PROTECTIVE EFFECT OF VITAMIN E ON BIOMARKERS OF OXIDATIVESTRESS AND INFLAMMATORY RESPONSE IN A RAT MODEL OF

ENDOTOXEMIAHUSSEIN, S.A.; EL-SENOSI, Y.A.; OMNIA, M. ABD EL-HAMID AND BAKR, W. M. T.Department of Biochemistry, Faculty of Veterinary Medicine, Moshtohor, Benha University, Qalioubeya, Egypt.1-ABSTRACT

The present study aimed to investigate the protective effect of vitamin E on theoxidative stress induced by lipopolysaccharide (LPS, endotoxin) in rats. This study wascarried out on 80 male rats divided into four equal groups of 20 rats each. Group I (control):rats administered corn oil (20 mg/kg/b.w/day )for 5 weeks. Group II,rats administeredvitamin E orally (100 mg/kg/b.w/day) for 5 weeks. Group III, rats injectedintraperitoneally(i.p.)with LPS (20 mg / kg/b.w.).Group IV, rats administered vitamin E (100mg/kg/b.w/day) for 5 weeks and then injected (i.p.) with LPS (20 mg / kg/b.w.).Bloodsamples for serum separation and liver tissues were collected two times at 2 and 5 hoursfrom the onset of injection with endotoxin. All sera were processed directly for glucose, UricAcid , Total Cholesterol, Triacylglycerols(TG), Phospholipids, Free Fatty Acids (FFA), Nitricoxide (NO), Aspartate aminotransferase (AST),Alanine aminotransferase ( ALT), γ-glutamyltransferase (GGT), Haptoglobin, α-1-acid glycoprotein (α-1-AGP), Vitamin C, VitaminE, Reduced glutathione (GSH) and L-Malondialdehyde (L-MDA) in addition to determinationof liver Glutathione peroxidase (GPx), Superoxide dismutase (SOD), Catalase(CAT),Glutathione reductase (GR) ,Vitamin C, Vitamin E, GSH and L-MDA were also analyzed.The obtained results revealed that, endotoxemia could potentially increase serum Glucose,Uric Acid, TG, Phospholipid, FFA, Haptoglobin, AGP, GSH, L-MDA concentrations in addition toALT, AST and GGT activities. Moreover, the values of SOD, CAT activities and GSH, L-MDAconcentrations in liver tissues were significantly decreased. Treatment with α- tocopherol(Vitamin E) decreased LPS-triggered pathogenic responses by mitigating liver damage andprevented the increase of lipid peroxidation (L-MDA),Glucose, Uric Acid, Triacylglycerols,Phospholipid, FFA, ALT, GGT, Haptoglobin and AGP. Moreover, treatment with vitamin E inendotoxin injected rats ameliorates the oxidative stress by increasing the concentrations ofserum and tissue GSH, SOD, CAT and Vitamin C. It could be concluded that, vitamin Eprotects against lipid peroxidation, oxidative stress and decrease the inflammatoryresponse to endotoxin injection.KEY WORDS: Endotoxin, Vitamin E, Oxidative stress, Inflammatory markers,Antioxidant status.

2- INTRODUCTION Lipopolysaccharide (LPS) is one component of gram-negative

bacterial cell wall, which released by destruction of cell wall and actsas a potent bacterial product in the induction of host inflammatoryresponses and tissue injury (50). Increased oxidative stress followingendotoxin administration is supported by the increased formation oflipid peroxidation products (19). Moreover, when bacteria are lysed byimmune effector cells and molecules, surges of endotoxin may be releasedinto the host, intensifying the inflammatory response and causingfurther activation of immune effector cells (46). Endotoxin inducesshock states in both humans and animals characterized by fever,hypotension, intravascular coagulation, and finally multi-organ failure

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system (MOFS) (80). Detoxification of endotoxin is considered to bemediated mainly by the reticuloendothelial system (RES), particularlyKupffer cells, in the liver (79).

The lipid soluble α-tocopherol (vitamin E) is a chain-breaking,free radical trapping, nonenzymatic, naturally occurring antioxidant incell membranes and plasma lipoproteins. Vitamin E is a fat-solublevitamin necessary in the diet of many species for normal reproduction,normal development of muscles, normal resistance to erythrocytes tohemolysis, and various other biochemical functions. Chemically, it isalpha-tocopherol, one of the three tocopherols (alpha, beta, and gamma)occurring in wheat germ oil, cereals, egg yolk, and beef liver. It isalso prepared synthetically. Tocopherols act as antioxidant (51).

Accordingly, this study was performed to investigate the protectiveeffect of oral supplementation of vitamin E on lipid peroxidation,biomarkers of oxidative stress and some inflammatory markers in serumand tissues in an endotoxemic rat model.

3- MATERIALS AND METHODS3.1. Experimental animals

Eighty white male albino rats of 8- 10 weeks old and weighing 150-200 gm were housed in separated metal cages and kept at constantenvironmental and nutritional conditions throughout the period ofexperiment. The animals were fed on constant ration and water wassupplied ad- libitum.

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3.2. Induction of Endotoxemia:Endotoxemia was induced by injecting the rats intraperitoneally

with a single dose of LPS* (from Esherichiacoli, serotype 026) in a dose20 mg / kg/ b.w. (17). LBS strain was obtained from Egyptian NationalInstitute of Animal Health and was extracted in biotechnology department.Faculty of veterinary, Cairo University. 3.3. Preparation and Dosage of Vitamin E: Vitamin E was administered orally in a daily dose of 100mg/kg/B.W. and prepared by dissolved in Corn oil for dilution (9). 3.4. Experimental design:

Animals were randomlydivided into 4 main groups (20 each) placed inseparate cages and classified as follow:Group I (control group): Rats administered with corn oil orally daily ata dose level of 20 mg/kg/b.w. for 5 weeks and served as normal group.Group II (vitamin E normal treated group): Animals received vitamin E(α-tocopherl), orally at a daily dose of 100 mg/kg/b.w for 5 weeks.Group III (endotoxin group): Rats injected intraperitoneally(I/P) with asingle dose of LPS (20 mg / kg/b.w.).Group IV (endotoxin pre- treated vitamin E group): Rats received vitaminE (α-tocopherol), orally in a daily dose of 100 mg/kg/b.w for 5 weeksbefore endotoxin injection. One hour after the last dose of vitamin Epretreatment rats were injected intraperitoneally (I/P) with a singledose of endotoxin (20 mg/ kgb.w.).3.5. Sampling:

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3.5.1. Blood samples: Blood samples for serum separation were collected by ocular vein

puncture in dry, clean, and screw capped tubes after overnight fastingfrom all animals groups (control and experimental groups) twice alongthe duration of experiment at 2 and 5hours from the onsetof LPSinjection. Serum was separated by centrifugation at 4000 r.p.m for 15minutes .The clean, clear serum was separated by Automatic pipette andreceived in dry sterile samples tube, and proceed directly for glucosedetermination, then kept in a deep freeze at - 20° C until used forsubsequent biochemical analysis.3.5.2. Liver specimens:

Rats were killed by decapitation. The liver specimen quicklyremoved, cleaned by rinsing with cold saline and stored at -20°C.Briefly, liver tissues was minced into small pieces, homogenized withice cold 0.05 M potassium phosphate buffer (pH7.4) to make 10%homogenates. The homogenates were centrifuged at 4000 r.p.m for 15minute at 4°C. The supernatant was used for the determination of L-malonaldehyde (L-MDA), vitamin E, Vitamin C and antioxidant enzymes. 3.5.3. Biochemical analysis:

Serum glucose, Uric Acid, Total cholesterol, TG, phospholipids,FFA, NO, AST, ALT, GGT, Haptoglobin, AGP, Vitamin C, Vitamin E , G-SH, L-MDA, SOD, GR, CAT and GPx were analyzed according to the methodsdescribed by(90), (30), (63); (84); (57); (21); (48) ; (12); (84); (60);(69); (11); (39);(8); (97); (65); (71); (36) and (3) ,respectively.3.6. Statistical analysis:

The obtained data were statistically analyzed by one-way analysisof variance (ANOVA) followed by the Duncan multiple test. All analyseswere performed using the statistical package for social science (SPSS,13.0 software, 2009). Values of P<0.05 were considered to besignificant.

