Interaction of furosemide with lipid membranes

Summary The interaction of furosemide with different phospholipids was investigated. Its influence on the lipid structure was inferred from its effect on the phase transition properties of lipids and on the conductance of planar bilayer membranes. The thermotropic properties of dipalmitoyl phosphatidylcholine, phosphatidylethanolamine (natural), dipalmitoyl phosphatidylethanolamine, brain sphingomyelin, brain cerebrosides and phosphatidylserine in the presence and absence of furosemide were investigated by differential scanning calorimetry,. The modifying effect of furosemide seems to be strongest on phosphatidylethanolamine (natural) and sphingomyelin bilayers. The propensity of furosemide to decrease the electrical resistance of planar lipid membranes was also studied and it is shown that the drug facilitates the transport of ions. Partition coefficients of furosemide between lipid bilayers and water were measured.Abbreviations DSC differential scanning calorimetry - PLM planar lipid membranes - DPPC dipalmitoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - PE phosphatidyl ethanol     

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.     

Mixed micelles of sphingomyelin and phosphatidylcholine with nonionic surfactants. Effect of temperature and surfactant polydispersity.

Mixed micelle formation of the polydisperse nonionic surfactant Triton X-100 as well as its homogeneous analogue, p-(1,1,3,3-tetramethylbutyl)-phenoxynonaoxyethylene glycol (OPE-9), with bovine brain sphingomyelin or dipalmitoyl phosphatidylcholine has been characterized by column chromatography on 6% agarose. At 40 degrees C, mixtures of OPE-9 and either sphingomyelin or dipalmitoyl phosphatidylcholine give a narrow size distribution for mixed micelles. A this temperature the size distribution of Triton X-100-containing mixed micelles is complicated because of the polydispersity of the oxyethylene chains. At 20 degrees C narrow size distributions are observed for mixed micelles of sphingomyelin/Triton X-100 and sphingomyelin/OPE-9 up to at least 0.06 mol fraction of lipid. For dipalmitoyl phosphatidylcholine this is observed only with OPE-9. At intermediate mol fractions of lipid (around 0.25), two populations of mixed micelles exist for sphingomyelin/Trition X-100, sphingomyelin/OPE-9, and dipalmitoyl phosphatidylcholine/OPE-9. At high mol fractions of lipid only one population of mixed micelles again exists. At 20 degrees C, sphingoymelin forms a clear solution with Triton X-100 and OPE-9 to a lipid mol fraction of at least 0.46 and 0.67, respectively. Dipalmitoyl phosphatidylcholine forms a clear solution with OPE-9 to a lipid mol fraction of at least 0.57 at the same temperature. Triton X-100 and dipalmitoyl phosphatidylcholine do not form stable, clear solutions at 20 degrees C unless the lipid mol fraction is extremely low. These results show that surfactant polydispersity and temperature are important determinants in the solubilization of lipids by nonionic surfactants. It is also shown that pure surfactant micelles and lipid/surfactant mixed micelles do not co-exist in the same solution.     

Lipid phase states influence glycophorin reconstitution

Reconstitution of glycophorin into dimyristoyl phosphatidylcholine and sphingomyelin vesicles was sub-maximal below the phase transition temperatures of these lipids. Reconstitution of glycophorin into diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine liposomes was maximal within a range of temperatures below the phase transition temperatures of dimyristoyl phosphatidylcholine and sphingomyelin but above the phase transition temperatures of diisostearoyl phosphatidylcholine and dioleoyl phosphatidylcholine. These findings indicate a greater tendency for reconstitution of glycophorin into fluid as opposed to solid lipid phases.     

Sphingomyelin exhibits greatly enhanced protection compared with egg yolk phosphatidylcholine against detergent bile salts

