The effect of astaxanthin and cadmium on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzymes activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro.

In this study, the effects of astaxanthin (AST) that belongs to carotenoid family and cadmium (Cd), which is an important heavy metal, on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzyme activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro were studied. In in vitro studies, 6PGD enzyme was purified from rat erythrocytes with 2',5'-ADP Sepharose4B affinity chromatography. Results showed inhibition of enzyme by Cd at IC50 ; 346.5 µM value and increase of 6PGD enzyme activity by AST. In vivo studies showed an increase in G6PD, 6PGD, and GR enzyme activities (P Ëƒ 0.05) and no chance in TrxR enzyme activity by AST. Cd ion inhibited G6PD, 6PGD, and GR enzyme activities (P Ë‚ 0.05) and also decreased TrxR enzyme activity (P Ëƒ 0.05). AST + Cd group G6PD enzyme activity was statistically low compared with control group (P Ë‚ 0.05). 6PGD and TrxR enzyme activities decreased without statistical significance (P Ëƒ 0.05); however, GR enzyme activity increased statistically significantly (P Ë‚ 0.05).

Autosomal phosphogluconic dehydrogenase polymorphism in the cat (Felis catus L.).

Three patterns of 6-phosphogluconic dehydrogenase activity were obtained by starch-gel electrophoresis of blood from domestic cats. Genetic analysis indicates control of these patterns by a pair of alleles at an autosomal locus. Presence of three enzymatically active bands in heterozygotes and of single bands in homozygotes is compatible with at least a dimeric structure for the enzyme.

6-phosphogluconate dehydrogenase: hemizygous manifestation in a patient with leukemia.

In a study of 41 patients with chronic myelocytic leukemia, two were found to have the 6-phosphogluconate dehydrogenase heterozygous phenotype A-B, and two had the phenotype characteristic of Pd(B) homozygosity. Since one of the two with Pd(B) homozygosity was the mother of two children with the A phenotype, it was presumed that she carried a Pd(A) gene not expressed in her blood cells. his was confirmed by electrophoretic analysis of her fibroblasts, which had the A-B phenotypic pattern. Gene deletion is considered to be the most likely explanation.

Impact of the Di(2-Ethylhexyl) Phthalate Administration on Trace Element and Mineral Levels in Relation of Kidney and Liver Damage in Rats.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used synthetic polymer in the industry. DEHP may induce reproductive and developmental toxicity, obesity, carcinogenesis and cause abnormal endocrine function in both human and wildlife. The aim of this study was to investigate trace element and mineral levels in relation of kidney and liver damage in DEHP-administered rats. Therefore, prepubertal male rats were dosed with 0, 100, 200, and 400 mg/kg/day of DEHP. At the end of the experiment, trace element and mineral levels, glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6-PGD), glutathione reductase (GR) and glutathione S-transferase (GST) enzyme activities were evaluated in the serum, liver, and kidney samples of rats. Furthermore, serum clinical biochemistry parameters, organ/body weight ratios and histological changes were investigated to evaluate impact of DEHP more detailed. Our data indicated that sodium (Na), calcium (Ca), potassium (K), lithium (Li), rubidium (Rb) and cesium (Cs) levels significantly decreased, however iron (Fe) and selenium (Se) concentrations significantly increased in DEHP-administered groups compared to the control in the serum samples. On the other hand, upon DEHP administration, selenium concentration, G6PD and GR activities were significantly elevated, however 6-PGD activity significantly decreased compared to the control group in the kidney samples. Decreased G6PD activity was the only significant change between anti-oxidant enzyme activities in the liver samples. Upon DEHP administration, aberrant serum biochemical parameters have arisen and abnormal histological changes were observed in the kidney and liver tissue. In conclusion, DEHP may induce liver and kidney damage, also result abnormalities in the trace element and mineral levels.

The effect of copper on red cell enzyme activities.