4- RESULTS AND DISCUSSIONFree radicals play a role in the pathogenesis of various diseases

(e.g., cardiovascular and cerebrovascular diseases, neurosensorydisorders,Parkinson’s disease ,inflammation ,cancer and others), and thepharmacological agents scavenging free radicals are considered important(10,52).LPS creates a status of oxidative stress which characterized byreleasing reactive oxygen species (ROS), neutrophils, phagocytic cellsand cytokines (89). Reactive oxygen species (ROS) are formed as a normalproduct of aerobic metabolism in mitochondrial and microsomal enzymaticreactions (69). However, these ROS can be generated at elevated ratesunder pathophysiological conditions (4).

The obtained data (Table1) revealed that, the mean value of serumglucose concentration significantly increased at 2 and 5hours afterendotoxin injection in rats when compared with control group. Theseresults came in accordance with the recorded data of Yelich and Janusek(95) who found that, the administration of endotoxin causedhyperglycemia as initial response to endotoxin followedbyhypoglycemia.These results may be related to that, hyperglycemia isassociated with protein glycation and simultaneously, with theproduction of reactive oxygen species and has been implicated in freeradical injury caused by hyperglycemia (40). In addition, bacterial

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endotoxins induced pancreatitis which caused an increase in serumamylase associated with increase in glucose level. On the other hand,the presented data

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showed that vitamin E Pretreatment endotoxin group significantly decreased serum glucose concentration at 5 hours after endotoxin injection when compared with rats injected with endotoxin only. These results are in a harmony with the recorded data of Kheir-Eldin et al., (50) who mentioned that, the endotoxin elevated the blood sugar level and vitamin E pretreatment shows decrease effect on the elevated level of plasma glucose because vitamin E improves the free radical defense system potential and may have a beneficial effect in improving glucose transport and insulin sensitivity (25).

significantly increased in endotoxin injected rats (5hr.) whencompared with control group. These resultsaresimilar to the recordeddata of Kahl and Elsasser, (49) who recorded that, uric acidconcentration was increased after LPS challenge in rats treated for 12days.This may be due to that, Uric acid is the product of the freeradical generating xanthine oxidase reaction, and enzymatic andnon-enzymatic lipid peroxidation so that uric acid protects againstoxidative damage of proteins and nucleobases by directly scavenginghydroxyl radical, singlet oxygen, hypochlorous acid, oxoheme oxidants,and hydroperoxyl radicals (25). In addition, the presented resultsdemonstrated that, serum uric acid concentration decreased significantlyin pretreatment vitamin E group when compared with LPS injected rats(5hr.).similarly, Zoroglu et al., (96) concluded that, decreased uricacid concentration after oral administration of vitamin E in ratsinjected with LPS. Because vitamin E has been indicated as the majorantioxidant that prevents the propagation of free radical damage inbiological membranes of xanathine oxidase during conversion of xanathineto uric acid (91).

The obtained data (Table 1) showed that, no significant change inserum total cholesterol concentration in rats injected LPS (2hr. and5hr.) group and in pretreatment vitamin E group when compared withcontrol group. These results came in accordance with the recorded dataof Lehr et al., (54) who concluded that, no effect of endotoxins wasobserved on the serum total cholesterol levels and no change afteradministration of vitamin E. Rather, there was no change in hepaticcholesterol levels after LPS administration. Moreover, as both LDLreceptor mRNA and protein levels were not significantly altered by LPSthis finding suggests that a decrease in hepatic clearance of LDL doesnot contribute to LPS-induced hypercholesterolemia (29).

The data displayed in (Table 1) stated that, the mean value ofserum TG concentration significantly increased in endotoxin injectedrats when compared with control group.These results are agreed well withthe recorded data of Adi et al., (1) who reported that, theadministration of endotoxin (LPS) has been used to mimic infections.Both infections and LPS administration produce hypertriglyceridemiaby increasing hepatic lipoprotein secretion and reducing lipoproteinclearance, presumably by decreasing lipoprotein lipase activity .It was proposed that, hypertriglyceridemia was due to the cataboliceffects of a circulating factor on fat cells that directly causedcachexia. Serum TG concentration was significantly decreased in

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pretreatment vitamin E group when compared with endotoxin treated ratsonly (5hr.).These results are agreed with Chunga et al., (18) whorecorded that, serum triacylglycerol increased in response to LPS andthese increases were, in part, reduced by α-tocopherol supplementationonly. Additionally, vitamin E can reduce triglycerides.Because vitamin Eenable to compete with triglycerides in the blood stream, therebyreducing their impact. It may also reduce the incidence of comorbiddiseases associated with high triglyceride levels, such asatherosclerosis (87).

Intraperitoneal injection of endotoxin to normal rats significantlyincreased serum phospholipids concentration at 5 hours after injectionas compared with control group (Table 1). These results are similar tothe recorded data of Gmeiner and Schlecht, (35) who stated that, theincrease of LPS injection was paralleled by increasing amounts ofphospholipid while the overall protein content in the outer membranedecreased in the rats. This is may be due to the increase of serumphospholipids after endotoxin which leads to a change in the activity ofone or more phospholipid transfer proteins, with decreased transfer ofphospholipids out of the plasma into tissues or increased transfer fromtissues into plasma (92). In addition, a significant decrease in thevalue of serum phospholipids concentration was reported in vitamin Epretreatment endotoxin group at 5 hours after endotoxin injection whencompared with rats treated with endotoxin. These results came inaccordance with the recorded data of Campbell et al., (15) who providedthat, decrease phospholipids concentration after 5 hr. from LPSinjection and treatment with oral administration of vitamin E. This isbecause vitamin E is an endogenous lipid soluble chain-breakingantioxidant that is known to protect cells from the diverse actions offree oxygen radicals by donating its hydrogen atom leading to decreasingof phospholipids concentration (13).

The mean value of serum FFA concentration increased significantlyin rats injected LPS when compared with control group as showed in(Table 1).These results are agreed with the recorded data of Chunga etal., (18) who recorded that, free fatty acid increased by 6 h. post-LPS,compared to 0 and 1.5 h, regardless of tocopherol supplementation. Thismay be related to that, LPS-triggered oxidative stress and inflammatoryresponses induced serum dyslipidemia and tocopherol supplementationdecreased or maintained these responses. Moreover, the LPS-mediatedincreases in α-TNF likely resulted free fatty acid

Effect of vitamin E on oxidative stress in endotoxemic rat

release from adipocytes by stimulating lipolysis. Likewise, hepatic freefatty acid accumulation induces oxidative stress through several ofintracellular pathways (29).The presented data demonstrated that, themean value of serum FFA was significantly decreased in vitamin Epretreatment group when compared with endotoxin treated rats only (5hr.)and these results came in accordance with the recorded data of Pascoeand Reed, (72) who demonstrated that, vitamin E protects fatty acids andprotein thiol groups against oxidation. Furthermore, the amount ofvitamin E present in tissues is related to fatty acid concentration(76). In addition, the majority of vitamin E in tissues is present inthose membranes with the highest fatty acid composition. It is a lipid-

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soluble vitamin and its main function is to protect polyunsaturatedfatty acids (PUFA) against oxidative stress. It can break the chain,prevent lipid peroxidation, trap peroxyl free radicals and preventdisruption of the membrane integrity(62).Vitamin E plays a role inprotecting FFA in cell membranes and LDL from oxidation by donating thehydrogen of the hydroxyl group, therefore, blocking the initiation andpropagation of lipid peroxidation (20).

