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.     

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Pseudopregnancy-dependent changes in rat ovarian LH/hCG receptors in relation to membrane lipid fluidity

The specific binding of [125I] hCG to ovarian membrane preparations as well as membrane fluidity have been investigated in immature rats during hormonally-induced pseudopregnancy. Membrane fluidity was monitored either by fluorescence polarization analysis of 1,6-diphenyl-1,3,5-hexatriene or by electron spin resonance of 16-, 12-, 5-doxyl stearic acid and CAT 16. A significant positive correlation was found between membrane lipid rigidity and the number of LH/hCG receptors. Luteinization of the ovary induced mobility of molecules in the hydrophobic membrane part at about the C16 carbon level. The changes in rigidity of membrane lipid were the apparent result of alterations in the cholesterol to phospholipids ratio. The results suggest that the increased rigidity of membrane lipid during pseudopregnancy may maximally expose ovarian LH/hCG receptors maintained in a cryptic form.     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

Azone类透皮剂作用机理及一种新型模型膜的研究

使用电子自旋共振波谱技术,采用5—doxyl stearic acid作为自旋标记物,新设计的镶嵌JPM和胆固醇的卵壳膜作为实验模型膜,进行Azone类透皮吸收促进剂的主要作用机理研究.实验证实上述膜是一种很有前途的模型膜.由于Azone透皮剂的作用,增大膜中类脂和自旋标记物的脂肪长链的运动速率,即增强类脂的流动性,使得类脂的序参数值减小,从而证实了前人有关角质化细胞间的类脂相是药物穿透角质层的主要通道的假设.为进一步探讨透皮剂对天然皮肤的作用,采用裸鼠皮肤角质层作为实验模型膜,得到与上述实验相符的结果.     

Organization of membrane lipids and proteins in human En(a-) erythrocytes that lack the major sialoglycoprotein, glycophorin A. A spin-label study.

Membrane fluidity was studied by electron-spin-resonance techniques in human En(a-) erythrocytes that lack the major membrane sialoglycoprotein, glycophorin A. By using stearic acid spin labels with a doxyl group in the C-12 or C-15 positions, we demonstrated that the hydrophobic core in these cells was more fluid than in normal cells. Surface-located regions in isolated En(a-) membranes, when probed with stearic acid labelled in the C-5 position, appeared more stable than in normal membranes. In isolated En(a-) membranes, protein motion was decreased when probed with a nitroxide derivative of maleimide. After incubation with anti-(glycophorin A) antibodies protein motion and membrane fluidity were increased in normal membranes. This effect was observed also after spectrin depletion, which by itself increased protein motion but decreased membrane fluidity in the hydrophobic core of the membrane. The results show that membrane proteins influence the fluidity of membrane lipids.     

The effect of Pycnogenol on the erythrocyte membrane fluidity

In the present study, the in vitro effect of polyphenol rich plant extract, flavonoid--Pycnogenol (Pyc), on erythrocyte membrane fluidity was studied. Membrane fluidity was determined using 1-[4-trimethyl-aminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 1,6-diphenyl-1,3,5-hexatriene (DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) fluorescence anisotropy. After Pyc action (50 microg/ml to 300 microg/ml), we observed decreases in the anisotropy values of TMA-DPH and DPH in a dose-dependent manner compared with the untreated erythrocyte membranes. Pyc significantly increased the membrane fluidity predominantly at the membrane surface. Further, we observed the protective effect of Pyc against lipid peroxidation, TBARP generation and oxidative hemolysis induced by H2O2. Pyc can reduce the lipid peroxidation and oxidative hemolysis either by quenching free radicals or by chelating metal ions, or by both. The exact mechanism(s) of the positive effect of Pyc is not known. We assume that Pyc efficacy to modify effectively some membrane dependent processes is related not only to the chemical action of Pyc but also to its ability to interact directly with cell membranes and/or penetrate the membrane thus inducing modification of the lipid bilayer and lipid-protein interactions.     

