Effects of 2-ethyl-6-methyl-3-hydroxypyridine succinate on lipid peroxidation and antioxidant defense in erythrocytes during storage of donor erythrocyte mass

The antioxidant activity of erythrocytes during long-term storage of donor erythrocyte mass was studied. It was demonstrated that the antioxidant activity of erythrocytes was retained for a long time if the antioxidant 2-ethyl-6-methyl-3-hydroxypyridine succinate was added to the blood preservative Glugicir.     

The effect of nitroxyl radicals and sodium nitrite on the methemoglobin content of the blood and on the enzymatic activity of erythrocyte antioxidant protection]

Preliminary introduction of nitroxyl radicals to mice decreases methaemoglobin-forming effect of sodium nitrite and diminishes the content of total SH-groups and restored glutathione as well as the activity of glutathione reductase and total activity of dehydrogenases of the pentosophosphate path of erythrocytes. High level of lipids peroxidation in case of sodium nitrite intoxication remains unchanged under preliminary administration of nitroxyl radicals as well. Activity of the key enzymes of antioxidant protection of erythrocytes, superoxide dismutase and catalase, is not recovered with sodium nitrite intoxication in presence of nitroxyl radicals.     

Synthesis and antioxidant efficiency of a new amphiphilic spin-trap derived from PBN and lipoic acid

The synthesis of a new amphiphilic antioxidant called PBNLP and derived from both alpha-phenyl-N-tert-butyl nitrone (PBN) and lipoic acid was described. Grafting a lactobionamide moiety onto the aromatic group of the PBN provided the water solubility of this compound. In vitro preliminary biological evaluations of its antioxidant capacity were performed using the KRL biological test based on free radical-induced hemolysis. The PBNLP induces a protection of erythrocytes against exogenous free radicals higher than that measured with lipoic acid or PBN alone or with lipoic acid or PBN derivatives in admixtures.     

Oxidative stress due to aluminum exposure induces eryptosis which is prevented by erythropoietin

The widespread use of aluminum (Al) provides easy exposure of humans to the metal and its accumulation remains a potential problem. In vivo and in vitro assays have associated Al overload with anemia. To better understand the mechanisms by which Al affects human erythrocytes, morphological and biochemical changes were analyzed after long-term treatment using an in vitro model. The appearance of erythrocytes with abnormal shapes suggested metal interaction with cell surface, supported by the fact that high amounts of Al attached to cell membrane. Long-term incubation of human erythrocytes with Al induced signs of premature erythrocyte death (eryptosis), such as phosphatidylserine externalization, increased intracellular calcium, and band 3 degradation. Signs of oxidative stress, such as significant increase in reactive oxygen species in parallel with decrease in the amount of reduced glutathione, were also observed. These oxidative effects were completely prevented by the antioxidant N-acetylcysteine. Interestingly, erythrocytes were also protected from the prooxidative action of Al by the presence of erythropoietin (EPO). In conclusion, results provide evidence that chronic Al exposure may lead to biochemical and morphological alterations similar to those shown in eryptosis induced by oxidant compounds in human erythrocytes. The antieryptotic effect of EPO may contribute to enhance the knowledge of its physiological role on erythroid cells. Irrespective of the antioxidant mechanism, this property of EPO, shown in this model of Al exposure, let us suggest potential benefits by EPO treatment of patients with anemia associated to altered redox environment.     

Lipid peroxidation and antioxidant enzyme activities in erythrocytes of type I and II alcoholics

Reduced and oxidized glutathione (GSH and GSSG), protein-bound glutathione, lipid peroxidation and antioxidant enzyme activities were determined in the erythrocyte lysates and membranes of type I and II alcoholics in order to clarify the effect of age-of-onset and the duration of the alcohol consumption on erythrocyte oxidant and antioxidant status. The osmotic fragility and susceptibility of the erythrocytes to haemolysis were also determined. Erythrocyte lipid peroxidation was significantly increased but, GSH and protein-bound GSH, GSH/GSSG ratio and antioxidant enzyme activities were markedly decreased in the erythrocytes of the alcoholic subgroups. Erythrocyte count and haemoglobin content in the blood of alcoholics were found to be decreased in accordance with the finding that erythrocytes were more fragile and less resistant to haemolysis particularly in type II alcoholics. The present study showed that ethanol-induced oxidative stress in erythrocytes can lead to haemolysis and membrane-specific injuries in erythrocytes of the alcoholic subtypes.     

