Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

A thermodynamic study of the effects of cholesterol on the interaction between liposomes and ethanol

The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8-45 degrees C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication.     

Effect of cholesterol on the structure and dynamic properties of unsaturated phospholipid bilayers

Molecular dynamics (MD) computer simulations of five different hydrated unsaturated phosphatidylcholine lipid bilayers built up by 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules with 40 mol% cholesterol, and the same five pure phosphatidylcholine bilayers have been performed at 303 K. The simulation box of a lipid bilayer contained 96 phosphatidylcholines, 64 cholesterols, and 3840 water molecules (48 phosphatidylcholine molecules and 32 cholesterols per layer and 24 water molecules per phospholipid or cholesterol in each case). The lateral self-diffusion coefficients of the lipids in these systems and mass density profiles with respect to the bilayer normal have been analyzed. It has been found that the lateral diffusion coefficients of phosphatidylcholine molecules increase with increasing number of double bonds in one of the lipid chains, both in pure bilayers and in bilayers with cholesterol. It has been found as well that the lateral diffusion coefficient of phosphatidylcholine molecules of a lipid bilayer with 40 mol% cholesterol is smaller than that for the corresponding pure phosphatidylcholine bilayer.     

Effects of lindane on membrane fluidity: intramolecular excimerization of a pyrene derivative and polarization of diphenylhexatriene.

Fluorescence polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH) have been compared with the excimer/monomer fluorescence intensity ratio (I'/I) of 1,3-di(2-pyrenyl)propane, (2Py(3)2Py). This ratio permits evaluation of changes in fluidity of the outer regions of the bilayer, where 2Py(3)2Py preferentially distributes. On the other hand, fluorescence polarization of DPH reports the structural order of the bilayer core. In the fluid phase of DMPC bilayers, for lindane concentrations higher than 25 microM, the excimer/monomer fluorescence intensity ratio (I'/I) decreases, thus reflecting an order increase of the probe environment. However, in the same conditions, the fluorescence polarization of DPH is almost insensitive to any perturbation. Identical results have been obtained in other pure lipid bilayers, namely DPPC and DSPC. However, both probes detect disordering effects of lindane in the gel phase of these lipids. The pyrene probe, unlike DPH, is very sensitive to the pretransitions of DPPC and DSPC, removed in the presence of lindane. Both probes fail to detect any apparent effect of lindane in DMPC bilayers enriched with high cholesterol content (greater than 30 mol%). However, in DMPC bilayers with low cholesterol content (less than 30 mol%), for temperatures below the phase transition of DMPC, both probes detect fluidizing effects induced by lindane. Nevertheless, above the phase transition of DMPC, 2Py(3)2Py detects ordering effects of lindane, whereas DPH detects hardly any effect. These results in DMPC bilayers with low cholesterol content are qualitatively similar to those described for DMPC without cholesterol.     

Glycosylation induces shifts in the lateral distribution of cholesterol from ordered towards less ordered domains

Several studies have indicated the involvement of steryl glycosides in the cellular stress response. In this work, we have compared the effect of 1-O-cholesteryl-beta-d-glucoside, 1-O-cholesteryl-beta-d-galactoside and cholesterol on the properties of glycerophospholipid and sphingolipid bilayers. The studies were performed in order to gain insight into the change in membrane properties that would follow upon the glycosylation of cholesterol in cells subjected to stress. DPH anisotropy measurements indicated that the cholesteryl glycosides (10-40 mol%) increased the order of the hydrophobic region of a POPC bilayer almost as efficiently as cholesterol. In a PSM bilayer, the cholesteryl glycosides were however shown to be much less effective compared to cholesterol in ordering the hydrocarbon chain region at temperatures above the gel to liquid-crystalline phase transition. Fluorescence quenching analysis of multicomponent lipid bilayers demonstrated that the cholesteryl glycosides, in contrast to cholesterol, were unable to stabilize ordered domains rich in PSM against temperature-induced dissociation. When the sterols were incorporated into bilayers composed of both POPC and PSM, the cholesteryl glycosides showed a higher propensity, compared to cholesterol, to influence the endothermal component representing the melting of POPC-rich domains, as determined by differential scanning calorimetry. Taken together, the results indicate that the glycosylation of cholesterol diminishes the ability of the sterol to reside in lateral domains constituted by membrane lipids having highly ordered hydrocarbon chains.     

