Redox metabolism in glucose-6-phosphate dehydrogenase deficient erythrocytes and its relation to antimalarial chemotherapy

Experiments in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes parasitized by Plasmodium falciparum proved that depletion of glutathione increased fluxes of reactive oxygen species and was detrimental to the parasite at various sites and developmental stages. Chloroquine is also considered an inducer of oxidant damage due to its role in preventing heme polymerization. Recently it has been found that GSH prevents cellular damage by degrading the toxic heme. Consequently, we suggest that the use of combinations of chloroquine and depletors of GSH would be highly efficient for the chemotherapy of malaria.     

Association of red cell glucose-6-phosphate dehydrogenase with haemoglobinopathies

A total of 1,112 randomly selected Saudi Arabs, of both sexes, living in Jeddah and the surrounding areas were screened for the phenotypic distribution of red cell glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). They were also investigated for haemoglobin and for thalassaemia. Phenotyping of the haemoglobins and the red cell enzymes was carried out by starch gel electrophoresis and the dye-decolouration screening test, while the investigation for thalassaemia was carried out by globin-chain biosynthesis, followed by column chromatography. The red cell Gd- alleles were significantly associated with the sickle-cell gene in both the males (chi 2(1): AS-28.80; SS-4.89) and females (chi 2(1): AS-10.99; SS-13.16). A similar association was also observed between G6PD deficiency and thalassaemias in males (chi 2(1): alpha-thalassaemia - 3.13; beta-thalassaemia - 11.06) and females (chi 2(1): alpha-thalassaemia - 6.63). However, no such association was detected between red cell 6PGD types and haemoglobin genes. The results suggest that the red cell G6PD deficiency, sickle-cell and thalassaemia genes might have evolved as a result of the same ecological factor, probably malaria.     

Primate red cell enzymes: glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase

Glucose-6-phosphate dehydrogenase (E. C.: 1.1.1.49) phenotypes and 6-phosphogluconate dehydrogenase (E. C.: 1.1.1.44) phenotypes were determined by starch-gel electrophoresis of red cell hemolysates of Galago crassicaudatus subspp., Propithecus verreauxi, Lemur spp., Hapalemur griseus, and Macaca mulatta. A single glucose-6-phosphate dehydrogenase (G6PD) phenotype was found in each species. A single 6-phosphogluconate dehydrogenase (6PGD) phenotype was found in Lemur spp., Hapalemur griseus, and Galago crassicaudatus argentatus. In a group of six Propithecus verreauxi, three 6PGD phenotypes, PGD A, PGD AB, and PGD B, were found. Three phenotypes, PGD A, PGD AB, and PGD B, were found in 38 G. c. crassicaudatus. The three phenotypes in each species are apparently the products of two codominant autosomal alleles, PGD^A and PGD^B. The frequency of PGD^A in G. c. crassicaudatus is 0.263. A population of 260 free-ranging macaques displays a polymorphism at the 6PGD locus. Three phenotypes, PGD A, PGD AB, and PGD B, were found. These also appear to be controlled by two codominant autosomal alleles, PGD^A and PGD^B the frequency of PGD^A = 0.913. Additional analysis of three well-defined troops within the macaque population indicated that there are no significant differences between the troops or within the population at the 6PGD locus.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Insulin secretion in patients deficient in glucose-6-phosphate dehydrogenase

Intravenous (IVGTT) and oral glucose tolerance tests (OGTT) were carried out in 12 men with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and in 11 normal men. The race, the mean age and body mass index were similar in the G-6-PD deficient and in the normal men. No significant differences were demonstrated between mean plasma glucose levels in the G-6-PD deficient subjects and those in the normal men during IVGTT and OGTT. In contrast the insulin levels were significantly lower for the G-6-PD deficient subjects as compared to the controls at 30 minutes (P less than 0.04) in the OGTT and at 1 min (P less than 0.001), 3 min (P less than 0.001), 5 min (P less than 0.001) and 10 minutes (P less than 0.002) in the IVGTT. All indexes of first phase insulin release were also significantly (P less than 0.001) lower in G-6-PD deficient men. These results emphasize the metabolic importance of G-6-PD in the process of glucose induced insulin release.     

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.     

Activity of glucose-6-phosphate dehydrogenase in black sea fish erythrocytes

The activity of glucose-6-phosphate dehydrogenase (G6PD) in erythrocytes of the dogfish and 5 other fish species from the Black Sea as well as the activity of monoamine oxidase in the seabream serum are investigated. A short-term intensive swimming, which is a stress for fish, as it produces a 10-fold rise of the monoamine oxidase activity, was the cause of a fall of theG6PD activity level by 43–45 % (p < 0.05) in erythrocytes of the horse mackerel and seabream. The stay of the scorpionfish under hypoxic conditions (15% saturation), which also is a stress for the fish, also produced a decrease of the enzyme activity level in erythrocytes by 62 % (p < 0.001).     

Plasmodium falciparum: thiol status and growth in normal and glucose-6-phosphate dehydrogenase deficient human erythrocytes

Thiol status and growth in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Experimental Parasitology 57, 239-247. The relationship of the thiol status of the human erythrocyte to the in vitro growth of Plasmodium falciparum in normal and in glucose-6-phosphate dehydrogenase (G6PD)-deficient red cells was investigated. Pretreatment with the thiol-oxidizing agent diamide led to inhibition of growth of P. falciparum in G6PD-deficient cells, but did not affect parasite growth in normal cells. Diamide-treated normal erythrocytes quickly regenerated intracellular glutathione (GSH) and regained normal membrane thiol status, whereas G6PD-deficient cells did not. Parasite invasion and intracellular development were affected under conditions in which intracellular GSH was oxidized to glutathione disulfide and membrane intrachain and interchain disulfides were produced. An altered thiol status in the G6PD-deficient erythrocytes could underlie the selective advantage of G6PD deficiency in the presence of malaria.     

Artifacts imitating aging of glucose-6-phosphate dehydrogenase in human erythrocytes.

Data in the literature based on the technique of graded osomotic hemolysis have been re-evaluated. Differences were previously found in the glucose-6-phosphate dehydrogenase/hemoglobin ratio and in the heat stability of the enzyme in hemolysates of 'old' and 'young' cells. These differences were believed to be due to the aging of the enzyme. As the erythrocyte membrane acts as a molecular sieve under hypotonic conditions [cf. Cseke, E., Váradi, A., Szabolcsi, G., and Biszku, E. (1978) FEBS Lett. 96, 15--18], the hemolysate obtained when a fraction is lysed does not properly represent the content of the lysed cells. As hemoglobin is lost from cells which are not yet lysed, the enzyme/hemoglobin ratio is underestimated in 'old' cells and overestimated in 'young' cells. It is further shown that the observed differences in the heat stability of glucose-6-phosphate dehydrogenase in the fractions obtained by graded hemolysis are due to the presence of different concentrations of endogeneous NADP. Therefore the published data obtained by graded osmotic hemolysis do not prove the assumption that the enzyme is aging during the lifetime of the erythrocyte.     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Red cell glucose-6-phosphate dehydrogenase deficiency among Andhras

Sixty-eight Andhra males and 45 Andhra females from Visakhapatnam town of Andhra Pradesh, India have been investigated for G-6-PD deficiency. The GdB- gene has a frequency of 4.41% among males. No G-6-PH deficient females were detected. The present data have been compared with the available tribal and non-tribal data from Andhra Pradesh. It is observed that the present sample, though non-tribal in nature, presents a relatively considerable frequency of the GdB- gene.