The characteristics of mixed micellar solutions with particular reference to bile

The Characteristics of Mixed Micellar Solutions with Particular Reference to Bile

MARTIN C. CAREY, M.D.~ DONALD M. SMALL, M.D.t Boston, Massachusetts

From the Divisions of Biophysics and Gastroenterology, Department of Medicine, Boston University Medical Center, 80 East Concord Street, Boston, Massachusetts 02118. This study was supported in part by N.I.H. Grants FO5 TW1212-02 and AM 11453-03. Requests for reprints should be addressed to Dr. Donald M. Small. ~' Traveling Student in Medicine, National University of Ireland, 1970. International Post-Doctoral Research Fellow. U.S. Public Health Service, 1968-1970. t Markle Scholar in Academic Medicine.

590

Biological systems are, for all intents and purposes, aqueous, and. water constitutes approximately 60 to 70 per cent of the body mass of higher animals including man. One of the most remarkable features of the physiology of the human organism is its capacity nevertheless to digest, absorb, transport, utilize and secrete lipids, which for the most part are water-insoluble. Ingested fats, mainly triglycerides of long-chain fatty acids, are emulsified in the gut lumen and subsequently hydrolyzed by pancreatic lipase to waterinsoluble monoglycerides and fatty acids [1]. In the watery milieu of the vertebrate gut a beautiful mechanism exists by which these insoluble products of lipolysis are solubilized and transported. This is mediated by the bile salts, a group of biologic lipids with extraordinary detergent properties [2-5], which form a protective watersoluble coating around these otherwise water-insoluble molecules. The water-soluble aggregates formed by bile salts and insoluble lipids are called "mixed micelles." These insoluble products of lipolysis are then shuttled in mixed micelles from the site of action of pancreatic lipase, that is the surface of the emulsion droplet, to the absorptive membrane of the intestinal cell where they are absorbed. Since the bile salts are not absorbed with the insoluble lipids, they can be used over and over again to aid the absorption of digested fat [6-8]. Insoluble lipids, once absorbed, can be transported effectively to and from cells in both lymph and blood of higher animals as lipoproteins [9]. The very large lipoproteins (chylomicrons and very low density lipoproteins) are really emulsion particles, that is, droplets of oil (triglycerides) stabilized by a surface layer of molecules (the "emulsifier") comprised of phospholipids and free cholesterol. The beta-lipoproteins are also spherical, but they are much smaller and resemble microemulsion particles. In these particles the ernulsifier is probably phospholipid and specific peptides. The smaller and more dense alpha-lipoproteins are made up of about half insoluble lipid and half specific peptides. The peptide of these lipoproteins apparently not only solubilizes the lipid, but also imparts a nonspherical shape to the particles [9]. Apart from the fact that the sizes of high density lipoproteins are in the range of mixed micelles, it is not known whether their propertie s are similar. In the bile, bile salts are present in high concentrations and are responsible for the solubilization of free cholesterol and phospholipid in mixed micelles [10-12]; a mechanism which, in normal bile, prevents the precipitation of these insoluble lipids and gallstone formation.

The American Journal of Medicine

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS~GAREY, SMALL

Mixed micellar solutions, including bile salt mixed micelles [3,4,12-14], contain particles of submicroscopic size (30 to 100 A). Since these small particles do not scatter light in the visible range appreciably, their solutions are optically clear. The ratio of the concentration of lipid solubilized (which we will call the "solubilizate") to the concentration of solubilizing agent ("solubilizer") is usually low, although exceptions occur, especially in the case of the bile saltlecithin and bile salt-monoglyceride mixed micelles. Micellar solutions form spontaneously and are stable. In any specific mixed micellar solution the micelle size varies only slightly, and thus mixed micellar solutions are "monodisperse" relative to particle size. Typical emulsions [15-17], on the other hand, generally contain particles which can be seen under the microscope. The diameters of these particles range from approximately 2,000 to 50,000 h.. Particles of this size scatter light in the visible spectrum so that the optical appearance of their suspensions is opalescent or frankly turbid. The ratio of "solubilizate" to "solubilizer" is high. in general, emulsions require an energy source for their formation and even though they may be relatively stable they will even-, tually separate into an oil phase and an aqueous phase. Within a given emulsion the particle size of most emulsions, unlike micellar solutions, varies over a rather wide range. Emulsion particles thus exhibit "polydispersity" in relation to size. The size of particles of microemulsions (particle diameters 100 to 2,000 A) fall in between those of true emulsions and mixed micelles [18,19]. Microemulsions with the smaller particle diameters (100 to 1,000 P,) are optically clear solutions. These may be considered to be highly swollen micelles. Microemulsions, however, behave more like true emulsions. Thus, they require external energy for their formation and are unstable thermodynamically. With the use of a powerful light source, microemulsions having particles greater than 1,000/~ diameter will give a bluish opalescence when viewed at an angle close to the incident beam (Tyndall effect). Although microemulsions may be quite monodisperse in relation to particle size, unlike true micelles they can be made to separate into two phases. We intend in this review to discuss micellar solubilization of insoluble lipids and to suggest that this form of solubilization has important physiologic significance. By way of background, the familiar chemical species of lipids will be classified in terms of how they interact with water [19,20]. Both the surface interactions of lipids with water and their interactions within water ("bulk" interactions) will be used to classify lipid species. In this classification we will emphasize the importance of the molecular structure, specifically that structure related to the water-soluble parts and to oil-soluble parts of the

