Erythrocyte oxidative stress markers in children with sickle cell disease / Marcadores de estresse oxidativo em eritrócitos de crianças com doença falciforme

Abstract 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 Student's 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 children's 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.

Resumo Objetivo Determinar parâmetros de estresse oxidativo em eritrócitos de crianças com doença falciforme e compará-los com os mesmos parâmetros em eritrócitos de crianças saudáveis, pois o estresse oxidativo desempenha um importante papel na fisiopatologia da doença falciforme, considerada um sério problema de saúde pública em muitos países. Métodos Foram obtidas amostras de sangue de 45 crianças com doença falciforme (21 meninos e 24 meninas com média de 9 anos, variação de 3 a 13) e 280 amostras de sangue de crianças sem hemoglobinopatias (137 meninos e 143 meninas com média de 10 anos, variação de 8 a 11), como grupo controle. Em todas as amostras foram determinados meta-hemoglobina, glutationa reduzida, substâncias reativas ao ácido tiobarbitúrico, porcentagem de hemólise, espécies reativas de oxigênio e atividade das enzimas glucose6-fosfato desidrogenase, superóxido dismutase e catalase. Os dados foram analisados com o teste t de Student e foram expressos como média ± desvio padrão. Um valor de p < 0,05 foi considerado significativo. Resultados Foram observadas diferenças significativas entre as crianças com doença falciforme e o grupo controle para os parâmetros meta-hemoglobina, substâncias reativas ao ácido tiobarbitúrico, porcentagem de hemólise, espécies reativas de oxigênio e atividade da enzima glucose6-fosfato desidrogenase, com níveis aumentados nos pacientes. Conclusões Foi possível determinar parâmetros de estresse oxidativo em eritrócitos de crianças, com técnicas laboratoriais simples e pequenos volumes de sangue. Esses biomarcadores podem ser úteis na avaliação da progressão e dos resultados de tratamentos da doença.

Validation of the rapid test carestart(tm) G6PD among malaria vivax-infected subjects in the Brazilian Amazon

Abstract: INTRODUCTION: In the Brazilian Amazon, malaria infections are primarily caused by Plasmodium vivax. The only drug that kills the hypnozoite form of P. vivax is primaquine, thereby preventing relapse. However, treating glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals with primaquine can lead to severe hemolysis. G6PD deficiency (G6PDd) affects approximately 400 million people worldwide, most of whom live in malaria-endemic areas. Therefore, clinicians need tools that can easily and reliably identify individuals with G6PDd. This study estimated the accuracy of the Carestart(tm) G6PD rapid test (Access Bio) in the diagnosis of G6PDd in male participants with and without P. vivax acute malaria. METHODS: Male participants were recruited in Manaus. Malaria diagnosis was determined by thick blood smear. G6PD quantitative analysis was performed spectro photometrically at a wave length of 340nm. The Carestart(tm) G6PD test was performed using venous blood. Genotyping was performed for individuals whose samples had an enzyme activity less than 70% of the normal value. RESULTS: Six hundred and seventy-four male participants were included in this study, of whom 320 had a diagnosis of P. vivax malaria. In individuals with enzyme activity lower than 30% (n=13), the sensitivity, specificity, positive predictive value, and negative predictive value of the Carestart(tm) G6PD test were as follows: 61.5% (95%CI: 35.5%-82.3%), 98.3% (95%CI: 97.0%-99.1%), 42.1% (95%CI: 23.1%-63.7%), and 99.2% (95%CI: 98.2%-82.3%), 98.3% (95%CI: 97.0%-99.1%), 42.1% (95%CI: 23.1%-63.7%), and 99.2% (95%CI: 98.2%-99.7%), respectively. Increases in sensitivity were observed when increasing the cut-off value. CONCLUSIONS: Despite low sensitivity, Carestart(tm) G6PD remains a good alternative for rapid diagnosis of G6PDd in malaria-endemic regions.