Previous studies have shown a marked effect of very high levels of copper on red cell glucose-6-phosphate dehydrogenase and glutathione. When the effect of more nearly physiological levels of copper were studied, red cell hexokinase, phosphofructokinase, phosphoglyceric kinase, pyruvate kinase, and 6-phosphogluconate dehydrogenase were found to be inhibited. Inhibition was observed both when copper was added directly to hemolysates or when hemolysates were prepared from red cells from whole blood which had been incubated with copper and washed. The inhibition of red cell enzymes by copper was completely reversed by the addition of EDTA.

Implication of lysine residues in the loss of 6-phosphogluconate dehydrogenase activity in aging human erythrocytes.

The activity of 6-phosphogluconate dehydrogenase (6-PGDH) decreases in aged human erythrocyte populations. The aged enzyme has 11 lysine residues less than the young enzyme, when they are measured with 2,4,6-trinitro-benzenesulfonic acid (TNBS). Treatment of young enzyme with ascorbate for 15 min produces the loss of 8 lysine residues and the diminution of enzymatic activity. These results suggest that there is a modification of lysine residue in human erythrocytes during senescence, probably caused by oxidation. This modification of lysine residue could imply the loss of enzymatic activity. This result is similar to that found in rat liver 6-PGDH during aging, described previously (Gordillo et al., J. Biol. Chem. 264 (1989) 17014-17019).

Adenine and pyridine nucleotides during rabbit reticulocyte maturation and cell aging.

In this paper we have used a new method which allows the simultaneous extraction and HPLC determination of ATP, ADP, AMP, NADP, NADPH, NAD and NADH to evaluate the changes in concentration of these compounds during maturation of rabbit reticulocytes and cell aging. The results show a significant increase of ATP concentration, higher ATP/ADP, ATP/AMP ratios and lower NADP+/NADPH, NAD+/NADH ratios in rabbit reticulocytes when compared to mature cells. Similar results were also obtained when the whole red blood cell population was separated into fractions of increasing mean age on discontinuous gradients of Percoll-BSA. These metabolic modifications are discussed in relation to the age-dependent metabolic decline of the erythrocyte.

Homeostasis between lipid peroxidation and antioxidant enzyme activities in healthy human aging.

In healthy people the plasma malondialdehyde increases with age, however there is a simultaneous rise in the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in red blood cells, although both enzymes show a biphasic behaviour, that is, reaching the lowest values at 40-50 years of age to rise remarkably later on. No significant changes were found in the case of glutathione peroxidase but the age-dependent behaviour is similar to the other enzymes mentioned above. The activity of glutathione reductase shows a clear increase depending on age, up to middle age, with or without flavin adenine dinucleotide. We conclude that the increase in the activities of the anti-oxidant enzymes of the red blood cells during aging, could be interpreted as a positive feedback mechanism in response to rising lipid peroxidation. Consequently, from the point of view of the parameters used the homeostasis between the production of free radicals and anti-oxidant systems seems to be maintained in the common, normal aging pattern.

Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP+, immunological and kinetic properties.

Human granulocyte 6 phosphogluconate dehydrogenase has been totally purified from a single patient with chronic granulocytic leukaemia. 48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds. Purified 6 phosphogluconate dehydrogenase was homogeneous when tested in acrylamide and acrylamide SDS gel electrophoresis or in immunodiffusion. The enzyme was immunologically identical in red blood cells, blood platelets and normal leukocytes. The fixation of both substrates, NADP-+ and 6 phosphogluconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibitor strictly competitive with respect to NADP-+ and non competitive with respect to 6 phosphogluconate. 2-3 Diphosphoglycerate seemed to be able to bind on both the fixation sites of NADP-+ and 6 phosphogluconate. The inhibition by ATP was competitive with 6 phosphogluconate and non competitive with NADP-+. 6 phosphogluconate dehydrogenase was inactivated by SH reagents and was partially protected against this inactivation by both substrates. Both substrates protected the enzyme against thermal inactivation. The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.

