Antioxidant therapy attenuates oxidative stress in chronic cardiopathy associated with Chagas' disease

International Journal of Cardiology 123 (2007) 43 – 49 www.elsevier.com/locate/ijcard

Antioxidant therapy attenuates oxidative stress in chronic cardiopathy associated with Chagas' disease Leonilda Banki Maçao a , Danilo Wilhelm Filho a,⁎, Roberto Coury Pedrosa b , Aline Pereira a , Patrícia Backes a , Moacir Aloisio Torres c , Tânia Silva Fröde d a b

d

Departamento Ecologia e Zoologia, Universidade Federal de Santa Catarina, Florianópolis, Brazil Hospital Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil c Departamento de Química, Universidade de São Paulo, São Paulo, Brazil Departamento de Análises Clínicas, Universidade Federal de Santa Catarina, Florianópolis, Brazil Received 11 September 2006; accepted 12 November 2006 Available online 27 February 2007

Abstract Oxidative stress is common in inflammatory processes of many diseases, including the Chagas' disease, which is characterized by chronic inflammation. The present study is a sequence of a related publication [Oliveira TB, Pedrosa RC, Wilhelm Filho D. Oxidative stress in chronic cardiopathy associated with Chagas' disease. Int J Cardiol in press.] on the same subjects, which showed an increase in oxidative stress associated with the progression of the severity of the disease. Components of the antioxidant system and oxidative biomarkers present in the blood were measured in the same chronic chagasic patients (n = 40), before and after vitamin E (800 IU/day) and vitamin C (500 mg/ day) supplementation for 6 months. Antioxidant enzymes and contents of reduced glutathione in erythrocytes and plasma TBARS contents were analyzed in four groups of patients in different stages of chronic Chagas heart disease (n = 10 each group, groups I, II, III, and IV) according to the Los Andes classification. After the combined vitamin supplementation, TBARS and protein carbonyl levels were decreased in plasma, whilst red cell GSH contents were increased in group I. The vitamin E contents found in the plasma were inversely related to the severity of the disease. No differences in gamma-glutamiltransferase activities were detected but the myeloperoxidase levels were decreased in patients at the initial stages, whilst seric nitric oxide levels were increased in groups II and III. After the antioxidant supplementation, CAT activity was increased in group II, GPx activity was increased in group I, GR activity was increased in groups I and II, whilst the GST activity was decreased in groups II, III and IV. The results clearly indicate that the antioxidant supplementation was able to counteract the progressive oxidative stress associated with the disease. New perspectives for the treatment of Chagas' disease might include an antioxidant therapy in order to attenuate the consequences of oxidative insult related to this disease. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Chagas' disease; Cardiopathy; Antioxidant supplementation; Vitamin E; Vitamin C; Oxidative stress

1. Introduction Recent evidences strongly suggest that damage is associated with the evolution of [1–5]. Oxidative stress is also implicated ment of heart failure [6,7], whilst chronic

oxidative stress Chagas' disease in the developinflammation is

⁎ Corresponding author. Departamento de Ecologia e Zoologia, CCB, UFSC, Trindade, Florianópolis, 88040-900, Brazil. Tel.: +55 48 3316917; fax: +55 48 3315156. E-mail address: [email protected] (D.W. Filho). 0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2006.11.118

implicated in several diseases [8,9]. In other words, chronic pathological processes and progressive inflammation can lead to alterations in the antioxidant status and to oxidative stress or redox imbalance [8]. As a consequence, the identification of changes in oxidative stress biomarkers involved in Chagas' disease can provide many important contributions to the understanding of cardiac damage in chronic chagasic patients. Chagas' disease is one of the most important medical problems in South America caused by a protozoan parasite, Trypanosoma cruzi [10], which in Brazil involves around

