Uncoupling of oxidative leak formation from lipid peroxidation in the human erythrocyte membrane by antioxidants and desferrioxamine

Human erythrocytes, briefly exposed to t-butylhydroperoxide and then incubated further in the absence of exogenous oxidant, undergo lipid peroxidation and formation of aqueous membrane leaks. Leak formation can be suppressed by various types of antioxidants and by desferrioxamine at concentrations at which lipid peroxidation still proceeds almost unaltered. This uncoupling of the two manifestations of an oxidative membrane damage indicates that loss of the barrier properties is not an obligatory consequence of the presence of peroxidized lipids in biological membranes.     

Oxidative Processes Induced by <Emphasis Type="Italic">tert</Emphasis>-Butyl Hydroperoxide in Human Red Blood Cells: Chemiluminescence Studies

The erythrocyte is a good model for investigation of the mechanisms of cell damage induced by oxidizing agents. Oxidative damage to cell components and cellular metabolism results in impaired rheological properties of circulating red blood cells and is involved in the development of some pathologies. The aim of the present study was to elucidate further the oxidative processes induced by tert-butyl hydroperoxide (tBOOH) in erythrocytes, identify cellular targets damaged by the oxidant, as well as estimate the energy and stoichiometry of the reactions that occur. The generation of free radicals in the cell was registered using the chemiluminescence technique. The products of oxyhemoglobin (oxyHb) oxidation, changes in intracellular glutathione (GSH) pool, and accumulation of the stable products of membrane lipid peroxidation were concurrently measured. The oxidative processes induced by tBOOH in red blood cells can be described as follows: 1) rapid GSH oxidation (30-60 sec) by glutathione peroxidase; 2) formation of radicals in the reaction between tBOOH and cellular Hb, which are then immediately consumed in lipid peroxidation reactions; 3) generation of chemiluminescence by the radicals formed. Several stages of the oxidative processes can be revealed. The order of the chemiluminescence reaction (n) with respect to oxidant was estimated to be equal to 2.5 at oxidant concentrations less than 0.5 mM and equal to 1.0 at higher oxidant concentrations. The order of the reaction of membrane lipid peroxidation was found to be n = 2.2 at 0.25-0.6 mM tBOOH and n = 0.5 at higher oxidant concentrations. The apparent activation energy of membrane lipid peroxidation was 55.8 +/- 6.4 kJ/mol, and that of oxyHb oxidation was 108 +/- 16 kJ/mol. It is shown that the interaction of tBOOH and HOCl in erythrocytes is accompanied by changes in both the total number of radicals generated in the cell and the time corresponding to the maximal rate of radical generation.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

Increase in vulnerability to oxidative damage in cholesterol-modified erythrocytes exposed to t-BuOOH

During the course of radical oxidation, cholesterol may exert seemingly contradictory effects. In order to gain a better understanding of the relationship between cholesterol levels and membrane susceptibility to oxidative damage induced by reactive oxygen species (ROS), here we analyze the integrity and structural stability of cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert-butyl hydroperoxide. The oxidant significantly increased ROS production, with almost complete oxidation of hemoglobin and a reduction in GSH content in the different erythrocyte groups at 2 mM concentration. These changes were accompanied by losses of cholesterol and total phospholipids, the main decreases being in phosphatidylethanolamine and phosphatidylcholine. The highest lipid loss was found in the cholesterol-depleted group. Fatty acid analyses revealed changes only in peroxidized cholesterol-modified erythrocytes, with decreases in linoleic and arachidonic acids. Fluorescence anisotropy studies showed an increase in the fluidity of the negatively charged surface of peroxidized control erythrocytes. Increased hemolysis and a positive correlation between cellular osmotic fragility and malondialdehyde contents were found in all peroxidized groups. These findings provide evidence that the modification of cholesterol levels in the erythrocyte membrane has provoking effects on peroxidation, with corresponding increases in oxidative damage in the treated cell, possibly as a consequence of lipid bilayer destabilization.     

