Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by alpha-tocopherol

The oxidation of rat red blood cells (RBC) by molecular oxygen was performed in an aqueous suspension with an azo compound as a free-radical initiator. The RBC were oxidized at a constant rate by a free-radical chain mechanism, resulting in hemolysis. The extent of hemolysis was proportional to the concentration of free radical. alpha-Tocopherol in RBC membranes suppressed the oxidation and hemolysis to produce an induction period. Tocopherol was constantly consumed during the induction period, and hemolysis developed when tocopherol concentrations fell below a critically low level. Among the membrane lipids, phosphatidylethanolamine, phosphatidylserine, and arachidonic acids were predominantly oxidized in the absence of tocopherol. In the presence of tocopherol, however, such lipid changes were suppressed during a 120-min incubation even when hemolysis started. Membrane proteins as well as lipids were oxidized. The formation of proteins with high molecular weight and concomitant decrease of the low-molecular-weight proteins were observed on gel electrophoresis with the onset of hemolysis. This study clearly showed the damage of RBC membranes caused by oxygen radical attack from outside of the membranes, and suggested that membrane tocopherol even below a critically low level could suppress lipid oxidation but that it could not prevent protein oxidation and hemolysis.     

Transbilayer distribution of alpha-tocopherol and lipid asymmetry in nerve tissue membranes

The alpha-tocopherol content and fatty acid composition of lipids in various types of nervous tissue membranes were studied. The transbilayer distribution of alpha-tocopherol and polyunsaturated fatty acids in liposomes and plasma membranes of synaptosomes was examined. It was shown that both phosphatidylethanolamine and phosphatidylserine are localized predominantly in the inner monolayer and they contain the bulk of polyenoic fatty acid residues. alpha-Tocopherol incorporated into liposomes from synaptosome plasma membrane lipids and present in synaptosome plasma membranes is also predominantly localized in the inner monolayers. No asymmetrical distribution of incorporated alpha-tocopherol was observed in liposomes prepared from a single phospholipid, e.g., dioleoylphosphatidylcholine.     

Lipid peroxidation modifies the picture of membranes from the "Fluid Mosaic Model" to the "Lipid Whisker Model"

The “Fluid Mosaic Model”, described by Singer and Nicolson, explain both how a cell membrane preserves a critical barrier function while it concomitantly facilitates rapid lateral diffusion of proteins and lipids within the planar membrane surface. However, the lipid components of biological plasma membranes are not regularly distributed. They are thought to contain “rafts” - nano-domains enriched in sphingolipids and cholesterol that are distinct from surrounding membranes of unsaturated phospholipids. Cholesterol and fatty acids adjust the transport and diffusion of molecular oxygen in membranes. The presence of cholesterol and saturated phospholipids decreases oxygen permeability across the membrane. Alpha-tocopherol, the main antioxidant in biological membranes, partition into domains that are enriched in polyunsaturated phospholipids increasing the concentration of the vitamin in the place where it is most required. On the basis of these observations, it is possible to assume that non-raft domains enriched in phospholipids containing PUFAs and vitamin E will be more accessible by molecular oxygen than lipid raft domains enriched in sphingolipids and cholesterol. This situation will render some nano-domains more sensitive to lipid peroxidation than others. Phospholipid oxidation products are very likely to alter the properties of biological membranes, because their polarity and shape may differ considerably from the structures of their parent molecules. Addition of a polar oxygen atom to several peroxidized fatty acids reorients the acyl chain whereby it no longer remains buried within the membrane interior, but rather projects into the aqueous environment “Lipid Whisker Model”. This exceptional conformational change facilitates direct physical access of the oxidized fatty acid moiety to cell surface scavenger receptors.     

Comparison of the photodynamic action of Rose Bengal and tetrachlorosalicylanilide on isolated porcine erythrocyte membranes

The light-mediated effects of Rose Bengal and 3,3',4',5-tetrachlorosalicylanilide (T4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.     

The cold but not hard fats in ectotherms: consequences of lipid restructuring on susceptibility of biological membranes to peroxidation,a review

The production of reactive oxygen species is a regular feature of life in the presence of oxygen. Some reactive oxygen species possess sufficient energy to initiate lipid peroxidation in biological membranes, self-propagating reactions with the potential to damage membranes by altering their physical properties and ultimately their function. Two of the most prominent patterns of lipid restructuring in membranes of ectotherms involve contents of polyunsaturated fatty acids and ratios of the abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine. Since polyunsaturated fatty acids and phosphatidylethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to lipid peroxidation. Although membranes from animals living at low body temperatures may be more prone to oxidation, the generation of reactive oxygen species and lipid peroxidation are sensitive to temperature. These scenarios raise the possibility that membrane susceptibility to lipid peroxidation is conserved at physiological temperatures. Reduced levels of polyunsaturated fatty acids and phosphatidylethanolamine may protect membranes at warm temperatures from deleterious oxidations when rates of reactive oxygen species production and lipid peroxidation are relatively high. At low temperatures, enhanced susceptibility may ensure sufficient lipid peroxidation for cellular processes that require lipid oxidation products.     

