Utilization of a differential hemoglobin elution procedure as a rapid assay for identification of embryonic and adult chicken red blood cells.

1. A hemoglobin elution-staining procedure has been developed for distinguishing embryonic chick red blood cells from adult chicken red blood cells. 2. Adult hemoglobin is eluted from red blood cells with 1.9 M potassium phosphate buffer, pH 7.2; whereas, embryonic hemoglobin is retained within the cells and gives positive staining with erythrosin B. 3. The hemoglobin elution-staining pattern during development can be correlated with two embryonic hemoglobins as detected by polyacrylamide gel electrophoresis. 4. The series of red blood cells staining with erythrosin B correspond to the primary erythrocyte series suggesting that hemoglobin expression during development is correlated with different cell populations.     

Stopped-flow kinetics of oxygen uptake and release by embryonic red blood cells.

The processes of O2 uptake and release by the three embryonic haemoglobins contained within early mouse embryonic red blood cells have been studied using dual-wavelength stopped-flow kinetic spectroscopy. The rate of O2 uptake in the pseudo-spherical, nucleated, embryonic red blood cells exhibits a greater than first-order dependence on O2 concentration. The time courses for the release from the red blood cells into dithionite-containing solutions tends towards a limiting rate at high dithionite concentrations. The rates of both the uptake and release processes observed in the embryonic cells are compared with those previously seen for adult mouse red blood cells. A new mathematical model is described which accurately simulates both uptake and release experimental data for the nucleated embryonic red blood cells.     

Is red blood cell survival limited by the activity of a critical enzyme?

Erythrocyte glutathione reductase is responsible for generating reduced glutathione, which has been implicated in maintaining the integrity of the red blood cell.Erythrocytes from peripheral blood were separated into fractions of increasing age and the activity of glutathione reductase and aspartate amino transferase determined in each fraction.The age-related decline in activity of both enzymes was confirmed, but with detailed resolution of the cells by age a significant secondary rise in only glutathione reductase activity was found in very old cells. As red blood cells from the same cohort survive in the circulation for varying periods they must vary in some way from one another. It is postulated that glutathione reductase is a critical enzyme which limits erythrocyte survival and that the rate of decline in activity varies from cell to cell. A simple mathematical model based on this postulate accounted quantitatively for both the pattern of glutathione reductase activity and the erythrocyte survival curve. In addition, a simplified model of the passage of erythrocytes through the circulation was designed and run. The predicted erythrocyte survival curve and pattern of glutathione reductase activity were very similar to observed patterns. This model may be useful in other situations where a finite resource is degraded at different rates by random passages through different pathways.     

Methylene blue directly oxidizes glutathione without the intermediate formation of hydrogen peroxide

Methylene blue stimulates the oxidation of glutathione in red blood cells in vitro and in vivo. This oxidation has been attributed to hydrogen peroxide that is generated from the autooxidation of leucomethylene blue arising from the reduction of methylene blue by NADPH. In this report we present evidence that methylene blue directly oxidizes glutathione and that oxidation of glutathione by hydrogen peroxide is a secondary reaction. Moreover, superoxide dismutase has no effect on the oxidation. Under aerobic conditions, methylene blue oxidizes glutathione 30 times faster than the spontaneous autooxidation of glutathione. Under anaerobic conditions the stoichiometry of the reaction of methylene blue with glutathione supports a direct chemical reaction. The reaction rates between glutathione and methylene blue suggest a second order reaction over the conditions tested. That neither oxygen radical formation nor significant amounts of hydrogen peroxide are produced by methylene blue, even in the presence of added glucose, is further confirmed by the failure to detect significant amounts of lipid peroxidation products, or hemolysis, in red blood cells incubated with the dye.     

Expression of an onco-developmental antegen among avian species.

