Molecular interactions in lecithin-retinol monolayers

Molecular Interactions in Lecithin-Retinol Monolayers 1 O. A. ROELS A:ND D. O. SHAH Marine Biology Division, Lamont Geological Observatory of Columbia University, Palisades, New Yor]~ 10964 Received J u n e 17, 1968 INTRODUCTION

The exact biochemical function of vitamin A aldehyde (retinal) in the visual cycle has been elucidated, but its mode of action outside vision remains obscure, although most animals cannot survive without vitamin A. Since the B-vitamins fulfill their biological function by acting as coenzymes, many workers in the vitamin A field have searched for an analogous role for vitamin A (retino]) or its derivatives, without success. Lately, the surface properties of retinol have received increasing attention following the observation that retinol, added in vitro, causes changes in the structure and permeability of biological membranes. Glauert st al. (1) have shown that the addition of retinol in vitro to suspensions of rabbit erythrocytes caused vacuolization and hemolysis of the cells. Blough and Ottewill (2) induced expansion and disintegration of chorioallantoic membrane particles by adding high doses of retinol in vitro to an aqueous suspension of these particles. The observed changes resembled those induced in erythrocytes by retinol. Similar disruptive effects of high concentrations of in vitro added retinol on lysosomal (3) and mitochondria] (4) membranes have been reported. Roels et al. (5) and Guha and Rods (6) have shown that dietary retinol regulates the stability of rat liver lysosomes in vivo. Anderson et al. (7) showed that dietary retinol influences the structure of the rat erythroeyte in vivo, probably because of its effect on the membrane of the cell. 1 L a m o n t Geological tion No. 1268.

Observatory

Contribu-

Ries and Walker (8) studied monolayers of binary mixtures of a horizontally oriented polar polymer polyvinyl acetate with 1-octadecanol, methyl stearate, cholesterol and three different long-chain fatty acids. They found that the films of the polymer with the fatty acids collapsed at pressures far greater than those for the components, possibly indicating an important effect of the polarity of the vertical components on the collapse pressure of the mixed films. Bangham et al. (9) used mixed monolayers as model systems to study the lipid-lipid and lipid-protein interactions governing the structure and function of biological membranes. They proposed a possible mechanism for the retinol-induced alterations in membrane structure and permeability by showing that lecithin-cholesterol monolayers at an air-water interface are penetrated and expanded when retinol is injected into the aqueous phase. Using a similar approach, Shah and Schulman (10, 11) studied the influence of induced dipoles, metal ions and cholesterol on the characteristics of phospholipid monolayers. They demonstrated that cholesterol liquefies a lecithin monolayer: the sterol occupies the cavities created by the thermal motion of the fatty acyl chains of the lecithin molecule; the size of the cavities depends on the degree of saturation of the fatty acids and on the compression of the monolayer. These authors found no interaction between the hydroxyl group of cholesterol and the polar group of lecithin. The presence of retinol in egg lecithin monolayers (12) and bilayers (13) stabilizes these lipid systems against oxidative degradation by osmium tetroxide, whereas pure egg

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ROELS AND SHAH

lecithin monolayers are destroyed by this agent. The same workers have also demonstrated that retinol increased the hydration of mixed lipid myelinics of egg lecithinretinol, compared to egg lecithin-cholesterol or egg lecithin-d-a-tocopherol: both cholesterol and d-a-tocopherol expand the distance between the egg lecithin molecules, allowing deeper penetration of the aqueous layer into the hydrophobic area, thereby decreasing the thickness of the hydrophobic layer. In contrast, retinol increases the thickness of the hydrophilic layer whilst maintaining the thickness of the hydrophobic layer compared to that of monolayers of egg lecithin alone. These studies indicate a possible surfaceactive role of retinol in the regulation of membrane structure and permeability. In an attempt to define the possible role of retinol in the lipid-lipid associations in membranes, we have studied the surface properties of mixed monolayers containing dipalmitoyl lecithin-retinol and egg lecithin-retinol. EXPERIMENTAL METHODS 1. Materials. l-a-Dipalmitoyl lecithin was bought from Mann Research Laboratories, New York, New York, and egg lecithin, prepared according to Pangborn et al. (14, 15) was supplied by the Sylvana Chemical Company, Orange, New Jersey. Both ]ecithins gave single spots on thin-layer plates coated with silica gel (Adsorbosil-2, Applied Science Laboratories, College Park, Pennsylvania) with chloroform/methanol/ water (60/35/5, v/v/v) as einent. Chromatographically pure all-trans-retinol was obtained from Distillation Products Industries, Rochester, New York. All lipid solutions were prepared in a nitrogen atmosphere in hexane-ethanol (80/20, v/v) both of spectroscopic grade. Inorganic chemicals of reagent grade and twice-distilled water were used. The fatty acid composition of the egg lecithin, which contains approximately equal amounts of saturated and unsaturated fatty acids, has been reported previously (16).