Volume 49, November

1970

lipid molecule. After discussing both water-insoluble lipids and soluble lipids (micelle-forming lipids), we will give an account of simple micelle formation, that is micelle formation by lipids without other lipid additives. Further, the structure and properties of "classic" micelles (soaps and aliphatic detergents) will be compared with bile salt micelles. We will then attempt to summarize the complex field of mixed micelles, that is, micelles composed of more than a single chemical species of lipid. We will suggest o

b

H,CICH'

>c.,

c

d

~

H,C\

~

jCH= H=C\

~

/CH=

To

AIR UPID SOLUBLE H=C\ PORTION OF /CH= MOLECULE

10 A

H,C.~

/CH,

WATER SOLUBLE PORTION OF MOLECULE

I (

l

WATER

Figure 1. Four (a-d) different schematic representations of a typical polar lipid molecule (Octadecanol). The mole. cules are pictured as they would lie at an air-water or oil-water interface. The polar group (-OH) of octadecanol in all representations is shown lying on the aqueous side of the interface indicated by the horizontal line. The long saturated hydrocarbon chain sticks upwards into the hydrophobic environment (air or oil). The traditional zig-zag configuration of the saturated carbon.carbon bonds is shown in a. The saturated hydrocarbon chain consists of covalently bonded carbon atoms in a tetrahedral coordination which allows considerable motion of one carbon atom on the next. An angle of approximately .109 de. grees is subtended by each carbon atom and any two neighboring bonds, i.e., C-C-C; H.C-C or H.C.H. In the solid state, motion about these bonds is limited, but in the liquid state motion is rather free. A photograph of a Stuart.Breigleb molecular model of octadecanol is shown in b. These models, often termed space-filling models, provide an accurate representation of the minimum Van der Waal's radii of the constituent atoms and their average bond angles. In c and d two short-hand representa. lions of this polar lipid are given. In c the hydrocarbon chain is represented by a straight line which is intended to indicate little or no motion of the chain, This" would represent the conformation of the chain part of the molecule in the crystalline state. The wavy line representation of the chain shown in d indicates that considerable thermal motion of the hydrocarbon chain part of the molecule is occurring, such as would be the case with octadecanol, above its melting point.

591

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS---CAREY, SMALL

how each class of micelle-forming lipid solubilizes each class of insoluble lipid, at least as far as is presently known. LIPIDS--DEFINITION AND GENERAL MOLECULAR STRUCTURE

Although lipid is a general word which refers to a heterogeneous group or organic compounds, we will define lipids as molecules having a large aliphatic or aromatic hydrocarbon component which may or may not have a water-soluble group(s) attached [19,20]. In Figure 1 are shown four structural representations (a, b, c and d) of an aliphatic lipid molecule, in this case the long-chain aliphatic alcohol, octadecanol. Figure la shows the conventional chemical representation of the long hydrocarbon chain of this molecule which is composed of covalently linked --CH2-- groups, one end of which terminates in a .--CH3 group whereas the other end terminates in a covalently linked water-soluble --OH group. Figure lb shows the same lipid represented, schematically by a space-filling molecular model which indicates the contours of the minimum Van der Waal's radii of the atoms. This type of model gives a better idea of the shape and volume of the molecule. Figures lc and ld are short-hand representations of the molecule. In Figure lc the chain is shown as a straight line indicating a rigid aliphatic ab

c

d ef

g

h

i

AIR

WATERSOLUBLE PORTIONOF MOLECULE

,,,/ "V POLAR LIPIDS

WATER

LIPID CLASSIFICATION BASED ON INTERACTION WITH WATER [19,20]

"~ "~ - -

%,,

%.

