IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Evaluation o f Erythrocyte Malondialdehyde, Glutathione Concentration and Serum Nitric Oxide Levels in Patients with Toxoplasma gondii A. A. M. Al-Azzauy Departme nt of Medical Microbiology, College of Pharmacy ,Unive rsity of Al- Mustansiriya Recei ved in ,1,March,2010 Accepted in , 3, June , 2010 Abstract The aim of this st udy was to evaluate the biological imp ortance of the magnitude of oxidative st ress, antioxidant and the levels of nitric oxide (NO) in the female p atients infected with Toxoplasma gondii by analyzing the levels of erythrocyte malondialdehy de (M DA) as an indicator for the oxidative st ress and erythrocyte reduced glutathione (GSH) level as indicator for the antioxidant st atus and serum nitric oxide levels. This p rosp ective st udy was conducted on fifty female p atients with toxop lasmosis and thirty normal healthy females of comp arable age and sex were considered as normal control. A st atist ically significant difference was found between p atients and control group in terms of M DA, GSH and NO levels. A decrease in erythrocyte GSH levels was detected, while erythrocyte M DA and serum NO levels increased significantly as comp ared with normal healthy control. Consequently , the results suggest that t he high infection vs control of increased erythrocyte M DA and serum NO levels p robably suggest the occurrence as a mechanism of tissue change in cases of toxop lasmosis. M oreover, it is recommended that the p atient levels of M DA, GSH, and NO should be evaluated in toxop lasmosis. Introduction Toxoplasma gondii is an obligate intracellular p rotozoan in birds and mammals [1]. Toxop lasmosis is the disease that occurs when T. gondii invades and multiplies asexually as tachy zoites within the cytop lasm of nucleated cells [2]. When host immunity develops, multiplication of tachy zoites ceases and tissue cyst s form, which remain latent, esp ecially in the brain and muscle. Sexual reproduction of T. gondii occurs only in the intest inal tract of cats; the resultant oocyst p asses in the feces remain infectious up to a year in soil, depending up on the temperature and moist ure content [3]. The two major routes of transmission in humans are oral and congenital. Humans become infected with T. gondii through direct contact with oocy st s in cat feces or through eating meat contaminated with the extraintestinal form of T. gondii [4]. The diagnosis of toxop lasmosis is most critical in four group s of p atients: p regnant women who acquire infection during gestation, immunocompromised p atients and p atients with chorioretinitis [5]. In this research p atients from the first group , were used. Reactive oxy gen sp ecies degrade p olyunsaturated lip ids, forming malondialdehyde (M DA) [6]. This comp ound is a reactive aldehyde and is one of the many reactive electrop hile sp ecies that causes toxic st ress in cells and forms covalent p rotein adducts which are referred to as advanced lip oxidation end p roducts, in analogy to advanced glycation end-products [7]. The p roduction of this aldehyde is used as a biomarker to measure the level of oxidative st ress in an organism [8, 9]. Glutathione (GSH) is a cyst eine-containing p eptide found in most forms of aerobic life [10]. It is not required in the diet and is instead sy nthesized in cells from its const ituent amino IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 acids [11]. Glutathione has antioxidant p rop erties since the thiol group in its cyst eine moiety is a reducing agent and can be reversibly oxidized and reduced. It is one of the most imp ortant cellular antioxidants; it defends the cell against oxidative damage by undergoing reaction with free radicals and peroxidase [10]. Nitric oxide (NO) is a free radical, an uncharged molecule with