Equimolar Cholesterol Research Articles

Use of liposome encapsulated sodium‐ 2,3‐dimercapto‐propane sulfonate (DMPS) in the treatment of mice loaded with cadmium

Abstract Sodium‐2, 3‐dimercaptopropane sulfonate (DMPS) is a potent thiol chelating drug used against heavy metal intoxication. With a view to enhance its efficacy at low dose DMPS was encapsulated in liposomes made up of equimolar egg phosphotidylcholine and cholesterol and then administered to mice loaded with cadmium. Both the free as well as liposomal drug enhanced urinary excretion of cadmium. While fecal excretion of cadmium was enhanced only by liposomal drug, liposomal DMPS was effective in mobilizing more cadmium from blood, liver, kidney and spleen by day 3 of treatment, Liposomal DMPS was also able to decorporate some cadmium from cadmium binding proteins with low molecular weight corresponding to metallothionein.

ESR study on synthetic glyceroglycolipid liposomal membranes

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmityl[ β-cellobiosyl-(1′→ 3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37°C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-DPPA in the Cel-DAG liposomes at 37°C showed broad resonance lines with the central peak being the highest, while that at 60°C gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37°C but not above T c. These results suggest that below T c the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.

Range of the solvation pressure between lipid membranes: dependence on the packing density of solvent molecules.

Well-ordered multilamellar arrays of liquid-crystalline phosphatidylcholine and equimolar phosphatidylcholine-cholesterol bilayers have been formed in the nonaqueous solvents formamide and 1,3-propanediol. The organization of these bilayers and the interactions between apposing bilayer surfaces have been investigated by X-ray diffraction analysis of liposomes compressed by applied osmotic pressures up to 6 X 10(7) dyn/cm2 (60 atm). The structure of egg phosphatidylcholine (EPC) bilayers in these solvents is quite different than in water, with the bilayer thickness being largest in water, 3 A narrower in formamide, and 6 A narrower in 1,3-propanediol. The incorporation of equimolar cholesterol increases the thickness of EPC bilayers immersed in each solvent, by over 10 A in the case of 1,3-propanediol. The osmotic pressures of various concentrations of the neutral polymer poly(vinylpyrrolidone) dissolved in formamide or 1,3-propanediol have been measured with a custom-built membrane osmometer. These measurements are used to obtain the distance dependence of the repulsive solvation pressure between apposing bilayer surfaces. For each solvent, the solvation pressure decreases exponentially with distance between bilayer surfaces. However, for both EPC and EPC-cholesterol bilayers, the decay length and magnitude of this repulsive pressure strongly depend on the solvent. The decay length for EPC bilayers in water, formamide, and 1,3-propanediol is found to be 1.7, 2.4, and 2.6 A, respectively, whereas the decay length for equimolar EPC-cholesterol bilayers in water, formamide, and 1,3-propanediol is found to be 2.1, 2.9, and 3.1 A, respectively. These data indicate that the decay length is inversely proportional to the cube root of the number of solvent molecules per unit volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes.

Pressure versus distance relationships have been obtained for egg phosphatidylcholine bilayers containing a range of cholesterol concentrations. Water was removed from between adjacent bilayers by the application of osmotic pressures in the range of 0.4-2600 atm (4 x 10(5)-2.6 x 10(9) dyn/cm2), and the distance between adjacent bilayers was obtained by Fourier analysis of X-ray diffraction data. For applied pressures up to about 50 atm and bilayer surface separations of 15-5 A, the incorporation of up to equimolar cholesterol has little influence on plots of pressure versus bilayer separation. However, for the higher applied pressures, cholesterol reduces the interbilayer separation distance by an amount that depends on the cholesterol concentration in the bilayer. For example, the incorporation of equimolar cholesterol reduces the distance between bilayers by as much as 6 A at an applied pressure of 2600 atm. At this applied pressure, electron density profiles show that the high-density head-group peaks from apposing bilayers have merged. This indicates that equimolar concentrations of cholesterol spread the lipid molecules apart in the plane of the bilayer enough to allow the phosphatidylcholine head groups from apposing bilayers to interpenetrate as the bilayers are squeezed together. All of these X-ray and pressure-distance data indicate that, by reducing the volume fraction of phospholipid head groups, cholesterol markedly reduces the steric repulsion between apposing bilayers but has a much smaller effect on the sum of the longer ranged repulsive hydration and fluctuation pressures. Increasing concentrations of cholesterol monotonically increase the dipole potential of egg phosphatidylcholine monolayers, from 415 mV with no cholesterol to 493 mV with equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effect of tomatine and its alleviation by cholesterol on larval growth and efficiency of food utilization inHeliothis zea andSpodoptera exigua