Inclusion of phosphatidylcholine within bile salt micelles protects against bile salt-induced cytotoxicity. In addition to phosphatidylcholine, bile may contain significant amounts of sphingomyelin, particularly under cholestatic conditions. We compared protective effects of egg yolk phosphatidylcholine (similar to phosphatidylcholine in bile), egg yolk sphingomyelin (mainly 16:0 acyl chains) and dipalmitoyl phosphatidylcholine against taurocholate in complementary in vitro studies. Upon addition of taurocholate-containing micelles to sonicated egg yolk phosphatidylcholine vesicles, subsequent micellization of the vesicular bilayer proved to be retarded when phospholipids had also been included in these micelles in the rank order: egg yolk phosphatidylcholine < dipalmitoyl phosphatidylcholine < sphingomyelin. Hemolysis of erythrocytes and LDH release by CaCo-2 cells after addition of taurocholate micelles were strongly reduced by including small amounts of sphingomyelin or dipalmitoyl phosphatidylcholine in these micelles (PL/(PL + BS) >/= 0.1), whereas egg yolk phosphatidylcholine provided less protection. Amounts of non-phospholipid-associated bile salts (thought to be responsible for cytotoxicity) in egg yolk phosphatidylcholine-containing micelles were significantly higher than in corresponding sphingomyelin- or dipalmitoyl phosphatidylcholine-containing micelles (tested at PL/(PL + BS) ratios 0.1, 0.15, and 0.2). LDH release upon incubation of CaCo-2 cells with taurocholate simple micelles at these so-called "intermixed micellar-vesicular" concentrations was identical to LDH release upon incubation with corresponding taurocholate-phospholipid mixed micelles. In conclusion, we found greatly enhanced protective effects of sphingomyelin and dipalmitoyl phosphatidylcholine compared to egg yolk phosphatidylcholine against bile salt-induced cytotoxicity, related to different amounts of non-phospholipid-associated bile salts. These findings may be relevant for protection against bile salt-induced cytotoxicity in vivo.     

The effect of gramicidin A on the temperature dependence of water permeation through liposomal membranes prepared from phosphatidylcholines with different chains lengths.

The permeation of water through liposomal membranes composed of various saturated phosphatidylcholine plus gramicidin A was studied as a function of temperature. 1. The presence of gramicidin in the liposomal bilayers caused an increase in water permeability. Below the phase transition temperature this effect could be measured quite clearly in all the systems we tested, but the extent of the increase was largely dependent on the length of the hydrocarbon chains. 2. Increasing amounts of gramicidin caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel-liquid crystalline phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. Differential scanning calorimetry analysis of the system containing these relatively small amounts of gramicidin still showed a clear transition from the liquid crystalline to the gel state with only a slight reduction in the enthalpy change. 3. In liposomes composed of dimyristoyl, dipalmitoyl and saturated egg phosphatidylcholine there was a concomitant decrease in the activation energy of water permeation in the presence of gramicidin below and above the phase transition temperature. The activation energy for water permeation through longer chained distearoyl phosphatidylcholine liposomal bilayers was the same with or without gramicidin in the bilayer. 4. It is concluded that the ability of gramicidin to form conducting channels in a gel state bilayer depends on the thickness of the paraffin core.     

Haptenic activity of galactosyl ceramide and its topographical distribution on liposomal membranes. I. Effect of cholesterol incorporation

The relation between the immune-reaction of phosphatidylcholine liposomes containing spin-labeled galactosyl ceramide with or without cholesterol and the topographical distribution of the glycolipid in membranes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and antibody binding occurred, irrespectively of the presence of cholesterol, though the motion of the fatty acyl chain of spin-labeled galactosyl ceramide was restricted by cholesterol. In dipalmitoyl phosphatidylcholine liposomes, unlike in egg yolk phosphatidylcholine liposomes, the immune-reaction depended on the cholesterol content. The electron spin resonance (ESR) spectra of spin-labeled galactosyl ceramide in dipalmitoyl phosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled galactosyl ceramide in the liposomes. Without cholesterol, most of the spin-labeled galactosyl ceramide was clustered on the dipalmitoyl phosphatidylcholine membrane, but with increase of cholesterol, random distribution of hapten on the membrane increased. The cholesterol-dependent change in the topographical distribution of hapten on the membranes was parallel with that of immune reactivity. 'Aggregates' composed solely of galactosyl ceramide did not show any binding activity with antibody. The findings suggest that the recognition of galactosyl ceramide by antibody depended on the topographical distribution of hapten molecules. Phosphatidylcholine and/or cholesterol may play roles as 'spacers' for the proper distribution of 'active' haptens on the membranes. The optimum density of haptens properly distributed on liposomal membranes is discussed.     

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269-282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37 degrees C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (approximately 1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization.     

Effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine 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.     