The mean value of serum ALT and AST activities significantlyincreased in rats injected with LPS when compared with control group asfound in (Table 1). These results came in accordance with the recordeddata of Hanan and Hagar, (38) who found that, induction of endotoxemiawith LPS resulted in increased serum activities of ALT and AST as ameasure of hepatic damage. Injection of LPS for 5 hr. before the onsetof endotoxin intoxication markedly attenuated LPS-induced hepaticdysfunction and accompanying oxidative stress in liver. In addition,high concentrations of AST and ALT were measured in the systemiccirculation as a result of acute destruction of hepatocytes andtreatment by vitamin E restored the levels of these indicators of liverdamage, con rming that reactive oxygen intermediates (ROIs) are involvedfi(22). Therefore, the increase in the serum ALT and AST activities mightperhaps be an indication of liver damage. This increase could beexplained that, free radical production which reacts withpolyunsaturated fatty acids of cell membrane leading to impairment ofmitochondrial and plasma membrane results in enzyme leakage (74).Theobtained data showed that, the mean value of serum ALT and ASTactivities were significantly decreased in pretreatment vitamin E groupwhen compared with endotoxin injected rats and these results are in aharmony with the data of Sushma et al., (85) who showed that, LPS causeda marked rise in serum level of ALT and AST where the activities ofliver enzymes were decreased significantly on supplementation withtocopherol. Furthermore, administration of tocopherol before LPSchallenge resulted in a significant reduction in the serum levels ofthese enzymes as a result of vitamin E has been reported to conferprotection against such changes in formaldehyde and monosodium glutamateinduced-hepatotoxicity and oxidative stress in rats(37)(68).

The presented data (Table 1) revealed that, mean value of serum GGTactivity increased significantly in endotoxin injected rats (5hr.) whencompared with control group.These results are agreed with the recordeddata of Hanan and Hagar, (38) who investigated that, induction ofendotoxemia with LPS resulted in increased serum levels of gammaglutamyltransferase (GGT), as a measure of hepatic damage. LPS inducedsignificant increase in the activity of GGT during the whole time courseof the experiment (8 days) (75). This could be attributed to increase inGGT occurred to restore glutathione used in the metabolism of drugswhich accounts for the elevated activity assayed (47).

The mean value of serum haptoglobin concentration increasedsignificantly in endotoxin injected rats (5hr.) when compared withcontrol group as displayed in (Table 1). These results are similar withNielsen et al., (64) who recorded that, the serum concentration ofhaptoglobin was increased by LPS and caused high mortality compared withthe control group. Likewise, plasma haptoglobin was increased by LPS

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treatment at 24 h. post injection in rats (27). The increase in plasmahaptoglobin after the initiation of the experiment indicates thepresence of an inflammatory condition in rats.Nevertheless, during theacute phase of inflammatory process there is a characteristic increasein some plasma proteins called collectively acute phase reactant (APR),LPS that induces a strong acute phase response, indicated by high levelof haptoglobin as confirmed by Silveira and Limaos, (83). Even though,the mean value of serum haptoglobin was significantly decreased inpretreatment vitamin E group (5hr.) when compared with endotoxininjected rats group only. These results are in a harmony with the dataof Carter et al., (16) who found that, serum haptoglobin concentrationswere significantly lower on days 0 and 7 than concentrations for ratthat required > 1 treatment with vitamin E administration. Serumhaptoglobin concentration on day 0 significantly correlated with thenumber of antimicrobial treatments required.

The data showed in (Table 1) illustrated that, intraperitonealinjection of endotoxin to normal rats significantly increased the valueof serum α-1-acid glycoprotein concentration after 2 and 5 hours incomparison with control group. These results are similar with therecorded data of Nielsen et al., (64) who recorded that, the serumconcentration of α-1-AGP was increased by LPS and induced high mortalitycompared with the control group. α-1-AGP has also been reported to beinvolved in nonspecific resistance to infection by gram-negativebacteria as well as in protection against LPS (42). The increase of α-1-AGP could be related to that, α-1-AGP interacts directly with LPS andthis α1-AGP–LPS complexes are removed by macrophages, enhancingclearance of LPS from the body (24). Furthermore, α-1-AGP an acute phase

Hussein et al., (2013)

reactant produced by the liver and elevated during inflammation tocontrol inappropriate or extended activation of the immune system (31).However, serum α-1-AGP was significantly decreased in pretreatmentvitamin E group (5hr.) when compared with LPS injected rats.Theseresults are in harmony with the data of Carter et al., (16) who foundthat, serum α-1-AGP concentrations were significantly lower on days 0and 7 than concentrations for rat that required > 1d.treatment withvitamin E administration. Serum α-1-AGP concentration on day 0significantly correlated with the number of antimicrobial treatmentsrequired.

The presented data (Table 1) stated that, no significant change inthe serum and liver vitamin E concentration in rats injected LPS (2hr.and 5hr.) group when compared with control group and significantincrease in pretreatment vitamin E (2 hr.) group in comparison with ratsinjected LPS (2 hr.). These results are similar with the data of Rojaset al., (77) who recorded that, LPS did not affect vitamin E levelseither in animals with low or with high liver vitamin E levels.Moreover, vitamin E the main lipid-soluble antioxidant, was not modifiedby the LPS treatment in rats when compared with control groups (70).Furthermore, tissue vitamin E level did not affect by LPS in animalseither with low or with high LPS concentrations and increasetissue vitamin E in pretreatment vitamin E group(43).

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No significant change in the serum and liver vitamin Cconcentration in rats injected LPS (2hr. and 5hr.) group when comparedwith control group and other groups as illustrated in (Table 1&2). Theseresults are in a harmony with the recorded data of Alessio et al., (5)who discussed that, vitamin C have little or no effect on oxidativestress, as measured by plasma thiobarbituric acid reactive substances(TBARS), at levels 1.0 gms and less. The differences in the reportedresults might be due to dose and time of 5 treatments.Likewise, LPS did not change the concentration of tissue vitamin C whencompared with all groups and significant increase in pretreatment withvitamin E after 6 hr. from LPS (66). This may be due to that, vitamin Cis a potent scavenger of free oxygen radicals and it has been shown thatmarginal LPS results in intracellular oxidative damage in the rats couldnot be make any change in vitamin C (88).

Intraperitoneal injection of endotoxin to normal rats caused non-significant increase in the value of serum L-MDA concentration after 2and 5 hours in comparison with control group as found in (Table 1).These results are agreed with the data of Ouchi et al., (70) who statedthat, lipid peroxidation in the guinea pig was not significantlymodified by endotoxin (LPS). Hence, Taillé et al., (14) showed that, L-MDA was nonsignificantly change in LPS treated animals at 6, 12, and 24h from injection.The obtained data showed that, a non-significant changein serum L-MDA concentration in pretreatment vitamin E group (2 and5hr.) when compared with LPS injected rats and other groups. Theseresults are similar with the recorded data of Ross and Moldeus, (78) whoproved that, after 5-week dietary supplementation of α-tocopherol don’tchange LPS-triggered lipid peroxidation, inflammation, and hepaticdamage. This is resulted in Intracellular vitamin E is associated withlipid rich membranes such as mitochondria and endoplasmic reticulum butvitamin E here hasn’t effect role in protecting against membrane lipidperoxidation by reactive lipid peroxyl and alkoxyl radicals.