J y NON-POLAR LIPIDS

Figure 2. Schematic shorthand representations of some typical lipid molecules mentioned in the text. Molecules are pictured as they would lie at an air-water or oil-water interface indicated by the horizontal line. Wavy lines represent the lipid.soluble hydrocarbon chain part of molecules in the liquid state; shaded areas, the lipid-soluble cyclic hydrocarbon part of molecules; open circle, polar head of an amphiphilic molecule; closed circle or oval, OH groups or ester groups as the case may be; 9 and O, the positively and negatively charged ionic polar groups. (a) The general configuration of an aliphatic amphiphilic molecule; (b) long.chained nonionic deter. gent; the closed circles represent a multiplicity of polyhydroxyl or polyether groups which acting in consort provide an effectively strong but bulky polar "head group"; (c) cholesterol; (d) lecithin; (e) lysolecithin; (f~ ionized fatty acid or anionic detergent; (g) bile salt; (h) nonpolar aliphatic lipid such as n-octadecane; (i) nonpolar aromatic lipid such as cholestane.

592

cha in configuration with little motion between atoms. This type of aliphatic chain structure occurs in the crystalline state. In Figure ld the hydrocarbon chain is shown as a wavy line which indicates that the chain has considerable molecular motion. Lipids in the liquid state or in micellar solutions have this "liquid like" aliphatic chain configuration. The OH group is shown as a closed circle. In the figure these molecules are drawn as they would lie at an air-water or oil-water interface. The water-soluble group, often called the "polar head group" or the "hydrophilic (water-loving) group" is shown lying in the water below the horizontal line, whereas the water.insoluble part of the molecule, or the "hydrophobic" (waterfearing) part, is shown sticking upwards into the hydrophobic environment, that is, air or oil. Other lipids with different polar head groups, such as amine, aldehyde, ether and ester groups, would be similarly represented. These types of molecules are also called amphiphiles ("loving both sides") because they are composed of two covalently linked parts with diametrically opposed solubilities. The polar lipids constitute the great majority of the biologically important lipids in man. Some examples of other amphiphiles are shown in shorthand form in Figure 2a through 2g. The lipid molecules represented by Figure 2h and 2i possess no hyd rophilic part and therefore lie above the surface of the water, that is, above the horizontal line. These lipids are called nonpolar lipids. Some molecular examples are long-chain paraffins such as octadecane (2h) or unsubstituted aromatic compounds such as cholestane (2i).

Polar lipids vary considerably in their behavior on and in water. Their behavior depends on the relative strength of the hydrophilic and hydrophobic portions of the molecule, or the "hydrophilic-hydrophobic balance" (HHB) of the molecule. If the hydrophilic part is strong compared to the hydrophobic part, the molecule tends to be water-soluble.* Conversely, if the HHB is tipped toward the hydrophobic side the molecule tends to be water-insoluble. The interactions of nonpolar lipids and of polar lipids in aqueous systems [19,20] are shown in Figure 3. The nonpolar lipids such as paraffin oil, phytane or cholestane are totally insoluble at the air-water interface and within water. For instance, if a drop of purified paraffin oil is placed on the surface of water Strong hydrophilic groups include ionized carboxyl, suffate and phosphate groups. Weak hydrophilic groups include alcohol, ester, aldehyde and amine groups. The strength of the hydrophobic (aliphatic or aromatic) portion of a molecule increases with the number of carbon atoms.