Protective effect of green tea on erythrocyte membrane of different age rats intoxicated with ethanol

It is known that aging is characterized by changes in cell metabolism resulting in modification of the structure and function of cell membrane components which is mainly the consequence of reactive oxygen species action. These disturbances are also enhanced by different xenobiotics, e.g. ethanol. Therefore, the aim of this paper is to examine green tea influence on total antioxidant status (TAS) and on composition and electric charge of erythrocyte membrane phospholipids in ethanol intoxicated rats of various ages. Antioxidant abilities of erythrocytes were estimated by measuring TAS. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC, while the extent of erythrocytes lipid peroxidation was estimated by HPLC measurement of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels. Electrophoresis was used to determine the surface charge density of the rat erythrocyte membrane. It was shown that the process of aging was accompanied by a decrease in TAS and in the total amount of phospholipids as well as by enhancement of lipid peroxidation and increase in surface charge density of erythrocyte membrane. Ethanol administration caused, in term, decrease in TAS and increase in the level of all phospholipids and lipid peroxidation products. Ethanol as well significantly enhanced changes in surface charge density of erythrocyte membrane. The ingestion of green tea partially prevented decrease in erythrocyte antioxidant abilities observed during aging and ethanol intoxication. Moreover, long-term drinking of green tea protects the structure of the erythrocytes membrane disturbed during aging process and/or chronic ethanol intoxication.     

Ascorbic acid recycling in human erythrocytes is induced by smoking in vivo

Tobacco smoke contains large numbers of radicals that burden the antioxidant defense and, thus, lower plasma antioxidants, in particular vitamin C or ascorbic acid, is commonly observed among smokers. Ascorbic acid recycling describes the process in which ascorbic acid is oxidized to dehydroascorbic acid by various pathways and subsequently reduced back to ascorbic acid intracellularly, e.g., in erythrocytes, thereby preserving the ascorbic acid pool. In humans who are unable to synthesize ascorbic acid, and in smokers in particular, who are prone to oxidation, this process must be very efficient and of great importance. It has previously been reported that isolated erythrocytes subjected to tobacco smoke in vitro had significantly lower ascorbic acid recycling as compared to controls. In contrast to these findings, we now report that freshly isolated erythrocytes from long-term smokers (n = 39) display a significantly increased rate of ascorbic acid recycling in vivo as compared to those isolated from nonsmokers (n = 31; p <.0001). Preliminary data suggests that the increase results from induction of dehydroascorbic acid reductase activity rather than from differences in energy status, glutathione content, or altered transport capacity. The induction of ascorbic acid recycling as a potential adaptation mechanism of the antioxidant defense to oxidative insults is discussed.     

Oxidative stress correction by nicotinamide and nicotynol-GABA in diabetic neuropathy

Concentration of lipid peroxidation products and antioxidant enzyme activities in rat brain and erythrocytes and the effects of nicotinamide and nicotinoyl-GABA administration on these parameters were estimated on 21st day of streptozotocin-induced diabetes. It was demonstrated more then two-fold diabetes-induced accumulation of conjugated dienes and malondialdehyde in tissues studied. Superoxide dismutase and glutathione reductase activities of both brain homogenate and erythrocytes as well as catalase and glutathione peroxidase activities of brain homogenate were shown to decrease significantly in diabetic rats, meanwhile, catalase activity of erythrocytes was increased and glutathione peroxidase unchanged. So the correlation between changes in enzymatic antioxidant system in brain and erythocytes failed to be found. Alterations observed were virtually prevented by the course of nicotinamide and nicotinoyl-GABA treatment. The results suggested that the suppression of antioxidant system could be primary biochemical disturbance in diabetic neuropathy progression. It was shown that the antioxidant efficacy of nicotinoyl-GABA is lower than that of nicotinamide. It was suggested that the mechanism of antioxidant action of nicotinamide and its structural analogue consists of both scavenging of lipid peroxides and NAD biosynthesis that leads to activation and normalization of altered energy and lipid metabolism.     

The antioxidant status during maturation of reticulocytes to erythrocytes in type 2 diabetics

Free radical-induced lipid peroxidation has been associated with numerous disease processes including diabetes mellitus. The extent of lipid peroxidation (LPO) and antioxidant defense system [i.e., levels of glutathione (GSH), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and catalase (CAT)] were evaluated in reticulocytes and erythrocytes of type 2 diabetic males and age-matched controls. Type 2 diabetics have shown increased lipid peroxidation and decreased levels of GSH, GR, GPx, G6PDH, and GST both in reticulocytes and erythrocytes compared to controls, indicating the presence of oxidative stress and defective antioxidant systems in these patients. CAT activity is found to be enhanced in both the reticulocytes and erythrocytes of diabetics, with a greater percentage enhancement in reticulocytes. The extent of increase in lipid peroxidation is greater in erythrocytes compared to reticulocytes in these patients. Furthermore, the maturation of reticulocytes to erythrocytes resulted in decreased GSH and decreased activities of all antioxidant enzymes (except CAT) both in normals and type 2 diabetes individuals, indicating decreased scavenging capacity as reticulocytes mature to erythrocytes. These maturational alterations are further intensified in type 2 diabetics. The present study reveals that the alterations in lipid peroxidation and antioxidant system lean toward early senescence of erythrocytes in type 2 diabetic patients.     