Fluidizing effects of centrophenoxine in vitro on brain and liver membranes from different age groups of mice

This study examined the effects of different concentrations of centrophenoxine on physical properties of synaptic plasma membranes and liver microsomes using electron spin resonance procedures. Membranes of different age groups of mice were labeled with the 5-doxyl stearic acid spin-label and membrane fluidity determined in the presence and absence of different concentrations of centrophenoxine. Centrophenoxine had a direct effect on membranes as shown by a significant increase in membrane fluidity. This effect was greatest in liver microsomes as compared to synaptic plasma membranes. Age differences were not observed in centrophenoxine-induced fluidization. Effects of centrophenoxine in vivo may be due in part to the drug acting directly on the physical properties of the membrane lipid environment.     

Altered cellular membrane fluidity levels and lipid peroxidation during experimental pancreas transplantation

Although the pathogenesis of ischemia reperfusion (IR) injury is based on complex mechanisms, free radicals play a central role. We evaluated membrane fluidity and lipid peroxidation during pancreas transplantation (PT) performed in 12 pigs (six donors and six recipients). Fluidity was measured by fluorescence spectroscopy, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations were used as an index of lipid oxidation. Pancreatic tissues were collected as follows: (A) donor, immediately before vascular clamping; (B) graft, following perfusion lavage with University of Wisconsin preservation fluid; (C) graft, after 16?h of cold ischemia; and (D) recipient, 30?min vascular postreperfusion. Fluidity and MDA and 4-HDA concentrations were similar in cases A, B, and C. However, there was significant membrane rigidity and increased lipid peroxidation after reperfusion (D). These findings suggest that reperfusion exaggerates oxidative damage and may account for the rigidity in the membranes of allografts during PT.     

Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.     

Ischemic Modification of Cerebrocortical Membranes: 5-Hydroxytryptamine Receptors, Fluidity, and Inducible In Vitro Lipid Peroxidation

The effect of ischemia on the properties of 5-hydroxytryptamine1A + B (5-HT1A+B) and 5-hydroxytryptamine1B (5-HT1B) binding sites, physical-state "fluidity" of the membrane, and its susceptibility to peroxidation in vitro was investigated in the cerebral cortex of gerbils. Ischemia was induced by bilateral carotid artery occlusion for 15 min alone or with release for 1 h. Ischemia both with and without reflow decreased the number of 5-HT1A + B and 5-HT1B binding sites, whereas ischemia and reflow altered the affinity for 5-HT1B binding sites. Resistance to the temperature-dependent increase in "fluidity" of the membrane was detected (by fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene as a probe) after ischemia and reflow but not in ischemia alone. Susceptibility of the membranes to Fe2+- and ascorbic acid-stimulated lipid peroxidation in vitro was decreased following ischemia and recirculation only. These findings strongly suggest that the composition and the function of the membrane are markedly disturbed during recirculation after ischemia.     

Lipid fluidity at different regions in LDL and HDL of beta-thalassemia/Hb E patients

Atherosclerosis-related vascular complications in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients may result from iron induced oxidation of lipoproteins. To identify the specific site of oxidative damage, changes in lipid fluidity at different regions in LDL and HDL particle were investigated using two fluorescence probes and two ESR spin probes. The magnitude of increased lipid fluidity in thalassemic lipoproteins was dependent on the location of the probes. In hydrophobic region, the rotational correlation times for 16-doxyl stearic acid and DPH anisotropy were markedly changed in LDL and HDL of the patients. In the surface region, there was only a slight change in the order parameter (S) for 5-doxyl stearic acid and TMA-DPH anisotropy. Lipid fluidity at the core of LDL and HDL showed good correlation with oxidative stress markers, the ratio of CL/CO, and the level of alpha-tocopherol, suggesting that hydrophobic region of thalassemic lipoprotein was a target site for oxidative damage.     

