Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.     

Encapsulation of hemoglobin in phospholipid liposomes: characterization and stability

Hemoglobin is encapsulated in liposomes of different lipid composition. The resulting dispersion consists primarily of multilamellar liposomes (hemosomes) of a wide particle size distribution (diameter ranging mainly between 0.1 and 1 micron). The encapsulation efficiency is significantly larger with liposomes containing negatively charged lipids as compared to liposomes made of phosphatidylcholine. The integrity of the phospholipid bilayer is maintained in the presence of hemoglobin. The reaction rate of CO binding to encapsulated hemoglobin is reduced compared to that of free hemoglobin, but it is still greater than that observed in red blood cells. Hemoglobin encapsulated in liposomes made from negatively charged phospholipids is less stable than hemoglobin entrapped in isoelectric phosphatidylcholine. The instability of hemoglobin is due to the protein interacting with the negatively charged lipid bilayer. This interaction leads in turn to hemoglobin denaturation, possibly involving the dissociation of the heme group from the heme-globin complex. The nature of the negatively charged phospholipid is important in promoting the interaction with hemoglobin, the effect being in the order phosphatidic acid greater than phosphatidylinositol congruent to phosphatidylglycerol greater than phosphatidylserine. The presence of equimolar amounts of cholesterol in the phospholipid bilayer has a stabilizing effect on hemoglobin. This effect is pronounced with saturated phospholipids, but it is also observed, though to a lesser extent, with unsaturated ones, indicating that the bilayer fluidity has a modulating effect. The presence of cholesterol possibly interferes with secondary interactions following the binding of hemoglobin to the negatively charged lipid bilayer.     

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.     

Influence of dicarboxylic phosphatidylcholines on phosphatidylcholine liposomes as revealed by gel chromatography and electron microscopy

The effect of dicarboxylic phosphatidylcholines (glutarylphosphatidylcholine) on the structural changes of phosphatidylcholine liposomes is examined by using multilamellar liposomes prepared with egg phosphatidylcholine or dipalmitoylphosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Investigations are performed by gel chromatography and electron microscopy. Glutarylphosphatidylcholine is in micellar form (rod-like micelles or globular micelles). The structures obtained depend on the fatty acid saturation of liposomes and on the charge of liposome (addition or not of dicetyl phosphate). With egg phosphatidylcholine/glutarylphosphatidylcholine dispersions, an aspect more similar to myelinic figures than liposomes is observed, while in the presence of dicetyl phosphate, liposomes similar to control egg phosphatidylcholine liposomes are obtained. Gel chromatography on Sepharose 4B and turbidity measurements prove that dicetyl phosphate increases the stability of egg phosphatidylcholine/glutarylphosphatidylcholine mixtures. On the other hand, in dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, incorporation of dicetyl phosphate destabilizes bilayer structure and the formation of mixed micelles occurs. Viscosity measurement shows, in the presence of dicetyl phosphate, an increased fluidity for dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, in agreement with the micellar organization. These data confirm that the disorganization of liposomal membranes by dicarboxylic phosphatidylcholine depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.     

Mechanism of Membrane Damage by Clostridium perfringens Alpha-Toxin

The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl-l -α-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N-terminal domain of alpha-toxin (AT_1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-l -α-phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT_1-246 did not. However, the C-terminal domain (AT_251-370) conferred binding to liposomes and the membrane-damaging activity on AT_1-246. These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.     

Methemoglobin reduction under near physiological conditions

Pure methemoglobin was prepared from fresh red cells and was used as substrate for methemoglobin reduction reaction. Two sources of methemoglobin reductase were used: (a) red cell hemolysate which was prepared by freezing and thawing of unwashed red cells; (b) purified methemoglobin reductase from bank blood. Methemoglobin reduction rate was measured in a mixture of pure methemoglobin (substrate) and hemolysate (enzyme). In other experiments the rate of methemoglobin reduction was measured in the above mixture with the addition of various other compounds such as NADH, cytochrome b5, and pure methemoglobin reductase. Only the addition of pure enzyme accelerated the rate of methemoglobin reduction. In other experiments, the rate of methemoglobin reduction was measured when the reduction reaction was carried out in the presence of various amounts of deoxyhemoglobin, globin, or albumin. It was shown that all proteins tested here decreased the reduction rate. It is concluded that (a) in the red cell, under normal conditions, only the activity of the methemoglobin reductase controls the speed of methemoglobin reduction, and (b) the inhibition of methemoglobin reduction by reduced hemoglobin is mostly nonspecific suggesting a noncompetitive reaction.     