Deficiencia de glucosa-6-fosfato deshidrogenasa eritrocitaria en Rosario / Erythrocyte glucose-6-phosphate dehydrogenase deficiency in Rosario / Deficiência de glicose-6-fosfato desidrogenase eritrocitária em Rosario

Se estudió la actividad enzimática (AE) de la enzima glucosa-6-fosfato deshidrogenasa eritrocitaria (G6PD) y la movilidad electroforética (ME) en una población de hombres y mujeres de la ciudad de Rosario (provincia de Santa Fe), Argentina y zona de influencia. Para la determinación de AE se utilizó la técnica cinética de Glock y McLean y para la electroforesis de la enzima, la técnica de M.C. Rattazzi y L.C. Bernini en acetato de celulosa. Los valores normales de actividad enzimática (AE) para hombres y mujeres adultos fueron de 8,1 ± 1,4 UI G6PD/g Hb. Se demostró que los valores de AE son independientes de la edad, sexo y concentración de hemoglobina. En todos los grupos etarios estudiados no se observaron diferencias significativas de AE con respecto a los adultos normales a excepción de los neonatos que presentaron un significativo aumento de la misma, lo cual está directamente relacionado con las características fisiológicas de los eritrocitos del recién nacido. Entre los 686 individuos estudiados se detectaron 2 pacientes deficientes de G6PD, lo que dio una prevalencia de 0,3% y el patrón electroforético correspondiente a esta población fue 98% (n: 672) para G6PD B y 2% (n: 14) para G6PD con movilidad rápida tipo A.

Enzymatic activity (EA) of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and electrophoretic mobility (EM) have been studied in a population of males and females in the city of Rosario and its area of influence. To determine EA, the Glock and McLean kinetic technique was used. Electrophoretic mobility assay was performed by M.C. Rattazzi and L.C. Bernini technique in cellulose acetate gel. Results demonstrated that the EA values in normal individual are independent of age, sex and hemoglobin values. The normal values of EA were: 8.1±1.4 IU of G6PD/g Hb. There were no significant differences in different age groups studied regarding healthy adults, except for neonatal group that yielded a significant EA increase which is directly related to the physiological characteristics of newborn erythrocytes. Two patients out of 686 individuals bearing G6PD deficiency were detected, corresponding to 0.3% prevalence. The electrophorectic mobility pattern was 98% (n: 672) for G6PD B, and 2% (n: 14) for G6PD A fast mobility variant.

Foi estudada a atividade enzimática (AE) da enzima glicose-6-fosfato desidrogenase eritrocitária (G6PD) e a mobilidade eletroforética (ME) numa população de homens e mulheres da cidade de Rosario, província de Santa Fe, Argentina e zona de influência. Para a determinação da AE foi utilizada a técnica cinética de GlocK e Mc Lean e para a eletroforese da enzima a técnica de M.C. Rattazzi e L.C. Bernini em acetato de celulose. Os valores normais de atividade enzimática (AE) para homens e mulheres adultos foram de 8,1 ± 1,4 UI G6PD/g Hb. Foi demonstrado que os valores da AE são independentes da idade, sexo e concentração de hemoglobina. Em nenhum dos grupos etários estudados foram observadas diferenças significativas de AE no que diz respeito aos adultos normais, com exceção dos neonatos que apresentaram um significativo aumento da mesma, o qual está diretamente relacionado com as características fisiológicas dos eritrócitos do recém-nascido. Entre os 686 indivíduos estudados foram detectados 2 pacientes deficientes de G6PD, o que deu uma prevalência de 0,3% e o padrão eletroforético correspondente a esta população foi de 98% (n: 672) para a G6PD B e 2% (n: 14) para G6PD com mobilidade rápida tipo A.

Metemoglobinemia em pacientes com malária por Plasmodium vivax em uso oral de primaquina / Methemoglobinemia in patients with Plasmodium vivax receiving oral therapy with primaquine

INTRODUÇÃO: A primaquina pode acarretar sérios eventos adversos, com destaque para a toxicidade ao sangue. O objetivo deste trabalho é determinar a metemoglobinemia de 20 pacientes com malária por Plasmodium vivax tratados com primaquina, comparando-os segundo o sexo e a expressão da glicose-6-fosfato desidrogenase. MÉTODOS: Quantificação da metemoglobina por espectrofotometria visível e avaliação qualitativa da glicose-6-fosfato desidrogenase. RESULTADOS: A metemoglobinemia variou de 2,85 a 5,45 por cento nos pacientes do sexo masculino e de 3,77 a 7,34 por cento no feminino. CONCLUSÕES: A instituição da terapia aumentou de maneira significativa os teores de metemoglobina, sem manifestação clínica evidente e independente do sexo e da atividade enzimática.

