Asymmetric antagonistic effects of an inhalation anesthetic and high pressure on the phase transition temperature of dipalmitoyl phosphatidic acid bilayers

The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.     

The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

A theoretical model of the temperature- and pressure-induced phase transition of phospholipid bilayers

A statistical thermodynamic model of phospholipid bilayers is developed. In the model, a new concept of a closely packed system is applied, i.e., a system of hard cylinders of equal radii, the radius being a function of the average number of gauche rotations in a hydrocarbon chain. Using this concept of a closely packed system, reasonable values are obtained for the change in specific volume at the order-disorder transition of lecithin bilayers. In addition to interactions between the lipid matrix and water molecules, between the head groups themselves and between hydrocarbon chains, as well as the intramolecular energy associated with chain conformation, the Hamiltonian of the membrane also includes the energy of the pressure field. Thus, the phase transition of phospholipid membranes induced not only by temperature hut also by hydrostatic pressure is described by this model simultaneously. In accordance with the experimental results, a linear relationship is obtained between the phase transition temperature and phase transition pressure. The other calculated phase transition properties of lecithin homologues. e.g., changes in enthalpy, surface area. thickness and gauche number per chain are in agreement with the available experimental data. The ratio of kink to interstitial conduction of bilayers is also estimated.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.     

The binding of organic ions to phospholipid bilayers

The binding of organic anions and cations, mainly tetraphenylboride and tetraphenylarsonium, to phospholipid membranes has been studied using an NMR method. Binding is appreciable and is affected by cholesterol in the membrane and counterions in solution. The passage of the organic anions through the membrane has also been followed. These measurements indicate that it is naive to use organic anions to measure membrane potentials in a simple manner.     

Hydration of phospholipid bilayers in the presence and absence of cholesterol

The number of water molecules bound (unfreezable) by a molecule of dipalmitoyl phosphatidylserine (DPPS) or by a molecule of dipalmitoyl phosphatidylcholine (DPPC) alone or in mixtures with cholesterol was determined by differential scanning calorimetry (DSC). When the phospholipids are in the gel state and in the absence of cholesterol, molecule of DPPS binds about 3.5 molecules of water and molecule of DPPC binds about 6 molecules of water. Number of water molecules bound increases when cholesterol crystallites are formed in the bilayer. For DPPS-cholesterol mixture at X(chol) -0.5, as well as for DPPC-cholesterol mixture at X(chol) -0.5 about 7 water molecules are bound.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

I. Phospholipid release from sonicated vesicles induced by a ‘critical’ fatty acid concentration

Dipalmitoyl phosphatidylcholine vesicles incubated in the presence of increasing amounts of myristic acid showed a progressive translocation of phospholipid molecules across a dialysis membrane. The rate of phospholipid translocation increased abruptly at a ‘critical’ value of myristic acid concentration. The translocation rate of mixed dipalmitoyl phosphatidylcholine/myristic acid vesicles obtained by cosonicating the two components was also dependent on a ‘critical’ fatty acid concentration. A marked release of K⁺ and different responses of fluorescent probes to the fatty acid addition were observed at this concentration.     

Phase behavior of freeze-dried phospholipid-cholesterol mixtures stabilized with trehalose

A study is presented of the role of cholesterol content on the gel-to-liquid crystalline phase transition of freeze-dried liposomes stabilized with trehalose, a well known lyoprotectant. The phospholipids considered in this work, DPPC and DPPE, belong to the two predominant phospholipid species found in numerous biological membranes. Cholesterol is found in abundance in mammalian plasma membranes. DSC measurements reveal that cholesterol-containing liposomes exhibit multiple phase transitions upon dehydration. Addition of trehalose to these systems lowers the phase transition temperature and limits the phase separation of the lipidic components upon freeze-drying. This work provides strong evidence for the effectiveness of trehalose in stabilizing cholesterol-containing membranes upon lyophilization.     

II. Phospholipid exchange and size enlargement in sonicated vesicles induced by a ‘critical’ fatty acid concentration

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level (‘critical’ concentration). This ‘critical’ concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement.Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidylcholine vesicles increased abruptly above a ‘critical’ fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the ‘critical’ concentration. The phospholipid exchange occurred both in fluid- and in solid-state vesicles. The ‘critical’ fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process.The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.     

Thermal transitions in dimyristoylphosphatidyleholine foam bilayers

Thermal transitions in the system dimyristoylphosphatidylcholine/water/ethanol/sodium chloride were studied in the temperature range 10–31 °C. The water-ethanol dispersions were investigated by differential scanning calorimetry and the foam bilayers by the microinterferometric method for investigation of thin liquid films. Calorimetry showed that an increase in ethanol content (up to 47.5 vol.% — the concentration used in the experiments with foam bilayers) did not significantly influence the temperature of the main phase transition and led to the disappearance of the pretransition. The microinterferometric study of the foam bilayer thickness showed that there were two thermal transitions — at 13 and 23 °C. An Arrhenius type dependence was obtained for the critical concentration of dimyristoylphosphatidylcholine (DMPC) in the solution, which was necessary for the formation of the foam bilayer. A steep change in the slope of the linearized Arrhenius dependence was found at 23 °C. Values of the binding energy of a DMPC molecule in the foam bilayers were calculated using the hole-nucleation theory of stability and permeability of bilayers. It was proved that the phase transition at 23 °C was due to melting of the hydrocarbon tails of phospholipid molecules. The low-temperature phase transition was assumed to be due to a change in the tilt of the hydrocarbon tails. These experiments demonstrate for the first time the occurrence of phase transitions in foam bilayers. Correspondence to: D. Exerowa     