44

L.B. Maçao et al. / International Journal of Cardiology 123 (2007) 43–49

5 million people [11]. The disease exhibits an acute phase followed by a chronic phase, which is separated by an indeterminate period during which the patient is relatively asymptomatic. The acute phase is characterized by an active infection, inflammation and myocardial damage [12,13]. The most important clinical manifestation of this disease is chronic myocarditis, and this may affect 30% of chronically infected patients in whom symptoms of cardiopathy appear years or decades after initial infection [11]. Despite some controversy related to results obtained in randomized control trials [14,15], vitamin E and C protect cellular homeostasis against ROS damage [8]. Therefore supranutritional doses of vitamins E and C have been reported to be beneficial in several disease such as cancer, chronic inflammation, Alzheimer, Parkinson, and cardiovascular diseases, among others [16,17]. Both vitamins seem to work synergistically to protect tissues against lipid peroxidation [8]. In vitro studies have been shown that the α-tocoferoxil radical is reduced by ascorbate [18] thus preventing propagation reactions in cellular membranes [19]. The main purpose of this study was to investigate the effect of antioxidant supplementation on oxidative stress biomarkers present in the blood of chronic chagasic patients at different stages of cardiopathy associated with the disease severity. 2. Experimental design 2.1. Selection of subjects Forty patients with chronic Chagas' heart disease (n = 10 each group) were studied, and were selected among 200 patients followed-up in the Cardiomyopathy Research Clinic for Chagas' disease of the Hospital Universitário Clementino Fraga Filho. The same patients were already been studied in a recent publication of our laboratories [1], in which the blood antioxidant status was evaluated (for exclusion/ inclusion criteria and other details see Ref. [1]). All patients were clinically stable for at least 3 weeks at the moment that the blood sample for this study was collected. Regarding the nutritional status of the chagasic patients, especially considering the ingestion of nutritional antioxidants such as vitamin C and E, no important variations were detected. Patients under medical treatment (e.g., digitalis, diuretic, and/or vasodilator agents) to prevent peripheral edema had their medication interrupted for 48 h before laboratory and clinical analyses, and no clinical problems occurred during the interruption of the drug treatment. Patients were grouped according to the clinicalhemodynamic modified classification of Los Andes [20] reviewed by [21], which is based on electrocardiographic, echocardiographic (M-mode and two-dimensional echocardiogram with intracavitary Doppler), and physical examination findings. World Health Organization and Helsinki Treaty regulations (1963), were followed. The institutional

review board approved the study protocol (071/05), and a fully informed written consent was obtained from each patient. The patients were classified into four groups as follows: 10 patients in group I (normal electrocardiogram and echocardiogram: no heart involvement); 10 patients in group II (normal/borderline electrocardiogram and abnormal echocardiogram: mild heart involvement), 10 patients in group III (abnormal electrocardiogram and echocardiogram, without congestive heart failure: moderate heart involvement), and 10 patients in group IV (abnormal electrocardiogram and echocardiogram with congestive heart failure: severe heart involvement) in functional class IV of the New York Heart Association (NYHA). Biomarkers of oxidative stress in the present study were measured at two intervals, before and after a 6 months period of daily vitamin supplementation. Antioxidant supplementation was carried out through a single daily ingestion of vitamin E 800UI and vitamin C 500 mg during dinner. 2.2. Sample preparation Blood samples were obtained from the antecubital vein in chilled tubes containing EDTA as anticoagulant (or without EDTA to obtain serum) in overnight fasting subjects. Immediately after blood collection a blood fraction (200 μl) was precipitated in trichloroacetic acid (TCA 12% 1:4 v/v) for glutathione assays. The remaining whole blood collected was centrifuged at 1500×g for 10 min to separate red cells and plasma. For enzymatic assays, red cells were washed twice with cold saline solution and then centrifuged again (5000×g for 5 min), and hemolysis was carried out by freezing/thaw procedure, using a buffer containing 0.1% Triton X-100, 0.12 M NaCl, 30 mM NaPO4, pH 7.4 (1:4, v/v). After this, samples were centrifuged at 15,000×g for 10 min and the supernatants and the acid extracts were stored in liquid nitrogen (− 170 °C) until analysis of the different parameters. Enzymatic evaluations were carried out in the supernatants, while the contents of GSH were obtained in whole blood extracts. GSH and TBARS contents were examined respectively only in blood extracts and plasma immediately after blood collection. Vitamin E and protein carbonyl contents were examined in plasma, whilst gamma-glutamil transferase and myeloperoxidase activities and •NO levels were examined in serum, which samples were kept in liquid nitrogen until analyses. 2.3. Antioxidant enzyme assays Catalase activity was determined by measuring the decrease in a 10 mM hydrogen peroxide solution at 240 nm [22]. Superoxide dismutase activity was measured at 480 nm according to the method of epinephrine autoxidation [23]. Glutathione reductase activity was measured at 340 nm according to [24], by measuring the rate of NADPH oxidation. Glutathione peroxidase was