The results displayed in (Table 1) proved that, no significantchange in the serum nitric oxide (NO) concentration in rats injected LPS(2hr. and 5hr.) group when compared with control group. These resultsare agreed with the data of Taylor et al., (89) who mentioned that, NOproduction by hepatocytes is not signi cantly enhanced by LPS, but wasfimarkedly enhanced by cytokines and even LPS-activated Kupffer cellsconditioned medium. Therefore, substimulatory doses of LPS caneffectively reprogram macrophages for altered responses to subsequentactivation by LPS and other microbial stimuli and no enhancement isobserved with NO production following LPS-dependent reprogramming.Theobserved enhancement of the NO response by LPS-dependent reprogrammingis not strictly dependent upon enhanced macrophage responsiveness for NOproduction, since neutralizing antibody to mouse NO only partially nochange for LPS response. Collectively, these data support the conclusionthat the LPS-dependent reprogramming event involved a more generalizedintracellular regulatory event cannot control NO production (41).

The obtained results (Table 2) revealed that, no significant changein the liver GPx activity in rats injected LPS (2hr. and 5hr.) groupwhen compared with control group. These results are agreed with the

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recorded data of Gilad et al., (34) who indicated that, no changes wereobserved in the activity of glutathione peroxidase in the brain of ratsexposed to LPS. Moreover, Aydin et al., (6) found that, no significantchange GPx enzyme activity in LPS rats determined compared to controlgroup. Decrease GPx activities or its hidden may indicate that highamount of hydrogen peroxide which may have accumulated in thecells. Increased hydrogen peroxide may be transform to hydroxylradicals and increase oxidative stress.

The presented data (Table 2) revealed that, no significant changein the liver GR activity in endotoxin injected rats (2hr. and 5hr.)group when compared with control group. These results are in a harmonywith the data of Rojas et al., (77) who recorded that, a dose of LPSwhich induced endotoxic shock did not alter any antioxidant enzymeor GR in the guinea pig myocardium, making the interpretation ofthe changes in the rest of the parameters studied more easy.GRis a key enzyme of the antioxidative system that protects cells againstfree radicals. The enzyme catalyzes the reduction of GSSG to GSH by theNADPH-dependent mechanism. No change of GSH/GSSG ratio may becontributes to

Effect of vitamin E on oxidative stress in endotoxemic rat

decrease oxidative stress and there is increasing evidence indicatingthat oxidative stress plays an important role in the pathogenesis ofmany diseases (86).

The obtained data (Table 1&2) demonstrated that, the mean value ofserum and tissue GSH concentration decreased significantly in endotoxininjected rats (2hr. and 5hr.) group when compared with control group.These results are in a harmony with the data of Lee et al., (53) andBallatori et al., (7) who observed that, increased oxidative stressfollowing endotoxin administration is supported by decreased levels ofreduced glutathione (GSH) in experimental animals. However, GSH is animportant regulator of cellular redox balance and plays a major role inprotecting against oxidative stress by reacting directly with ROS. Theimpact of GSH on immune function has been investigated as confirmed byAdler et al., (2) who provided that, GSH depletion in rats impairs T-cell and macrophage immune function and the level of GSH is depressed inthe serum (58). Moreover, serum and liver GSH was significantlyincreased in pretreatment vitamin E group (5hr.) when compared withendotoxin injected rats only and these results are similar with the dataof Nadia et al., (61) and Gilad et al., (34) who observed that,treatment with α- Tocopherol increased GSH by approximately 15.4%compared with control. This is because of glutathione in the reducedstate (GSH) is present in human plasma intracellular, it has antioxidantproperties to inhibit free radical formation and functions moregenerally as a redox buffer (23).

The data found in (Table 2) stated that, intraperitoneal injectionof endotoxin to normal rats significantly increased liver L-MDAconcentration after 5 hours in comparison with control group. Theseresults are in a harmony with the recorded data of Freeman and Crapo(33) who reported that, L-MDA is a major oxidation product ofperoxidized poly-unsaturated fatty acids and increased L-MDA content is

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an important indicator of lipid peroxidation and his study has shown asignificant elevation in L-MDA levels with significant decline in ratliver homogenates after LPS administration. Formation of lipid peroxidesin the crude homogenates resulted in response to the administration ofLPS. This may be due to an enhanced generation of O2

.- and H2O2 thataccelerated peroxidation of native membrane lipids. Peroxidation ofmitochondrial membrane led to loss of cell integrity, increase inmembrane permeability and alteration of Ca2+ homeostasis that contributeto cell death due to alteration in the inner membrane potential (44).Nevertheless, L-MDA an end product of lipid peroxidation was shown toincrease in the mitochondria from rat liver in response to exhaustivetreadmill running in rats. Rise in L-MDA could be due to increasedgeneration of reactive oxygen species (ROS) due to the excessiveoxidative damage generated in these rats (56). These oxygen species inturn can oxidize many other important biomolecules including membranelipids. The lipid peroxides and free radicals may be important inpathogenesis of sepsis (82).

The mean value of liver L-MDA concentration decreased significantlyin vitamin E pretreatment group (5hr.) when compared with endotoxininjected rats (5hr.). These results are agreed with the data ofMcDowell, (59) who indicated that, positive effect of vitamins E isevidence for liver MDA level decreased and it is known that vitamin E isthe rst line of defense against lipid peroxidation. Vitamin E and otherfihydrophobic antioxidants function mainly in and around themembrane/lipid bilayers, which halt lipid peroxidation by acting asperoxyl radical trapping, chain breaking antioxidants (45).

The presented data (Table 2) proposed that, liver SOD activity wasdecreased significantly in rats injected LPS (2hr. and 5hr.) group whencompared with control group. These results are in a harmony with therecorded data of Pilkhwal et al., (73) who reviewed that, LPS markedlydecreased liver SOD levels indicating oxidative stress in rats ascompared with control group after injection 4 hours and this decreasepersist after 8 hours. Decrease of SOD activities may indicate thathigh amount of hydrogen peroxide which may have accumulated inthe cells. Increased hydrogen peroxide may be transform to hydroxylradicals and increase oxidative stress (6).While, Pretreatment withvitamin E significantly increased liver SOD activity at 2 and 5hoursafter endotoxin injection when compared with endotoxin treated ratsonly. These results came in accordance with the recorded data of Sushmaet al., (85) who investigated that, tocopherol supplementation in LPS-challenged rats increased the SOD level in both pre and post-LPSchallenged groups which were supplemented with tocopherol for 15 days,possibly because α-Tocopherol decreased lipid peroxidation, leading todecreased levels of (o•-2) and endotoxemia is also accompanied bysignificant changes in the reductive oxidative balance of criticaltarget organs (93).