The effect of tomatine on larval growth ofHeliothis zea andSpodoptera exigua was assessed by rearing larvae on diets with different concentrations of the chemical added. When reared from neonates, linear dose-response relationships were obtained for both species, withS. exigua being three times more sensitive to tomatine thanH. zea. Tomatine toxicity was completely alleviated inH. zea by the addition of equimolar cholesterol into the diet; however, inS. exigua some toxicity was maintained. Larvae ofS. exigua that were started on control diet were insensitive to tomatine after five days; larvae started on diet with an EC50 of tomatine and then switched to control diet after five days failed to recover from toxicosis. Larval growth ofH. zea, on the other hand, was affected both at the neonate and third-instar stage, but normal growth resumed when the larvae were transferred to control diet. Tomatine had little or no affect on food consumption, assimilation, or dietary utilization of the food by third-instar larvae ofS. exigua, except at a concentration 10 times the EC50. In contrast, the efficiency of food utilization ofH. zea larvae decreased with increasing tomatine concentrations. Assimilation of the food tended to increase, although not significantly, as tomatine levels increased. Food consumption ofH. zea larvae also increased when the tomatine concentration was greater than an EC50. The addition of equimolar cholesterol to diets with an EC50 of tomatine restored weight gain and nutritional indices values to control values. These results are related to the utility of using tomatine in host-plant resistance programs.

A non-covalent method of attaching antibodies to liposomes

A novel non-covalent method of attaching antibodies to liposomes which exploits the high affinity of streptavidin for biotin, is described. The two-step coupling protocol involves the initial attachment of streptavidin to liposomes containing biotin PE, followed by the coupling of biotinated antibodies to streptavidin-liposomes. The association of streptavidin with liposomes containing biotinated PE is rapid (< 5 min), resulting in a maximum association of 40 molecules of streptavidin per 100 nm vesicle. In the presence of equimolar cholesterol, the amount of streptavidin bound is twice that observed when biotin PE/egg PC liposomes are used. Irrespective of the mole ratio of biotin to antibody (e.g. for 1–6 biotins per antibody), or the molar ratio of antibody to streptavidin in the second incubation step, equimolar amounts of antibody bind to streptavidin. It is shown that anti-rat-erythrocyte IgG or F(ab′) 2 complexed to liposomes via the streptavidin linker bind specifically to rat erythrocytes but not to human erythrocytes. This coupling protocol can be readily extended to other biotinated antibodies.

Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential

Many drugs exhibit lipophilic and cationic (basic) characteristics. Previous studies have shown that lipophilic cations can be accumulated into model membrane ‘liposomal’ (vesicular) systems in response to establishing a membrane potential (inside negative) across the vesicle membrane. We demonstrate here that the anticancer drugs, adriamycin and vinblastine, can be rapidly accumulated into egg phosphatidylcholine large unilamellar vesicles in response to a valinomycin-dependent K + diffusion potential (Δψ) to achieve high effective interior concentrations. Further, trapping efficiencies approaching 100% can be easily achieved. The influence of lipid composition and the requirement for valinomycin have been examined for adriamycin. Equimolar cholesterol levels inhibit the uptake process at 20°C. However, incubation at higher temperature results in enhanced uptake. Similarly, the presence of egg phosphatidylserine or incubation at elevated temperatures results in significant adriamycin uptake in the absence of valinomycin. It is shown that the adriamycin retention time in the vesicles is enhanced by an order of magnitude or more when actively trapped by the presence of a membrane potential in comparison to passive trapping procedures. It is suggested that such active trapping procedures may be of use for loading liposomal systems for drug delivery applications, and may provide avenues for controlled release of encapsulated material.

