Monolayers lecithin-cholesterol

This lamellar phase is formed of alternate sheets of lipid and water. The lipidic sheets containing the lecithin and the cholesterol are made of two superposed layers of oriented molecules. Each of these two monolayers is mixed and consists of lecithin and cholesterol molecules arranged side by side with their paraffinic ends turned toward the inside of the sheet and their polar groups (phosphatidyl choline group for lecithin and hydroxyl group for the cholesterol) outward—i.e., toward the adjacent sheet of water. This constitution of each of the two mono-layers forming the lipidic sheet is in conformity with the conclusion arising from the study of mixed monolayers of cholesterol and lecithin spread on the free surface of water (1). 

Surface Potential. Shah and Schulman have proposed that interaction between dipoles of uncharged lipids in mixed monolayers should result in a change in surface potential, AV. Linearity of the relation of AV to composition of the lecithin-cholesterol monolayer was taken to indicate absence of interaction (17). We do not agree with Shah and Schulman, since surface potential does not appear to be a valid criterion for assaying interaction between dipoles of uncharged lipids. Except for the speculations of Shah and Schulman (17, 18), there is neither theoretical nor experimental evidence that dipole-dipole interactions have... 

The infrared evidence for hydrogen bonding between cholesterol and lecithin in chloroform solution is no evidence of a similar complex in the monolayer but suggests such a possibility. It does not exclude the hydrophobic bonding suggested by Chapman from NMR studies of the aqueous suspensions of equimolar mixtures of cholesterol and lecithin (3). 

Figure I. Schematic of interaction of calcium ion with dioleoyl, egg, and di-palmitoyl lecithins, and of egg lecithin-cholesterol monolayers...

B Egg lecithin-cholesterol monolayers. Increased spacing between phosphate groups results in strong internal salt linkage preventing binding of Ca2+... 

Concept of Intermolecular Cavities in Mixed Monolayers. In mixed monolayers a deviation in average area per molecule occurs if one component forms expanded and the other condensed monolayers. This reduction in average area per molecule has been attributed by previous workers to an interaction between components in the mixed monolayer. However, this need not be true in all cases where condensation occurs. In several instances the condensation can be explained on the basis of steric considerations in the mixed monolayers. Although the following discussion is based on lecithin-cholesterol monolayers, it is equally applicable to other mixed monolayers. 

Dipalmitoyl Lecithin—Cholesterol Monolayers. The average area per molecule in dipalmitoyl lecithin-cholesterol monolayers shows deviation at low surface pressures, whereas at 30 dynes per cm. it follows the additivity rule (Figures 8 and 9) (42). The surface pressure—area curve of dipalmitoyl lecithin monolayers is liquid-expanded up to 30 dynes per cm., whereas above this surface pressure it is relatively incompressible (42). Figures 10b and c represent the structures of the dipalmitoyl... 

Figure 8. Average area per molecule of dipalmitoyl lecithin-cholesterol monolayers at various surface pressures...

Egg Lecithin—Cholesterol Monolayers. The average area per molecule in egg lecithin-cholesterol monolayers shows deviation from the additivity rule at all surface pressures (42). The deviation in this case could be explained by the presence of molecular cavities caused by the kink in the oleoyl chain of egg lecithin, which would reduce the average area per molecule at low as well as high surface pressures (Figure lOg). 

The optimum condensation at molecular ratios of 3 to 1 and 1 to 3 in egg lecithin-cholesterol monolayers and 1 to 1 in dipalmitoyl lecithin-cholesterol monolayers (42) do not imply complex formation between lecithin and cholesterol but rather suggest average geometrical arrangements of these molecules. 

