Mixed monolayers of cholesterol with fatty acids

Mixed Monolayers of Cholesterol with Fatty Acids KINSI MOTOMURA, TAKASHI TERAZONO, HIROSI MATUO, AND RYOHEI MATUURA Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812, Japan

Received September 30, 1975; accepted February 7, 1976 The surface pressure versus mean area curves of the mixed monolayers of cholesterol with n-long chain fatty acids was obtained at various compositions and temperatures. I t was observed that the area occupied by the expanded monolayer of fatty acid becomes somewhat smaller by addition of cholesterol. The surface pressure of the transition from expanded to condensed state varied with the composition and temperature, whereas the area occupied by the condensed monolayer composed of cholesterol and fatty acid satisfied the additivity rule. The two-dimensional phase diagram of the cholesterol-myristic acid system was constructed and the partial molar enthalpy of myristic acid was evaluated. Itwas concluded that cholesterol and fatty acid are miscible in the expanded state and immiscible in the condensed state and that the interaction between them is not specific but large enough to form the homogeneous expanded monolayer.

INTRODUCTION

MATERIALS AND METHODS

Many investigations have been made on mixed monolayers of cholesterol and lipids since Leathes reported that cholesterol caused a diminution of the area occupied by lipids in monolayers (1-3). The significant effect of cholesterol has been interpreted in terms of the packing of hydrocarbon chains (4), the molecular association (2), or the phase transition (3, 5) in the mixed monolayers. Thermodynamic studies by Cadenhead and Phillips (5) and by Gershfeld and Pagano (6, 7) are still insufficient to explain the condensing effect of cholesterol. In order to make this effect clear it is necessary to reveal the thermodynamic state and miscibility of cholesterol and lipids in monolayers. This is carried out in a manner similar to that applied by us in the study of mixed monolayers of fatty acids (8-11). In the present paper we investigate the surface pressure of mixed monolayers of cholesterol with n-long chain fatty acids and clarify their miscibility and state in the monolayers with the aid of thermodynamics of monolayers.

Myristic, pentadecanoic, and stearic acids were purified by fractional distillation and recrystallization from petroleum ether. Cholesterol was obtained from Fluka A. G., Switzerland, and recrystallized from ethanol. Benzene purified carefully by the standard procedure was used as the spreading solvent. As the substrate 0.001 N hydrochloric acid was employed which was prepared from twice distilled water and hydrochloric acid (Wako super special grade). The surface pressure of monolayers was measured by a Wilhelmy type surface balance. The temperature of substrate was being kept constant within +0.2°C during the experiment. RESULTS AND DISCUSSION It is well established that the transition surface pressure from expanded to condensed phase of a film-forming substance is affected by addition of other film-forming substance. It is also known that the area of expanded film is generally decreased by addition of

cholesterol. Therefore, the measurement of 52

Journal of Colloid and ln~erlau Science, Vol. 57,

No. 1, October 1976

Copyright O 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.

CHOLESTEROL-FATTY ACID MONOLAYERS transition surface pressure of the mixed monolayer of myristic acid, which forms a wellcharacterized expanded film, with cholesterol, which forms a condensed film, may be expected to provide information regarding the action and thermodynamic state of cholesterol in the mixed monolayer. Surface pressure, % versus mean area per molecule, A, curves of the cholesterol-myristic acid system were measured at various compositions at 25°C and some of them are shown in Fig. 1. The condensing effect of cholesterol is seen at large values of the mole fraction of myristic acid in the monolayer, x2~, though not so remarkable. It is also observed that the kink point on the curve, which is regarded as the starting point of transformation to the condensed phase, varies with the composition. A small amount of cholesterol increases the surface pressure at the kink point. With further addition of cholesterol, it becomes constant. Finally the mixed monolayer becomes a condensed fihn analogous to that of pure cholesterol. Cadenhead and Phillips have reported such results for systems of cholesterol and expanded components (5). The variation of the transition surface pressure, ~-% against x2" is depicted by the curve 1 in Fig. 2. The value of 7r~q is almost constant below x2"

53

30 G

E 20

2 ,

¢3

v

c -o

o

3

~0

u 45~'O--'o''°O o

N

0.90

0.95

1.0,

x2

FIG. 2. Transition pressure versus mole fraction of myristic acid curves of the cholesterol-myristic acid monolayer: 1, 25°C; 2, 20°C; 3, 15°C.

