J. De Gier

Lipid composition and permeability of liposomes.

Abstract 1. 1. Layered latticed liquid crystals (liposomes) in dilute electrolyte solutions have been prepared from a number of synthetic lecithins with variations in length and number of double bonds of the paraffin chains. Using the property of these structures to act as practical, ideal osmometers, their permeability behaviour towards glycerol and erythritol has been studied. The penetration of such non-electrolytes into the liposome appears to be strongly temperature dependent in a way similar to that demonstrated for glycerol permeation into erythrocytes. 2. 2. Introduction of double bonds into the paraffin chains causes a marked enhancement of the permeability. The diffusion rate of glycerol into the liposomes of (distearoyl)lecithin, (1-stearoyl-2-oleoyl)lecithin, (dioleoyl)lecithin and (dilineoyl)lecithin, increases in that order. 3. 3. Decreasing chain length also increases the permeability which is demonstrated by comparing the swelling rate of liposomes prepared from (distearoyl)lecithin, (dipalmitoyl)lecithin and (dimyristoyl)lecithin and that of those prepared from (1-stearoyl-2-myristoyl)lecithin and (1-stearoyl-2-decanoyl)lecithin. 4. 4. Comparison of the permeability of the liposomes from (1-stearoyl-2-decanoyl)lecithin and (1-stearoyl-2-myristoyl)lecithin with those obtained from (dimyristoyl)lecithin and (dipalmitoyl)lecithin, respectively, suggests that, certainly at lower temperatures, the model structures of phospholipids with asymmetric chains are much more permeable than those of lecithins with chains of equal length but with the same total number of paraffin carbon atoms. 5. 5. Liposomes of mixtures of phospholipid and cholesterol normally demonstrate a decrease in permeability which is proportional to the concentration of cholesterol.

Osmotic properties and water permeability of phospholipid liquid crystals

Abstract A smectic mesophase (myelin-like structure, layer-latticed liquid crystal) of charged phospholipid behaves as an almost perfect osmometer when alkali metal salts, glucose, sucrose or mannitol are used as solutes. Other solutes show graded permeabilities (ethylurea, methylurea, ethylene glycol, ammonium acetate, propionamide, glycerol > urea > malonamide > erythritol). Osmotically driven swelling and shrinkage were followed by means of the changes in optical extinction; the validity of this was confirmed by determination of the volumes and interstitial spaces of centrifuged pellets. The rapid volume changes, determined optically, combined with the measured total external surface areas of the phospholipid dispersions, were used to calculate osmotic water permeability coefficients (0.8–16 μsec−1). The complementary effects of surface charge and electrolyte concentration on the equilibrium volumes of smectic mesophases were examined optically and by centrifugation. The volume of the particles decreased with increasing concentration of the electrolyte solution in which they formed. The intramellar spacings were not consistent with a single Hamaker constant over the range of electrolyte concentration and area-charge ratio studied. The constant was high (10-11 ergs) with low electrolyte concentrations and lower by a factor of about 100 with a high electrolyte concentration.

The preference of cholesterol for phosphatidylcholine in mixed phosphatidylcholine-phosphatidylethanolamine bilayers.

The following phosphatidylethanolamines were studied by differential scanning calorimetry: 1,2-dipalmitoleoyl-, 1,2-dioleoyl-, 1,2-dilauroyl-, 1,2-dielaidyl-, 1,2-dimyristoyl- and 1,2-dipalmitoyl-sn-glycero-3-phosphoryl-ethanolamine. The saturated and trans-unsaturated species underwent thermotropic phase transitions at temperatures about 20-30 degrees C higher than the corresponding phosphatidylcholines but the enthalpy changes were nearly identical. The transition temperatures for the cis-unsaturated species were about the same as those of the corresponding phosphatidylcholines but here the enthalpy change was markedly decreased as compared with the phosphatidylcholines. Freeze-fracture electron microscopy revealed phase changes from a lamellar to a hexagonal phase for 1,2-dipalmitoleoyl- and 1,2-dioleoyl-sn-glycero-phosphorylethanolamine at 20 and 0 degrees C respectively. At these temperatures no transitions were apparent in the calorimeter scan. Incorporation of increasing amounts of cholesterol into phosphatidylethanol-amine bilayers gradually decreased the enthalpy changes of the phase transition in the same manner as was demonstrated before for phosphatidylcholine/cholesterol mixtures. This was studied both for 1,2-dipalmitoleoyl- and 1,2-dimyristoyl-sn-glycerophosphorylethanolamine. In an equimolar mixture of 1,2-dioleoyl- and 1,2-dipalmitoylphosphoryl-ethanolamine, which showed phase separation, cholesterol preferentially decreased the transition of the lowest melting component. In equimolar mixtures of phosphatidylethanolamines and phosphatidylcholines, which showed phase separation, cholesterol preferentially abolished the transition of the phosphatidylcholine component present. This occurred both in experiments where the phosphatidylcholine was the lowest melting and where it was the highest melting component present in the mixture. These experiments strongly suggest that in phosphatidylcholine-phosphatidylethanolamine mixtures at temperatures where both components are in the liquid-crystalline state cholesterol is preferently associated with the phosphatidylcholine component in the mixture.