L.B. Maçao et al. / International Journal of Cardiology 123 (2007) 43–49

assayed at 340 nm according to [25] through the glutathione/ NADPH/glutathione reductase system, by the dismutation of tert-butylhydroperoxide, and glutathione S-transferase activity (GST) was determined at 340 nm using CDNB (1-chloro2,4-dinitrobenzene) as substrate and a 0.15 M GSH concentration [26]. All activities were measured in duplicate and expressed in milliliters of whole blood. 2.4. Glutathione assay Reduced glutathione (GSH) was measured at 412 nm according to [27], using Elmann's reagent (DTNB: 2dithionitrobenzoic acid). Immediately after the blood was collected, the acid extracts were obtained by the addition of whole blood portions to trichloroacetic acid 12% (1:4 v/v), and then stored in liquid nitrogen until assay. After being centrifuged at 5000×g for 5 min, the supernatants from the acid extracts were added to 0.25 mM DTNB in 0.1 M sodium phosphate buffer pH 8.0, and the formation of thiolate anion was immediately determined. Glutathione determinations were measured in duplicate and expressed in micromoles per milliliter of whole blood. 2.5. Vitamin E evaluation Determination of vitamins E in plasma was carried out by high performance liquid chromatography (HPLC) with UV detection at 292 nm [28]. An aliquot of 100 μl of plasma was added to 100 μl of ethanol and vortexed for 10 s and added to 100 μl of hexane, and again vortexed for 45 s. After centrifugation at 8000×g for 5 min, 75 μl of the supernatant (hexane) was transferred to an Eppendorf and the hexane was evaporated by nitrogen flow. 125 μl dietilether and 375 μl methanol were added and this mixture was injected in HPLC. Isocratical elution was carried out with methanol using a flow rate of 1 ml/min. Samples were measured in duplicate and the plasma concentration of α-tocoferol was determined through a standard curve and expressed as μg vitamin E ml − 1 . Vitamin C determinations were not presented due to methodological problems. 2.6. Lipoperoxidation (TBARS levels) evaluation The lipid peroxidation extent was spectrophotometrically determined at 535 nm in plasma by the thiobarbituric acid method described by [29]. The thiobarbituric reactive species amount (TBARS) was determined using an extinction coefficient of 1.56 × 105 M− 1 cm− 1. Briefly, the method consists in the plasma precipitated with trichloroacetic acid (TCA) followed by the incubation with TBA 0.76%, at 100 °C, for 60 min in a phosphate buffer at pH 7.4 (60 mM Tris–HCl and 0.1 mM DPTA). At room temperature the samples were then centrifuged (5 min, 10,000×g) and the absorbance of a pink chromophore was measured in triplicate and values were expressed in nmol TBARS ml− 1.

45

2.7. Protein carbonyl contents Protein carbonyl contents were determined by the method of [30], where 200 μl of plasma were added to 800 μl DPNH (2,4-dinitrophenyl-hydrazine), a classic carbonyl-specific reagent. Ethanol/ethyl acetate (1:1; v:v) was used to remove hydrazine excess. After this, incubation with guanidine chloride 6 mol l− 1 was performed and then the maximum absorbance in the range of 360–370 nm was recorded. The final carbonyl protein values were expressed using the extinction coefficient of 22 mmol l− 1 cm− 1. 2.8. Gamma-glutamiltransferase assay Conventional kit (GAMA GT PP, Gold Analisa Diagnóstica Ltda) was used for the determination of gamma-glutamiltransferase activity through a colorimetric method at 405 nm [31]. Four volumes of buffer (glicilglicine 206.25 mmol l− 1, sodium hydroxide 130 mmol l− 1, pH 7.9) were added to one volume of substrate (γ-glutamil3-carboxi-4-nitroanilide 32.5 mmol l− 1), at constant 37 °C. 100 μl of serum was added to 1000 μl of this reaction media and the initial absorbance was recorded together with others at 1, 2 and 3 min, and the absorbances differences (ΔA min− 1) were recorded at 405 nm and the concentrations expressed in U GGT l− 1. 2.9. Nitric oxide assay Nitric oxide (•NO) levels were measured indirectly by the method of Griess [32,33]. Serum samples were deproteinized by adding 2.5 N sodium hydroxide and zinc sulfate (10%). An aliquot of 300 μl was diluted in a solution of ammonium formate (30 μl), sodium phosphate (30 μl) and suspension of Escherichia coli EC ATCC 25922 (30 μl) diluted (1:10) in a PBS buffer. The solution was incubated for 2 h at 37 °C and centrifuged at 5000×g, for 5 min, and 250 μl of the supernatant was transferred to a cuvette containing the same volume of Griess solution [sulfanilamide 1% (p/v), phosphate acid 5% (v/v) and N-(1naftil) etilenodiamine 0.1% (p/v)] and incubated for 10 min at room temperature. The pink chromophore produced by the reaction of this reagent with NO2− was measured in an Elisa plaque (Organon-Tecknica, Roseland, New Jersey, EUA) at 540 nm. Standard curves were obtained to quantify nitrate/nitrite (•NO) in μM concentrations [34]. 2.10. Myeloperoxidase assay Aliquots of 40 μl of serum from patients or standards (MPO from human neutrofiles 0.7–140 mU ml− 1) were added to 360 μl of a buffer containing 0.167 mg ml− 1 dianisidine 2HCl and 0.0005% of H2O2). After 15 min of incubation at room temperature the enzymatic reaction was interrupted with the addition of 30 μl of sodium azide 1% [35]. After centrifugation (5000×g, for 5 min), the

