Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.     

Antioxidant Activity of Vitamin E and Trolox: Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro

Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the processing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO^•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information to that obtained currently from assays performed in aqueous buffer media.     

Evaluation of extracellular lipid peroxidation in brain cortex of anaesthetized rats by microdialysis perfusion and high-performance liquid chromatography with fluorimetric detection

A method for in vivo evaluation of lipid peroxidation in the extracellular space of anaesthetized rat brain cortex was developed. This method involved the use of microdialysis perfusion and high-performance liquid chromatography. The microdialysates, eluted from implanted probes, were reacted with thiobarbituric acid (TBA) prior to analysis by an HPLC system equipped with a fluorescence detector (excitation and emission wavelengths were 515 and 550 nm, respectively). Lipid peroxidation in the extracellular space was evaluated as the concentration of malondialdehyde, a lipid peroxidation end product which reacts with TBA to form a fluorescent conjugate. Significantly increased production of malondialdehyde following hydrogen peroxide perfusion (0.03%, 0.3% at a flow-rate of 1 μl/min) was observed in the brain cortex of anaesthetized rats.     

Accumulation of ceroid-like pigments in macrophages cultured with phosphatidylcholine liposomes in vitro

When mouse peritoneal macrophages as well as P388D1 cells, an established macrophage-like cell line, were cultured with liposomes composed of rat liver phosphatidylcholine and phosphatidylserine, storage of fluorescent products, ceroid-like pigments, within those cells was observed with light and fluorescence microscopy, and fluorescence spectrophotometry. The amounts of thiobarbituric acid-reactive substances and fluorescent products in macrophages were increased gradually to reach a maximal level to between 6 and 8 days of culture. The involvement of peroxidation of liposomal lipids in the formation of the pigments was further suggested by the 6 days that incorporation of alpha-tocopherol into liposomes decreased the storage of the pigments. No appreciable formation of the pigments was observed in macrophages cultured with liposomes containing dipalmitoylphosphatidylcholine instead of rat liver phosphatidylcholine. The fluorescent products formed in cultured cells were found in lipid-soluble and -insoluble fractions. Lipid-insoluble fluorescent products had an excitation maximum at 360 nm and a fluorescence maximum at 430 nm in SDS-aqueous solution (pH 7.4) and the intensity of the fluorescence was quenched at base pH, but it was not changed in acidic media. These findings indicate that the macrophages can store Schiff base fluorescent substances formed by the reaction between peroxidation products of exogenous lipids and amino compounds in the cells, under some pathological conditions.     

Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury

Increasing appreciation of the causative role of oxidative injury in many disease states places great importance on the reliable assessment of lipid peroxidation. Malondialdehyde (MDA) is one of several low-molecular-weight end products formed via the decomposition of certain primary and secondary lipid peroxidation products. At low pH and elavated temperature, MDA readily participates in nucleophilic addition reaction with 2-thiobarbituric acid (TBA), generating a red, fluorescent 1:2 MDA:TBA adduct. These facts, along with the availability of facile and sensitive methods to quantify MDA (as the free aldehyde or its TBA derivative), have led to the routine use of MDA determination and, particularly, the “TBA test” to detect and quantify lipid peroxidation in a wide array of sample types. However, MDA itself participates in reactions with molecules other than TBA and is a catabolic substrate. Only certain lipid peroxidation products generate MDA (invariably with low yields), and MDA is neither the sole end product of fatty peroxide formation and decomposition nor a substance generated exclusively through lipid peroxidation. Many factors (e.g., stimulus for and conditions of peroxidation) modulate MDA formation from lipid. Additional factors (e.g., TBA-test reagents and constituents) have profound effects on test response to fatty peroxide-derived MDA. The TBA test is intrinsically nonspecific for MDA: nonlipid-related materials as well as fatty peroxide-derived decomposition products other than MDA are TBA positive. These and other considerations from the extensive literature on MDA, TBA reactivity, and oxidative lipid degradation support the conclusion that MDA determination and the TBA test can offer, at best, a narrow and somewhat empirical window on the complex process of lipid peroxidation. The MDA content and/or TBA reactivity of a system provides no information on the precise structures of the “MDA precursor(s),” their molecular origins, or the amount of each formed. Consequently, neither MDA determination nor TBA-test response can generally be regarded as a diagnostic index of the occurrence/extent of lipid peroxidation, fatty hydroperoxide formation, or oxidative injury to tissue lipid without independent chemical evidence of the analyte being measured and its source. In some cases, MDA/TBA reactivity is an indicator of lipid peroxidation; in other situations, no qualitative or quantitative relationship exists among sample MDA content, TBA reactivity, and fatty peroxide tone. Utilization of MDA analysis and/or the TBA test and interpretation of sample MDA content and TBA test response in studies of lipid peroxidation require caution, discretion, and (especially in biological systems) correlative data from other indices of fatty peroxide formation and decomposition.     

Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate

An iron chelate, ferric nitrilotriacetate (Fe3+-NTA), is nephrotoxic and also carcinogenic to the kidney in experimental animals. Iron-promoted lipid peroxidation in the proximal tubules is thought to be responsible for the pathologic process. In the present study, iron-promoted lipid peroxidation, with thiobarbituric acid (TBA) formation as an indication, in the tubular surface was simulated in vitro using rat kidney brush border membrane vesicles and the results were compared with those using linoleate micelles and rat liver microsomal lipid liposomes. Addition of ascorbate, cysteine, or dithiothreitol to the Fe3+-NTA solution resulted in consumption of dissolved oxygen and promoted the lipid peroxidation in the micelles and in the liposomes. In contrast, addition of glutathione to the Fe3+-NTA solution caused only sluggish oxygen consumption and far less peroxidation in these lipid systems. When the brush border membrane vesicles were used for the peroxidation substrate, Fe3+-NTA and glutathione could promote TBA formation at a rate comparable to that elicited by Fe3+-NTA with cysteine or dithiothreitol. Acivicin, a gamma-glutamyl transpeptidase inhibitor, suppressed the peroxidation of the brush border membrane vesicles promoted by Fe3+-NTA and glutathione. These results suggest the following mechanism of proximal tubular cell lipid peroxidation promoted by Fe-NTA: Fe3+-NTA filtered through glomeruli is rapidly reduced by cysteine and Fe2+-NTA starts lipid peroxidation at the site, leading to proximal tubular necrosis. Cysteine is amply supplied by the decomposition of glutathione within the lumen by the action of gamma-glutamyl transpeptidase and dipeptidase situated at the proximal tubular brush border membrane.     

Carnosine,homocarnosine and anserine: could they act as antioxidants in vivo?

Carnosine, homocarnosine and anserine have been proposed to act as antioxidants in vivo. Our studies show that all three compounds are good scavengers of the hydroxyl radical (.OH) but that none of them can react with superoxide radical, hydrogen peroxide or hypochlorous acid at biologically significant rates. None of them can bind iron ions in ways that interfere with 'site-specific' iron-dependent radical damage to the sugar deoxyribose, nor can they restrict the availability of Cu2+ to phenanthroline. Homocarnosine has no effect on iron ion-dependent lipid peroxidation; carnosine and anserine have weak inhibitory effects when used at high concentrations in some (but not all) assay systems. However, the ability of these compounds to interfere with a commonly used version of the thiobarbituric acid (TBA) test may have led to an overestimate of their ability to inhibit lipid peroxidation in some previous studies. By contrast, histidine stimulated iron ion-dependent lipid peroxidation. It is concluded that, because of the high concentrations present in vivo, carnosine and anserine could conceivably act as physiological antioxidants by scavenging .OH, but that they do not have a broad spectrum of antioxidant activity, and their ability to inhibit lipid peroxidation is not well established. It may be that they have a function other than antioxidant protection (e.g. buffering), but that they are safer to accumulate than histidine, which has a marked pro-oxidant action upon iron ion-dependent lipid peroxidation. The inability of homocarnosine to react with HOCl, interfere with the TBA test or affect lipid peroxidation systems in the same way as carnosine is surprising in view of the apparent structural similarity between these two molecules.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Protection of liposomal lipids against radiation induced oxidative damage.