INTRODUCTION: Primaquine can produce adverse reactions as toxicity to blood when used in the treatment of vivax malaria. This work aimed to determine methemoglobinemia in patients with vivax malaria receiving oral therapy with primaquine. METHODS: Spectrophotometric quantification of methemoglobinemia and qualitative assay for glucose-6-phosphate dehydrogenase. RESULTS: Methemoglobinemia ranged from 2.85 to 5.45 percent in male patients and 3.77 to 7.34 percent in female patients. CONCLUSIONS: A statistically significant increase in methemoglobinemia was observed following oral therapy with primaquine, with no clinical manifestations, and independent of sex and the qualitative expression of glucose-6-phosphate dehydrogenase.

Malária e aspectos hematológicos em moradores da área de influência dos futuros reservatórios das hidrelétricas de Santo Antônio e Jirau, Rondônia, Brasil / Malaria and hematological aspects among residents to be impacted by reservoirs for the Santo Antônio and Jirau Hydroelectric Power Stations, Rondônia State, Brazil

Em Rondônia, prevê-se a construção de mais duas usinas hidrelétricas (UHE) no rio Madeira, a montante da cidade de Porto Velho, Rondônia, Brasil (de Santo Antônio e Jirau). O objetivo deste trabalho foi analisar a prevalência da malária antes do início da implantação das obras civis e fazer considerações sobre os impactos da doença com o ingresso de milhares de trabalhadores e agregados atraídos pelas oportunidades de emprego e comércio. Os resultados obtidos mostram que a malária se faz presente em toda região, em variados graus de prevalência. Além disso, a existência de potenciais portadores assintomáticos de malária entre a população nativa pode ter relevância epidemiológica e deve ser considerada nos programas de controle da malária, vinda tanto das autoridades públicas quanto das empresas responsáveis pela instalação das UHE, visando o diagnóstico e tratamento precoce, controle vetorial, abastecimento de água e aplicação de infra-estrutura nos centros urbanos.

In Rondônia State, Brazil, two new hydroelectric plants, Santo Antônio and Jirau, are scheduled for construction on the Madeira River, upriver from the State capital, Porto Velho. The current study analyzes malaria prevalence before the construction and provides information on the possible impacts of malaria burden related to the influx of thousands of persons attracted by direct and indirect employment opportunities. According to the findings, malaria is present throughout the region, with varying prevalence rates. The existence of potential asymptomatic malaria carriers among the local population may be epidemiologically relevant and should be considered in the malaria control programs organized by public authorities and companies responsible for building the power plants, aimed at early diagnosis and treatment, vector control, water supply, and infrastructure in the urban areas.

Changes in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and reduced glutathione (GSH) activities in the development of senile and diabetic cataracts.

Oxidative and osmotic stress have been implicated in the pathogenesis of cataracts. Reactive oxygen intermediates (ROI) mediate peroxidation of membrane lipids and cause irreversible damage to lens proteins. The purpose of this study was to assess the changes in erythrocyte glucose- 6-phosphate dehydrogenase enzyme (G6PD) and reduced glutathione (GSH) levels in the development of senile and diabetic cataracts. The activity of erythrocyte G6PD and the concentration of GSH were measured to assess changes in oxidation-reduction status. The oxidation-reduction status of 26 non-diabetic non-cataract (control) subjects were compared with 24 diabetic non-cataract, 30 diabetic cataract and 28 non-diabetic cataract subjects. The results revealed that the GSH and G6PD levels of the subjects with senile cataracts were significantly lower than the subjects without cataracts. The present study reveals the risk of developing senile cataracts is associated with decreased levels of erythrocyte G6PD and GSH. In the formation of diabetic cataracts an adequate supply of NADPH (G6PD activity) is essential to produce osmotically active sorbitol in the lens.