46

L.B. Maçao et al. / International Journal of Cardiology 123 (2007) 43–49

Table 1 Enzymatic activities in the blood of chagasic patients before and after antioxidant supplementation Group I

Group II

Not supplemented

Supplemented

Group III

Group IV

Not Supplemented Not Supplemented Not supplemented supplemented supplemented

Supplemented

CAT (mmol min− 1 ml− 1) 8.50 ± 0.74 9.97 ± 0.82 7.12 ± 0.79 10.33 ± 1.13⁎ 7.96 ± 0.94 7.86 ± 1.60 7.64 ± 0.60 9.21 ± 1.00 SOD (U SOD ml− 1) 177.94 ± 12.05 211.94 ± 29.49 164.33 ± 11.72 192.78 ± 24.78 159.08 ± 11.56 223.85 ± 35.51 187.57 ± 32.81 218.36 ± 12.04 GPx (μmol min− 1 ml− 1) 2.71 ± 0.45 4.46 ± 0.40⁎⁎ 3.31 ± 0.39 4.14 ± 0.27 3.03 ± 0.57 4.44 ± 0.59 3.22 ± 0.37 3.51 ± 0.36 0.40 ± 0.04 0.77 ± 0.16⁎⁎⁎ 0.44 ± 0.04 0.59 ± 0.13 0.35 ± 0.05 0.52 ± 0.05⁎ 0.33 ± 0.03 0.52 ± 0.09 GR (μmol min− 1 ml− 1) GST (μmol min− 1 ml− 1) 2.79 ± 0.42 2.83 ± 0.55 3.79 ± 0.78 2.09 ± 0.53⁎ 4.38 ± 0.94 2.56 ± 0.36⁎ 4.10 ± 0.76 2.02 ± 0.32⁎ GGT (U l− 1) 36.00 ± 8.33 39.40 ± 6.64 21.00 ± 3.16 26.00 ± 4.66 24.50 ± 5.14 33.50 ± 6.35 25.00 ± 6.87 27.99 ± 5.83 n = 10 in each group; values represent mean ± S.E.M.; ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 represent significant differences between each chagasic group before and after antioxidant supplementation, by Student t-test.

supernatants and standard concentrations of MPO (0.7– 140 mU ml− 1) were measured at 450 nm in an Elisa (Organon-Tecknica, Roseland, New Jersey, EUA) plaque, and values were expressed in mU ml− 1. 2.11. Chemicals All the chemicals were of analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.12. Statistical analysis Comparisons among the different chagasic groups (I to IV) at the two different conditions, before and after supplementation, were carried out by ANOVA, with post hoc analysis using the Tukey test. Student t-test was used to compare each chagasic group before and after antioxidant supplementation. TBARS levels were evaluated in triplicate and all other parameters in duplicate, and the values were expressed in ml of plasma or whole blood. 3. Results After vitamin supplementation an increase in catalase and glutathione peroxidase activities was observed in the blood of patients from group II (p = 0.0306) and group I (p = 0.0097), respectively, whilst no differences were found

in superoxide activity (Table 1). The glutathione reductase activity was also increased in supplemented patients from groups I and III (p b 0.0001 and p = 0.0324, respectively; Table 1). Conversely, the glutathione S-transferase activity was decreased after vitamin supplementation in patients from groups II, III e IV (p = 0.0448, p = 0.0436 and p = 0.0210, respectively, Table 1). In the other hand, no differences were found in the activity of gamma-glutamiltransferase before and after antioxidant supplementation (Table 1). Reduced glutathione contents in blood were increased in patients of group I after supplementation (p = 0.0225), whilst in group II this difference was very near to the significant level (p = 0.0550). After supplementation, excepting in group IV, an increase in vitamin E levels was found in the plasma of supplemented patients (I p = 0.0119, II p = 0.0254, and III p = 0.0439; Table 2). The vitamin E profile found in the plasma of chagasic patients after supplementation showed a higher consumption of vitamin E parallel to the severity of the disease (Table 2). The vitamin C determinations did not reveal such profile probably due to methodological problems (data not shown). Lipoperoxidation measured as TBARS contents in plasma was strongly inhibited after antioxidant supplementation in all groups examined (I p = 0.0026, II p = 0.0073, III p b 0.0001, and IV p = 0.0061; Table 2). As found in TBARS levels, the protein carbonyl contents in plasma were also