An EPR investigation of surfactant action on bacterial membranes

The effects of the surfactants, alcohol ethoxylate, amine ethoxylate, amine oxide and SDS on cell membranes were investigated using the lipid soluble spin label 5-doxyl stearic acid (5-DS). Electron paramagnetic resonance (EPR) spectroscopy revealed that the action of the surfactants was to significantly increase membrane fluidity of Proteus mirabilis, Staphylococcus aureus and Saccharomyces cerevisiae. The action of these surfactants as biocides was investigated and found to be dependent on the type of organism tested. There was, however, no direct correlation between enhanced membrane fluidity observed due to the action of the surfactants and biocidal activity. Data presented suggest that perturbing the fluidity of the cytoplasmic membrane is not immediately responsible for cell death.     

Biological activity of pentachlorophenol on the digestive gland cells of the freshwater mussel Unio tumidus

Many chlorinated phenols and their derivatives are used extensively as insecticides, fungicides and herbicides by industrial and agricultural users throughout the world. Among these substances, pentachlorophenol (PCP) is a broad-spectrum biocide, which is still used as a wood preservative. In this paper, the digestive gland cells were used to assess the effect of PCP in the range of concentrations 3.75-75 microM (0.01-0.2 ppm) on oxidative DNA damage, fluidity changes and peroxidation activity in the plasma membrane. The toxic property of PCP on DNA strand breakage was studied using the comet assay. The results showed that pentachlorophenol in the range of 37.5-75 microM contributed to these lesions. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric method using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS). It was shown that PC did not influence the surface of plasma membrane but contributed to the increase in the fluidity of the internal region of the lipid bilayer in the range of concentrations 18.75-75 microM (0.05-0.2 ppm). We also examined the effect of PCP on the lipid peroxidation. To imply its peroxidation properties the spectrophotometry method was used to measure the level of malondialdehyde (MDA), one of the endpoints of the peroxidation of polyunsaturated fatty acids. The obtained results showed that PCP in the used doses did not initiate the formation of lipid peroxides. Thus, our investigation indicates that PCP can behave as a prooxidant agent but its action depends on the used doses and parameters chosen for the research.     

Effect of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus

It has been previously reported that Nigella sativa oil (NSO) and thymoquinone (TQ), active constituent of N. sativa seeds oil, may prevent oxidative injury in various models. Therefore, we considered the possible effect of TQ and NSO on lipid peroxidation level following cerebral ischemia-reperfusion injury (IRI) in rat hippocampus. Male NMRI rats were divided into nine groups, namely, sham, control, ischemia and ischemia treated with NSO or TQ. TQ (2.5, 5 and 10 mg/kg), NSO (0.048, 0.192 and 0.384 mg/kg), phenytoin (50 mg/kg, as positive control) and saline (10 ml/kg, as negative control) were injected intraperitoneally immediately after reperfusion and the administration was continued every 24h for 72 h after induction of ischemia. The transient global cerebral ischemia was induced using four-vessel-occlusion method for 20 min. Lipid peroxidation level in hippocampus portion was measured as malondialdehyde (MDA) based on its reaction with thiobarbituric acid (TBA) following ischemic insult. The transient global cerebral ischemia induced a significant increase in TBA reactive substances (TBARS) level (p<0.001), in comparison with sham-operated animal. Pretreatment with TQ and NSO were resulted a significant decrease in MDA level as compared with ischemic group (66.9+/-1.5 vs. 297+/-2.5 nmol/g tissue for TQ, 10 mg/kg; p<0.001 and 153.5+/-1.3 nmol/g tissue for NSO, 0.384 mg/kg; p<0.001). Using a reversed-phase HPLC system, the amount of TQ in NSO was also quantified and was 0.58% w/w. These results suggest that TQ and NSO may have protective effects on lipid peroxidation process during IRI in rat hippocampus.     

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.     

Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli

The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.     

The distribution of lipid attached spin probes in bilayers: application to membrane protein topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.     

Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl-L-alpha phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.