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.

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 æg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease

Increased fetal hemoglobin levels in patients infected with human immunodeficiency virus (HIV1/2)

Fetal hemoglobin was measured in HIV1/2 patients under treatment with combined therapy (zidovudine and a protease inhibitor). A total of 143 patients and 103 normal individuals were investigated by the quantitative method of Betke and the semi-quantitative acid elution method of Kleihauer. In the normal person, hemoglobin F makes up less than 1 percent and an increase higher than 1.5 percent was observed in 21.4 percent of HIV patients by the method of Betke and in 24.8 percent of HIV-infected patients by the method of Kleihauer. The quantitative biochemical method of Betke showed that the populations were significantly different (two-tailed Mann-Whitney test). The reason for this hemoglobin F increase might be ascribed to the effect of zidovudine or to direct viral action on gamma chain expression. The finding of a higher F cell frequency indicated by the method of Kleihauer rather suggests that there is an increased F cell clone proliferation rather than an increase in hemoglobin F level in every cell

Correlation of discocyte frequency and ATP concentration in preserved blood: a morphological indicator of red cell viability

Red blood cells (RBC) are viable if kept in an adequate preservative solution, although gradual changes in morphology and metabolism may occur. There is a gradual decrease in adenosine-5'-triphosphate (ATP) cocentration, pH, glucose consumption, and enzyme activity during preservation. The normal discocyte shapes are initially replaced by echimocytes and stomatocytes and, at final stages, by spherocytes, the last before eplenic sequestration. Post-transfusional survival has been correlated with the ATP concentration. RBC preserved in ADSOL, a solution containing adenine, dextrose, sodium chloride, and mannitol, are viable for transfusion for up to 6 weeks. Erythrocytes from 10 blood units taken from healthy adult donors were preserved for 12 weeks in ADSOL at 4 graus Celsius. We now report a significant correlation (r2 = 0.98) between the percentage of discocytes (89 to 7 percent) and ATP (100 to 10 percent) concentration in ADSOL-preserved RBC. The results suggest that the percent of discocyte shapes used as an indicator OF ATP concentration may be a useful indicator for quality control of RBC viability in centers which have limited assay facilites.

Alteraçoes bioquimicas durante a preservaçao de eritrocitos in vitro em meio CE com vitamina E / Biochemical changes during red blood cells preservation in CE medium plus vitamin E in vitro

Ja foi demonstrado que o meio CE com vitamina E mantem estaveis durante 100 dias os valçores hematimetricos de eritrocitos parcialmente fixados em glutaraldeido (Leonart,M.S.S. et al. Rev. Bras. Anal. Clin., 21:111, 1989). Neste trabalho, partiu-se de 20 unidades de sangue nenoso de individuos saudaveis e estudou-se o comportamento bioquimico dos eritrocitos durante 12 semanas de preservaçao a 4 C em meio CE com vitamina E (CE), em meio CE com vitamina E apos fixaçao parcial em glutaraldeido (GACE), e em ADSOL (Heaton, A et al. Transfusion, 21:600, 1981). Os valores de adenosina trifosfato (ATP), que se mantiveram acima de 80% dos iniciais durante 2 semanas de estoque, decairam rapidamente, e se exauriram em cerca de 4 semanas, sem diferenças significativas entre a s tres formas de preservaçao. As concentraçoes de 2,3-difosfoglicerato (2,3-DPG) permaneceram acima de 90% das iniciais na 1 semana. Nas amostras em ADSOL, os valores de 2,3-DPG foram significativamente mais baixos entre 14 e 28 dias (p< 0,05) e se exauriram ao final da 3 semana; nas CE e GACE, ao final da 5 semana. Observou-se decaimento na concentraçao extracelular de ions sodio e aumento na de ions potassio em 12 semanas de preservaçao. A entrada de sodio e a saida de potassio ocorreram mais lentamente nos eritrocitos preservados em ADSOL que em meio CE (p<0.05). A percentagem de hemolise observada durante o periodo de preservaçao foi significativamente diferente entre os tres grupos (p<0,05). Ao final de 12 semanas, ocorreu menos de 1% de hemolise em GACE, cerca de 2% em CE, e cerca de 7% em ADSOL. Concluiu-se que eritrocitos humanos conservados em meio CE mais vitaminas E, com ou sem fixaçao parcial em glutaraldeido, mantem o sistema de transporte de sodio e potassio e o metabolismo ativo de consumo de ATP e 2,3-DPG, e baixo grau de hemolise durante 12 semanas; portanto, adequados ao uso em laboratorio