Table 2 Non-enzymatic antioxidants, TBARS, protein carbonyl and nitric oxide levels and myeloperoxidase activity in the blood of chagasic patients before and after antioxidant supplementation Group I

Group II

Group III

Group IV

Not Supplemented Supplemented

Not Supplemented supplemented

Not Supplemented supplemented

Not Supplemented supplemented

1.22 ± 0.23 46.35 ± 4.59 37.70 ± 8.24 0.080 ± 0.006 8.83 ± 1.72 439.12 ± 63.11

1.13 ± 0.24 1.32 ± 0.34 32.09 ± 5.41 58.66 ± 11.44⁎ 50.75 ± 7.39 11.95 ± 0.90⁎⁎⁎ 0.084 ± 0.011 0.031 ± 0.005⁎⁎ 4.90 ± 0.16 8.54 ± 1.85⁎ 466.35 ± 63.11 335.03 ± 30.01⁎

1.41 ± 0.16 42.37 ± 3.04 40.30 ± 7.81 0.074 ± 0.008 5.14 ± 0.15 358.17 ± 13.07

GSH (μmol ml− 1) 0.82 ± 0.19 Vitamin E (μg ml− 1) 42.90 ± 7.98 TBARS (nmol ml− 1) 36.34 ± 8.91 PC (nmol mg− 1) 0.100 ± 0.020 • NO (μM) 8.39 ± 1.66 MPO (mU ml− 1) 366.00 ± 26.31

1.53 ± 0.20⁎ 88.97 ± 12.73⁎ 13.41 ± 3.13⁎⁎ 0.033 ± 0.004⁎⁎ 7.07 ± 1.23 316.45 ± 18.38

1.72 ± 0.19 78.62 ± 11.41⁎ 12.23 ± 1.73⁎⁎ 0.028 ± 0.005⁎⁎⁎ 8.64 ± 1.62 332.81 ± 26.68⁎

1.21 ± 0.12 51.92 ± 8.14 14.42 ± 2.89⁎⁎ 0.023 ± 0.005⁎⁎ 8.46 ± 1.82⁎ 343.13 ± 47.30

n = 10 in each group; PC = protein carbonyl; values represent mean ± S.E.M.; ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 represent significant differences between each chagasic group before and after antioxidant supplementation by Student t-test.

L.B. Maçao et al. / International Journal of Cardiology 123 (2007) 43–49

strongly decreased after antioxidant supplementation in all patients (group I p = 0.0092, II p b 0.0001, III p = 0.0021 and IV p = 0.0007; Table 2). In the other hand, •NO levels in patients after supplementation were increased in groups III and IV (p = 0.0328 and p = 0.0413, respectively; Table 2), whilst myeloperoxidase levels in patients belonging to groups II and III were decreased after supplementation (p = 0.0205 and p = 0.0382, respectively; Table 2). 4. Discussion After the antioxidant supplementation some antioxidant enzymes showed enhanced activities. Catalase activity was enhanced in group II whilst in groups I and IV the increases were not statistically significant. Interestingly, patients of group I examined 1 year before showed decreased activities compared to healthy subjects [1]. Red cells of vitamin Edeficient rats showed decreased catalase activity [36,37], indicating a protection of vitamin E against elevated intracellular levels of hydrogen peroxide, which might elicit apoptotic processes [38]. Despite that superoxide dismutase activities did not show significant differences, all groups of patients showed a tendency of increase activities after vitamin supplementation. However, compared to the levels found 1 year before [1], the activities were elevated in all groups after vitamin supplementation. The activities of glutathione peroxidase in blood showed a similar response compared to catalase and superoxide dismutase after antioxidant supplementation, but the only significant increase was observed in group I. GPx is associated to membranes and is able to neutralize in loco hydrogen peroxide and hydroperoxides derived from lipoperoxidation process, in a process where GSH is oxidized to generate the very toxic GSSG [39,40]. Furthermore, this enzyme has also an important role against the cytotoxicity of enhanced •NO levels, regarding both the host and the parasite [2,41]. After the antioxidant supplementation the activities of glutathione reductase showed increased values in patients from groups I and III, also suggesting an improvement in the blood reducting capacity of these patients, probably through the elevation of red cell GSH concentrations [8], which was reflected in the higher GSH values found in group I. Except for group I patients, the glutathione S-transferase activities showed decreased values in all groups after supplementation, thus reverting the profile of increased activities parallel to the severity of the disease showed in notsupplemented patients. This response was the opposite of that observed for the other antioxidant enzymes, which showed generally enhanced activities after supplementation. Considering that GST is also involved in the detoxification of hydroperoxides produced in lipoperoxidation processes [8], the decreased TBARS contents found in patients from groups II, III and IV, are probably responsible for the decreased GST activities showed by these patients.