End products of lipid peroxidation in erythrocyte membranes in Alzheimer's disease

Alzheimer's disease (AD) is accompanied by oxidative stress in the brain. Because the brain tissue is rich in polyunsaturated fatty acids, it is prone to the free radical attack resulting in lipid peroxidation. Intermediates of lipid peroxidation may diffuse from the primary site, cross the blood-brain barrier and modify erythrocyte membranes in the bloodstream. We exposed isolated erythrocyte membranes from patients with AD and the control group to in vitro free radical damage and monitored the accumulation of the end products of lipid peroxidation, lipofuscin-like pigments (LFPs), by fluorescence spectroscopy. LFPs were analyzed by means of tridimensional and synchronous fluorescence spectroscopy. The levels of LFP formed during in vitro peroxidation were significantly higher in erythrocyte membranes from patients with AD compared with the control group. Furthermore, the chemical composition of LFP in AD was different from the control group. The analysis of the specific modifications of erythrocyte membranes in AD is of great medical importance regarding the need of a diagnostic blood biomarker.     

The lipid whisker model of the structure of oxidized cell membranes

An essential feature of the innate immune system is maintaining cellular homeostasis by identifying and removing senescent and apoptotic cells and modified lipoproteins. Identification is achieved through the recognition of molecular patterns, including structurally distinct oxidized phospholipids, on target cells by macrophage receptors. Both the structural nature of the molecular patterns recognized and their orientation within membranes has remained elusive. We recently described the membrane conformation of an endogenous oxidized phospholipid ligand for macrophage scavenger receptor CD36, where the truncated oxidized sn-2 fatty acid moiety protrudes into the aqueous phase, rendering it accessible for recognition. Herein we examine the generality of this conformational motif for peroxidized glycerophospholipids within membranes. Our data reveal that the addition of a polar oxygen atom on numerous peroxidized fatty acids reorients the acyl chain, whereby it no longer remains buried within the membrane interior but rather protrudes into the aqueous compartment. Moreover, we show that neither a conformational change in the head group relative to the membrane surface nor the presence of a polar head group is essential for CD36 recognition of free oxidized phospholipid ligands within membranes. Rather, our results suggest the following global phenomenon. As cellular membranes undergo lipid peroxidation, such as during senescence or apoptosis, previously hydrophobic portions of fatty acids will move from the interior of the lipid bilayer to the aqueous exterior. This enables physical contact between pattern recognition receptor and molecular pattern ligand. Cell membranes thus "grow whiskers" as phospholipids undergo peroxidation, and many of their oxidized fatty acids protrude at the surface.     

Effect of a hyperlipidic diet on lipid composition, fluidity, and (Na+-K+)ATPase activity of rat erythrocyte membranes

Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.     

Fluorenyl fatty acids as fluorescent probes for depth-dependent analysis of artificial and natural membranes.

The main objective of depth-dependent fluorescent probes is to provide information at a distinct position in the membrane hydrophobic core. We report here a series of fluorenyl fatty acids which can probe both artificial and natural membranes at different depths. Long-chain acids (C4, C6, and C8) are attached to fluorene chromophore on one side, and a hydrophobic tail (C4) is attached on the other side, so that on incorporation in membranes the carboxyl end of the molecule is oriented toward the membrane-water interface and the hydrophobic tail points toward the membrane interior. These acids can be readily partitioned into membranes. The disposition of these fluorenyl fatty acids in membranes was studied by fluorescence quenching using iodide as a water-soluble and 9,10-dibromostearic acid as a lipid-soluble quencher. The results obtained indicate that attachment of a hydrophobic tail is essential for effective alignment of depth-dependent fluorescent probes. The length of the hydrophobic tail was varied and an n-butyl chain was found to be most effective. In all cases, the compounds with a hydrophobic tail were found to be probing the membrane deeper than their counterparts with no hydrophobic tail. Further, the compounds with hydrophobic tails were more strongly immobilized in the membrane as indicated by fluorescence polarization studies. However, the effect of such a tail varied with membrane type. Thus in artificial membranes an n-butyl chain was found to be extremely important for effective monitoring by shallow probes like 4-(2'-fluorenyl)butyric acid, whereas in erythrocyte ghost membranes the same n-butyl tail was found to be more desirable for deeper probes like 8-(2'-fluorenyl)octanoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.     