Interaction of Digitonin and Its Analogs with Membrane Cholesterol1

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation and denaturation of hemoglobin encapsulated in liposomes

A study was made of the in vitro stability of hemoglobin-containing liposomes (‘hemosomes’) prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in ‘hemosomes’ prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken ‘hemosomes’. The denaturation of hemoglobin in ether-injection ‘hemosomes’ was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N 2 or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken ‘hemosomes’ the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine ‘hemosomes’ than in equivalent liposomes containing no hemolysate. The ‘hemosome’ seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.

Interaction of hapten-sensitized liposomes with cells bearing hapten-specific receptors.

The binding of liposomes sensitized with 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) to MOPC-315 cells, which secrete and bear on their surfaces anti-DNP immunoglobulins, was studied. The binding was affected by cholesterol content, phospholipid composition and hapten density of liposomes: The binding of distearoylphosphatidylcholine liposomes sensitized with 5 mol% hapten to the cells increased with increasing cholesterol content in liposomes. The amount of liposomes composed of phospholipid with a higher transition temperature (such as distearoylphosphatidylcholine), which bound to MOPC-315 cells, was much higher than that of liposomes composed of phospholipid with a lower transition temperature (such as egg yolk phosphatidylcholine). The amount of distearoylphosphatidylcholine liposomes containing equimolar cholesterol, which bound to the cells at 0 degrees C, increased with increasing amount of the hapten in liposomes up to 2.5 mol%. The binding became maximum at 2.5 mol% and decreased with higher hapten density in liposomes. The immunogenicity of hapten-sensitized liposomes is known to be affected by the liposomal composition such as cholesterol content, phospholipid composition and hapten density. This model study suggests that the binding of liposomes to cells is important for expressing the immunogenicity of hapten-sensitized liposomes.

Fate of cholesterol-rich liposomes after subcutaneous injection into rats

The fate of small (30–60 nm) and large (about 400 nm diameter) liposomes composed of phosphatidylcholine or sphingomyelin and equimolar cholesterol and containing quenched carboxyfluorescein and 111In-labelled bleomycin after footpad injection of rats was investigated. Judging from the values of latent carboxyfluorescein in the plasma, phosphatidylcoline and spingomyelin small liposomes entered the blood circulation intact, presumably via the lymphatics to reach 3 h after injection a peak of 16.9 and 23.1% of the dose per total blood, respectively. Of the 111In radioactivity given in phosphatidylcholine liposomes, about 32% was recovered in the liver. Hepatic uptake of 111In for sphingomyelin liposomes was lower (about 9%) reflecting their slower rate of clearance. No latent carboxyfluorescein could be detected in the blood after injection with phosphatidylcholine or sphingomyelin large liposomes and levels of 111In in the liver (and spleen) were very low. A proportionally much greater amount of liposomal 111In was intercepted by the popliteal and to a lesser extent, the lumbar lymph nodes. Such uptake was significantly higher (e.g. 463%) for small than for large (e.g. 195% per g popliteal nodes) liposomes. After foot pad injection of large liposomes into Walker 256 tumour-bearing rats, localization of 111In in the popliteal nodes was similar to that seen with control animals. However, 111In localization in the lumbar nodes was augmented more than 5-fold. These results suggest that large liposomes as used in this study, characterized by efficient drug entrapment, inability to reach the liver and spleen and improved localization in the lymph nodes of tumour-bearing animals may be preferable to vesicles of small size for the local treatment of lymphatic metastases.

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1 c phosphatidylcholine and 16:0/18:1 c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using 31P-NMR, freeze fracturing and differential scanning calorimetry (DSC). The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with 31P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H II phase, 16:0/15:1 c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.

