Maria Suely Soares Leonart

Oxidative process in erythrocytes of individuals with hemoglobin S

Abstract The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AA<AS<SS, by the increase in the content of Heinz bodies, methemoglobin, hemolysis, GSH depletion and lowering activities of the enzymes G6-PD and GR. The protective actions of the vitamins C and E for the oxidative stress induced by TBHP were observed for the erythrocytes in the lowering Heinz bodies, methemoglobin, hemolysis, and partial recovery of GSH more efficiently in AS and SS erythrocytes. Recovery was not observed in the levels of the activities of the enzymes G6-PD and GR, under the vitamins actions. The results obtained confirmed the higher susceptibility of the sickle erythrocyte to oxidation which necessitates precaution in the transfusion adequacy of AS erythrocytes. On the other hand, the protective effect of the vitamins C and E over the oxidative stress observed in erythrocytes AS and SS open perspectives for their use for treatment of patients with sickle cell anemias, as well as in the preservation of transfusional erythrocyte bag units.

Effect of vitamin C, deferoxamine, quercetin and rutin against tert-butyl hydroperoxide oxidative damage in human erythrocytes

Abstract The mature human erythrocyte, when submitted to oxidative stress, can demonstrate depletion of reduced glutathione, oxidation of the hemoglobin molecule and aggregation of complexes of iron close to the membrane. These can produce abnormalities in the erythrocyte membrane and hemolysis. The aim of this work was to study the antioxidative action of vitamin C (vit. C), deferroxamine (DFO) and the flavonoids quercetin and rutin in normal human erythrocytes, submitted to in vitro oxidative stress induced by tert-butylhydroperoxide (tBHP). Venous blood was colleted in citrate–phosphate–dextrose (CPD) solution, as anticoagulant, from healthy adult individuals after informed consent. The erythrocytes were resuspended in PBS to obtain 35% globular volume, and then submitted to the oxidative action of tBHP for up to 30 min, with or without previous incubation for 60 min with vit. C, DFO, quercetin and rutin. Decrease in the GSH concentration, G6-PD and GR activities, and increase in the methemoglobin and Heinz bodies (HB) formation, occurred with the increase in tBHP concentration. tBHP did not effect on the membrane proteins detected by SDS-PAGE. Quercetin, partially prevented the GSH decrease and the formation of HB, but did not prevent MetHb formation from oxidative damage by tBHP. Rutin, after tBHP induction, prevented the GSH decrease and the formation of HB. Vit. C, had no influence on the depletion of GSH, inhibited partially the metHb formation, and it protected GR, but not G6-PD from oxidative damage by tBHP. DFO partially inhibited the metHb formation and GSH decrease, but it did not protect GR and G6-PD from oxidative damage by tBHP. The results obtained suggest that vit. C, DFO and the flavonoids quercetin and rutin contribute to the decrease in the oxidative stress caused by tBHP.

Erythrocyte oxidative stress markers in children with sickle cell disease

Objective 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, 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.OBJECTIVE 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, 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. METHODS Blood samples were obtained from 45 children with sickle cell disease (21 males and 24 females with a mean age of 9 years; range: 3-13 years) and 280 blood samples were obtained from children without hemoglobinopathies (137 males and 143 females with a mean age of 10 years; range: 8-11 years), as a control group. All blood samples were analyzed for methemoglobin, reduced glutathione, thiobarbituric acid reactive substances, percentage of hemolysis, reactive oxygen species, and activity of the enzymes glucose 6-phosphate dehydrogenase, superoxide dismutase, and catalase. Data were analyzed using Students t-test and were expressed as the mean±standard deviation. A p-value of <0.05 was considered significant. RESULTS Significant differences were observed between children with sickle cell disease and the control group for the parameters methemoglobin, thiobarbituric acid reactive substances, hemolysis, glucose 6-phosphate dehydrogenase activity, and reactive oxygen species, with higher levels in the patients than in the controls. CONCLUSIONS Oxidative stress parameters in childrens erythrocytes were determined using simple laboratory methods with small volumes of blood; these biomarkers can be useful to evaluate disease progression and outcomes in patients.

