Does hydrogen bonding contribute to lipoperoxidation-dependent membrane fluidity variation? An EPR-spin labeling study.

This study is aimed at making clear the relationship between oxidative stress of the phospholipid bilayer and membrane fluidity. Di-(hydroperoxylinoleoyl)-phosphatidylcholine (diHpLPC) was used as a highly hydroperoxidized and unsaturated phospholipid species in order to investigate the issue. Hydrophylic Interaction Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry (HILIC-ESI-MS) and NMR spectroscopy were employed to define the structure of the peroxidized phospholipid as 1-(9-hydroperoxy-10c,12t)octadecadienoyl-2-(9t,11c-13-hydroperoxy)octadecadienoyl-sn-glycero-3-phosphorylcholine. This phospholipid's ability to form vesicular structures was confirmed by Sepharose 4B gel filtration and Dynamic Light Scattering (DLS) of its aqueous suspensions. Fatty acid misalignment and fluidity gradient were studied in the bilayer of both supported planar bilayers (SPB) and multilamellar vesicles (MLV) made of different DLPC/diHpLPC mixtures by means of spin labelling-EPR spectroscopy of either n-DSPC or 3-doxylcholestane spin labels embedded in the membranes. It was found that diHpLPC increases both fatty acid misalignment and rigidification with increasing molar ratio in spite of increasing unsaturation of the fatty acid core. Basing on our observations, the observed ability of pure diHpLPC to form rigid and disordered SPB and MLV bilayers is proposed to be dependent on the cross bridging of oxidized linoleoyl chains by mutual hydrogen bonding of hydroperoxyl groups. However, the contribution to the observed overall rigidification of the model membranes by trans double bonds in the peroxidized chains should not be neglected, as a second membrane fluidity effector also arising from lipid peroxidation.

Biophysics of lipid bilayers containing oxidatively modified phospholipids: insights from fluorescence and EPR experiments and from MD simulations.

This review focuses on the influence of oxidized phosphatidylcholines (oxPCs) on the biophysical properties of model membranes and is limited to fluorescence, EPR, and MD studies. OxPCs are divided into two classes: A) hydroxy- or hydroperoxy-dieonyl phospatidylcholines, B) phospatidylcholines with oxidized and truncated chains with either aldehyde or carboxylic group. It was shown that the presence of the investigated oxPCs in phospholipid model membranes may have the following consequences: 1) decrease of the lipid order, 2) lowering of phase transition temperatures, 3) lateral expansion and thinning of the bilayer, 4) alterations of bilayer hydration profiles, 5) increased lipid mobility, 6) augmented flip-flop, 7) influence on the lateral phase organisation, and 8) promotion of water defects and, under extreme conditions (i.e. high concentrations of class B oxPCs), disintegration of the bilayer. The effects of class A oxPCs appear to be more moderate than those observed or predicted for class B. Many of the abovementioned findings are related to the ability of the oxidized chains of certain oxPCs to reorient toward the water phase. Some of the effects appear to be moderated by the presence of cholesterol. Although those biophysical alternations are found at oxPC concentrations higher than the total oxPC concentrations found under physiological conditions, certain organelles may reach such elevated oxPC concentrations locally. It is a challenge for the future to correlate the biophysics of oxidized phospholipids to metabolic studies in order to define the significance of the findings presented herein for pathophysiology. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study.

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.

Comparative 5-doxylstearoyllecithin and 3-doxylcholestane EPR spin labeling study of phospholipid bilayer perturbation by different oxidized lecithin species.