The American Journal of Medicine

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS--CAREY,

the bulk phase. Excess lipid is present as a lens floating on the water [19]. In Class II lipids, the "insoluble swelling amphiphiles," we can place such important biologic lipids as phospholipids, cerebrosides and monoglycerides [19,20] (Figure 3). These lipids are also insoluble in water, but water, on the other hand, is soluble in them. The water molecules penetrate between the polar head groups of these lipids provided the lipid is above a temperature at which the hydrocarbon chains are in the liquid state (see Figure 1). These lipids are not solubilized by the water as the chains remain adherent to one another. The penetration of water between the polar groups results in a swollen lipid structure which usually consists of alternating lipid bilayers and layers of water. This type of structure is called a lamellar liquid crystal and has been described for a number of lipid-water mixtures [13,14,22-36]. When a large amount of water is present these liquid crystals separate from the excess water as a second phase [22]. This phase may be recognized as the familiar "myelin figures" under the microscope.* The formation of a liquid crystalline phase in water is called "lyotropic mesomorphism"; lyotropic because it occurs when a solvent (water) is added and mesomorphic because a change in physical state results. These lipids also spread on the surface of water to form stable monolayers.

in a vessel, it will form a lens of oil and will not spread [19]. Most polar lipids, t on the other hand, have surface solubility in water, that is, when the lipid is added to water, molecules will be concentrated at the surface of the water as a layer one molecule thick (monolayer or monomolecular layer) [19]. However, different types of polar lipids have different bulk interactions with water and form different types of monolayers and therefore may be conveniently divided into three classes [20] (Figure 3). Class I lipids are "insoluble nonswelling amphiphiles" as they are insoluble within water and will not swell in water. Their HHB is tipped strongly toward the hydrophobic side. Included in this class are molecules such as triglycerides, diglycerides and longchain alcohols (Figure 3). If one places a drop of liquid long-chain alcohol or triglyceride on the surface of water irl a vessel it will spread to cover the surface completely provided enough oil is present (Figure 3). The aqueous surface becomes covered by a single layer of lipid molecules one molecule thick (monolayer, monomolecular layer or film). This monolayer is stable because the lipid cannot be solubilized in t As exceptions to this, certain important polar lipids which have very large :hydrophobic parts such as esters of long-chained or large aromatic alcohols and long-chained fatty acids (e.g., palmitoyl oleate, cholesteryl oleate) will not spread to form a monolayer and thus behave as nonpolar lipids [19]. The physical properties of cholesteryl esters are discussed elsewhere [21].

CLASS

Excellent photographs of myelin figures will be found in the text by Nageotte [37].

SURFACEAND BULK INTERACTIONS WITH WATER

NON-POLAR LIPIDS

EXAMPLES

Dodecane, octadecane, paraffin oil, phytane, carotene, squalene. Cholestane, benzypyrenes, coprostane, benzphenanthracenes. Cholesteryl oleate, cholesteryl Iinoleafe, palmltoyl oleate, etc.

WILL NOT SPREAD TO FORM A MONOLAYER INSOLUBLE IN BULK

POLAR LIPIDS I ; Insoluble nonswelling amphiphlles

FORMS A STABLE MONOLAYEB

k0::J

"IT: Insoluble swelling omphiphiles

INSOLUBLE IN BULK FORMS A STABLE MONOLAYER

.....

LI,,D ~. ;,'~ 1~~" 1~"• I~.PRO~pR BILAYERs[Ig~l J~s ~..J~~ ~ .. ~' ~, ; ~ . ~ ,

Trl':Soluble omphiphiles FORMS AN UNSTABLE MONOLAYER

B)withouf lyotr opic mesomorphism

FORMS AN UNSTABLE MONOLAYER

~

---')mlcelle

.C.H.A.,N.S

BULK PHASE- pure liquid crystals in pure water LIQUID ~ " - CRYSTAL(L -'C)

A)with lyotropic mesomcrphlsm -') L.C:-) mlcelle

SMALL

Phospholipids, monoglycerldes, "acid soaps", glyceryl, monoethers, alpha-hydroxy fatty acids.

G R O U P S ~H~ 'AOLRP ! ~T~O)NSodium end potassium salts of long-chain fatty acids, many of the ordinary anionic, cationic and nonionlc detergents, lysoleclthln, palmltoyl coenzyme A.

BULK PHASE-o mlcenor solution above CMC

~ BULK PHASE-0 micellar solution above CMC

Trlglycerides, diglycerldes, long-chain protonated fatty acids, long-chain alcohols, amines and aldehydes. Vitamins A, D, E and K, cholesterol, sitosterol.