Ginkgo biloba extract (EGb 761) attenuates oxidative stress induction in erythrocytes of sickle cell disease patients

A doenca falciforme promove anemia hemolitica e oclusao dos pequenos vasos, causados pela presenca de altas concentracoes de hemoglobina S, cujas consequencias incluem a producao aumentada de especies reativas de oxigenio e diminuicao da capacidade de defesa antioxidante. O objetivo desse estudo foi avaliar a acao protetora de um extrato padronizado de Ginkgo biloba (EGb 761), selecionado devido ao seu alto conteudo de flavonoides e terpenoides, em eritrocitos de pacientes com anemia falciforme (HbSS, eritrocitos SS) submetidos ao estresse oxidativo usando terc-butil-hidroperoxido e 2,2-azobis-(amidinopropano)-diidrocloreto, in vitro. Indices de hemolise, glutationa reduzida, concentracao de metemoglobina, peroxidacao lipidica e especies reativas de oxigenio foram determinados. Eritrocitos de pacientes com anemia falciforme apresentaram taxas aumentadas de oxidacao da hemoglobina e peroxidacao lipidica e a concentracao de EGb 761 necessaria para atingir o mesmo efeito antioxidante foi pelo menos duas vezes maior em relacao aos eritrocitos normais (HbAA, eritrocitos AA), inibindo a formacao de especies reativas de oxigenio (IC50 de 13.6 µg/mL), prevenindo parcialmente a peroxidacao lipidica (IC50 de 242.5 µg/mL) e prevenindo a hemolise (IC50 de 10.5 µg/mL). Portanto, EGb 761 possui um efeito benefico no estado oxidativo dos eritrocitos SS. Entretanto, o EGb 761 nao preveniu a oxidacao da hemoglobina e da glutationa reduzida, nas concentracoes examinadas.

Estudo de métodos laboratoriais para o controle de qualidade de unidades transfusionais eritrocitárias no Centro de Hematologia e Hemoterapia do Paraná (Hemepar), Brasil

O sistema publico de hemoterapia no Brasil esta estruturado em hemocentros estaduais e regionais, cujos procedimentos preconizados pelo Ministerio da Saude para a garantia da qualidade envolvem selecao e cuidados com os doadores, producao e preservacao de hemocomponentes, analises laboratoriais e procedimentos para garantir a destinacao adequada aos receptores. As unidades transfusionais eritrocitarias (UTE) devem ser avaliadas quanto a funcionalidade dos eritrocitos apos a transfusao. O presente trabalho visou estudar: porcentual de hemolise, morfologia eritrocitaria, teores de meta-hemoglobina (metHb), glutationa reduzida (GSH), adenosina trifosfato (ATP), volume corpuscular medio (VCM), hemoglobina corpuscular media (HCM), concentracao de hemoglobina corpuscular media (CHCM) e atividade de glicose 6-fosfato desidrogenase (G6-PD). Foram estudadas 19 UTE mantidas em bolsas com salina-adenina-glucose-manitol (SAGM), preparadas a partir de unidades de sangue total (ST), da rotina do Laboratorio de Controle de Qualidade de Hemocomponentes do Centro de Hematologia e Hemoterapia do Parana (Hemepar). Os eritrocitos apresentaram indices adequados de morfologia e integridade celular e baixos teores de hemolise. A partir de uma analise do conhecimento cientifico atualizado, das normas preconizadas pela RDC 153/2004 da Anvisa, das atividades rotineiras praticadas no Hemepar e dos resultados obtidos, recomenda-se o monitoramento da lesao de estoque. Sugere-se a implementacao, a cada duas semanas, de preservacao das UTE, das determinacoes de porcentagem de discocitos, indice de hemolise a partir da dosagem de cianometa-hemoglobina extracelular, VCM, HCM, CHCM, metaHb e GSH, considerando-se que, desta forma, se podera contribuir para a melhoria da qualidade da hemoterapia.

Protective action of deferiprone and deferoxamine in erythrocytes isolated from patients with β-thalassemias

One of the most deleterious consequences of iron overload in thalassemia is the presence of non-transferrin bound iron (NTBI), a free radical that acts as a catalyst for free oxygen radicals, in particular for hydroxyl free radicals (OH.). These radicals oxidize both membrane lipids and proteins causing irreversible damage to biologically important molecules and cellular structures. Treatment with iron chelators has been important to improve survival of these individuals. The aim of this work was the study on the effects of deferoxamine (DFO) and deferiprone (DFP) on erythrocytes under the pro-oxidative action of TBHP isolated from normal individuals and patients with β-thalassemia. The in vitro action of deferoxamine and deferiprone on the oxidative metabolism of erythrocytes from β-thalassemic patients treated at the Centro de Hematologia e Hemoterapia do Parana (HEMEPAR), Brazil, under the pro-oxidative action of TBHP was studied. Methemoglobin concentrations, reduced glutathione (GSH), hemolysis indexes and the enzyme activities of G6-PD and GR were determined. The oxidation indexes were higher in erythrocytes of β-thalassemic individuals than those from normal individuals. Treatment of the normal and β-thalassemic erythrocytes with DFO and/or DFP protected against the formation of GSH promoted by TBHP.