A 3-doxylcholestane spin label was employed in addition to 5-doxylstearoyl lecithin for a more detailed study of the different effects exerted by variously oxidized lecithins on fatty acid alignment in phospholipid planar bilayers. Either spin label was enclosed in oriented PLPC planar samples also containing in turn a variety of conjugated-dienes lecithins and cleaved chain lecithins, in order to monitor EPR spectral angular dependence loss. Data obtained by use of arachidonoyl-hydroxystearoyl-PC and palmitoyl-hydroxylinoleoyl-PC confirm that the 5-DSPC nitroxide ring almost completely retains its orientation in CD-PCs-containing planar membranes, in contrast with angular dependence loss observed in the presence of the CC-PC molecular species palmitoyl-oxononanoyl-PC and palmitoyl-oxovaleroyl-PC, already seen with cleaved-chain palmitoyl-glutaroyl-PC and palmitoyl-azelaoyl-PC. However, the 3-DC nitroxide ring also loses its orientation with CD-PCs, in addition to being disoriented by cleaved chain-lecithins, similarly to 5DSPC. Joint information from the two spin labels will help to clarify whether OXPC-related disordering involved the whole bilayer structure or only the hydrophobic core. In addition, the propensity of different OXPCs to form bilayer vesicles in water suspension was also determined by Sepharose 4B gel-chromatography. The results suggest that CD-PCs might yield SPB bilayer structures with a disordered hydrophobic core, while pure CC-PC more probably forms non-bilayer disordered structures, possibly micelles or mixed micelle/bilayers.

Spin labeling EPR studies of the properties of oxidized phospholipid-containing lipid vesicles.

This study aims at characterizing the structure and some properties of phospholipid multi-lamellar vesicles (MLVs) containing the oxidized species gamma-palmitoyl-beta-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin (HPPLPC), gamma-palmitoyl-beta-(9-hydroxy-10,12-octadecanedienoyl)-lecithin (HOPLPC), gamma-palmitoyl-beta-glutaroyl-lecithin (GlPPC) and gamma-palmitoyl-beta-azelaoyl-lecithin (AzPPC). Sepharose 4B gel-chromatography was used to ensure and check that only MLVs are used in EPR measurements. Gel-solid to gel-liquid transition temperature (Tm), lateral phase separation, fluidity gradient and polarity profile were studied by use of EPR spectroscopy of enclosed n-doxylstearoyl lecithin spin labels. Contrarily to conjugate dienes and normal phospholipids, pure carboxyacyl species yielded aqueous suspensions showing gel-chromatography elution profile resembling that of lysolecithin micelles. Conjugate dienes/DPPC MLVs showed lateral phase separation at room temperature and Tm value lower than pure DPPC MLVs. Pure conjugate dienes MLVs resembled more PLPC MLVs and displayed free miscibility with PLPC in mixed MLVs. Pure HPPLPC MLV bilayer appeared to be slightly more rigid, while that of HOPLPC and the polarity profile of MLVs made of the pure conjugate dienes species were similar to those of normal PLPC. It is concluded that carboxyacyl lecithins in MLVs tend to disrupt vesicle structure, while conjugated dienes lecithins are more able to affect some physical properties of the bilayer, and that DPPC in MLVs enhances these effects while PLPC shows a better compatibility with the lipoperoxides.

Different oxidized phospholipid molecules unequally affect bilayer packing.

The aim of this study was to gain more detailed knowledge about the effect of the presence of defined oxidized phospholipid molecules in phospholipid bilayers. After chromatographic and mass spectrometry analysis, the previously used product of the Fenton reaction with unsaturated lecithins proved to consist of a plethora of oxidatively modified lecithins, useless either for the detailed study of the effects brought about in the bilayer or as the source of defined oxidized phospholipid molecules. The latter, particularly 2-(omega-carboxyacyl)- and 2-(n-hydroperoxyacyl)-lecithins, can be more conveniently prepared by chemical or enzymatic synthesis rather than by chemical or physical oxidation. The effect of those molecules and of commercially available 12-hydroxy-stearic and dodecanedioic acid was studied in planar supported phospholipid bilayers (SPBs) by use of EPR spectrometry. The SPBs also contained 2-(5-doxylstearoyl)-lecithin as the spin probe, and the EPR spectral anisotropy loss, indicative of bilayer disordering, was measured as a function of the molar percentage of oxidized lipid. Most oxidized lipid molecules examined in this study were able to induce bilayer disordering, while hydroperoxyl group-bearing acyl chains appeared to be much less effective. It is concluded that the effects of different oxidized phospholipids on phospholipid bilayer structure cannot be generalized, as happens with batch-oxidized phospholipids, and that the use of defined oxidized phospholipid molecular species for membrane oxidative stress guarantees a more reliable and detailed response.

Oxidized phospholipids induce phase separation in lipid vesicles.

The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 degrees C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids' heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 degrees C or less), and almost disappeared with increasing temperature (36 degrees C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 degrees C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.

