Oxidative erythrocyte membrane damage in hereditary spherocytosis.

The occurrence, in Hereditary Spherocytosis, of an oxidative damage to red blood cell membranes was studied by "in vitro" treatment of the erythrocytes with tert-butylhydroperoxide, methylene blue, or phenylhydrazine. Spherocytes were found to be more sensitive than normal erythrocytes to the action of these drugs. Tert-butylhydroperoxide caused a more intense lipid peroxidation as well as more extensive membrane protein alterations, namely spectrin degradation, formation of high molecular weight aggregates, and globin binding to the membrane. Marked spectrin degradation was also induced by methylene blue and by phenylhydrazine, which differed from each other for their effects on the generation of membrane-bound globin and of intermediate proteolysis products. Spectrin appeared therefore to be, in Hereditary Spherocytosis, a highly sensitive target to oxidative stress, a phenomenon which may, also "in vivo", increase the rate of spectrin loss thus enhancing erythrocyte fragility.     

Melatonin reduces phenylhydrazine-induced oxidative damage to cellular membranes: evidence for the involvement of iron

Phenylhydrazine and iron overload result in augmented oxidative damage and an increased likelihood of cancer. Melatonin is a well known antioxidant and free radical scavenger. The aim of this study was to determine whether melatonin would protect against phenylhydrazine-induced oxidative damage to cellular membranes and to evaluate the possible role of iron in this process. Changes in lipid peroxidation and microsomal membrane fluidity were estimated after the treatment of rats with phenylhydrazine (15 mg/kg body weight, daily, 7 days) alone and melatonin or ascorbic acid (15 mg/kg body weight, two times daily, 8 days), or their combination. Additionally, lipid peroxidation was measured in liver homogenates from untreated and melatonin or ascorbic acid-treated rats in vivo and exposed to iron in vitro. Melatonin, but not ascorbic acid, reduced phenylhydrazine-induced lipid peroxidation in vivo in spleen (3.16+/-0.06 vs. 3.83+/-0.12 nmol/mg protein, P<0.05) and plasma (7. 73+/-0.52 vs. 9.96+/-0.71 nmol/ml, P<0.05) and attenuated the decrease in hepatic microsomal membrane fluidity (1/polarization, 3. 068+/-0.007 vs. 3.027+/-0.008, P<0.05). In vitro exposure to iron significantly enhanced the lipid peroxidation in liver homogenates from untreated (3.34+/-0.75 vs. 1.25+/-0.28, P<0.05) or ascorbic acid-treated rats (2.72+/-0.39 vs. 0.88+/-0.06, P<0.05) but not from melatonin-treated rats (1.49+/-0.55 vs. 0.68+/-0.20, NS). It is concluded that free radical mechanisms are involved in the toxicity of phenylhydrazine and that the antioxidant melatonin, but not ascorbic acid, reduces the toxic affects of phenylhydrazine in vivo and of iron in vitro in cell membranes. Therefore, melatonin co-treatment in conditions of iron overload may prove beneficial.     

Measurement of the diene conjugated form of linoleic acid in plasma by high performance liquid chromatography: a questionable non-invasive assay of free radical activity?

It has been previously reported that the main diene-conjugated fatty acid in human plasma is a non-oxygen containing linoleic acid isomer (PL-9, 11-LA'). It has also been proposed that this isomer can be used as a specific marker of free radical-mediated lipid peroxidation in humans. Here we report that the in vitro induction of lipid peroxidation in human and rat blood with either UV irradiation or phenylhydrazine failed to increase the plasma levels of this isomer. The induction of lipid peroxidation in vivo in rats pretreated with either phenylhydrazine or bromotrichloromethane also failed to increase the plasma levels of this isomer. These findings demonstrate that PL-9, 11-LA' cannot be used as an in vivo marker of free radical-mediated lipid peroxidation in rats and casts doubts on its validity as a specific marker in humans.     

Inhibitory effect of the flavonoid silymarin on the erythrocyte hemolysis induced by phenylhydrazine

The flavonoid silymarin, which is used as a therapeutical agent in the treatment of liver diseases, can inhibit the hemolysis and lipid peroxidation induced by phenylhydrazine on erythrocytes obtained from rats treated with the flavonoid. This effect is ascribed to the antioxidant properties as a free radical scavenger exhibited by the flavonoid. Silymarin failed to inhibit the glutathione depletion induced by phenylhydrazine on erythrocytes. It is proposed that the flavonoid acts at the membrane level of the cell avoiding the lipid peroxidative and fluidizing effect of phenylhydrazine.     