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.     

Lipids favoring inverted phase enhance the ability of aerolysin to permeabilize liposome bilayers

Channel formation by the bacterial toxin aerolysin follows oligomerization of the protein to produce heptamers that are capable of inserting into lipid bilayers. How insertion occurs is not understood, not only for aerolysin but also for other proteins that can penetrate membranes. We have studied aerolysin channel formation by measuring dye leakage from large unilamellar egg phosphatidylcholine vesicles containing varying amounts of other lipids. The rate of leakage was enhanced in a dose-dependent manner by the presence of phosphatidylethanolamine, diacylglycerol, cholesterol, or hexadecane, all of which are known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. Phosphatidylethanolamine molecular species with low L-H transition temperatures had the largest effects on aerolysin activity. In contrast, the presence in the egg phosphatidylcholine liposomes of lipids that are known to stabilize the lamellar phase, such as sphingomyelin and saturated phosphatidylcholines, reduced the rate of channel formation, as did the presence of lysophosphatidylcholine, which favors positive membrane curvature. When two different lipids that favor hexagonal phase were present with egg PC in the liposomes, their stimulatory effects were additive. Phosphatidylethanolamine and lysophosphatidylcholine canceled each other's effect on channel formation.     

Influence of phosphatidylcholine/cholesterol molar ratios in liposomes on cholesterol reactivity with cholesterol:oxygen oxidoreductase]

The reactivity of sonicated phosphatidylcholine-cholesterol liposomes with cholesterol : oxygene oxydoreductase, an enzyme which catalyses the oxidation of the 3 beta hydroxyl group of cholesterol to a ketone group, is compared with that of ternary system phosphatidylcholine-cholesterol-Thesit. Regardless to the phosphatidylcholines nature and the phosphatidylcholine/cholesterol molar ratio (R), the enzymatic oxidation rate of liposomal cholesterol is slower than when the reaction is developed in the present of Thesit, a surfactif agent which destroyes the lamellar particles. This is true whether Thesit is added during preparation of dispersions or during incubation with cholesterol oxydase. The enzymatic oxydation rate of cholesterol of ternary systems phosphatidylcholine-cholesterol-Thesit is independent of the (R) value and the phosphatidylcholine fatty acid unsaturation, whereas that of phosphatidylcholine-cholesterol dispersions depends on these two parameters. The reaction rate increases in the order: dipalmitoylphosphatidylcholine to yolk egg phosphatidylcholines, and dioleylphosphatidylcholine. The optimal conditions for cholesterol oxidation were found to be R = 0.5. This result is not affected by the phosphatidylcholines nature. In order to explain these data, various hypotheses are considered. In particular, the weak liposomal cholesterol reactivity with cholesterol oxidase could result from an inhibitory effect on the enzyme-substrate combination due to the polar phosphorylcholine groups.     

Phosphatidylcholine composition of emulsions influences triacylglycerol lipolysis and clearance from plasma

Sonicated emulsions containing triolein, a specific phosphatidylcholine and cholesterol were prepared. Bolus doses were injected intravenously into rats and plasma clearance kinetics and organ uptakes were determined. Emulsion triacylglycerol lipolysis by rat heart lipoprotein lipase was measured in vitro. Phosphatidylcholine molecular species influenced emulsion metabolism in vivo and in vitro. Emulsions containing saturated phosphatidylcholines at temperatures below their melting points were poor substrates for lipoprotein lipase, compared with those stabilized by mixed chain phosphatidylcholines. Distearoylphosphatidylcholine stimulated hepatic uptake compared with emulsions made with egg yolk phosphatidylcholine, which modeled chylomicrons closely. Emulsion populations with the same surface compositions but with mean diameters of 700-800 A and 1100-1300 A were metabolized similarly, suggesting that, within the normal chylomicron size range, size alone does not determine the disposition of triacylglycerol-rich emulsions or lipoproteins.     

Action of pancreatic phospholipase A2 on phosphatidylcholine bilayers in different physical states.