Membrane cholesterol contents influence the protective effects of quercetin and rutin in erythrocytes damaged by oxidative stress

Flavonoids are potent scavengers of reactive oxygen species (ROS) that effectively prevent erythrocyte oxidation. Their antioxidant activities are governed by their structural characteristics and their ability to interact with and penetrate lipid bilayers. In order to gain a better understanding of the relationship between cholesterol contents and the antioxidant effectiveness of flavonoids against oxidative damage induced by ROS in cells, here we analyzed the integrity and structural stability of cholesterol-modified (enriched or depleted) and control erythrocytes exposed to tert-butyl hydroperoxide in the presence of quercetin or rutin. In control and cholesterol-enriched erythrocytes, quercetin provided greater protection against lipid peroxidation, ROS formation, and it preserved better cellular integrity than rutin. Both antioxidants suppressed the alterations in membrane fluidity and lipid losses with similar efficiency, reducing hemoglobin oxidation by 30% and GSH losses by 60% in the above-mentioned erythrocytes. Cholesterol depletion reduced the efficiency of the antioxidant power of both flavonoids against oxidative damage induced in the erythrocyte membrane, while a stronger degree of protection of GSH and hemoglobin contents was observed, mainly in the presence of rutin. These findings suggest a preferential incorporation of the antioxidants into the membranes from erythrocytes with normal and high cholesterol contents, whereas they would mainly be located in the cytoplasm of cholesterol-depleted erythrocytes.     

Probe-assisted flow cytometric analysis of erythrocyte membrane response to site-specific oxidant stress

BACKGROUND: Probe-assisted flow cytometry was used to monitor the response of membranes of living cells to oxidant stress in the presence and absence of antioxidants. Test conditions (fluorophore loading, oxidant concentration) were investigated and storage-related changes in erythrocyte response to oxidant stress explored. METHODS: Erythrocytes were incubated with a lipophilic fluorescent probe and exposed to site-specific oxidant challenge, induced by cumene hydroperoxide, in the presence and absence of urate, ascorbate, or alpha tocopherol in physiological amounts. Fluorescence of labeled and treated erythrocytes was measured for 120 min using a Coulter EPICS Elite ESP flow cytometer. RESULTS: Probe loading was dose and time dependent. Cumene hydroperoxide exhibited a potent and dose-dependent oxidant effect on erythrocyte membranes. Alpha tocopherol slowed, but did not prevent, membrane oxidation. Ascorbate appeared to have no effect on peroxidation initially, but then slowed and stopped propagation of membrane oxidation. The effect of urate was slight. CONCLUSIONS: This technique can provide insight into oxidative processes at the cellular level. Results indicated that lipophilic alpha tocopherol was the most effective antioxidant in slowing membrane peroxidation, but ascorbate appears to stop chain propagation. This effect may be owing to vitamin C/E interaction. Further study is needed.     

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.     

Iron-mediated oxidative stress in erythrocytes.

Erythrocytes subjected extracellularly to iron-mediated oxidant stress undergo haemoglobin oxidation and membrane damage, which can be modulated by maintaining the energy requirements of the cells. The results presented here suggest that a balance exists between the oxidation state of the haemoglobin and the oxidative deterioration of the membrane lipids, which is dependent on the metabolic state of the erythrocytes. These findings have important implications for thalassaemic erythrocytes that may be exposed to excess plasma iron levels, in which excessive membrane-bound iron in the form of haemichromes is a characteristic feature and in which cellular ATP levels are lowered.     

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.     

Hypochlorous acid-derived modification of phospholipids: characterization of aminophospholipids as regulatory molecules for lipid peroxidation

Hypochlorous acid (HOCl), an inflammatory oxidant derived from neutrophil myeloperoxidase, can chlorinate cytosolic proteins and nuclear DNA bases of target cells by passing through the cell membrane. However, little is known about the consequences of HOCl-derived modification of cell membrane components, including phospholipids. In this study, we characterize the reaction of HOCl with phospholipid molecules and found that aminophospholipids are the key molecules that chemically regulate lipid peroxidation. Upon incubation with HOCl, the peroxidation of egg yolk phosphatidylcholine was significantly enhanced in the presence of phosphatidylethanolamine (PE). In contrast, the peroxidation was significantly inhibited in the presence of phosphatidylserine (PS). On the basis of mass spectrometric and electron paramagnetic resonance characterization, the initiator of the peroxidation was identified as the nitrogen-centered radical originating from PE-derived chloramines, especially N,N-dichlorinated PE, a major product in the HOCl-modified PE. Although PS was also chlorinated upon reaction with HOCl, the formed chloramine rapidly decomposed to phosphatidylglycolaldehyde, a novel class of lipid aldehyde. Formation of phosphatidylglycolaldehyde was also confirmed in the porcine brain PS and erythrocyte cell membrane ghost exposed to HOCl. These results provide a novel mechanism for the HOCl-induced oxidative damage and its endogenous protection in the cell membrane at the site of inflammation.     