Perturbation of phospholipid membranes by juvenile hormone

The effects of juvenile hormone and its analogs Altozar 4E and ZR-777 5E on the phase properties of liposomes prepared from dipalmitoyl phosphatidyl-choline (DPPC) have been examined by differential scanning calorimetry. Each of these compounds reduced the co-operativity of the gel to liquid-crystalline phase transition, which is manifest as a distinct broadening of the main transition endotherm, and split the transition into two distinguishable components centered at 34 and 37°C. However, there was no significant change in enthalpy of the main phase transition, suggesting that juvenile hormone and its analogs perturb the bilayer primarily in the vicinity of the phospholipid headgroups. Moreover, this perturbation does not appear to influence bilayer permeability since the osmotic swelling rates of liposomes prepared from either phosphatidylcholine or dipalmitoyl phosphatidylcholine that contained up to 33 mol% juvenile hormone were not significantly different from the swelling rates of corresponding liposomes containing no juvenile hormone. Splitting of the transition endotherm into two peaks appeared to be peculiar to compounds possessing juvenile hormone activity. A mixture of fatty acid methyl esters broadened the main transition of DPPC, but did not split the endotherm or shift the transition midpoint, and the insect hormone ecdysone had no discernible effect on the DPPC transition apart from eliminating the pretransition. The data indicate that juvenile hormone and its analogs can modulate the physical properties of phospholipid bilayers, and raise the prospect that some of the physiological effects of this hormone may be achieved through its influence on the molecular organization of membrane lipid.     

The affinity of cholesterol for phosphatidylcholine and sphingomyelin.

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for N-palmitoyl sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.     

Intrinsic perturbing ability of alkanols in lipid bilayers

The proportionality constant between the equipotency concentrations of a series of solutes and the fraction of a solute in the membrane phase is directly related to the solute to lipid mol ratio. Experimental measurements of partition coefficient and of several alkanol-induced effects show that the solute/lipid mol ratlos for a series of alkanols are not constant at their equipotency concentrations. The deviations in the solute/lipid ratios are similar in the various systems, and these deviations seem to depend primarily upon the chain length and branching in alkanols. It is suggested that such intrinsic differences in the perturbing ability of alcohols arise from a specificity of interaction between alkanols and lipid bilayer. We have correlated partition coefficients (in n-octanol, in egg phosphatidylcholine liposomes, and in dipalmitoyl phosphatidylcholine liposomes) for thirteen alkanols to the equipotency concentrations for their ability to modify the order-disorder thermotropic transition in dipalmitoyl phosphatidylcholine, ability to stimulate the hydrolysis of phosphatidylcholine in a bilayer by bee venom phospholipase A₂, and for the activation of the galactoside transport system in Escherichia coli. Significant correlation is found between equipotency concentrations for perturbing the order-disorder transition, the activation of phospholipase A₂-catalyzed hydrolysis and the activation of galactoside transport system.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Effect of liposome composition on the activity of detergent-solubilized acylcoenzyme A: cholesterol acyltransferase

Acylcoenzyme A:cholesterol acyltransferase (ACAT) was solubilized from Ehrlich ascites cell microsomes with Triton X-100. After removal of the detergent, ACAT activity per mg protein was reduced by 50 to 65% as compared with untreated microsomes. When this microsomal extract was combined with liposomes composed of cholesterol and egg phosphatidylcholine, the ACAT activity increased 5.4- to 6.7-fold. Under these conditions sucrose density gradient centrifugation indicated that more than 50% of the added lipid was incorporated into vesicles having the same density as the ACAT activity, suggesting the formation of a complex. ACAT activity increased 2.9-fold when the phosphatidylcholine content of the liposomes was raised from 0.5 to 5.0 mumol/mg microsomal protein. By contrast, the ACAT activity increased only 42% when the cholesterol content of the liposomes was raised from 0.17 to 0.57 mumol/mg microsomal protein. Addition of phosphatidylethanolamine to the liposomes produced little change in ACAT activity, whereas the activity was reduced by 25 and 50%, respectively, when sphingomyelin or phosphatidylserine was added. ACAT activity was five times higher when the liposomes were prepared from dioleoylphosphatidylcholine than from saturated phosphatidylcholines, including hydrogenated egg yolk, dimyristoyl or dipalmitoyl phosphatidylcholine. Likewise, the ACAT activity with liposomes made from soybean or egg yolk phosphatidylcholine was almost 3.5-fold greater than with those prepared from the saturated phosphatidylcholines. These results are consistent with the view that the activity of ACAT can be modified by changes in the composition of the membrane lipids with which the enzyme is associated.     

Use of liposomes in probing the uptake of liposomal phosphatidylcholine by rabbit lung in vitro.

The purpose of this study was to characterize the uptake of liposomal phosphatidylcholine by lung tissue and its subcellular organelles. Multilamellar liposomes were prepared from egg yolk phosphatidylcholine, dicetyl phosphate, and cholesterol (molar ratio 7 : 2 : 1). Liposomal phosphatidylcholine labeled with [1-14C]dipalmitoyl phosphatidylcholine was taken up by lung slices and incorporated into subcellular organelles including lamellar bodies, mitochondria, and microsomes. In addition, when liposomes were incubated with lamellar bodies, mitochondria, or microsomes, the transfer of liposomal phosphatidylcholine to these subcellular fractions was facilitated by the cytosolic fraction. In tissue slice experiments after 1 h of incubation, about 86% of the total radioactivity absorbed by lung slices and subcellular organelles was recovered in phosphatidylcholine. The ratio of the radioactivity of fatty acids at 1- and 2-positions of dipalmitoyl phosphatidylcholine recovered from all fractions was nearly 1 : 1. This suggests that most phosphatidylcholine molecules were taken up intact. In conclusion, this study provides a method using liposomes as a tool for probing the phosphatidylcholine transfer mechanism in lung.     

