Micellar solubilization of cholesterol and of mixtures of cholesterol with cholesteryl esters of C18 fatty acids by a nonionic surfactant

Micellar Solubilization of Cholesterol and of Mixtures of Cholesterol with Cholesteryl Esters of C18 Fatty Acids by a Nonionic Surfactant H A N S S C H O T T AND F A K R U L A. A. S A Y E E D 1

School of Pharmacy, Temple University, Philadelphia, Pennsylvania 19140 Received May 17, 1985; accepted September 4, 1985 Cholesterol and its binary mixtures with cholesteryl esters of four C~s fatty acids having from zero to three ethylenic double bonds were solubilized by aqueous micellar solutions of the nonionic suffactant, nonoxynol 10, between 27 and 51 °C. The equilibrium form of cholesterol under those conditions is the monohydrate. Its sohibilization limit increased linearly with temperature and nearly tripled between 27 and 51°C. In binary mixtures of cholesterol monohydmte and an ester, the esters did not reduce the solubilization limit of cholesterol monohydrate, unless the surfactant solution was saturated first with the ester. Solubilization ratios (solubility of ester in the presence of cholesterol monohydrate to solubility of ester solubilizedby itself) were independent of unsaturation but decreased with increasing temperature, from 0.7 at 27°C to 0.5 at 37°C and to 0.35 at 44°C. Higher unsaturation and higher temperatures resulted in increases in the combined amounts of lipids solubilized from binary mixtures of cholesterol monohydrate and an ester. Nonionic micelles bear some similarity to lipoproteins, which transport cholesterol and its esters in blood. If the solubilizing capacity of lipoproteins for these lipids is a factor in the formation and/or removal of lipid atheroscleroticdeposits, our observationsmay offeran explanation for the fact that foodstuffshigh in polyunsaturated fatty acids cause lessatherosclerosisthan those containing monounsaturated and saturated fatty acids. © 1986AcademicPress,Inc. INTRODUCTION The micellar solubilization o f cholesteryl esters o f C18 fatty acids by a representative nonionic surfactant, n o n o x y n o l I 0, has been reported (1). Its micelles have a structure resembling globular proteins (2) and lipoproteins (3) near their isoelectric points. Lipoproteins are the carders o f cholesterol and cholesteryl esters in blood (4). Atherosclerotic deposits in the tunica intima o f blood vessels are formed from lipids solubilized in low-density lipoproteins, and m a y be r e m o v e d by solubilization in high-density lipoproteins. T h e mechanism(s) o f lipid deposition and removal is u n k n o w n (4, 5). The capacity oflipoproteins to solubilize lipids m a y well affect the extent o f formation of atherosclerotic deposits by lipids a n d / o r their rel Present address: Hospital Products Division, Abbott Laboratories, North Chicago, III. 60064.

moval. The lipids being transported by lipoproteins in blood and deposited as fatty streaks and atherosclerotic plaque contain a mixture o f cholesterol and cholesteryl esters (6). D a t a on the solubility o f these lipids in lipoproteins are scarce. Using nonionic micelles as a model for lipoproteins, the micellar solubilization studies o f cholesteryl esters with C18 fatty acids by n o n o x y n o l 10 (1) are therefore being extended to mixtures o f these esters with cholesterol in the present work. The first step was to investigate the solubilization o f cholesterol alone by n o n o x y n o l 10. Most studies o f the micellar solubilization o f cholesterol relate to the formation and dissolution o f gallstones and, therefore, e m p l o y e d bile salts with or without lecithin ((7-9) and references cited therein). Synthetic ionic and nonionic surfactants and mixtures thereof, whose micellar properties differ considerably from those o f bile salts ( 10, 11) are also capable o f solubilizing cholesterol (12-20). Except for

144 0021-9797/86 $3.00 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

Journal of Colloid and Interface Science, Vol. 112, No. 1, July 1986

MICELLAR SOLUBILIZATION OF CHOLESTEROL (1), the micellar solubilization of cholesteryl esters of fatty acids has not been reported. EXPERIMENTAL