Rabbit antisera directed against an onco-developmental antigen on chicken red blood cells have been serologically dissected through specific adsorptions. It is now possible to detect 13 antigenic determinants with the fractionated antisera. The onco-developmental antigen referred to as chicken fetal-leukemic antigen (CFA) is fetal-specific in the white Leghorn chicken, being present on the embryonic but not adult peripheral red blood cells of non-being present on the embryonic but not adult peripheral red blood cells of non-leukemic birds. However, one or more of the onco-developmental antigenic determinants have been detected on adult peripheral red blood cells of non-Gallus avian species, as well as on red blood cells from two adult chicken varieties. For phylogenetic purposes, red blood cells from avian species were characterized for their combinations of CFA determinants. Comparisons among species revealed specific patterns of antigenic expression within phylogenetic groups. Several CFA determinants were restricted in their occurrence to species within a single family, and one determinant was found in all cases where CFA was expressed. The distribution of CFA determinants was used to determine immunological distances among four Galliform species. These distances agreed with the immunological relationships established using different serological markers.     

Anion transport in embryonic and adult chicken red blood cells

The rates of Cl and SO₄ transport at 0° and 37°C, respectively, have been measured under exchange conditions for red blood cells of embryonic and adult chickens. It was found that the rate of self-exchange of SO₄ in embryonic red cells decreases as the embryo matures, and that the SO₄ transport rate was lower in adult compared to embryonic red cells. In contrast, no difference in the rate of Cl self-exchange was found between adult and embryonic red cells.     

Molecular bases of the treatment of Alzheimer's disease with antioxidants: prevention of oxidative stress

Alzheimer's disease is associated with a systemic oxidative stress situation which can be followed in vivo by determining biomarkers such as plasma lipoperoxides and TBARS levels and the oxidation degree of glutathione in red blood cells. It has been observed that Alzheimer's patients show an increased level of plasma TBARS, which indicates a higher free radical oxidation of plasma unsaturated phospholipids, and an increased oxidation of red blood cells glutathione, which indicates oxidative stress in peripheral cells. This latter, glutathione oxidation, was found to correlate statistically with the cognitive status of the patients. Treatment with vitamin E resulted in an improved cognitive performance only of those patients in which the tocopherol acted as an antioxidant, according to blood indicative markers of oxidative stress. Indeed, the effect of vitamin E on Alzheimer's disease patients showed considerable variations both in its antioxidant function and in its capacity to improve cognitive functions. An important conclusion from the reported results is that epidemiological or clinical studies that aim to test the effect of antioxidant supplementation on given functions should include the determination of the antioxidant status of the patients by the measurement of blood markers of oxidative stress.     

Effect of omega-3 polyunsaturated fatty acids on activity of glutathione-dependent enzymes in the liver cytosol and blood erythrocytes in rats with experimental chronic bronchitis and in the norm

The influence of omega-3 polyunsaturated fatty acids (omega-3 PUFA) on the activity of glutathione reductase, glutathione transferase and glutathione peroxidase in the liver cytosole and red blood cells of normal rats and animals with experimental chronic bronchitis. omega-3 PUFA ("Tekom" medication) activate glutathione reductase of liver cytosole and glutathionperoxidase in the red blood cells in rats. In the rats with chronic inflammatory process in bronchia omega-3 PUFA corrects the glutathione-dependent systems of detoxication. Effects were more expressed in the liver cytosole in comparison with the red blood cells. The using of omega-3 PUFA as a means for treatment and prophylaxis was more effective than for treatment only.     

Blood groups and biochemical polymorphism in the Namaqua sheep breed

Summary. The Namaqua is an indigenous fat-tailed African breed of sheep which has remained relatively isolated and which at one time dwindled to near extinction. Frequency data are given for blood group antigens, red cell glutathione and potassium types, for electrophoretic variants of red cell haemoglobin, 'X' protein, nucleoside phosphorylase, NADH-diaphorase, lysine and carbonic anhydrase and of plasma esterase, transferrin and albumin. Of particular interest was the occurrence of the i blood group, a bimodal distribution in red cell glutathione concentrations and red cell potassium concentrations of around 57 mmo1/1 cells, i.e. neither typically LK nor HK type.     

Glutathione peroxidase and oxidative hemolysis in trout red blood cells

Red blood cells from the trout Salmo irideus contain several hemoglobin components that are prone to oxidation with production of oxygen radicals. The rate of hemolysis has been correlated to the extent of methemoglobin formation. A difference in the rate of hemolysis between red blood cells saturated with either CO or O2 was evident only when diminished glutathione peroxidase activity was observed. These results confirm the important role of this enzyme in providing protection against or repair of oxidative damage to the red cell membrane.     

