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associated with episodes of acute illness and progressive organ damage&#44; which begins in infancy and is primarily responsible for a shortened life expectancy in affected patients&#46;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">3</span></a> Rates of morbidity and mortality are still high for patients with sickle cell disease&#46; In Brazil&#44; up to 25&#37; of the children affected died during their first 5 years of life&#44; but early diagnosis and treatment might reduce these rates and improve their quality of life&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">4</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Sickle hemoglobin results from a substitution of glutamic acid to valine at the sixth amino acid position of the &#946;-globin chain&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">5</span></a> This ostensibly minor change is the origin of hemoglobin S&#44; and is responsible for significant changes in the stability and solubility of the molecule&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">6</span></a> The tendency of deoxygenated hemoglobin S to undergo polymerization underlies the innumerable expressions of the sickling syndromes with intravascular hemolysis&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">7</span></a> Free plasma hemoglobin is able to initiate lipid peroxidation&#44; and the heme&#44; which readily dissociates from methemoglobin&#44; may contribute significantly to oxidative stress&#44;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">8</span></a> which might play a significant role in the pathophysiology of sickle cell disease-related microvascular dysfunction&#44; vaso-occlusion&#44; and development of organ damage&#46;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">9</span></a> Biomarkers of oxidative stress can therefore be potentially useful&#44; 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2-mercaptoethanol&#44; pyrogallol&#44; 2&#44;2-azobis&#40;2-amidinopropane&#41;hydrochloride &#40;AAPH&#41;&#44; ethylenediaminetetraacetic acid &#40;EDTA&#41;&#44; and 5&#44;5-dithiobis-2-nitrobenzoic acid &#40;DTNB&#41; were obtained from Sigma&#8211;Aldrich &#40;St&#46; Louis&#44; MO&#44; USA&#41;&#46; Sodium and potassium phosphates&#44; saponin&#44; trichloroacetic acid&#44; and thiobarbituric acid were supplied by Vetec Ltda &#40;Rio de Janeiro&#44; RJ&#44; Brazil&#41;&#46; Sodium citrate&#44; tris&#40;hydroxymethyl&#41;aminomethane&#44; and methanol were obtained from Merck &#40;Darmstadt&#44; Germany&#41;&#46; G6-PD activity was determined using a PD410 kit by Randox Laboratories &#40;Antrim&#44; United Kingdom&#41;&#46; All organic solvents were of high quality and were double-distilled&#44; and all the other chemicals were of analytical grade&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Blood samples</span><p id="par0030" class="elsevierStylePara elsevierViewall">Blood samples were obtained from 45 children diagnosed with sickle cell disease &#40;21 males and 24 females with a mean age of 9 years&#59; range&#58; 3&#8211;13&#41; at the hematopediatric department of Hospital de Cl&#237;nicas&#44; Universidade Federal do Paran&#225; &#40;UFPR&#41;&#46; A control group consisted of 280 children without hemoglobinopathies &#40;137 males and 143 females with a mean age of 10 years old&#59; range&#58; 8&#8211;11 years&#41; who were participants of the university extension project entitled &#8220;Incidence of anemia and parasitic infections in school-aged children in municipal schools of metropolitan region of Curitiba-Parana &#8211; Brazil&#44;&#8221; from UFPR&#46; The use of human subjects was approved by the Ethical Committee for Research Involving Humans&#44; Hospital de Cl&#237;nicas&#44; UFPR&#46; Informed consent was obtained from the guardians for all the children&#46; Children with any hematological alteration were excluded from the study&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">A venous blood sample of 5<span class="elsevierStyleHsp" style=""></span>mL was collected from each patient in K3-EDTA coated tubes&#46; Aliquots &#40;200<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of whole blood were separated for determination of G6-PD activity&#46; Then&#44; samples were centrifuged at 3000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min&#46; The plasma and the buffy coat were removed by aspiration&#44; and the erythrocytes were washed with phosphate buffered saline &#40;PBS&#41; &#40;NaCl&#44; 150<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#59; NaH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span>&#44; 1&#46;9<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#59; and Na<span class="elsevierStyleInf">2</span>HPO<span class="elsevierStyleInf">4</span>&#44; 8&#46;1<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#41; three times&#46; Finally&#44; red blood cells were suspended in PBS solution and water to obtain suspensions with hematocrits of approximately 10&#37; and 40&#37; for PBS solution and of approximately 40&#37; for water solution&#46; Hemoglobin concentration was measured in all suspensions&#46; Not all analyses were performed in each specimen due to the limited volumes available&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Hematologic parameters</span><p id="par0040" class="elsevierStylePara elsevierViewall">The complete blood count was determined using the Pentra 80 electronic cell counter &#40;Horiba Medical&#44; Japan&#41;&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Methemoglobin concentration</span><p id="par0045" class="elsevierStylePara elsevierViewall">Methemoglobin concentration was determined according to a method based on Naoum et al&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">11</span></a> adapted to small volumes&#46; Aliquots &#40;100<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of 10&#37; erythrocyte suspensions were hemolyzed with 100<span class="elsevierStyleHsp" style=""></span>&#956;L of 1&#37; saponin and were stabilized in 1000<span class="elsevierStyleHsp" style=""></span>&#956;L of 60<span class="elsevierStyleHsp" style=""></span>mmol&#47;L phosphate buffer&#59; the absorbance was then determined at 630<span class="elsevierStyleHsp" style=""></span>nm &#40;for methemoglobin&#41; and at 540<span class="elsevierStyleHsp" style=""></span>nm &#40;for oxyhemoglobin&#41;&#46; Methemoglobin concentration was expressed as a percentage in relation to hemoglobin concentration&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Reduced glutathione determination</span><p id="par0050" class="elsevierStylePara elsevierViewall">Reduced glutathione &#40;GSH&#41; concentration was determined by a method previously described by Beutler&#44;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> by evaluating the reduction of 5&#44;5&#8242;-dithiobis&#40;2-nitrobenzoic acid&#41; &#40;DTNB&#41; by sulfhydryl compounds from the formation of a yellow colored anionic product whose absorbance was measured at 412<span class="elsevierStyleHsp" style=""></span>nm&#46; Aliquots of 50<span class="elsevierStyleHsp" style=""></span>&#956;L of 40&#37; suspension of red blood cell in PBS were used&#46; The GSH concentration was expressed in &#956;mol&#47;gHb&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Lipid peroxidation</span><p id="par0055" class="elsevierStylePara elsevierViewall">Lipid peroxidation of red blood cell membranes was assessed based on Cesquini et al&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">13</span></a> Aliquots &#40;600<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of a 10&#37; suspension of red blood cell were added to 250<span class="elsevierStyleHsp" style=""></span>&#956;L of 25&#37; trichloroacetic acid and 600<span class="elsevierStyleHsp" style=""></span>&#956;L of 1&#37; thiobarbituric acid&#44; boiled for 15<span class="elsevierStyleHsp" style=""></span>min at 100<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and cooled for 5<span class="elsevierStyleHsp" style=""></span>min at 0<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The absorbance of the thiobarbituric acid reactive substances &#40;TBARS&#41; formed was then read at 532<span class="elsevierStyleHsp" style=""></span>nm using <span class="elsevierStyleItalic">¿</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>156&#47;&#40;mmole<span class="elsevierStyleHsp" style=""></span>cm&#41; and the concentrations are expressed in nmol&#47;gHb&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Measurement of hemolysis</span><p id="par0060" class="elsevierStylePara elsevierViewall">Hemolysis of red blood cell was carried out as described by Banerjee et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">14</span></a> adapted to microplates by mixing 10&#37; suspension of red blood cell in PBS with varying amounts of AAPH solution &#40;providing final concentrations of 50&#44; 100&#44; and 