Oxidative hemolysis of erythrocytes and its inhibition by free radical scavengers

The oxidative hemolysis of rabbit erythrocytes induced by free radicals and its inhibition by chain-breaking antioxidants have been studied. The free radicals were generated from either a water-soluble or a lipid-soluble azo compound which, upon its thermal decomposition, gave carbon radicals that reacted with oxygen immediately to give peroxyl radicals. The radicals generated in the aqueous phase from a water-soluble azo compound induced hemolysis in air, but little hemolysis was observed in the absence of oxygen. Water-soluble chain-breaking antioxidants, such as ascorbic acid, uric acid, and water-soluble chromanol, suppressed the hemolysis dose dependently. Vitamin E in the erythrocyte membranes was also effective in suppressing the hemolysis. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without phytyl side chain, incorporated into dimyristoylphosphatidylcholine liposomes, suppressed the above hemolysis, but alpha-tocopherol did not suppress the hemolysis. Soybean phosphatidylcholine liposomes also induced hemolysis, and a lipid-soluble azo initiator incorporated into the soybean phosphatidylcholine liposomes accelerated the hemolysis. The chain-breaking antioxidants incorporated into the liposomes were also effective in suppressing this hemolysis.     

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, alpha-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.     

Antioxidant Action of 2,2,4,6-Tetra-Substituted 2,3-Dihydro-5-hydroxybenzofuran Against Lipid Peroxidation: Effects of Substituents and Side Chain

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di- tert -butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di- tert -butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), &#102 -tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of &#102 -tocopherol on the efficacy of radical scavenging within the membranes. Di- tert -butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert -butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.     

Antioxidant action of 2,2,4,6-tetra-substituted 2,3-dihydro-5-hydroxybenzofuran against lipid peroxidation: effects of substituents and side chain

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di-tert-butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of alpha-tocopherol on the efficacy of radical scavenging within the membranes. Di-tert-butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert-butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

Do spin traps also act as classical chain-breaking antioxidants? A quantitative kinetic study of phenyl tert-butylnitrone (PBN) in solution and in liposomes

Free radical spin traps such as phenyl tert-butylnitrone (PBN) are often reported to provide protection of the central nervous system of animal models against free radical damage, and the effects are attributed to its "antioxidant activity." The effects of PBN and p-CH(3)O-PBN were compared with known antioxidants, alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), in quantitative kinetic studies of lipid peroxidation thermally initiated under controlled conditions. Results obtained on the spin traps in organic solvents and in dilinoleoyl phosphatidylcholine (DLPC) bilayers indicated that the spin traps do not act as peroxyl radical trapping antioxidants but rather act only as moderate "retarders" of oxygen uptake at relatively high concentration. At low oxygen partial pressures, e.g., 14 torr, which better reflect oxygen partial pressures in biological systems, PBN provides a more significant reduction in oxygen uptake (up to 50%) by DLPC bilayers but still did not act as a typical antioxidant. However, at low partial pressures, PBN does act cooperatively with PMHC. It is suggested that its role in biological fluids and tissues may be to extend the suppressed oxidation by natural antioxidants expected to be present. The combination of antioxidant/spin trap, alpha-(3, 5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butylnitrone did not exhibit any enhanced antioxidant efficiency compared with the related hindered phenol, 2,6-di-tert-butyl-4-methoxyphenol.     

Mechanism accounting for the induction of nonspecific permeability of the inner mitochondrial membrane by hydroperoxides

The effect of antioxidants on the nonspecific permeability of the inner mitochondrial membrane induced by cumene hydroperoxide or Ca(2+) has been studied. Butylated hydroxytoluene, butylated hydroxyanisole and 2,2,5,7,8-pentamethyl-6-chromanol, taken at a concentration up to 50 microM, suppress the cumene hydroperoxide-induced accumulation of lipid peroxidation products. In the same range of concentrations, these antioxidants inhibit the activation of nonspecific permeability by cumene hydroperoxide or Ca(2+). Propyl gallate, being less effective under such conditions, fails to affect the induction of nonspecific permeability. Additionally, 2,2,5,7,8-pentamethyl-6-chromanol at a concentration decreasing the accumulation of lipid peroxidation products by 70% has been shown not to increase the lag period of nonspecific permeability induction. Higher antioxidant concentrations, while leading to an increase in the lag period of nonspecific permeability induction, cause but minor suppression of lipid peroxidation. From the results obtained we can assume that free radicals formed in the course of hydroperoxide decomposition or on mitochondrial redox complex interact directly with a system responsible for nonspecific permeability or with regulating components of this system.     