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)     

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging

The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.     

Alterations of red cell membranes from phenylhydrazine-treated rabbits

Reticulocytosis was induced in rabbits by two methods: phlebotomy and injection of phenylhydrazine. Normal erythrocytes, reticulocytes from bed rabbits, reticulocytes from phenylhydrazine-treated rabbits, and erythrocytes treated in vitro with phenylhydrazine were compared with respect to their plasma membrane labeling by galactose oxidase and NaB³H₄, and lactoperoxidase-catalyzed incorporation of ¹²⁵I. Normal erythrocyte membranes and membranes from reticulocytes of bled rabbits showed almost identical labeling patterns, the presence of 2–3 glycoproteins with moderate to low mobilities on dodecyl sulfate acrylamide gel electrophoresis. Labeling in the absence of enzyme was negligible. In contrast, the reticulocytes from phenylhydrazine-treated rabbits exhibited a large incorporation of tritium without prior treatment with galactose oxidase. Even after prereduction with unlabeled NaBH₄ to remove this nonspecific labeling, the labeled glycoprotein components found in normal erythrocytes were not detectable. Normal erythrocytes treated in vitro with phenylhydrazine, washed, and labeled with galactose oxidase had labeling patterns, including high nonspecific incorporation of ³H, similar to those observed with in vivo phenylhydrazine treatment.Solubilization of membranes with lithium diiososalicylate followed by partitioning with phenol showed that the same glycoproteins were presented in normal or phenylhydrazine membranes, although only the former extract was labeled by galactose oxidase. Individual carbohydrates from the membranes were analyzed by gas-liquid chromatography and, in the case of hexosamines, on the amino acid analyzer. The results of these analyses indicated a slight decline in galactose content with phenylhydrazine treatment. Reticulocyte membranes from bled rabbits also showed a decrease in galactose content, although it was less pronounced.Most of the label incorporated by nonspecific borohydride labeling of membranes from phenylhydrazine-treated animals was found associated with protein. The modified amino acids from labeled proteins are similar to those formed in reactions of oxidized lipids and proteins in model systems.     

Membrane alterations in phenylhydrazine-induced reticulocytes

It is known that reticulocytes formed in animals in response to phenylhydrazine treatment have a shorter life span than those formed as a consequence of bleeding. The experiments presented illustrate that the reticulocytes formed consequent to the action of this drug exhibit membrane alterations, in the absence of intracellular oxidative changes (e.g., to hemoglobin), which might be expected to contribute to their decreased survival. These membrane alterations include the formation of flourescent chromolipids, a decrease in spectrin polypeptides, and an increase in high molecular weight membrane protein polymers. It is suggested that these effects are unique to the reticulocytes formed as a response to phenylhydrazine since they are a consequence of the peroxidation of membrane phospholipids initiated by this agent.     

Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes

It has been recently hypothesized that in PIH a placental oxidant-antioxidant imbalance might cause the release of lipoperoxidation products into the circulation, with subsequent damage of endothelial cell membranes. In this hypothesis the endothelial cell and further increase in circulating lipoperoxide levels, which are by themselves able to induce smooth muscle constriction and increased pressor responsiveness to angiotensin II. In order to investigate this issue, we studied the basal content of lipid peroxides in terms of malondialdehyde (MDA) in the syncytiotrophoblast plasma membranes (SPM) from PIH women. Moreover, we investigated the susceptibility to peroxidation of SPM using anin vitro oxidative stress as a tool to verify the predisposition to thein vivo development of peroxidation products. The fatty acid composition of the membranes was also analyzed. Microvillus membrane lipoperoxide concentrations were significantly increased in PIH women (62.8±7.6 ng MDA/mg prot) compared with healthy pregnant subjects (37.6±4.8 ng MDA/mg prot; p<0.01).The formation of TBARS under the action of phenylhydrazine was significantly greater in PIH women (90.3±7.4 mmol MDA/mol cholesterol) than in normal pregnant subjects (68.6±6.4 mmol MDA/mol cholesterol; p<0.01). In PIH microvillus membrane we also observed a significant increase of the content of polyunsaturated arachidonic acid.The increased susceptibility to oxidative stress of SPMs from PIH women might be due either to reduced antioxidant systems or to an abnormality of the lipid composition of the membrane. The present work also demonstrated in PIH a reduction in the SPM content of saturated fatty acids with an increase in polyunsaturated fatty acids, which are the major substrate for peroxidation. On the other hand, the higher lipoperoxidation may be due to the observed increased susceptibility to peroxidative stress, to a primary reduction in placental perfusion with tissue hypoxia or to both factors, which can potentiate each other.     

Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anaemia

Temporal changes in the levels of denatured haemoglobin (Heinz bodies) and fluorescent lipid peroxidation products in the red cells of rabbits administered phenylhydrazine have been followed. Heinz bodies were maximal just before the period when most of the cell destruction occurred, whereas lipid peroxidation products were maximum when reticulocyte levels were highest. This implies that lipid peroxidation occurs mainly in immature cells and that haemoglobin denaturation is more likely than lipid peroxidation to be a major contributor to haemolysis.     

On the mechanism of phenylhydrazine-induced hemolytic anemia

The thiobarbituric acid (TBA)-reactants have been measured in the circulating red blood cells (RBC) and RBC trapped in the spleens of normal and phenylhydrazine-treated rats. There was significant TBA-reactivity in the circulating RBC of phenylhydrazine-treated rats which was increased 3-fold in RBC obtained from the spleen. Since lipid peroxidation accompanies formation of TBA-reactive malonyldialdehyde, it is suggested that phenylhydrazine induces anemia as a consequence of peroxidation of RBC membrane lipids and this effect may be a result of the autoxidation of the drug and the interaction of oxygen radicals with membrane lipids.     

Structural modification of erythrocyte membranes during oxidative stress and activity of membrane bound NADH-methemoglobin reductase

The activity of NADH-methemoglobin reductase (metHb-reductase) in membranes isolated from human erythrocytes treated with phenylhydrazine at its sublytic concentration was studied. A decrease in the activity of membrane-bound metHb-reductase was shown to depend on the concentration of phenylhydrazine. Simultaneously, an increase in the level of membrane-bound methemoglobin and a change in the fluorescence parameters of membrane-bound 4,4'-diisothiocy-anatostilbene-2,2'-disulfonic acid were registered. In the case when Hb-free erythrocyte ghosts were treated with 0.2-2.0 mM phenylhydrazine, the activity of metHb-reductase did not change. The obtained results indicate that the inhibition of the activity of membrane-bound metHb-reductase by phenylhydrazine-induced oxidative stress in human erythrocytes is not caused by the direct action of the oxidant on the enzyme. The reason for this is the interaction of the products of hemoglobin oxidation with erythrocyte membrane (protein band 3) and structural changes in membrane proteins.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Fluorescence emitted from microsomal membranes by lipid peroxidation

The fluorescence emitted from rat liver microsomal membranes which had undergone enzymatic and nonenzymatic lipid peroxidation was detected directly. This fluorescence produced in peroxidized membranes increased progressively with peroxidation reaction time, and the fluorescent substances produced were retained in the membranes without being released into the aqueous phase. Extracts of the peroxidized membranes with organic solvents (chloroform/methanol) emitted fluorescence which was also dependent on the peroxidation reaction time. The generation profiles of fluorescence emitted from both the peroxidized membranes and their extracted membrane lipids differed essentially from that of thiobarbituric acid-reactive substances which reached a plateau at a relatively early stage of peroxidation reaction. These results indicate that lipid peroxidation induces stepwise chemical and physical changes in membranes and that the fluorescence from peroxidized membranes will be useful in studying such changes occurring in biological membranes.     