150<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#41;&#46; This reaction mixture was incubated for 3<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>&#176;C with shaking&#46; The extent of hemolysis was determined spectrophotometrically by measuring the absorbance of the hemolysate at 540<span class="elsevierStyleHsp" style=""></span>nm in a microplate reader &#40;Thermo Scientific&#44; Thermo Plate&#44; USA&#41;&#46; Red blood cells in a solution of 200<span class="elsevierStyleHsp" style=""></span>mmol&#47;L of AAPH were used as the 100&#37; hemolysis control&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Activity of glucose6-phosphate dehydrogenase &#40;G6-PD&#41;</span><p id="par0065" class="elsevierStylePara elsevierViewall">Aliquots &#40;200<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of whole blood before erythrocyte isolation were washed with 2<span class="elsevierStyleHsp" style=""></span>mL PBS three times&#46; G6-PD activity was determined using the Cobas Mira automated analyzer &#40;Roche&#44; Mannheim&#44; Germany&#41; with the PD410 commercial kit &#40;Randox&#44; Antrim&#44; United Kingdom&#41; as described in the manufacturer&#39;s manual&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Superoxide dismutase activity</span><p id="par0070" class="elsevierStylePara elsevierViewall">The enzyme activity was based on a method adapted from Beutler<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> of the auto-oxidation of pyrogallol&#46; Aliquots of 200<span class="elsevierStyleHsp" style=""></span>&#956;L of packed red blood cell were hemolyzed with 300<span class="elsevierStyleHsp" style=""></span>&#956;L of cold deionized water&#44; and a chloroform-ethanol extract was prepared&#46; The mixture was centrifuged at 2300<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min&#46; Varying amounts of the clear supernatant extract &#40;0&#44; 20&#44; 40&#44; 60&#44; 80&#44; 100 and 300<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; were added to a solution of tris&#8211;HCl and water&#46; After 10<span class="elsevierStyleHsp" style=""></span>min&#44; 20<span class="elsevierStyleHsp" style=""></span>&#956;L of a 1<span class="elsevierStyleHsp" style=""></span>mmol&#47;L pyrogallol solution was added to each tube and the absorbance was measured at 412<span class="elsevierStyleHsp" style=""></span>nm in a microplate&#46; The amount of extract required to inhibit pyrogallol auto-oxidation by 50&#37; was used to determine the level of enzyme activity&#46;</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Catalase activity</span><p id="par0075" class="elsevierStylePara elsevierViewall">The enzyme activity was determined by a method adapted from Beutler<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> that measures the rate of decomposition of hydrogen peroxide by catalase spectrophotometrically at 240<span class="elsevierStyleHsp" style=""></span>nm&#46; Aliquots of 50<span class="elsevierStyleHsp" style=""></span>&#956;L of 40&#37; suspension of red blood cell were added to 450<span class="elsevierStyleHsp" style=""></span>&#956;L of a hemolyzing solution of &#946;-mercaptoethanol &#40;0&#46;7<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#41; and EDTA &#40;0&#46;27<span class="elsevierStyleHsp" style=""></span>mol&#47;L&#41;&#46; This solution was diluted 1&#58;100 in PBS and 10<span class="elsevierStyleHsp" style=""></span>&#956;L of the final solution was added to 990<span class="elsevierStyleHsp" style=""></span>&#956;L of hydrogen peroxide solution&#46; The decrease in absorbance of the system was measured for 10<span class="elsevierStyleHsp" style=""></span>min&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Intracellular reactive oxygen species</span><p id="par0080" class="elsevierStylePara elsevierViewall">Reactive oxygen species were determined according to a method based on L&#243;pez-Revuelta et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">15</span></a> adapted to small volumes of blood samples in a microplate&#46; Erythrocytes &#40;995<span class="elsevierStyleHsp" style=""></span>&#956;L of 10&#37;&#44; v&#47;v suspension in PBS&#41; were incubated with 5<span class="elsevierStyleHsp" style=""></span>&#956;L of dichlorodihydrofluorescein-diacetate &#40;DCFDA&#44; 10<span class="elsevierStyleHsp" style=""></span>mol&#47;L&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 30<span class="elsevierStyleHsp" style=""></span>min&#46; This suspension was diluted in 9&#46;0<span class="elsevierStyleHsp" style=""></span>mL of PBS and 37&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L of this was then added to 112&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L of PBS in 96-well plates&#46; Determination of reactive oxygen species was performed using a GloMax<span class="elsevierStyleSup">&#174;</span>-Multi Microplate Multimode Reader fluorimeter &#40;Promega Corporation&#44; USA&#41;&#46; Under these conditions&#44; DCFDA was hydrolyzed to 2&#8242;&#44;7&#8242;-dichlorodihydrofluorescein &#40;DCFH<span class="elsevierStyleInf">2</span>&#41;&#44; which then became available for oxidation by reactive oxygen species to produce fluorescent 2&#44;7-dichlorofluorescein &#40;DCF&#41;&#46; Fluorescence was determined at 530<span class="elsevierStyleHsp" style=""></span>nm after excitation at 495<span class="elsevierStyleHsp" style=""></span>nm&#46; Reactive oxygen species formation was expressed as fluorescence units &#40;UF&#41;&#47;gHb&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Statistical analyses</span><p id="par0085" class="elsevierStylePara elsevierViewall">Statistical analysis was performed using Statistica 8&#46;0 software &#40;StatSoft&#44; USA&#41;&#46; No outliers were identified&#46; The Kolmogorov&#8211;Smirnov test was used to assess the normality and all parameters were distributed normally&#46; Data were expressed as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation and compared between groups using Student&#39;s <span class="elsevierStyleItalic">t</span>-test&#59; a <span class="elsevierStyleItalic">p</span>-value &#60;0&#46;05 was considered significant&#46;</p></span></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Results</span><p id="par0090" class="elsevierStylePara elsevierViewall">Data from blood counts of healthy children and patients with sickle cell disease are illustrated in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#46; Statistically significant differences were observed for all parameters&#44; except for medium corpuscular hemoglobin &#40;MCH&#59; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall">Data from oxidative stress parameters are illustrated in <a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>&#44; comparing patients with sickle cell disease with healthy children&#46; Statistically significant differences were observed for methemoglobin&#44; TBARS&#44; percentage of hemolysis&#44; G6-PD activity&#44; and reactive oxygen species &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Discussion</span><p id="par0100" class="elsevierStylePara elsevierViewall">Normal erythrocytes suffer oxidative stress due to the production of reactive oxygen species that results from oxygen metabolism&#46; However&#44; this is efficiently repaired by the highly powerful antioxidant systems of the cell without any problematic effect&#46; Oxidative stress occurs as a result of an imbalance between reactive oxygen species production and antioxidant defenses&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">16</span></a></p><p id="par0105" class="elsevierStylePara elsevierViewall">In sickle cell disease&#44; oxidative stress may result from high levels of meta hemoglobin S&#44; which is less stable than meta hemoglobin A&#44; leading to intravascular hemolysis&#44;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">17</span></a> ischemia-reperfusion injury&#44; chronic inflammation&#44; and higher auto-oxidation of sickle hemoglobin&#46;<a class="elsevierStyleCrossRefs" href="#bib0240"><span class="elsevierStyleSup">18&#44;19</span></a> Many potential antioxidants are of interest in relation to sickle cell disease&#44;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> and several studies have demonstrated significant increases in stress markers and differing behavior in antioxidant defense systems in patients with sickle cell disease when compared to those in healthy subjects&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">21</span></a></p><p id="par0110" class="elsevierStylePara elsevierViewall">The present results for blood counts confirm several features of sickle cell disease that are already known&#44; such as the hemolytic anemia&#44;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">21</span></a> evidenced by low levels of hemoglobin<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">7</span></a> and increased levels of white blood cells and platelets&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">6</span></a></p><p