an unp aired electron. NO p lay s multiple roles in both intracellular and extracellular signaling mechanisms [12]. This highly reactive, y et simple molecule is p roduced in the body by the isoenzy me nitric oxide sy nthase (NOS) using L-arginine as a substrate. Three iso forms of NOS have been characterized, two of them are constitut ive NOS (cNOS) and the third is inducible (iNOS) by endotoxins and cy tokines [13]. T he aim of the p resent study was t o evaluate the magnitude of oxidative st ress by estimating the level of erythrocyte M DA levels, erythrocyte GSH concentration as indicators for the antioxidative st atus and levels of serum nitric oxide in p atients with toxop lasmosis. Material and methods -S ubjects: The blood samples were collected from fifty female p atients, infected with Toxoplasma gondii and found to be positive in ELISA t est. The samples were collected from Kamal Al-Samurai hosp ital, Al-Ilwia hosp ital for maternity . None of those p atients was on a sp ecial diet or taking any antioxidant (vitamin E, C, etc) or treated with antioxidant drugs, no smoking or drinking habits and did not take any hormonal medication. Thirty normal healthy females of comp arable age and sex were considered as normal control. -Blood sample s: blood samples (5ml) transferred into p lain tubes containing (acid-citrate- dextrose) (ACD) as anticoagulant. Tubes were mixed and p laced immediately in crushed ice, then assayed within (1-2) hrs. of blood collection. Blood samples were centrifuged at (5000) rp m for (10) min., then p lasma and buffy coat were removed by asp iration. Ery throcytes were washed three times with p hosp hate buffered saline (PBS) p H= 7.4 (0.02 M p hosp hate; 0.123M NaCl). The packed cell volume (PCV) after the final wash was used for the assay of M DA and GSH concentration. Serum was st ored at (-20˚C) and used for the determination of nitric oxide level. -Chemicals: the chemicals and reagents used in this st udy were of annular grade unless otherwise sp ecified and were obtained from BDH chemicals Ltd., England; Sigma, chemicals USA; Fluka A.G., Germany . Assays -M DA: M DA was assay ed according to the method of Ohkawa et al. [14] with minor modification from Hirayama et al. [15]. The reaction to form thiobarbituric acid-reactive substances (TBA-RS) depends on the condensation of two molecules of (TBA) with one molecule of M DA to generate a reddish chromogen that absorbs light at (532) nm wave length. -Glutathione concentration: determination of erythrocyte glutathione concentration was p erformed according to the method of Virgil [16] which is a modified version of that of Beulter [17]. Virtually , all of the non protein sulfhy dry l group s of ery throcyte are in the form of reduced GSH. 5,5-Dithiobis (2-nitrobenzoic acid) DT NB is a disulfide chromogen that is readily reduced by sulfhy dry l comp ounds to an intensely y ellow comp ound. The absorbance of the reduced chromogen is measured at (412) nm and is directly p rop ortional to the GSH concentration [17]. -Nitric oxide level: serum nitrite p lus nitrate concentration as an index of serum NO levels were determined by the method described p reviously [18]. Quantification of nitrite and nitrate was based on the Griess reaction, in which chromop hore with a strong absorbance at (450)nm is formed by reaction of nitrite with a mixture of naphthy l ethy lenediamine and sulp hanilamide. The absorbance was measured in a sp ectrop hotometer to give the nitrite concentration. For nitrate detection, a second sample was treated with cop p orised cadmium in glycine buffer at p H (9.7) to reduce nitrate to nitrite, the concentration of which thus represented the tot al nitrite plus nitrate. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 A standard curve was established with a set of serial dilutions (10 -8 – 10 -3 mol/l) of sodium nitrite. All samples were assay ed in dup licate. 