The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains.     

The gel phase of dipalmitoyl phosphatidylcholine. An infrared characterization of the acyl chain packing

It is shown, by infrared spectroscopy, that the packing in the gel phase of fully-hydrated dipalmitoyl phosphatidylcholine is not uniform over a large temperature range. With decreasing temperature, starting at that of the pretransition, there is a gradual change in the molecular packing of the acyl chains, from near hexagonal to orthorhombic of monoclinic.     

Evidence for acyl chain trans/gauche isomerization during the thermal pretransition of dipalmitoyl phosphatidylcholine bilayer dispersions

In order to clarify, in dipalmitoyl phosphatidylcholine multilayers, the effect of the 34 degrees C thermal pretransition on the acyl chain intramolecular disordering process, Raman spectra of dipalmitoyl phosphatidylcholine gels at 20 and 34 degrees C were compared in the 1000--1200 cm-1 skeletal C-C stretching region. In addition to an overall intensity decrease associated with a change in chain packing characteristics, the growth of intensity in the 1080--1090 and 1122 cm-1 regions in the 34--20 degrees C) difference spectrum clearly indicates that the thermal pretransition is accompanied by an increase in the population of hydrocarbon chain gauche rotamers toward the center of the bilayer.     

Influence of phospholipid peroxidation on the phase behavior of phosphatidylcholine and phosphatidylethanolamine in aqueous dispersions

The influence of oxygen-induced phospholipid peroxidation on the phase behavior of aqueous dispersions of both egg phosphatidylcholine (egg-PC) and egg phosphatidylethanolamine (egg-PE) has been investigated. Phospholipid peroxidation was followed via malondialdehyde formation and analyses of acyl chain compositions. 13C nuclear magnetic resonance spectroscopy (NMR) and the amino-indicating probe trinitrobenzenesulfonic acid were used to study the effect of peroxidation on the chemical structure of hydrated egg-PE. The macroscopic organization of the phospholipids was monitored by 31P NMR and small-angle X-ray diffraction. Differential scanning calorimetry was employed to study the influence of peroxidation on the thermotropic behavior of egg-PE. The results show that egg-PE is more sensitive to the effects of peroxidation than egg-PC. In the latter, no changes in the macromolecular organization were observed. However, peroxidation strongly influenced the polymorphic phase behavior of PE. Initial peroxidation stabilized hydrated egg-PE in a lamellar system up to 70 degrees C, presumably by modification of the head group. Such modifications were confirmed by 13C NMR experiments, which indicated the formation of Schiff bases between PE head groups and aldehydes. Furthermore, quantitative analyses of trinitrobenzenesulfonic acid reactable egg-PE and the corresponding fatty acid compositions revealed the presence of cross-links between the ethanolamine head groups, likely involving the bifunctional malondialdehyde. Prolonged peroxidation of egg-PE resulted in a loss of order in the system, possibly by the formation of intermediate nonbilayer structures.     

X-ray analysis and calorimetry on phosphatidylcholine model membranes. The influence of length and position of acyl chains upon structure and phase behaviour

The effect of variation of the acyl chain composition of phosphatidylcholines upon thermal behaviour of multilamellar liposomes was evaluated by calorimetry and X-ray studies. A total of thirteen different phosphatidylcholines were examined. They differed from each other in the length as well as in the position of the acyl chains in the glycerol backbone. The experimental results show that the hitherto accepted phase scheme for phosphatidylcholine-water systems is incomplete and has to be extended to include the behaviour of samples that have been stored for long times at low temperatures. The X-ray results show that the structure of the new low-temperature phase is not in agreement with the hexagonal packing of the acyl chains. To explain the X-ray results, a two-dimensional orthorhombic unit cell has to be assumed in order to fit all the observed reflexes in the wide-angle region.     

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.     

Effect of α-tocopherol on the phase transition of phosphatidylcholine and on water transport through phosphatidylcholine liposome membranes

The interaction between α-tocopherol and phosphatidylcholine was studied in liposomes by differential scanning calorimetry and osmotic water transport studies. Addition of α-tocopherol to phosphatidylcholine resulted in a reduction in enthalpy at the transition temperature, a rise in osmotic water permeability of the liposomes below the phase transition temperature and disappearance of the discontinuity of osmotic water transport at the phase transition. Also the temperature dependence of osmotic water transport was reduced below the transition temperature. A comparison between cholesterol and α-tocopherol in regulation of permeability was made and the physiological relevance of tocopherol in regulation of membrane permeability is discussed.     