The phospholipidomic signatures of human blood microparticles, platelets and platelet-derived microparticles: a comparative HILIC-ESI-MS investigation.

The phospholipidomic signatures of human blood microparticles and platelets, evaluated by hydrophilic interaction liquid chromatography coupled to electrospray ionization--mass spectrometry, were compared. The phospholipidome of platelet-derived microparticles, obtained by platelets stimulation with a mixture of Ca(II), thrombin and collagen, was also considered for the comparison. Platelets, blood microparticles and platelet-derived microparticles displayed qualitatively similar phospholipidomes, all based on eight major phospholipid classes, namely: phosphatidylcholines, diacyl- and plasme(a)nyl-phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, sphingomyelins and lyso forms of phosphatidylcholines and phosphatidylethanolamines. However, while the phospholipidomes of platelets and platelet-derived microparticles were found to be generally similar also from a quantitative point of view, a higher relative incidence of species bearing polyunsaturated side chains, especially in phospholipid classes sharing the choline head (i.e. phosphatidylcholines and lyso-phosphatidylcholines), was observed in the case of blood microparticles. As a further peculiar feature, never reported before, the relative abundance of lyso-phosphatidylcholines among the eight identified phospholipid classes was found to be significantly higher in the lipid extracts of blood microparticles.

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo.

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.

Respiration state IV-generated ROS destroy the mitochondrial bilayer packing order in vitro. An EPR study.

The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, beta-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.

EPR studies of phospholipid bilayers after lipoperoxidation. 1. Inner molecular order and fluidity gradient.

The molecular order of fatty acyl chains in oriented lipid bilayers on solid support (SPB), made of either natural or synthetic phospholipids oxidized by Fenton reagent and probed with spin labeled lecithin (5-DSPC) was studied by means of EPR spectrometry. Phospholipids (ASPC, EYPC, mitochondrial extract) were oxidized as either aqueous buffer/methanol dispersions or reverse-phase evaporation vesicles (REV) suspensions. Oxidation was preliminarily revealed both by assaying MDA and by detecting conjugated dienes. Oxidized phospholipid species was quantified by preparative TLC. The degree of order in oriented lipid bilayers of samples containing oxidized phospholipids was estimated by the loss of EPR spectral anisotropy, and an empirical index of the related bilayer disorder was calculated from the second derivative spectra. Bilayers made of each non-oxidized phospholipid species from either ethanol solutions or REV suspensions showed the highest anisotropy, while the increasing presence of oxidized lipids in the samples resulted in progressive loss of EPR spectral anisotropy. In contrast, vesicles containing 40% of the oxidized species maintained an unaltered fluidity gradient, while REV formation was hindered by oxidized phospholipid percentages higher than 45% for ASPC and EYPC, and 35% for Mitochondrial lipids (MtL). It is concluded that the early stages of lipoperoxidation bring about disordering of the phospholipid bilayer interior rather than fluidity alterations, and that prolonged oxidation may result in loss of structural and chemical properties of the bilayer until the structure no longer holds.

Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC.

The properties of two oxidatively modified phospholipids viz. 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), were investigated using a Langmuir balance, recording force-area (pi-A) isotherms and surface potential psi. In mixed monolayers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) a progressive disappearance of the liquid expanded-liquid condensed transition and film expansion was observed with increasing content of the oxidized phospholipids. The above is in agreement with fluorescence microscopy of the monolayers, which revealed an increase in the liquid expanded region of DPPC monolayers. At a critical pressure pi(s) approximately 42 mN/m both Poxo- and PazePC induced a deflection in the pi-A isotherms, which could be rationalized in terms of reorientation of the oxidatively modified acyl chains into aqueous phase (adaptation of the so-called extended conformation), followed upon further film compression by solubilization of the oxidized phospholipids into the aqueous phase. Surface potential displayed a discontinuity at the same value of area/molecule, corresponding to the loss of the oxidized phospholipids from the monolayers. Our data support the view that lipid oxidation modifies both the small-scale structural dynamics of biological membranes as well as their more macroscopic lateral organization. Accordingly, oxidatively modified lipids can be expected to influence the organization and functions of membrane associated proteins.