Psychoactive substance use and the risk of motor vehicle accidents

The driving performance is easily impaired as a consequence of the use of alcohol and/or licit and illicit drugs. However, the role of drugs other than alcohol in motor vehicle accidents has not been well established. The objective of this study was to estimate the association between psychoactive drug use and motor vehicle accidents requiring hospitalisation. A prospective observational case-control study was conducted in the Tilburg region of The Netherlands from May 2000 to August 2001. Cases were car or van drivers involved in road crashes needing hospitalisation. Demographic and trauma related data was collected from hospital and ambulance records. Urine and/or blood samples were collected on admission. Controls were drivers recruited at random while driving on public roads. Sampling was conducted by researchers, in close collaboration with the Tilburg police, covering different days of the week and times of the day. Respondents were interviewed and asked for a urine sample. If no urine sample could be collected, a blood sample was requested. All blood and urine samples were tested for alcohol and a number of licit and illicit drugs. The main outcome measures were odds ratios (OR) for injury crash associated with single or multiple use of several drugs by drivers. The risk for road trauma was increased for single use of benzodiazepines (adjusted OR 5.1 (95% Cl: 1.8-14.0)) and alcohol (blood alcohol concentrations of 0.50-0.79 g/l, adjusted OR 5.5 (95% Cl: 1.3-23.2) and >or=0.8 g/l, adjusted OR 15.5 (95% Cl: 7.1-33.9)). High relative risks were estimated for drivers using combinations of drugs (adjusted OR 6.1 (95% Cl: 2.6-14.1)) and those using a combination of drugs and alcohol (OR 112.2 (95% Cl: 14.1-892)). Increased risks, although not statistically significantly, were assessed for drivers using amphetamines, cocaine, or opiates. No increased risk for road trauma was found for drivers exposed to cannabis. The study concludes that drug use, especially alcohol, benzodiazepines and multiple drug use and drug-alcohol combinations, among vehicle drivers increases the risk for a road trauma accident requiring hospitalisation.

The effect of partial replacements of membrane cholesterol by other steroids on the osmotic fragility and glycerol permeability of erythrocytes

Abstract 1. 1. (A) Sonicated dispersions of lecithins were incubated with aliquots of washed human erythrocytes, leading to the removal of part of the cholesterol complement, eventually followed by lysis. (B) Sonicated dispersions of lecithin and cholesterol with one of a range of other steroids were prepared and incubated with aliquots of washed human erythrocytes. By a simple exchange process, the steroid composition of the erythrocytes were modified. 2. 2. (A) The osmotic fragility of erythrocytes was considerably increased after removal of part of the cholesterol complement with pure lecithin dispersions. (B) The osmotic fragilities of the modified erythrocytes were measured, and it was found that relatively small differences were detectable in view of the substantial replacement of part of the cholesterol with one of the other steroids. 3. 3. Significant differences in the glycerol permeabilities of the modified human and pig erythrocytes were demonstrated. In the presence of 3-ketosteroids, the rates of penetration of glycerol were significantly higher than those containing 3β-hydroxy steroids. Cholesterol-depleted erythrocytes also exhibited considerably increased glycerol permeability.

Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.

Effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidycholine liposomes

Aspects of osmotic properties of liposomes, prepared from synthetic lecithin, above, at and below the gel to liquid crystalline phase transition temperature are described. The experiments show that liposomal membranes with their lipids in the gel state are still permeable to water. The rate of water permeation changes drastically on passing the transition temperature. The water permeation has activation energies of 9.5 +/- 1.28 and 26.4 +/- 0.9 kcal/mol above and below the transition temperature, respectively, indicating that the diffusion processes take place by different mechanisms. With respect to the barrier properties of the liposomes in the vicinity of the transition temperature, the following conclusions can be made. (1) Studying the osmotic shrinkage of liposomes at a fixed temperature near the transition point, the experiments indicate that dimyristoyl phosphatidylcholine membranes are highly permeable to glucose under these conditions, where liquid and solid domains co-exist. Under the same conditions the osmotic experiments did not indicate a strong increase in glucose permeability of dipalmitoyl phosphatidylcholine membranes as compared to the situation above and below the transition temperature. (2) On the other hand, perturbations of the phase equilibrium by temperature varations resulted in a marked increase of the glucose permeation through dipalmitoyl phosphatidylcholine bilayers. Once a new phase equilibrium of liquid and solid regions is established the permeation rate of glucose is much less.

The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy

A new type of lipid organization is observed in mixtures of phosphatidyl-choline with cardiolipin (in the presence of Ca2+), monoglycosyldiglyceride and phosphatidylethanolamine (in the presence of cholesterol). This phase is characterised by an isotropic 31P NMR signal and is visualised by freeze-fracturing as particles and pits on the fracture faces of the lipid bilayer. As the most favourable model for this phase we propose the inverted micelle sandwiched in between the two monolayers of the lipid bilayer.

Hydrolysis of phosphatidylcholine liposomes by pancreatic phospholipase A2 at the transition temperature.

Abstract Dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine dispersed as liposomes in water are hydrolysed by phospholipase A 2 from pig pancreas only near the transition temperature of these lipids. Both above and below the transition temperature of the lipids the rate of hydrolysis in these model membranes is negligible. By contrast, hydrolysis of these liposomes with bee venom phospholipase A 2 occurs below, at the above the transition temperature. The concept of lateral compressibility of lipids at the transition temperature is firmly substantiated by these observations.

Some lipid characteristics of red cell membranes of various animal species.

Determinations were carried out of some lipid classes of red cell ghosts of sheep, ox, pig, man, rabbit and rat. In the studied red cell ghosts only small differences appeared to exist in the amounts of total lipids, phosphatides and free cholesterol. On the other hand, determinations of the individual types of phosphatides revealed a most pronounced deviation in the quantitative proportions of the major phosphatides. In the given order of animals a significant increase of the lecithin percentage of the ghost phosphatides was observed. Possible relations between the specific liquid composition and the properties of the red cell membranes are discussed.