id="par0115" class="elsevierStylePara elsevierViewall">As previously demonstrated&#44;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">8</span></a> methemoglobin levels are increased in individuals with sickle cell disease&#46; There is an electron transfer in the bonding interaction between the heme and the oxygen &#40;O<span class="elsevierStyleInf">2</span>&#41; in oxygenated hemoglobin&#46; When hemoglobin deoxygenates&#44; the heme iron normally remains in the ferrous state&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> In this exchange&#44; alterations wherein hemoglobin autoxidizes result in methemoglobin&#44; with the heme iron in ferric state&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">8</span></a> Alterations in erythrocyte function or structure can lead to an enhanced flow of methemoglobin that can lead to oxidative stress&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">15</span></a></p><p id="par0120" class="elsevierStylePara elsevierViewall">The increased intra- and extra-erythrocytic oxidative stress induces lipid peroxidation and membrane instability&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">14</span></a> TBARS is one of the existing biomarkers&#44; and this evaluation is an indirect quantification of lipid peroxidation processes&#44; which makes it a good indicator of pro-oxidant stimuli&#46; In accordance with results reported previously&#44;<a class="elsevierStyleCrossRefs" href="#bib0245"><span class="elsevierStyleSup">19&#44;20&#44;22</span></a> the present study observed significantly higher levels of TBARS in patients with sickle cell disease than in the controls&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Rigid and deformed sickle erythrocytes have a shortened lifespan and undergo both intravascular and extravascular hemolysis&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">23</span></a> Higher percentages of hemolysis in erythrocyte from children with sickle cell disease than in the control group were observed&#44; both in basal suspensions of erythrocytes and in suspensions incubated with an oxidizing agent&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">G6-PD is an important enzyme related to the antioxidant defense in erythrocytes&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> Higher activity of this enzyme in patients with sickle cell disease was found than in the control group&#46; It was previously reported that erythrocytes from patients with sickle cell disease have an increased percentage of reticulocytes&#44; while the activity of G6-PD in reticulocytes is normal&#44; but declines exponentially as the red cells age&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">24</span></a></p><p id="par0135" class="elsevierStylePara elsevierViewall">Sickle cells spontaneously generate approximately two times more reactive oxygen species than normal red blood cells&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">25</span></a> In accordance with the findings of George et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">26</span></a> elevated levels of reactive oxygen species in sickle erythrocytes were also demonstrated&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">Reduced glutathione &#40;GSH&#41; is present at high concentrations in erythrocytes and acts by itself or <span class="elsevierStyleItalic">via</span> glutathione peroxidase as a major reducing source to maintain cell integrity&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">17</span></a> The measurements of GSH and its oxidized form glutathione disulfide &#40;GSSG&#41; have been considered useful indicators of <span class="elsevierStyleItalic">in vivo</span> oxidative stress&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">27</span></a> The majority of studies of adults with sickle cell disease reported some deficits of endogenous synthesis of GSH&#44; probably due to its consumption by increased oxidant production&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">26&#44;28</span></a> Although Rusanova et al&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">22</span></a> showed high levels of GSH in pediatric patients with sickle cell disease&#44; the present study found no difference in GSH levels between children with sickle cell disease and the control group&#46;</p><p id="par0145" class="elsevierStylePara elsevierViewall">Superoxide dismutase can convert superoxide to hydrogen peroxide&#44; and catalase can remove excess hydrogen peroxide&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">16</span></a> According to Silva et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> the increased pro-oxidant generation in sickle cell disease results in an antioxidant deficiency&#46; However&#44; there are some discrepancies between studies on superoxide dismutase and catalase levels in this disease&#44; with some studies observing increased activity and others observing decreased levels&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">29</span></a> An increase in these enzymes activity potentially constitutes a defense mechanism in response to increased oxidative stress&#44;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">19</span></a> or might be a consequence of increased reticulocyte content in blood samples from patients with sickle cell disease&#46; However&#44; a decrease in enzyme levels was related to disease severity in patients&#46;<a class="elsevierStyleCrossRefs" href="#bib0250"><span class="elsevierStyleSup">20&#44;22</span></a> These seemingly contradictory findings could be due to differences in the extent of oxidative stress&#44; disease severity&#44; enzyme polymorphism&#44; and the enzyme co-factor&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">29</span></a> The present results showed no difference between the activities of these enzymes in children with sickle cell disease and those in healthy children&#44; according with Cho et al&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">30</span></a> with regard to catalase&#46; These results may be due to large individual variability found among patients&#46;</p><p id="par0150" class="elsevierStylePara elsevierViewall">In light of evidence suggesting that an excess of oxidative stress has implications in sickle cell disease pathophysiology&#44; the assessment of oxidative stress parameters in these patients may provide useful information regarding the use of current medications and may lead to the development of new therapeutic strategies&#46;<a class="elsevierStyleCrossRefs" href="#bib0200"><span class="elsevierStyleSup">10&#44;19&#44;20</span></a> Monitoring the oxidative stress involves the observation of different parameters associated with pro-oxidant and antioxidant biomarkers&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">27</span></a> However&#44; the use of an isolated biomarker and the measurement of individual antioxidants are not likely to be useful indexes of oxidative status&#46; The oxidant&#8211;antioxidant balance involves biochemical reactions that require the evaluation of many endpoints&#46;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">26</span></a></p><p id="par0155" class="elsevierStylePara elsevierViewall">The present study evaluated eight oxidative stress markers&#44; including pro-oxidant and antioxidant parameters&#46; The results indicate the presence of a hyperoxidative status in children with sickle cell disease&#44; which can be observed by their high levels of methemoglobin&#44; TBARS&#44; hemolysis&#44; reactive oxygen species&#44; and G6-PD activity&#46; Simple techniques were used to determine these parameters using small volumes of blood&#46; These parameters that appeared altered in children with sickle cell disease can be useful in the evaluation of disease progression and treatment&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Conflicts of interest</span><p id="par0160" class="elsevierStylePara elsevierViewall">The authors declare no conflicts of interest&#46;</p></span></span>"
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              "identificador" => "sec0025"
              "titulo" => "Hematologic parameters"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Methemoglobin concentration"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Reduced glutathione determination"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Lipid peroxidation"
            ]
            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Measurement of hemolysis"
            ]
            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "Activity of glucose6-phosphate dehydrogenase &#40;G6-PD&#41;"
            ]
            8 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "Superoxide dismutase activity"
            ]
            9 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Catalase activity"
            ]
            10 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Intracellular reactive oxygen species"