2. Methods. The surface pressure was measured by a modified Wilhelmy plate method, and the surface potential was determined with the use of a radioactive

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FIG. 1. Surface pressure: area curves for mixed monolayers of dipalmitoyl lecithin-retinol at different molar ratios. electrode, as described previously by Shah and Schulman (16). The surface measurements were taken on subsolutions of O.02M NaCI at pH 6.0, 25°C. The state of the films was inferred from the mobility of talc particles sprinkled on the monolayer when a gentle stream of air was blown at the particles by means of a dropper. If the talc moves very little or not at all, the monolayer is in the gel or solid state, respectively. If the talc moves freely in the air stream, the monolayer is in the liquid state. The principle underlying these observations is that molecules in solid monolayers do not move past one another; in the gel state they move very little, and in the liquid state they move freely. Although this method is less quantitative than surface viscosity measurements to determine the state of monolayers, it is reproducible within 1 dyne/cm in determining changes in the state of monolayers (17). RESULTS Dipalmitoyl Lecithin-Retinol Monolayers. The surface pressure: area curves and the

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M O L E C U L A R I N T E R A C T I O N S IN L E C I T H I N - R E T I N O L M O N O L A Y E R S

surface potential: area curves of dipalmitoyl leeithin-retinol monolayers are shown in Figs. 1 and 2. The average area per molecule versus the molar ratio of the lipids in the monolayer at different surface pressures is shown in Fig. 3. The average potential per molecule for dipalmitoyl leeithin-retinol monolayers versus the molar ratio of the lipids in the monolayer at different surface pressures is shown in Fig. 4. The broken lines between the end points in Figs. 3 and 4 represent the additivity rule of molecular areas and of average potentials. Egg Lecithin-Retinol Monolayers. For egg lecithin-retinol monolayers, the average area per molecule versus the molar ratio of the lipids in the monolayer is shown in Fig. 5. The average area shows a deviation from the additivity rule at all surface pressures. The average potential versus the molar ratio of the lipids in these monolayers is

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given in Fig. 6, which shows a deviation from the additivity rule at all surface pressures. The State of the Monolayers. Monolayers of retinol are in the liquid state. Mixed monolayers of egg lecithin-retinol and dipalmitoyl leeithin-retinol are in the liquid state. Egg lecithin monolayers are in the liquid state. Although cholesterol monolayers are highly incompressible (i.e., they have steep surface pressure-area curves), they are in the liquid state. The dipMmitoyl lecithin monolayers are in the liquid state up to a surface pressure of 36-38 dynes/era, and in the gel state from 38-42 dynes/em.

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F i g u r e 1 shows t h a t all-trans-retinol has a limiting a r e a of 20A 2 p e r molecule. T h i s a r e a is s t r i k i n g l y s m a l l e r t h a n t h a t of isoste~ric acid, for w h i c h t h e l i m i t i n g a r e a is 2 8 . 2 A 2 p e r molecule, according to Hies a n d C o o k (18). T h e retinol m o n o l a y e r can b e compressed so t h a t t h e a v e r a g e a r e a of each molecule is o n e - t h i r d less t h a n t h a t o c c u p i e d b y one molecule of isostearic acid a t collapse pressure, a l t h o u g h b o t h molecules h a v e v e r y low collapse pressures: 15 d y n e s / c m for isostearic acid a n d 22 -4-1 d y n e s / c m for all-trans-retinol. T h e m o s t likely e x p l a n a t i o n for this d i s c r e p a n c y w o u l d a p p e a r to be t h e higher p o l a r i t y of retinol c o m p a r e d to isos-

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MOLECULAR INTERACTIONS IN LECITHIN-RETINOL MONOLAYERS

tearic acid. This higher polarity results from the system of conjugated double bonds in the aliphatic side chain of retinoh on compression of the monolayer, some retinol molecules may slip their polar chain of conjugated systems more deeply into the aqueous phase, resulting in an alternating sequence of retinol molecules penetrating EGG LECITHIN- RETINOL 28