4-Hemoglobin level: Hb was determined using hemoglobin kit (Randox) p rocedure no. 540- UV 1996. The Hb levels were measured in the p atients and control individuals to determine the M DA level and GSH concentration. Hb, erythrocyte M DA level and GSH concentration measurements of the matched sets were performed on the same day. The results were analyzed by st udent's (t-test) to find out level of significance. P value ≤0.05 was considered as statist ically significant. Re sults Table (1) rep resents the samp le size (n), mean ± SD and significance of erythrocyte M DA level exp ressed in (n mol/g Hb) of normal healthy control and the p atients of toxop lasmosis. Erythrocyte M DA level was significantly increased in group (2) as comp ared with group (1) normal healthy control (P< 0.001). Table (2) demonst rates the mean ± SD of erythrocyte GSH concentration exp ressed in (nmol/g Hb) of normal healthy control and the p atients of toxop lasmosis. Erythrocyte GSH concentration was significantly decreased in group (2) as comp ared with group (1) normal healthy control (P< 0.001). Table (3) shows the mean ± SD of serum nitric oxide level exp ressed in (μ mol/l) of normal healthy control and the p atients of toxop lasmosis. Serum nitric oxide level was significantly increased in group (2) as comp ared with group (1) normal healthy control (P< 0.001). Discussion Toxoplasma gondii is a highly frequent obligate intracellular p rotozoan p arasite. It is reported that about one-third of the world p op ulation is infected with T. gondii; the disease has asy mptomatic p rogress in 90% of the patients with sound immune sy stems [19, 20]. It is assumed that t he malondiald ehyde (M DA) arising from the lip id peroxidation is an indicator of the oxidative st ress in tissue and cells. Lip id peroxidase is a deriv ative enzy me of feeble unsaturated fatty acid which is p roduced as a result of d ecomposition of a set of complex comp onents [21]. In this st udy , the findings revealed an incr ease in the M DA level in the erythrocy te of Toxoplasma gondii p atients as comp ared with normal healthy control (table 1). These findings are in agreement with Ulku et a l. [22] and Yazar et al. [23]. The increase of M DA level in the erythrocyte of Toxoplasma gondii p atients demonst rates the increase of lipid peroxidation. The results of the p resent study st rongly suggest that one of the main r easons for h igh M DA levels in the p atients infected with toxop lasmosis could be the decreased activity of the defense sy stem p rotecting the tissues from free radical dama ge. The p otentially harmful effects of reactive oxy gen sp ecies ar e controlled by the cellular antio xidant defense sy stem. GSH is an imp ortant constituent of intracellular p rotective mechan isms against a number of noxious st imuli including o xidative st ress. It p lay s a role in p reventing the transformation of hemo globin into methemoglobin due t o o xidation. M oreover, it maintains the sulfhydry l (- SH) group s in proteins in a reduced state and protects t hese group s against oxidation [24]. In the p resent study , erythrocy te GSH concentration was significantly decreased in the p atients infected with toxop lasmosis as comp ared with healthy control (table 2). The lower GSH concentration in p atient group can be exp lained with the o xidative st ress caused by lip id p eroxidation and depletion in the GSH concentration, which is an endo gen antioxidant. The results in table (3) showed increased level of serum nitric o xide in the Toxoplasma gondii p atients. NO is the p roduct of arginin e metabolism and one of the most effective O2- free toxins. There are also p revious st udies rep orting an increase in the NO level in p arasitic diseases [19, 22, 25]. It can be stated that the NO level increase as a defensive mechan ism to p rotect the p atient against the harmful effects of the parasite. