Ocena wpływu kwercetyny na aktywność wybranych enzymów u szczurów doświadczalnych w stresie oksydacyjnym wywołanym tłuszczami pokarmowymi - PDF

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ORIGINAL PAPERS Adv Clin Exp Med 2008, 17, 5, ISSN X Copyright by Silesian Piasts University of Medicine in Wrocław BOŻENA REGULSKA ILOW, RAFAŁ ILOW Assessment of the Influence of Quercetin

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ORIGINAL PAPERS Adv Clin Exp Med 2008, 17, 5, ISSN X Copyright by Silesian Piasts University of Medicine in Wrocław BOŻENA REGULSKA ILOW, RAFAŁ ILOW Assessment of the Influence of Quercetin on the Activities of Selected Enzymes in Experimental Rats Under Oxidative Stress Due to Oxidized Dietary Fats Ocena wpływu kwercetyny na aktywność wybranych enzymów u szczurów doświadczalnych w stresie oksydacyjnym wywołanym tłuszczami pokarmowymi Department of Food Science and Dietetics, Silesian Piasts University of Medicine in Wrocław, Poland Abstract Background. The kind and quality of dietary fat may influence the bioactivity of quercetin manifested as its effect on the activities of selected enzymes. Objectives. The aim of the study was to evaluate the effect of quercetin on the oxidative potential of blood and liver functions in rats under oxidative stress due to dietary fats. Material and Methods. The rats were fed diets with 8% content of fat and 0.5% addition of cholesterol. Subsequent evaluations involved the effect of quercetin on the activities of superoxide dismutase (SOD) in ery throcytes and glutathione peroxidase (GPx) in full blood as well as those of alanine and asparginate aminotrans ferases (ALT and AST, respectively) and alkaline phosphatase in plasma and liver extracts. The dietary source of prooxidants included oxidized sunflower oil and lard. The four week experiment was performed on 80 male Buffalo rats, of which 40 were given a 0.075% addition of quercetin as a dietary supplement (60 quercetin per mg/kg of rat body mass). Results. Quercetin as well as the quality and kind of fat in the diet were found to exert a significant effect on the investigated parameters. The addition of quercetin resulted in a significant activation of SOD in the rats on a diet with fresh oil and decreased SOD activity in the rats fed oxidized lard. It decreased the activity of GPx in rats fed oxidized lard and oil. The activity of plasma alanine and asparginate aminotransferases was in the physiological range and did not indicate damage to the liver cells. Quercetin decreased significantly the activity of ALT in the plasma of rats on a diet containing oxidized oil. Quercetin reduced significantly plasma AP activity in rats fed fresh lard and increased its activity in rats on a diet containing oxidized oil. The changes in AP activity in plasma and in liver extracts followed the same directions. Quercetin exerted an unfavorable effect in the form of increased accu mulation of fat in the liver cells, which was indicated by high levels of the de Ritis index. The degree of fatty degen eration of the liver was differentiated and it depended on the kind and quality of the dietary fat. Conclusions. The direction of quercetin activity was determined by the kind of fat in the diet. Beneficial effects were observed only in selected groups of animals, i.e. decreased ALT activity was observed only in animals fed a diet with oxidized oil and increased SOD activity only in rats on a diet with fresh oil (Adv Clin Exp Med 2008, 17, 5, ). Key words: aminotransferases, SOD, GPx, oxidized fat, quercetin, rats. Streszczenie Wprowadzenie. Rodzaj i jakość tłuszczu w diecie mogą warunkować aktywność biologiczną kwercetyny, przeja wiającą się jej wpływem na aktywność wybranych enzymów. Cel pracy. Ocena wpływu kwercetyny na potencjał oksydacyjny krwi i czynność wątroby u szczurów w stresie oksydacyjnym wywołanym tłuszczami pokarmowymi. Materiał i metody. Zastosowano u szczurów diety z 8% zawartością tłuszczu i 0,5% dodatkiem cholesterolu, i oceniano wpływ kwercetyny na aktywność