Liposomes were prepared from phospholipids extracted from biological membranes. A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes. The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation. High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation. Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation. The naturally occurring compounds reduced glutathione (GSH) and vitamin E(alpha-T) and the powerful radiation protector cysteamine (MEA). All three molecules could protect the liposomes against peroxidation. The membrane-soluble compound vitamin E was by far the most powerful. About 50 per cent protection was achieved by using 5 X 10(-6) M alpha-T, 10(-4) M GSH and 5 X 10(-4) M MEA. The fatty acid composition of the lipids altered drastically as a result of the irradiation. Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids. Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes.     

Plasma thiobarbituric acid reactivity: reaction conditions and the role of iron, antioxidants and lipid peroxy radicals on the quantitation of plasma lipid peroxides

The effects of Fe3+, lipid peroxy radicals and the antioxidant butylated hydroxytoluene on the 2-thiobarbituric (TBA) acid quantitation of plasma lipid peroxides were investigated. Whole plasma and plasma fractions prepared by trichloroacetic acid (TCA) protein precipitation and lipid extraction, demonstrated markedly differing TBA reactivities in the presence or absence of added Fe3+. Examination of the spectral profiles of the TBA reacted whole plasma and TCA precipitated fractions demonstrated the presence of interfering compounds which gave rise to an artifactual increase in lipid peroxide concentrations. In contrast the TBA reacted lipid extracts had low levels of interfering compounds that could be removed by our previously described high pressure liquid chromatographic method (Wade, Jackson and van Rij (1985) Biochem. Med. 33, 291-296). Further characterization of the TBA reactivity of the lipid extract showed that Fe3+ at an optimal concentration of 0.5 mM was necessary for the quantitative decomposition of the lipid peroxides to the TBA reactive product malondialdehyde (MDA). However the presence of Fe3+ resulted in further peroxidation of any unsaturated lipids present. Butylated hydroxytoluene (BHT) at an optimal concentration of 1.4 mM inhibited Fe3+ stimulated peroxidation without affecting the formation of the MDA-TBA chromogen. Using a standardized TBA test with plasma lipid extracts and the addition of optimal concentrations of Fe3+ and BHT, we have determined the mean concentration of lipid peroxides in 30 healthy human subjects to be 102.7 +/- 20.0 ngm/ml.     

Generation of toxic phospholipid(s) during oxyhemoglobin-induced peroxidation of phosphatidylcholines

When egg yolk diacylglycerophosphocholine (PC) liposomes were incubated with human oxyhemoglobin, peroxidation of liposomal lipid was induced, as monitored by an increase of thiobarbituric acid (TBA)-reactive substances, an increase of lipid hydroperoxides and the generation of chemiluminescence in the presence of luminol. During the reaction, cytotoxic substance(s), which induced shedding of acetylcholinesterase-enriched vesicles from human erythrocytes, were produced. Formation of TBA-reactive substances and lipid hydroperoxides preceded generation of chemiluminescence, conversion of oxyhemoglobin to methemoglobin and production of the toxic substances. Either superoxide dismutase or catalase could suppress generation of chemiluminescence, but not other events. Methemoglobin or ferrous ion plus ascorbate could induce peroxidation of the liposomes without production of the cytotoxic substance(s). Synthetic PCs containing both saturated and polyunsaturated fatty acyl chains caused the production of cytotoxic products which induced shedding of vesicles from erythrocytes, whereas those containing only polyunsaturated fatty acyl chains did not, suggesting that the molecular species which can produce cytotoxic products may be phospholipids containing both saturated and polyunsaturated fatty acids. The mechanism of oxyhemoglobin-induced peroxidation of lipids will be also discussed.     

Effect of cisplatin, carboplatin and stobadine on lipid peroxidation of kidney homogenate and phosphatidylcholine liposomes.