Effect of hyperhomocysteinemia on erythrocyte function and its impact on erythropoiesis in experimental rats

The current work was conducted to study the possible effect of hyperhomocysteinemia [HHcy] on some erythrocytic functions as a cause of anemia and its impact on erythropoietin [EPO] release in experimental rats. Forty adult male albino rats were divided into two main groups, the control group [Group I] and the hyperhomocysteinemic group [HHcy] [Group II]. Hyperhomocysteinemia was induced by subcutaneous injection of DL- homocysteine at a dose of 100 mg/kg/day for 4 weeks. At the end of the fourth week each main group was subdivided into two subgroups, [Ia] control, [Ib] Hypoxic-control [IIa] and HHcy, [IIb] Hypoxic-HHcy. Hypoxia was induced by a single intra-peritoneal injection of desferrioxamine [200 mg/kg] 22 hours before decapitation. Whole blood was used for determination of erythrocytic fragility, hematological parameters, reticulocyte percentage. Homocysteine, erythropoietin [EPO], urea and creatinine were estimated in plasma. Erythrocytes were used for estimation of lipid peroxidation, G6PDH activity and membrane separation for deten-nination of Na[+] K[+] ATPase enzymatic activity. Homocysteine, EPO and creatinine plasma levels, and reticulocyte% and erythrocytic lysis in addition to erythrocyte lipid peroxidation were all significantly higher, while G6PDH and Na[+] K[+]- ATPase enzymatic activities were significantly lower in the HHcy group as compared to the control group. A significant positive correlation was found between total Hcy plasma levels and erythrocyte lipid peroxidation. EPO plasma levels showed a significant increase in response to hypoxic stimulation in the control group, while blunted response was observed in the HHcy group. It could be concluded that disturbance in erythrocyte membrane and enzymatic functions in HHcy increases the susceptibility of RBCs to hemolysis and reduces its life span. Hyperhomocysteinemia also impacts EPO release to hypoxic stimulation which may be due to damaging effect of HHcy on renal cells

Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37°C, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37°C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50 percent higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 æM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 æM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

Neonatal screening for glucose-6-phosphate dehydrogenase deficiency.

OBJECTIVE: This study was carried out to detect the incidence of erythrocytic Glucose-6 -Phosphate dehydrogenase (G-6-PD) deficiency, to compare the incidence of hyperbilirubinemia in G-6-PD deficient neonates as compared to G-6-PD normal neonates and to asses the usefulness of neonatal screening for G-6-PD deficiency. METHOD: In a retrospective hospital based study 2,479 male and female neonates consecutively born at Indraprastha Apollo hospital between July 1998 to June 2003 who were screened for G-6-PD levels were evaluated for the incidence of G-6-PD deficiency. RESULTS: Incidence of G-6-PD deficiency was found to be 2.0%. Incidence in males was 283% and female was 1.05%. The incidence of hyperbilirubinemia was found to be 32% in G-6-PD deficient neonates which was significantly higher than the incidence of hyperbilirubinemia in neonates with normal G-6-PD, which was 12.3% (P< 0.001). CONCLUSION: Our data suggests that neonatal screening for G-6-PD deficiency is a useful test for preventing and early treatment of complications associated with it.

effect of aluminum on human erythrocyte glucose 6-phosphate dehydrogenase

Glucose 6-phosphate dehydrogenase [G6PD], the first enzyme in initiating the pentose phosphate shunt, is an important component in generation of NADPH. Although innumerable studies have been performed on human erythrocyte G6PD, however, the effect of trace elements on the enzyme activity requires further investigations. To study the effect of aluminum on human erythrocyte G6PD. In this experimental research, following the purification of G6PD using chromatographic methods, the effect of different concentrations of Al[3+] [up to 100 micro-molar] on G6PD activity was studied. The enzyme activity was measured at different concentrations of glucose 6-phosphate and NADP[+] to determine the type of inhibitory action. Aluminum at the concentration of 100 microM showed a considerable inhibitory effect on G6PD activity [60%]. The type of inhibitory action, depending on the use of glucose-6-phosphate or NADP[+], was competitive and noncompetitive, respectively. Aluminum exerts an inhibitory action on human erythrocyte G6PD activity

Synergistic enhancement of a copper chelator, bathocuproine disulphonate, and cysteine on in vitro growth of Plasmodium falciparum in glucose-6-phosphate dehydrogenase-deficient erythrocytes.