1. Saturated and unsaturated phosphatidylcholines, dispersed as liposomes in water, can be hydrolysed by phospholipase A2 from pig pancreas. A pure saturated phosphatidylcholine is hydrolysed only near its transition temperature. An unsaturated phosphatidylcholine is hydrolysed preferentially near its transition temperature, but hydrolysis can occur also above the transition temperature, albeit at a much lower rate. 2. An equimolar mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, which shows cocrystallization of the paraffin chains, is hydrolyzed between 25 and 40 degrees C with a maximum at 32 degrees C, in agreement with the calorimetric scan of the phase transition. 3. An equimolar mixture of dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine, which shows a monotectic behaviour, is hydrolysed at all temperatures. Hydrolysis is maximal at 0 and 40 degrees C, at which temperatures dilauroyl phosphatidylcholine and distearoyl phosphatidylcholine undergo their phase transition, respectively. 4. Both in the mixture showing cocrystallization and in the mixture in which phase separation occurs, the phosphatidylcholine species with the shorter fatty acid chains is hydrolysed at a higher rate than the longer chain fatty acid species. 5. Hydrolysis is inhibited by the presence of cholesterol in liposomes prepared of saturated phosphatidylcholine. Inhibition is complete at a cholesterol concentration of 35 mol %. Subsequent addition of filipin and amphotericin B to the mixed cholesterol-phosphatidylcholine liposomes overcomes the inhibitory effect of cholesterol.     

Regulation of trout gill AMP deaminase by lipid bilayers. Effects of phospholipid composition and temperature

Trout gill AMP deaminase is inhibited by liposomes made of synthetic phosphatidylcholines containing higher saturated fatty acids. A preincubation of 1 hr, at 4 degrees C, was necessary to obtain the maximal effect. At 4 or 25 degrees C, these phospholipids modified essentially the substrate affinity of the enzyme by increasing the Michaelis constant proportionally to the length of the fatty acid chain. At 13 degrees C, the liposomes decreased the Hill coefficient also, thus inducing a negative cooperativity. Natural phosphatidylcholine and phosphatidylserine were without significant effect on gill AMP deaminase while natural sphingomyelin exhibited a similar effect to that shown in the presence of synthetic phosphatidylcholines. These results are discussed in relation to a possible effect of sphingomyelins in vivo.     

A method for determination of saturated phosphatidylcholine

Phosphatidylcholine preparations containing saturated and unsaturated molecular species were subjected to KMnO(4)-NaIO(4) oxidation in aqueous acetic acid, which left only disaturated species intact. After the oxidation, the remaining intact phosphatidylcholine was separated by thin-layer chromatography. The procedure could be used as a simple and rapid method for microdetermination of the saturated species in phosphatidylcholine preparations containing more than 0.1 micro mole of the saturated species. The contents of the saturated species in native phosphatidylcholines obtained from rat lung tissue and washings by this procedure were 35.7% and 58.3%, respectively.     

Permeability properties of liposomes prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin

Liposomes have been prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin.Permeability properties of liposomes thus prepared were studied toward glucose. The glucose permeability of liposomes with saturated lecithins (dipalmitoyllecithin and dimyristoyllecithin) and sphingomyelin appears to be more strongly temperature dependent than that of liposomes with lecithin containing unsaturated fatty acyl chains (egg and rat liver lecithins). The permeability of glucose through vesicles of dipalmitoyllecithin or dimyristoyllecithin was enhanced drastically at their transition temperatures, while the incorporation of about 25 mole% of egg lecithin into liposomes of saturated lecithins suppressed the enhanced permeation rates of glucose above the transition temperatures.The incorporation of small amounts of cholesterol enhanced the temperature-dependent permeability of glucose through the bilayer of saturated lecithins or sphingomyelin. This tendency was best shown in the case of dipalmitoyl-lecithin, in which 20 mole% of cholesterol had the most stimulating effect on the temperature-dependent permeability. The introduction of more than 33 mole% of cholesterol showed, however, reduced effects on the temperature-dependent permeability through liposomes with saturated lecithins or sphingomyelin. It was also shown that cholesterol had a much larger effect on the regulation of the temperature-dependent permeability of liposomes prepared with saturated lecithins or sphingomyelin than on that of liposomes prepared with phospholipids containing unsaturated fatty acids.     

Influence of physical conditions on the oxidation of hemoglobin during freeze-drying

The potential influence of some physical conditions--dialysis, crystallization, concentration, pH, and temperature--on the amount of methemoglobin obtained after freeze-drying of hemoglobin has been studied. Among these parameters, pH and crystallization influence the oxidation. In acid medium (pH 5), the oxygen saturation is better than that obtained for pH 8. Crystalline hemoglobin leads to a methemoglobin rate significantly lower (29% versus 49%) than untreated hemoglobin. Methemoglobin is continuously formed during desiccation even at the lowest temperature. Although it has been possible to lessen the denaturation of hemoglobin by the choice of a definite preliminary treatment of the samples, we were not able to reduce methemoglobin to low and physiological values. However, the results obtained with crystalline hemoglobin make it possible to propose mechanisms for the oxidation of the hemoprotein.     