Alterations in the organization of phosphatidylcholine/cholesterol bilayers by tetrahydrocannabinol

The interactions of delta 9-tetrahydrocannabinol (THC) with various phosphatidylcholines (PCs) was studied in model membranes by differential scanning calorimetry. THC present in PC bilayers above a certain concentration complexed stoichiometrically with phospholipids containing both saturated and unsaturated fatty acids. When the bilayer PCs were sufficiently dissimilar for phase separation to occur, THC preferentially associated with the lower melting point lipid. The presence of cholesterol below 20 mol% in dipalmitoylphosphatidylcholine bilayers enhanced THC X PC complex formation. Above 20 mol% cholesterol, there was no indication of THC X dipalmitoylphosphatidylcholine complex formation. This is in agreement with a phase rearrangement occurring in PC bilayers at concentrations of cholesterol of approximately 20 mol%. These studies suggest several possible mechanisms for the modulation of membrane activities by hydrophobic drugs such as THC.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Differential scanning calorimetric studies of the thermotropic phase behavior of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC-DPPC bilayers at any composition, and that SOPC-DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC-DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC-DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC-DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.     

Glycosylation induces shifts in the lateral distribution of cholesterol from ordered towards less ordered domains

Several studies have indicated the involvement of steryl glycosides in the cellular stress response. In this work, we have compared the effect of 1-O-cholesteryl-β-d-glucoside, 1-O-cholesteryl-β-d-galactoside and cholesterol on the properties of glycerophospholipid and sphingolipid bilayers. The studies were performed in order to gain insight into the change in membrane properties that would follow upon the glycosylation of cholesterol in cells subjected to stress. DPH anisotropy measurements indicated that the cholesteryl glycosides (10-40 mol%) increased the order of the hydrophobic region of a POPC bilayer almost as efficiently as cholesterol. In a PSM bilayer, the cholesteryl glycosides were however shown to be much less effective compared to cholesterol in ordering the hydrocarbon chain region at temperatures above the gel to liquid-crystalline phase transition. Fluorescence quenching analysis of multicomponent lipid bilayers demonstrated that the cholesteryl glycosides, in contrast to cholesterol, were unable to stabilize ordered domains rich in PSM against temperature-induced dissociation. When the sterols were incorporated into bilayers composed of both POPC and PSM, the cholesteryl glycosides showed a higher propensity, compared to cholesterol, to influence the endothermal component representing the melting of POPC-rich domains, as determined by differential scanning calorimetry. Taken together, the results indicate that the glycosylation of cholesterol diminishes the ability of the sterol to reside in lateral domains constituted by membrane lipids having highly ordered hydrocarbon chains.     

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H(II) hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.     

On the importance of the phosphocholine methyl groups for sphingomyelin/cholesterol interactions in membranes: a study with ceramide phosphoethanolamine

In this study, we have examined how the headgroup size and properties affect the membrane properties of sphingomyelin and interactions with cholesterol. We prepared N-palmitoyl ceramide phosphoethanolamine (PCPE) and compared its membrane behavior with D-erythro-N-palmitoyl-sphingomyelin (PSM), both in monolayers and bilayers. The pure PCPE monolayer did not show a phase transition at 22 degrees C (in contrast to PSM), but displayed a much higher inverse isothermal compressibility as compared to the PSM monolayer, indicating stronger intermolecular interactions between PCPEs than between PSMs. At 37 degrees C the PCPE monolayer was more expanded (than at 22 degrees C) and displayed a rather poorly defined phase transition. When cholesterol was comixed into the monolayer, a condensing effect of cholesterol on the lateral packing of the lipids in the monolayer could be observed. The phase transition from an ordered to a disordered state in bilayer membranes was determined by diphenylhexatriene steady-state anisotropy. Whereas the PSM bilayer became disordered at 41 degrees C, the PCPE bilayer main transition occurred around 64 degrees C. The diphenylhexatriene steady-state anisotropy values were similar in both PCPE and PSM bilayers before and after the phase transition, suggesting that the order in the hydrophobic core in both bilayer types was rather similar. The emission from Laurdan was blue shifted in PCPE bilayers in the gel phase when compared to the emission spectra from PSM bilayers, and the blue-shifted component in PCPE bilayers was retained also after the phase transition, suggesting that Laurdan molecules sensed a more hydrophobic environment at the PCPE interface compared to the PSM interface both below and above the bilayer melting temperature. Whereas PSM was able to form sterol-enriched domains in dominantly fluid bilayers (as determined from cholestatrienol dequenching experiments), PCPE failed to form such domains, suggesting that the size and/or properties of the headgroup was important for stabilizing sphingolipid/sterol interaction. In conclusion, our study has highlighted how the headgroup in sphingomyelin affect its membrane properties and interactions with cholesterol.     

Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration

Model membranes composed of cholesterol plus one of two phosphatidylcholines (PC), each containing a saturated and a dienoic acyl chain, have been studied by differential scanning calorimetry. The gel to liquid-crystalline phase transition temperature of 1-palmitoyl-2-linoleoyl PC was -19.5 degrees C and that of 1-stearoyl-2-linoleoyl PC was -13.7 degrees C. The addition of cholesterol to the phosphatidylcholines in aqueous dispersion resulted in the progressive removal of the phase transition as observed by differential scanning calorimetry. Per mole of sterol in the membrane, cholesterol was more effective at reducing the enthalpy change of the phase transitions of these bilayers containing dienoic phosphatidylcholines than it is in eliminating the transition of membranes made with other phospholipids that contain more saturated chains. No transitions in membranes made with palmitoyl-linoleoyl PC or stearoyl-linoleoyl PC could be detected calorimetrically when 17 mol% cholesterol was present.     

The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ?ngstr?ms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.     

AFM study of the thermotropic behaviour of supported DPPC bilayers with and without the model peptide WALP23

Temperature-controlled Atomic Force Microscopy (TC-AFM) in Contact Mode is used here to directly image the mechanisms by which melting and crystallization of supported, hydrated DPPC bilayers proceed in the presence and absence of the model peptide WALP23. Melting from the gel L_β′ to the liquid-crystalline L_α phase starts at pre-existing line-type packing defects (grain boundaries) in absence of the peptide. The exact transition temperature is shown to be influenced by the magnitude of the force exerted by the AFM probe on the bilayer, but is higher than the main transition temperature of non-supported DPPC vesicles in all cases due to bilayer–substrate interactions. Cooling of the fluid L_α bilayer shows the formation of the line-type defects at the borders between different gel-phase regions that originate from different nuclei. The number of these defects depends directly on the rate of cooling through the transition, as predicted by classical nucleation theory.The presence of the transmembrane, synthetic model peptide WALP23 is known to give rise to heterogeneity in the bilayer as microdomains with a striped appearance are formed in the DPPC bilayer. This striated phase consists of alternating lines of lipids and peptide. It is shown here that melting starts with the peptide-associated lipids in the domains, whose melting temperature is lowered by 0.8–2.0 °C compared to the remaining, peptide-free parts of the bilayer. The stabilization of the fluid phase is ascribed to adaptations of the lipids to the shorter peptide. The lipids not associated with the peptide melt at the same temperature as those in the pure DPPC supported bilayer.     

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.     

New structural model for mixed-chain phosphatidylcholine bilayers

Multilamellar suspensions of a mixed-chain saturated phosphatidylcholine with 18 carbon atoms in the sn-1 chain and 10 carbon atoms in the sn-2 chain have been analyzed by X-ray diffraction techniques. The structural parameters for this lipid in the gel state are quite different than usual phosphatidylcholine bilayer phases. A symmetric and sharp wide-angle reflection at 4.11 A indicates that the hydrocarbon chains in hydrated C(18):C(10)PC bilayers are more tightly packed than in usual gel-state phosphatidylcholine bilayers and that there is no hydrocarbon chain tilt. The lipid thickness is about 12 A smaller than would be expected in a normal bilayer phase, and the area per molecule is 3 times the area per hydrocarbon chain. In addition, the bilayer thickness increases upon melting to the liquid-crystalline state, whereas normal bilayer phases decrease in thickness upon melting. On the basis of these data, we propose a new lipid packing model for gel-state C(18):C(10)PC bilayers in which the long C(18) chain spans the entire width of the hydrocarbon region of the bilayer and the short C(10) chain aligns or abuts with the C(10) chain from the apposing molecule. This model is novel in that there are three hydrocarbon chains per head group at the lipid-water interface. Calculations show that this phase is energetically favorable for mixed-chain lipids provided the long acyl chain is nearly twice the length of the shorter chain. In the liquid-crystalline state C(18):C(10)PC forms a normal fluid bilayer, with two chains per head group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasonic evidence for structural relaxation in large unilamellar liposomes

