The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

Penetration of the insect defensin A into phospholipid monolayers and formation of defensin A-lipid complexes.

Defensin A is an inducible cationic protein secreted in the hemolymph of fleshfly Phormia terranovae larvae in response to bacterial or septic injuries. Defensin A is known to permeabilize the bacteria cell membranes by forming voltage-dependent channels. The penetration of this small protein into lipid monolayers was studied as a function of the polar head and acyl chain length of phospholipids. The extent of penetration by defensin A is higher in monolayers made of anionic phospholipids than in monolayers made of zwitterionic phospholipids (phosphatidylcholines), because of electrostatic interactions. From the analysis of the compression isotherm parameters of mixed defensin A/phospholipid monolayers, it appears that defensin A interacts with phospholipid by forming 1:4 complexes. These complexes are not miscible in the lipid phase and induce microheterogeneity in the lipid membrane. These clusters might be related to the ion-channel structures responsible for the biological activity of defensin A.     

Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear.

In this study, we have used cholesterol oxidase as a probe to study cholesterol/phospholipid interactions in mixed monolayers at the air/water interface. Mixed monolayers, containing a single phospholipid class and cholesterol at differing cholesterol/phospholipid molar ratios, were exposed to cholesterol oxidase at a lateral surface pressure of 20 mN/m (at 22 degrees C). At equimolar ratios of cholesterol to phospholipid, the average rate of cholesterol oxidation was fastest in unsaturated phosphatidylcholine mixed monolayers (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylcholine), intermediate in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and slowest in sphingomyelin monolayers (egg yolk or bovine brain sphingomyelin). The average oxidation rate in mixed monolayers was not exclusively a function of monolayer packing density, since egg yolk and bovine brain sphingomyelin mixed monolayers occupied similar mean molecular areas even though the measured average oxidation rate was different with these two phospholipids. This suggests that the phospholipid acyl chain composition influenced the oxidation rate. The importance of the phospholipid acyl chain length on influencing the average oxidation rate was further examined in defined phosphatidylcholine mixed monolayers. The average oxidation rate decreased linearly with increasing acyl chain lengths (from di-8:0 to di-18:0). When the average oxidation rate was examined as a function of the cholesterol to phospholipid (C/PL) molar ratio in the monolayer, the otherwise linear function displayed a clear break at a 1:1 stoichiometry with phosphatidylcholine mixed monolayers, and at a 2:1 C/PL stoichiometry with sphingomyelin mixed monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol displacement from membrane phospholipids by hexadecanol

Adding cholesterol to monolayers of certain phospholipids drives the separation of liquid-ordered from liquid-disordered domains. The ordered phases appear to contain stoichiometric complexes of cholesterol and phospholipid. Furthermore, it has been suggested that the cholesterol in these complexes has a low chemical activity compared to that of the free sterol; i.e., that in excess of the phospholipid binding capacity. We have now tested the hypothesis that the membrane intercalator 1-hexadecanol (HD) similarly associates with phospholipids and thereby displaces the complexed cholesterol. HD introduced into monolayers of pure dimyristoylphosphatidylcholine generated highly condensed (stable and solid) domains. In contrast, the phase behavior of mixed monolayers of the phospholipid, sterol, and alcohol suggested that HD could substitute for cholesterol mole for mole in promoting liquid-ordered domains. We also found that the transfer of cholesterol from mixed monolayers to aqueous cyclodextrin was greatly stimulated by the presence of HD, but only at levels sufficient to competitively displace the sterol from the phospholipid. This enhanced efflux was interpreted to reflect an increase in uncomplexed cholesterol. We conclude that HD forms complexes with dimyristoylphosphatidylcholine that are surprisingly similar to those of cholesterol. HD competitively displaces cholesterol from the phospholipid and thereby increases its chemical activity.     

Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on mixed monolayers of phospholipids and fusogenic lipids.

1. The behaviour of mixed monolayers of 14 different lipids with preparations of erythrocyte lipids, purified natural and synthetic phospholipids, cholesterol and galactosylceramide was investigated. 2. The mean areas occupied per molecule in mixed films containing lipids that are fusogenic for hen erythrocytes were compared with those for corresponding films containing lipids that are inactive as fusogens. 3. Fusogenic lipids were found to exhibit interactions, which were not shown by non-fusogenic lipids, in mixed monolayers with several species of phospholipid, particularly those containing a choline head group. 4. Heterogeneity in the hydrophobic chains of phosphatidylcholine, their degree of unsaturation and the presence of cholesterol had little effect on the interaction of phosphatidylcholine with fusogenic lipids. 5. Fusogenic lipids showed little specific interaction with natural or synthetic preparations of phosphatidylethanolamine. 6. The possible significance of these observations in relation to the action of fusogenic lipids on biological membranes is discussed in the light of the asymmetrical distribution of phospholipids in erythrocyte membranes.     