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 As a conclusion, the increase of seru m NO lev el in the p atients infected with toxop lasmosis can be associated with t he stimulation of the cell mediated immune resp onse. Acknowle dgement: I'd like to thank the resp onsible st aff of clinical b iochemist ry laboratory in the College of Pharmacy, Al-M ust ansiriy a University for their advices and help . Re ferences 1- M otoy a, J. G. and Liesenfeld, O.,( 2004). Toxop lasmosis. T he Lancet, 363: 1965-1976. 2- Ry an, K. J.and Ray , C. G., 2004. Sherris M edical M icrobiolo gy , 4 th ed., M cGraw Hill,.722- 727. 3- Nishikawa, Y., Kawasa, O., Vielemeyer, O., Suzuki, H., Joiner, K. A., Xuar, X. and Nagasawa, H., (2007). Toxoplasma gondii infection induces apoptosis in noninfected macrophages: role of n itric oxide and other soluble factors. Parasite immunol., 29: 375-385. 4- Calderaro, A., Peruzzi, S., Piccolo, G., Gorrini, C., M ontecchini, S., Rossi, S., Ch ezz i, C. and Dettori, G.,(2009). Laboratory diagnosis of Toxoplasma gondii infection. Int. J. M ed. Sic., 6(3) : 135-136. 5- Kravetz , J. D. and Federman, D. G., (2005). Toxop lasmosis in p regnancy. Am. J. M ed., 118(3): 212-216. 6- Pry or, W. A. and Stanley, J. P., (1975). "Letter: A suggested mechanism for the production of malonaldehyde during the antoxidation of p olyunsaturated fatty acids. None enzy matic p roduction of p rostaglandin endoperoxides during antio xidation". J. Org. Ch em., 40(24) : 3615-3617. 7- Farmer, E.E. and Davo ine, C.,( 2007). Reactive electrop hile sp ecies. Curr. Op in. Plant Biol., 10(4): 380-386. 8- M oore, K. and Roberts, L. J., (1998). M easurement of lip id peroxidation. Free Radic. Res., 28(6): 659-671. 9- Del Rio, D., Stewart, A. J. and Pellegrin, N.,( 2005). A review of recent st udies on malondialdehy de as toxic molecule and biological mark er of oxidative st ress. Nutr. M etab. Cardiovasc. Dis., 15(4): 316-328. 10- M eister, A. and Anderson, M . E., (1983). Glutathione, Annu. Rev. Biochem, 52 : 711-760. 11- M eist er, A., 1988. Glutathione metabolism and its selective modification. J. Biol. Chem., 263(33): 17205-17208. 12- M oncada, S., Palmer, R. M . and Higgs, E., (1991). Nitric oxide: Phy siology , p athop hysiology and pharmacology . Pharmacol. R ev., 43: 109-112. 13- Jen Kin, D. C., Charles, I.G., T homson, L. L., Moss, D.W., Holmes, L. S. and Baylis, S. A.,( 1995). Role of nitric o xide in tumor growt h. Proc. Natl. Acad. Sci. USA, 92:4392-4396. 14- OhKawa, H., Ohishi, N. and Ya gi, K., (1979). Assay for lip id peroxides in animal tissues by thiobarbituric acid r eaction, Anal Biochem., 95: 351-358. 15- Hira Yama, A.,( 2000). He modialysis does not influence p eroxidative st ate already p resent in uremia. Nep hron, 86:436-440. 16- Virgil, F. and Geor ge, G.,(1998). Biochemical asp ects of hematolo gy , Tietz textbook of clinical chemistry by Carl A.Burt is and Edward, R., Ashwood, 2 nd ed., W.B. Saunders Company, USA ch.37: 1982-1994. 17- Beutler, E., Duron, O. and Kelly , B. M ., (1963). Improved method for the determination of blood glutathione, J. lab. Clin. M ed., 61: 882-888. 18- Cortas, N. K. and Wakid, N.W., (1990). Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin. Chem., 36: 1440-1443. 19- Kang, K. M ., Lee, J. H., Choi, I. W., Shir, D. W. and Lee, Y. H., (2004). Eff ects of INOS inhibitor on IFN-γ production and up op tosis of sp lenocytes in genetically different st rains of mice infected with Toxoplasma gondii. Korean J. Parasitol., 42: 175-183. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 20- Deorari, A. K., Broor, S., M aitreyi, R. S., Agarwal, D., Kumar, H., Paul, V. K. and Sin gh, M ., (2000). Incidence, clinical sp ectrum and outcome of intrauterine infections in neonates. J. Trop .p ediatr., 46: 155-159. 