            ]
            11 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Statistical analyses"
            ]
          ]
        ]
        6 => array:2 [
          "identificador" => "sec0075"
          "titulo" => "Results"
        ]
        7 => array:2 [
          "identificador" => "sec0080"
          "titulo" => "Discussion"
        ]
        8 => array:2 [
          "identificador" => "sec0085"
          "titulo" => "Conflicts of interest"
        ]
        9 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2015-07-06"
    "fechaAceptado" => "2015-10-16"
    "PalabrasClave" => array:2 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec706078"
          "palabras" => array:3 [
            0 => "Oxidative stress"
            1 => "Sickle cell disease"
            2 => "Children"
          ]
        ]
      ]
      "pt" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Palavras-chave"
          "identificador" => "xpalclavsec706077"
          "palabras" => array:3 [
            0 => "Estresse oxidativo"
            1 => "Doen&#231;a falciforme"
            2 => "Crian&#231;as"
          ]
        ]
      ]
    ]
    "tieneResumen" => true
    "resumen" => array:2 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Objective</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">To determine eight parameters of oxidative stress markers in erythrocytes from children with sickle cell disease and compare with the same parameters in erythrocytes from healthy children&#44; since oxidative stress plays an important role in the pathophysiology of sickle cell disease and because this disease is a serious public health problem in many countries&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Blood samples were obtained from 45 children with sickle cell disease &#40;21 males and 24 females with a mean age of 9 years&#59; range&#58; 3&#8211;13 years&#41; and 280 blood samples were obtained from children without hemoglobinopathies &#40;137 males and 143 females with a mean age of 10 years&#59; range&#58; 8&#8211;11 years&#41;&#44; as a control group&#46; All blood samples were analyzed for methemoglobin&#44; reduced glutathione&#44; thiobarbituric acid reactive substances&#44; percentage of hemolysis&#44; reactive oxygen species&#44; and activity of the enzymes glucose 6-phosphate dehydrogenase&#44; superoxide dismutase&#44; and catalase&#46; Data were analyzed using Student&#39;s <span class="elsevierStyleItalic">t</span>-test and were expressed as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; A <span class="elsevierStyleItalic">p</span>-value of &#60;0&#46;05 was considered significant&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Significant differences were observed between children with sickle cell disease and the control group for the parameters methemoglobin&#44; thiobarbituric acid reactive substances&#44; hemolysis&#44; glucose 6-phosphate dehydrogenase activity&#44; and reactive oxygen species&#44; with higher levels in the patients than in the controls&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Oxidative stress parameters in children&#39;s erythrocytes were determined using simple laboratory methods with small volumes of blood&#59; these biomarkers can be useful to evaluate disease progression and outcomes in patients&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Objective"
          ]
          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
          ]
          2 => array:2 [
            "identificador" => "abst0015"
            "titulo" => "Results"
          ]
          3 => array:2 [
            "identificador" => "abst0020"
            "titulo" => "Conclusions"
          ]
        ]
      ]
      "pt" => array:3 [
        "titulo" => "Resumo"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Determinar par&#226;metros de estresse oxidativo em eritr&#243;citos de crian&#231;as com doen&#231;a falciforme e compar&#225;-los com os mesmos par&#226;metros em eritr&#243;citos de crian&#231;as saud&#225;veis&#44; pois o estresse oxidativo desempenha um importante papel na fisiopatologia da doen&#231;a falciforme&#44; considerada um s&#233;rio problema de sa&#250;de p&#250;blica em muitos pa&#237;ses&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Foram obtidas amostras de sangue de 45 crian&#231;as com doen&#231;a falciforme &#40;21 meninos e 24 meninas com m&#233;dia de 9 anos&#44; varia&#231;&#227;o de 3 a 13 anos&#41; e 280 amostras de sangue de crian&#231;as sem hemoglobinopatias &#40;137 meninos e 143 meninas com m&#233;dia de 10 anos&#44; varia&#231;&#227;o de 8 a 11 anos&#41;&#44; como grupo controle&#46; Em todas as amostras foram determinados meta-hemoglobina&#44; glutationa reduzida&#44; subst&#226;ncias reativas ao &#225;cido tiobarbit&#250;rico&#44; porcentagem de hem&#243;lise&#44; esp&#233;cies reativas de oxig&#234;nio e atividade das enzimas glucose6-fosfato desidrogenase&#44; super&#243;xido dismutase e catalase&#46; Os dados foram analisados com o teste <span class="elsevierStyleItalic">t</span> de Student e foram expressos como m&#233;dia<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>desvio padr&#227;o&#46; Um valor de <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;05 foi considerado significativo&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Foram observadas diferen&#231;as significativas entre as crian&#231;as com doen&#231;a falciforme e o grupo controle para os par&#226;metros meta-hemoglobina&#44; subst&#226;ncias reativas ao &#225;cido tiobarbit&#250;rico&#44; porcentagem de hem&#243;lise&#44; esp&#233;cies reativas de oxig&#234;nio e atividade da enzima glucose6-fosfato desidrogenase&#44; com n&#237;veis aumentados nos pacientes&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclus&#245;es</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Foi poss&#237;vel determinar par&#226;metros de estresse oxidativo em eritr&#243;citos de crian&#231;as&#44; com t&#233;cnicas laboratoriais simples e pequenos volumes de sangue&#46; Esses biomarcadores podem ser &#250;teis na avalia&#231;&#227;o da progress&#227;o e dos resultados de tratamentos da doen&#231;a&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0025"
            "titulo" => "Objetivo"
          ]
          1 => array:2 [
            "identificador" => "abst0030"
            "titulo" => "M&#233;todos"
          ]
          2 => array:2 [
            "identificador" => "abst0035"
            "titulo" => "Resultados"
          ]
          3 => array:2 [
            "identificador" => "abst0040"
            "titulo" => "Conclus&#245;es"
          ]
        ]
      ]
    ]
    "NotaPie" => array:1 [
      0 => array:2 [
        "etiqueta" => "&#9734;"
        "nota" => "<p class="elsevierStyleNotepara" id="npar0020">Please cite this article as&#58; Hermann PB&#44; Pianovski MA&#44; Henneberg R&#44; Nascimento AJ&#44; Leonart MS&#46; Erythrocyte oxidative stress markers in children with sickle cell disease&#46; J Pediatr &#40;Rio J&#41;&#46; 2016&#59;92&#58;394&#8211;9&#46;</p>"
      ]
    ]
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      0 => array:8 [
        "identificador" => "tbl0005"
        "etiqueta" => "Table 1"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "at1"
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          "leyenda" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">RBC&#44; red blood cells&#59; MCV&#44; medium corpuscular volume&#59; MCH&#44; medium corpuscular hemoglobin&#59; MCHC&#44; medium corpuscular hemoglobin concentration&#59; WBC&#44; white blood cells&#59; PLA&#44; platelets&#59; CV&#44; Pearson&#39;s coefficient of variation &#40;&#37;&#41;&#46; Data are presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46;</p>"
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                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col"><span class="elsevierStyleItalic">p</span>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">RBC &#40;10<span class="elsevierStyleSup">6</span>&#47;mm<span class="elsevierStyleSup">3</span>&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
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                        0 => array:2 [
                          "etal" => false
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                            1 => "H&#46;M&#46; Souza"
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                      "doi" => "10.1002/pbc.22269"
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                            0 => "M&#46; Aslan"
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                      "doi" => "10.1016/j.freeradbiomed.2007.08.014"
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Original article
Erythrocyte oxidative stress markers in children with sickle cell disease
Marcadores de estresse oxidativo em eritrócitos de crianças com doença falciforme
Priscila Bacarin Hermanna,
Corresponding author
prihermann@hotmail.com

Corresponding author.
