Species differences in membrane susceptibility to lipid peroxidation

The susceptibility of liver microsomes to lipid peroxidation was evaluated in seven species: rat, rabbit, trout, mouse, pig, cow, and horse. Lipid peroxidation was measured as thiobarbituric acid reactive substances formed in the presence of either FeCl₃-ADP/ascorbate or FeCl₂/H₂O₂ initiating systems. For rat, rabbit, and trout microsomes, the order of susceptibility to peroxidation was rat > rabbit >> trout. The lack of peroxidation in trout microsomes could be explained by high microsomal vitamin E levels. Membrane fatty acid levels differed between species. Docosahexaenoic acid predominated in the trout, arachidonic acid in the rat, and linoleic acid in the rabbit. The contribution of individual fatty acids to lipid peroxidation reflected the degree of unsaturation with docosahexaenoic > arachidonic >>> linoleic. For all species except trout, the predicted susceptibility to peroxidation, based on the response of individual fatty acids, agreed well with directly measured microsomal peroxidation. With the exception of the trout, vitamin E content ranged from 0.083–0.311 nmol/mg microsomal protein between species, and low levels did not influence susceptibility to peroxidation. Trout microsomes peroxidized only after vitamin E depletion by prolonged incubation. The data indicate that below a vitamin E threshold, species differences in membrane susceptibility to peroxidation can be reasonably predicted based only on content of individual peroxidizable fatty acids.     

Calcium potentiates the peroxidation of erythrocyte membrane lipids

A clonal cell line from rat osteosarcoma was found to possess parathyroid hormone and isoproterenol sensitive adenylate cyclase. This study examines the relationship between the two hormones and triphosphoguanine nucleotide with respect to enzyme activation. Concentration-dependence curves, analyzed by computer-aided curve fitting, revealed: (1) in the presence of 5 microM GTP there were two apparent affinities for parathyroid hormone (Km 9 and 89 nM) and isoproterenol (Km 72 and 340 nM; (2) and two affinities for guanosine-5' (beta, gamma-imido)triphosphate (Km 0.25 and 1.3 microM); (3) hormones and guanine nucleotides reciprocally shifted each other's concentration dependence curve to the high affinity sites; (4) parathyroid hormone and isoproterenol interacting with high affinity sites competed for the same adenylate cyclase; (5) parathyroid hormone and isoproterenol, acting on low affinity sites had additive effects and also stimulated adenylate cyclase in the absence of added guanine nucleotides. The findings are consistent with (i) competition of parathyroid hormone and isoproterenol for the activation of the high (hormone) affinity complex containing: receptors, nucleotide subunit, triphosphoguanine nucleotide, catalytic unit (ii) the apparent presence of receptor-nucleotide sub-unit GDP-catalytic unit complexes with low hormone affinity which are stimulated by parathyroid hormone and isoproterenol separately.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Alterations in erythrocyte membrane fluidity by phenylhydrazine-induced peroxidation of lipids

The addition of phenylhydrazine (.05 – 0.5 mM) to hemoglobin-free human erythrocyte membranes results in the peroxidation of endogenous phospholipids as measured by the thiobarbaturic acid reaction. The incorporation of 1,6-diphenyl-1,3-5-hexatriene into these membranes revealed a decrease in bulk lipid fluidity. Additionally, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate was decreased and red-shifted after phenylhydrazine treatment of the membranes. The results obtained are consistent with the view that changes in the physical state of plasma membranes subsequent to the peroxidation of membrane lipids may be a determinant of the mechanical properties of drug-treated as well as aging cells.     

Fatty acids of plant vacuolar membrane lipids

Fatty acid (FA) composition of vacuolar membrane lipids from storage tissues of umbelliferous plants, viz., parsnip (Pastinaca sativa L.), parsley (Petroselinium crispum L.), and carrot (Daucus carota L.) is studied by gas-liquid chromatography and the FA biosynthetic pathways are considered. Vacuolar membrane lipids are characterized by high (78% of the total FA pool) content of unsaturated FA among which linoleic acid is predominant. Its content in vacuolar lipids of parsnip, parsley and carrot is 53.5, 55.1, and 54.9%, respectively. Parsnip and parsley vacuolar lipids contain large amounts of hexadecadienoic C16:2ω6 acid (8.0 and 4.6%, respectively). The content of α-linolenic acid in vacuolar lipids of tested plants varies from 4.8 to 7.3%. Palmitic acid (18.0–20.7%) predominates among saturated FA. High content of linoleic and hexadecadienoic acid in parsnip and parsley vacuolar lipids is suggestive of a crucial role of the microsomal ω6 fatty-acid desaturase fad2 gene in resistance and acclimation of plants to low temperatures.     