= 0.96, but it decreases with increasing x2" in the range from 0.96 to 1. Similar observations are made in the cases of 20 and 15°C, which are also given by the curves 2 and 3, respectively, in Fig. 2. The above results indicate that cholesterol and myristic acid interact with each other to such an extent that they are miscible in the expanded state. The information about their miscibility in the condensed state can be obtained from thermodynamic consideration on the behavior of 7r eq v e r s u s

x27r c u r v e .

x2. . . .

1

[-13

or

~2o

x~*'¢ > x~"'e,

c

[2]

where the superscripts c and e indicate the condensed and expanded phases, respectively, when the relation

~1o

a ~-

20

30

40

F I 6 . 1. S u r f a c e p r e s s u r e cholesterol-myristic

versus mean

acid

monolayer

a r e a c u r v e s of at

25°C:

a e-

(x2 . . . .

x2*"O

x (oaVax2-,%,~,~ < 0

50

A ( ,~2/molec. )

1,

1 (myristic acid); 2, 0.98; 3, 0.94; 4, 0.88; 5, 0.50; 6, 0.30; 7, 0 (cholesterol).

x~ '~=

o

According to (11, Eqs. [223 and E35"]), the fact that Ozreq/OX2"~'e< 0 in the range from x2. . . . 0.96 to 1 requires either

30

the

1

D]

is assumed to hold. a designates the mean molar area. In the former case, cholesterol and myristic acid are immiscib]e with each other in the condensed state and their twodimensional state is represented by a phase

Journal of Colloid and Interface Science, Vol. 57, No. 1, October 1976

54

MOTOMURA E T AL.

2oi 1

20

30 A(

~2/molec.

40 )

FIG. 3. Surface pressure versus m e a n area curves of the cholesterol-stearic acid monolayer at 25°C: 1,

x~~ = 1 (stearic acid); 2, 0.83; 3, 0.67; 4, 0.50; S, 0.33; 6, 0.17; 7, 0 (cholesterol). diagram similar to that of the eutectic system. In the latter case, on the other hand, the condensed film emerging at the transition point is a homogeneous mixture of the two components and the phase diagram possesses a feature similar to that of the azeotropic system. Taking account of the experimental observation that the transition point has a nearly constant surface pressure and becomes vague with decrease in x2~ in the range below x2~ = 0.96, it seems reasonable to presume that the cholesterol-myristic acid system has a state in which an expanded two-component monolayer exists in equilibrium with condensed monolayers of cholesterol and myristic acid, the composition being in the vicinity of x2~ = 0.96. In order to further assure the immiscibility of cholesterol and myristic acid in the condensed state, the mixed monolayer of cholesterol with stearic acid which forms a condensed film was examined at various compositions at 25°C. The results are shown in Fig. 3. The mean area per molecule taken at a fixed surface pressure is plotted against the mole fraction of stearic acid in Fig. 4. I t is seen that the cholesterol-stearic acid system satisfies the additivity rule A = Xl*Al Jr- x~'M2,

[-4]

where Ai is the area per molecule of the component i in the pure monolayer, within experimental errors. Cholesterol and stearic acid are so different in the chemical structure that they cannot form an ideal mixed monolayer. So, we m a y now say that they are immiscible in the condensed state in all compositions. We already know that the monolayer of myristic acid is immiscible even with the condensed monolayers of behenic and cerotic acids (8, 9). In the case of the cholesterol-myristic acid system, the additivity rule is satisfied in the range of small x~ "~, as shown in Fig. 5. These facts prove that cholesterol and myristic acid are immiscible in the condensed film. The two-dimensional behavior of the cholesterol-myristic acid system appeals to be markedly different from that of the cholesterolstearic acid system, but the difference is not a qualitative one. In Figs. 6 and 7, the ~r versus A curves for the mixed monolayers of cholesterol and pentadecanoic acid at 25 and

35 30

3

.-. 25

"x,q,q

25 '20

2o o

4d

~35<

35

30

30

25

25

0

0.5

1.0

x7 FIG. 4. M e a n area versus mole fraction of stearic acid curves of the cholesterol-stearic acid monolayer at 25°C: 1, 5 dyne/era; 2, 15 dyne/era.

Journal of Colloid and Interface Sdence. Vol. 57. No.rt, October 1976

CHOLESTEROL-FATTY ACID MONOLAYERS 40

55

40 1

35 ~ \

35

30

30

2

4

5

6

20 E

< C

>,10 25

25

20

20

d \

40"

2'0

'40

< 35

25

4b

30 A ( ,~2 / m o l e c . )

(

°°°

I

35

FIG. 7. Surface pressure versus mean area curves of the cholesterol-pentadecanoic acid monolayer at 15°C: 1, x2" = 1 (pentadecanoic acid); 2, 0.91; 3, 0.67; 4, 0.50; 5, 0.33; 6, 0.09; 7, 0 (cholesterol).