Association of cholesterol with lysophosphatidylcholine

With equimolar cholesterol, lysophosphatidylcholine (lysoPC) or 1-ether-2-deoxylyso-phosphatidylcholine (etherdeoxylysoPC) form unilamellar vesicles of identical dimensions. 13C-NMR spectra of such vesicles are interpreted on the premise that suppression of a signal by broadening (i.e. decrease of T 2 ∗ relaxation time) indicates a decrease of motion of the carbon atom relative to its surroundings. The signals for sn-glycerol C-1 and C-2 are completely suppressed in the lysoPC-cholesterol vesicles. In contrast, in the vesicles containing etherdeoxylysoPC, all three glycerol carbon signals make their appearance, with the T 2 ∗ of C-2 approaching the T 2 ∗ in the monomolecularly dissolved lysolipid. This result argues for lipid-lipid complexing in the ‘hydrogen belts’ of the lysoPC-cholesterol bilayer, specifically, for hydrogen bonding involving the hydroxyl and carbonyl groups of lysoPC and the hydroxyl of cholesterol.

Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration

We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.

Preparation and structural studies of cholesterol bilayers

Bilayers consisting, in their hydrophobic core, entirely of cholesterol can be constructed if a hydrophilic molecular anchor is supplied. O- Methoxyethoxyethoxyethylcholesterol and cholesterol sulfate form multilayered liposomes in water. With equimolar cholesterol added, cholesterol sulfate, cholesterolphosphocholine, and O- methoxyethoxyethoxyethylcholesterol form small unilamellar liposomes on prolonged sonication. The dimensions of cholesterol-cholesterolphosphocholine vesicles are comparable to those of phospholipid vesicles. 13C-NMR spectra suggest that the centers of the bilayers are liquid. The permeability of the cholesterol-cholesterolphosphocholine bilayer against glycerol is lower than that of dipalmitoylphosphatidylcholine-cholesterol bilayer; the activation energy of permeation is two times larger, an indication of a higher degree of structural organization in the ‘hydrogen belts’ of the cholesterol-cholesterolphosphocholine bilayer.

The bilayer stabilizing role of sphingomyelin in the presence of cholesterol. A 31P NMR study

1. 1. The polymorphic phase behaviour of bovine brain sphingomyelin alone and in the presence of soya phosphatidylethanolamine (which prefers the hexagonal (H II) configuration in isolation) has been investigated employing 31P NMR techniques. The influence of cholesterol on the phase behaviour of these systems has also been characterized. 2. 2. The 31P NMR spectra obtained from dry and hydrated sphingomyelin indicate that the local motion and conformation in the phosphate group region is similar to that of glycerol-based phospholipids. In addition, in the presence of excess water the bulk of the sphingomyelin displays 31P NMR spectra consistent with the bilayer configuration. Further, a hydrocarbon phase transition is apparent as the temperature is increased through 35°C, in agreement with calorimetric results. This transition is removed by equimolar cholesterol. 3. 3. In the absence of cholesterol, bovine brain sphingomyelin has a similar ability to stabilize the bilayer configuration when mixed with soya phosphatidylethanolamine as does 16 : 0/16 : 0 or egg yolk phosphatidylcholine, and relatively complete bilayer stabilization occurs at 30 mol% or higher sphingomyelin contents. The mixed systems containing sphingomyelin display much less tendency to adopt non-bilayer phases when the temperature is raised to 70°C, in contrast to their phosphatidylcholine-containing counterparts. 4. 4. In the presence of cholesterol, 30 mol% sphingomyelin/70 mol% soya phosphatidylethanolamine dispersions display a much greater affinity for the bilayer configuration than do similar phosphatidylcholine/soya phosphatidylethanolamine systems, which tend to adopt non-bilayer configurations at low and/or equimolar cholesterol levels.

Effects of divalent cations and pH on phosphatidylserine model membranes: A 31P NMR study

The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing 31P-NMR techniques. The addition of Ca 2+ results in the observation of a “rigid lattice” 31P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T 1. This corresponds to a strong and specific headgroup immobilization by Ca 2+, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (H II) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.