A. Materials The surfactant, nonoxynol 10 NF, tradename Igepal CO-710 of GAF Corporation, was an anhydrous liquid. Its cloud point in 1 or 2% solutions is 71°C. Its HLB is 13.6. The CMC, expressed as molarity, determined from surface tension versus log concentration plots, is given by the least-squares equation log CMC

- 4 . 9 9 8 + 249.3/T

[1]

(N = 4, r = 0.995) between T = 300 and 324 K. Cholesterol, cholesteryl oleate, linoleate, and linolenate were Kodak laboratory chemicals. Anhydrous cholesterol was primary standard, purified through the dibromide. Its monohydrate was prepared by recrystallization from 95% ethanol, followed by washing with water (21, 22). It was stored over water in the dark. Cholesteryl stearate was supplied by ICN Pharmaceuticals, Inc. The purity of cholesterol and its esters was determined by thin-layer chromatography on silica gel sheets by Kodak, which were activated at 110°C for 30 min. A spot of the solution of each lipid in chloroform was developed in a hexane-ether (94:6 v/v) mixture. After drying, the plates were sprayed with a 2,7-dichlorofluorescein solution. Illumination with ultraviolet light revealed single spots in every case. Glacial acetic acid used for the colorimetric assay was "aldehyde-free, suitable for cholesterol determination" (J. T. Baker Chemical Co.). All other chemicals were ACS reagent grade. The water used was double distilled.

B. Colorimetric Cholesterol Assay The method of Zlatkis and Zak (23), which determines total (i.e., free and esterified) cholesterol, is based on the formation of a stable, purple tetraenylic carbonium ion produced by oxidation with Fe 3+ in an acetic acid-sulfuric

145

acid environment (23, 24). It was adapted to solubilization studies as follows. Aliquots of 0.3 ml of aqueous nonoxynol solutions saturated with cholesterol or an ester are pipetted into dry 30-ml test tubes containing 2.7 ml glacial acetic acid. The original procedure (23) utilized anhydrous conditions. According to it, pure cholesterol or one of the esters is dissolved in 3.0 ml acetic acid. The rest of the assay follows closely the ZlatkisZak procedure. Two milliliters of the color reagent, containing ferric chloride hexahydrate in glacial acetic acid diluted with concentrated sulfuric acid (23), are poured down the side of the test tube to form a lower layer. Shortly after mixing, the solution changes from light brown to purple. At that point, the test tubes are cooled in an ice bath to room temperature. The absorbance of the absorption maximum at 558 n m begins to decrease only about 30 min after the solution reached room temperature. Less water than 0.3 ml increases the initial absorbance and the wavelength of maximum absorption, larger water volumes reduce both. The blank solution contains 0.3 ml water + 2.7 ml glacial acetic acid + 2.0 ml color reagent. Nonoxynol 10 did not interfere with the color reaction: Using 0.3 ml aqueous 3.0% nonoxynol solution instead of 0.3 ml water in the blank solution gave identical results. Beer-Lambert plots of absorbance at 558 n m versus lipid concentration were straight lines going through the origin. The range of linearity extended at least to 7.4 × 10 -5 M. Molar absorption coefficients + their standard deviations, obtained by regression analysis of the slopes, were as follows: cholesterol 8,950 + 42 liter/mole cm (N = 11); cholesteryl oleate 8,520 + 60 (N = 25); linoleate 8,580 + 61 (N = 24); linolenate 7,870 + 23 (N = 16). The absorbance at 558 nm divided by the molar absorption coefficient must be multiplied by a factor of 5.0/0.3 to obtain the molar concentration of the solubilized species, because 0.3-ml aliquots of filtrate were diluted to 5.0 ml with glacial acetic acid and color reagent. In the absence of surfactant, cholesteryl stearate was not soluble enough in the mixture Journal of Colloid and Interface Science, Vol. 112, No, I, July 1986

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of 0.3 ml water + 2.7 ml glacial acetic acid to permit determining its molar absorption coefficient. Since cholesteryl stearate and oleate had identical molar absorption coefficients at 563 n m in an anhydrous solvent containing only glacial acetic acid, it was assumed that they also had identical values at 558 n m in the presence of water. Therefore, the molar absorption coefficient of 8,520 of cholesteryl oleate was used for the stearate as well. Absorbances of the various esters and of cholesterol at 558 nm were additive in binary lipid mixtures.