dysmutazy ponadtlenkowej (SOD) w krwinkach czerwonych i pero ksydazy glutationowej (GPx) we krwi pełnej oraz aktywności aminotransferaz: alaninowej (ALT) i asparaginiano wej (AST) i fosfatazy zasadowej (AP) w osoczu i ekstraktach wątrobowych. Źródłem prooksydantów w diecie 504 B. REGULSKA ILOW, R. ILOW szczurów były utlenione: olej słonecznikowy i smalec. Czterotygodniowe doświadczenie żywieniowe przeprowa dzono u 80 szczurów, samców, rasy Buffalo, 40 z nich otrzymywało 0,075% dodatek kwercetyny jako suplement diety (60 mg kwercetyny/kg masy ciała). Wyniki. Kwercetyna oraz jakość i rodzaj tłuszczu w diecie miały istotny wpływ na badane wskaźniki. Kwercety na istotnie aktywizowała SOD w grupie szczurów otrzymujących dietę ze świeżym olejem oraz zmniejszała ak tywność SOD w grupie otrzymującej smalec utleniony. Obniżała aktywność GPx u szczurów karmionych utlenio nymi: smalcem i olejem. Aktywność aminotransferaz, alaninowej i asparaginianowej w osoczu, z zakresu warto ści fizjologicznych nie wskazywała na uszkodzenie komórek wątroby. Kwercetyna istotnie obniżała aktywność ALT w osoczu szczurów na diecie z utlenionym olejem. Kwercetyna istotnie obniżała aktywność AP w osoczu szczurów na diecie ze świeżym smalcem i podwyższała aktywność na diecie z olejem utlenionym. Kierunek zmian aktywności AP w osoczu i ekstraktach wątrobowych pokrywał się. Kwercetyna miała niekorzystne działanie uboczne związane ze wzmożonym gromadzeniem tłuszczu w komórkach wątrobowych, na co wskazywała wyso ka wartość współczynnika de Ritisa. Stłuszczenie wątroby było zróżnicowane i zależne od rodzaju i jakości tłusz czu w diecie. Wnioski. O kierunku działania kwercetyny decydował rodzaj tłuszczu w diecie. Korzystny wpływ, polegający na obniżeniu aktywności ALT, dotyczył tylko grup zwierząt na diecie z utlenionym olejem oraz polegający na akty wizacji SOD tylko grupy szczurów na diecie ze świeżym olejem (Adv Clin Exp Med 2008, 17, 5, ). Słowa kluczowe: aminotransferazy, SOD, GPx, tłuszcz utleniony, kwercetyna, szczury. Fats which have undergone oxidative changes are dangerous to the health of people who consume foods containing such products. This is associated with the introduction of free radicals, primary and secondary products of oxygenation, into the organ ism with the diet as well as with the depletion of the bioactivity of unsaturated fatty acids. Free rad icals and lipid peroxides damage biological mem branes and cause their permeability [1]. Aldehydes, formed from peroxides, damage internal organs, among others the liver [2]. Polymers of fatty acids decrease the digestibility of fat. Quercetin, a common bioflavonoid most abun dantly occurring in foodstuff [3], has a multidirec tional activity [4, 5], exerting, among others a chol agogic antioxidative effect and affects the activities of many enzymes. Due to its antioxidative proper ties, it may alleviate the harmful effects of oxidized dietary fats. However, the fact that the activity is so multidirectional hampers the evaluation of its potential effects on metabolic parameters in oxida tive stress. The literature contains numerous reports on the hepatoprotective and antioxidative effects of quercetin [6, 7], although its hepatopro tective activity under conditions of oxidative stress caused by the consumption of oxidized fats has not been fully investigated. The aim of the study was to evaluate the effect of quercetin on the condition and excretory function of the liver (through estima tion of the activity of index and excretive enzymes) and the oxidative status of blood (through mea surement of SOD and GPx activity). Material and Methods Experimental Design The study was carried out on eighty male Buffalo rats, with an initial mean body mass of 147 ± 28.3 g and a final body mass of ± 28.4 g. For the four weeks of the experiment the rats were kept in appropriate conditions (room temperature, 12 hour light dark rhythm). All the procedures for the animal experiments were approved by the local authorities. The animals were divided into eight groups of 10 