Gd(-) Carapicuiba, a rare glucose-6 phosphate dehydrogenase variant associated with moderate enzyme deficiency and chronic hemolysis

A 27 year old Brazilian male of both Portuguese and Spanish origin presenting nonspherocytic chronic hemolytic anemia was found to have a rare glucose-6-phosphate dehydrogenase variant herein named Gd(-) Carapicuiba. The red blood cell enzyme variant is characterized by a moderate enzyme deficiency (47%), high Km for its substrates G6P and NADP, decreased activity against deamino-NADP, increased Ki for NADPH and decreased heat stability. The clinical signs of the patient are probably related to these properties of the enzyme variant

Viabilidade de sangue conservado em recipientes de varias procedencias. / Viability of blood stored in recipients of different sources

Os autores estudaram os sangues contidos em recipientes de plastico e de vidro de cinco procedencias diferentes, utilizando para isto as determinacoes de 2-3-difosfoglicerato (2-3-DPG), adenosina trifosfato eritrocitaria (ATP), hemoglobina (Hb), latico desidrogenase (LDH), sodio e potassio plasmaticos, e pressao parcial sanguinea do oxigenio e do dioxido de carbono e pH sanguineo. Quanto ao 2-3-DPG e ATP, hemoglobina e LDH plasmatica e pH, houve paralelismo, mas houve leves diferencas nos demais parametros entre as diversas procedencias

Solubilization of erythrocyte stroma-bound enzymes by Triton X-100.

Os autores estudaram o efeito solubilizador do octilfenoxipolietoxietanol (Triton X-100) sobre cinco enzimas ligadas ao estroma, a fosfofruto quinase, a gliceraldeido-3-fosfato desidrogenase, aldolase, glutationa redutase e glutationa peroxidase. Nao foram detectadas diferencas entre as atividades enzimaticas de hemolisados obtidos com agua contra Triton X-100 a 1%, antes e depois de centrifugacao, significando que o Triton X-100 nao aumentou a solubilizacao das enzimas estudadas. As maiores atividades de hemolisados desprovidos de estroma foram obtidos com Triton X-100, quando nao utilizado previo tratamento pelo congelamento e descongelamento.As maiores atividades foram obtidas em hemolisados nao centrifugados, submetidos ao congelamento e descongelamento, em agua ou Triton X-100 a 1%

Efeito da anfotericina B sobre a viabilidade eritrocitaria.Perspectiva do seu uso na prevencao da doenca de Chagas induzida pela transfusao de sangue. / Effect of amphothericin B on the erythrocyte viability. Perspective of its use in the prevention of Chagas' disease induced by blood transfusion

O efeito do antibiotico anfotericina B sobre o sangue armazenado em recipientes plasticos foi estudado. Foram determinados os parametros adenosina-trifosfato, 2-3-difosfoglicerato, pressao parcial de oxigenio, pressao parcial de dioxido de carbono, pH, hemoglobina e latico-desidrogenase plasmaticas e sodio e potassio plasmaticos. Leves diferencas foram notadas em relacao aos controles, mas houve significativo aumento do potassio e diminuicao do sodio nos recipientes com anfotericina B

Determinacao da lisozima no liquido cefalorraqueano normal e patologico. / Determination of lisozymes in the normal and pathological cerebrospinal fluid

As concentracoes da LZM foram determinadas nas amostras de LCR normal e patologico; o metodo utilizado foi o turbidimetrico melhorado. A LZM foi mensuravel em todas as amostras normais; apresentou-se aumentada em 60% e 83% dos casos de meningites virais e bacterianas, respectivamente. Foram, tambem observadas elevacoes em neurocisticercose, leucemia linfoblastica aguda, mieloma multiplo e neurocriptococose, nos dois casos de neurolues, a LZM do LCR foi normal

Deficiencia de glicose-6-fosfato desidrogenase em recem-nascidos. / Glucose-6-phosphate dehydrogenase deficiency in newborn

Os autores apresentam 8 casos de recem-nascidos portadores de deficiencia de glicose-6-fosfato desidrogenase, detectados entre 67 pacientes portadores de ictericia na primeira semana de vida, sem incompatibilidade sanguinea materno-infantil (11,9%), de um total de 405 pacientes com qualquer tipo de ictericia (6,29%). E comentada a evolucao dos pacientes especialmente dos que apresentaram comprometimento neurologico