Effects of cold water swimming on blood rheological properties and composition of fatty acids in erythrocyte membranes of untrained older rats

This is the first report on the effects of a single bout of swimming to exhaustion in cold water on rat erythrocyte deformability, aggregation and fatty acid composition in erythrocyte membranes. The results indicate that there was a significant decrease in body temperature of experimental rats swimming in water at 4 degrees C and 25 degrees C when compared to the control. Erythrocyte aggregation indices did not change after swimming in water at 4 degrees C whereas erythrocyte deformability increased at shear stress 1,13 [Pa] and 15,96 [Pa]. Physical effort performed in water at 4 degrees C when compared to the control group resulted in an increase in monounsaturated and polyunsaturated n-3 fatty acid content in erythrocyte membranes that influenced the increase in their fluidity and permeability even though that of polyunsaturated n-6 fatty acids decreased. Physical effort performed in 25 degrees C water resulted in an increase in saturated fatty acid content and a decrease in all polyunsaturated fatty acids and polyunsaturated n-6 fatty acids when compared to the control group. Swimming of untrained old rats in cold water affected rheological properties oferythrocytes in a negligible way while changes in the fatty acid composition of erythrocyte membranes were more pronounced.     

Photodynamic effect of chlorine e6 on erythrocyte membranes

Photodestruction of erythrocyte membranes sensitized by water soluble chlorophyll derivative chlorine e6 (Chl e6) was studied. It has been determined that light irradiation of erythrocyte ghosts with wave length lambda-660 nm in the presence of Chl e6 caused in protein and lipid components of the membrane deep destructive changes which were expressed in cross-linking of the membrane polypeptides and accumulating in the membrane products of peroxidation of unsaturated fatty acids residues, in phospholipids mainly. It has been shown that these processes were realized only in the presence of molecular oxygen and consequently possess pure photodynamic character.     

Membrane changes in rat erythrocyte ghosts on ghee feeding

Alterations in membrane lipid composition is known to result in functional and structural changes in the membrane, and dietary lipids play an important role in this change. It was of interest to study the influence of ghee feeding to the rat on membrane structure and function. The activities of membrane bound enzymes Na⁺ K⁺ ATPase and Acetylcholinesterase were studied as an index of membrane changes. Male weanling rats were fed 2.5% fresh or thermally oxidized ghee in the diet for a period of 8 weeks. The control rats were fed groundnut oil. A decrease of 28% in the membrane fluidity of erythrocyte ghost membranes was observed in the oxidized ghee fed group at 37°C, by fluorescence polarization measurements using 1,6-Diphenyl-1,3,5-hexatriene as a probe. The activities of Na⁺ K⁺ ATPase and Acetylcholinesterase showed an increase of 65% and 200% respectively after feeding oxidized ghee (2.5%). Also changes in Na⁺, K⁺ and ATP kinetics were observed in these rats. Increased membrane lipid peroxidation (80%) and C/PL ratio (11%) in the oxidized ghee fed group was observed. Marginal changes in the fatty acid composition were also seen. Further, an increase in the osmotic fragility of erythrocytes was observed in the oxidized ghee fed rats. It is inferred from these experiments that consumption of oxidized ghee with the diet affects the erythrocyte ghost membrane structure and function at 2.5% level, whereas consumption of fresh ghee has no effect on the erythrocyte membrane.     

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N₂ atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin.We have found a large seasonal variation in the endogenous level of α-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of α-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol.This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.     

Proteoliposome interaction with human erythrocyte membranes. Functional implantation of gamma-glutamyl transpeptidase

The transfer of detergent solubilized and purified gamma-glutamyl transpeptidase (gamma-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes has been studied. The transfer of gamma-glutamyl transpeptidase was observed upon incubation of gamma-GTase incorporated dipalmitoylphosphatidylcholine vesicles with erythrocyte ghost membranes at 37 degrees C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when dipalmitoylphosphatidylcholine proteoliposomes were used compared to dimyristoylphosphatidylcholine, and intermediate values were observed when binary mixtures of DMPC and DPPC were used. Moreover, the transfer of gamma-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of gamma-GTase has been observed to be associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin, and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. However, the inability of entrapped [14C]-sucrose delivery from proteoliposomes into ghost membrane vesicle suggest that fusion per se is not primarily involved in the transfer process. It appears that the transfer of gamma-glutamyl transpeptidase occurs by a collisional transfer process resulting in intermembrane protein transfer. The gamma-glutamyl transpeptidase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. In addition, the uptake of L-glutamate was inhibited by the dipeptide gamma-glutamyl-L-glutamate, thus supporting the proposed role of gamma-glutamyl transpeptidase in the uptake of amino acids in biological membranes.     