animals. Half of the rats (n = 40) were given a diet with an 8% sunflower oil content and the other half (n = 40) were given a diet with an 8% pork lard content. Within these two subgroups receiving sunflower oil or lard, half (n = 20) had a diet with oxidized sunflower oil or lard and the other half fresh fat. Half of the rats eating a diet with fresh fat (n = 10) and half of the rats eating a diet with oxidized fat also had a bioflavonoid quercetin. The supplement amounted to 0.75 g per kilo of diet (60 mg of quercetin per kg of rat body mass). The experiment was performed using Quercetin dihy drate reagent (Fluka, cat. no ). The rats had unlimited access to the fodder and water. Their consumption of fodder and water was checked every two days and their body mass measured once a week. The diet was prepared according to the method described in [8] (Table 1). Preparation of Oxidized Fat for Use as an Ingredient of the Diet Two equal portions of pork lard and two of refined sunflower oil were prepared. One portion of each was kept fresh, while the other was sub jected to thermal oxidation. The oxidation process was carried out by heating the fat under a quartz lamp for 65 hours. A 2.5 cm thick layer of fat was placed in a porcelain dish, cm. The quartz lamp was kept 14 cm from the surface of the fat. The initial temperature of the fat was 50ºC and its final temperature was no higher than 75ºC. The The Influence of Quercetin on the Activities of Selected Enzymes 505 Table 1. Ingredients of the diets used in the experiments, containing 8% fat Tabela 1. Skład diety doświadczalnej zawierającej 8% tłuszczu Diet ingredients Amount in g/kg diet (Składniki diety) Zawartość w g/kg diety Casein 252 Fat (oil or lard) 80 Grain starch 100 Potato starch 50 Sucrose Vitamin supplement (solid) 5 Vitamin supplement (liquid) 5* Mineral supplement 50 Vitamin E 0.3* Cholesterol 5 Distilled water 100* Components of the mineral supplement: (Skład mieszanki mineralnej:) NaH 2 PO 4 2 H 2 O Mg SO NaCl 1.27 KCl 3.43 FeSO 4 7 H 2 O 0.62 CuSO 4 5 H 2 O 0.31 MnSO 4 1 H 2 O 0.15 ZnCO Na 2 MoO 4 2 H 2 O KJO CaCO Components of the solid vitamin supplement: amount in g/60 kg diet: (Skład mieszanki witaminowej stałej:) (zawartość w g/60 kg diety:) inositol 6.60 p aminobenzoic acid 6.60 Nicotinic acid (vitamin PP) 6.60 Riboflavin (vitamin B 2 ) 1.32 Pyridoxal (vitamin B 6 ) 1.32 Folic acid 0.12 D calcium pantothenate 4.08 Thiamin (vitamin B 1 ) 1.32 Vitamin K 0.60 Grain starch complete to 300 g Components of the liquid vitamin supplement: amount in U/60 kg diet: (Skład mieszanki witaminowej płynnej:) (zawartość U/60 kg diety:) Vitamin A Vitamin D Vitamin B mg Distilled water to 300 cm 3 * Amount in cm 3. * Zawartość w cm 3. oxidation conditions were established based on the report of Ziemlański et al. [9]. In the fresh and oxidized fats, the content of the polar fraction was determined, the fatty acid component, and the content of primary and secondary products of oxi dation were measured as the peroxide and ani sidine values (Table 2). Preparation of the Biological Material for Analysis After completion of the feeding stage of the experiment, the rats underwent light ether anesthe sia and blood was taken directly from their hearts into test tubes containing heparin. After the rats had been sacrificed, their livers were prepared. The organ was rinsed in a physiological salt solution, blotted dry, and weighed. Institutional approval for the described animal experiment was obtained. 