Synergistic inhibition of oxidation in dispersed phosphatidylcholine liposomes by a combination of vitamin E and cysteine

Oxidations of soybean phosphatidylcholine liposomes in an aqueous dispersion initiated by free radicals generated initially either in the aqueous phase or in the lipid phase were efficiently suppressed by vitamin E in the membranes. Vitamin E was consumed linearly with time and, when the inhibition period was over the oxidation proceeded rapidly at a rate similar to that in the absence of vitamin E. L-Cysteine was also effective by itself in scavenging radicals in the aqueous region, but it was consumed more rapidly than vitamin E. On the other hand, cysteine could not scavenge the radicals efficiently in a lipid region. Nevertheless, when vitamin E was incorporated into liposomes, the addition of cysteine in the aqueous phase prolonged the inhibition period and it reduced the rate of decay of vitamin E markedly even when the radicals were generated initially in the lipid bilayer. Furthermore, it was found by an electron spin resonance study that chromanoxyl radical disappeared quite rapidly when it was mixed with cysteine and that the spin adduct of cysteine radical was observed in the presence of alpha-(4-pyridyl-N-oxide)-N-tert-butyl nitrone. It was concluded that L-cysteine located in an aqueous region could regenerate vitamin E by reacting with vitamin E radical formed in a lipid region and show a synergistic antioxidant effect, although its efficiency of vitamin E regeneration was lower than that by vitamin C.     

The specificity of lipoxygenase-catalyzed lipid peroxidation and the effects of radical-scavenging antioxidants

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenase-mediated lipid peroxidation proceeds in general via regio-, stereo- and enantio-specific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio-, stereo-, and enantio-random. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radical-scavenging antioxidants such as alpha-tocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenase-catalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physico-chemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.     

The transfer of antioxidants between liposomes

Abstract

A quantitative study is reported on a comparison of antioxidant action of the a-tocopherol model 2,2,5,6,7-pentamethyl-6-hydroxychroman (PMHC), 2,6-di-tert-butyl-4-methoxvphenol (DBHA) and a-tocopherol when these antioxidants are delivered to peroxidizing dilinoleoylphosphatidylcholine (DLPC) bilayers by (a) intermembrane transfer between donor dimyristoylphosphatidylcholine (DMPC) liposomes and acceptor DLPC liposomes, versus (b) the conventional coevaporation methods. The profiles of inhibited oxidation using method (a) were comparable with those of method (b) during inhibited peroxidation of DLPC liposomes, initiated by lipid-soluble azo-bis-dimethylvaleronitrile (ADVN), and quantitative determinations of the rate of chain initiation, R_i, were the same for the two methods. Differences observed, between antioxidant action by methods (a) and (b), when using the water-soluble initiator azo-bis-amidinopropane hydrochloride (ABAP), are attributed to slow diffusion of ABAP through the multilamellar DLPC system and a resultant non-uniform initiation. PMHC underwent facile intermembrane transfer through a barrier of dialysis tubing, from donor DMPC liposomes to acceptor DLPC or DMPC liposomes, based on analytical and quantitative inhibition studies. α-Tocopherol was comparatively slow to undergo intermembrane transfer by direct contact between liposomes and transferred only slightly through a barrier. The slight but measurable solubility of PMHC and DBHA in the aqueous phase supports a pathway of intermembrane transfer involving a water-soluble intermediate.     

Co-oxidation of 2,2,5,7,8-pentamethyl-6-chromanol and diphenyl disulfide with 3-chloroperoxybenzoic acid

The aim of this study was to determine whether disulfides could serve as protective antioxidants for alpha-tocopherol or vice versa. The chosen reaction system was a co-oxidation of the model compound of alpha-tocopherol, 2,2,5,7,8-pentamethyl-6-chromanol (PMC), and diphenyl disulfide (1) by 3-chloroperoxybenzoic acid. The rate of oxidation of the disulfide was approximately twice as fast as that of PMC when each compound was oxidised separately. However, when they were co-oxidised, the rate of loss of PMC increased while that of the disulfide decreased. The reason appeared to be that the disulfide was preferentially oxidised to the thiosulfinate (2) and the thiosulfonate (3) which then reacted with unchanged PMC to form compound (4), the major product, and benzenethiol. Benzenethiol was then re-oxidised to the disulfide.     