, Mara Albonei Dudeque Pianovskib, Railson Henneberga, Aguinaldo José Nascimentoa, Maria Suely Soares Leonarta
a Department of Clinical Analysis, Clinical Laboratory, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil
b Department of Pediatric Hematology and Oncology, Hospital de Clínicas, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil
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    "cabecera" => "<span class="elsevierStyleTextfn">Original article</span>"
    "titulo" => "Erythrocyte oxidative stress markers in children with sickle cell disease"
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        "autoresLista" => "Priscila Bacarin Hermann, Mara Albonei Dudeque Pianovski, Railson Henneberg, Aguinaldo Jos&#233; Nascimento, Maria Suely Soares Leonart"
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            "nombre" => "Mara Albonei Dudeque"
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            "nombre" => "Maria Suely Soares"
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            "entidad" => "Department of Clinical Analysis&#44; Clinical Laboratory&#44; Universidade Federal do Paran&#225; &#40;UFPR&#41;&#44; Curitiba&#44; PR&#44; Brazil"
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            "entidad" => "Department of Pediatric Hematology and Oncology&#44; Hospital de Cl&#237;nicas&#44; Universidade Federal do Paran&#225; &#40;UFPR&#41;&#44; Curitiba&#44; PR&#44; Brazil"
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    "titulosAlternativos" => array:1 [
      "pt" => array:1 [
        "titulo" => "Marcadores de estresse oxidativo em eritr&#243;citos de crian&#231;as com doen&#231;a falciforme"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Sickle cell disease is one of the most common hematologic disorders in the world and is a serious public health problem in many countries&#44; including Brazil&#46;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">1</span></a> There are over 2 million Brazilian carriers of the sickle gene&#44; and this disease is estimated to have an incidence of one in every 1000 live births&#46; In 2001&#44; a decree of the Ministry of Health included screening for hemoglobinopathies in the pre-existing screening programs&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">2</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Sickle cell disease has been characterized as a multi-system disease&#44; associated with episodes of acute illness and progressive organ damage&#44; which begins in infancy and is primarily responsible for a shortened life expectancy in affected patients&#46;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">3</span></a> Rates of morbidity and mortality are still high for patients with sickle cell disease&#46; In Brazil&#44; up to 25&#37; of the children affected died during their first 5 years of life&#44; but early diagnosis and treatment might reduce these rates and improve their quality of life&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">4</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Sickle hemoglobin results from a substitution of glutamic acid to valine at the sixth amino acid position of the &#946;-globin chain&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">5</span></a> This ostensibly minor change is the origin of hemoglobin S&#44; and is responsible for significant changes in the stability and solubility of the molecule&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">6</span></a> The tendency of deoxygenated hemoglobin S to undergo polymerization underlies the innumerable expressions of the sickling syndromes with intravascular hemolysis&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">7</span></a> Free plasma hemoglobin is able to initiate lipid peroxidation&#44; and the heme&#44; which readily dissociates from methemoglobin&#44; may contribute significantly to oxidative stress&#44;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">8</span></a> which might play a significant role in the pathophysiology of sickle cell disease-related microvascular dysfunction&#44; vaso-occlusion&#44; and development of organ damage&#46;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">9</span></a> Biomarkers of oxidative stress can therefore be potentially useful&#44; both to identify patients who are at high risk of oxidative damage and to evaluate the effects of anti-oxidative therapies&#46;<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">10</span></a></p><p id="par0020" class="elsevierStylePara elsevierViewall">The purpose of this work was to evaluate the parameters of oxidative stress in erythrocytes from children with sickle cell disease&#44; including percentages of hemolysis&#44; methemoglobin&#44; reduced glutathione&#44; thiobarbituric acid-reactive substances&#44; glucose 6-phosphate dehydrogenase activity&#44; reactive oxygen species&#44; and the anti-oxidant enzymes catalase and superoxide dismutase&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Chemicals</span><p id="par0025" class="elsevierStylePara elsevierViewall">Meta-phosphoric acid&#44; 2-mercaptoethanol&#44; pyrogallol&#44; 2&#44;2-azobis&#40;2-amidinopropane&#41;hydrochloride &#40;AAPH&#41;&#44; ethylenediaminetetraacetic acid &#40;EDTA&#41;&#44; and 5&#44;5-dithiobis-2-nitrobenzoic acid &#40;DTNB&#41; were obtained from Sigma&#8211;Aldrich &#40;St&#46; Louis&#44; MO&#44; USA&#41;&#46; Sodium and potassium phosphates&#44; saponin&#44; trichloroacetic acid&#44; and thiobarbituric acid were supplied by Vetec Ltda &#40;Rio de Janeiro&#44; RJ&#44; Brazil&#41;&#46; Sodium citrate&#44; tris&#40;hydroxymethyl&#41;aminomethane&#44; and methanol were obtained from Merck &#40;Darmstadt&#44; Germany&#41;&#46; G6-PD activity was determined using a PD410 kit by Randox Laboratories &#40;Antrim&#44; United Kingdom&#41;&#46; All organic solvents were of high quality and were double-distilled&#44; and all the other chemicals were of analytical grade&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Blood samples</span><p id="par0030" class="elsevierStylePara elsevierViewall">Blood samples were obtained from 45 children diagnosed with sickle cell disease &#40;21 males and 24 females with a mean age of 9 years&#59; range&#58; 3&#8211;13&#41; at the hematopediatric department of Hospital de Cl&#237;nicas&#44; Universidade Federal do Paran&#225; &#40;UFPR&#41;&#46; A control group consisted of 280 children without hemoglobinopathies &#40;137 males and 143 females with a mean age of 10 years old&#59; range&#58; 8&#8211;11 years&#41; who were participants of the university extension project entitled &#8220;Incidence of anemia and parasitic infections in school-aged children in municipal schools of metropolitan region of Curitiba-Parana &#8211; Brazil&#44;&#8221; from UFPR&#46; The use of human subjects was approved by the Ethical Committee for Research Involving Humans&#44; Hospital de Cl&#237;nicas&#44; UFPR&#46; Informed consent was obtained from the guardians for all the children&#46; Children with any hematological alteration were excluded from the study&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">A venous blood sample of 5<span class="elsevierStyleHsp" style=""></span>mL was collected from each patient in K3-EDTA coated tubes&#46; Aliquots &#40;200<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of whole blood were separated for determination of G6-PD activity&#46; Then&#44; samples were centrifuged at 3000<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min&#46; The plasma and the buffy coat were removed by aspiration&#44; and the erythrocytes were washed with phosphate buffered saline &#40;PBS&#41; &#40;NaCl&#44; 150<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#59; NaH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span>&#44; 1&#46;9<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#59; and Na<span class="elsevierStyleInf">2</span>HPO<span class="elsevierStyleInf">4</span>&#44; 8&#46;1<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#41; three times&#46; Finally&#44; red blood cells were suspended in PBS solution and water to obtain suspensions with hematocrits of approximately 10&#37; and 40&#37; for PBS solution and of approximately 40&#37; for water solution&#46; Hemoglobin concentration was measured in all suspensions&#46; Not all analyses were performed in each specimen due to the limited volumes available&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Hematologic parameters</span><p id="par0040" class="elsevierStylePara elsevierViewall">The complete blood count was determined using the Pentra 80 electronic cell counter &#40;Horiba Medical&#44; Japan&#41;&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Methemoglobin concentration</span><p id="par0045" class="elsevierStylePara elsevierViewall">Methemoglobin concentration was determined according to a method based on Naoum et al&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">11</span></a> adapted to small volumes&#46; Aliquots &#40;100<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of 10&#37; erythrocyte suspensions were hemolyzed with 100<span class="elsevierStyleHsp" style=""></span>&#956;L of 1&#37; saponin and were stabilized in 1000<span class="elsevierStyleHsp" style=""></span>&#956;L of 60<span class="elsevierStyleHsp" style=""></span>mmol&#47;L phosphate buffer&#59; the absorbance was then determined at 630<span class="elsevierStyleHsp" style=""></span>nm &#40;for methemoglobin&#41; and at 540<span class="elsevierStyleHsp" style=""></span>nm &#40;for oxyhemoglobin&#41;&#46; Methemoglobin concentration was expressed as a percentage in relation to hemoglobin concentration&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Reduced glutathione determination</span><p id="par0050" class="elsevierStylePara elsevierViewall">Reduced glutathione &#40;GSH&#41; concentration was determined by a method previously described by Beutler&#44;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> by evaluating the reduction of 5&#44;5&#8242;-dithiobis&#40;2-nitrobenzoic acid&#41; &#40;DTNB&#41; by sulfhydryl compounds from the formation of a yellow colored anionic product whose absorbance was measured at 412<span class="elsevierStyleHsp" style=""></span>nm&#46; Aliquots of 50<span class="elsevierStyleHsp" style=""></span>&#956;L of 40&#37; suspension of red blood cell in PBS were used&#46; The GSH concentration was expressed in &#956;mol&#47;gHb&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Lipid peroxidation</span><p id="par0055" class="elsevierStylePara elsevierViewall">Lipid peroxidation of red blood cell membranes was assessed based on Cesquini et al&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">13</span></a> Aliquots &#40;600<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of a 10&#37; suspension of red blood cell were added to 250<span class="elsevierStyleHsp" style=""></span>&#956;L of 25&#37; trichloroacetic acid and 600<span class="elsevierStyleHsp" style=""></span>&#956;L of 1&#37; thiobarbituric acid&#44; boiled for 15<span class="elsevierStyleHsp" style=""></span>min at 100<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and cooled for 5<span class="elsevierStyleHsp" style=""></span>min at 0<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The absorbance of the thiobarbituric acid reactive substances &#40;TBARS&#41; formed was then read at 532<span class="elsevierStyleHsp" style=""></span>nm using <span class="elsevierStyleItalic">¿</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>156&#47;&#40;mmole<span