Reaction of xanthine oxidase-derived oxidants with lipid and protein of human plasma

Xanthine oxidase and purines have recently been detected in the circulation during acute viral infection and following hepatotoxicity and shock. Reactions of xanthine oxidase-generated oxidants with human plasma or bovine serum albumin (BSA) and egg phosphatidylcholine (PC) liposomes have been studied by measuring protein sulfhydryl oxidation and two markers of free radical-mediated lipid peroxidation, thiobarbituric acid reactive substances (TBARS) and conjugated dienes. Plasma incubated with 5 mU/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (Hx) for 2 h at 37 degrees C had 25-53% oxidation of sulfhydryl groups, with greater than 80% of the oxidation occurring during the first 20 min of the reaction. Concentrations of BSA similar to those present in serum, when exposed to XO/Hx-mediated oxidative stress, showed an even greater decrease in sulfhydryl concentration than that of plasma. No significant increase in plasma TBARS and conjugated dienes was observed during the 2-h incubation period in the presence of XO. Egg PC liposomes, suspended to a plasma phospholipid-equivalent concentration, showed a minor increase in TBARS and conjugated dienes under similar XO/Hx incubation conditions. In the presence of 0.23 mM BSA, lipid peroxidation was completely inhibited. A similar inhibition of lipid peroxidation was induced by cysteine but not by uric acid. Electrophoretic and arsenite-mediated sulfur reduction analysis revealed that BSA was oxidized beyond the disulfide form, with sulfenic acid formed during the initial period of oxidation. Protein sulfhydryls served as sacrificial antioxidants, preventing plasma lipid peroxidation, as well as being targets for oxidative damage. Plasma protein thiol oxidation was determined to be a more sensitive and specific indication of oxidant stress to the vascular compartment than assessment of lipid oxidation byproducts.     

Oxidative damages in erythrocytes of patients with metabolic syndrome

The aim of the study was to estimate the changes caused by oxidative stress in structure and function of membrane of erythrocytes from patients with metabolic syndrome (MS). The study involved 85 patients with MS before pharmacological treatment and 75 healthy volunteers as a control group. Cholesterol level, lipid peroxidation, glutathione level (GSH), and antioxidant enzyme activities in erythrocytes were investigated. The damage to erythrocyte proteins was also indicated by means of activity of ATPase (total and Na⁺,K⁺ ATPase) and thiol group level. The membrane fluidity of erythrocytes was estimated by the fluorescent method. The cholesterol concentration and the level of lipid peroxidation were significantly higher, whereas the concentration of proteins thiol groups decreased in the patient group. ATPase and GSH peroxidase activities diminished compared to those in the control group. There were no differences in either catalase or superoxide dismutase activities. The membrane fluidity was lower in erythrocytes from patients with MS than in the ones from control group. These results show changes in red blood cells of patients with MS as a consequence of a higher concentration of cholesterol in the membrane and an increased oxidative stress.     

Oxygen-related processes in red blood cells exposed to tert-butyl hydroperoxide

The correlation between the oxidative processes in tert-butyl hydroperoxide (tBHP)-exposed red blood cells and the reactions of oxygen consumption and release were investigated. Red blood cell exposure to tBHP resulted in transient oxygen release followed by oxygen consumption. The oxygen release in red blood cells was associated with intracellular oxyhaemoglobin oxidation. The oxygen consumption proceeded in parallel with free radical generation, as registered by chemiluminescence, but not to membrane lipid peroxidation. The oxygen consumption was also observed in membrane-free haemolyzates. The order of the organic hydroperoxide-induced reaction of oxygen release with respect to the oxidant (tBHP) was estimated to be 0.9 +/- 0.1 and that of the oxygen consumption reaction was determined to be 2.4 +/- 0.2. The apparent activation energy values of the oxygen release and oxygen consumption were found to be 107.5 +/- 18.5 kJ/mol and 71.0 +/- 12.5 kJ/mol, respectively. The apparent pKa value for the functional group(s) regulating the cellular oxyHb interaction with the oxidant in tBHP-treated red blood cells was estimated to be 6.7 +/- 0.2 and corresponded to that of distal histidine protonation in haemoprotein. A strong dependence of tBHP-induced lipid peroxidation on the oxygen concentration in a red blood cell suspension was observed (P50 = 32 +/- 3 mmHg). This dependence correlated with the oxygen dissociation curve of cellular haemoglobin. The order of the membrane lipid peroxidation reaction with respect to oxygen was found to be 0.5 +/- 0.1. We can conclude that the intensity of the biochemical process of membrane lipid peroxidation in tBHP-exposed erythrocytes is controlled by small changes in such physiological parameters as the oxygen pressure and oxygen affinity of cellular haemoglobin. Neither GSH nor oxyhaemoglobin oxidation depended on oxygen pressure.     