The inverted hexagonal phase is more sensitive to hydroperoxidation than the multilamellar phase in phosphatidylcholine and phosphatidylethanolamine aqueous dispersions.

The effect of phase behaviour (hexagonal II phase and lamellar phase) on the peroxidation of membrane phospholipids has been investigated in dilinoleoyl phosphatidylcholine (DLPC)/dilinoleoyl phosphatidylethanolamine (DLPE) aqueous dispersions. Peroxidation was initiated with a water-soluble radical inducer 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPN). The phospholipid morphology was monitored by 31P-nuclear magnetic resonance (NMR). Phospholipid hydroperoxides (PCOOH and PEOOH) were determined by chemiluminescence high-performance liquid chromatography (CL-HPLC). In pH-induced phase transition systems, DLPE in the bilayer state was much less oxidized than in the hexagonal II state. In composition-induced phase transition systems, the formation of total hydroperoxides and the consumption of alpha-tocopherol in the hexagonal II phase were greater than in the bilayer phase. These data suggest that the hexagonal II phase is more sensitive to hydroperoxidation than the bilayer phase in phospholipid aqueous dispersions.     

Influence of dicarboxylic phosphatidylcholines on the stability and phase transition of phosphatidylcholine liposomes

Utilizing reflectance spectrophotometry, hemoperfusion, rate of oxygen consumption and redox level of mitochondrial cytochrome c (+c1) in livers in situ of anesthetized rats were measured. The transition to the anoxic state was induced by raising the pressure on the liver surface to more than the hepatic blood pressure by pressing with the tip of the optical guide of the reflectance spectrophotometer. During this transition, the average oxygen saturation of hemoglobin in the liver in situ decreased linearly with time until it became 10--20% of the total. This was followed by reduction of mitochondrial cytochrome c (+c1), which reached completion in 10--20 s. The measured O2 consumption rate remained constant until the percentage of oxyhemoglobin in situ decreased to a critical level. There was then a decrease in the rate of O2 consumption which was accompanied by a progressive reduction of cytochrome c (+c1). It was shown that amounts of hemoglobin and mitochondrial respiratory chain cytochromes in the liver in situ could be measured non-invasively and could provide important signals for vital cellular functions. The changes in hemoperfusion and rate of O2 consumption of the liver in situ following ethanol ingestion were also shown in rats, and are briefly discussed with respect to possible application of this method to study the pathophysiology of tissues.     

Evidence for acyl chain trans/gauche isomerization during the thermal pretransition of dipalmitoyl phosphatidylcholine bilayer dispersions

In order to clarify, in dipalmitoyl phosphatidylcholine multilayers, the effect of the 34°C thermal pretransition on the acyl chain intramolecular disordering process, Raman spectra of dipalmitoyl phosphatidylcholine gels at 20 and 34°C were compared in the 1000–1200 cm⁻¹ skeletal C-C stretching region. In addition to an overall intensity decrease associated with a change in chain packing characteristics, the growth of intensity in the 1080–1090 and 1122 cm⁻¹ regions in the (34-20°C) difference spectrum clearly indicates that the thermal pretransition is accompanied by an increase in the population of hydrocarbon chain gauche rotamers toward the center of the bilayer.     

Gramicidin promotes formation of the hexagonal HII phase in aqueous dispersions of phosphatidylethanolamine and phosphatidylcholine

It is shown by ³¹P-NMR and electron microscopy that gramicidin promotes the formation of the hexogonal H_II phase in aqueous dispersions of dielaidoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine, when present in molar ratios of 1 : 200 and higher. In addition gramicidin also induces the hexogonal H_II phase in aqueous dispersions of dioleoylphosphatidylcholine, when present in molar ratios of 1 : 25 and higher.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).     

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines.

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.     

Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.     

The effects of acyl chain ordering and crystallization on the main phase transition of wet lipid bilayers

The main gel-fluid phase transition of wet lipid bilayers is examined in terms of a microscopic interaction model which incorporates both trans-gauche isomerism of the lipid acyl chains and crystal orientation variables for the lipid molecules. The model gives two scenarios for the phase behavior of wet lipid bilayers in terms of temperature: (i) chain melting occurs at a higher temperature than crystallization, or (ii) chain melting and crystallization occur at the same temperature. Experimental data for lipid bilayers is consistent with the second scenario. In this case, computer simulation is used to investigate the non-equilibrium behaviour of the model. The numerical data is intepreted in terms of interfacial melting on heating and grain formation on cooling through the main phase transition. Interfacial melting is a non-equilibrium process in which the grains of a polycrystalline bilayer melt inwards from the boundaries. The prediction of interfacial melting in wet lipid bilayers is examined in relation to data from both equilibrium and nonequilibrium measurements, to corresponding phase behavior in monolayers, and to previous theoretical work.Abbreviations DHPE dihexadecyl phosphatidylethanolamine - DMPA dimyristoyl phosphatidic acid - DMPC dimyristoyl phosphatidylcholine - DPPC dipalmitoyl phosphatidylcholine - DSC differential scanning calorimetry - MCS/S Monte Carlo steps per site Supported in part by the NSERC of Canada and FCAC du QuébecSupported by the Danish Natural Science Research Council under grant J.nr. 5.21.99.72     

