Acatalasemic Erythrocytes Research Articles

Effect of hydrogen peroxide on normal and acatalasemic mouse erythrocytes

ObjectivesNormal and acatalasemic mouse erythrocytes were used to clarify the relationship between oxidative damage in H2O2-treated erythrocytes and catalase activity. Design & MethodsGeneration of hydrolysis-resistant erythrocytes and hemolysis were examined. The osmotic fragility test, the negative charges and the number of membrane-flickering erythrocytes among the H2O2-treated erythrocytes were investigated. ResultsSmall amounts of hydrolysis-resistant mouse erythrocytes were generated by treatment with 0.1 mM H2O2, and the amount of acatalasemic erythrocytes was larger than untreated controls. Hemolysis in the acatalasemic erythrocytes was observed 30 min after the addition of the H2O2. A drastic increase in hydrolysis-resistant erythrocytes and a loss of membrane proteins in the acatalasemic erythrocytes were found as a result of the addition of 1 mM H2O2. Hemolysis in normal erythrocytes was observed at 3 mM H2O2. ConclusionsCatalase is a potent H2O2-scavenger even in acatalasemic mouse erythrocytes. It is concluded that the drastic increase of hydrolysis-resistant erythrocytes is induced by a loss of membrane function and is associated with the low catalase activity in these cells.

Characterization of Acatalasemic Erythrocytes Treated with Low and High Dose Hydrogen Peroxide: HEMOLYSIS AND AGGREGATION

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.

Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes

A new method of hydrogen peroxide determination for the measurement of catalase activity and rates of hydrogen peroxide removal by erythrocytes was described. Hydrogen peroxide was determined by converting it to the indamine dye with a water-soluble ironporphyrin and measuring the absorbance at 590 nm. This method was applied to the assay of catalase in hemolysates from human, rat and mouse blood. The activities obtained were in agreement with those obtained by other methods including UV method. The present method was also applied to the determination of rates of hydrogen peroxide removal by intact erythrocytes from human subjects, rats and mice. Data suggested that normal erythrocytes have substantial capacity to remove extracellular hydrogen peroxide. From the measurement of catalase activity in erythrocytes treated with 3-amino-1,2,4-triazole and rates of hydrogen peroxide removal by the erythrocytes, it is deduced that rate constants related to the hemoglobin content (k/g Hb) for hydrogen peroxide removal by catalase in normal and acatalasemic erythrocytes are 42.0 ± 6.0 and 8.0 ± 3.0, respectively.

Impaired in vitro accumulation of mercury in erythrocytes of acatalasemic mice.

In order to elucidate the role of erythrocyte catalase in the accumulation of mercury in erythrocytes, labeled erythrocytes and plasma were prepared by exposing normal and acatalasemic mice to radioactive mercury vapor (203Hg0: 6.8mg/m3) for 30 min. Labeled erythrocytes (or plasma) were mixed with unlabeled plasma (or erythrocytes) of normal or acatalasemic mice and incubated at 0 degrees C for 1 h. After incubation, the radioactivity of mercury in the erythrocytes and the plasma was measured by a gammascintillation counter. When labeled erythrocytes were incubated with unlabeled plasma, the ratio of mercury transferred from acatalasemic erythrocytes to normal plasma (11.6%) or to acatalasemic plasma (13.3%) were significantly higher than that from normal erythrocytes to normal plasma (1.8%) or to acatalasemic plasma (2.2%). When labeled normal (or acatalasemic) plasma was incubated with unlabeled normal or acatalasemic erythrocytes, the uptake of mercury by acatalasemic erythrocytes from normal plasma was 2.0%, and 1.2% from acatalasemic plasma, which tended to be lower than that by normal erythrocytes from normal plasma (3.4%) or from acatalasemic plasma (2.2%). The results indicated impaired accumulation of mercury in acatalasemic erythrocytes, suggesting the importance of catalase in taking up mercury in erythrocytes and protecting other organs from toxic effects of metallic mercury.

Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase.

To investigate the relative importance of catalase and glutathione in erythrocyte oxidant defense, human and mouse (normal and acatalasemic) erythrocytes were reversibly lysed and resealed in the presence of exogenous catalase or glutathione. This resulted in an increase in intracellular catalase activity or glutathione concentration in the resealed erythrocytes while normal cellular structure, hemoglobin concentration, cell volume, cellular deformability, and adenosine triphosphate concentration were maintained. Resealing alone had no effect on oxidant sensitivity. In human cells, a threefold increase in catalase activity resulted in the maintenance of glutathione levels in response to hydrogen peroxide (H2O2) challenge. Reconstitution of congenitally acatalasemic mouse erythrocytes, which were extremely sensitive to even micromolar concentrations of H2O2 with purified catalase resulted in complete protection against H2O2. Indeed, the catalase-reconstituted acatalasemic cells were less sensitive to H2O2-mediated damage than were normal, catalase-replete mouse cells. In contrast, alteration of the glutathione status of human and mouse (normal and acatalasemic) cells had no significant effect on oxidant sensitivity. Even a five-fold increase in intracellular glutathione concentration (greater than 30 micromoles glutathione per gram of hemoglobin) in normal or catalase-deficient (azide-treated or acatalasemic) red blood cells had no protective effect against H2O2-mediated lipid peroxidation or methemoglobin generation. Similarly, depletion of glutathione by 1-chloro-2,4-dinitrobenzene also had no effect on erythrocyte H2O2 sensitivity. These results suggest an important role for catalase in protection against H2O2-mediated damage at physiologic levels and that catalase is as at least as important as glutathione in cellular defense against H2O2.

Characterization of human residual catalase of an acatalasemic patient by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunodetection.

