The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Vitamin E and its function in membranes

Vitamin E is a fat-soluble vitamin. It is comprised of a family of hydrocarbon compounds characterised by a chromanol ring with a phytol side chain referred to as tocopherols and tocotrienols. Tocopherols possess a saturated phytol side chain whereas the side chain of tocotrienols have three unsaturated residues. Isomers of these compounds are distinguished by the number and arrangement of methyl substituents attached to the chromanol ring. The predominant isomer found in the body is alpha-tocopherol, which has three methyl groups in addition to the hydroxyl group attached to the benzene ring. The diet of animals is comprised of different proportions of tocopherol isomers and specific alpha-tocopherol-binding proteins are responsible for retention of this isomer in the cells and tissues of the body. Because of the lipophilic properties of the vitamin it partitions into lipid storage organelles and cell membranes. It is, therefore, widely distributed in throughout the body. Subcellular distribution of alpha-tocopherol is not uniform with lysosomes being particularly enriched in the vitamin compared to other subcellular membranes. Vitamin E is believed to be involved in a variety of physiological and biochemical functions. The molecular mechanism of these functions is believed to be mediated by either the antioxidant action of the vitamin or by its action as a membrane stabiliser. alpha-Tocopherol is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions. The unpaired electron of the tocopheroxyl radical thus formed tends to be delocalised rendering the radical more stable. The radical form may be converted back to alpha-tocopherol in redox cycle reactions involving coenzyme Q. The regeneration of alpha-tocopherol from its tocopheroxyloxyl radical greatly enhances the turnover efficiency of alpha-tocopherol in its role as a lipid antioxidant. Vitamin E forms complexes with the lysophospholipids and free fatty acids liberated by the action of membrane lipid hydrolysis. Both these products form 1:1 stoichiometric complexes with vitamin E and as a consequence the overall balance of hydrophobic:hydrophillic affinity within the membrane is restored. In this way, vitamin E is thought to negate the detergent-like properties of the hydrolytic products that would otherwise disrupt membrane stability. The location and arrangement of vitamin E in biological membranes is presently unknown. There is, however, a considerable body of information available from studies of model membrane systems consisting of phospholipids dispersed in aqueous systems. From such studies using a variety of biophysical methods, it has been shown that alpha-tocopherol intercalates into phospholipid bilayers with the long axis of the molecule oriented parallel to the lipid hydrocarbon chains. The molecule is able to rotate about its long axis and diffuse laterally within fluid lipid bilayers. The vitamin does not distribute randomly throughout phospholipid bilayers but forms complexes of defined stoichiometry which coexist with bilayers of pure phospholipid. alpha-Tocopherol preferentially forms complexes with phosphatidylethanolamines rather than phosphatidylcholines, and such complexes more readily form nonlamellar structures. The fact that alpha-tocopherol does not distribute randomly throughout bilayers of phospholipid and tends to form nonbilayer complexes with phosphatidylethanolamines would be expected to reduce the efficiency of the vitamin in its action as a lipid antioxidant and to destabilise rather than stabilise membranes. The apparent disparity between putative functions of vitamin E in biological membranes and the behaviour in model membranes will need to be reconciled.     

Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes

The effect of lipid peroxidation on membrane fluidity was examined in sonicated soybean phospholipid vesicles. Following iron/ascorbate dependent peroxidation, the vesicles were labeled with a series of doxyl stearate spin probes which differed in the site of attachment of the nitroxide free radical to the fatty acid. Comparison of motional and partitioning parameters derived from electron spin resonance spectra of the probes indicated that the membranes were less fluid following peroxidation. However, the magnitude of the fluidity decrease was markedly dependent on the intramembrane location, as well as on the extent of lipid peroxidation. The effect of lipid peroxidation on fluidity was maximal in the membrane microenvironment sampled by 12-doxyl stearate, whereas other regions of the bilayer were less affected. These findings indicate that lipid peroxidation leads to an alteration of the transbilayer fluidity gradient.     

Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.     

Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction

Unilamellar liposomes are used as a simple two-compartment model to study the interaction of antioxidants. The vesicle membrane can be loaded with lipophilic compounds such as carotenoids or tocopherols, and the aqueous core space with hydrophilic substances like glutathione (GSH) or ascorbate, mimicking the interphase between an aqueous compartment of a cell and its surrounding membrane.

Unilamellar liposomes were used to investigate the interaction of GSH with the carotenoids lutein, β-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2'-azo-bis-[2,4-dimethylvaleronitrile] (AMVN). Malondialdehyde (MDA) formation was measured as an indicator of oxidation; additionally, the loss of GSH was followed. In liposomes without added antioxidant, MDA levels of 119 ± 6 nmol/mg phospholipid were detected after incubation with AMVN for 2 h at 37°C. Considerably lower levels of 57 ± 8 nmol MDA/mg phospholipid were found when the liposomal vesicles had been loaded with GSH. Upon incorporation of β-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. An optimal further protection was observed with 0.02 nmol β-carotene/mg phospholipid or 0.06 nmol lycopene/mg phospholipid. At higher levels both these carotenoids exhibited prooxidant effects. Lutein inhibited lipid peroxidation in a dose-dependent manner between 0.02 and 2.6 nmol/mg phospholipid. With increasing levels of lycopene and lutein the consumption of encapsulated GSH decreased moderately, and high levels of β-carotene led to a more pronounced loss of GSH.

The data demonstrate that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. Different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids.     

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.     

Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction

Unilamellar liposomes are used as a simple two-compartment model to study the interaction of antioxidants. The vesicle membrane can be loaded with lipophilic compounds such as carotenoids or tocopherols, and the aqueous core space with hydrophilic substances like glutathione (GSH) or ascorbate, mimicking the interphase between an aqueous compartment of a cell and its surrounding membrane.

Unilamellar liposomes were used to investigate the interaction of GSH with the carotenoids lutein, β-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2′-azo-bis-[2,4-dimethylvaleronitrile] (AMVN). Malondialdehyde (MDA) formation was measured as an indicator of oxidation; additionally, the loss of GSH was followed. In liposomes without added antioxidant, MDA levels of 119 ± 6 nmol/mg phospholipid were detected after incubation with AMVN for 2 h at 37°C. Considerably lower levels of 57 ± 8 nmol MDA/mg phospholipid were found when the liposomal vesicles had been loaded with GSH. Upon incorporation of β-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. An optimal further protection was observed with 0.02 nmol β-carotene/mg phospholipid or 0.06 nmol lycopene/mg phospholipid. At higher levels both these carotenoids exhibited prooxidant effects. Lutein inhibited lipid peroxidation in a dose-dependent manner between 0.02 and 2.6 nmol/mg phospholipid. With increasing levels of lycopene and lutein the consumption of encapsulated GSH decreased moderately, and high levels of β-carotene led to a more pronounced loss of GSH.

The data demonstrate that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. Different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids.     

The location and function of vitamin E in membranes (review)

Vitamin E is a fat-soluble vitamin that consists of a group of tocols and tocotrienols with hydrophobic character, but possessing a hydroxyl substituent that confers an amphipathic character on them. The isomers of biological importance are the tocopherols, of which alpha-tocopherol is the most potent vitamin. Vitamin E partitions into lipoproteins and cell membranes, where it represents a minor constituent of most membranes. It has a major function in its action as a lipid antioxidant to protect the polyunsaturated membrane lipids against free radical attack. Other functions are believed to be to act as membrane stabilizers by forming complexes with the products of membrane lipid hydrolysis, such as lysophospholipids and free fatty acids. The main experimental approach to explain the functions of vitamin E in membranes has been to study its effects on the structure and stability of model phospholipid membranes. This review describes the function of vitamin E in membranes and reviews the current state of knowledge of the effect of vitamin E on the structure and phase behaviour of phospholipid model membranes.     

Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes

The role of the hydroxyl groups of cholesterol and tocopherol in mediating their interaction with phospholipid bilayers has been a subject of considerable interest. We have examined this question by using derivatives of cholesterol and tocopherol in which the hydroxyl group is esterified to succinate. The hemisuccinate esters of cholesterol and alpha-tocopherol can be readily incorporated into phospholipid membranes and in fact can by themselves form closed membrane vesicles as demonstrated by the encapsulation of [3H]sucrose. The thermotropic behavior of mixtures containing each succinate ester and phospholipid was studied by differential scanning calorimetry. The effect of cholesteryl hemisuccinate on the thermotropic properties of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylethanolamine is very similar to that of cholesterol. This indicates that the 3 beta-OH is not required for the formation of a cholesterol-phospholipid complex. In mixtures of tocopherol acid succinate and phospholipids the peak transition temperature is progressively shifted to lower temperatures as the mole fraction of alpha-tocopherol succinate is increased, while the enthalpy of the transition is only slightly affected. At a tocopherol succinate/phospholipid molar ratio of 9/1 a phase transition is still detectable. A comparison between tocopherol succinate and tocopherol indicates that the substitution of the hydroxyl group reduces the interaction of tocopherol with phospholipids to a small but measurable extent. Thus, the hydroxyl group of tocopherol is more important than the hydroxyl group of cholesterol in influencing their interactions with phospholipids.     

Probe-assisted flow cytometric analysis of erythrocyte membrane response to site-specific oxidant stress

BACKGROUND: Probe-assisted flow cytometry was used to monitor the response of membranes of living cells to oxidant stress in the presence and absence of antioxidants. Test conditions (fluorophore loading, oxidant concentration) were investigated and storage-related changes in erythrocyte response to oxidant stress explored. METHODS: Erythrocytes were incubated with a lipophilic fluorescent probe and exposed to site-specific oxidant challenge, induced by cumene hydroperoxide, in the presence and absence of urate, ascorbate, or alpha tocopherol in physiological amounts. Fluorescence of labeled and treated erythrocytes was measured for 120 min using a Coulter EPICS Elite ESP flow cytometer. RESULTS: Probe loading was dose and time dependent. Cumene hydroperoxide exhibited a potent and dose-dependent oxidant effect on erythrocyte membranes. Alpha tocopherol slowed, but did not prevent, membrane oxidation. Ascorbate appeared to have no effect on peroxidation initially, but then slowed and stopped propagation of membrane oxidation. The effect of urate was slight. CONCLUSIONS: This technique can provide insight into oxidative processes at the cellular level. Results indicated that lipophilic alpha tocopherol was the most effective antioxidant in slowing membrane peroxidation, but ascorbate appears to stop chain propagation. This effect may be owing to vitamin C/E interaction. Further study is needed.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

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.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

In vitro evaluation of the antioxidant activity and biomembrane interaction of the lazaroid U-74389G

The aim of this paper was to clarify whether the interaction of the lazaroid U-74389G with phospholipid membranes might be relevant as to its antioxidant activity. Thus we evaluated the "in vitro" antioxidant activity of U-74389G in two experimental models: 1) bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical; 2) peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidino-propane) hydrochloride, on mixed dipalmitoyl-phosphatidylcholine/linoleic acid unilamellar vesicles. Moreover, given that biophysical techniques may help in explaining the role of a drug in its interaction with the microenvironment of the model lipid membranes, we used a classical approach to investigate the U-74389G/model membrane interaction: the differential scanning calorimetry technique on dimyristoylphosphatidylcholine multilamellar and unilamellar vesicles and the Langmuir-Blodgett technique on dimyristoylphosphatidylcholine monolayers. The results evidenced the strong antioxidant activity of U-74389G (especially in a membranous system) and its capability to interact with and be transported across model membranes. Thus one can speculate that U-74389G can act as scavenger of chain-propagating lipid peroxyl radicals within the membranes and may be able to protect not only cell membranes, but also intracellular components against peroxidative attack. Furthermore, also if there is no certain proof that the effect on the lipid packing order may play a key role in its antioxidant activity, the fluidifying effect on phospholipid bilayers of U-74389G favourably complements its free radical scavenging characteristics.     