High-throughput fluorescence screening of antioxidative capacity in human serum.

Diphenylhexatriene-labeled phosphatidylcholine and propionic acid have been established as selective fluorescence markers for the continuous determination of oxidation processes in the lipid and aqueous phases of unfractionated human serum. Oxidation of the respective fluorophores leads to a decrease in fluorescence intensity from which the time-dependent degradation of the marker molecule can be determined. The lag times preceding the propagation of oxidation are representative for the antioxidative capacity of the system, which may be influenced by exogenous factors, e.g., the antioxidants from the diet. Supplementation of human serum by quercetin, rutin, vitamin E, vitamin C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the oxidation marker and the hydrophobicity of the antioxidant. Quercetin and vitamin E showed a higher in vitro capacity of protecting lipoproteins against oxidation. In contrast, rutin and vitamin C were more efficient as inhibitors in the aqueous phase. The same effect on serum was found after dietary consumption of apples. This result is in line with the known observation that intake of plant polyphenols leads to an increase in serum levels of hydrophilic antioxidants.     

Reaction of 2,2,5,7,8-pentamethyl-6-chromanol, an alpha-tocopherol analogue, with NO in the presence of oxygen

An alpha-tocopherol model compound, 2,2,5,7,8-pentamethyl-6-chromanol, reacted with nitric oxide (NO) in the presence of various amounts of oxygen to afford four major products. Distribution of the products was varied depending on the ratio of NO and O2, and the preincubation time of NO and O2.     

Relation of lipid peroxidation to loss of cations trapped in liposomes

Lipid peroxidation and alterations in cation loss have been induced in liposomes by ferrous ion, ascorbic acid, reduced and oxidized glutathione, and gamma radiation. Modifications of these effects by tocopherol and 2,6-di-tert-butyl-4-methylphenol (BHT) were studied when these antioxidants were either incorporated in the membrane or were added to already formed liposomes prior to the addition of the chemical agent or to irradiation. Lipid peroxidation, as indicated by the thiobarbituric acid test for malonic dialdehyde, did not correlate with alterations in cation loss. The largest amounts of lipid peroxidation induced by ascorbic acid and glutathione were associated with decreased cation loss. Inhibition of Fe(2+)- and radiation-induced lipid peroxidation by antioxidants did not inhibit the associated increase in cation loss. Tocopherol was a more effective antioxidant than BHT when it was incorporated in the membrane, whereas BHT was more effective when it was added to the liposomes after formation.     

Prooxidant and antioxidant properties of Trolox C, analogue of vitamin E, in oxidation of low-density lipoprotein

Trolox C (Trolox), a water-soluble analogue of vitamin E lacking the phytyl chain, was investigated with respect to its effect on the oxidation of low-density lipoprotein (LDL). Trolox was added at different time points of LDL oxidation induced by Cu2+ and aqueous peroxyl radicals. In the case of Cu2+ -induced LDL oxidation, the effect of Trolox changed from antioxidant to prooxidant when added at later time points during oxidation; this transition occurred whenever alpha-tocopherol was just consumed in oxidizing LDL. Thus, in the case of Cu2+ -dependent LDL oxidation, the presence of lipophilic antioxidants in the LDL particle is likely to be a prerequisite for the antioxidant activity of Trolox. When oxidation was induced by peroxyl radicals, as a model of metal-independent oxidation, the effect of Trolox was always antioxidant, suggesting the importance of Cu2+ /Cu+ redox-cycling in the prooxidant mechanism of Trolox. Our data suggest that, in the absence of significant amounts of lipophilic antioxidants, LDL becomes highly susceptible to oxidation induced by transition metals in the presence of aqueous reductants.     

Reduction of 8a-hydroxy-2,2,5,7,8-pentamethyl-6-chromanone

Two-electron oxidation of 2,2,5,7,8-pentamethyl-6-chromanol (PH), a model compound of alpha-tocopherol, gives the unstable 8a-hydroxy-2,2,5,7,8-pentamethyl-6-chromanone (POH) which rearranges to form stable 2-(3-hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ). POH and PQ are isomers which have the same oxidation state. The aim of this work was to compare the ease of reduction of POH and PQ at room temperature using a variety of biological and chemical reductants in a reductant:POH (PQ) ratio of 20:1 (or 16:1). Ascorbic acid completely reduced POH to PH in 20 min, but had no effect on PQ after 40 min. Sodium ascorbate did not reduce POH or PQ at all after 40 min. Sodium dithionite reduced POH to PH (85%) in 20 min, but reduced PQ to its hydroquinone, PQH2 (67%) in 40 min. Dithiothreitol produced a slight reduction of POH to PH (21%) but reduced PQ to PQH2 (69%). NADH/FAD reduced POH and PQ to PQH2 (73% and 42%, respectively) in 10 min. It was concluded that POH is easier to reduce than PQ and more likely to form PH as a product, particularly under conditions of mild acidity.     