47

Decreased GSH levels in plasma were already described in patients with dilated cardiomyopathy and cardiac failure [42,43], as well as in blood of chagasic patients examined in another related study [5]. In chronic chagasic patients from group I, the red cell contents of GSH were increased after vitamin supplementation, a response that might reflect a partial recovery of their blood reducing capacity. Chow [37] showed that GSH levels decreased faster in vitamin Edeficient rats. Accordingly, in patients from group IV (cardiac failure), which was also characterized by low levels of GSH, there was a higher vitamin E consumption compared to other groups. The combined results suggest a synergic interaction between GSH and vitamin E [8]. After supplementation all patients but those from group IV showed increased vitamin E in plasma. The patients from that group display heart failure and they probably have used more the supplemented nutritional antioxidants to counteract the higher oxidative stress associated with the severity of the disease. Accordingly, the decline in vitamin E contents observed in plasma was also correlated with the severity of the disease. In a recent study, Carvalho and collaborators [44] showed that vitamin E-deficient rats after acute infection with T. cruzi increased myocardiyte, sympatic denervation, leucopenia and enhanced monocyte differentiation toward macrophages. TBARS levels found in the plasma of all patients showed a strong decrease after supplementation. Vitamin E is considered the most effective chain-breaker of propagation reactions in membranes [8,45]. Accordingly, oral administration of vitamin E was able to decrease oxidative stress in patients with Hanseniasis, who also showed decreased contents in TBARS and protein carbonyl [46], and similar results were obtained in other diseases [47]. According to the results observed in lipoperoxidation, protein carbonyl contents were also strongly decreased after vitamin supplementation. The combined decreased of both biomarkers of oxidative stress after supplementation is a strong indication of the efficiency of vitamin E and C to counteract the progressive oxidative insult related to chronic chagasic patients [1,48]. Wen and Garg [4] demonstrated that these biomarkers were enhanced in the heart mitochondria of mice infected by T. cruzi. Although speculative, the antioxidant supplementation was probably able to attenuate oxidative stress in the heart and other organs affected by the parasite. No differences were found in gamma-glutamiltransferase activities before and after vitamin supplementation in the present study. In this regard, this result is not in agreement with recent reports that have shown that serum GGT could be a biomarker of oxidative stress [49,50]. Augmented •NO levels parallel to the progression of Chagas' disease were reported by Péres-Fuentes and collaborators [2], a correlation that was not found in our previous study [1]. Other study demonstrated that in cardiac failure iNOS expression is inversely related thereby decreasing •NO generation leading to higher production of

48

L.B. Maçao et al. / International Journal of Cardiology 123 (2007) 43–49

nitrotirosine, and also to hypertrophy of the left ventricule [51]. Naviliat and collaborators [52] also observed increased 3-nitrotirosine production in myocardium of chagasic mice, which promoted a decline in parasitism but also promoted increased damage in myocardium. Interestingly, patients with orovalvar cardiopathy also showed increased •NO levels compared to healthy subjects [1], but both exhibiting higher levels compared to the cardiopathy of chronic chagasic patients [[1], this study]. The myeloperoxidase expression was decreased after vitamin supplementation in patients from groups II and III, suggesting that the inflammatory process was attenuated by vitamin E and C. Decreasing the inflammatory process is beneficial because • NO contents in blood might be recovered, as found for patients of group III. In short, an increase condition of oxidative stress seem to accompany chronic chagasic patients [1,2]. The combined antioxidant supplementation of daily intake of 800 IU of vitamin E and 500 mg of vitamin C is able to attenuate such oxidative insult. The profile of vitamin E contents in plasma was inversely related to the severity of the disease. The antioxidant therapy sharply decreased TBARS and protein carbonyl contents in plasma essentially in all groups examined. Also, after vitamin supplementation •NO contents in blood were elevated in patients from groups III and IV. Furthermore, after supplementation, the myeloperoxidase activity decreased in groups II and III, therefore attenuating the deleterious consequences related to the chronic inflammatory process associated with the disease. Acknowledgements This work was supported in part by grants from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). DWF is a recipient of a research fellowship from CNPq (Proc. 307485/2003-0), and AP and PB are recipients of PIBIC-CNPq scholarships. We also thank the Laboratório Neo Química Com. and Ind. Ltda, which provided Vitamin C (Citroplex), and also the Grupo EMS Sigma Pharma, which provided Vitamin E (E-TABS) for antioxidant supplementation. References [1] Oliveira TB, Pedrosa RC, Wilhelm Filho D. Oxidative stress in chronic cardiopathy associated with Chagas' disease. Int J Cardiol 2007;123:43–9. [2] Péres-Fuentes R, Juegan JF, Barnabé C, et al. Severity of chronic Chagas' disease is associated with cytokine/antioxidant imbalance in chronically infected individuals. Int J Parasitol 2003;33:293–9. [3] Rivera MT, De Sousa AP, Moreno AHM, et al. Progressive Chagas' cardiomyopathy is associated with low selenium levels. Am J Trop Med Hyg 2002;66(6):706–12. [4] Wen JJ, Garg N. Oxidative modifications of mitochondrial respiratory complexes in response to the stress of Tripanosoma cruzi infection. Free Radic Biol Med 2004;37:2072–81. [5] Wen JJ, Yachelini PC, Sembaj A, Manzur RE, Garg N. Increased oxidative stress is correlated with mitochondrial dysfunction in chagasic patients. Free Radic Biol Med 2006;41(2):270–6.