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283

more or less deeply into the aqueous phase, enabling much closer packing than with isostearic acid, where the saturated aliphatic chain is highly hydrophobic. The deviation from additivity in average area per molecule at low pressures in dipalmitoyl ]ecithinretinol monolayers is similar to that in dipalmitoyl lecithin-cholesterol monolayers previously reported by Shah and Schulman (10). These deviations can be explained by consideration of the intermolecular cavities present in the monolayers: the height of these cavities depends upon the length and degree of saturation of the hydrocarbon chains of the f a t t y acids in lecithin, as well as on the state of compression of the monolayers and the temperature of the subsolution (10). The apparent reduction in average area per molecule at low surface pressures can be accounted for by the steric characteristics of f a t t y acyl chains. At low surface pressures, intermolecular cavities between the fatty acyl chains of dipalmitoyl lecithin can be occupied by the cholesterol or retinol molecules. At higher pressures, the fatty acyl chains of dipalmitoyl lecithin are compressed so that they would occupy an almost vertical position, perpendicular to the water surface. The cholesterol or the retinol molecules then obviously need space between the parallel f a t t y acyl chains of the dipalmitoyl lecithin molecules, and additivity is the rule. At the lower pressures, the cholesterol or retinol molecules can be accommodated between the f a t t y acyl chains of adjacent dipalmitoyl lecithin molecules and, therefore, the average area per molecule of the mixed monolayer at low surface pressures is less than what the additivity rule would predict. The surface potential of all-trans-retinol monolayers, shown in Fig. 2, is about 460 my at 21A 2 per molecule. This potential is considerably higher than that for long-chain alcohols such as octadecanol, which is 400410 mv at collapse pressure, according to Harkins and Fisher (19). All long-chain alcohols, including cholesterol, give a surface potential of 400-410 my at their limiting area. This increase of 50 mv in the surface potential of retinol is due to the presence of conjugated double bonds in the retinol molecules. These double bonds, which are located

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ROELS AND SHAH

near the polar hydroxyl group of the molecule, can act as induced dipoles in relation to surface potential. Hughes and Rideal (20) found that a double bond located near a polar group acts as an induced dipole in relation to the surface potential of the molecule. Shah and Schulman (10, 17) confirmed that the presence of a double bond near the polar group strikingly influences the surface potential of sphingomyelin and plasmalogen monolayers. I t has also been shown that a deviation in the average potential per molecule indicates an ion-dipole or ion-ion interaction in the mixed monolayer (10). As shown in Fig. 4, dipalmitoyl lecithin-retinol monolayers show a deviation from additivity in the average potential per molecule at all surface pressures. This suggests that there is interaction between the polar groups of these molecules, thereby changing the average surface dipole of the molecules in the mixed monolayer. I t is important to note that for cholesterol-dipalmitoyl lecithin monolayers, the average potentials per molecule follow the additivity rule at all surface pressures (10), indicating a lack of interaction between the polar groups of cholesterol and lecithin. In contrast, there clearly is interaction between the polar groups of lecithin and retinol in their mixed monolayers. This is due to the presence of four conjugated double bond systems in retinol, which increase the polarity of the hydroxyl group of the molecule causing ion-dipole interaction between this hydroxyl group and the polar group of lecithin. Egg Lecithin-Retinol Monolayers. In contrast to dipalmitoyl lecithin-retinol monolayers, egg lecithin-retinol monolayers showed deviation from additivity in average area per molecule at all surface pressures. This can be explained on the basis of intermolecular cavities caused by the presence of unsaturated f a t t y acyl chains in the egg lecithin molecules. Similar results were reported with mixed monolayers of egg lecithin-cholesterol (10). The deviation from additivity can be explained by the presence of the molecular cavity caused by the kink in the oleoyl chain of egg lecithin which would reduce the average area per molecule at low as well as at high surface pressures: the kinks

