selected two different doses (1 μg and 50 μg Se/kg/ body weight) on HCD-induced oxidative stress was investigated. The hemolysis of the erythrocytes of the HCD rats as well as by high levels of selenium or their combination was markedly increased. Likewise, atherogenic index and plasma glutathione peroxidase (GPx) activity were significantly increased in the same groups of rats compared to control ones. In contrast, paraoxonase activity, glutathione levels and protein thiol levels, catalase, GPx, and superoxide dismutase activities were significantly decreased in rats that received the HCD, high selenium dose, or their combination. Malondialdehyde and protein carbonyl levels in the plasma and red blood cells were significantly increased by HCD and high selenium dose administration. Co-administration of selenium at low dose with or without an HCD restored all of the investigated parameters to near-normal values. The results of this study suggest that G. I. Harisa (*) : G. Shazly Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia e-mail:
[email protected] G. I. Harisa Department of Biochemistry, College of Pharmacy, Al-Azhar University (Boys), Nasr City, Cairo, Egypt O. M. Abo-Salem : E.-s. M. El-sayed Department Pharmacology and Toxicology, College of Pharmacy, Al-Azhar University (Boys), Nasr City, Cairo, Egypt O. M. Abo-Salem Department of Medical Laboratories, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia G. Shazly Department of Industrial Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt
excess selenium administration with HCD worsens the atherogenic index and enhances formation of oxidized red blood cells. At dosage levels in the nutritional range such as 1 μg Se/kg body weight, selenium ameliorates the atherogenic index and preserves the antioxidant capacity of the erythrocytes. Keywords Oxidized red blood cells . Selenium . Rats . Paraoxonase . Erythrocytes integrity
Introduction Selenium is an integral component of selenoproteins, which are involved in the redox control of biological systems [1]. Selenoproteins include glutathione peroxidases, thioredoxin reductases, iodothyronine deiodinases, selenoprotein P, and selenophosphate synthetase [2]. Glutathione peroxidase (GPx) and thioredoxin reductase protect cells against oxidative damage [3]. An appropriate dose of selenium exerts an anti-atherogenic effect by lowering the lipid level and suppressing oxidative stress [4]. High levels of dietary selenium are known to cause tissue damage due to the increased production of reactive oxygen species (ROS) [5, 6]. In this case, selenium interacts with cellular thiols, leading to an increase in the production of reactive oxygen species (ROS) [6].The cytotoxicity of selenite and of other reducible selenium compounds is believed to be linked to their ability to catalyze the oxidation of thiols (R-SH) to disulfides (R-S-S-R), which under aerobic conditions occurs with the production of ROS [6]. ROS can attack membrane lipids, proteins, and nucleic acid, which results in the breakdown of cells. The membrane of red blood cells (RBCs) is a potential target for these reactive species [7]. Therefore, optimal hemostatic regulation of selenium is critical [8]. In hypercholesterolemia (HC), there is an increase in the cholesterol level in the blood, which means that cholesterol may also be accumulated in the tissues [9]. The vascular
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endothelium and erythrocyte membranes become enriched with cholesterol in response to a prolonged elevated blood cholesterol level [10, 11]. The accumulation of cholesterol in the erythrocytes under aerobic conditions may cause an increase of their osmotic fragility as well as the inactivation of membrane-bound enzymes. These events are associated with changes in erythrocytes’ rheological properties that are known to promote the development of plaques and of atherosclerotic lesions [10]. As a result of the cholesterol accumulation, the free-radical-generating enzymes (xanthine oxidase and NADPH oxidase) are activated, which leads to an increase in the formation of ROS [12]. Erythrocytes can detoxify ROS through the action of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and GPx. In addition, reduced glutathione (GSH) and other nonenzymatic antioxidants are involved in the removal of ROS from circulation by erythrocytes [7]. Under conditions of high oxidative stress, the antioxidative protection system of the erythrocytes is overwhelmed, causing them to be converted into their oxidized form, oxidized red blood cells (Ox-RBCs), and to become catalytic generators of ROS [13]. The excessive production of the ROS causes vascular damage as the Ox-RBCs exhibit higher aggregability and adhesiveness to the endothelium than the normal RBCs [13]. Furthermore, they contain lipid–protein conjugates that are closely related to some of the conjugates found in oxidized low-density lipoprotein which can function as the ligands for the macrophage scavenger receptors [13]. Although paraoxonase 1 (PON1) protects the lipoproteins and the blood components from some of the damage caused by the ROS [14], its protective effects are limited; especially additional ROS are formed by the metal-inactivated PON1 [15]. As to the role of selenium in atherosclerosis, the available evidence is contradictory and no data are as yet available that describe the effect of selenium on the activity of PON1. Since selenium exhibits its antioxidant properties at low levels but acts as a pro-oxidant at higher doses, the present study was conducted to explore its effects on the atherogenic index and oxidative stress markers of rat erythrocytes at a low but not deficient as well as at a high but yet not toxic level. Also investigated were the effects of different levels of selenium on the osmotic fragility and integrity of the erythrocytes under high cholesterol conditions.