Liver catalase activity decreased significantly in rats injectedLPS (2hr. and 5hr.) group when compared with control group as confirmedby (Table 2).These results are in a harmony with the recorded data ofSushma et al., (85) who recorded that, LPS significantly reduced thelevels of liver catalase as compared to the control group. Although,

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decreased liver catalase after LPS administration in mice. It may besuggested that after LPS administration in the mice there may bedecreased H2O2 in the liver, and not scavenge enough those increasedoxidant burden liver tissue so not more catalaseexpression(27).Meanwhile, liver catalase activity was significantlyincreased in vitamin E pretreatment group (2 and 5hr.) when comparedwith endotoxin injected rats. These results came in accordance with therecorded data of Sushma et al., (85) who investigated that, catalaseactivity was also significantly increased at only in tocopherol

Hussein et al., (2013)

supplemented and LPS challenged group. Moreover, Gilad et al., (34)provided that, vitamin E treatment (pre or post) signi cantly reversedfidepleted catalase activity in liver and brain while no protective effectwas observed in kidney tissue. It had been thought that intracellularsuperoxide amount is increased as a result of increased NADPHoxidase enzyme of stimulated phagocytic leucocytes. At the sametime, increased SOD activity, protects the oxygen metabolizingcells against the harmful effects of free radicals such as lipidperoxidation and take place in the intracellular termination ofphagocytic bacteria. It can be thought that based on the increaseof SOD enzyme activity, hydrogen peroxide can increase as well.

From the obtained results it could be concluded that, endotoxemiacould potentially influence serum and liver tissues biochemicalparameters. So that, LPS has been reported to disrupt the cell membrane.Indeed, this work demonstrated the action of LPS is mainly due to theformation of free radicals that attack the cell membranes. This studyprovides novel evidence that, α- tocopherol (Vitamin E) decreased LPS-triggered pathogenic responses by mitigating liver damage, beingcounteracted the oxidative stress and prevented the increase of lipidperoxidation (MDA). Moreover, vitamin E protects against lipidperoxidation, oxidative stress and decrease the inflammatory response toendotoxin injection.

5- REFERENCES1. Adi,S., Pollock,A.S.,Shigenaga,J.K., Moser, A.H., Feingold, K.R.

and Grunfeld, C.1992:Role for monokines in the metabolic effects ofendotoxin. Interferon gamma restoresresponsiveness of C3H/HeJ mice invivo.JClin. Invest.89: 1603-1609.

2. Adler, V., Yin, Z.,Tew, K.D. and Ronai, Z. 1999: Role ofredoxpotential and reactive oxygen species in stresssignaling.Oncogene;18:6104–11.

3. Aebi , H. 1984:Catalase in vitro, Methods Enzymol 105: 121 – 126.4. Ahmed, R.S., Seth, V.,Pasha, S.T. and Banerjee, B.D. 2000:

Influence of dietary ginger (zingiberofficinalesrosc) onoxidativestress induced by malathion in rats. Food Chem.Toxicol. 38:443–450.

5. Alessio, H.M., Goldfarb, A.H. and Cao, G. 1997:Exercise-inducedoxidative stress before and after vitamin C supplementation.International Journal of Sports Nutrition,7: 1-9.

6. Aydin, H., Yildiz, G., Engin, A., Yilmaz, A., Çelik, K. and Bakir,S. 2010:Malondialdehyde, vitamin E, and anti-oxidant enzyme

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activity levels in patients with crimean-congo hemorrhagicfever,African Journal of MicrobiologyResearch.4: 2402-2409.

7. Ballatori, N., Hammond, C.L., Cunningham, J.B., Krance, S.M. andMarchan, R. 2005: Molecular mechanisms of reduced glutathionetransport:role of the MRP/CFTR/ABCC and OATP/SLC21A families ofmembrane proteins. Toxicol Appl Phamacol 204:238-55.

8. Baumann, H. and Gauldie, J. 1994: The acute phase response. Immunol.Today15:74-80.

9. Bella, D.L., Schock, B.C., Lim, Y., Leonard, S.W., Berry, C. andCross, C.2006: Regulation of the alpha-tocopherol transfer proteininmice: lack of response to dietary vitamin E or oxidative stress.Lipids41:105–12.

10. Benito, E. and Bosch, M.A.1997: Impaired phosphatidylcholinebiosynthesis and ascorbic acid depletion in lung duringlipopolysaccharide-induced endotoxemia in guinea pigs. Mol. CellBiochem175:117-23.

11. Beutler, E., Duron, O., Kelly MB. 1963:Improved method for thedetermination of blood glutathione.J. Lab Clin. Med. 61, 882-88.

12. Breuer, J. 1996: Report on the symposium “drug effect in clinicalchemistry methods .Eur J ClinChemclinBiochem34;385-386.

13. Burton, G.W., Joyce, A. and Ingold, K.U. 1999: Is vitamin E theonly lipid-soluble, chain-breaking antioxidant in human bloodplasma and erythrocyte membranes? Arch BiochemBiophys,221: 281-90.

14. Taillé,C., Foresti, R., Lanone, S., Zedda, C., Green, C., Aubier,M.,Motterlini, R. and Boczkowski, J. 2001:Protective Role of HemeOxygenases against Endotoxin-induced Diaphragmatic Dysfunction inRats, Am J RespirCrit Care Med.163. pp 753–761.

15. Campbell, N. A., Williamson, B. and Heyden, R.J. 2006: Biology:Exploring Life. Boston, Massachusetts: Pearson Prentice Hall. 

16. Carter, J.N ., Meredith, G.L. , Montelongo, M., Gill,D.R., Krehbiel, C.R., Payton, M.E. and Confer, A.W. 2002:Relationship of vitamin E supplementation and antimicrobialtreatment with acute-phase protein responses in cattle affected bynaturally acquired respiratory tract disease. Am J Vet Res . ; 63:1111-7.

17. Cassatella, M. A.,Bazzoni, F., Flynn, R.M., Dusi, S., Trinchieri,G. and Rossi, F. 1990: Molecular basis of interferon-gamma andlipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPHoxidase components.J. Biol. Chem., 265, 20241-20246.

Effect of vitamin E on oxidative stress in endotoxemic rat

18. Chunga, M-Y., Yeunga, S.F., Parka, H.J., Volekb, J.S. and Bruno,R.S.2010: Dietary α- and γ-tocopherol supplementation attenuateslipopolysaccharide-induced oxidative stress and inflammatory-related responses in an obese mouse model of non-alcoholic steatohepatitis,.Journal of Nutritional Biochemistry3;242-244.

19. Clements, N.C. and Habib, M.P.1995: The early pattern of conjugateddienes in liver and lung after endotoxin exposure. Am J Respir CritCare,Med,151:780–784.

14

20. Cobbold, C. A.,Sherratt, J. A. and Maxwell, S. R. J. 2002:Lipoprotein oxidation and its significance for atherosclerosis: Amathematical approach. Bulletin of Mathematical Biology, 64; 65–95.

21. Connerty,H.V.,Briggs,A.R. and Eaton,E.H.Jr.1961:Determination ofSerum, phospholipids, lipid phosphorous. In Practical ClinicalBiochemistry, 4th edn, Varley, H. ed, pp. 319–320. CBS Publishers,India

22. Coskun, O.A.F., Kanter, M. and Kuzey, G.M. 2005: Protection ofendotoxin-induced oxidative renal tissue damage of rats by vitaminE or/and EGb 761 treatment. J ApplToxicol; 25:8-12.

23. Dancygier, H. and Schirmacher, P. 2010: Free Radicals, ReactivOxygen Species, Oxidative and Endoplasmic Reticulum Stress, Springer-Verlag Berlin Heidelberg14;173-180.

24. David, F., Moore , Myrna, R., Rosenfeld, Philip,M.,Gribbon, C.,Winlove, P. and Tsai, C. M. 1997:Alpha-1-Acid (AAG,Orosomucoid) Glycoprotein: Interaction with BacterialLipopolysaccharide and Protection from Sepsis, INFLAMMATION ,21 , 69-82.