~

MICELLE

P O LA R G R O U P S Y D R O C A R D O A R N O (C M C O )R A T IE

Conjugated and free blle salts, sulfated bile alcohols, sodlum salt of fusldlc acid, rosin soaps, eaponins, sodium salts of phenanthrene sulfonic acld, possibly penlcllllns and phenothiazines.

Figure 3. Classification of biologically active lipids (modified from [19,20].). "On the left the major lipid classes are listed. The center depicts the physical state of the lipid in the bulk aqueous system (i.e., within water) and on the surface of water. The insets on the right represent the authors' conception of the arrangement of the molecules in: a l a m e l lar liquid crystal ("myelin figures") formed by class II lipids (insoluble swelling amphiphiles, e.g., lecithin) in an excess of water; a spherical micelle formed by class Ilia lipids (soluble amphiphiles, e.g., typical detergents); the micelle formed by class IIIB lipids (soluble amphiphiles, in this case the bile salts). Examples of lipids typical of each class are given in the column on the far right.

Volume 49, November 1970

593

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

The Class III lipids are the "soluble amphiphiles." Molecules of this class may be divided into two subclasses: Class II IA, made up of aliphatic amphiphiles, including soaps and many of the ordinary anionic, cationic and nonionic detergents and the important biologic compound lysolecithin [19,20]. The HHB of these lipids is tipped strongly towards the hydrophilic side; thus they are very soluble in water, form micelles and in very high concentration undergo lyotropic mesomorphism, that is, their micelles pack to form liquid crystals. Class I IIB amphiphiles usually have bulky aromatic hydrocarbon moieties. This subclass includes the bile salts, sodium fusidate, rosin soaps and saponins [19]. These compounds are also very soluble in water and readily form micelles; however, even at their highest concentrations they do not exhibit lyotropic mesomorphic behavior. The monolayers formed by molecules of this class are very unstable as the molecules are soluble and go into the bulk aqueous phase. Class III lipids are the only lipids that can form micelles. Other lipids (nonpolar and polar) can be solubilized and transported by these micelle-forming

CMC

X

~.D I

SOLUTION OF" /

MONOMERS/ I

/ I q'~

i

/

I '""~o ;"'~

A

Y

/

KRAFFr POINT

S~

~

C

CRYSTLLI ANE s

CONCENTRATIONOF DETERGENT

Figure 4. The behavior of soluble lipids (class III) as a function of temperature and concentration. The temperature is plotted on the ordinate and increasing concentra. tions of soluble lipid (amphiphile) on the abscissa. Mi. celles occur only in region Y. In this region the amphiphile is above a concentration indicated by the line BD and above a temperature indicated by BC. BD is the critical micellar concentration (CMC). Thus, the CMC varies along BD with the temperature. The solution must also be above a certain temperature (BC) which indicates the transition temperature from the crystalline state to a micellar solution. This temperature is called the critical' micellar temperature (CMT). The point B where the CMC and CMT curves meet is termed the "Krafft point" [40]. It can be considered a triple point and indicates the CMT at the CMC.

594

lipids. Although it has not yet been clearly demonstrated, other biologically occurring compounds such as certain peptides might behave like class III lipids and be responsible for solubitizing the insoluble lipids of high density lipoproteins [9]. CHARACTERISTICS OF SIMPLE MICELLES (MICELLES CONTAINING ONLY ONE LIPID SPECIES)