In vitro growth of Plasmodium falciparum is restricted in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes (RBC), as a result of oxidative stress. Bathocuproine disulphonate (BCS), a copper chelator, as well as cysteine have been shown to synergistically stimulate the in vitro growth of various mammalian cells and Trypanosoma under oxygenated conditions. We examined the effects of these two chemicals on the in vitro growth of P. falciparum in G6PD-deficient RBC, and found that addition of BCS and cysteine synergistically enhanced the growth of the P. falciparum FCR-3 strain in these RBC to the same level as in normal RBC. However, BCS or cysteine alone had no stimulatory effect. To explain this synergistic enhancement, changes in thiol, NADPH and glutathione contents were investigated. After addition of cysteine alone, thiol content in the medium decreased rapidly, but when BCS was added, it was maintained at about 35% at 24 hours after incubation, suggesting that BCS stimulates parasite growth in G6PD-deficient RBC by inhibiting copper-mediated oxidation of cysteine in the medium. In these RBC, no increase in NADPH level, but a slight increase in glutathione, was observed in the presence of both BCS and cysteine. The slight increase of glutathione, was probably due to incorporation of cysteine from the medium, although this could not fully explain the synergistic growth enhancement. These findings taken together suggest that cysteine incorporated into G6PD-deficient RBC may help maintain the thiol groups in many proteins, such as membrane proteins, hemoglobin and enzymes, and plays an important role in maintaining an appropriate culture state necessary for parasite growth. We also examined the effects of BCS and cysteine on adaptation of wild isolates of P. falciparum to in vitro cultivation using the candle jar method. Although there was no drastic effect on growth enhancement, the presence of BCS and cysteine accelerated the appearance of schizonts in many isolates.

Glucose-6-phosphate dehydrogenase deficiency in blood donors: screening by micromethaemoglobin reduction test.

500 blood donors were screened for G6PD deeiciency using micromethaemoglobin reduction (microMRT) test. Most of the blood donors were young adult males (95.8%). The overall incidence of G6PD deficiency was found to be 0.8%. There, was apparently decreased frequency of G6PD deficient blood donors with increasing age, and no relation could be ascertained between G6PD) deficiency and blood groups.

Effect of dietary garlic supplementation on lipid profile, oxidant-antioxidant status and aortic mechanical properties in experimental atherosclerosis

Experimental atherosclerosis was induced in a group of chicken [1 day old] by feeding a standard chicken diet supplemented with 1% cholesterol for twelve weeks. Another group was given the same diet supplemented with garlic [80 mg/kg body weight/day] for twelve weeks. The third group received standard poultry diet and served as controls. The alterations of lipids, lipid peroxidation products and antioxidant parameters of plasma and liver as well as the changes in the mechanical properties of the arterial walls were investigated. Hypercholesterolemia, a significant increase in low density lipoprotein cholesterol [LDL-C], triglycerides [TG] and phosphatidylcholine phospholipid fraction [PC] together with the significant decrease in high density lipoprotein cholesterol [HDL-C], very low density lipoprotein cholesterol [VLDL-C], total phospholipids and lysophosphatidylcholine [LPC] percent, induced by cholesterol feeding, were almost corrected by garlic administration. Lipid peroxidation products concentrations were significantly higher in atherosclerotic chicken and returned to almost control values by dietary garlic supplementation. In a corollary fashion, pronounced effect of garlic on antioxidants including liver glutathione [GSH], glutathione peroxidase [GPx], glucose-6-phosphate dehydrogenase [G6PDH], superoxide dismutase [SOD] and plasma antioxidant activity, expressed as D[max] was clearly noticed through restoration of their levels towards normal values after impairment under hypercholesterolaemia. As regards the mechanical properties, ultimate strain [U.S] of the thoracic aorta was significantly increased in the atherosclerotic group of chicken while the tensile strength [T.S] decreased significantly. The inner diameter of both thoracic and abdominal aortae decreased significantly in atherosclerotic group of chicken. These changes were returned to almost control values by addition of garlic powder to diet. These results suggest that garlic supplemented diet corrected not only pathological, lipids and antioxidant parameters but also the mechanical properties of the aortae