506 B. REGULSKA ILOW, R. ILOW The following parameters were assessed from the biological material: the activity of glutathione peroxidase (GPx, U/g Hb) via the kinetic method, using a set of Ransel Glutathione Peroxidase reagents (Randox UK, cat. no. RS 504). This method is based on that of Paglia and Valentine [10]; the activity of superoxide dismutase (SOD, U/g Hb) via the kinetic method, using a set of Ransod Superoxid Dismutase reagents (Randox UK, cat. no. SD 125). This method is based on that of Oyanagui [11]; the activities of alanine [12] and aspartate [13] aminotransferases using kinetic enzymatic diagnostic tests from BioSystems (cat. nos. COD and COD 11561); the activity of alkaline phosphatase [14] using enzymatic diagnostic tests from Biochemtest (cat. no ); the de Ritis index was calculated as the quo tient of asparginate and alanin aminotransferase activity. Statistical Assessment of the Results The normality of the distribution in the exam ined groups was assessed with Shapiro Wilk s W test. In case of lack of a normal distrib ution, logarithmic transformation was applied. The significance of the differences between the aver age values for the examined groups was assessed using one way analysis of variance (p 0.05). Data were tested for homogeneity of variances with Levene s test. To assess the influence of dif ferences on the analysis of variance (average com parison post hoc), Tukey s Honest Significant Difference test (HSD) was applied. A Cochrane Cox test was used in case of a normal distribution of variables but a lack of homogeneity of the vari ance. The nonparametric Kruskal Wallis test was used for comparisons in cases of lack of a normal distribution as well as a lack of homogeneity of the variance of variables. All the statistical calcula tions were done with the STATISTICA 6.0 PL (StatSoft. Inc., USA). Results Fat Added to Diet The changes in the quality of dietary fat are presented in Table 2. The peroxide number of the sunflower oil increased 194 times as a result of oxidation and in lard 114 times. The anisidine number increased 15 times in oxidized oil and 87 times in oxidized lard. The percentage of polyunsaturated fatty acids decreased after oxida tion of oil by 25%. The composition of fatty acids in lard changed slightly by oxidation. Oxidation resulted in an increase in the polar fraction: in lard from 1.3 to 16.9% and in oil from 2.1 to 36.5%. Table 2. Changes in quality parameters of fresh and oxidized fats Tabela 2. Zmiany wskaźników jakości tłuszczów świeżych i utlenionych Kind of fat/quality parameter Fresh lard Oxidized lard Fresh Oxidized (Rodzaj tłuszczu/wskaźniki jakości) (Świeży smalec) (Utleniony sunflower oil sunflower oil smalec) Świeży olej (Utleniony olej słonecznikowy) słonecznikowy) Superoxide number: (Liczba nadtlenkowa:) [meq O 2 /kg] 1.5 ± ± ± ± 0.0 [mg O 2 /100g] Anisidine number 0.5 ± ± ± ± 0.8 (Liczba anizydynowa) Polar fraction (Frakcja polarna) [%] Fatty acid type [%]: (Suma kwasów tłuszczowych:) Saturated (Nasycone) Monounsaturated (Jednonienasycone) Polyunsaturated (Wielonienasycone) Unidentified (Niezidentyfikowane) The Influence of Quercetin on the Activities of Selected Enzymes 507 The Effect of Quercetin on the Activities of Liver and Antioxidative Enzymes Decreased plasma ALT activity was observed in the group fed oxidized oil and quercetin (37.4 ± ± 14.1 U/L) compared with controls (59.5 ± 14.7 U/L). Increased AP activity was observed in the plasma of rats fed oxidized sunflower oil and quercetin (196.0 ± 24.3 U/L) compared with the group fed oxidized oil (109.0 ± 17.2 U/L) and in the plasma of animals given oxidized oil and quercetin (196.0 ± 24.3 U/L) compared with the group fed fresh oil and quercetin (117.9 ± 20.0 U/L). Quercetin significantly reduced the level of AP in the plasma of rats fed fresh lard (90.0 ± 20.5 U/L) compared with controls (233.4 ± 95.2 U/L). In the groups on diet containing oxidized oil, the addition of quercetin resulted in an elevation of the de Ritis index from 1.5 to 2.5. In the group fed a diet containing oxidized lard, the addition of quercetin increased the level of the index from 2.4 to 3.7. High levels of the de Ritis index were observed in animals receiving quercetin and fresh (2.5 ± 0.7) or oxidized lard (3.7 ± 0.6). The results are summarized in Table 3. The activities of AST and ALT increased in the livers of the animals on a diet with fresh oil and quercetin to 58.0 ± 12.6 U/g of liver and (53.7 ± ± 14.1 U/g of liver, respectively, compared with the animals without quercetin (25.7 ± 4.4 U/g of liver and 31.7 ± 5.3 