25. Davies, K. and Seranian, A. 1996: Uric acid-iron ion complexes.Biochem J 235:747 .

26. Dutta, K. andBishayi, B.2009: Escherichia coli lipopolysaccharideadministration alters antioxidant profile duringhypercholesterolemia,Indian Journal of Clinical Biochemistry,24 :179-183.

27. Elizabeth, A., Koutsos,´aLo ´pez, J.C.G.andKlasingy, K.C.2006:Carotenoids from In Ovo or Dietary Sources Blunt SystemicIndices of the In ammatory Response in Growing Chicks (Gallusflgallusdomesticus).J. Nutr.136;1031-1027.

28. Faure, P., Rossini, E., Lafond, J.L., Richard, M.J., Favier, A. andHalimi, S. 1997: Vitamin E improves the free radical defense systempotential and insulin sensitivity of rats fed high fructose diets. JNutr.127:103–107.

29. Feingold, K. R., Staprans,I., Memon,R., Moser,A.H., Shigenaga,J.K, Doerrler,W., Dinarello,C.A, and GrunfeldC.1992 : Endotoxinrapidly induces changes in lipid metabolism that producehypertriglyceridemia: low doses stimulate hepatic triglycerideproduction while high doses inhibit clearance. J Lipid Res. 33: 1765-1776.

30. Fossati, P.,Prencipe, L. and Berti, G.1980:Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system indirect enzymic assay of uric acid in serum andurine.Clin.Chem.26,227-31

31. Fournier, T.,Medjoubi-N, N. and Porquet, D.2000: Alpha-1-acidglycoprotein . Biochimic Biophysic Acta,1482:157-171.

32. Fragaakis, S.A. and Thomson, C. 2007: The Health Professional'sGuide to Popular Dietary Supplements. 3rd ed. Chicago, IL.AmericanDietetic Association.

33. Freeman, B.A. and Crapo, J.D. 1981: Hyperoxia increases oxygenradical production in rat lungs and lung mitochondria. J.Biol. Chem.256, 10 986–10 992.

34. Gilad, E., Zingarelli, B., O’Connor, M., Salzman, A.L. ,Bert ´ ok,L. and Szab´ o, C. 1996:Effects of radio detoxi ed endotoxin onfinitric oxide production in J774 macrophages and in endotoxin shock,J. Endotoxin. Res.3, 513–519.

15

35. Gmeiner , J. and Schlecht, S. 2000:Molecular composition of theouter membrane of Escherichia coli and the importance of proteinlipopolysaccharide interactions ,Archives of Microbiology ,127; 81-86.

36. Goldberg, D.M. and Spooner, R.J. 1983: Glutathione reductase.Methods of Enzymatic Analysis (Bergmeyen, H.V. Ed.) 3rd. edn. 3: 258– 265.

37. Gulec, M.,Gurel, A. and Armutcu, F. 2006: Vitamin E protectsagainst oxidative damage caused by formaldehyde in the liver andplasma of rats, Mol Cell Biochem290, 61–67.

38. Hanan and Hagar, H. 2000: Leukotriene Receptor Blocker, ZafirlukastAttenuates Lipopoly-saccharide Induced Multiple Organ DysfunctionIn Rats.Xenobiotica18, 1305–1310.

39. Harris, L.J., and Ray, S.N. 1935: Diagnosis of Vitamin C inSubnutrition by Urine Analysis Lancet1,71 .

40. Hayaishi, G.O. and Shimizu, T. 1999: Metabolic and functionalsignificance of prostaglandins in lipid peroxide research . Inlipid peroxide and medicine ,Yagi K(ed) .Academic Press:New York,pp.41-43.

41. Hirohashi, N. and Morrisonm, D. C.1996: production in vitromodulates LPS-dependent interleukin-6pretreatment of mousemacrophages Low-dose lipopolysaccharide (LPS), 64:1011. Infect.Immun.

Hussein et al., (2013)

42. Hochepied, T., Berger, F.G., Baumann, H. and Libert, C. 2003: alpha(1)-Acid glycoprotein: an acute phase protein with inflammatory andimmunomodulating properties. Cytokine & Growth Factor Reviews 14:25-34.

43. Ibrahim, W.H., Blagavan, H.N., Chopra, R.K. and Chow, C.K. 2000: Dietary coenzyme Q10 and vitamin E alter the status of these compounds in rat tissues and mitochondria. J Nutr130: 2343-2349.

44. Igbavboa, U., Zwizinski, G.W., Pferiffer, D.R. 1989: Release ofmitochondrial matrix protein through a Ca2+requiringcyclosporin-sensitive pathway. Biochem. Biophys. Res. Commun. 161, 619–625.

45. Janero, D.R., Burghardt, B. 1989: Cardiac membrane vitamin E andmalondialdehyde levels in heart muscle of normotensive andspontaneouslyhypertensive rats. Lipids24, 33–38.

46. Janeway, J.r.C.A. and Medzhitov, R.2002: Innate immunerecognition. Annu. Rev. Immunol.20:197–216.

47. John, B.H.1991: Clinical diagnosis and management by laboratorymethods.18th. ed. W. B. Saunders company. USA.

48. Johnson, A.M.,Rohlfs,E.M. and Silverman,L.M.1999:Proteins.In:BurrtisCA,Ashwood ,E.R.,eds.Tietz Text-book ofclinical chemistry.Philadelphia :WB Saunders Company ,pp.477-540.

49. Kahl, S. and Elsasser, T.H 2004: Endotoxin challenge increasesxanthine oxidaseactivity in cattle: effect of growth hormoneandvitamin E treatment,Domestic Animal Endocrinology26 ; 315–328.

50. Kheir-Eldin, A.A.,Motawi, T.K., Gad, M.Z., Abd-ElGawad, H.M.2001: Protective effect of vitamin E, β-carotene andN-acetylcysteine from the brain oxidative stress induced in

16

rats by lipopolysaccharide. The International Journal of Biochemistry & CellBiology33: 475–482.

51. Konukoglu, D., Iynem, H. and Ziylan, E.1999:Antioxidant status inexperimentalperitonitis: Effects of alpha tocopherolandtaurolin.Pharmacol. Res39:247-51.

52. La Londe, C., Nayak, U., Hennigan, J. and Demling, R.H.1997:Excessive liver oxidantstress causes mortality in response toburninjury combined with endotoxinand is prevented withantioxidants.J.Burn Care Rehabil18:187-92.

53. Lee, K.J., Andrejuk, T.,Dziuban, S.W. J.r. and Goldfarb, R.D. 1995:Deleterious effects of buthioninesulfoximine on cardiac functionduring continuous endotoxemia. ProcSocExpBiolMed ,209:178–184.

54. Lehr, H.A., Sagban, T.A., Ihling, C. Zähringer, U., Hungerer,K.D.,Blumrich, M., Reifenberg, K. and Bhakdi,S.2001:Immunopathogenesisof atherosclerosis: endotoxin acceleratesatherosclerosis inrabbits on hypercholesterolemic diet.Circulation104:914-20.

55. Leyva, F., Anker, S.D., Godsland, I.F., Teixeira, M., Hellewell,P.G., Kox, W.J.,Poole-Wilson, P.A. and Coats, A.J. 1998: Uric acidin chronic heart failure: a markerof chronic inflammation. EurHeartJ.19:1814 –1822.