[6] Dieterich S, Bieligk U, Beulich K, Hasenfuss G, Prestle J. Gene expression of antioxidative enzymes in the human heart: increased expression of catalase in the end-stage failing heart. Circulation 2000;101:33–9. [7] Mak S, Newton GE. The oxidative stress hypothesis of congestive heart failure. Radical thoughts. Chest 2001;120:2035–46. [8] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. 3rd ed. Oxford: Oxford University Press; 1999. [9] Sukkar SG, Rossi E. Oxidative stress and nutritional prevention in autoimmune rheumatic disease. Autoimmun Rev 2004;3:199–206. [10] Schmuñis GA. A tripanossomíase americana e seu impacto na saúde pública das Américas. In: Brener Z, Andrade ZA, Barral-Neto M, editors. Trypanosoma cruzi e Doença de Chagas. 2 ed. Rio de Janeiro: Guanabara Koogan; 2000. p. 1–15. [11] Rassi Jr A, Marin-Neto JA. Cardiopatia chagásica crônica. Rev Soc Cardiol Estado de São Paulo 2000;10(4):06–32. [12] Koberle F. Chagas' disease and Chagas syndromes: the pathology of American Trypanosomiasis. Adv Parasitol 1968;6:63–116. [13] Andrade ZA. Mechanisms of myocardial damage in T. cruzi infection. Ciba Foundation Symposium 99. Cytophatology of parasitic diseases. London: Pitmann Books; 1983. p. 214–33. [14] Miller ER, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005;142:37–46. [15] Stanner SA, Hughes J, Kelly CNM, Buttriss J. A review of the epidemiological evidence for the ‘antioxidant hypothesis’. Public Health Nutr 2003;7(3):407–22. [16] Brigelius-Flohé R, Kelly FJ, Salonen JT, et al. The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr 2002;76(4):703–16. [17] Padayatty SJ, Katz A, Wang Y, et al. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 2003;22(1): 18–35. [18] Ayaori M, Hisada T, Suzukawa M, et al. Plasma levels and redox status of ascorbic acid and levels of lipid peroxidation products in active and passive smokers. Environ Health Perspect 2000;108:105–8. [19] Naidoo D, Lux O. The effect of vitamin C and supplementation on lipid and urate oxidation products in plasma. Nutr Res 1998;18(6):953–61. [20] Carrasco HA. Diagnóstico de daño miocárdico en la enfermedad de Chagas. Textos de la Universidad de Los Andes: Cosenjo de Publicaciones de la Universidad de Los Andes, Merida; 1983. [21] Xavier SS. Estudo longitudinal da morbi-mortalidade cardíaca da doença de Chagas em uma coorte de um grande centro urbano: análise clínica eletrocardiográfica, radiológica e ecocardiográfica de 604 casos. Rio de Janeiro:UFRJ, 181pp. Tese. (Doutorado em Cardiologia). Programa de pós-graduação em Medicina, Universidade Federal do Rio de Janeiro; 1999. [22] Aebi H. Catalase in vitro. Methods Enzymol 1984;204:234–54. [23] Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:188–92. [24] Flohé L, Gunzler WA. Assays of glutathione peroxidase. Methods Enzymol 1984;105:114–21. [25] Calberg I, Mannervik B. Glutathione reductase from rat liver. Methods Enzymol 1985;113:484–90. [26] Habig WH, Pabst MJ, Jacoby WB. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 2000;249: 7130–9. [27] Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882–90. [28] Nicoletti G, Crescibene M, Scornaienchi M, et al. Plasma levels of vitamin E in Parkinson's disease. Arch Gerontol Geriatr 2001;33:7–12. [29] Bird RP, Draper AH. Comparative studies on different methods of malondyhaldehyde determination. Methods Enzymol 1984;90:105–10. [30] Levine RL, Garland D, Oliver CN, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186: 464–78.