The effects of the nephrotoxic, anticancer agents cisplatin (CDDP) and carboplatin (CBDCA), and the free radical scavenger, stobadine, were investigated on lipid peroxidation (LPO) of rat kidney homogenates and phosphatidylcholine (PC) liposomes. Kidney homogenates were incubated in air at 37 degrees C for 6-48 h and lipid peroxidation was detected spectroscopically as absorbance (533 nm) of the thiobarbituric acid-malondialdehyde (TBA-MDA) complex. CDDP (0.3-10 mmol.l-1) increased LPO of the homogenate. CBDCA decreased the TBA-MDA absorbance, yet was found to interfere with MDA, TBA and/or with the TBA-MDA complex. Thus when CBDCA is involved, the TBA-MDA method for detection of LPO is not suitable. Stobadine (0.1 mmol.l-1 and 1 mmol.l-1) inhibited LPO either in the control homogenate and in the homogenate where peroxidation was increased by CDDP. The effect of CDDP and CBDCA on peroxidation of PC liposomes was monitored as oxygen consumption using a Clark-type oxygen electrode. CDDP increased but CBDCA decreased the rate of oxygen consumption during the peroxidation of liposomes induced by FeSO4. The results suggest that the effects of CDDP and CBDCA on LPO may be linked with their nephrotoxicity.     

The effect of calcium on phospholipid peroxidation

The effect of calcium ions on the peroxidation of ox-brain phospholipid liposomes in different free-radical catalysing systems has been assessed. Using thiobarbituric acid-reactivity (TBA) as a measure of lipid peroxidation, calcium ions both inhibited and enhanced peroxidation in the different systems.Changing the composition of the ox-brain phospholipid liposome with synthetic non TBA-reactive phosphatidylcholine, significantly altered its susceptibility to peroxidation both in the presence and absence of calcium ions.The results are discussed with reference to the possibility that calcium ions induce conformational changes in membrane phospholipids. Susceptibility to peroxidation is then influenced by a complex interrelationship between the qualitative lipid composition of the membrane, the pro-oxidant catalyst and the presence of calcium or other active ions.     

Antioxidant profile of dihydroxy- and trihydroxyphenolic acids-A structure–activity relationship study

Eight structurally similar dihydroxy and trihydroxyphenolic acids (protocatechuic acid, 3,4-dihydroxyphenylacetic acid, hydrocaffeic acid, caffeic acid, gallic acid, 3,4,5-trihydroxyphenylacetic acid, 3-(3,4,5-trihydroxyphenyl)propanoic acid and 3-(3,4,5-trihydroxyphenyl)propenoic acid) were examined for their total antioxidant capacity (TAC). Furthermore, their ability to scavenge peroxyl radicals, generated by AAPH in liposomes, was determined. The antioxidant/pro-oxidant activity of the compounds was screened using the 2′-deoxyguanosine assay. All compounds behave as radical scavengers, with 3,4,5-trihydroxyphenylacetic acid being the most potent. Nevertheless, in the lipid peroxidation assay an inverse ranking order was observed, 3,4-dihydroxyphenylacetic acid being the most effective compound. All the dihydroxylated compounds showed a pro-oxidant behaviour leading to an increase of 50% in 8-OH-dG induction. From the structure–antioxidant activity relationship studies performed it may be concluded that the number of phenolic groups and the type of the alkyl spacer between the carboxylic acid and the aromatic ring strongly influence the antioxidant activity.     

Antioxidant profile of dihydroxy- and trihydroxyphenolic acids--a structure-activity relationship study

Eight structurally similar dihydroxy and trihydroxyphenolic acids (protocatechuic acid, 3,4-dihydroxyphenylacetic acid, hydrocaffeic acid, caffeic acid, gallic acid, 3,4,5-trihydroxyphenylacetic acid, 3-(3,4,5-trihydroxyphenyl)propanoic acid and 3-(3,4,5-trihydroxyphenyl)propenoic acid) were examined for their total antioxidant capacity (TAC). Furthermore, their ability to scavenge peroxyl radicals, generated by AAPH in liposomes, was determined. The antioxidant/pro-oxidant activity of the compounds was screened using the 2'-deoxyguanosine assay. All compounds behave as radical scavengers, with 3,4,5-trihydroxyphenylacetic acid being the most potent. Nevertheless, in the lipid peroxidation assay an inverse ranking order was observed, 3,4-dihydroxyphenylacetic acid being the most effective compound. All the dihydroxylated compounds showed a pro-oxidant behaviour leading to an increase of 50% in 8-OH-dG induction. From the structure-antioxidant activity relationship studies performed it may be concluded that the number of phenolic groups and the type of the alkyl spacer between the carboxylic acid and the aromatic ring strongly influence the antioxidant activity.     