Oxidative stress and neonatal jaundice: an etiological study

This study aimed at investigating the role of oxidative stress in the development of neonatal jaundice. The enzyme activities of erythrocyte glucose-6-phosphate dehydrogenase [G6PD], plasma glutathione peroxidase [GSHPx] and glutathione-S-transferase [GST] were measured by quantitative determination of enzyme activity in 40 jaundiced full term newborns with different peak bilirubin levels [12.5 - 20 mg/dl] [not attributable to any known etiology] and 20 control newborns. The level of plasma advanced oxidation protein products [AOPP], an index of oxidative stress was also measured in these newborns. None of the jaundiced newborns needed phototherapy or exchange blood transfusion before the study. Plasma GSHPx activity was significantly lower in infants with hyperbilirubinaemia compared to control group. However, the enzyme activities of both plasma GST and erythrocyte G6PD as well as plasma AOPP concentrations were significantly higher in jaundiced newborn infants than in the control group. Plasma GSHPx activity demonstrated a significant negative correlation with GST activity, bilirubin concentration and AOPP levels. A significant positive correlation was also evident between serum bilirubin and plasma AOPP concentrations. The results of this study suggest that low GSHPx activity in jaundiced newborn infants might predispose these infants to oxidative stress. This may result in the development of mild oxidative hemolysis and jaundice

Neonatal screening for glucose-6-phosphate dehydrogenase deficiency in Taiwan.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathic disease in Taiwan. The mass neonatal screening of G6PD deficiency by fluorometric spot test in Taiwan was started with a pilot program in 1984. The nationwide screening was started on July 1, 1987, and a follow-up system comprising of eighteen referral hospitals, including outlying islands, was organized for confirmatory test, medical care and genetic counseling. From July 1987 to December 1997, 2,971,192 heel blood samples collected on filter paper from 1,143 delivery units were screened by four neonatal screening centers. 46,570 cases were confirmed as G6PD deficiency is estimated to be around 2.1% (male 3.1%, female 0.9%) in Taiwan. The coverage rate of neonatal screening was 99% in 1997. To assess the reliability of the confirmatory test, an external quality assurance (QA) program for G6PD assay was developed. Periodically, 3 or 5 lyophilized quality control materials with different activities of G6PD were sent to each referral hospital by speed post delivery in dry ice. From January 1988 to June 1998, 85 QA services were performed. Two hundred and seven (13.5%) abnormal QA results were found, which were attributed to clerk (11.6%), procedural (16.4%), and instrumental errors (47.3%). In aid to confirm G6PD deficiency, a method to detect the G6PD mutation by using the dried blood samples was developed. The frequencies of the mutant alleles in Taiwan were determined to be 46.8% (1376G > T), 16.2% (1388G > A), 7.9% (95A > G), 6.5% (493A > G), 5.6% (392G >T), 4.6% (1024C > T), 0.5% (487G > A) and 0.5% (519C > G), respectively.

G6PD deficiency in an unselected Brazilian population.

Data about glucose-6-phosphate dehydrogenase (G6PD) deficiency are not available in Brazil, a country characterized by a great mix of races. The disease is associated with ethnic groups. High prevalence (5 to 25%) has been reported in Africa, Asia, Middle East and the Mediterranean. We present here the first report of our one year experience testing for G6PD in an unselected population in the south of Brazil.