Membrane cholesterol contents modify the protective effects of quercetin and rutin on integrity and cellular viability in oxidized erythrocytes

Flavonoids protect cells damaged by oxidative stress. This, together with other biological activities, is governed by structural features of flavonoids and the nature and physical state of the cell membrane. We have previously proved that membrane cholesterol contents modify the protective power of quercetin and rutin against oxidative stress in erythrocytes. Here we analyzed the lipid asymmetry, the integrity, and cell viability of native and cholesterol-modified erythrocytes exposed to tert-butyl hydroperoxide in presence of both antioxidants. Our results provides clear evidence that quercetin affords better protection than rutin against lipid peroxidation, ROS generation, erythrophagocytosis and cellular instability in oxidized erythrocytes with normal and modified cholesterol contents. Both antioxidants provided a high of protection for the transbilayer aminophospholipid asymmetry, only partly preserving cell morphology in oxidized control and cholesterol-depleted erythrocytes. Cholesterol depletion reduced the protection provided by both antioxidants against phosphatidylserine externalization, erythrophagocytosis and hemolysis, which is in accordance with the lower degree of preservation against lipid peroxidation observed in oxidized cholesterol-depleted erythrocytes. This lower degree of preservation is presumably attributable to the low antioxidant contents in these erythrocyte membranes, or even to a lower efficiency of the antioxidant in a modified lipid environment due to the removal of cholesterol.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.     

Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate

In crude synaptosomal fractions from rat brain exposed to iron and ascorbate, enhanced lipid peroxidation (more than 3-fold compared to control), loss of protein thiols up to the extent of 40% compared to control, increased incorporation of carbonyl groups into proteins (more than 4.5-fold compared to control) and non-disulphide covalent cross-linking of membrane proteins have been observed. The phenomena are not inhibited by catalase or hydroxyl radical scavengers like mannitol or dimethyl sulphoxide. However, chain breaking antioxidants like alpha-tocopherol and butylated hydroxytoluene prevent both lipid peroxidation and accompanying protein oxidation. It is suggested that in this system lipid peroxidation propagated by the decomposition of preformed lipid hydroperoxides by iron and ascorbate is the primary event and products of the peroxidation process cause secondary protein damage. In view of high ascorbate content of brain and availability of several transition metals, such ascorbate mediated oxidative damage may be relevant in the aetiopathogenesis of several neurodegenerative disorders as well as ageing of brain.     

Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture.

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.     

Oxidant induced injury of erythrocyte—Role of green tea leaf and ascorbic acid

Oxidant and free radical-generating system were used to promote oxidative damage in erythrocytes. Among the oxidants used, phenylhydrazine represents one of the most investigated intracellular free radical-generating probes, which in the presence of haemoglobin autooxidises and give rise to hydroxyl radical, a marker for cellular damage. Erythrocyte, as a single cell, is a good model to be used for studying the haemolytic mechanism of anaemia. Our present investigations reveal increased lipid peroxidation of erythrocyte using phenylhydrazine as well as other oxygen-generating systems (hydrogen peroxide, iron with hydrogen peroxide). It has further been observed that not only lipid peroxidation, phenylhydrazine causes significant elevation in methemoglobin formation, catalase activity and turbidity, in the above system, which are the typical characteristics of haemolytic anaemia. However, exogenous administration of green tea leaf extract and ascorbic acid as natural antioxidants and free radical scavengers were shown to protect separately increased lipid peroxidation caused by phenylhydrazine, though the degree of protection is more in case of green tea leaf extract than ascorbic acid. Results suggest that oxidative damage in vivo due to haemolytic disease may be checked to some extent by using natural antioxidants. (Mol Cell Biochem 276: 205–210, 2005)