C. GLC Assay for Cholesterol and Cholesteryl Esters The GLC procedure for determining cholesterol and its esters singly and in binary mixtures has been described (1). The results are in satisfactory agreement with those obtained by the colorimetric assay. For instance, the solubilization limit of cholesterol in 3.0% nonoxynol solution at 42°C was 1.010 + 0.012 mg/ml (N = 4) by colorimetry and 0.990 + 0.035 mg/ml (N = 2) by GLC. The difference is not significant at the 5% probability level. Table I compares the solubilization limits of cholesterol with a cholesteryl ester in binary

mixtures determined by colorimetry and by GLC. GLC analysis gave the separate concentrations of cholesterol and of the ester. Such pairs of concentrations were converted into the two corresponding absorbances at 558 n m according to the Zlatkis-Zak colorimetric method by means of the molar absorption coefficients listed in the previous section, and added. The sum of the two calculated absorbances is compared in Table I with the combined absorbance of cholesterol plus the ester at 558 nm determined by the colorimetric method. The agreement is satisfactory: The differences between the added absorbances calculated from GLC values and the combined absorbances determined directly vary between 0 and 3%. In four of the eight pairs, the GLC method gave higher calculated absorbances than the colorimetric method; in the other four, it gave lower values.

D. Solubilization Procedures For systems containing only one lipid, an excess solid was mixed with the nonoxynol solution. After flushing the amber, 125-ml, standard-taper bottles with nitrogen, they were shaken in a constant-temperature bath at 100 oscillations/min for various lengths of time.

TABLE I Comparison of Combined Solubilization Limits of Cholesterol and Cholesteryl Esters in 3.0% Nonoxynol Determined Colorimetrically and by GLC

Ester

Linolenate Linoleate Linolenate Linoleate Stearate Oleate Oleate Oleate

Temperature and method of solubilization a

27°C, 27°C, 37°C, 37°C, 37°C, 37°C, 37°C, 37°C,

III III III III I III IV V

Absorbance at 558 nm determined by colorimetry b

1.010 0.857 1.721 1.602 1.213 1.301 1.298 1.008

+ 0.014 ___0.011 ___0.013 + 0.014 + 0.001 + 0.001 + 0.018 + 0.025

Methods described in next section. b Average of four determinations ___SD. c Average of two determinations _ SD. a

Journal of Colloid and Interface Science, Vol. 112, No. 1, July 1986

Separate determinations of cholesterol and ester by GLC, converted into absorbances and added c

0.994 0.833 1.723 1.650 1.235 1.258 1.312 0.997

+ 0.021 + 0.009 _ 0.035 ___0.027 + 0.043 _ 0.039 + 0.025 + 0.037

Difference of absorbances, % based on absorbance measured colorimetdcally

1.6 2.8 -0.1 -3.0 -1.8 3.3 -1.1 1.1

MICELLAR SOLUBILIZATION OF CHOLESTEROL Equilibrium was attained when the colorimetric assays on two samples withdrawn 5 or 10 days apart and filtered gave the same absorbance. Filtration was carried out through Millipore membranes in a jacketed holder for temperature control. The effect of pore size was investigated to ensure that any colloidal lipid that may have been formed during solubilization was retained by the filter while surfactant micelles passed freely. In one experiment, an undersaturated solution containing 10 mg cholesterol in 50 ml of 2.0% nonoxynol solution was shaken for 45 days at 37 ° to ensure complete dissolution, and filtered through Millipore membrane filters of cellulose acetate + nitrate with different pore sizes. The first few milliliters of filtrate were always discarded because of possible membrane sorption of the solubilizate. In another experiment, a colloidal cholesterol dispersion was prepared by adding 2.0 ml acetone containing 36 mg cholesterol to 50 ml of 1.0% nonoxynol solution. Aliquots of the milky dispersion were withdrawn after 1 and 5 weeks shaking at 37°C, and filtered through different Millipore membranes. The average pore size did not significantly affect the absorbance of the filtrates treated with the Zlatkis-Zak reagent (see Table II). All