Fluorescence formation and heme degradation at different stages of lipid peroxidation

Secondary oxidative products of autoxidized methyl linoleate were divided into three groups (SP-I, SP-II and SP-III), which were then compared as to their abilities to form fluorescent substances and to degrade heme. SP-III showed a marked ability to produce two fluorescent substances exhibiting an excitation maximum at 350-360 nm and an emission maximum at 410-430 nm, while SP-I showed a more strongly degradative effect on heme than SP-III. The heme degradation was observed in parallel with the changes of TBA value in an early stage of lipid peroxidation and the fluorescence formation markedly increased according to the decrease of TBA value in a later stage. The results suggested that there are different reactive substances which bring about fluorescence formation and heme degradation and that they are produced at different stages of lipid peroxidation.     

The reaction of 2-thiobarbituric acid with biologically active alpha,beta-unsaturated aldehydes

The standard assay for lipid peroxidation is the measurement of the pink, 532 n, absorbing chromogen which is formed upon reaction of 2-thiobarbituric acid (TBA) with the lipid peroxidation product malonaldehyde (MDA). The present studies indicate that the toxic lipid peroxidation product trans-4-hydroxynonenal and its dehydration product trans, trans-nonadienal react with TBA to form chromogens which absorb maximally at 530 and 532 nm, respectively. Other biologically active alpha, beta-unsaturated aldehydes, such as acrolein and crotonaldehyde, short-chain homologs of alkenals formed during lipid peroxidation, and trans,trans-muconaldehyde, a novel diene dialdehyde, react with TBA to form products which absorb maximally at 495 nm. The molar extinction coefficients of the aldehyde: TBA chromogens formed were found to vary widely, suggesting that only small contributions to the 532 nm absorption by TBA adducts of reactive aldehydes other than MDA may be encountered during the use of the TBA assay.     

Inhibition of copper-induced lipid peroxidation by sinapic acid and its derivatives in correlation to their effect on the membrane structural properties

The efficiencies of sinapic acid and its derivatives syringic acid, syringaldehyde, three sinapoyl esters (ethyl, propyl, butyl sinapates), 4-vinylsyringol and sinapine were investigated for prevention of lipid peroxidation in correlation with their interactions with model lipid membrane systems. Significant antioxidant activities of propyl and butyl sinapates were seen by fluorimetric assay in phosphatidylcholine liposomes as model membrane using C11-BODIPY^581/591 lipophilic fluorescent probe. The sinapic acid esters also had the highest impact on membrane structural properties, as observed by differential scanning calorimetry and fluorescence polarisation measurements. The greatest protection of phospholipids from peroxidation by these esters correlated well with their polarity and insertion into the lipid bilayer.     

The combined effect of pycnogenol with ascorbic acid and trolox on the oxidation of lipids and proteins

Pycnogenol (PYC), a procyanidin-rich extract of French maritime pine bark (Pinus pinaster) has strong antioxidant potential and promotes cellular health. The aim of this study was to investigate a possible cooperation of natural antioxidant PYC with synthetic antioxidants ascorbic acid and trolox in the model system of lipid peroxidation determined as conjugated dienes formation in liposomes and on the oxidation of proteins (in BSA and plasma proteins) determined as protein carbonyls. The present study shows that PYC and trolox significantly increased inhibition of lipid peroxidation initiated by copper acetate and tert-butylhydroperoxide in concentration and time dependence compared with untreated unilamellar liposomes. PYC and trolox added simultaneously to the oxidized liposomes exerted an additive preventive effect. PYC s inhibitory effect on formation of carbonyl compounds in BSA and plasma proteins, oxidized by two oxidative systems--H2O2/FeSO4 and HOCl, were studied in co-operation with other synthetic antioxidants--ascorbic acid and trolox. We found the synergistic or additive effect of PYC with mentioned antioxidants.     

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.