Susceptibility of plasma lipids to peroxidation

Lipid peroxidation is an old and yet novel subject. It induces membrane disturbance and damage and its products are known to induce the generation of various cytokines and cell signaling. In the present work, the susceptibility and specificity of human plasma lipids to oxidation were studied, aiming specifically at elucidating the effects of oxidation milieu and oxidants. Cholesteryl esters (CEs) and phosphatidylcholines (PCs) were more readily oxidized in plasma than in organic solution under similar conditions. The susceptibilities of PC and free cholesterol (FC) relative to CE to free radical-mediated lipid peroxidation induced by peroxyl radicals and peroxynitrite were smaller in plasma than in organic solution. The higher rate of CE oxidation by free radicals than PC may be accounted for by the physical effects as well as higher content of polyunsaturated lipids in CE than PC. On the contrary, PC was more readily oxidized than CE by lipoxygenases. The lipid hydroperoxides were stable in organic solution but reduced to the corresponding hydroxides in plasma, the rate being much faster for PC hydroperoxides than for CE and FC hydroperoxides. It was confirmed that free radical-mediated oxidation gave both cis,trans and trans,trans, racemic, random hydroperoxides, while that by lipoxygenase gave only regio- and stereo-specific cis,trans-hydroperoxide.     

Initiation of peroxidation of liposome membrane lipids by activated polymorphonuclear leukocytes

The influence of active oxygen forms produced by zymosan-stimulated polymorphonuclear leukocytes (PMNL) on the initiation of liposome lipid peroxidation has been studied. It has been shown, by measuring the concentration of TBA-active products, that lipid peroxidation induced by PMNL stimulation occurs only in the presence of Fe-ADP. This fact demonstrates that OH'-radicals are responsible for the initiation of lipid peroxidation. Superoxide dismutase and catalase almost completely inhibited PMNL-stimulated peroxidation. The results obtained suggest that active oxygen forms that appear during PMNL stimulation can migrate at a considerable distance from the place of their origin, initiating peroxidation of cell membrane lipids and lipoproteins in the presence of Fe ions, which seems to underlie bacteriocidal and cytotoxic action of phagocytes.     

Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.     

The increase in viscosity and peroxidation of sarcoplasmic reticulum membrane lipids in isadrine myocarditis

The viscosity of membranes isolated from sarcoplasmic reticulum of rabbits with isadrine myocarditis was studied, using pyrene as a hydrophobic fluorescent probe. The increase in the viscosity of membranes from injured heart occurred at lower temperatures and was sharper than in the case of intact heart in both "free" and "bound" lipid domains. The increase in the lipid viscosity under myocarditis was associated with decreased Ca++, Mg++ -ATPase and cAMP-dependent protein kinase activities and with an elevated content of lipid peroxidation products.     

Inactivation of chlorophyllase by negatively charged plant membrane lipids

Chlorophyllide combines spontaneously not only with phosphatidylcholine (PC) liposomes but also with various other (plant) lipids dispersed in an aqueous medium. The lipid-associated chlorophyllide is highly fluorescent and the fluorescence yield is virtually independent of the nature of the lipid. Chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) activity assays that are based on the determination of this chlorophyllide fluorescence show that phosphatidylglycerol (PG), and also sulphoquinovosyldiacylglycerol (SQDG), associate with isolated chlorophyllase, thereby inactivating the enzyme in a co-operative way. The extent of this inactivation depends on the pH and ionic strength of the reaction medium and can be completely reversed by divalent cations (Mg2+). The inhibition of chlorophyllase effected by free PG liposomes can be counteracted by electrically neutral lipids at relatively high concentration (PC and also chloroplast lipids). Digalactosyldiacylglycerol (DGDG) is not effective in this respect. When PG has been incorporated in PC or DGDG liposomes, its ability to inhibit chlorophyllase activity is reduced. Whereas the remaining chlorophyllase-inactivating effect of PG, incorporated in PC, can still be reversed by Mg2+, this is not found when enzyme inactivation is caused by PG incorporated in DGDG. The results reported here are consistent with those obtained earlier concerning the stabilization of chlorophyllase by PG and PG/galactolipid mixtures (Lambers, J.W.J., Verkleij, A.J. and Terpstra, W. (1984) Biochim. Biophys. Acta 786, 1-8). They are discussed in terms of the regulation of chlorophyllase activity by lipids surrounding the enzyme and by divalent cations.     

Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane.

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.     

Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes

The increased generation of reactive oxygen species that occurs in the condition of obesity may be responsible for oxidative injury to erythrocyte membranes, which could lead to a decrease in tissue oxygenation. Therefore, we have looked into the effects of obesity on both indexes of oxidative damage and physical-chemical properties of erythrocyte membranes in 50 overweight or obese [25 < body mass index (BMI) < 33], normotensive, nondiabetic women and 50 age-matched lean healthy women (BMI < 25). In the obese group compared with the lean group, we found that a) the onset of free radical-induced erythrocyte hemolysis and the ratio between reduced and oxidized glutathione were reduced, whereas the rate of free radical-induced damage increased; b) the n-3 fatty acid and the phospholipid contents decreased; c) the ratio between cholesterol and phospholipids increased; and d) the membrane fluidity decreased. These findings suggest an impairment of erythrocyte membrane physical-chemical properties in overweight and obese people as a consequence of oxidative injury that might be part of a pathogenetic mechanism responsible for obesity-related pathologies such as atherosclerosis and hypertension.     

Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration

Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.     

Effects of phytic acid on the myoglobin-t-butylhydroperoxide-catalysed oxidation of uric acid and peroxidation of erythrocyte membrane lipids

Phytic acid stimulated the myoglobin-t-butylhydroperoxide (TBHP)-catalysed oxidation of uric acid, but inhibited the peroxidation of erythrocyte membrane lipids induced by the same system. Butylated hydroxy-toluene, a free radical chain reaction-terminating antioxidant, also suppressed the myoglobin-TBHP-induced lipid peroxidation. Moreover, phytic acid inhibited the hydroxyl radical-induced degradation of deoxyribose, but the extent of inhibition in this system was reduced by increasing the ferric ion concentration, suggesting that these effects of phytic acid on the myoglobin-TBHP-mediated oxidation are more likely attributable to its metal chelating properties rather than to a free radical scavenging action. The effectiveness of phytic acid, a naturally occurring antioxidant, in the inhibition of both iron- (as previously shown) and myoglobin-dependent lipid peroxidation suggests its possible therapeutic application as a non-toxic antioxidant for ameliorating the extent of oxy-radical-mediated myocardial ischemia/reperfusion damage.Abbreviations ASC Ascorbic acid - BHT Butylated Hydroxytoluene - DMSO Dimethyl Sulfoxide - TBHP t-Butylhydroperoxide - TBA Thiobarbituric Acid - TBARS Thiobarbituric Acid-reactive Substances     

All fatty acids are not equal: discrimination in plant membrane lipids

Plant membrane lipids are primarily composed of 16-carbon and 18-carbon fatty acids containing up to three double bonds. By contrast, the seed oils of many plant species contain fatty acids with significantly different structures. These unusual fatty acids sometimes accumulate to >90% of the total fatty acid content in the seed triacylglycerols, but are generally excluded from the membrane lipids of the plant, including those of the seed. The reasons for their exclusion and the mechanisms by which this is achieved are not completely understood. Here we discuss recent research that has given new insights into how plants prevent the accumulation of unusual fatty acids in membrane lipids, and how strict this censorship of membrane composition is. We also describe a transgenic experiment that resulted in an excessive buildup of unusual fatty acids in cellular membranes, and clearly illustrated that the control of membrane lipid composition is essential for normal plant growth and development.     

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

Lipid peroxidation contributes to age-related membrane rigidity

The aim of this work was to assess the relative contributions of lipid peroxidation and cholesterol content to the increase in membrane rigidity observed during senescence. Membrane fluidity was manipulated through exposure to peroxidized or cholesterol-loaded liposomes. Small unilamella liposomes were prepared and either peroxidized by Fe⁺⁺-ADP-ascorbic acid or loaded with cholesterol. After incorporation of the liposomes into rat liver microsomal membranes, membrane fluidity was quantitated by measuring changes in polarization. Membranes exhibited a greater sensitivity to peroxidation than cholesterol in that incorporation of peroxidized liposomes induced microsomal membrane rigidity substantially more than did cholesterol-loaded liposomes. Thus it is proposed, based on data from the present and earlier studies, that membrane fluidity can be modulated readily by lipid peroxidation of membrane phospholipids, irrespective of the influences of cholesterol. These results support the proposal that alterations of lipid structure are more potent and effective than compositional changes in cholesterol in inducing age-related increases in membrane rigidity.     

Adaptation of membrane lipids to alcohols.

The effects of alcohols of different chain lengths on the fatty acid composition of Escherichia coli K-12 have been examined. My results indicate that these cells radically change their fatty acid composition when grown in the presence of alcohols. These changes represent an adaptive membrane alteration compensating for the direct physicochemical interaction of alcohols with the membrane. Similar adaptive responses of membrane lipids are proposed as a possible biochemical basis for tolerance to alcohol and related drugs.