TABLE II Effectof Membrane Filter on Absorbanceof Filtrate Treated with Zlatkis-Zak Reagent Filtrate from Undersaturated solution in 2.0% nonoxynol Weeks shaken at 37°C:

9

Averagepore size, #rn 0.220 a 0.050 b 0.025 c

Suspension in 1.0% nonoxynol 1

5

Absorbance at 558 nm 0.143 0.141 0.142

0.310 0.313 0.305

0.412 0.408 0.410

,,b.c Catalog n u m b e r s : a G S W P 04700, b V M W P 04700, c V S W P 04700, Millipore Corp., Bedford, Mass.

147

samples in subsequent studies were filtered through membranes with average pore size of 0.050 #m. The time required to attain equilibrium solubilization of cholesterol and its esters was 2-3 weeks, presumably due to the formation of viscous lyotropic liquid crystalline phases surrounding the particles. Only at the highest equilibration temperature, namely, 51 °C, was there slight ester hydrolysis. None occurred at the lower temperatures (1).

E. Preparation of Binary Mixtures of Cholesterol Monohydrate and Cholesteryl Esters Cholesterol monohydrate rather than the anhydrous form was used in simultaneous solubilization experiments with cholesteryl esters because it is the stable form in the presence of water in the temperature range employed (see below). The following five methods were used to prepare the binary mixtures. (I) Equal weights of cholesterol monohydrate and a cholesteryl ester were triturated together thoroughly with mortar and pestle. (II) Since preliminary experiments indicated that the esters were not solubilized to the full extent expected, one part of cholesterol monohydrate was thoroughly ground together with nine parts of an ester. (III) To obtain more intimate mixing, the 1:9 mixtures which had been triturated together were heated to 75°C under nitrogen. The clear melts were cooled to room temperature and ground up. In this connection, it has been found that cholesterol or its monohydrate and cholesteryl esters with C18 fatty acids do not form solid solutions (25). The mixtures of cholesterol monohydrate and esters prepared by these three methods were shaken at constant temperatures with 3.0% nonoxynol solutions, filtered, and analyzed by the colorimetric method. Solubilization equilibrium was indicated by a constant absorbance at 558 n m in samples withdrawn 10 days apart. Aliquots of the filtrates were Journal of Colloid and Interface Science, Vol. 112, No. 1, July 1986

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SCHOTT AND SAYEED

then dried at room temperature over Drierite. The residues were dissolved in chloroform and analyzed by GLC. The last two methods involved sequential solubilization.

~ _

2.5

2.0

..J

(IV) Nonoxynol (3.0%) solutions were saturated with cholesterol monohydrate, leaving excess solid in suspension. After solubilization equilibrium had been reached, excess solid cholesteryl oleate was added and the resultant suspension was shaken at the same temperature until a new equilibrium was established. (V) Nonoxynol (3.0%) solutions were saturated with cholesteryl oleate, leaving an excess solid ester present after solubilization equilibrium had been reached. Excess solid cholesterol monohydrate was then added, and the suspensions were shaken until a new equilibrium was attained. RESULTS AND DISCUSSION

A. Micellar Solubilization of Cholesterol Starting with anhydrous cholesterol, the plot of concentration of cholesterol solubilized in 3.0% nonoxynol at 37°C versus time went through a maximum in about I0 days and then dropped to a plateau equilibrium value 20% below the maximum (Fig. 1). This is ascribed to gradual transformation of the bulk anhydrous cholesterol, which has a higher solubility, to the monohydrate, which is the equilibrium form in aqueous media. Similar observations have been described in other cholesterol dissolution studies with aqueous solvents (26), and natural (7) and synthetic (27) surfactant solutions. The lower curve in Fig. 1 was obtained when the starting material was the monohydrate. After about 1 month in the 37°C shaking bath, the two curves coincided. The solubilization limit of cholesterol monohydrate at 37°C was proportional to the surfactant concentration. The equation of the linear plot, obtained by regression analysis, is S = 0.0113 + 0.7357(% nonoxynol - 0.0044) Journal of Colloid and Interface Science, Vol. 112, No. 1, July 1986

