Peroxidative modification of phospholipids in myocardial membranes

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.     

Involvement of lipid peroxidation in regression of heart hypertrophy

The regression of hypertrophied heart presupposes disassembling of all cardiomyocyte components including membrane structures. The involvement of free radical oxidation of membrane phospholipids was studied in the cardiac regression. Altitude hypertrophy was developed in barocamera (7000 m, 6 hours daily). 3 weeks of periodical hypoxia leads to 1.5-fold increase of heart weight (right ventricle weight was 2-fold increased). In 7-10 days after adaptation the heart weight reduced to normal. Both, the development and regression of myocardial hypertrophy was accompanied by changes in Mb and Hb organ concentration proportional to weight changes. In lipid extraction of maximal hypertrophied heart, the 30% decrease of lipid peroxidation product (diene conjugates) regularly occurred. The rate of regression had negative correlation with peroxidation products accumulation. Intraperitoneal injection of free radical scavenger BHT attenuate the regression rate. The results suggest that unlike the common knowledge about the membrane injury effect, lipid peroxidation can play positive role in disassembling of superfluous cell membrane structures.     

Lipid fluorophores of the crystalline lens in cataracts

Microcolumn liquid and column chromatography technique is conjunction with UV-spectrophotometry and spectrofluorescent analysis were used to study lipid peroxidation products accumulated in human lenses during cataract formation by means of chromatographic separation in regard to the molecular weight and polarity properties. Cataract is characterized by the appearance of certain substances changing UV-absorption lipid spectra in the region of 230 and 274 nm and having special fluorescence (excitation--320-370 nm), (emission--405-460 nm). The same changes were observed by ultrasoundinduced lipid peroxidation of model lipid samples. The accumulated lipid peroxidation products are concentrated in the same chromatographic fractions that are responsible for the change of UV-absorption and fluorescent spectra of lipids of cataractous lenses. It is the evidence of free radical lipid peroxidation products accumulation in human lenses at cataract formation. Along with the formation of diene and triene conjugates in the lens lipids, cataract is characterized by the formation of cetodienes and of low molecular weight lipid fluorescent products of fatty acids oxidation with low polarity due to the appearance of tetraene derivatives of polyunsaturated fatty acids. The particular features of mature cataract are an increased intensity of long-wave lipid fluorescence in the blue-green region (430-460 nm) of the spectrum, formation of high molecular weight fluorescent lipid peroxidation products with high polarity, and smooth decrease in absorbance in the region of 220-330 nm. During cataract formation products of deep lipid peroxidation resulting from radical phospholipids and fatty acids polymerisation are accumulated. It is supposed that lipid peroxidation is an initial phase of membrane desintegration and formation of HMW-proteins in cataract.     

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.     

Oxidative stress induced by beta-amyloid peptide(1-42) is involved in the altered composition of cellular membrane lipids and the decreased expression of nicotinic receptors in human SH-SY5Y neuroblastoma cells

The neurotoxic effects and influence of beta-amyloid peptide (Aβ)_1–42 on membrane lipids and nicotinic acetylcholine receptors (nAChRs) in human SH-SY5Y neuroblastoma cells were investigated in parallel. Exposure of the cultured cells to varying concentrations of Aβ_1–42 evoked a significantly decrease in cellular reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide), together with enhanced lipid peroxidation and protein oxidation. Significant reductions in the total contents of phospholipid and unbiquinone-10, as well as in the levels of the 3 and 7 subunit proteins of nAChRs were detected in cells exposed to Aβ_1–42. In contrast, such treatment had no effect on the total cellular content of cholesterol. Among these alterations, increased lipid peroxidation and decreased levels of cellular phospholipids were most sensitive to Aβ_1–42, occurring at lower concentrations. In addition, when SH-SY5Y cells were pretreated with the antioxidant Vitamin E, prior to the addition of Aβ_1–42, these alterations in neurotoxicity, oxidative stress, composition of membrane lipids and expression of nAChRs were partially prevented. These findings suggest that stimulation of lipid peroxidation by Aβ may be involved in eliciting the alterations in membrane lipid composition and the reduced expression of nAChRs associated with the pathogenesis of AD.     

Red cell membrane alterations in human chronic fluoride toxicity.