The ultrasonic absorption of large unilamellar vesicles (average diameter 0.2 micron) was determined in the frequency range 0.5-5 MHz. The liposomes were composed of a 4:1 mixture by weight of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol. They were studied with and without cholesterol or gramicidin incorporated into the bilayer. A large increase in absorption occurs at the solid to liquid-crystalline phase transition temperature (42 degrees C) of the pure lipid vesicles. This increase in absorption is interpreted as a structural relaxation of the 'melting' fatty acid chains occurring with an average relaxation time of 76 ns. The liposomes were also found to be extremely permeable near the transition temperature. Essentially complete release of cytosine arabinoside, a small water-soluble molecule, occurred at 42 degrees C. Addition of cholesterol or gramicidin to the bilayer of the liposomes broadened the ultrasonic absorption and reduced the efflux of cytosine arabinoside at the phase transition. No increase in absorption was observed at the transition temperature in the presence of 50 mol% of cholesterol. Gramicidin, in addition to broadening the transition, slows the isomerization of bonds in the hydrocarbon chains of the lipids. A concentration of 5 mol% gramicidin increased the average relaxation time to 211 ns.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

Effects of cholesterol and temperature on the permeability of dimyristoylphosphatidylcholine bilayers near the chain melting phase transition

The passive leakage of glucose across bilayers of dimyristoylphosphatidylcholine (DMPC), cholesterol (variable), and dicetyl phosphate (constant 5.9 mol%) has been measured as efflux over 30 min from multilamellar vesicles. Bilayer cholesterol was varied from 20 mol% to 40 mol%. Glucose permeation rates were measured from 10 degrees C to 36 degrees C, and showed a maximum in permeability at 24 degrees C, the DMPC phase transition temperature. Increasing the bilayer cholesterol content above 20 mol% reduced that permeability peak. These results are quite consistent with a large number of similar bilayer permeability studies over the past 25 years. However, they are not consistent with a previous study of these same systems, which reported increased glucose permeability with temperature, without any maximum at or near the lipid chain melting temperature (K. Inoue, Biochim. Biophys. Acta 339 (1974) 390-402).     

The Barrier Domain for Solute Permeation Varies With Lipid Bilayer Phase Structure

The chemical selectivities of the transport barriers in lipid bilayers varying in composition and phase structure (gel-phase DPPC and DHPC bilayers and liquid-crystalline DPPC/CHOL/50:50 mol% bilayers) have been investigated by determining functional group contributions to transport of a series of α-substituted p-toluic acid analogs obtained in vesicle efflux experiments. Linear free energy relationships are established between the free energies of transfer for this series of compounds from water to the barrier domain and corresponding values for their transfer from water into six model bulk solvents (hexadecane, hexadecene, decadiene, chlorobutane, butyl ether, and octanol) determined in partitioning experiments to compare the barrier microenvironment to that in these model solvents. The barrier microenvironment in all bilayers studied is substantially more hydrophobic than octanol, thus establishing the location of the barrier beyond the hydrated headgroup interfacial region, as the interface is expected to be more hydrophilic than octanol. The chemical nature of the barrier domain microenvironment varies with bilayer phase structure. The barrier regions in non-interdigitated DPPC and interdigitated DHPC gel-phase bilayers exhibit some degree of hydrogen-bond acceptor capacity as may occur if these domains lie in the vicinity of the ester/ether linkages between the headgroups and the acyl chains. Intercalation of 50 mol% cholesterol into DPPC bilayers, which induces a phase transition to a liquid-crystalline phase, substantially increases the apparent barrier domain hydrophobicity relative to gel-phase bilayers to a nonhydrogen bonding, hydrocarbonlike environment resembling hexadecene. This result, combined with similar observations in liquid-crystalline egg-PC bilayers (J. Pharm. Sci. (1994), 83:1511–1518), supports the notion that the transition from the gel-phase to liquid-crystalline phase shifts the barrier domain further into the bilayer interior (i.e., deeper within the ordered chain region). Received: 16 September 1997/Revised: 14 May 1998