[2]

o I.~ 0 n-" B.I

~ I.O b.l ..-I

°o.~

,'o zb 3; 4'o 5b ~0i TIME,days

FIG. 1. Solubilization rates of anhydrous cholesterol and of cholesterol monohydrate in 3.0% nonoxynol solutions at 37°C.

(N = 6, r = 0.9999), where the solubilization limit S is expressed in mmole/liter. The percent nonoxynol is w/v; the CMC at 37°C is 0.0044%. The solubilization limit of cholesterol monohydrate increased linearly with temperature between 27 and 51 °C, the range investigated. For 1.5%' nonoxynol solutions (neglecting the CMC), the least-squares equation is S = - 0 . 7 5 0 + 0.0500(t°C) [3] (N = 5, r = 0.9960). For 3.0% nonoxynol solutions, the least-squares equation is S = - 1.433 + 0.0979(t°C)

[4]

(N = 5, r = 0.9990). Doubling the surfactant concentration doubled slope and intercept. In the temperature range investigated, between 300 and 324 K, there was also a linear relation between log solubilization limit and the reciprocal of the absolute temperature T. For 1.5% nonoxynol solutions, the leastsquares equation is 1865 log S = 6.032 - T

[5]

(N = 5, r = -0.9942). For 3.0% nonoxynol solutions, the equation is

149

MICELLAR SOLUBILIZATION OF C H O L E S T E R O L

1860 log S = 6.314 - - T

[6]

(N = 5, r = -0.9910). On a semilogarithmic plot, the two straight lines are parallel. By means of the van't Hoff equation for equilibrium processes, d log K d(1/T)

AH 2.303R

[7]

one can calculate an enthalpy ~ H of - ( - 1 8 6 3 ° K ) (1.987 cal/mole °K) (2.303) = 8523 cal/mole or 8.5 kcal/mole. Cholesterol is very slightly soluble in water (although the published solubility values (e.g., (28, 29)) differ from each other and from that of Eq. [2]). Therefore, the process to which the equilibrium constant K and the calculated enthalpy refer is the transfer of cholesterol monohydrate from water to nonoxynol micelles (micellar binding). The enthalpy value is only approximate since one of the assumptions underlying the use of Eq. [7], namely, that size and hydration of the micelles do not change in the temperature range investigated, is not completely valid. Endothermic enthalpies of transfer from water to nonionic micelles have been reported for another compound which is very slightly soluble in water and

consists of a large and rigid molecule, namely, the antibiotic griseofulvin (30).

B. Joint Solubilization of Cholesterol Monohydrate and a Cholesteryl Ester 1. Effect of method of solubilization on sol ubilization ratios. All experiments were made with 3.0% nonoxynol solutions. The various methods of preparing the binary mixtures were investigated in detail for cholesteryl oleate. The data are reported in Table III as solubilization ratios of cholesterol and of cholesteryl oleate. The former are the ratios of the solubilization limits of cholesterol monohydrate solubilized jointly with cholesteryl oleate to the solubilization limits of cholesterol monohydrate solubilized singly. The latter are the ratios of the solubilization limits of cholesteryl oleate solubilized jointly with cholesteryl monohydrate to the solubilization limits of cholesteryl oleate solubilized singly, which are 0.432 + 0.004 mmole/liter at 37°C and 1.892 _+ 0.006 mmole/liter at 44°C. Table IV lists the solubilization ratios of the other three esters, based on published single solubilization limits (1). The solubilization ratios of cholesterol are close to unity, indicating that the esters did not interfere with the uptake of cholesterol by