56. Liu, J., Yeo, H.C., Overvik-Douki, E., Hagen, T.,Doniger, S.J.,Chu, D.W., Brooks, G.A. and Ames, B.N.2000:Chronically and acutelyexercised rats: biomarkers of oxidative stress and endogenousantioxidants.Journal of Applied Physiology, 89: 21-28.

57. Matsubara, C., Neshikawa, Y., Yoshida, Y. and Tateamura, K.1983: Aspectrophotometric method for thedetermination of free fatty acidin serum using acyl-coenzyme A synthetase and acyl-coenzyme Aoxidase.Anal. Bioc.130, 128–133.

58. Maurice, M.M., Nakamura, H., Voort, V.E.A., VlietAI, V., Staal,F.J. and Tak, P.P. 1997: Evidence for the role of an altered redoxstate in hyporesponsiveness of synovial T cells in rheumatoidarthritis. J. Immunol ; 158:1458–65.

59. McDowell, L.R.1989: Vitamins in Animal Nutrition -ComparativeAspects to Human Nutrition. In: McDowellLR (ed.).Vitamin A and E.London: Academic Press. pp. 93–131.

60. Montgomery, H. A. C. and Dymock, J .F. 1961: Colorimetricdetermination of nitric oxide. Analyst, 86:414.

61. Nadia Z. Shaban, Madiha H. Helmy, Mohamed A.R. El-Kersh , BothainaF. Mahmoud 2003:Effects of Bacillus thuringiensis toxin on hepaticlipid peroxidationand free-radical scavengers in rats given alpha-tocopheroloracetylsalicylate,Comparative Biochemistry and Physiology PartC135 ; 405–414.

62. Nathens, A.B.,Neff, M.J. and Jurkovich, G.J. 2002: Randomizedprospective trial of antioxidant supplementation in critically illsurgical patients.Ann. Surg. 236: 814-822.

63. NCEP expert panel 1988:(NCEP) Expert Panel on Detection,Evaluation, and Treatment of High Blood Cholesterol inAdults .circulation,148:36-69.

64. Nielsen, S.S., Grøfte, T., Tygstrup, N. and Vilstrup, H. 2006:Synthesis of acute phase proteins in rats with cirrhosis exposed tolipopolysaccharide, BioMedCentralComparativeHepatology,5:3.

17

65. Nishikimi, M., Roa, N.A. and Yogi, K.1972: Measurement ofsuperoxide dismutase.Biochem. Bioph. Res. Common., 46, 849 – 854.

Effect of vitamin E on oxidative stress in endotoxemic rat

66. Ognjanović, B.I., Pavlović,S. Z., Maletić, S. D., éikić, R.V.,ätajn, A. ä., Radojičić, R. M. ,Saičić, Z.S. and Petrovic, V.M.2003:Protective Influence of Vitamin E on Antioxidant DefenseSystem in the Blood of Rats Treated with Cadmium .Physiol. Res.52: 563-570.

67. Ohkawa, H., Ohishi, W., and Yagi, K.1979: Assay for lipid peroxidesin animal tissues by thiobarbituric acid reaction. Anal .Biochem ,95,351-8.

68. Onyema, O.O, Farombi, E. O., Emerole, G. O., Ukoha, A. I. andOnyeze,G.O.2006:Effect of Vitamin E on Mono-sodium GlutamateInduced Hepatoxicity and Oxidative Stress in Rats.Indian Journal ofBiochemistry & Bio- physics, 43:20-24.

69. Oruc, E.O. and Uner, N.2000: Combined effects of 2,4-D andazinphosmethyl on antioxidant enzymes and lipid peroxidation inliver of Oreochromisniloticus. Comp. Biochem. Physiol. C 127, 291–296.

70. Ouchi, K.; Tanabe, J.; Tominaga, T.; Ito, K. ; Saijo, S. andMatsuno,S.1993:Altered energy metabolism and oxidative injuryfollowing endotoxemia in rats with normal or cirrhotic livers. Res.Exp.Med.193:81-88 .

71. Paglia, D.E and Valentine,W.N.1967: Studies on the quantitative andqualitative characterization of erythrocyte glutathione peroxidaseJ. Lab. Clin. Med.70: 158 – 169.

72. Pascoe, G.A. and Reed, D.i.1988: Cell calcium, vitamin E, and thethiolredoxsystem in cytotoxicity. J Free Radic Biol Med ,6:209-24.

73. Pilkhwal, S., Tirkey, N.,Kuhad, N. and Chopra, K.2010:Effect ofBioflavonoid Quercetin on Endotoxin Induced Hepatotoxicity andOxidative Stress in Rat Liver. IJPT 9: 47-53.

74. Poli, G., Albano, E.and Dianzani, M. U. 1990: Lipid Peroxidationand Covalent Binding in the Early Function Impairment of GolgiApparatus by Carbon Tetrachloride, 8:1-10.

75. Rajpert-De Meyts, E.,Heisterkamp, N.,GroVen, J.2000: Cloning andnucleotide sequence of human gamma-glutamyltranspeptidase. Proc NatlAcad Sci USA 85:8840–8844

76. Rice,D. and Kennedy,S.1988:Vitamin E function and effects ofdeficiency .Br Vet J 144;482-495.

77. Rojas, C., Cadenas, S., Herrero, A., MCndez, J. and Barja,G.1996:endotoxin depletes ascorbate in the guinea pig heart.protective effects of vitamins C and E against oxidativestress ,Life Science,59:649-657.

78. Ross, D. and Moldeus, P.1991: Antioxidant defense systems andoxidative stress.In: C. Vigo-Pelfrey (ed). Membrane LipidOxidation. CRC, Boca Raton,Fl, pp 151–170.

79. Sakaguchi, O., Abe, H., Sakaguchi, S. and Hsu, C.C.1982:Effect of lead acetate on superoxide anion generation and itsscavengers in mice given endotoxin. Microbiol Immunol; 26: 767–778.

18

80. Sakaguchi, S. and Furusawa, S.2006: Oxidative stress andseptic shock: metabolic aspects ofoxygen derived freeradicals generated in the liver during endotoxemia, FEMSImmunolMed Microbiol;47 :167–177

81. Saw, M., Stromme, J.H.,Iondon, J.L. and Theodorsen, L.1983:IFCCmethod for g-glutamyletransferase (g-glutamyl)-peptide :aminoacidg-glutamyletransferase.ClinChem Acta . 135; 315F-338F.

82. Sharma, J.B., Sharma, A.,Bahadur, A., Vimala, N., Satyam, A. andMittal, S.2006:Oxidative stress markers and antioxidant levels innormal pregnancy and pre – eclampsia. Int J GynecolObstet . 94:23-27.

83. Silveira,V.L.F. and Limaos, E.A. 1990:Effect of bacteria endotoxinon plasma concentration of haptoglobin and fibrinogen in ratstreated with metopyrone,Agents and Actions,31,1/2.

84. Stein, E.A.1987: Lipids ,lipoproteins,and apolipoproteins. In :NWTietz, ed Fundementals of clinical chemistry.3 rded. Philadelphia :WB Saunders;448.

85. Sushma B, Kanwaljit C. and Praveen R. 2010: Vitamin ESupplementation Modulates Endotoxin-induced Liver Damage in a RatModel .Am. J. Biomed. Sci. , 2, 51-62.

86. Tandogan, B. and Ulusu, N.N.2006:Kinetic Mechanism and MolecularProperties of Glutathione Reductase, FABAD J. Pharm. Sci.,31, 230-237.