Isoelectric points and subunit sizes of catalases in human blood and human cultured skin fibroblasts from acatalasemic and normal subjects were analyzed by isoelectric focusing in agarose gel and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, followed by electroblotting to polyvinylidene difluoride membranes for immunodetection. The results indicated that the isoelectric point of residual catalase in the C fraction prepared from acatalasemic erythrocytes was identical with that of catalase prepared from normal erythrocytes. The residual catalase in homogenates of acatalasemic cultured skin fibroblasts also reacted with anticatalase rabbit serum and had an isoelectric point identical with that of normal catalase. Subunit sizes of normal and acatalasemic catalases in the C fractions of erythrocytes were also found to be identical on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting and immunoenzymatic amplification. The results indicated no substantial difference in molecular size and charge of catalase proteins between normal and acatalasemic erythrocytes and fibroblasts.

Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes

Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H2O2 in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H2O2). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H2O2 in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H2O2 when H2O2 is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.

Catalase and Glutathione Peroxidase Are Equally Active in Detoxification of Hydrogen Peroxide in Human Erythrocytes

Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H2O2 in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H2O2). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H2O2 in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H2O2 when H2O2 is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.

In vitro mercury uptake by human acatalasemic erythrocytes.

Human acatalasemic erythrocytes had only 0.01 to 0.06X the uptake found in normal erythrocytes with hydrogen peroxide, and 0.06 to 0.24X the uptake without hydrogen peroxide in regard to their ability of in vitro mercury uptake from air saturated with mercury vapor. Normal erythrocytes with hydrogen peroxide took up 9.3 to 37.4X more mercury than cells without hydrogen peroxide, whereas acatalesemic cells with hydrogen peroxide took up 2.4 to 5.8X more mercury than cells without hydrogen peroxide. Similar results were obtained from normal and acatalasemic hemolysates. In addition, the concentration of hydrogen peroxide affected the uptake of mercury by both normal and acatalasemic erythrocytes. Data indicate that the catalase-hydrogen peroxide system plays a role in mercury uptake, presumably by mercury oxidation, since oxidation states of the mercury were never determined in these studies.

Interrelationship of catalase and C-reactive protein in acatalasemic erythrocytes

Immunological reactions of catalase in the erythrocytes of normal, hypocatalasemic, and acatalasemic individuals were investigated using specific antisera prepared against purified human erythrocyte and hepatic catalases and against human C-reactive protein. Acatalasemic erythrocytes examined with anti-erythrocyte catalase antiserum were totally deficient in one of the three components which were regularly observed in normal erythrocytes, while hypocatalasemic erythrocytes revealed only a partial deficiency of this same component. Antihuman hepatic catalase antiserum failed to develop a visible precipitin reaction with lysates of acatalasemic erythrocytes while those of normal erythrocytes gave distinct precipitation in the agar medium. Immunologic reactions between erythrocyte catalase and C-reactive protein also were examined. The results of these studies suggest that molecular subunits of erythrocyte catalase and C-reactive protein are antigenically similar. The hypothetical implications of the above observations are discussed.

無カタラーゼ血液症患者血液に対する2, 3細菌の作用について

As previously reported in Part I the author found that hemoglobin had tendency to show the production of MetHb and the decolorization remarkably when Streptococcus hemolyticus, Streptococcus viridans, and Diprococcus pneumoniae I, II and III were cultured in the medium using the blood of acatalasemic patients.These changes clearly have indicated that the blood of these patients lacking in the catalase is unable to dispose of hydrogen peroxide (H2O2) produced by bacteria and subsequently Hb is oxidized to form MetHb and with progress of oxidation the constituents of the blood seem to turn to decolorization substances such as propentodyopent.This time with a view to clarify this point still further, the author studied action of still bacteria en erythrocytes of the normal and the patients, and from the results of this study arrived at the following conclusion.1) Diprococcus pneumoniae II, III and Streptococcus viridans markedly accumulate H2O2 during oxidation process of glucose.2) When glucose is used as substrate loaded with acatalasemic erythrocytes and shaken, in the case of Diprococcus pnemoniae I, II, or Streptococcus viridans, a marked production of MetHb has been observed, when normal erythrocytes are loaded, the production of MetHb has been extremely small as compared with each of these bacteria in the case of the acatalasemic erythrocytes.When pyruvate, succinate, or aspartate is used as substrate, the influences of each of these bacteria on erythrocytes are on the whole quite small.3) Productivity of MetHb and decolorization by bacteria have a parallel relationship with the accumulation of H2O2.4) Using glucose as substrate, influences of Diprococcus pneumoniae II on the respiration of erythrocytes are as follows.a. In the case where erythrocytes are not loaded (the control) O2-consumption decreases by 60-90 minutes.b. In the case where acatalasemic erythrocytes are loaded, so long as Hb exists, O2-consumption continues to rise.c. In the case where normal erythrocytes are loaded, up to 60 minutes O2-consumption is comparatively lower than that of the control but it does not fall even 120 minutes later.d. On examining H2O2 in solution after these reactions, an extremely minutes quantity of it has been traced in the case of loading acatalasemic erythrocytes, while none can be traced in the case of loading normal erythrocytes.e. Thiourea and cysteine have been found to completely recompensate whatever influences exerted upon O2-comsumption of bacteria by acatalasemic blood.5) After studying color changes of the erythrocytes to which H2O2 of various concentrations had been adden, the results thus obtained were quite identical with those which erythrocytes had been influenced by respiring bacteria.6) From these faets as far mentioned it may be assumed that the influences of respiring bacteria upon the acatalasemic erythrocytes are due to the action of H2O2 produced by bacteria.