Red cells from humans exposed chronically to toxic levels of fluoride through drinking water showed significant increase in lipid peroxidation and membranous cholesterol and phospholipids. Additionally, electrophoretic patterns of ghost membrane proteins revealed the presence of a new band in the range of congruent to 66 Kd and increase in the high molecular weight protein and predominance of bands with a molecular weight of congruent to 93 Kd and congruent to 20 Kd. The activities of total, Na(+)-K(+)-, Mg(2+)- and Ca(2+)-ATPases were significantly decreased in the red cell ghosts of fluorotic patients.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

Effects of L-carnitine on RBC membrane composition and function in hyperinsulinemic rats

The purpose of the study was to investigate the effects of L-carnitine (CA) on the susceptibility of erythrocyte (RBC) to peroxide-induced lipid oxidation, RBC membrane composition, ATPases activity and oxidative stress in fructose-fed hyperinsulinemic rats. The rats were subjected to experimental hyperinsulinemia and hyperglycemia by feeding a high fructose diet (60 g/100 g) for 6 weeks. The rats showed significant alterations in the RBC membrane composition. The protein content was lower than control animals, while cholesterol, phospholipids and free fatty acids were higher in fructose-fed animals. Significant differences in the total carbohydrate and relative proportions of hexose, hexosamine, sialic acid and fucose of membranes were observed. In these rats, membrane-bound ATPases (total ATPase, Na+, K+ ATPase, Mg2+ and Ca2+ ATPases) were significantly lower while thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LHP) in RBC membrane were significantly higher than those of control rats. The red cells were more susceptible to peroxide-induced oxidative stress that correlated with reduced levels of vitamin E found RBC membrane. When fructose-diet fed rats were treated simultaneously with CA (300 mg/kg b.w/day, i.p.), such alterations in membrane composition and enzyme activities did not occur. Effects of fructose loading on lipid peroxidation was also alleviated by CA. These findings suggest that high levels of dietary fructose is detrimental to RBC membrane integrity and that CA may have membrane stabilizing effects in this diet-induced model of type 2-diabetes.     

An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.     

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.     

Involvement of phospholipase A2 in neurodegeneration

Phospholipases A₂ are a heterogeneous class of enzymes that hydrolyse fatty acids from the sn-2 position of membrane phospholipids. Prolonged stimulation of phospholipase A₂ may damage membrane integrity, not only because of the loss of essential phospholipid from the lipid bilayer but also as a result of an uncontrollable Ca²⁺ influx. The increased levels of intracellular Ca²⁺ may be responsible for enhanced lipolysis, proteolysis and DNA fragmentation. This process along with the accumulation of lipid peroxidation products may be associated with neurodegeneration in acute neural trauma (ischemia, head and spinal cord injuries) and neurodegenerative diseases (Alzheimer's disease).     

Caloric restriction results in phospholipid depletion, membrane remodeling, and triacylglycerol accumulation in murine myocardium

Herein, we utilize the power of shotgun lipidomics to demonstrate that modest caloric restriction results in phospholipid depletion, membrane remodeling, and triacylglycerol (TAG) accumulation in murine myocardium. After brief periods of fasting (4 and 12 h), substantial decreases occurred in the choline and ethanolamine glycerophospholipid pools in murine myocardium (collectively, a decrease of 39 nmol of phospholipid per milligram of protein at 12 h representing approximately 25% of total phospholipid mass and approximately 20 cal of Gibbs free energy per gram wet weight of tissue). Remarkably, the selective loss of long-chain polyunsaturated molecular species was present in the major phospholipid classes thereby altering the physical properties of myocardial membranes. No decrease in TAG mass was present in myocardium during fasting, but rather myocardial TAG increased during 12 h of refeeding nearly 3-fold returning to baseline levels only after 24 h of refeeding. No alterations in other examined lipid classes were present during fasting. In contrast to these lipid alterations in myocardium, no decreases in phospholipid mass were present in skeletal muscle myocytes and a dramatic decrease in skeletal muscle (or skeletal muscle associated) TAG mass was prominent after 12 h of fasting. These results identify phospholipids as a rapidly mobilizable energy source during modest caloric deprivation in murine myocardium, while triacylglycerols are a major source of energy reserve in skeletal muscle. Collectively, these results demonstrate dramatic alterations in the membrane composition of mildly fasted mammalian myocardium that identify the unanticipated plasticity of myocardial phospholipids to adapt to modest chemical and physical perturbations.     

Effect of oxidants on small intestinal brush border membranes and colonic apical membranes--a comparative study

This study compares composition of the rat small intestinal brush border membranes (BBM) and colonic apical membranes (CAM) and their susceptibility to in vitro exposure to various oxidants. Differences were observed between BBM and CAM in their lipid composition, sugar content, alkaline phosphatase (ALP) activity and cholesterol/phospholipid ratio. BBM and CAM were exposed to superoxide generated by xanthine+xanthine oxidase (X-XO) or peroxides such as tertiary butyl hydroperoxide (tBuOOH) and hydrogen peroxide (H(2)O(2)) and alterations in ALP activity, peroxidation parameters and membrane lipids were analyzed. Exposure of BBM and CAM to superoxide resulted in decrease in ALP activity and increase in peroxidation parameters such as protein carbonyl content, malondialdehyde and conjugated diene. Superoxide exposure also resulted in lipid alterations specifically in certain phospholipids. These alterations were prevented either by superoxide dismutase or by allopurinol. Peroxides did not have any significant effect. These results suggest that both BBM and CAM are susceptible to superoxide, which can bring about peroxidation and degradation of membrane lipids specifically, certain phospholipids.     