class="elsevierStyleHsp" style=""></span>cm&#41; and the concentrations are expressed in nmol&#47;gHb&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Measurement of hemolysis</span><p id="par0060" class="elsevierStylePara elsevierViewall">Hemolysis of red blood cell was carried out as described by Banerjee et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">14</span></a> adapted to microplates by mixing 10&#37; suspension of red blood cell in PBS with varying amounts of AAPH solution &#40;providing final concentrations of 50&#44; 100&#44; and 150<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#41;&#46; This reaction mixture was incubated for 3<span class="elsevierStyleHsp" style=""></span>h at 37<span class="elsevierStyleHsp" style=""></span>&#176;C with shaking&#46; The extent of hemolysis was determined spectrophotometrically by measuring the absorbance of the hemolysate at 540<span class="elsevierStyleHsp" style=""></span>nm in a microplate reader &#40;Thermo Scientific&#44; Thermo Plate&#44; USA&#41;&#46; Red blood cells in a solution of 200<span class="elsevierStyleHsp" style=""></span>mmol&#47;L of AAPH were used as the 100&#37; hemolysis control&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Activity of glucose6-phosphate dehydrogenase &#40;G6-PD&#41;</span><p id="par0065" class="elsevierStylePara elsevierViewall">Aliquots &#40;200<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; of whole blood before erythrocyte isolation were washed with 2<span class="elsevierStyleHsp" style=""></span>mL PBS three times&#46; G6-PD activity was determined using the Cobas Mira automated analyzer &#40;Roche&#44; Mannheim&#44; Germany&#41; with the PD410 commercial kit &#40;Randox&#44; Antrim&#44; United Kingdom&#41; as described in the manufacturer&#39;s manual&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Superoxide dismutase activity</span><p id="par0070" class="elsevierStylePara elsevierViewall">The enzyme activity was based on a method adapted from Beutler<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> of the auto-oxidation of pyrogallol&#46; Aliquots of 200<span class="elsevierStyleHsp" style=""></span>&#956;L of packed red blood cell were hemolyzed with 300<span class="elsevierStyleHsp" style=""></span>&#956;L of cold deionized water&#44; and a chloroform-ethanol extract was prepared&#46; The mixture was centrifuged at 2300<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 10<span class="elsevierStyleHsp" style=""></span>min&#46; Varying amounts of the clear supernatant extract &#40;0&#44; 20&#44; 40&#44; 60&#44; 80&#44; 100 and 300<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; were added to a solution of tris&#8211;HCl and water&#46; After 10<span class="elsevierStyleHsp" style=""></span>min&#44; 20<span class="elsevierStyleHsp" style=""></span>&#956;L of a 1<span class="elsevierStyleHsp" style=""></span>mmol&#47;L pyrogallol solution was added to each tube and the absorbance was measured at 412<span class="elsevierStyleHsp" style=""></span>nm in a microplate&#46; The amount of extract required to inhibit pyrogallol auto-oxidation by 50&#37; was used to determine the level of enzyme activity&#46;</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Catalase activity</span><p id="par0075" class="elsevierStylePara elsevierViewall">The enzyme activity was determined by a method adapted from Beutler<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">12</span></a> that measures the rate of decomposition of hydrogen peroxide by catalase spectrophotometrically at 240<span class="elsevierStyleHsp" style=""></span>nm&#46; Aliquots of 50<span class="elsevierStyleHsp" style=""></span>&#956;L of 40&#37; suspension of red blood cell were added to 450<span class="elsevierStyleHsp" style=""></span>&#956;L of a hemolyzing solution of &#946;-mercaptoethanol &#40;0&#46;7<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#41; and EDTA &#40;0&#46;27<span class="elsevierStyleHsp" style=""></span>mol&#47;L&#41;&#46; This solution was diluted 1&#58;100 in PBS and 10<span class="elsevierStyleHsp" style=""></span>&#956;L of the final solution was added to 990<span class="elsevierStyleHsp" style=""></span>&#956;L of hydrogen peroxide solution&#46; The decrease in absorbance of the system was measured for 10<span class="elsevierStyleHsp" style=""></span>min&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Intracellular reactive oxygen species</span><p id="par0080" class="elsevierStylePara elsevierViewall">Reactive oxygen species were determined according to a method based on L&#243;pez-Revuelta et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">15</span></a> adapted to small volumes of blood samples in a microplate&#46; Erythrocytes &#40;995<span class="elsevierStyleHsp" style=""></span>&#956;L of 10&#37;&#44; v&#47;v suspension in PBS&#41; were incubated with 5<span class="elsevierStyleHsp" style=""></span>&#956;L of dichlorodihydrofluorescein-diacetate &#40;DCFDA&#44; 10<span class="elsevierStyleHsp" style=""></span>mol&#47;L&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 30<span class="elsevierStyleHsp" style=""></span>min&#46; This suspension was diluted in 9&#46;0<span class="elsevierStyleHsp" style=""></span>mL of PBS and 37&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L of this was then added to 112&#46;5<span class="elsevierStyleHsp" style=""></span>&#956;L of PBS in 96-well plates&#46; Determination of reactive oxygen species was performed using a GloMax<span class="elsevierStyleSup">&#174;</span>-Multi Microplate Multimode Reader fluorimeter &#40;Promega Corporation&#44; USA&#41;&#46; Under these conditions&#44; DCFDA was hydrolyzed to 2&#8242;&#44;7&#8242;-dichlorodihydrofluorescein &#40;DCFH<span class="elsevierStyleInf">2</span>&#41;&#44; which then became available for oxidation by reactive oxygen species to produce fluorescent 2&#44;7-dichlorofluorescein &#40;DCF&#41;&#46; Fluorescence was determined at 530<span class="elsevierStyleHsp" style=""></span>nm after excitation at 495<span class="elsevierStyleHsp" style=""></span>nm&#46; Reactive oxygen species formation was expressed as fluorescence units &#40;UF&#41;&#47;gHb&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Statistical analyses</span><p id="par0085" class="elsevierStylePara elsevierViewall">Statistical analysis was performed using Statistica 8&#46;0 software &#40;StatSoft&#44; USA&#41;&#46; No outliers were identified&#46; The Kolmogorov&#8211;Smirnov test was used to assess the normality and all parameters were distributed normally&#46; Data were expressed as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation and compared between groups using Student&#39;s <span class="elsevierStyleItalic">t</span>-test&#59; a <span class="elsevierStyleItalic">p</span>-value &#60;0&#46;05 was considered significant&#46;</p></span></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Results</span><p id="par0090" class="elsevierStylePara elsevierViewall">Data from blood counts of healthy children and patients with sickle cell disease are illustrated in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#46; Statistically significant differences were observed for all parameters&#44; except for medium corpuscular hemoglobin &#40;MCH&#59; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall">Data from oxidative stress parameters are illustrated in <a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>&#44; comparing patients with sickle cell disease with healthy children&#46; Statistically significant differences were observed for methemoglobin&#44; TBARS&#44; percentage of hemolysis&#44; G6-PD activity&#44; and reactive oxygen species &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Discussion</span><p id="par0100" class="elsevierStylePara elsevierViewall">Normal erythrocytes suffer oxidative stress due to the production of reactive oxygen species that results from oxygen metabolism&#46; However&#44; this is efficiently repaired by the highly powerful antioxidant systems of the cell without any problematic effect&#46; Oxidative stress occurs as a result of an imbalance between reactive oxygen species production and antioxidant defenses&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">16</span></a></p><p id="par0105" class="elsevierStylePara elsevierViewall">In sickle cell disease&#44; oxidative stress may result from high levels of meta hemoglobin S&#44; which is less stable than meta hemoglobin A&#44; leading to intravascular hemolysis&#44;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">17</span></a> ischemia-reperfusion injury&#44; chronic inflammation&#44; and higher auto-oxidation of sickle hemoglobin&#46;<a class="elsevierStyleCrossRefs" href="#bib0240"><span class="elsevierStyleSup">18&#44;19</span></a> Many potential antioxidants are of interest in relation to sickle cell disease&#44;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> and several studies have demonstrated significant increases in stress markers and differing behavior in antioxidant defense systems in patients with sickle cell disease when compared to those in healthy subjects&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">21</span></a></p><p id="par0110" class="elsevierStylePara elsevierViewall">The present results for blood counts confirm several features of sickle cell disease that are already known&#44; such as the hemolytic anemia&#44;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">21</span></a> evidenced by low levels of hemoglobin<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">7</span></a> and increased levels of white blood cells and platelets&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">6</span></a></p><p id="par0115" class="elsevierStylePara elsevierViewall">As previously demonstrated&#44;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">8</span></a> methemoglobin levels are increased in individuals with sickle cell disease&#46; There is an electron transfer in the bonding interaction between the heme and the oxygen &#40;O<span class="elsevierStyleInf">2</span>&#41; in oxygenated hemoglobin&#46; When hemoglobin deoxygenates&#44; the heme iron normally remains in the ferrous state&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> In this exchange&#44; alterations wherein hemoglobin autoxidizes result in methemoglobin&#44; with the heme iron in ferric state&#46;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">8</span></a> Alterations in erythrocyte function or structure can lead to an enhanced flow of methemoglobin that can lead to oxidative stress&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">15</span></a></p><p id="par0120" class="elsevierStylePara elsevierViewall">The increased intra- and extra-erythrocytic oxidative stress induces lipid peroxidation and membrane instability&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">14</span></a> TBARS is one of the existing biomarkers&#44; and this evaluation is an indirect quantification of lipid peroxidation processes&#44; which makes it a good indicator of pro-oxidant stimuli&#46; In accordance with results reported previously&#44;<a class="elsevierStyleCrossRefs" href="#bib0245"><span class="elsevierStyleSup">19&#44;20&#44;22</span></a> the present study observed significantly higher levels of TBARS in patients with sickle cell disease than in the controls&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Rigid and deformed sickle erythrocytes have a shortened lifespan and undergo both intravascular