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37°C without vesicle fusion.The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing ¹³C NMR using N-¹³CH_3? labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (halftime 4 h) in the temperature range at which the hydrocarbon phase transition occurs.The activation energy of the flip-flop rate above the phase transition is 23.7 ± 2.0 kcal/mol.     

Delayed luminescence of pyrene in phosphatidylcholine dispersions

Delayed luminescence of pyrene in phosphatidylcholine dispersions was studied over the temperature range 5–60°C. It was found that the pyrene delayed-luminescence spectrum is similar to its fluorescence spectrum. The transient behavior of the luminescence was also studied and was found to show a drastic change when the lipid matrix undergoes phase transition. This method will be a new tool for membrane studies, especially for the detection of the phase transition.     

The influence of dioxan on the bilayer and monolayer phase transition of phospholipids

The influence of 1.4.-dioxan on the bilayer phase transition of various phospholipids was studied by differential scanning calorimetry and turbidity measurements. The addition of 1.4.-dioxan to lipid bilayers decreases the transition temperature T_m increases the transition enthalpy of the transition. The cooperativity of the transition is unaffected. The phospholipid monolayer transition from the liquid-condensed to the liquid-expanded phase was measured by recording area versus temperature curves at constant surface pressure (isobars). The monolayer transition temperature at constant surface pressure is increased when 1.4.-dioxan is added to the subphase. The change in molecular area becomes larger. A comparison of monolayer isobars on water and water/dioxan as subphase at constant surface tension rather than surface pressure leads to a decrease of the transition temperature on water/dioxan as subphase. This decrease as well as the larger change in molecular area at the monolayer transition can be correlated to the decrease in T_m and the increase in the transition enthalpy of the corresponding bilayer system. 1.4.-Dioxan seems to accumulate at the lipid head group/water interface, thus lowering the tension of the bilayer membrane. This cyclic ether can be used for altering the characteristics of bilayer membranes without disturbing the lipid chain organization.     

Small phosphatidylcholine vesicles appear to be faceted below the thermal phase transition

Summary We have studied the main thermal transition in dipalmitoyl phosphatidylcholine (DPPC) multilayers and a similar transition in small (300 Å diameter), single-walled vesicles by X-ray diffraction. As judged by the large-angle diffraction, the transition in the multilayers is narrow; aside from small tails, the transition occurs over a range of 0.5°C. In contrast, the transition in the vesicles is quite broad; the range is about 7°C. These observations are in agreement with recently published data.Referring to the small vesicles below the thermal transition, a bilayer structure in which the C₁₆ chains are all straight and pointed radially is inconsistent with the large-angle diffraction. Assuming instead that the chains are packed in a regular, planar array, it is clear from their small size that the vesicles can have only limited regions of planar packing. The X-ray data indicate that the planar regions are 75 Å across on the average. In view of the 75-Å size and the average vesicle diameter of about 300 Å, we propose that the small vesicles are faceted below the transition, i.e., that the vesicles are polygonal. The small-angle diffraction pattern from the vesicles below the transition provides support for the faceted structure.     

Thiocyanate and bromide ions influence the bilayer structural parameters of phosphatidylcholine bilayers

The influence of monovalent cations and anions on the structural parameters of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined at 25 degrees C using X-ray diffraction. It was shown that monovalent salts, in general, have little effect on lipid packing within the bilayer. However, fully hydrated DPPC bilayers in 1 M KSCN pack in an interdigitated acyl chain phase. This is the first observation of an ion-induced interdigitated bilayer phase in a zwitterionic lipid. In addition, gel state DPPC bilayers in 1 M KBr imbibe approx. 10 A more solvent than bilayers in water. The influence of these same salts on the phase transitions of DPPC bilayers was also examined using high-resolution differential scanning calorimetry. These results are discussed in terms of ion-induced changes in solvent and solvent/bilayer structure.