Phenotyping of eight erythrocytic enzymes in one acrylamide gel.

A procedure has been developed to phenotype eight erythrocytic enzymes, phosphoglucomutase (PGM1), adenylate kinase (AK), 6-phosphogluconate dehydrogenase (6PGD), adenosine deaminase (ADA), glyoxalase (GLO), esterase-D (EsD), acid phosphatase (AcP), and glutamic pyruvate transaminase (GPT) in one acrylamide gel and also to detect the presence of common abnormal hemoglobins. The agar overlay technic has been eliminated. This simplified procedure renders the phenotyping of erythrocytic enzymes practical in paternity testing.

Glucose-6-phosphate dehydrogenase deficiency and chronic haemolysis in an English family.

Three male members of an English family with chronic haemolytic anaemia due to glucose-6-phosphate dehydrogenase deficiency are reported. The disease was symptomless in one adult, but crises, caused either by increased haemolysis or failure of marrow compensation, occurred in two children. They were typically precipitated by trivial infection. Two normal female members of the family were obligatory heterozygotes. A hitherto undescribed ;slow' variant of the enzyme was identified electrophoretically.

Glutathione-linked enzyme activities in red cell aging.

The enzyme activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phospho-gluconate dehydrogenase (6PGD), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were measured in normal human red cells separated centrifugally in a discontinuous density gradient of Percoll. Activities of G6PD, GSH-Px and GR decreased with red cell aging.

Isoelectrofocusing in acetate membrane: the method and application.

Acetate membrane as a new medium for isoelectrofocusing was used in this study. The acetate membrane was specially produced for isoelectrofocusing, and contains a surface active agent. The ampholine solution system and isoelectric condition for this purpose are described. Polymorphic traits as alpha1-antitrypsin in human serum and 6-phosphogluconate dehydrogenase (6-PGD) in human erythrocyte could be identified by this method. The isoelectric patterns and the isoelectric points of these proteins were demonstrated on the acetate membrane.

Oxidative haemolysis in protein malnutrition.

A study of the haemolytic anaemia observed in protein-energy malnutrition (PEM) in Kivu disclosed the following results. The in vitro resistance to oxidative aggressions of PEM patients' erythrocytes was decreased: when incubated with acetylphenylhydrazine, a higher percentage of the cells showed Heinz bodies, as compared with erythrocytes of local controls. Normal or increased activities were found for certain erythrocyte enzymes involved in the detoxification of activated oxygen: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase. The level of reduced glutathione was not decreased. Reduced activities were observed for two enzymes containing trace elements: glutathione peroxidase and superoxide dismutase. It is suggested that the shortened erythrocyte lifespan observed in PEM patients corresponds to an oxidative process which results from the decrease of both enzyme activities. The hypothesis that depletion of trace elements could be responsible for the decreased activity of those enzymes is discussed.

Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency. II. Effect of pyrimidine nucleotides and their derivatives on glycolytic and pentose phosphate shunt enzyme activity.

We evaluated the glycolytic intermediate concentrations from the erythrocytes of a patient with hereditary pyrimidine 5'-nucleotidase (P5'N) deficiency. Conclusive evidence for a metabolic block was not found. We evaluated the effects of the pyrimidine (cytidine and uridine) tri- and diphosphate nucleotides (CTP, CDP, UTP, UDP) and the choline and ethanolamine derivatives of CDP (CDP-choline, CDP-ethanolamine) on the activities of key enzymes of the Embden-Meyerhof pathway. CTP and UTP inhibited fructose-6-phosphate competitively for phosphofructokinase and phosphoenolpyruvate competitively for pyruvate kinase. In both cases, the Ki of the pyrimidine nucleotide and Km of the glycolytic substrate were above their intraerythrocytic concentrations. CTP was a competitive inhibitor of ADP for pyruvate kinase with a Ki near its intraerythrocytic concentration. CDP-choline and CDP-ethanolamine had no effect on the activities of Embden-Meyerhof or pentose phosphate shunt enzymes. Thus, the nature of the hemolytic anemia in hereditary P5'N deficiency remains enigmatic.