30 25

0

0.5

1.0

FIG. 5. Mean area versus mole fraction of myristic acid curves of the cholesterol-myristic acid monolayer at 25°C: 1, 5 dyne/cm; 2, 15 dyne/cm. 15°C, respectively, are shown for comparison. I t is clear that the behavior of cholesterolpentadecanoic acid system at higher temperatures is similar to that of cholesterol-myristic acid system and at lower temperatures to that of cholesterol-stearic acid system. We can now construct a phase diagram for

the cholesterol-myristic acid system. Taking up the data at 25°C from Fig. 2 and drawing a line through the transition surface pressures, we obtain the phase diagram shown in Fig. 8. The line AE represents the condition of equilibrium of the expanded two-component monolayer composed of cholesterol and myristic acid with the condensed monolayer of myristic acid. At the point E which is designated by 7req = 23.7 d y n e / c m and x2~ = 0.96, the expanded two-component monolayer is in equilibrium with the condensed monolayer of each component. Unfortunately, a line representing the equilibrium of the expanded two-component monolayer with the 30

20

E

D -41 E

E 20

B

ll ll

~o

=>,

t! l

v

iI

1o iI

20

30

40

/

50

t

A ( ,~,2/molec. )

c

FIG. 6. Surface pressure versus mean area curves of the cholesterol-pentadecanoic acid monolayer at 25°C: 1, x~*= 1 (pentadecanoic acid); 2, 0.98; 3, 0.91; 4, 0.83; 5, 0.50; 6, 0 (cholesterol).

~'

o.~o

o.~5

too

FIG. 8. Phase diagram of the cholesterol-myristic acid monolayer at 25°C.

Journal of Colloid and Interface Science,

Vol. 57, No. 1, October1976

56

MOTOMURA E T AL.

condensed monolayer of cholesterol could not be decided experimentally. It may be the reason for the unsuccessful observation of transition surface pressure that the slope of this line is too steep for the kink point to appear on the 7r versus A curves. However the slope of this line can be calculated with the aid of the thermodynamics developed in the preceding papers (10, 11). According to (11, Eq. 1,13]), the desired slope is expressed in the form (a~-~'Vax~'~,%,~ = ( a u l V a X , ' , O r , p , # (a~ . . . . a,-,e),

[5]

where 51 . . . .

[6]

al ~

and dl . . . .

a e -- x 2 " ' * ( a a ' / O x 2 " " ) r 4 , , , .

1'7]

The slope of line AE, on the other hand, is derived from (10, Eq. 1,106]) as = (a#~e/ax~',e)~,~,J(a~ . . . .

a~"e),

I-8]

where a2 . . . .

a2e

[9~

and

a2 . . . .

ae + x.-,e(0ao/ax2-,%,~,..

[IO]

Making use of the equation Xlll"e ( 0 # I V 0X2V' e) T, p, i-

+ X~.e(O~,e/OX~"e)~.~,~ = O,

[11]

which is an analog of the Gibbs-Duhem equation, Eq. 1,5] can be combined with Eq. E8] to give the expression 1 ] - -

\ax2",e/T,p

~

--

slope of the curve of the expanded twocomponent film in equilibrium with the condensed cholesterol film from the slope of curve AE at the point E. Since Oae/Ox2 '~'e cannot be evaluated without serious errors from the ~r versus A curves in Fig. 1, the exact values of al *,e and c~2=,e are hardly obtained. Therefore, we introduce the assumption that

which is rewritten in the form (a2. . . .

•2.,e)/(ai . . . .

[14]

where Eqs. 1'6] and [9] have been used. Supposing further the line AE to be a straight line, then the calculation of Eq. [-12] can be carried out easily. The line bearing this slope is depicted by the dotted line CE in Fig. 8. Now we can say that the area surrounded by AEC is the one-phase region representing the expanded two-component monolayer composed of cholesterol and myristic acid, the area AEB is the two-phase region representing the coexistence of the expanded two-component and condensed myristic acid Inonolayers, the area CED is the two-phase region of the expanded two-component and condensed cholesterol monolayers, and the area above the line BD is the two-phase region of the condensed monolayers of cholesterol and myristic acid. The point E can be called a two-dimensional eutectic point. Now we can determine the entropy change associated with the transformation of myristic acid from the expanded two-component monolayer to the condensed myristic acid monolayer. The variation of equilibrium surface pressure with temperature is related to the change of the partial molar entropy, ~2, by

=

~

al 't'e) = a2c/al c,

( o,~eq/OT) ~,~2.,.

xi *,e dl~, c ~ dlf,e x

1,13]

al*,e/alc ~_ a2*,e/a2e,

[12]

LkOxgy'eIT,pJAE

(~ . . . .