TABLE III Joint Solubilization of Cholesterol Monohydrate and Cholesteryl Oleate by 3.0% Nonoxynol Solutions Solub'dization ratios a (nag. liter-l/mg - liter -1) Temperature and method of solubilization

37°C, III 37°C, IV 37°C, V 44°C,I 44°C, II 44°C, III 44°C, IV 44°C, V

Cholesterol concentration i~ mixt.____~e Cholesterol concentration singly J

0.96 1.01 0.73 0.99 0.97 0.93 0.95 0.55

Oleate concentration in mixture Oleate concentration singly J

147'281 133'28l 159'281 447'1232 44411232 436q232 441q232 693q232

= = = = = = = =

0.52 0.47 0.56 0.36 0.36 0.35 0.36 0.56

a Concentrations in numerators are averages of duplicate G L C measurements; concentrations in denominators are averages of four eolorimetric measurements.

Journal of Colloid and Interface Science, Vol. 112, No. 1, July 1986

150

SCHOTT AND SAYEED TABLE IV Joint Solubilization of Cholesterol Monohydrate and Cholesteryl Esters by 3.0% Nonoxynol Solutions Solub'dizationratiosa (rag.liter-l/rag•liter-1)

Ester Linolenate Linoleate Stearate

Temperatureand methodof solubilization 27°C, 37°C, 27°C, 370C, 37°C,

Cholesterolconcentrationin mixture Cholesterolco.ncentratio~ singly ]

III III III III I

0.98 0.98 0.94 1.02 1.00

Ester concentrationin mixture Ester concentrationsingly ) 479/613 761/1570 245/376 548/1160 62/134

= = = = =

0.78 0.48 0.65 0.47 0.46

"Concentrations in numerators are averages of duplicate GLC measurements; concentrations in denominators are averages of four colorimetric measurements.

the micelles. The exception is Method V, where the surfactant solution was first saturated with cholesteryl oleate. This procedure reduced the solubilization limit of cholesterol to 73% of the amount observed when cholesterol was solubilized alone at 37°C and to 55% at 44°C. The higher of the two ratios was observed at 37oc, which is below the melting point of cholesteryl oleate. At that temperature, the solubilization limit of the ester by itself was 4.4× lower than at 44°C, where the ester is in a liquid crystalline form. Being more extensively solubilized by itself at 44°C than at 37°C, the ester offered greater competition to cholesterol for the solubilization site at the higher temperature. All esters were solubilized less extensively in the presence of cholesterol than in its absence. Even in Method V, where the surfactant solution was first saturated with cholesteryl oleate and cholesterol monohydrate was added afterwards, the final concentration of the solubilized ester was only 56% of its concentration when the ester was solubilized in the absence of cholesterol. Thus, cholesterol monohydrate displaced nearly 1/2 of the cholesteryl oleate previously solubilized from the micelles. When added simultaneously with or before the oleate, cholesterol monohydrate reduced the solubilization limit of the oleate by nearly 2/3. The solubilization ratios of the other esters ranged from 0.46 to 0.78. Journal of Colloid and Interface Science, Vol. 112,No. 1, July 1986

The solubilization ratios of cholesterol monohydrate by Methods I-IV were practically the same, namely, unity. The solubilization ratios of cholesteryl oleate at 44°C obtained by Methods I-IV were identical and the ratios at 37°C obtained by Methods III, IV, and even V differed only by +9%. Thus, at constant temperature, the proportions of cholesterol monohydrate and esters in the binary mixtures and the method of blending had only minor effects on the solubilization ratios, except for Method V on that of cholesterol. The effect of the order of addition on the extent ofsolubilization of two solubilizates was reported to be more pronounced when some pairs of steroids were solubilized in polysorbate 40 solutions. While in some instances, each steroid dissolved independently of the other, in other instances the extent of solubilization depended markedly on the order of addition. The more polar steroid, which was solubilized more extensively by itself, generally suffered the greater solubility loss during joint solubilization (31, 32). The explanation, in terms of location of the steroids in the micellar hydrocarbon core or in the hydrated polyoxyethylene shell, was not supported by thermodynamic studies (33). Cholesterol monohydrate is most soluble in solvents with solubility parameters in the range of 8.8 ___ 1.5 (cal/cm3) 1/2 (34). This range is considerably below the solubility parameter of the hydrated polyoxyethylene shell but over-