Alpha-tocopherol protection of erythrocytes from hemolysis induced by thermal injury

It is shown that skin burn is accompanied by activation of lipid peroxidation (accumulation of TBA-reactive substances and of fluorescent end-products) in the blood of experimental animals. The decrease in red blood cell membrane stability was demonstrated exerting as increase in the rate of spontaneous hemolysis, content of extraerythrocyte++ haemoglobin and increased sensitivity to exogenous oleic acid. It is estimated that alpha-tocopherol possesses protective stabilizing effect on red blood cell membrane. This stabilizing action is observed when alpha-tocopherol was injected both before the skin burn and immediately after it. It is concluded that two different mechanisms are responsible for stabilizing effect of tocopherol, namely: 1) antiradical, realized via inhibition of lipid peroxidation, and 2) non-antioxidant, caused by interaction of tocopherol with phospholipid hydrolysis products by phospholipases A2 (free fatty acids and lysophospholipids).     

The effect of α tocopherol,all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation of rod outer segments

The effect of tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation induced by ascorbate-Fe²⁺ of rod outer segment membranes isolated from bovine retina was examined. The inhibition of light emission (maximal induced chemiluminescence) by tocopherol, all-trans retinol and retinyl palmitate was concentration dependent. All trans retinol showed a substantial degree of inhibition against ascorbate-Fe²⁺ induced lipid peroxidation in rod outer segment membranes that was 10 times higher than the observed in the presence of either tocopherol or retinyl palmitate. Inhibition of lipid peroxidation of rod outer segment membranes by tocopherol and retinyl palmitate was almost linear for up to 0,5 mol vitamin/mg membrane protein, whereas all-trans retinol showed linearity up to 0,1 mol vitamin/mg membrane protein. Incubation of rod outer segments with increasing amounts of low molecular weight cytosolic proteins carrying 1-[¹⁴C] linoleic acid, [³H] retinyl palmitate or [³H] all-trans retinol during the lipid peroxidation process produced a net transfer of ligand from soluble protein to membranes. Linoleic acid was 4 times more effectively transferred to rod outer segment membranes than all-trans retinol or retinyl palmitate. Incubation of rod outer segments with delipidated low molecular weight cytosolic proteins produced inhibition of lipid peroxidation. The inhibitory effect was increased when the soluble retinal protein fraction containing a tocopherol was used. These data provide strong support for the role of all-trans retinol as the major retinal antioxidant and open the way for many fruitful studies on the interaction and precise roles of low molecular weight cytosolic retinal proteins involved in the binding of antioxidant hydrophobic compounds with rod outer segments.     

Interaction of aspirin (acetylsalicylic acid) with lipid membranes

We studied the interaction of Aspirin (acetylsalicylic acid) with lipid membranes using x-ray diffraction for bilayers containing up to 50 mol% of aspirin. From 2D x-ray intensity maps that cover large areas of reciprocal space we determined the position of the ASA molecules in the phospholipid bilayers and the molecular arrangement of the molecules in the plane of the membranes. We present direct experimental evidence that ASA molecules participate in saturated lipid bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and preferably reside in the head group region of the membrane. Up to 50 mol% ASA molecules can be dissolved in this type of bilayer before the lateral membrane organization is disturbed and the membranes are found to form an ordered, 2D crystal-like structure. Furthermore, ASA and cholesterol were found to co-exist in saturated lipid bilayers, with the ASA molecules residing in the head group region and the cholesterol molecules participating in the hydrophobic membrane core.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.