~2.,*)/(a~ . . . .

as',O, [15]

in which

the system being in the state indicated by the point E. It is now possible to calculate the Journal of Colloid and Interface Science, Vol. 57, No. 1, October 1976

+ a2°(o'r°/OT)p

[16]

CHOLESTEROL-FATTY ACID MONOLAYERS

-3.0 qa

"6 E "-4 t,9

-2.0

el,' ¢"4

it"-

t

-1.o

I.~CM it-

0

0.96

0.98

1.00

3'r, e

x2 FIG. 9. Partial molar enthalpy change of myristic acid versus mole fraction of myristic acid curve of the cholesterol-myristic acid monolayer at 20°C.

and

X

(Or'/OT)p,x~.,.~.,.+a2e(O~'°/aT),,

E173

where we have used (11, Eqs. ]-2-], Ell], [31"]). In the above equation, di denotes the partial molar area and V° the surface tension of pure water. Rearrangement of Eq. E15] results in

~2e-~2*=[a~ c - a e-xI',e(oaVox2*,e),,~,~-] X (&r°q/OT).,.2 . . . . (a2 e - ~2")(&r*,/OT).,~,,.

+ Ea'+ xl "'e(oaVox~''e) ~ , ~ , _ a,e3 x (o.~o/OT).,~.,,.,.,. (a:o-a~')(av°/aT)~.

E18-1

Estimating the right-hand side of Eq. [-18-] by making use of experimental results on the assumption that A2*,e is adequately replaced by the extrapolated area of the myristic acid monolayer, A2* by the corresponding area of stearic acid, A2 * by 18 A2/molecule, A2 * by 20/~2/molecule, and (O~e/OT)p,~ o by 0, we can obtain the partial molar entropy change of myristic acid. Moreover, the partial molar enthalpy change is calculated by the relation /~2~--/z2" = T ( ~ ° -

~2").

El9-]

57

The numerical result for /~2e - /~e is plotted against x~~,* in Fig. 9. The values are not so different as to discuss their distinction. Therefore, we may say that there is no specific interaction between cholesterol and myristic acid in the monolayer state, though the concentration range is too narrow. In conclusion, cholesterol and fatty acid are miscible with each other in the expanded state, while they are immiscible in the condensed state. The two-dimensional phase diagram of cholesterol-myristic acid system is constructed by reading the transition surface pressure and applying the thermodynamics of monolayers. It is clarified that the interaction between cholesterol and fatty acid in the monolayer is not specific but large enough to form the homogeneous expanded monolayer. REFERENCES 1. LEATI~S, J. B., Lancet 208, 853 (1925). 2. DERVlCHIAN, D. G., "Surface Phenomena in Chemistry and Biology," (J. F. Danielli, K. G. A. Pankhurst, and A. C. Riddiford, Eds.), p. 70. Pergamon Press, London, 1958. 3. PHILLIPS, M. C., "Progress in Surface and Membrane Science," (J. F. Danielli, M. D. Rosenberg, and D. A. Cadenhead, Eds.) 5, p. 139. Academic Press, New York, 1972. 4. ADAm,N. K. ANDJESSOI%G., Proc. Roy. Soc. A120, 473 (1928). 5. CADENIIEAD,D. A. AND PHILLIPS, M. C., Advan. Chem. Ser. 84, 131 (1968). 6. PAGANO, R. E. AND GERSrlFELD, N. L., J. Phys. Chem. 76, 1238 (1972). 7. GERSHFELD, N. L. AND PAGANO, R. E., J. Phys. Chem. 76, 1244 (1972). 8. KURAMOTO,N., SEKITA, K., MOTOMURA,K., AND MATUURA, R., Mere. Fac. Sci. Kyushu Univ. C8, 67 (1972). 9. MATUURA, R., MOTOMURA,K., AND KURAMOTO, N., Proc. Int. Congr. Surface Active Substances 6th, 2, 351 (1972). 10. MOTOMURA,K., J. Colloid Interface Sci. 48, 307 (1974). 11. MOTO•URA, K., SEKITA, K., AND MATUURA, R., J. Colloid Interface Sci. 48, 319 (1974).

Journal of Colloid and Interface Science. Vol. 57, No. 1, October 1976