Peroxidative damage to cardiac mitochondria: identification and purification of modified adenine nucleotide translocase.

Rat myocardial membranes exposed to free radical-generating systems exhibit both lipid peroxidation and protein alterations. The most sensitive protein, a 28-kDa polypeptide, was previously shown to increase slightly in apparent molecular weight before disappearing completely from the protein profile [N. L. Parinandi, C. W. Zwizinski, and H. H. O. Schmid (1991) Arch. Biochem. Biophys. 289, 118-123]. We now report that isolated cardiac mitochondria contain a 28-kDa protein which responds in the same manner to treatment with Cu2+/t-butylhydroperoxide. The protein exhibits several characteristic properties of the mitochondrial adenine nucleotide translocase. This assignment is supported by the finding that carboxyatractyloside, a specific inhibitor of the adenine nucleotide translocase, can prevent the oxidant-induced changes in the 28-kDa protein. Efficient purification schemes for the isolation of milligram quantities of unmodified and oxidatively altered adenine nucleotide translocase from rat heart mitochondria are described.     

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.     

Functional changes in bladder tissue from type III collagen-deficient mice

The present work investigates the protective effects of N-acetylcysteine (NAC) on carbofuran-induced alterations in lipid composition and activity of membrane bound enzymes (Na⁺-K⁺-ATPase and Ca²⁺-ATPase) in the rat brain. Animals were exposed to carbofuran at a dose of 1 mg/kg body weight, orally, for a period of 28 days. A significant increase in lipid peroxidation in terms of TBARS was observed in brain after carbofuran exposure. NAC administration (200 mg/kg body weight) on the other hand lowered the carbofuran-induced lipid peroxidation to near normal. The increased lipid peroxidation following carbofuran exposure was accompanied by a significant decrease in the levels of total lipids, which is attributed to the reduction in phospholipid levels. Furthermore, NAC administration had a beneficial effect on carbofuran-induced alterations in lipid composition. The ratio of cholesterol to phospholipid, a major determinant of membrane fluidity, was increased in response to carbofuran exposure. This was associated with decreased activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase. NAC was observed to offer protection by restoring the cholesterol to phospholipid ratio along with the activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The results clearly suggest that carbofuran exerts its neurotoxic effects by increasing lipid peroxidation, altering lipid composition and activity of membrane bound enzymes. NAC administration ameliorated the effects of carbofuran suggesting its potential therapeutic effects in carbofuran neurotoxicity.     

t-butyl hydroperoxide-induced perturbations of human erythrocytes as a model for oxidant stress

Erythrocytes were incubated with t-butyl hydroperoxide in the presence and absence of hemoglobin as a model system for oxidative stress and the alterations in the structure and integrity of the membranes were investigated. The results showed that in the presence of hemoglobin a significant modification in the membrane surface charge was induced but no such alteration was observed in peroxidized hemoglobin-free membranes. As increased hemoglobin oxidation occurred in the erythrocytes, membrane lipid peroxidation diminished, suggesting a protective role for methemoglobin in t-butyl hydroperoxide-induced lipid peroxidation. Electrophoresis on polyacrylamide gels showed modification of the cytoplasmic protein region but no high molecular weight aggregates formed at the concentrations of the hydroperoxide used in this work. The results suggest that the t-butyl hydroperoxide/normal erythrocyte system seems to be an instructive model for membrane perturbations characteristic of oxidative disorders.     

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.     

Effects of melittin on adipocyte metabolism unrelated to lysophospholipid accumulation

Melittin addition to rat or hamster adipocytes resulted in inhibition of lipolysis, cyclic AMP accumulation and glucose oxidation. Low concentrations of melittin were not insulin-like with respect to either stimulation of glucose metabolism or inhibition of lipolysis. Higher concentrations of melittin lysed adipocytes. In the presence of melittin, cellular phospholipids were released to the medium and hydrolyzed with little accumulation of lysophospholipids. Only in adipocytes incubated with melittin contaminated with phospholipase A2 was any appreciable accumulation of lysophospholipids seen and this was in the medium. These data suggest that the toxic effects of melittin on adipocytes are not due to the accumulation of lysophospholipids but rather to the loss of membrane phospholipids or alterations in membrane proteins.     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.