L.B. Maçao et al. / International Journal of Cardiology 123 (2007) 43–49 [31] IFCC Methods for the Measurement of Catalytic Concentration of Enzymes—Part 4. IFCC Method for γ-Glutamyltransferase. J Clin Chem Clin Biochem 1983;21:633–45. [32] Green IC, Wagner DA, Glowski J, et al. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Biochem 1982;126:131–8. [33] Di Rosa M, Lalenti A, Ianaro A, Sautenbin L. Interaction between nitric oxide and cyclooxygenase pathway. Prostaglandins Leukot Essent 1996;54:229–38. [34] Fröde-Saleh TS, Calixto JB, Medeiros YS. Effects of anti-inflammatory drugs upon nitrate and myeloperoxidase levels in the mouse pleurisy induced by carrageenan. Peptides 1999;20:949–56. [35] Rao TS, Currie JL, Shaffer AF, Isakson PC. Comparative evaluation of arachidonic acid (AA) - and tetradecanoylphorbol acetate (TPA)induced dermal inflammation. Inflammation 1993;17:723–41. [36] Chow CK. Glucose and dietary vitamin E protection against catalase inactivation in the red cells of rats. Int J Vitam Nutr Res 1980;50(4): 364–9. [37] Chow CK. Oxidative damage in the red cells of vitamin E-deficient rats. Free Radic Res Commun 1992;16(4):247–58. [38] Boveris A, Cadenas E. Cellular sources and steady-state levels of reactive oxygen species. In: Clerck L, Massaro D, editors. Oxygen, gene expression and cellular, vol. 105. New York: Marcel Dekker; 1997. p. 1–25. [39] Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527–602. [40] Gaté L, Paul J, Nguyen BG, et al. Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 1999;53:169–80. [41] Romao PR, Fonseca SG, Hothersall JS, et al. Glutathione protects macrophages and Leishmania major against nitric oxide-mediated cytotoxicity. Parasitology 1999;118:559–66.

49

[42] Belch JJ, Bridges AB, Scott N, Chopra M. Oxygen free radicals and congestive heart failure. Br Heart J 1991;65:245–8. [43] Macmurray J, Chopra M, Abdullah I, et al. Evidence of oxidative stress in chronic heart failure in humans. Eur Heart J 1993;14:1493–8. [44] Carvalho LSC, Camargos ERS, Almeira CT, et al. Vitamin E deficiency enhances pathology in acute Tripanosoma cruzi-infected rats. Trans R Soc Trop Med Hyg 2006;100(11):1025–31. [45] Pryor WA. Vitamin E and heart disease: basic science to clinical intervention trials. Free Radic Biol Med 2000;28:141–64. [46] Vijayaraghavan R, Suribabu CS, Sekar B, et al. Protective role of vitamin E on the oxidative stress in Hansen's disease (Leprosy) patients. Eur J Clin Nutr 2005;59(10):1121–8. [47] Esposito E, Rotilio D, Di Matteo V, et al. A review of specific dietary antioxidants and the effects on biochemical mechanisms related to neurodegeneratives processes. Neurobiol Aging 2002;23(5):719–35. [48] Zacks MA, Wen J, Vyatkina G, Bhatia V, Garg N. An overview of chagasic cardiomyopathy. An Acad Bras Cienc 2005;77(4):695–715. [49] Lim JS, Yang JH, Chun BY, Kan S, Jacobs DR, Lee DH. Is serum γ-glutamyltransferase inversely associated with serum antioxidants as a marker of oxidative stress? Free Radic Biol Med 2004;37(7):1018–23. [50] Lee DH, Blomhoff R, Jacobs DR. Is serum gamma-glutamyltransferase a marker of oxidative stress? Free Radic Res 2004;38(6):535–9. [51] Vanderheyden M, Bartunek J, Knaapen M, et al. Hemodynamic effects of inducible nitric oxide synthase and nitrotyrosine generation in heart failure. J Heart Lung Transplant 2004;23:723–8. [52] Naviliat M, Gualco G, Cayota A, Radi R. Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice. Braz J Med Biol Res 2005;38(12):1825–34.