Fluorescence characteristics of peroxidation products in porcine intestinal brush-border membranes

Treatment of the porcine intestinal brush-border membranes with 100 microM ascorbic acid and 10 microM Fe2+ in the presence of various concentrations of tert-butyl hydroperoxide (t-BuOOH) resulted in a marked fluorescence development at 430 nm, depending on the hydroperoxide concentration. This fluorescence formation was closely related to lipid peroxidation of the membranes as assessed by formation of conjugated diene. However there is no linear relation between thiobarbituric acid-reactive substances (TBARS) and fluorescence formation. On the other hand, fluorescence formation in the membranes by treatment with ascorbic acid/Fe2+ or t-BuOOH alone was negligible. The results with antioxidants and radical scavengers suggest that ascorbic acid/Fe2+/t-BuOOH-induced lipid peroxidation of the membranes is mainly due to t-butoxyl and/or t-butyl peroxy radicals. Most TBARS produced during the peroxidation reaction were released from the membranes, but fluorescent products remained in the membrane components. The fluorescence properties of products formed by lipid peroxidation of the membranes were compared with those of products derived from the interaction of malondialdehyde (MDA) or acetaldehyde with the membranes. The fluorescence products in the acetaldehyde-modified membranes also exhibited the emission maximum at 430 nm, while the emission maximum of MDA-modified membranes was 470 nm. The fluorescence intensity of MDA-modified membranes was markedly decreased by treatment with 10 mM NaBH4 but that of the peroxidized or acetaldehyde-modified membranes was enhanced by about two-fold with the treatment. In addition, a pH dependence profile revealed that the fluorescence intensity of the peroxidized or acetaldehyde-modified membranes decreases with increasing pH of the medium, whereas that of MDA-modified ones did not change over the pH range from 5.4 to 8.0. On the basis of these results, the fluorescence properties of products formed in the intestinal brush-border membranes by lipid peroxidation are discussed.     

Effect of a zinc-deficient diet on lipid peroxidation in liver and tumor subcellular membranes

The effect of a zinc-deficient diet on lipid peroxidation in liver and tumor mitochondrial and microsomal membrane preparations from BALB/c mice was investigated. Mitochondrial and microsomal membranes from both tissues displayed increased rates of in vitro peroxidation, both enzymatic and nonenzymatic. Measurement of in vivo peroxidation, using diene conjugation as an index of measurement revealed slight increases in tissues from zinc-deficient animals that were not statistically significant. Serum lipoperoxides analyzed from all three groups revealed no significant differences. The results point to an alteration in the peroxidation potential of mitochondrial and microsomal membranes due to zinc deficiency which may be related to an alteration in fatty acid composition.     

Effect of phenylhydrazine on red blood cell metabolism

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.     

Effects of oxidative stress on the Dolichol content of isolated rat liver cells

Dolichol, a long-chain polyisoprenoid broadly distributed in all tissues and cellular membranes with unknown function(s), might have a role in free radical metabolism [it accumulates in older tissues and decreases after CCl₄ (in liver) or phenylhydrazine (in spleen and liver) administration]. The effects of the NADPH-ADP-Fe system on Dolichol levels in isolated hepatocytes were explored and the time-course of changes was compared with the release of MDA in the incubation medium and the decrease in CoQ 9 and 10 and Vitamin E levels. Results showed that the system increased lipid peroxidation and decreased Dolichol and CoQ levels in_parallel fashions and lowered Vitamin E levels with shorter latency. Meanwhile, no increase in dead cells and no Dolichol release in the medium were detected. In conclusion, an increase in oxidative stress possibly caused a rapid degradation of dolichol by the same (unknown) mechanism responsible for the breakdown of_Ubiquinone isoprenoid chains.     

Effects of oxidative stress on the Dolichol content of isolated rat liver cells

Dolichol, a long-chain polyisoprenoid broadly distributed in all tissues and cellular membranes with unknown function(s), might have a role in free radical metabolism [it accumulates in older tissues and decreases after CCl₄ (in liver) or phenylhydrazine (in spleen and liver) administration]. The effects of the NADPH-ADP-Fe system on Dolichol levels in isolated hepatocytes were explored and the time-course of changes was compared with the release of MDA in the incubation medium and the decrease in CoQ 9 and 10 and Vitamin E levels. Results showed that the system increased lipid peroxidation and decreased Dolichol and CoQ levels in_parallel fashions and lowered Vitamin E levels with shorter latency. Meanwhile, no increase in dead cells and no Dolichol release in the medium were detected. In conclusion, an increase in oxidative stress possibly caused a rapid degradation of dolichol by the same (unknown) mechanism responsible for the breakdown of_Ubiquinone isoprenoid chains.     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.