Ubiquinones have surface-active properties suited to transport electrons and protons across membranes

Surface-active properties of ubiquinones and ubiquinols have been investigated by monomolecular-film techniques. Stable monolayers are formed at an air/water interface by the fully oxidized and reduced forms of the coenzyme; collapse pressures and hence stability of the films tend to increase with decreasing length of the isoprenoid side chain and films of the reduced coenzymes are more stable than those of their oxidized counterparts. Ubiquinone with a side chain of two isoprenoid units does not form stable monolayers at the air/water interface. Mixed monolayers of ubiquinol-10 or ubiquinone-10 with 1,2-dimyristoyl phosphatidylcholine, soya phosphatidylcholine and diphosphatidylglycerol do not exhibit ideal mixing characteristics. At surface pressures less than the collapse pressure of pure ubiquinone-10 monolayers (approx. 12mN.m(-1)) the isoprenoid chain is located substantially within the region occupied by the fatty acyl residues of the phospholipids. With increasing surface pressure the ubiquinones and their fully reduced equivalents are progressively squeezed out from between the phospholipid molecules until, at a pressure of about 35mN.m(-1), the film has surface properties consistent with that of the pure phospholipid monolayer. This suggests that the ubiquinone(ol) forms a separate phase overlying the phospholipid monolayer. The implications of this energetically poised situation, where the quinone(ol) is just able to penetrate the phospholipid film, are considered in terms of the function of ubiquinone(ol) as electron and proton carriers of energy-transducing membranes.     

Comparison of different vehicles to study the effect of tocopherols on gene expression in intestinal cells

Recent studies have focused on the ability of tocopherols to regulate gene expression. For such experiments, the methodology used to deliver molecules to the cells is crucial and could lead to different results depending on the vehicle used. The objective of the present study was to compare commonly used tocopherol vehicles (ethanol, BSA and mixed micelles) in terms of toxicity, stabilization of tocopherols, uptake efficiency of tocopherols by cells and effect on gene expression. Lactate dehydrogenase measurements did not reveal cytotoxicity of any of the tested vehicles. Tocopherol recovery measurements showed that approximately 80% of the tocopherol was lost in ethanolic solutions, while only approximately 30% and 10% were lost in BSA and mixed micelles, respectively. After 24 h incubation, Caco-2 cell monolayers treated with mixed micelles exhibited the highest alpha-tocopherol intracellular concentrations (5-times those measured with the two other vehicles). Similar results were obtained with gamma-tocopherol. Vehicles, except mixed micelles that activate the FXR/bile acids signalling pathway, did not affect expression of nuclear receptors involved in lipid metabolism or their target genes. This study establishes mixed micelles as the best vehicle to deliver tocopherols to intestinal cell monolayers in culture.     

A spin probe study of the influence of cholesterol on motion and orientation of phospholipids in oriented multibilayers and vesicles

Spin probes have been used to study at the molecular level the influence of cholesterol on bilayers of egg lecithin and dipalmitoyl lecithin. Distinct differences between the two lecithin systems were revealed. Increasing amounts of cholesterol result in extension of the fatty acid chains and decreased amplitude of motion of the long axes of the fatty acids in egg lecithin. In dipalmitoyl lecithin cholesterol causes an increase in the mobility and amplitude of motion of the fatty acid side chains, presumably due to alteration of the molecular interactions between phospholipids by relaxing the close packing of these molecules. These data provide an explanation for the condensing and fluidizing effects of cholesterol in water-containing phases and monolayers of egg lecithin and dipalmitoyl lecithin, respectively, and for the permeability behavior of egg lecithin and dipalmitoyl lecithin liposomes in the presence and absence of cholesterol. Differences are revealed between the spin bilayer environments in hydrated phospholipid films and vesicles.     

The interaction of alpha-tocopherol with phospholipid liposomes: the absence of transbilayer mobility

The interaction of alpha-tocopherol with liposomes obtained from saturated and unsaturated phospholipids and the rate of its flip-flop were studied using fluorescent technique. It was found that the amount of alpha-tocopherol introduced into outer and inner monolayers remained unchanged for many hours. No migration from the outer to the inner monolayers and vice versa was observed. The effect did not depend on the fatty acid phospholipid composition. The results obtained are considered in view of the optimal conditions of membrane tissue saturation with liposome-incorporated tocopherol.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Movement of cholesterol between vesicles prepared with different phospholipids or sizes

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.     