TABLE I THICKNESS OF LAMELLAE IN MYELINICS

Parametera

Myelinic mean hydrophobic layer thick-

Egg lecithin only

Egg Egg lecithin: lecithin: retinol cholesterol 1:1 molar 1:1 molar

46.2 A 44.8 A 40.6 A 0.42b 0.45 0.68

ness Myelinic mean hydrophilic layer thickness

17.2 A 23.8 A 24.5 A 0.28 0.08 0.77

:~ of (hydrophilic + hydrophobic) layer thickness

63.4 A 68.6 A 65.1 A

a The mean hydrophobic layer thickness is based on at least 100 measurements. The mean hydrophilic layer thickness was determined from 50 measurements. b Standard deviation. in the unsaturated fatty acid chains of egg lecithin would provide cavities adquate to accommodate retinol or cholesterol molecules at these different surface pressures, thereby reducing the average area per molecule in the mixed monolayers. The changes in surface potential of egg lecithin-retinol monolayers versus the molar proportions of these compounds in the monolayer are comparable to those of dipalmitoyl lecithin-retinol. However, the deviation from the additivity rule of molecular areas is smaller in egg lecithin-retinol monolayers than in egg lecithin-cholesterol monolayers. Cholesterol forms highly condensed, liquid monolayers: their surface pressure:area curves are very stable, almost incompressible, but their surface viscosity is very low. In contrast, retinol forms expanded liquid monolayers, causing the smaller deviation from the additivity rule. Conclusion. The observations reported in this paper, together with data published earlier on mixed cholesterol-lecithin monolayers (10), give a simple explanation for observations made in studies of both lecithincholesterol and lecithin-retinol myelinics reported earlier from this laboratory: Anderson et al. (1967) prepared myelinics by dispersing egg lecithin alone, egg lecithin

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285

FIG. 7. Negatively-stained all-trans-retinol-egg lecithin myelinics have a mean thickness of 44.8A2 for the hydrophobic layer (A) and 23.8A~ for the hydrophilic layer (B). (X 414,296) and retinol, and egg lecithin and cholesterol, in water. Electron microscopic observations of the negatively-stained myelinics revealed parallel layers of clear hydrophobic and darkly-stained hydrophilic layers. A typical example of such a myelinic of all-trans-retinol and egg lecithin is shown in Figure 7. The authors found the thickness of the hydrophobic and hydrophilic layers in mixed myelinics reported in Table I. It is quite apparent from Table I that the addition of cholesterol to egg lecithin decreases the mean hydrophohic layer thickness compared to a myelinic prepared from egg lecithin alone, indicating a deeper penetration of the water layer into the hydrophobic region, with relatively little change

(1.7A) in the total width of the (hydrophobic + hydrophilic) layer thickness. The thickness of the hydrophilic layer of course increased proportionately. There was relatively little change in the mean hydrophobic layer thickness of the egg lecithinretinol myelinics, compared to pure egg lecithin, but the total width of the (hydrophobic + hydrophilic) thickness is considerably increased compared to the myelinics prepared from egg lecithin alone. This increase in over-all width is due to the significant increase of the hydrophilic layer thickness, compared to myelinics prepared from egg lecithin alone. The ion-dipole interaction between the hydroxyl group of retinol and the phosphate group of lecithin indicates the

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ROELS AND SHAH ,

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FIG. 8. Graphic representation of changes in the thickness of the hydrophilic and hydrophobic layers of mixedlipid myelinics. (/)--Pure egg lecithin myelinic. (2)--Egg lecithin-cholesterol myelinic. (3)--Egg lecithin-retinol myelinic. [] = hydrophilic layer. high polarity of the retinol hydrophilic group. This results in deeper penetration of the retinol hydroxyl group in the water layer than of the cholesterol hydroxyl group, and, thus, a thicker layer of water in this area is immobilized. Figure 8 illustrates graphically the differences in dimensions between the hydrophobic and hydrophilic layers of myelinics prepared from egg lecithin alone, from egg lecithin -k retinol, or from egg lecithin -k cholesterol. The schematic representation of the bilayer structure of the myelinic shown in Figure 8 was drawn from three-dimensional scale models of the molecules. It indicates how the insertion of cholesterol molecules between two molecules of egg lecithin enables deeper penetration of the aqueous layer into the hydrophobie area by forcing the egg lecithin molecules apart. (Fig. 8, No. 2). In contrast, the insertion of a retinol molecule between two adjacent egg lecithin molecules (Fig. 8, No. 3) does not allow deeper penetration of the aqueous layer, but binds a thicker layer of water to the polar groups of both retinol and the adjacent phospholipid molecules. The observation reported here, that there is a significant increase in surface potentil of alltrans-retinol monolayers compared to the potentials for long-chain alcohols, including cholesterol, and the ion-dipole or ion-ion

interaction in the retinol-lecithin monolayers, explains the strong binding of water molecules around the polar area of retinollecithin myelinics. The conjugated systems of double bonds close to the hydroxyl group of retinol explain its interaction with phospholipids. This greater polarity of the retinol hydroxyl group, resulting in ion-dipole formation between retinol and phospholipid in monolayers and in greater hydration of the polar groupsinretinol-phospholipid myelinics, may further contribute to our understanding of the role of retinol in membranes. The presence of retinol in the lipid moiety of membranes may greatly enhance the polarity of certain areas of the membrane, increasing the possibility of lipid-protein interaction. Moreover, such areas in membranes may also be particularly important for transport phenomena across membranes. SUMMARY The surface pressure and surface potential of mixed monolayers of dipalmitoyl lecithinretinol and egg lecithin-retinol were measured on subsolutions of O.02M NaCl. For dipalmitoyl lecithin-retinol monolayers, the average area per molecule versus the molar ratio of the components shows a deviation from the additivity rule at low