Activity of antioxidant enzymes in enterocytes of the small intestine and blood cells after ionizing irradiation and administration of vitamin E in rats

The long-term influence of low X-ray irradiation increases lipid peroxidation (LP) in radiosensitive (bone marrow, enterocytes of small intenstine) and in relatively radioresistant blood cells (erythrocytes). The activation of antioxidant system enzymes in observed cells does not decrease LP intensity. We concluded that additional administration of alpha-tocopherol provided the decrease of the first and end products of LP in the observed tissues mostly in the beginning of the experiment. Antioxidant effect of the preparation is more significant in cells with high proliferative activity but normal activity of enzymes was not determined.     

Clotrimazole as an amplifier of hemin-induced damage to human erythrocytes. Antioxidant-independent protective effects of flavonoids

This study is focused on the effect of the antifungal drug clotrimazole (CLT), also possessing anti-malarial and anticancer activities, on hemin-induced hemolysis and changes in ion permeability and filter-ability of human erythrocytes. In the presence of 10 μM clotrimazole, the hemolytic response of erythrocytes to exogenous hemin at concentrations as low as 2–8 μM was considerably potentiated and their filterability, as measured by passing them through a 5-μm nuclepore filter, dropped sharply. Flavonoids quercetin (Q) and taxifolin (DHQ), unlike the standard antioxidant Trolox, abolished the effects of clotrimazole, suggesting that protection of hemin-treated erythrocytes by flavonoids is not related to their antioxidant properties.     

Regulation of Free Radical Processes by Delta-Sleep Inducing Peptide in Rat Tissues under Cold Stress

An intraperitoneal injection of an exogenous delta-sleep inducing peptide (DSIP) at a dose of 12 g/100 g body weight shifted the prooxidant–antioxidant balance of free radical process (FRP) in tissues and erythrocytes of rats: the activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and the concentrations of antioxidants (reduced glutathione in particular) increased. The DSIP stimulated the myeloperoxidase activity in blood neutrophils and had no effect on the activity of xanthine oxidase, a prooxidant enzyme, in the brain and liver. Cold stress displaced the prooxidant–antioxidant balance by increasing the xanthine oxidase activity in tissues and decreasing the myeloperoxidase activity in blood neutrophils; it also inhibited the enzyme antioxidant activities in tissues and erythrocytes that was neutralized by an increased ceruloplasmin activity in blood plasma and by an elevated level of antioxidants in rat blood and tissues. Preliminary administration of DSIP to animals exposed to cold stress restored the prooxidant–antioxidant balance: it normalized the myeloperoxidase activity in blood neutrophils, decreased the xanthine oxidase activity, and increased the activity of antioxidant enzymes in tissues and erythrocytes restoring the antioxidant level. The molecular regulation mechanism of free radical processes by DSIP in tissues under stressful conditions is discussed.     

Erythrocyte lipid peroxidation and antioxidants in cigarette smokers

The present study has analysed the relationship between lipid peroxidation and antioxidant status in erythrocytes from 30 adult male cigarette smokers and an equal number of age and sex-matched normal subjects. Erythrocyte lipid peroxidation was markedly increased. The enzymic antioxidants were decreased in erythrocytes of cigarette smokers. The present study highlights the occurrence of lipid peroxidation and possible breakdown of antioxidant status in cigarette smoking.     

Lipid peroxidation and antioxidant system in the blood of cancerous patients with metastasis

Free-radical-mediated damages may play an important role during metastasis. To investigate their relevance in the metastatic process MDA levels, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, and selenium, zinc and copper contents were determined in plasma and erythrocytes from 20 cancerous patients with metastasis and 30 age-matched controls. Significantly higher concentrations of MDA in plasma as well as in erythrocytes were found comparing to the control group. In both plasma and erythrocytes, GPX activity and selenium and zinc levels were significantly lower in patients than in controls. However, SOD activity in erythrocytes and copper levels in both plasma and erythrocytes were significantly higher in patients. The impaired antioxidant system may favor accumulation of free radicals which may induce the process of metastasis. On the other hand, it is possible that the antioxidant system is impaired as a consequence of abnormality in the antioxidative metabolisms due to the cancer process.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Using chemiluminescence to determine whole blood antioxidant capacity in rheumatoid arthritis and Parkinson's disease patients