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.     

Lipid asymmetry and alpha-tocopherol distribution in outer and inner monolayers of bilayer lipid membranes

Using chemical modification of aminophospholipids (phosphatidylethanolamine, PEA; phosphatidylserin, PS), the location of these phospholipids and their fatty acids has been studied in the inner and outer monolayers of unilayer liposomes. It has been shown that both PEA and PS were located predominantly in the inner monolayer and contained most of the polyenoic fatty acid residues. alpha-tocopherol incorporated into these liposomes was also located predominantly in the inner monolayers. In the liposomes, prepared from a single phospholipid (dioleoylphosphatidylcholine) no asymmetrical distribution of incorporated alpha-tocopherol was observed.     

The condensing effect of cholesterol in lipid bilayers

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.     

Electron spin resonance study of the interaction of alpha-tocopherol with phospholipid model membranes.

The effect of up to 20 mol% incorporation of alpha-tocopherol on acyl chain order and dynamics in liquid crystalline phosphatidylcholine (PC) membranes was studied as a function of acyl chain unsaturation by electron spin resonance (ESR) of 5-, 7-, 12- and 16-doxyl spin labelled stearic acids intercalated into the membrane. Order parameters S in the upper portion of the chain (positions 5 and 7) and correlation times tau C in the lower portion (positions 12 and 16) determined from the ESR spectra indicate that in general alpha-tocopherol restricts acyl chain motion within the membrane. The magnitude of the increases in order appears to be dependent upon phospholipid molecular area, being the greatest (up to 15%) in saturated dimyristoylphosphatidylcholine (14:0-14:0 PC) which possesses a relatively small area per molecule as opposed to much smaller increases (less than 3%) in unsaturated PC membranes of larger molecular area. This behavior is interpreted as incompatible with the hypothesis of Lucy and coworkers (A.T. Diplock and J.A. Lucy (1973) FEBS Lett. 29, 205-210), who proposed that membranes are structurally stabilized by interactions between the phytyl side chain of alpha-tocopherol and the polyunsaturated chains of phospholipids.     

The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure.

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Mixtures of analogues and phospholipid were also studied in monolayers. In all the bilayer systems studied cholesterol caused the greatest 'rigidifying' effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.     

Transbilayer distribution of alpha-tocopherol and lipid asymmetry in nerve tissue membranes

The alpha-tocopherol content and fatty acid composition of lipids in various types of nervous tissue membranes were studied. The transbilayer distribution of alpha-tocopherol and polyunsaturated fatty acids in liposomes and plasma membranes of synaptosomes was examined. It was shown that both phosphatidylethanolamine and phosphatidylserine are localized predominantly in the inner monolayer and they contain the bulk of polyenoic fatty acid residues. alpha-Tocopherol incorporated into liposomes from synaptosome plasma membrane lipids and present in synaptosome plasma membranes is also predominantly localized in the inner monolayers. No asymmetrical distribution of incorporated alpha-tocopherol was observed in liposomes prepared from a single phospholipid, e.g., dioleoylphosphatidylcholine.     

N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel-->liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.     

Influence of calcium, cholesterol, and unsaturation on lecithin monolayers

Surface pressures and potentials of mixed monolayers of dicetyl phosphate-cholesterol, dipalmitoyl lecithin-cholesterol, egg lecithin-cholesterol, and phosphatidic acid-cholesterol were measured. The surface potential is shown to be a more reliable parameter for the study of interactions in monolayers than the surface pressure. Monolayers of dicetyl phosphate-cholesterol follow the additivity rule for area/molecule whereas lecithin-cholesterol monolayers deviate from it. The reverse is true for the additivity rule with regard to surface potential/molecule. Thus, the surface potential indicates that there is no interaction (or complex formation) between lecithin and cholesterol, but that there is ion-dipole interaction between dicetyl phosphate and cholesterol, as well as between phosphatidic acid and cholesterol. The apparent condensation of mixed monolayers of lecithin when cholesterol is added is explained by a consideration of molecular cavities or vacancies caused by thermal motion of the fatty acyl chains, the size of these cavities being influenced by the length and degree of saturation (especially the proportion of monounsaturation) of the fatty acyl chains and the extent of compression of the monolayer. The cholesterol molecules occupy these cavities and therefore cause no proportional increase in area/molecule in the mixed monolayers. Monolayers are liquefied by the presence of cholesterol as well as of unsaturated fatty acyl chains; in contrast, Ca(++)tends to solidify lecithin monolayers. The available evidence suggests that cholesterol can both impart fluidity to the monolayer and occupy the molecular cavities caused by the fatty acyl chains.