Recently Bourges, Small, and Dervichian (5) reported that a para-crystalline lamellar structure of egg lecithin can solubilize cholesterol up to a maximum of one molecule of cholesterol per molecule of lecithin. However, they conclude that this should not be considered as a molecular association but rather the consequence of the relative arrangement of the molecules in the lamellar structure which is a mutual (solid) solution of lecithin and cholesterol. They also reported that the state of compression in the lamellar structure corresponds to that of a highly compressed mixed monolayer of lecithin-cholesterol. The NMR results of Chapman and Penkett (8) also appear to indicate that solubilization of cholesterol in egg lecithin dispersions results in a highly packed structure in which fatty acyl chains possess little molecular motion. Our results from lecithin-cholesterol monolayers also suggest that these mixed mono-layers are two-dimensional solutions with no specific interaction and that the apparent condensation in some instances is caused by the steric factors of the fatty acyl chains and not by the interaction or association between lecithin and cholesterol. 

In addition, it is generally observed for mixtures of aliphatic hydrocarbons that if there is a difference in molecular size, a negative excess volume of mixing results (21). For the lecithin-cholesterol mixtures molecular area decreases (Figure 7), but it does not decrease with the mixture of the lecithins which forms an ideal mixture. Thus, the mixing of cholesterol and lecithin monolayers is consistent with bulk hydrocarbon mixtures with respect to both positive excess heats and negative excess volumes of mixing. 

Cholesterol and lecithin form completely miscible solutions in mono-layers at very low surface pressures, characterized by excess positive heats and excess negative areas of mixing. At elevated surface pressures, phase separation occurs. Since these solutions conform to regular solution theory, the hydrocarbon domain of the monolayer makes the major contribution to the heats of mixing. The polar region of the monolayer may... 

Table II. Comparison of Monolayer Properties for Stearic Acid, Cholesterol and Lecithin at 25°C...

J. Tinoco and D. J. McIntosh, Interactions Between Cholesterol and Lecithin in Monolayers at an Air-Water Interface, Chem. Phys. Lipids 4, 72-84 (1970). 

Funasaki and coworkers [12] studied intermolecular interactions in mixed micelle formation of polyoxyethylene (15) dihydrocholesterol (DHC-E015) with polyoxyethylene (7) dodecanol (C12E07), by means of volumetric methods. The objective of their study was to investigate the condensing effect of cholesterol on phospholipid monolayers and the membrane-thickening action of cholesterol on lecithin bilayers. The volume... 

Our present ideas about the nature of biological membranes, which are so fundamental to all biochemical processes, are based on the Singer-Nicholson mosaic model. This model of the membrane is based on a phospholipid bilayer that is, however, asymmetrical. In the outside monolayer, phosphatidylcholine (lecithin) predominates, whereas the inner monolayer on the cytoplasmic side is rich in a mixture of phos-phatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Cholesterol molecules are also inserted into the bilayer, with their 3-hydroxyl group pointed toward the aqueous side. The hydrophobic fatty acid tails and the steran skeleton of cholesterol... 

Figure 5. Mixed monolayers of cholesterol-1,2-dimyristoyl-3-lecithin system at 23°C., pH 6, and 5 dynes per cm.

Discontinuities are seen in the relationship between increase in film pressure, An, and lipid composition following the injection of globulin under monolayers of lecithin-dihydro-ceramide lactoside and lecithin-cholesterol mixtures. The breaks occur at 80 mole % C 16-dihydrocaramide lactoside and 50 mole % cholesterol. Between 0 and 80 mole % lactoside and between 0 and 50 mole % cholesterol the mixed films behave as pure lecithin. Two possible explanations are the formation of complexes, having molar ratios of lecithin-lactoside 1 to 4 and lecithin-cholesterol 1 to 1 and/or the effect of monolayer configurations (surface micelles). In this model, lecithin is at the periphery of the surface micelle and shields the other lipid from interaction with globulin. 