Membrane composition modulates thiopental partitioning in bilayers and biomembranes

The nonspecific interaction of thiopental with erythrocyte ghosts, synaptic membranes, microsomes and mitochondria has been measured at 25°C and pH 6.6. In cholesterol-depleted erythrocyte ghosts the partition coefficient decreases with increasing cholesterol content. In sonicated liposomes made from egg lecithin and cholesterol the partition coefficient also decreases with increasing cholesterol content. The dependence of the partition coefficient on cholesterol content in the biological membranes, on average, parallels that in the lipid bilayers. The partition coefficient in lipid bilayers made from lipids extracted from erythrocyte ghosts was comparable to that in the corresponding egg lecithin/cholesterol bilayer. The partition coefficients of all the biomembranes are consistently lower than those in the corresponding egg lecithin/cholesterol bilayer, the free energy of transfer between biomembrane and corresponding bilayer being ?1 kcal/mol.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe₂PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L_β) or ripple gel (P_β′) phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa⁻¹ depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L_c) phase to the lamellar gel (L_β or L_β′) phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L_c/L_β transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L_c/L_α transition was observed at a temperature higher than the main-transition temperature. The main (L_β/L_α) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L_β′, P_β′ and pressure-induced interdigitated gel (L_βI) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe₂PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and $\text{n-}\text{propane}$ (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006°C/atm, respectively, compared with 0.024°C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58°C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1°C are estimated, in mol%: He, 10.2; N₂, 13.2; N₂O, 9.04; $\text{n-}\text{C}{}_3\text{H}{}_8$, 6.3 and cyclopropane, 12.8.     

Kinetic investigations on the phase transition of phospholipid bilayers

Pressure-jump experiments were performed on vesicles and liposomes of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine following the time course of solution turbidity. For both lipids two relaxation effects were evaluated the time constants of which exhibit clear maxima at the midpoint of the phase transition. The time constants lie for vesicles in the 100 μs and 1 ms ranges and for liposomes in the 1 ms and 10 ms ranges. The processes are slightly faster for dimyristoyl phosphatidylcholine than for dipalmitoyl phosphatidylcholine. All relaxation times are concentration-independent. The time constant and amplitude behaviours indicate that all processes are cooperative in agreement with previous interpretations. It is demonstrated that cooperative units can be evaluated from the relaxation amplitudes. These are of the same order of magnitude as those obtained from static experiments. On the grounds of the present kinetic investigation we can state that the application of the linear Ising model to two-dimensional processes as attempted for the static lipid phase transition is inadequate.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

A simple theoretical model for the effects of cholesterol and polypeptides on lipid membranes

A simple theoretical model for the effects of impurities on biomembranes is proposed. The model accounts for the cholesterol-induced decrease of membrane phase transition temperature, membrane condensation above the gel to liquid crystalline phase transition, and increase in lateral compressibility. The model also predicts that addition of molecules such as cholesterol and polypeptides to membranes results in unmasking of a continuous phase transition. This results in a second broad peak in the calorimetric curves for melting of lipid-cholesterol mixtures, and the appearance of a second melting transition in membranes modified by the incorporation of polypeptides. The theory assumes that the membrane may be adequately described by a kink model, and that impurities are randomly distributed in the membrane. The difference in size and shape of impurity molecules, compared to membrane lipids, results in a spatial disordering in the membrane which in turn causes increased chain disorder and membrane condensation, as well as a decrease in the cooperativity of melting. The second transition results from a second expansion of the condensed, partially disordered membrane, which takes place over a several degree temperature range. This transition, although unmasked by boundary effects of non-lipid molecules, does not correspond to melting of a boundary annulus or phase separation.     

Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles Effect of membrane composition and temperature

Small unilamellar vesicles were used to measure the permeability of saturated phosphatidylcholine bilayers to glucose. The presented method circumvents most of the common restrictions of classical permeability experiments. Increasing the fatty acid chain length of the lipids reduced the permeation rate significantly. Raising the temperature above that of the lipid phase transition drastically increased membrane permeability. Arrhenius plots demonstrated the activation energy to be independent of membrane composition and the phase-state of the lipids. The permeation process is discussed in terms of a constant energy to disrupt all hydrogen bonds between permeant and aqueous solvent prior to penetrating the membrane. The magnitude of the permeability coefficient is partly determined by a unfavourable change in entropy of activation on crossing the water/lipid interface. All results indicate that the penetration of the dehydrated permeant into the hydrophobic barrier is the rate-limiting step in the permeation of glucose.