and extravascular hemolysis&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">23</span></a> Higher percentages of hemolysis in erythrocyte from children with sickle cell disease than in the control group were observed&#44; both in basal suspensions of erythrocytes and in suspensions incubated with an oxidizing agent&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">G6-PD is an important enzyme related to the antioxidant defense in erythrocytes&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> Higher activity of this enzyme in patients with sickle cell disease was found than in the control group&#46; It was previously reported that erythrocytes from patients with sickle cell disease have an increased percentage of reticulocytes&#44; while the activity of G6-PD in reticulocytes is normal&#44; but declines exponentially as the red cells age&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">24</span></a></p><p id="par0135" class="elsevierStylePara elsevierViewall">Sickle cells spontaneously generate approximately two times more reactive oxygen species than normal red blood cells&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">25</span></a> In accordance with the findings of George et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">26</span></a> elevated levels of reactive oxygen species in sickle erythrocytes were also demonstrated&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">Reduced glutathione &#40;GSH&#41; is present at high concentrations in erythrocytes and acts by itself or <span class="elsevierStyleItalic">via</span> glutathione peroxidase as a major reducing source to maintain cell integrity&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">17</span></a> The measurements of GSH and its oxidized form glutathione disulfide &#40;GSSG&#41; have been considered useful indicators of <span class="elsevierStyleItalic">in vivo</span> oxidative stress&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">27</span></a> The majority of studies of adults with sickle cell disease reported some deficits of endogenous synthesis of GSH&#44; probably due to its consumption by increased oxidant production&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">26&#44;28</span></a> Although Rusanova et al&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">22</span></a> showed high levels of GSH in pediatric patients with sickle cell disease&#44; the present study found no difference in GSH levels between children with sickle cell disease and the control group&#46;</p><p id="par0145" class="elsevierStylePara elsevierViewall">Superoxide dismutase can convert superoxide to hydrogen peroxide&#44; and catalase can remove excess hydrogen peroxide&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">16</span></a> According to Silva et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">20</span></a> the increased pro-oxidant generation in sickle cell disease results in an antioxidant deficiency&#46; However&#44; there are some discrepancies between studies on superoxide dismutase and catalase levels in this disease&#44; with some studies observing increased activity and others observing decreased levels&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">29</span></a> An increase in these enzymes activity potentially constitutes a defense mechanism in response to increased oxidative stress&#44;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">19</span></a> or might be a consequence of increased reticulocyte content in blood samples from patients with sickle cell disease&#46; However&#44; a decrease in enzyme levels was related to disease severity in patients&#46;<a class="elsevierStyleCrossRefs" href="#bib0250"><span class="elsevierStyleSup">20&#44;22</span></a> These seemingly contradictory findings could be due to differences in the extent of oxidative stress&#44; disease severity&#44; enzyme polymorphism&#44; and the enzyme co-factor&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">29</span></a> The present results showed no difference between the activities of these enzymes in children with sickle cell disease and those in healthy children&#44; according with Cho et al&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">30</span></a> with regard to catalase&#46; These results may be due to large individual variability found among patients&#46;</p><p id="par0150" class="elsevierStylePara elsevierViewall">In light of evidence suggesting that an excess of oxidative stress has implications in sickle cell disease pathophysiology&#44; the assessment of oxidative stress parameters in these patients may provide useful information regarding the use of current medications and may lead to the development of new therapeutic strategies&#46;<a class="elsevierStyleCrossRefs" href="#bib0200"><span class="elsevierStyleSup">10&#44;19&#44;20</span></a> Monitoring the oxidative stress involves the observation of different parameters associated with pro-oxidant and antioxidant biomarkers&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">27</span></a> However&#44; the use of an isolated biomarker and the measurement of individual antioxidants are not likely to be useful indexes of oxidative status&#46; The oxidant&#8211;antioxidant balance involves biochemical reactions that require the evaluation of many endpoints&#46;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">26</span></a></p><p id="par0155" class="elsevierStylePara elsevierViewall">The present study evaluated eight oxidative stress markers&#44; including pro-oxidant and antioxidant parameters&#46; The results indicate the presence of a hyperoxidative status in children with sickle cell disease&#44; which can be observed by their high levels of methemoglobin&#44; TBARS&#44; hemolysis&#44; reactive oxygen species&#44; and G6-PD activity&#46; Simple techniques were used to determine these parameters using small volumes of blood&#46; These parameters that appeared altered in children with sickle cell disease can be useful in the evaluation of disease progression and treatment&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Conflicts of interest</span><p id="par0160" class="elsevierStylePara elsevierViewall">The authors declare no conflicts of interest&#46;</p></span></span>"
    "textoCompletoSecciones" => array:1 [
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          "identificador" => "xres696342"
          "titulo" => "Abstract"
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            1 => array:2 [
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          "titulo" => "Introduction"
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          "secciones" => array:12 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Chemicals"
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            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Blood samples"
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            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Hematologic parameters"
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            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Methemoglobin concentration"
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            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Reduced glutathione determination"
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            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "Lipid peroxidation"
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            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Measurement of hemolysis"
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            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "Activity of glucose6-phosphate dehydrogenase &#40;G6-PD&#41;"
            ]
            8 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "Superoxide dismutase activity"
            ]
            9 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Catalase activity"
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            10 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Intracellular reactive oxygen species"
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            11 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Statistical analyses"
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          "titulo" => "Results"
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          "titulo" => "Discussion"
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    "fechaRecibido" => "2015-07-06"
    "fechaAceptado" => "2015-10-16"
    "PalabrasClave" => array:2 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec706078"
          "palabras" => array:3 [
            0 => "Oxidative stress"
            1 => "Sickle cell disease"
            2 => "Children"
          ]
        ]
      ]
      "pt" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Palavras-chave"
          "identificador" => "xpalclavsec706077"
          "palabras" => array:3 [
            0 => "Estresse oxidativo"
            1 => "Doen&#231;a falciforme"
            2 => "Crian&#231;as"
          ]
        ]
      ]
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    "resumen" => array:2 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Objective</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">To determine eight parameters of oxidative stress markers in erythrocytes from children with sickle cell disease and compare with the same parameters in erythrocytes from healthy children&#44; since oxidative stress plays an important role in the pathophysiology of sickle cell disease and because this disease is a serious public health problem in many countries&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Blood samples were obtained from 45 children with sickle cell disease &#40;21 males and 24 females with a mean age of 9 years&#59; range&#58; 3&#8211;13 years&#41; and 280 blood samples were obtained from children without hemoglobinopathies &#40;137 males and 143 females with a mean age of 10 years&#59; range&#58; 8&#8211;11 years&#41;&#44; as a control group&#46; All blood samples were analyzed for methemoglobin&#44; reduced glutathione&#44; thiobarbituric acid reactive substances&#44; percentage of hemolysis&#44; reactive oxygen species&#44; and activity of the enzymes glucose 6-phosphate dehydrogenase&#44; superoxide dismutase&#44; and catalase&#46; Data were analyzed using Student&#39;s <span class="elsevierStyleItalic">t</span>-test and were expressed as the mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; A <span class="elsevierStyleItalic">p</span>-value of &#60;0&#46;05 was considered significant&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Significant differences were observed between children with sickle cell disease and the control group for the parameters methemoglobin&#44; thiobarbituric acid reactive substances&#44; hemolysis&#44; glucose 6-phosphate dehydrogenase activity&#44; and reactive oxygen species&#44; with higher levels in the patients than in the controls&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Oxidative stress parameters in children&#39;s erythrocytes were determined using simple laboratory methods with small volumes of blood&#59; these biomarkers can be useful to evaluate disease progression and outcomes in patients&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Objective"
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          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