Hydrolysis of phosphatidylcholine liposomes by phospholipases A2. Effects of the local anesthetic dibucaine.

(1) Dibucaine evokes a downward shift in the phase transition temperature of saturated phosphatidylcholines, while it also affects the pretransition. (2) The binding of dibucaine to phosphatidylcholine liposomes increases sharply when the lipid is transformed from the gel phase to the liquid-crystalline phase. (3) The activity of Naja naja phospholipase A2 towards dimyristoyl phosphatidylcholine liposomes is either stimulated or inhibited by dibucaine, depending on whether the substrate is in the gel or the liquid-crystalline state, respectively, whereas the activity of pancreatic phospholipase A2 is inhibited by the anesthetic irrespective of the physical state of the substrate. This observation is further substantiated by the results of studies on liposomes prepared from mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine. (4) The uptake of dibucaine by positively charged liposomes composed of phosphatidylcholine and stearylamine is considerably reduced in comparison with pure phosphatidylcholine liposomes. This decrease is paralleled by a reduction of the inhibitory and stimulatory effects of dibucaine on the hydrolysis of such liposomes by pancreatic and Naja naja phospholipase, respectively. (5) The inhibitory action of dibucaine towards the pancreatic phospholipase is lowered by increasing CaCl2 concentrations. This reduction is accompanied by a decreased uptake of anesthetic by the liposomes.     

The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure.

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Mixtures of analogues and phospholipid were also studied in monolayers. In all the bilayer systems studied cholesterol caused the greatest 'rigidifying' effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.     

Physicochemical properties of dipalmitoyl phosphatidylcholine after interaction with an apolipoprotein of pulmonary surfactant

We studied the interaction between an apolipoprotein of pulmonary surfactant and the principal lipid found in this material, dipalmitoyl phosphatidylcholine. The apolipoprotein was extracted from canine surfactant and purified to greater than 90% homogeneity. The apolipoprotein was mixed for 16 h at room temperature with dipalmitoyl phosphatidylcholine dispersed in a buffer containing 0.1 M NaCl and 3mM CaCl2. Unbound lipid, unbound protein, and recombinants of lipid and protein were separated by density gradient centrifugation. 71% of the apolipoprotein was found associated with dipalmitoyl phosphatidylcholine. In comparable experiments using bovine plasma albumin about 13% of the albumin was recovered with the lipid. The physicochemical state of the lipid in the apolipoprotein-lipid complex was modified after binding of the protein. A distinct phase transition at 42 degrees C could no longer be detected, and the rate of adsorption to an air-liquid interface of the apolipoprotein-lipid complex was greater than that of the lipid alone. Surface tension vs. surface area isotherms of the dipalmitoyl phosphatidylcholine-apolipoprotein materials, however, were similar to those exhibited by pure dipalmitoyl phosphatidylcholine. The results suggest a physiological role for this apolipoprotein. It may bind to dipalmitoyl phosphatidylcholine under conditions expected in vivo, and may modify the physical properties of the aggregated dipalmitoyl phosphatidylcholine to form domains of lipid in a liquid-crystalline array. The complex dipalmitoyl phosphatidylcholine and apolipoprotein would have the physical properties necessary for its physiological function, allowing it to absorb to the alveolar interface and reduce its surface tension to less than 10 dynes/cm. Dipalmitoyl phosphatidylcholine, by itself, is in a gel-crystalline array below its phase transition temperature (42 degrees C) and would be incapable of effecting these actions.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

Phospholipase A2 and reacylation of phospholipids

Transacetylation of labeled CoA-oleate and oleate into liposomes from dipalmitoyl phosphatidylcholine catalyzed by phospholipase A2 from rat liver mitochondria was studied. It was shown that the efficiency of CoA-oleate incorporation slightly exceeded that of oleic acid both in the phosphatidylcholine and lysophosphatidylcholine fractions. It was found also that some amount of the labeled substrate remains bound to the enzyme; the type of oleate and CoA-oleate binding differs, depending on their concentrations. The autonomy of lipid component formation in mitochondrial membranes is discussed.