151

MICELLAR SOLUBILIZATION OF C H O L E S T E R O L

laps that of the nonylbenzene core of the nonoxynol micelles. Thus, both cholesterol monohydrate and the cholesteryl esters are probably solubilized in the micellar hydrocarbon core and compete for this location. By virtue of its hydroxyl group, which can become hydrated, the former can probably be located even in the outermost region of the core, into which some water has penetrated, whereas the more hydrophobic esters are probably confined to the innermost, anhydrous region of that core. The greater adaptability of cholesterol monohydrate would give it an edge during joint solubilization with an ester. 2. Effect of temperature on solubilization ratios. Increasing the temperature of solubilization of mixtures of cholesteryl oleate and cholesterol monohydrate from 37°C, 2°C below the melting point of the oleate, to 44°C, where the bulk ester exists in a liquid crystalline state, tripled the absolute amount ofoleate solubilized. However, its solubilization ratio decreased by 27%. Similar observations apply to the two polyunsaturated esters. Both are solid at 27°C and liquid crystalline at 37°C (1). While the solubilization limits in the presence of cholesterol monohydrate increased substantially when the temperature was increased above their melting points, the solubilization limits in the absence of cholesterol monohydrate increased even more, so that the solubilization ratios actually decreased. 3. Effect of unsaturation on solubilization ratios. At a given temperature, the amount of ester solubilized in the presence of cholesterol monohydrate increased with increasing unsaturation, as did the amount of ester solubilized in its absence (see Tables III and IV). For instance, at 37°C, the ester concentrations in the presence of cholesterol monohydrate were 62, 147, 548, and 761 mg/ml for fatty acids with zero, one, two, and three ethylenic double bonds, respectively. However, their solubilization ratios were essentially the same. At constant temperature, the total amount of lipids solubilized from binary mixtures of cholesterol monohydrate with an ester also in-

creased with increasing unsaturation of the fatty acid, because the amount of ester solubilized by itself increased with unsaturation while the solubilization ratios of the four esters were essentially the same regardless of unsaturation. Moreover, the solubilization ratio of cholesterol monohydrate was constant and practically unity at all temperatures. The exception, namely, Method V, is unrealistic under physiological conditions. 4. Maximum occupation numbers. Average maximum occupation numbers of lipid molecules solubilized per micelle are listed in Table V. They were calculated from published average micellar aggregation numbers Z for nonoxynol which, in the range of 10.5-35°C, are proportional to temperature (35). The linear regression equation is Z = 36.0 + 3.23(t°C)

[8]

(N -- 29, r = 0.9944). The equation was extended to 37°C but not to 44°C because, on approaching the cloud point, the increase of Z with temperature is often greater than linear.

TABLE V M a x i m u m N u m b e r of Lipid Molecules Solubilized per Nonoxynol Micelle in 3.0% Nonoxynol Solutions Solubilized singly Estersolubilizedjointlywith cholesterolmonohydrate N~ Lipid Cholesterol monohydrate Cholesteryl linolenate Cholesteryl linoleate Cholesteryl oleate Cholesteryl stearate

N~.~

N~

27°C 37°C 27°C 37°C 27°C

37°C

3.4

7.75

2.65

8.55

2.1

4.15

5.4

11.75

1.6

6.3

1.05

3.0

4.2

10.9

0.6

1.5

0.8

8.5

0.2

0.7

0.3

8.1

a M a x i m u m n u m b e r of lipid molecules solubilized per micelle for single lipids. b N u m b e r of molecules of cholesterol + n u m b e r of molecules of ester solubilized per micelle at saturation in binary systems by Methods I-IlL Journal of Colloid and Interface Science, Vol. 112,No. 1, July 1986