U/g of liver, respectively). The results are summarized in Table 4. The activity of AP in the liver increased signif icantly in the group receiving oxidized oil and quer cetin (16.2 ± 2.0 U/g of liver) compared with the animals on a diet with oxidized oil (3.1 ± 0.7 U/g of liver) without quercetin. Quercetin caused a significant increase in the activity of SOD in the erythrocytes of the rats on a diet containing fresh oil and a decrease in SOD activity in the groups on a diet containing oxidized lard. Quercetin decreased the activity of GPx in full blood of the rats on a diet with oxidized oil (41.4 ± ± 2.5 vs ± 2.9 U/g Hb) and decreased the activity of SOD and GPx in the rats on a diet with oxidized lard (2954 ± 334 vs ± 251 U/g Hb and 47.4 ± 3.0 vs ± 3.9 U/g Hb, respectively). Discussion The polar fraction content in fat should not exceed 24% and the content of polymers 12%. Frying fat achieves such parameters after 5 6 days of frying in the same batch [15, 16]. According to the accepted criteria, oxidized oil added to the diet in our experiment achieved the parameters dis qualifying it as consumable. The polar fraction has a liquid consistency and it is not possible to remove it from fat. It is therefore absorbed by the fried food and becomes a dietary constituent [17]. The Effect of Dietary Quercetin on Liver Function Aminotransferases are index enzymes and an increase in their activity suggests damage to hepa tocyte structure, with discontinuity or increased permeability of its cell membrane. ALT is charac terized by higher organ specificity in relation to the liver, while AST may also originate from injured heart, skeletal muscles, lungs, and kidneys. The de Ritis index has a diagnostic value in diseases of the liver. This is the quotient of asparginate and alanin aminotransferase activity. A de Ritis index of about 1.2 may indicate cirrhosis of the liver, while suggests acute hepatitis. A de Ritis index approaching 2 suggests fatty degeneration of the liver. Increased AP activity is observed in liver damage of cholestatic origin. In animals on diets containing fresh fats, sun flower oil and lard, the addition of quercetin did not induce changes in the activity of the plasma aminotranferases. Quercetin s beneficial effect of decreasing effect ALT activity was observed in the plasma of the animals on a diet with oxidized oil. This beneficial effect was not observed in the ani mals with oxidized lard as the dietary source of fat. The effect of quercetin depended on the kind of oxidized dietary fat and the content of polyunsatu rated fatty acids, which were 4.4 times more abun dant in oxidized oil than in oxidized lard. The hepatoprotective effect of quercetin has been observed by numerous authors [18 21]. Positive effects were obtained by authors using quercetin and diosmine in a total amount of 60 mg/kg of rat body mass (in the present experiment: 60 mg of quercetin per kg of rat body mass) [18]. The detrimental effect of a diet containing lard on the liver structure was observed regardless of the quality of the fat. The high level of the de Ritis index in the groups fed a diet containing lard sug gested fatty degeneration of the liver. An increased AST/ALT ratio in all the groups of animals receiving quercetin may also be evi dence of fatty degeneration of the liver, and the changes reached the level of statistical signifi cance in the groups fed oxidized fat and quercetin. Rats on the diet with oxidized lard and quercetin revealed an unfavorable synergistic 508 B. REGULSKA ILOW, R. ILOW Table 3. The influence of the investigated diets on the activities of alanine (ALT) and aspartate (AST) aminotransferases and those of alkaline phosphatase (AP) in the plasma, dismutase super oxide (SOD) in red blood cells, and glutathion peroxide (GP) in the blood of the laboratory rats Tabela 3. Wpływ diet na aktywność: aminotransferaz alaninowej (ALT) i asparaginianowej (AST), fosfatazy zasadowej (AP) w surowicy, dysmutazy ponadtlenkow
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