The consequences of oxidative stress and inflammation are implicated in a wide range of diseases, including rheumatoid arthritis and Parkinson's disease. The status of antioxidant capacity in rheumatoid arthritis and Parkinson's disease remains unclear, in part due to common practice of assaying erythrocytes separately to plasma. This method removes any synergistic interactions between plasma and erythrocyte‐based antioxidants. The experiments in this report tested antioxidant capacity in whole blood, erythrocytes and plasma by group and disease stage. Medically diagnosed patients were recruited along with appropriate control group participants. Fasting venous blood was assayed using chemiluminescence methods for: time to maximum light emitted, maximum light emitted, and plasma antioxidant capacity in vitamin E analogue units. Here we demonstrate that whole blood exhibits higher antioxidant capacity than either plasma or erythrocytes assayed separately. We report increased oxidative stress in the blood of rheumatoid arthritis patients by group (p = 0.018, p = 0.049). We show increased antioxidant capacity in Parkinson's disease patients by group (p < 0.001). For later stage Parkinson's disease patients, we report reduced oxidative stress (p = 0.025), and increased antioxidant capacity and for erythrocytes (p < 0.001, p = 0.004) and whole blood (p < 0.001, p = 0.003). Early stage Parkinson's disease showed higher antioxidant capacity on only one measure (p = 0.008). Whole blood chemiluminescence is a useful technique for determining redox status in disease and might help clarify the role of oxidative stress in rheumatoid arthritis and Parkinson's disease.     

Interaction of ascorbate and alpha-tocopherol enhances antioxidant reserve of erythrocytes during anemia in visceral leishmaniasis

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and alpha-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and alpha-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and alpha-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and alpha-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous alpha-tocopherol. The results suggest an interacting role of ascorbate and alpha-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L.     

Bioactivity of biflorin, a typical o-naphthoquinone isolated from Capraria biflora L

Capraria biflora L. (Scrophulariaceae) is a perennial shrub widely distributed in several countries of tropical America. The present work verified the cytotoxic and antioxidant potential of biflorin, an o-naphthoquinone isolated from C. biflora collected in the northeast region of Brazil. The cytotoxicity was tested on three different animal cell models: mouse erythrocytes, sea urchin embryos and tumor cells, while the antioxidant activity was assayed by the thiocyanate method. Biflorin lacked activity on mouse erythrocytes as well as on the development of sea urchin eggs, but strongly inhibited the growth of all five tested tumor cell lines, especially the skin, breast and colon cancer cells with IC50 of 0.40, 0.43 and 0.88 micro/ml for B16, MCF-7 and HCT-8, respectively. Biflorin also showed potent antioxidant activity against autoxidation of oleic acid in a water/alcohol system.     

Eryptosis and oxidative damage in type 2 diabetic mellitus patients with chronic kidney disease

It has been suggested that oxidative stress may participate in the progression of diabetes and its complications. Long-term complications of type 2 diabetes mellitus (T2DM) include retinopathy, atherosclerosis, shortened life span of erythrocytes, nephropathy, and chronic kidney disease (CKD). Oxidative damage has been associated with erythrocyte apoptosis induction in other pathological conditions. Our aim was to study the presence of eryptosis and its possible relationship with oxidative damage in patients with T2DM without CKD (T2DM/CKD(-)) and in patients with T2DM and CKD (T2DM/CKD(+)).Oxidative damage of lipids erythrocytes were increased in diabetic patients. The highest lipoperoxidation was found in T2DM/CKD(+). Likewise, the lower plasma total antioxidant capacity, GSH/GSSG ratio, and GSH in erythrocytes were found in T2DM/CKD(+) patients. A negative correlation was found between plasma total antioxidant capacity and oxidative damage. Phosphatidylserine (PS) externalization was measured in erythrocytes to evaluate eryptosis. Annexin binding in erythrocytes of T2DM/CKD(+) patients was higher than in healthy subjects and T2DM/CKD(-) patients. A positive correlation between lipoperoxidation and PS externalization in erythrocytes was found. This work showed that the erythrocytes of diabetic patients have increased oxidative damage, a reduction of antioxidant systems and more erythrocyte PS externalization. The duration of diabetes and the presence of CKD increase both oxidative damage and eryptosis. It is possible that a longer time of evolution induces an increase in erythrocyte oxidative damage and the consumption of blood antioxidant systems, adding to the osmotic stress in CKD and so contributes to an increase in PS externalization in diabetic patients.     

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.