Adaptation of the Blood Antioxidant Defence Mechanisms of Sheep with a Genetic Lesion Resulting in Low Red Cell Glutathione Concentrations

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.     

Effect of vitamin E on the oxidative state of glutathione in plasma

Reduced and oxidized glutathione levels in red blood cells and plasma from humans were determined after oral vitamin E treatment. The experiments confirmed that vitamin E enhances reduced glutathione levels in red blood cells. Moreover, vitamin E supplement resulted in a significant reduction of the plasma oxidized glutathione content. Thus, it seems that the effect of vitamin E on the reduced glutathione content is not exerted via direct modulation of the glutathione-synthesizing enzymes, but rather by a more general mechanism of preserving reduced glutathione consumption by reducing the burden of the glutathione system.     

Blood antioxidative enzymes during epileptic activity

It has been shown that the development of generalized epileptic activity in rats led to the decrease in superoxide dismutase (SOD) activity without affecting glutathione peroxidase (GP) and glutathione reductase (GR) activity. Long-term examination of 13 patients suffering from generalized forms of epilepsy has shown an about 30% decrease in SOD and GP activity in red blood cells. It is assumed that the functional insufficiency of the antioxidative system plays an essential role in the development of lipid peroxidation disturbances during epilepsy.     

Methionine 35 oxidation reduces toxic effects of the amyloid beta-protein fragment (31-35) on human red blood cell

Amyloid beta-peptide, the central constituent of senile plaques in Alzheimer's disease brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of Alzheimer's disease, since it represents the residue in the beta-amyloid peptide most susceptible to oxidation "in vivo". In this study, the fragment 31-35 of the beta-amyloid peptide, which has a single methionine at residue 35, was used to investigate the influence of the oxidation state of methionine-35 on the beta-amyloid peptide (31-35) mediated cytotoxic effects. Because no extensive studies have yet addressed whether amyloid beta peptides-mediated toxic effects can occur in the absence of mitochondria, human red blood cells were used as cell model. Exposure of intact red blood cells to beta-amyloid peptide (31-35) induced a marked stimulation (approximately 45%) of the pentose phosphate pathway and a significant inhibition of the red cell enzyme catalase, compared with the results observed in control red blood cells. In contrast, exposure of red blood cells to the beta-amyloid peptide (31-35)-Met35OX i.e. in which the sulfur of methionine is oxidised to sulfoxide, induced a slight activation of PPP (approximately 19%), and an inhibition of catalase activity lower with respect to the results observed in beta-amyloid peptide (31-35)-treated red blood cells. Since the activities of red cell phosphofructokinase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase and the functionality of hemoglobin were not modified within the red cell following to beta-amyloid peptides exposure, it is likely that beta-amyloid (31-35)-catalase interaction may represent a selective toxic event. Together, these results support the hypothesis that Abeta peptide and the oxidative state of Met-35 may be involved in the mechanisms responsible of neurodegeneration in Alzheimer's disease.     

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.     

Glutathione activation of a cysteine proteinase from Schistosoma mansoni

A cysteine proteinase isolated from Schistosoma mansoni adults requires reduction by thiols for activation. The proteinase is located in the parasite digestive tract where it degrades hemoglobin released from host red blood cells. Reduced glutathione (GSH) has been shown to be effective in activation. Total glutathione concentration and the GSH/oxidized glutathione (GSSG) ratio were measured in whole blood lysate (3.2 mM, 284), serum (24 microM, 9.8) and material collected from the parasite digestive tract (4.2 mM, 137). The ratio of GSH/GSSG at which the enzyme displays half-maximal activity (Kox) is 1.0. Proteinase activation as a function of glutathione concentration and time was determined. The first-order reaction yielded a half-time of activation of 13 min at 5 mM. The second-order rate constant was 12.7 M-1 X min-1. The function of the proteinase and its possible regulation by glutathione activation are discussed.     

Human red blood cells as bioreactors for the inactivation of harmful xenobiotics

Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione. This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione. By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state. These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells. These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles.     

Different metabolizing ability of thiol reactants in human and rat blood: biochemical and pharmacological implications

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position beta125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.