Materials and Methods Chemicals Glutathione, sodium selenite, paraoxon, cholic acid, thiobarbituric acid, tetraethoxypropane, guanidine hydrochloride, and Tris–HCl were obtained from Sigma Chemical
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Co. (St. Louis, MO, USA). The remaining chemicals were of analytical grade. Instrumentation The instruments used in the study included a JENWAY spectrophotometer (model6105 UV/VIS) and a Sigma 3K 20 centrifuge (Germany), which can be used for speeds of up to 10,000×g. Animals and Diets Male Wistar albino rats with a body weight of 180±210 g were housed under standard laboratory conditions (12 h:12 h light/dark cycles at 25±2 °C) with free access to standard pellet food and water ad libitum. The rats were maintained under normal conditions for 1 week to acclimatize them to the laboratory environment. All of the animals received care in compliance with the guidelines set by the Animal Care and Use Committee of our institute and the National Institutes of Health. Experimental Design The rats were randomly divided into six groups, each of which was composed of six animals. In group 1, the rats received standard diet pellets (control). The rats in group 2 (hypercholesterolemic group) were fed a HCD, which included 4 % cholesterol, 1 % cholic acid, and 0.5 % propylthiouracil (HC) [16]. The composition of normal diet and HCD is shown in Table 1. The rats in group 3 (high-dose selenium group) received a daily 50 μg/kg/body weight dose of sodium selenite (HD-Se). The required dose of selenium was prepared by dissolving the required amount of sodium selenite in distilled water and administered daily through an oral tube for 1 month. The rats in group 4 received an HCD plus HD-Se (HD-Se-HC). The rats in group 5 (low-dose selenium group) received daily doses of Table 1 The composition of normal diet and diet induced hypercholesterolemia (high cholesterol diet (HCD) Ingredients
Normal diet
High cholesterol diet
Carbohydrate Fats Proteins Cellulose Vitamins and mineral Salts Cholesterol Cholic acid Propyl thiouracil
72.2 % 3.4 % 19.8 % 3.6 % 0.5 % 0.5 % – – –
72.2 % 3.4 % 19.8 % 3.6 % 0.5 % 0.5 % 4% 1% 0.5 %
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Determination of the Atherogenic Index and the Levels of Total Bilirubin and Lactate Dehydrogenase Activity The plasma levels of the total cholesterol (TC) and the highdensity lipoprotein cholesterol (HDLC) were used to calculate the atherogenic index (AI) according to the equation AI = (TC − HDLC)/HDLC. The total bilirubin (TBL) and lactate dehydrogenase (LDH) levels were determined spectrophotometrically using a commercially available kit (Biocon Diagnostic, Germany). The membrane cholesterol of the erythrocytes was extracted using the method described by Folch et al. [18]. Determination of Oxidative Stress Markers The levels of protein thiols (PSH) were estimated using the method developed by Koster et al. [19]. The GSH and oxidized glutathione (GSSG) levels were measured using a previously described method [20]. The degree of lipid peroxidation was demonstrated by spectrophotometrically measuring the levels of malondialdehyde (MDA) [21]. The degree of protein oxidation was determined by measuring the protein carbonyl (PCO) levels [22]. The total protein content was assayed according to the method described by Lowry et al. [23]. Determination of the Enzymatic Antioxidants Activity The activity of PON1 was determined by measuring the initial rate of the hydrolysis of paraoxon to p-nitrophenol [24]. The GPx activity was measured using the method described by Rotruck et al. [25]. The GR activity was measured according to the method used by Pinto and Bartley [26]. The GST activity was measured using the method
described by Habig et al. [27]. The activity of SOD was determined according to the method developed by Marklund and Marklund [28]. The CAT activity was measured using the method reported by Aebi [29]. Determination of Erythrocytes Osmotic Fragility Erythrocyte hemolysis was determined by the measurement of osmotic fragility behavior regarding the NaCl solution as follows: 25-μl blood samples were added to a series of 2.5ml saline solutions (0.0–0.9 % of NaCl) in test tubes. After gentle mixing and standing for 15 min at room temperature, the samples were centrifuged at 1,500 rpm for 5 min. The absorbance of released Hb was measured at 540 nm [30]. Statistical Analysis The data were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post-analysis test to compare all of the groups; this analysis was performed using GraphPad Prism®. The data are expressed as the mean ± standard deviation (SD). A value of P ≤0.05 was used as the criterion for significance.