Chemistry of Synthetic Bilayer Membranes

Abstract The preparation of bilayer membranes from synthetic dialkyl amphiphiles is described. According to the electron microscopic observation, the bilayer structure similar to that of biomembranes is formed spontaneously when dialkyldimethyl-ammonium bromides with C10-C20 alkyl groups are dispersed in water by sonication. The line broadening in the NMR spectrum strongly suggests that these synthetic bilayers constitute liquid crystalline or solid phases. The hydrophilic head group may be sulfonium and modified ammonium moieties. The bilayer assembly is also formed from dialkyl amphiphiles with anionic head groups: (phosphate, sulfonate, and carboxylate), from nonionic dialkyl amphiphiles (oligomeric ethylene oxide) moiety) and from zwitterionic dialkyl amphiphiles. These bilayers incorporate equimolar cholesterol fairly readily. The redistribution of catalyst (a cholesterol derivative) and substrate (p-nitrophenyl palmitate) molecules is very slow when they are tightly bound to the aggregate. The intra...

Polymorphic phase behaviour of lipid mixtures as detected by 31P NMR. Evidence that cholesterol may destabilize bilayer structure in membrane systems containing phosphatidylethanolamine

1. 1. 31P NMR is a useful analytical technique for the study of the polymorphic phase behaviour of hydrated phospholipids in excess water. Such possibilities arise due to lateral diffusion of phospholipids which, in non-bilayer phases, produce additional motional averaging mechanisms. This results in distinctive 31P NMR spectra for lipids in the bilayer phase, hexagonal (H 11) phase, or phases such as the inverted micellar, cubic or rhombic. 2. 2. The polymorphic phase behaviour of soya phosphatidylethanolamine and mixtures of soya phosphatidylethanolamine with saturated and unsaturated phosphatidylcholines has been investigated. As the temperature is decreased below −10°C soya phosphatidylethanolamine tends to enter the bilayer phase. At higher temperatures (0–50°C) the hexagonal (H 11) phase is observed. The addition of 50 mol% egg yolk or (liquid-crystalline) 16 : 0 16 : 0 phosphatidylcholine is sufficient to stabilize the bilayer phase. 3. 3. The addition of equimolar cholesterol to 18 : 1 c 18 : 1 c phosphatidylethanolamine results in the bilayer-hexagonal (H 11) transition moving to lower temperatures. Similarly, the addition of equimolar cholesterol to soya phosphatidylethanolamine/35 mol% egg yolk phosphatidylcholine mixtures destabilizes the component in the bilayer phase. Alternatively, the addition of cholesterol to mixtures containing 30 mol% 16 : 0 16 : 0 phosphatidylcholine stabilizes the bilayer configuration. These results are discussed with regard to the observation that biological membranes containing high concentrations of phosphatidylethanolamine contain little or no cholesterol. Itis suggested that cholesterol may be excluded because its presence would disrupt bilayer structure. 4. 4. It is proposed that the bilayer structure of lipids in biomembranes may be in dynamic equilibrium with other available phases. In the light of such possibilities a mechanism of “flip-flop” phenomena in biological membranes is suggested.

Lateral diffusion in phospholipid multibilayers measured by fluorescence recovery after photobleaching.

The method of fluorescence recovery after photobleaching has been used to measure the temperature dependence of the lateral diffusion coefficients (D) of two fluorescent lipid analogues in phospholipid multibilayers of various compositions. The probes employed were 3,3-dioctadecyloxocarbocyanine (diO-C18(3) and N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE). In fluid egg phosphatidylcholine multibilayers at 25 degrees C, D was about 4 X 10(-8) cm2/s for NBD-PE and 1.5 X 10(-7) cm2/s for diO-C18(3) and was moderately temperature dependent (2-fold change over 10 degrees C). Equimolar cholesterol reduced D for NBD-PE in these multibilayers by a factor of 2. A greater than 100-fold decrease in D was detected in dimyristoylphosphatidylcholine multibilayers at approximately 23 degree C, which coincides with the gel-to-liquid-crystalline transition temperature, Tm (D 5 X 10(-8) cm2/s at T greater than Tm to D less than 5 X 10(-10) cm2/s at T less than Tm). Equimolar cholesterol abolished this transition behavior, raising D below Tm and decreasing D above Tm. These results confirm and extend previous studies of lateral diffusion employing magnetic resonance and other optical techniques and give additional confidence in the fluorescence methods.