A simple, rapid fluorometric assay for the determination of glucose 6-phosphate dehydrogenase activity in dried blood spot specimens.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy of human beings and is the most common cause of jaundice and acute hemolytic anemia in South East Asia. The deficiency causes acute hemolytic anemia following ingestion of 6-amino quinoline antimalarials, phenacetin, and other substances. The rapid identification of infants or patients with this deficiency would help to prevent their exposure to these substances and subsequent risk to health. The assay is relatively simple. A 3mm punch from a dried blood spot sample is placed in a well of a black fluorescent microtiter plate containing calibrators and controls in duplicate. 100 microl of reagent is added and the sample is allowed to react for 30 minutes at ambient temperature after which 200 microl of stop reagent is added. The plate may be read immediately or up to one hour in a fluorescent reader (ex 355 nm: em 460 nm). Glutathione. ascorbate and bilirubin do not affect the assay. hemoglobin does quench the fluorescence by about 1.1 fluorescence units/g/dHb. This would not cause any false negatives and deficients would not be missed. G6PD activity in whole blood normal samples was examined at -20, 6 and 37 degrees C over 14 days. The samples lost about 20% activity after 48 hours and 31% by the end of 14 days. The samples stored at -20 degrees C and 6 degrees C remained relatively stable over this period. In a preliminary study eight diagnosed G6PD deficient samples had a mean value of 2.0 U/gHb (range 0.8 to 4.4) and fell within 3 SD units of the mean. Forty one normal samples had a mean of 6.6 micromol/min/gHb. Only one sample with a low hemoglobin level fell outside of 3 SD units of the mean. The Wallac assay was compared to the Sigma G6PD assay and although the values appeared lower at normal levels, the deficient samples compared well.

Effect of experimental diabetes on the activities of hexokinase, glucose-6-phosphate dehydrogenase and catecholamines in rat erythrocytes of different ages.

Hexokinase and glucose-6-phosphate dehydrogenase were assayed in various circulating age fractions i.e., young, middle-aged and old red cell from control, diabetic and insulin-treated diabetic rats. An increase in the activity of hexokinase was observed in three age-wise separated fractions of red cells from diabetic animals in comparison to control. The activity of glucose-6-phosphate dehydrogenase on the other hand decreased in separated ageing fractions of diabetic red cells when compared to control. Reversal of these two enzymes were observed in insulin-treated diabetic rats. The levels of glycosylated haemoglobin and catecholamines were found to increase with ageing red cells in controls and also increased in red cells plasma.

Leukocyte glucose 6 phosphate dehydrogenase as prognostic indicator in ANLL and CML.

Glucose 6 phosphate dehydrogenase (G6PD) was estimated in the leucocytes of 35 patients with acute non-lymphocytic leukemia (ANLL) and 10 patients with chronic myeloid leukemia (CML). G6PD levels were found to be significantly decreased in majority of the patients with ANLL while it was increased in all CML patients. Variation in G6PD was found to be dependent on the percentage of myelocytes inANLL. Cytogenetic analysis was also carried out in these patients. Correlation analysis of leucocyte G6PD activity and karyotype with prognostic assessment clearly indicated the association of (s) high percentage of chromosomal abnormalities especially translocations, (b) low survival and remission rates, with patients having decreased G6PD activity when compared to patients with normal activity in ANLL. The studies indicate that leucocyte G6PD may be useful as a diagnostic and prognostic tool.

Epidemiology of G-6-PD deficiency in Saudi Arabia

Glucose-6-phosphate dehydrogenase deficiency is the most frequently encountered red cell enzymopathy affecting the pentose phosphate pathway of glucose metabolism. The aim of this study is to conduct a comprehensive epidemiological survey of G-6-PD deficiency in Saudi Arabia Twenty-seven thousand, four hundred and seven Saudis living in 31 different areas were included in the study. The activity of G-6-PD was estimated in red cell hemolysates using commercially available kits from Boehringer Mannheim GmbH and the units of G-6-PD activity were calculated as mU/109 erythrocytes. Overall frequency of G-6-PD deficiency was 0.0905 and 0.041 in Saudi males and females, respectively. When separated on the basis of the provinces, the highest frequency was in the eastern province in both males and females and the lowest was in the northern province. Further separation of the data was carried out and significant differences were encountered in the different areas within each province. In each area the deficient females encountered were significantly more that the expected number calculated using Hardy Weinburg equilibrium. G-6-PD deficiency frequently occurs in several areas of Saudi Arabia. In general, this corresponds to malaria endemicity in the past. The Hardy Weinburg equilibrium is disturbed either due to the high rate of consanguinity or to the inactivation of the normal X-chromosome [Lyons phenomenon] in the heterozygote females. Awareness programs about this frequent enzymopathy are needed in Saudi Arabia to prevent the consequences of the deficiency state