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SCHOTT AND SAYEED

Small occupation numbers like those of Table V are typical of solubilizate molecules which contain large, rigid, and asymmetric moieties (36). The lipid occupation numbers increase with increasing temperature. At constant temperature, they increase with increasing unsaturation of the esters, both when the esters were solubilized by themselves and together with cholesterol. The molar ratio, (moles micellar surfactant/ mole solubilized cholesterol), is 20.0 for nonoxynol at 37°C (compare Table V and Eq. [8]). In the case of bile salts, it is ca. 14 for sodium deoxycholate regardless of its concentration, 16-20 for 0.15 M sodium cholate, and ca. 40 for 0.05 M sodium cholate (37). Thus, the capacity for solubilizing cholesterol is comparable for the typical nonionic surfactant, nonoxynol 10, and for bile salts. 5. Potential implications for atherosclerosis. The mechanism(s) involved in the formation of atherosclerotic deposits in blood vessels is (are) not known. However, food intake oftriglycerides high in polyunsaturated fatty acids is known to decrease the formation of such deposits or increase their removal (4, 5). Table VI shows the increase in micellar solubilization limits of the combined amounts of cholesterol monohydrate plus an ester with increasing unsaturation of the fatty acid at body temperature. If the analogy between lipid

TABLE VI Effect of Unsaturation on Combined Solubilization Limits of an Ester and Cholesteryl Monohydrate during Joint Solubilization by Method III in 3.0% Nonoxynol at 37°C

Ester

Linolenate Linoleate Oleate Stearate

Total absorbance at 558 n m a

1.721 1.602 1.301 1.213

± ± + +

0.013 0.014 0.001 0.001

Combined solubilization limits of ester + cholesterol (mmole/liter) b

3.347 3.018 2.434 2.263

Average of four determinations + SD. b Average of duplicate determinations by GLC.

a

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solubilization in nonoxynol micelles and in lipoproteins is valid, and provided that the solubilizing capacity oflipoproteins is a factor affecting the formation and/or removal of atherosclerotic deposits, the above fact would explain in part the lesser tendency of polyunsaturated triglycerides in foodstuffs to cause atherosclerosis. In vivo, cholesteryl esters derive most of their fatty acids, particularly the polyunsaturated essential linoleic and linolenic acids, from triglycerides in food. Atherosclerotic deposits contain both cholesterol monohydrate and cholesteryl esters. The former reduces the solubilizing capacity of micelles and probably also of lipoproteins for the latter. However, the extent of solubilization of the esters by themselves increases with unsaturation while their solubilization ratio in the presence of cholesterol is not affected by it. Since the polyunsaturated esters are more extensively solubilized than the monounsaturated and saturated esters in the presence as well as in the absence of the ubiquitous cholesterol monohydrate, they would be less prone to become supersaturated in plasma and to form atherosclerotic deposits. ACKNOWLEDGMENTS Adapted from a dissertation submitted by F. A. A. Sayeed to Temple University in partial fulfillment of the requirements for the Doctor of Philosophy degree. Presented at the Division of Colloid and Surface Chemistry, American Chemical Society Annual Meeting, Philadelphia, Pa., August 1984. REFERENCES 1. Sayeed, F. A. A., and Schott, H., J. Colloid Interface Sci. 109, 140 (1986). 2. Schott, H., Z Amer. Oil Chem. Soc. 45, 823 (1968). 3. Mantulin, W. W., and Gotto Jr., A. M., in "International Conference on Atherosclerosis" ( L A. Carlson, Ed.), p. 57-69. Raven, New York, 1978. 4. Schettler, G., and Moefl, H., Naturwissensch. 65, 130 (1978). 5. Lehninger, A. L., "Principles of Biochemistry," Chap. 12, 26. Worth, New York, 1982. 6. Katz, S. S., Shipley, G. G., and Small, D. M., J. Clin. Invest. 58, 200 (1976).

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Journal of Colloid and Interface Science, Vol. I 12, No. 1, July 1986