Results Effects of Selenite and HCD on Body Weight The results of the present work indicated that the mean body weight of the HCD rats fed an HCD was significantly higher, by 86 %, than that of the control rats. However, the body weight of the rats that received the HD-Se treatment alone, or in conjunction with a cholesterol-enriched diet, was significantly lower than that of the control rats. The rats that received the LD-Se treatment alone, or in conjunction with an HCD, attained a body weight that was similar to that of the rats maintained on a normal diet (see Fig. 1).
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Body wight change/gm
1 μg/kg/body weight sodium selenite (LD-Se). In group 6, the rats were treated with an HCD plus LD-Se (LD-Se-HC). The selenium doses were selected based on the study published by Sreekala and Indira [17]. At the end of the experiment, all of the rats were allowed to fast for 12 h prior to being sacrificed under diethyl-ether anesthesia. Blood samples were collected in heparinized tubes and incubated at room temperature for 10 min. The erythrocyte number, hemoglobin (Hb), and hematocrit (Hct) were measured using a Coulter® AC.T diff™ hematology analyzer. Furthermore, the erythrocyte osmotic fragility test was performed calorimetrically. Afterwards, the blood samples were centrifuged at 1,500 rpm for 10 min to separate the plasma. The packed erythrocytes were separated after removal of buffy coat and washed three times with a 0.9 % NaCl solution. The erythrocytes were then lysed by adding an equal volume of distilled water. The isolated samples were then stored at −20 °C until analysis.
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a
15
b,c
10 5 0
b,c
Control HC HD-Se HD-Se-HC LD-Se LD-Se-HC
-5 -10
a,b
a,b
Fig. 1 Body weight change of control rats as well as rats treated low dose of Se, high dose of Se, high cholesterol diet, and their combination. Data were expressed as mean ± SD (n=6). a Significantly different from control group, b significantly different from HC group, c significantly different from HD-Se-HC, P ≤0.05
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Effects of Selenite and HCD on the Atherogenic Index, TBL, and LDH In the present study, the rats that were fed a HCD, HD-Se, or HD-Se-HC exhibited a 256 %, 235 %, and 274 % higher atherogenic index, respectively, than rats that received a normal diet. In contrast, the rats that received a diet that included LD-Se with or without additional cholesterol maintained an atherogenic index similar to that of the control rats (see Table 2). Effects of Selenite and HCD on Plasma GSH, PSH, PCO, MDA, and GPx The administration of selenium at a high dose in conjunction with a high-cholesterol diet resulted in a significantly higher (44 %) TBL than that of the rats that received a normal diet, LD-Se, or LD-Se-HC. Similarly, LDH activity was higher (12 %) in rats that received a HCD and HD-Se than in rats that received a standard diet, whereas the intake of LD-Se with a normal diet or an HCD resulted in an LDH activity similar to that of control rats. The high-cholesterol, HD-Se, and HD-Se-HC diets resulted in significantly lower plasma GSH levels, by 32 %, 38 %, and 48 %, respectively, than those in the control group. Furthermore, the PSH levels in the rats that received cholesterol-rich, HD-Se, and HD-SeHC diets were lower, respectively, than those of the control group. In contrast, the administration of cholesterol-rich, HD-Se, and HD-Se-HC diets resulted in significantly higher PCO levels, by 49 %, 55 %, and 65 %, respectively, than in rats fed a standard diet; similarly, the MDA level in these three groups was significantly higher, by 187 %, 190 %, and 377 %, respectively, than that of rats that received a normal diet. The results of the present study indicate that plasma
GPx activity is 51 %, 96 %, and 116 % higher in rats fed the HCD, HD-Se, and HD-Se-HC diets, respectively, than in the control group. However, in rats that were fed LD-Se or LDSe-HC diets, plasma GPx activity was similar to that of rats fed the HCD or HD-Se-HC diets (Table 2). Effects of Selenite and HCD on Plasma PON1 Activity The activity of PON1 was significantly lower in the experimental groups that received HCD, HD-Se, and HD-Se-HC, by 26 %, 37 %, and 43 %, respectively, than in the control group. The supplementation of the diet with LD-Se resulted in PON1 activity at a level similar to that observed in the control group. Consequently, the treatment of rats with a diet supplemented with LD-Se and HCD maintains PON1 activity, as shown in Fig. 2. Effects of Selenite and HCD on the Erythrocytes Membrane Cholesterol The changes of the membrane cholesterol levels of the erythrocytes in the different groups studied are depicted in Fig. 3. The HCD, HD-Se, or HD-Se-HC diet resulted in markedly higher total cholesterol content in the membrane of the erythrocytes by 27 %, 22 %, and 31 %, respectively, than in the erythrocytes of rats fed a standard diet. Effects of Selenite and HCD on the Hematological Parameters A significant decrease was observed in the number of erythrocytes and the levels of Hb and Hct in the group that received HD-Se in addition to HCD (P