Metabolic changes in red cells in response to adhesion of porcine K88 fimbriated Escherichia coli.

Red cells glycolytic enzymes attached and nonattached to K88+ Escherichia coli were assayed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase activities, were measured. E. coli with K88ab fimbriae, E. coli with K88ac fimbriae, and isolated K88ab fimbriae were investigated for their effect on the above enzymes. Different changes were obtained with K88ab + bacteria compared with K88ac + bacteria. Purified fimbriae gave a third set of responses.

Mechanisms of the toxic effect of lead. I. Free lead in erythrocyte.

In this work, a selection of children was examined based on their free erythrocyte protoporphyrin (FEP) and total blood lead (PbB) contents. Two groups with clear differences in lead sensibility were selected. One group with high FEP levels (deep lead alteration), named "normal," and one group with low FEP levels (discrete lead alteration), named "lead tolerant," and both with similar PbB concentration, were formed. The selection, based on FEP level, showed a correlation with other indicators (neuromotor alterations). A lower activity of the enzymes Glyceraldehyde 3-phosphate dehydrogenase (GA3PD), Glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and Lactate dehydrogenase (LDH) was found in the normal group when compared to the tolerant group. Therefore, the influence of the lead acetate upon the same erythrocyte enzyme activities was investigated. Lead inhibition of the enzymes GA3PD and G6PD was found, and this inhibition could be related to the free lead content found in the erythrocytes. Inhibition of the enzymes 6PGD and LDH was also found, but these enzymes were not inhibited by exposure to lead acetate. Erythrocyte free lead content was high in the normal group when compared to the lead tolerant group and could be a determinant factor in the biochemical alterations found. Low erythrocyte free lead contents could be an indicator of lead tolerance. TCA-precipitation was found to be a useful method to evaluate free lead content; it gave similar results to gel filtration molecular chromathography.

Clinical utility of fractionating erythrocytes into "Percoll" density gradients.

Two rapid methods for fractionating the RBC into five or nine layers of increasing density are reported. These procedures have been used to monitor the decline of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activity during the process of red cell aging in normal subjects and in beta-thal carriers, to study transfused patients with G6PD and pyruvate kinase (PK) deficiency and to test the effects of inositol hexaphosphate (IHP) encapsulation on RBC subpopulations.

Rapid purification of human erythrocyte 6-phosphogluconate dehydrogenase by means of affinity chromatography.

A rapid two-step procedure is reported by which homogeneous 6-Phosphogluconate dehydrogenase can be isolated from human erythrocytes. This method is based upon direct affinity chromatography of the hemolysates on adenosine 2',5'-bisphosphate-agarose (yielding a 4,500-fold purification), followed by anion exchange chromatography on a micro-column of DEAE-Sephadex. The present method represents a considerable simplification over previously available procedures for the purification of this enzyme protein from human erythrocytes.

Role of pentose-phosphate pathway in haemolytic crisis of chronic copper toxicity of sheep.

Although the rise in blood copper is associated with onset of the acute haemolytic crisis of chronic copper poisoning in sheep, the sudden fall in erythrocyte glutathione is apparently not due to a direct action of the copper. Moreover the reduced glutathione of the red cells is converted to some form that is not capable of regeneration by the pentose-phosphate mechanism. Only negligible inhibition of the pentose-phosphate enzymes occurs. As the haemolysis proceeds, there is a rapid recovery of erythrocyte glutathione levels, and a marked increase in pentose-phosphate enzyme activity, consistent with influx of young red cells. It seems that the release of copper into blood from liver at the haemolytic crisis is associated with an increase of the oxidative state of the blood, possibly by simultaneous release of other components from the liver.