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          2 => array:2 [
            "identificador" => "abst0015"
            "titulo" => "Results"
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          3 => array:2 [
            "identificador" => "abst0020"
            "titulo" => "Conclusions"
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      "pt" => array:3 [
        "titulo" => "Resumo"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Objetivo</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Determinar par&#226;metros de estresse oxidativo em eritr&#243;citos de crian&#231;as com doen&#231;a falciforme e compar&#225;-los com os mesmos par&#226;metros em eritr&#243;citos de crian&#231;as saud&#225;veis&#44; pois o estresse oxidativo desempenha um importante papel na fisiopatologia da doen&#231;a falciforme&#44; considerada um s&#233;rio problema de sa&#250;de p&#250;blica em muitos pa&#237;ses&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Foram obtidas amostras de sangue de 45 crian&#231;as com doen&#231;a falciforme &#40;21 meninos e 24 meninas com m&#233;dia de 9 anos&#44; varia&#231;&#227;o de 3 a 13 anos&#41; e 280 amostras de sangue de crian&#231;as sem hemoglobinopatias &#40;137 meninos e 143 meninas com m&#233;dia de 10 anos&#44; varia&#231;&#227;o de 8 a 11 anos&#41;&#44; como grupo controle&#46; Em todas as amostras foram determinados meta-hemoglobina&#44; glutationa reduzida&#44; subst&#226;ncias reativas ao &#225;cido tiobarbit&#250;rico&#44; porcentagem de hem&#243;lise&#44; esp&#233;cies reativas de oxig&#234;nio e atividade das enzimas glucose6-fosfato desidrogenase&#44; super&#243;xido dismutase e catalase&#46; Os dados foram analisados com o teste <span class="elsevierStyleItalic">t</span> de Student e foram expressos como m&#233;dia<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>desvio padr&#227;o&#46; Um valor de <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;05 foi considerado significativo&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Foram observadas diferen&#231;as significativas entre as crian&#231;as com doen&#231;a falciforme e o grupo controle para os par&#226;metros meta-hemoglobina&#44; subst&#226;ncias reativas ao &#225;cido tiobarbit&#250;rico&#44; porcentagem de hem&#243;lise&#44; esp&#233;cies reativas de oxig&#234;nio e atividade da enzima glucose6-fosfato desidrogenase&#44; com n&#237;veis aumentados nos pacientes&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclus&#245;es</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Foi poss&#237;vel determinar par&#226;metros de estresse oxidativo em eritr&#243;citos de crian&#231;as&#44; com t&#233;cnicas laboratoriais simples e pequenos volumes de sangue&#46; Esses biomarcadores podem ser &#250;teis na avalia&#231;&#227;o da progress&#227;o e dos resultados de tratamentos da doen&#231;a&#46;</p></span>"
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        "etiqueta" => "&#9734;"
        "nota" => "<p class="elsevierStyleNotepara" id="npar0020">Please cite this article as&#58; Hermann PB&#44; Pianovski MA&#44; Henneberg R&#44; Nascimento AJ&#44; Leonart MS&#46; Erythrocyte oxidative stress markers in children with sickle cell disease&#46; J Pediatr &#40;Rio J&#41;&#46; 2016&#59;92&#58;394&#8211;9&#46;</p>"
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          "leyenda" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">RBC&#44; red blood cells&#59; MCV&#44; medium corpuscular volume&#59; MCH&#44; medium corpuscular hemoglobin&#59; MCHC&#44; medium corpuscular hemoglobin concentration&#59; WBC&#44; white blood cells&#59; PLA&#44; platelets&#59; CV&#44; Pearson&#39;s coefficient of variation &#40;&#37;&#41;&#46; Data are presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46;</p>"
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                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col">Control group&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col"><span class="elsevierStyleItalic">p</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr><tr title="table-row"><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>280&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">RBC &#40;10<span class="elsevierStyleSup">6</span>&#47;mm<span class="elsevierStyleSup">3</span>&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">3&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">29&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Hemoglobin &#40;g&#47;dl&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">13&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;6&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">8&#46;9<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">21&#46;5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Hematocrit &#40;&#37;&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">39&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2&#46;7&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;7&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">26&#46;7<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;6&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">20&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MCV &#40;fl&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">82&#46;3<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">4&#46;8&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">86&#46;3<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>11&#46;8&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">13&#46;7&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;05&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MCH &#40;pg&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">28&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;6&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">5&#46;5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">28&#46;9<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>4&#46;64&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">16&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#62;0&#46;05&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MCHC &#40;g&#47;dl&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">34&#46;3<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">3&#46;6&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">33&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;01&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">WBC &#40;10<span class="elsevierStyleSup">3</span>&#47;mm<span class="elsevierStyleSup">3</span>&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">21&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">13&#46;8<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>6&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">44&#46;3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">PLA &#40;10<span class="elsevierStyleSup">3</span>&#47;mm<span class="elsevierStyleSup">3</span>&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">292&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>58&#46;3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">19&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">458&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>199&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">43&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
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          "leyenda" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">METHb&#44; methemoglobin&#59; GSH&#44; reduced glutathione&#59; TBARS&#44; thiobarbituric acid reactive substances&#59; HEMO&#44; hemolysis&#59; G6-PD&#44; glucose 6-phosphate dehydrogenase&#59; SOD&#44; superoxide dismutase&#59; CAT&#44; catalase&#59; ROS&#44; reactive oxygen species&#46; Data presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46;</p>"
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head  " align="" valign="top" scope="col">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col">Control group&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col">Patients&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col"><span class="elsevierStyleItalic">p</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr><tr title="table-row"><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>100&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>45&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">METHb &#40;&#37;&#41;<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">2&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">4&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">GSH &#40;&#956;mol&#47;gHb&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2&#46;3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#62;0&#46;05&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">TBARS &#40;nmol&#47;gHb&#41;<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">24&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;8&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">41&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>20&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">HEMO 0<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">b</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">1&#46;1<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">4&#46;7<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;7&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">HEMO 50<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">b</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">25&#46;0<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>7&#46;9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">49&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>19&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">HEMO 100<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">b</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">55&#46;5<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>10&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">80&#46;7<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>13&#46;6&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">HEMO 150<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a><span class="elsevierStyleSup">&#44;</span><a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">b</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">80&#46;1<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>7&#46;5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">92&#46;6<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">G6-PD &#40;U&#47;gHb&#41;<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">6&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">13&#46;2<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#60;0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">SOD &#40;U&#47;gHb&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">1846&#46;1<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>457&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">1832&#46;4<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>647&#46;1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">&#62;0&#46;05&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">CAT &#40;U&#47;gHb&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="char" valign="top">2&#46;6 10<span class="elsevierStyleSup">5</span><span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>6&#46;6 10<span class="elsevierStyleSup">4</span>&nbsp;\t\t\t\t\t\t\n
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