87. Tarascio, J.R.2009: Does Vitamin E Lower Triglycerides??. Am JClinNutr .; 31 :100-5.

88. Tatara, M. and Ginter, E. (1994): Erythrocyte membrane fluidity andtissue lipid peroxides in female guinea-pigs on graded vitamin Cintake. Physiol Res 43: 101-105.

89. Taylor, B.S., Alarcon, L.H. and Billiar, T.R.1998: Inducible nitricoxide synthase in the liver: regulation and function. Biochemistry 63,766–781.

90. Tietz, N.W.1995: Clinical guide to laboratory tests .3rd

ed.philadelphia :WB saunders ,pp.268-273. 91. Traber, M.G. and Packer, L.1995: Vitamin E: beyond antioxidant

function. Am J ClinNutr ;62(Suppl):1501S–9S.

Hussein et al., (2013)

92. Viviano, C.J., Bakewell, W.E., Dixon, D., Dethloff, L.A. andHookGER 1995: Altered regulation of surfactant phospholipid andprotein .A during acute pulmonary in ammation. fl Biochim Biophys Acta1259: 235–244.

93. Wang, H.H., Hung, T.M., Wei, J. and Chiang, A.N. 2004: Fish oilincreases antioxidant enzyme activities in macrophages and reducesatherosclerotic lesion in apo E knock outmice.CardiovascularRes61:169-76.

94. Williams, L. and Wilkins 2008: Nutrition and Diagnosis-Related Care.6th ed. Philadelphia, Pa: Escott-Stump S, ed.

95. Yelich, M.R. and Janusek, W.L. (1994): Glucose, lactate, insulinand somatostatin responses to endotoxin in developing rats Shock 2,438-444.

96. Zoroglu,S.S., Armutcu, F., Ozen, S., Gurel, A., Sivasli, E.,Yetkin, O., Meram, I. 2004:Increased oxidative stress and altered

19

activities of erythrocyte free radical scavenging enzymes inautism, Eur. Arch.Psychiatry Clin. Neurosci. 254: 143–147.

97. Zsila, F. , Bikadi,Z. and Hazai, E. 2008: Organogold complexesprobe a large beta-barrel cavity for human serum alpha1-acidglycoprotein, Biochimica et Biophysic aActa,1784: 1106–1114.

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ون� ي# ئ%; ا لوب�� لج�� ي�ر� ن� ه�اء م�ج� ام�ي# ت� ي# ن� سى - ق�� ام�ي# ت� ي# الان�#ر� - ق�� دB ذاى م�ال�ون�,ال- - ك�ت� وال�ده�ت# ار� دك�ت� #%BBون� رب ي# �BBاس لوب�� ي#روك�سBBدار� ., ج�� ون� ث�* ي# ئ%; ا لوب�� دج�� وق��ف�رت� �BBBBBس �ح� ا �ات% �BBBBBت ل ئ�� خلت# ب%�ى ال�ي� ا #BBBBBمت وك�ي# ي# وذ ع�ن� ال�ب* ��BBBBBاذه� وج #%BBBBBب ى ر� ر� م�ن� ك�لا ف� ي# �BBBBBكر ن��رك �BBBBBدم- س �BBBBBك� ال #BBBBBول�ت ة� - ح�مص� ال�ي� #BBBBBي ئ%; لا ون� ب�; �BBBBBده�ون�–ذه �BBBBBال

ة� ورث�# ق� �BBBBوس ة� –ال�ق� #BBBBي ره� اح�مBBBBاض� ذه�ب� �BBBBات� - ج رب�� و اسBBBBي� �BBBBي راي��ام�ب# ��BBBBن� س ن ي# رار� - الاي�� ي# �BBBBو ف �BBBBي راي��ام�ب# ��BBBBل س ن #BBBBام�ت لوب�� ا ج�� �BBBBام ار� - ج�� �BBBBت ي#رار�ام�ب# �BBBBس ف ران�� ��BBBBن-ا يØن� - ال�ف� لوي�� وج�� ي� ئ%� ا �BBBن� -1-ه ي# كBBBون��روي�� لت# ون�–ج�� ي# ئ%; ا ��BBBلوب ل ج�� ي�ر� �BBBال�مصل م�ج ة� ب�� اف� ��BBBض �الا لى ب�� اذه� ا� #%BBBب ى ر� ون� م�عBBBدل ف� ي# ئ%; ا ��BBBلوب - ج�� ر� ي# وث%� سBBBمي# د ذن�# س�ون��راك�سBBBت#

ل ي�ر� الان�#ر� م�ج� دB ذاى م�ال�ون�ال-و- - ك�ت� ى ال�ده�ت# ح� ف� Øسي د ن�� . ال�كت�21

لصBBب� ن� ع�لى ال�دراسBBة� وج�� �سBBمم ا ي#رى# ال�ت� �BBكت ر لة ال�ت� ي# ث%; �ا ��BBار ب ��BBات� ع�لى ض ��BBة� ال�مكوب #BBي �ئ% ا مت# ة� ال�كي# #�BBوث ة� ل�لمصل ال�حي# سBBج� ن�� �د وا ك� ال�كت� �BBل م�ن� وذ�لال رات� ج�� �BBض لل ا� ى وج�� ب� ف� رك�ي# ��BBاء ن ;BBس ة� ع�� #BBلي ى# ال�خ� د� �BBع ال س�اس�ا ن�#رح�� �لى ا ن� ا� ي»# كBBو ور ب�� د� �BBره� ال�خBBى# ال�ج ت� �BBم ال هBBاج�� اء ن�� ;BBس ا. وان� ع�� #�BBلاب ن� ال�خ� ح�ف�

ن� ه�اء( ام�ي# ت� ي# ي#رول )ق�� وك�وف� ا-ت�� ل�ف� �ذىا �لى ا ار م�ن� ال�خد ا� ب%; اره� الا¬ مة� ال�ص� اح�� سمم وال�ت� ي#رى# ل�لت� كت� ة� ال�ت� ي� لBف� ال�مسBب* ر ل�ت� دمي# �%Bا وب لاب�# د ج�� ل�Bك� ال�كت� وذ�لال م�ن� ع ج�� ك�سBBBده� م�ت� �ة� الا #BBBي وق�� هBBBاذ ل�لBBBده�ون� ال�ق� ج�� ك�سBBBدى والا� �ا ل ال�ت� لت# ف� ات� م�ن� وال�ت� هاب�� الال�ت� ة� اث�� خ� ي� �BBBة� اس �BBBج ي# ب� ن� ي�� ن� ح�ف� وك�سBBBي# دوت�� ب%� �ل�ك� .الا د� �BBBل

صح ي� اول ئ�# �BBBت ب� ن� ي�� ام�ي# �BBBت ي# اء ق�� �BBBى# ه دوذ ف� �BBBة� جBBBة� ال�ج�رع #BBBي ها ال�موصى ال�طي* ك� ن�� �BBBل ة� وذ� #�BBBاث لBBBف� م�ن� ل�لوق�� ا ب�� لاب�# ى# ال�خ� ت� �BBBم ال ج� ي� ور ن�#سBBBمى ع�ما ئ�� د� �BBBره� ال�خBBBو ال�ج �ارات� د� �BBBBال ، ة� �BBBف مل ال�طلت# ;BBBBس لBBBBك� ون�� مBBBBراض� ب�� �هBBBات� ال�شBBBBرط�ان� الا اض�ل وال�ت� مBBBراض� ال�مف� �ة� وا ي# �BBBلت�و ال�حساس ي#ره�ا ال�ف� مBBBراض� م�ن� وع� �الا

اره� . ال�ص�

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