Original articleErythrocyte oxidative stress markers in children with sickle cell diseaseMarcadores de estresse oxidativo em eritrócitos de crianças com doença falciforme

Objective 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, 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.OBJECTIVE 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, 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. METHODS Blood samples were obtained from 45 children with sickle cell disease (21 males and 24 females with a mean age of 9 years; range: 3-13 years) and 280 blood samples were obtained from children without hemoglobinopathies (137 males and 143 females with a mean age of 10 years; range: 8-11 years), as a control group. All blood samples were analyzed for methemoglobin, reduced glutathione, thiobarbituric acid reactive substances, percentage of hemolysis, reactive oxygen species, and activity of the enzymes glucose 6-phosphate dehydrogenase, superoxide dismutase, and catalase. Data were analyzed using Students t-test and were expressed as the mean±standard deviation. A p-value of <0.05 was considered significant. RESULTS Significant differences were observed between children with sickle cell disease and the control group for the parameters methemoglobin, thiobarbituric acid reactive substances, hemolysis, glucose 6-phosphate dehydrogenase activity, and reactive oxygen species, with higher levels in the patients than in the controls. CONCLUSIONS Oxidative stress parameters in childrens erythrocytes were determined using simple laboratory methods with small volumes of blood; these biomarkers can be useful to evaluate disease progression and outcomes in patients.

Analysis of manual reticulocyte counts in the clinical laboratories of Ponta-Grossa and Campos Gerais, PR, Brazil

A contagem do reticulocitos e usada extensamente na rotina laboratorial para avaliar a atividade eritropoietica da medula ossea, e e de grande importância no diagnostico e no prognostico na terapia de anemias hemoliticas. Sao coradas com o azul de metileno novo e o azul cresil brilhante, o que conferem o aspecto caracteristico de reticulo quando observado ao microscopiootico. Criterios conhecidos foram observados para um bom desempenho da contagem manual de reticulocitos, principalmente, atencao especial nas peliculas do sangue durante a montagem das lâminas; contagem nos campos que nao contem sobreposicao celular; e tambem no numero de celulas avaliadas. O objetivo deste estudo foi avaliar a variacao interobservadores, analisar o erro estatistico da contagem manual dos reticulocitos, e demonstrar as limitacoes deste metodo. A analise de correlacao intraclasses segundo Bartko [Psychol Rep 19:3,1966; 34:418,1974] foi usada para avaliar a concordância entre 12 observadores em um total de 25 lâminas do sangue, com contagens variadas de reticulocitos. Os resultados das analises estatisticas indicam que o erro casual, calculado como (1-r2) variou de 4 a 60% entre os observadores. Embora ocorra imprecisao entre os observadores, o perfil geral entre eles e similar, e o coeficiente de correlacao intraclasse indicou que os resultados obtidos sao clinicamente uteis.

GENOTOXICIDADE E CITOTOXICIDADE DOS RAIOS X EM CRIANÇAS SUBMETIDAS À RADIOGRAFIA PANORÂMICA

ABSTRACT Objective: To assess the genotoxicity and cytotoxicity produced by X-rays in the epithelium of the oral mucosa of infants exposed to panoramic radiography. Methods: The sample consisted of 30 healthy children, 19 females and 11 males, ranging in age from 4 to 10 years (average of 7 years of age). Oral mucosa cells were collected by liquid-based cytology immediately before and after seven days following the exposure to panoramic radiography. Smears were processed and stained using the modified Feulgen Rossenbeck technique. Bud and broken egg nuclear projections, changes in the form of micronuclei, and genotoxic and cytotoxic changes of pyknosis, karyorrhexis and karyolysis were analyzed and quantified. Results: The frequency of pyknosis, buds and broken eggs was significantly higher after exposure to X-rays (p<0.05), but there was no statistically significant difference regarding gender, as well as in the other changes studied. Conclusions: Exposure to X-rays emitted during submission to panoramic radiography may induce cell death in the epithelium of children’s oral mucosa. No evidence was found for a significant genotoxic effect.

Controle de qualidade na preservação de eritrócitos para transfusão

Desde as primeiras transfusoes sanguineas, ainda noseculo XVIII, pesquisadores e hemoterapeutas tem buscadoa forma de transmitir a vida atraves de celulas que mantenhama sua essencia, mas que nao provoquem efeitos deleterios aoreceptor. Nas transfusoes diretas entre doador e receptor, damesma forma que se observava a vida retornando a corpos jaquase inertes, ocorriam reacoes adversas inesperadas e, mui-tas vezes, letais. A partir das dificuldades enfrentadas, apro-fundaram-se estudos no sentido de garantir a qualidade.