Proteolysis in human erythrocytes is triggered only by selected oxidative stressing agents

Human erythrocytes have been treated with different agents producing oxidative stress. Diamide, tetrathionate, chromate, cystamine and iodate preferentially influenced the cellular redox state by oxidation of free and protein thiol groups leaving the redox state of hemoglobin virtually unaffected. None of these compounds was able to stimulate the proteolysis. By contrast, phenylhydrazine, nitrite, hydrogen peroxide, ter-butylhydroperoxide, cumene hydroperoxide and copper-ascorbate caused a noticeable oxidation of hemoglobin to methemoglobin. These latter agents, except nitrite and copper-ascorbate, triggered proteolysis. Identical results have been obtained in a ghost-free hemolysate. The fraction containing the proteolytic activity was isolated from hemolysate and tested on native or oxidant-treated hemoglobin. The proteolysis was stimulated by all agents able to produce methemoglobin. It is concluded that proteolysis correlated to an unbalance of cellular redox state. The results obtained with isolated and recombined fractions suggests that increased proteolysis does not depend on the removal of the effect of protease(s) inhibitor(s). Since all agents stimulating proteolysis are able to generate free radicals, it seems that protein breakdown is triggered by the direct effect of these intermediates on proteins (mostly hemoglobin) without the involvement of radical species produced in the membranes by action of organic hydroperoxides. In addition, since nitrite and copper-ascorbate, which also oxidize hemoglobin by radical generation, are unable to stimulate proteolysis, it should be concluded that protein degradation induced by oxidative stress is a complex event induced only by specific agents.     

Nuclear magnetic resonance studies on liposomes: Effects of steroids on lecithin fatty acyl chain mobility

Summary The effects of fourteen sterols on the NMR spectra of liposomes derived from egg yolk phosphatidylcholines were studied by continuous-wave and Fourier-transform measurements at 60 MHz. Sterols were compared for their ability to broaden the acyl methylene resonances of phosphatidylcholine, when incorporated into liposomes at 25% molar ratio. The ratio of the phosphatidylcholine peak heights (acyl methylene: cholinen-methyl) was used as a criterion of the relative condensing activity for the different sterols. This ratio was inversely proportional to the molar volume of the incorporated sterol, as measured by the parachor of the compound. Small sterols had little condensing effect, and the larger sterols such as cholesterol and ergosterol had maximum condensing effects. The study confirmed the importance of the sterol side-chain at C-17 as a requirement for sterol-phospholipid interaction.     

Temperature-dependent spectral changes of a hemin-lipophilic imidazole complex incorporated into liposomes prepared from dipalmitoyl-, dimyristoyl- and egg yolk-phosphatidylcholine

The optical spectra of the hemin-lipophilic imidazole complex incorporated into liposomes prepared from three types of phosphatidylcholine were studied at various temperatures. The Soret peak of the system with dipalmitoyl- and dimyristoyl-phosphatidylcholine liposomes showed a marked bathochromic shift at the phase transition temperature, but in egg yolk phosphatidylcholine liposomes no spectral change was observed in the temperature range of 10° to 50°.     

Effects of the acyl chain composition of phosphatidylcholines on the stability of freeze-dried small liposomes in the presence of maltose.

The effects of the acyl chain composition of phosphatidylcholines (PCs) on the stability of small unilamellar vesicles during freeze-drying and rehydration in the presence of maltose were studied by monitoring the retention of a trapped marker, calcein, in the internal liposome compartment. In dipalmitoyl PC, beta-oleoyl-gamma-palmitoyl-PC and egg yolk PC liposomes, good or fair retentions (>50%) were observed in the presence of maltose, but maltose was ineffective in preserving retention in the dioleoyl PC (DOPC) liposomes (<10%). The extremely low retention in the DOPC liposome was ascribed to neither a formation of the inverted hexagonal phase of the liposomal membrane nor the fusion/aggregation of the liposomes in the drying-rehydration process. Differential scanning calorimetry measurements suggested that interactions of maltose with PC headgroups were essential to stabilizing the dry liposomes. These interactions were significant in the saturated or mixed chain liposomes but were markedly reduced in the DOPC liposomes.