Reaction of 2,2,5,7,8-pentamethyl-6-chromanol, an α-tocopherol analogue, with NO in the presence of oxygen

An α-tocopherol model compound, 2,2,5,7,8-pentamethyl-6-chromanol, reacted with nitric oxide (NO) in the presence of various amounts of oxygen to afford four major products. Distribution of the products was varied depending on the ratio of NO and O₂, and the preincubation time of NO and O₂.     

Synergistic interaction between vitamin E and the bile pigments bilirubin and biliverdin

The oxidation of soybean phosphatidylcholine (PC) liposomes initiated with a lipid-soluble azo compound within the liposomal membranes has been studied in the absence and presence of membrane-bound vitamin E and water-soluble bile pigments. In the absence of vitamin E, lipid peroxidation proceeded linearly and without delay. Low micromolar amounts of bilirubin ditaurine (BR-DT, a model compound of conjugated bilirubin) or biliverdin (BV) inhibited the oxidation of PC significantly and in a concentration-dependent way. In contrast, neither taurine, ascorbic acid nor reduced glutathione inhibited significantly under these conditions. Both bile pigments were consumed during their protective action. Vitamin E incorporated into the liposomal membranes suppressed the oxidation initially almost completely, thereby producing an induction period. In the combined presence of vitamin E and either of the two bile pigments at 10 microM each, this induction period was increased by at least 200%. In contrast, when 10 microM vitamin E was combined with an equimolar concentration of reduced glutathione, the induction period increased by only about 30%. BR-DT and BV both spared the consumption of vitamin E during the oxidation of PC liposomes. These results demonstrate that conjugated bilirubin and BV located in the aqueous phase can directly scavenge lipid radicals to some extent. Furthermore, both bile pigments can act synergistically with membrane-bound vitamin E to prevent lipid peroxidation initiated in the lipid phase, most likely through regeneration of the vitamin from its chromanoxyl radical.     

Antioxidant activity of 5-alkoxymethyl-6-chromanols

The 5-alkoxymethyl-2,2,7,8-tetramethyl-6-chromanols (II) are excellent antioxidants against autoxidising safflower oil (ASO), although not as good as 2,2,5,7,8-pentamethyl-6-chromanol (I), the model compound of alpha-tocopherol. The aim of this work was to determine whether the rate of reaction of (II) with the radicals diphenylpicrylhydrazyl (DPP*) and galvinoxyl (ArO*) was directly proportional to their antioxidant activity against ASO. Compounds (II) reacted faster with DPP* than with ArO* but, in each case, slower than compound (I). The rates of reaction of I and II with both radicals followed the order I > II (R = H) > II (R = CH3) > II (R = other alkyls) and were directly proportional to their antioxidant activity against ASO.     

Hepatic uptake and antihepatotoxic properties of vitamin E and liposomes in the mouse

Intravenous administration of soybean phosphatidylcholine liposomes containing different amounts of tocopherol acetate leads to a dose and time dependent increase of mouse liver tocopherol content, which was not observed when the preparation was given orally. When benzo[a]pyrene pretreated mice intoxicated with 400 mg/kg AAP were pretreated 2 h before with 1 g/kg phosphatidylcholine liposomes containing 4 mg/kg vitamin E acetate, these animals were protected against liver damage. Vitamin E alone or liposomes lacking vitamin E showed no protection. In an inflammatory liver disease model, i.e. fulminant hepatitis induced by intraperitoneal administration of 700 mg/kg galactosamine and 1 microgram/kg lipopolysaccharide phosphatidylcholine liposomes protected at a dose of 1 g/kg i.v. In this case, however, the protection was not due to the presence of vitamin E. These findings demonstrate the usefulness of phosphatidylcholine for liver protection and show that the protective spectrum is improved when they contain vitamin E. The data suggest that phosphatidylcholine is an excellent carrier for delivery of vitamin E to the liver.