Interaction of alpha-tocopherol with iron: antioxidant and prooxidant effects of alpha-tocopherol in the oxidation of lipids in aqueous dispersions in the presence of iron

The dual functions of alpha-tocopherol in the oxidation of lipids in aqueous dispersions in the presence of iron were studied, aiming specifically at elucidating the effect of interaction between alpha-tocopherol and iron. Ferrous ion decomposed hydroperoxide rapidly and induced the free radical chain oxidation of soybean phosphatidylcholine liposomes. alpha-Tocopherol acted primarily as a radical scavenger in the oxidation induced by ferrous ion and acted as an antioxidant. Ferric ion decomposed hydroperoxide much more slowly than ferrous ion, but it also induced the oxidation of liposomal membranes. alpha-Tocopherol incorporated into artificial liposomal membranes reduced ferric ion rapidly to give more reactive ferrous ion, and alpha-tocopherol acted either as an antioxidant or as a prooxidant depending on the experimental conditions. When alpha-tocopherol was depleted by the interaction with ferric ion, it acted solely as a prooxidant, whereas if some alpha-tocopherol remained, it acted as an antioxidant. On the other hand, alpha-tocopherol residing in the intact erythrocyte membranes did not reduce ferric ion in the aqueous region.     

Fatty acid and alcohol partitioning with intestinal brush border and erythrocyte membranes

Summary Relative partition coefficients of fatty acids and alcohols between aqueous buffers and biological membranes have been determined from the linear relationship between isotope content of sedimented membranes and aqueous concentration. This technique allows study of highly lipid soluble compounds such as long-chain saturated fatty acids. Rat intestinal brush border membranes and erythrocyte ghost membranes were studied by using homologous series of saturated fatty acids mono-unsaturated fatty acids and 10, 12, and 14 carbon normal alcohols. The influence of chain length on partitioning was similar in the three series with an incremental, free energy of –820 cal/mole per methylene group in brush borders for the saturated fatty acids. Incremental enthalpy and entropy were –1331 cal/mole and –1.64 cal/mole,^oK respectively. Decrease in the partition coefficient due to the double bond (monounsaturated relative to saturated) had an incremental free energy of +1178 cal/mole, incremental enthalpy of –3453 cal/mole, and incremental entropy of –7.34 cal/mole,^oK, while substitution of the hydroxyl for the ionized carboxyl group (pH 7.4) increased the partition coefficient by 72-fold. From these data it must be concluded that the lipid phase of the membrane bilayer is extremely hydrophobic, similar to heptane or polyethylene in polarity.     

脂质过氧化对人红细胞膜脂流动性的影响

研究枯稀过氧化氢/高铁血红素体系所产生的烷基过氧自由基对红细胞的损伤。测定了脂质过氧化的产物——丙二脂的生成,并证明阿魏酸钠对脂质过氧化的抑制。荧光偏振的结果指出,膜脂过氧化以后降低了膜脂的流动性。人红细胞用5DSA和16DSA标记并用ESR检测膜脂流动性,结果表明,序参数S几乎没有发生变化,旋转相关时间τ值的增加证明膜脂过氧化以后,疏水尾部的物理状态发生了改变。经脂质过氧化以后,红细胞膜中的不饱和脂防酸的减少,可能是降低膜脂流动性的原因之一。     

Peroxidation of liposomes in the presence of human erythrocytes and induction of membrane damage of erythrocytes by peroxidized liposomes

Hemolysis (Kobayashi, T., Takahashi, K., Yamada, A., Nojima, S. and Inoue, K. (1983) J. Biochem. 93, 675-680) and shedding of acetylcholinesterase-enriched membrane vesicles (diameter 150-200 nm) were observed when human erythrocytes were incubated with liposomes of phosphatidylcholine which contained polyunsaturated fatty acyl chains. These events occurring on erythrocyte membrane were inhibited by radical scavengers or incorporation of alpha-tocopherol into liposomes, suggesting that lipid peroxidation is involved in the process leading to membrane vesiculation and hemolysis. The idea was supported by findings that generation of chemiluminescence, formation of thiobarbituric acid reactive substance, accumulation of conjugated diene compounds in liposomes and decrease of polyunsaturated fatty acids in liposomes occurred concomitantly during incubation. Hemolysis was also suppressed by the addition of extra liposomes, insensitive to peroxidation, or of serum albumin even after the completion of peroxidation of liposomes. These results suggest that peroxidized lipids, responsible for vesiculation and hemolysis, may be formed first in liposomes and then gradually transferred to erythrocyte membranes. The accumulation of these lipids peroxides may eventually cause membrane vesiculation followed by hemolysis.