Although glycosphingolipids are the specific lipid components in the antigen-antibody complex, their activity is markedly enhanced by other (auxiliary) lipids such as lecithin and lecithin-cholesterol mixtures (15). The present study deals with the effect of lipid composition on the penetration of lactoside—cholesterol and lactoside—lecithin monolayers by rabbit y-globulin. We also investigated the lecithin-cholesterol system. Furthemore, since criteria for the existence of lipid-lipid complexes in monolayers are still few (8, 17), we have used infrared spectroscopy to examine lipid mixtures for the presence of complexes. 

On the one hand, the cholesterol-lactoside system did not show film contraction (unpublished data). This was expected since the monolayers of dihydroceramide lactosides (7) and of cholesterol are not compressible. On the other hand, lecithin-lactoside and lecithin-cholesterol systems did show contraction, which could have been predicted since the lecithin monolayer is of the expanded type and is very compressible. (The area per molecule of lecithin at 2 dynes per cm. is large, 110 sq. A., as opposed to 52 sq. A. for Ci -dihydroceramide lactoside and 40 sq. A. for cholesterol.)... 

The nature of the lecithin-cholesterol association is probably also that of a complex (1 to 1), in which the OH group of cholesterol is involved through hydrogen bonding. It is not known, however, whether the monolayer consists of a uniform population of bimolecular complexes or of configurations (surface micelles) in which cholesterol molecules are surrounded by an equal number of lecithin molecules. 

Van Deenen has reported (48) that the mixed monolayers of dide-canoyl lecithin-cholesterol follow the additivity rule of molecular areas even though this lecithin forms expanded monolayers. This can be explained similarly by an intermolecular cavity of smaller height, which cannot accommodate cholesterol (Figures lOd and 4d). 

The concept of ion-dipole interaction between lecithin and cholesterol has been suggested by many workers for the packing of these lipids in myelin or in the cell membrane (18, 19, 52). This concept is not supported by the surface potential measurements of mixed monolayers of lecithin and cholesterol. In contrast to dicetyl phosphate-cholesterol... 

Similar reasoning accounts for the condensation of monolayers of dioleoyl lecithin by cholesterol reported by Van Deenen (48) (Figure lOh). 

Even though 1,2-dilinoleoyl and l-palmitoyl-2-linolenoyl lecithins form more expanded monolayers than egg lecithin, their mixed mono-layers with cholesterol follow the additivity rule (48). This can be explained as follows. At low surface pressures, these lecithins have greater intermolecular spacing and hence form intermolecular cavities of smaller height which cannot accommodate cholesterol molecules (Figure 4e). At high surface pressure, the linoleoyl and linolenoyl chains, as opposed to oleoyl chains, do not form cavities in the monolayer (Figure lOi). 

Monolayers of dicetyl phosphate-cholesterol follow the additivity rule for average area per molecule, whereas lecithin—cholesterol mono-layers deviate from it. The reverse is true for the additivity rule of average potential per molecule. Thus, the surface potential indicates that there is no interaction (or complex formation) between lecithin and cholesterol, but there is ion-dipole interaction between dicetyl phosphate and cholesterol as well as between phosphatidic acid and cholesterol. 

The apparent condensation of mixed monolayers of lecithin in the presence of cholesterol is explained by a consideration of molecular cavities or vacancies caused by thermal motion of fatty acyl chains, the height of these cavities being influenced by the length, inclination, and degree of unsaturation (especially the proportion of monounsaturation) of the fatty acyl chains and the extent of compression of the monolayer. Mono-layers are liquefied by the presence of unsaturated fatty acyl chains or by the addition of cholesterol. 

Since cholesterol is an important component of many biological membranes mixtures of polymerizable lipids with this sterol are of great interest. In mixed monolayers of natural lipids with cholesterol a pronounced condensation effect , i.e. a reduction of the mean area per molecule of phospholipid is observed68. This influence of cholesterol on diacetylenic lecithin (18, n = 12), however, is not very significant (Fig. 32). Photopolymerization indicates phase separation in this system. Apparently due to the large hydrophobic interactions between the long hydrocarbon chains of... 

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