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MOLECULAR INTERACTIONS IN LECITHIN-I~ETINOL MONOLAYERS

2S7

surface pressures, but follows the additivity rule at surface pressures greater than 15 d y n e s / c m ; the surface potential per molecule versus the molar ratio of the components shows a deviation from the additivity rule at all surface pressures. For egg leeithin-retinol monolayers, the average area and the average potential per molecule show deviations from the additivity rule at all surface pressures. The deviation in average area can be explained on the basis of intermolecular cavities in the monolayer. The deviation in average potential per molecule suggests a strong ion-dipole interaction between the hydroxyl group of retinol and the polar group of lecithin. A comparison of mixed monolayers of lecithin-cholesterol and lecithin-retinol suggests t h a t the hydroxyl group of retinol associates with the polar group of lecithin, whereas the hydroxyl group of cholesterol does not. The difference in the surface properties of retinol and cholesterol is due to their different hydrophilic-hydrophobic balance. The surface potential of retinol is about 50 m v higher than t h a t of cholesterol or octadecanol. This is due to the induced dipoles of the double bonds located about the polar group of retinol. The interaction of the hydroxyl group of retinol with the polar group of lecithin indicates the high polarity of this group, explaining increased hydration in this area, enhancing the possibility of lipid-protein interaction. This observation m a y explain the influence of retinol on the structure and stability of biological membranes. ACKNOWLEDGMENTS

Health Service NIH gra~tts AM-04464 and GM13660. REFERENCES

We express our most sincere gratitude to the late Professor J. H. Schulman, whose inspiring stimulus made this work possible. This work was supported by the U.S. Public

19. I-IARKINS,W. D., ANDFISHER, E. K., J. Chem. Phys. 1,852 (1933). 20. HUGHES, A., AND RIDE&L, E. K., Proc. Roy. Soc. (London) Ai40, 253 (1933).

1. GLAUERT, A. M., DANIEL, M. R., LucY, J. A., AND DINGLE, J. T., Or. Cell Biol. 17,

111 (1963). 2. BLOUGH,H. A., AND OTTEWILL, R. I-I., Exptl. Cell Res. 44, 46 (1966). 3. DEDUVE, C., WATTIAUX, R., AND WIBO, M., Biochem. Pharmacol. 9, 97 (1962).

4. LucY, J. A., LUSCOMBE, M., AND DINGLE, J. T., Biochem. J. 89,419 (1963). 5. ROELS, O. A., TROUT, M., AND GUHA, A., Biochem. J. 97,353 (1965). 6. GUHA, A. AND ROELS, O. A., Biochim. Biophys. Acta 111,364 (1965). 7. ANDERSON,O. R., ROELS, O. A., AND PFISTER, R. M., Nature 213, 47 (1967). 8. RIGS, H. E. AND WALKER, D. C., J. Colloid. Sci. 16, 361 (1961). 9. BANGHAM, A. D., DINGLE, J. T., AND LUCY, J. A., Bioehem. J. 90,133 (1964). 10. SHAH, D. 0., AND SCHULMAN.,J. H., J. Lipid Res. 8,215 (1967). 11. SHAH, D. O., AND SCHULMAN, J. H., Advan. Chem. Ser., in press (1968). 12. DREHER, K. D., SCHULMAN,J. I~I., ANDERSON, O. R., AND ROELS, 0. A., J. Ultrastruct. Res. 19, 586 (1967). 13. ANDERSON, O. R., ROELS, O. A., DREHER, K. D., AND SCHULMAN,J. I-I., J. Ultrastruct. Res. 19, 600 (1967). 14. PANGBORN, M. C., J. Biol. Chem. 188, 471

(1951). ]5. PANGBORN,M. C., ALMEIDA,J. O., MALTANER, F., SILVERSTEIN, A. 1~'~., AND THOMPSON, W. R., "Cardiolipid Antigens," W.H.O. Monographs, No. 6, 2nd ed., Geneva (1955). 16. SHAH, D. O., AND SCHULMAN, ,~. I~I., J. Lipid Res. 6,341 (1965). 17. SHAH,D. O., AND SCHULMAN, J. H., Lipids 2,

21 (1967). 18. RIGS, H. E., AND COOK, H. D., Or. Colloid Sci.

9,535 (1954).

Journal of Colloidand Interface Science, Vo[.29, No. 2, February 1969