21- Koltas, I. S., Yucebilgic, G., Bilgin, R., Parsak, C. K. and Sakman, G., (2006). Serum malondialdehy de level in patients with cy stic echinoco ccosis. Saudi M ed. J., 27:1703-1705. 22- Ulku, K., Tuncay , C., Tugba, R. K., Cemil C. and Ni lgun, U. D., (2008). M alondialdehy de, Glutathione and nitric o xide levels in Toxoplasma gondii serop ositive p atients. Korean J. Parasitol., 46(4): 293-295. 23- Yazar, S., Kilic, E., Saraymen, R. and Ozbige, H., (2004). Serum malondialdehyde levels in patients infected with Plasmodium. West Indian M ed., 53:147-149. 24- Akkus, I., (1995). Effect of fre e radicals and pathop hysiological. Kony a, Turkey . M imoza p ublisher. 32, ISBN 975-543-038-5, 1-76. 25- Daubener, W., Posdziech, V., Hadding, U. and M ackenzie, C. R., (1999). Inducible anti- p arasitic effector mechanisms in human uroepethelial cells: tryp top han degradation vs. NO p roduction. M ed. M icrobiol. Immunol. 187: 143-147. Table (1) Biostatisti cal calculations and student t-test of e rythrocyte MDA level for normal healthy control (group 1) and the patients of toxoplasmosis (group 2). Erythrocyte M DA level (n mol/g Hb) Normal healthy control (group 1) Toxop lasmosis p atients (group 2) Samp le size (n) 30 50 M ean ± SD 4.43 ± 1.65 20.75 ± 2.06 Probability < 0.001* *normal healthy control (group 1) versus t oxop lasmosis p atients (group 2) Table (2) Biostatisti cal calculations and student t-test of e rythrocyte GS H concentration for normal healthy control (group 1) and the patients of toxoplasmosis (group 2). Erythrocyte GSH concentration (n mol/g Hb) Normal healthy control (group 1) Toxop lasmosis p atients (group 2) Samp le size (n) 30 50 M ean ± SD 6.95 ± 1.21 2.10 ± 0.10 Probability < 0.001* *normal healthy control (group 1) versus t oxop lasmosis p atients (group 2) Table (3) Biostatisti cal calculations and student t-test of Se rum nitric oxide level for normal healthy control (group 1) and the patients of toxoplasmosis (group 2). Serum n itric oxide level (μ mol/l) Normal healthy control (group 1) Toxop lasmosis p atients (group 2) Samp le size (n) 30 50 M ean ± SD 42.38 ± 1.51 48.47 ± 0.30 Probability < 0.001* *normal healthy control (group 1) versus t oxop lasmosis p atients (group 2) 2011) 1( 24مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد تقدیر مستوى المالون ثنائي االلدهاید وتركیز الجلوتاثایون في كریات الدم الحمر و مستوى اوكسید النتریك في مصل الدم لدى النساء المصابات بداء المقوسات زّاويـد العــمــي محـد علـمـأح ة المستنصریةـعـامـالج،فرع االحـیاء المجهریة الطبیـة ، كلیــة الصیدلـة 2010،أذار ،1البحث في استلم 2010، حزیران،3، قبل البحث في خالصةال إنَّ الهدف من هذه الدراسة هو بیان االهمیة الحیویة للزیادة الحاصلة في مستویات االجهـاد التأكسـدي ومسـتوى اوكسـید ى المـالون ثنـائي االلدیهایـد النتریك وٕانخفاض مضادات االكسدة عند النساء المصابات بداء المقوسات مـن خـالل تقـدیر مسـتو لمضــادات فـي كریــات الــدم الحمــر الــذي یعـد دلــیال لالجهــاد التأكســدي، وتركیــز الجلوتاثــایون فـي كریــات الــدم الحمــر دلــیال عینــة مـن النســاء المصــابات بــالمرض والمثبتــة 50اجریــت الدراســة علــى . االكسـدة ومســتوى اوكســید النتریــك فــي مصـولهن . عینة من نساء غیر مصابات بالمرض مجموعة سیطرة سویة 30اصابتهم مختبریا، و للنسـاء المصـابات بـالمرض اءفي مستوى المالون ثنائي االلدیهاید في كریات الدم الحمر اً ملحوظً اً أظهرت النتائج ارتفاع اءن فـي كریـاتهن الحمـر هن، كما اظهرت النتائج انخفاضًا في مسـتوى الجلوتاثـایو ئوفي مستوى اوكسید النتریك في مصول دما .سلیماتمقارنة بالنساء ال للمصـابات ومســتوى اءویمكـن القـول مـن خـالل النتـائج ان زیــادة مسـتوى المـالون ثنـائي االلدیهایـد فــي كریـات الـدم الحمـر حـدثت اوكسید النتریك في مصول دم النساء المصابات اصابة شدیدة بالمقارنـة مـع مجموعـة السـیطرة مـن المحتمـل ان تكـون . نتیجة لتغیراالنسجة في حالة االصابة بداء المقوسات