The soluble amphiphiles (classes IliA and IIIB) are often termed "association colloids." In very dilute aqueous solutions these lipids exist as ideal solutions of unassociated molecules. As the concentration of the lipid molecules is increased, however, micelles form spontaneously by association of the hydrophobic parts of the molecules. The hyd rophilic groups remain in the aqueous environment at the surface of the aggregates, thus forming a water-soluble shell around the enclosed hydrophobic parts. Micelle formation can occur only above a certain solute concentration, the critical micellar concentration (CMC), and at solution temperatures above the critical micellar temperature (CMT). The CMC and CMT can best be explained by considering the effects of temperature and concentration on the physical state of a soluble amphiphile (detergent, soap, bile salt, etc.) in water (Figure 4). For instance, at low temperatures little detergent is soluble (Figure 4, point A). However, as the temperature is increased the amount of detergent in solution (as individual molecules) increases slightly in a nearly linear fashion along AB. After point B there is a rather sharp increase in solubility over a narrow range of temperature (BC). Why does this occur? Consider a solution in zone X ata temperature well above B. Under these conditions the individual molecules of detergent are freely distributed throughout the water, forming a true solution. As the concentration of detergent is increased (moving toward zone Y) a point of maximum molecular solubility is reached at line BD. Any further addition of detergent results in the formation of micelles (zone Y). Therefore, the concentration at which micelle formation first occurs (BD) is the CMC. It is influenced slightly by temperature. Consider now a mixture falling in zone Z with a concentration of detergent greater than B. This mixture is a cloudy suspension of two phases, a liquid phase having a composition lying along line AB and a solid phase composed of the crystalline detergent. As the temperature is increased a point is reached on BC where the solution clears. This clearing is due to melting of the rigid hydrocarbon chains in the crystal, the penetration of water around the polar group and the subsequent dispersion of detergent into micelles. Thus, BC gives the relation between temperature and the maximum solubility of the detergent in a micellar solution whereas ABD gives the relation between temperature and the maximum solubility of the detergent as individual mole-

The American Journal of Medicine

CHARACTERISTICS

cules. The temperature of clearing (BC) for solutions of salts of long-chain fatty acids increases only slightly with increases in the concentration of amphiphiles above the CMC [38]. This has led previous investigators to ignore the effect of detergent concentration on solubility above the CMC. In fact, the " K r a f f t point," often used synonomously with CMT, originally referred to the slight temperature variation in BC for soaps of long-chain fatty acids. In contrast, the CMT of certain bile salts (lithocholates) increases significantlywith increases in the concentration above the CMC [39]. For this reason we have chosen not to use the terms CMT and Krafft point synonomously, as is comm3n practice. Rather, we have restricted the use of the latter term to the temperature at which clearing occurs in solutions in which the concentration of amphiphile is at the CMC [40]~ Therefore, the Krafft point represents a single point (B) and the CMT a range of temperature which varies with the concentration of amphiphile present along BC. In the case of typical detergents, the micelles are, in most cases, spherical and generally of a single micellar weight in solution concentrations just above the CMC. Thus, each micelle contains the same number of molecules of the lipid. This number is called the aggregation number (Ag#). The transformation from an ideal to a micellar solution is indicated by the line BD in Figure 4, indicating an abrupt transition. Nevertheless, it can be shown by most physicochemical means that this transition (CMC) does, in fact, occur over a narrow concentration range [41]. Note that the CMC of any detergent solution is also approximately equal to the maximum concentration of the molecularly dispersed molecules in the solution, even at concentrations well above the CMC. These unassociated molecules are in rapid dynamic equilibrium with the molecules in the micelles. A detergent with a relatively large hydrophobic part will have a lower CMC than a detergent with a relatively small hydrophobic moiety [41]. Of course, if the hydrophobic part is very large such as is present in long-chain lecithins, the lipid will fall into class II and liquid crystals, not micelles, form in water [22]. On the other hand, lecithins with very short fatty acid chains (C.~-C4) or with one long and one short chain can form aggregates of micellar dimensions in water [42]. Even di- and triglycerides with very short chains such as triacetin, tripropionin and 1,3 dibutyrin aggregate spontaneously in aqueous solutions to form micelle-like particles, which provide an effective substrate for pancreatic lipase [43]. The CMT is also the temperature at which the aliphatic chain of a soluble lipid melts, that is, becomes liquid instead of solid. Thus, the longer or larger the hydrophobic moiety of a detergent, the higher the CMT. For many soaps and detergents this temperature range lies well above room temperature [38]. Conversely, short-chained, cis-unsaturated, branched-chain and polar substituted lipids have lower CMTs, some below

Volume 49, November 1970

OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

0~ The common dihydroxy and trihydroxy bile salts (cholates and deoxycholates) have CMTs lying far below the freezing point of water [3,39]. Micelles formed by long-chain aliphatic soaps and detergents are usually spherical in shape, as was first proposed by Hartley [44]. This type of micelle is illustrated schematically in the inset in Figures 3 and 4. In some cases increasing the concentration of detergent only increases the number of micelles, with little or no effect on the size or shape of the micelle. However, in certain cases, as the concentration of the detergent increases, spherical micelles elongate into rod-shaped micelles.* The aliphatic chains on the inside of the spherical micelles exist in a liquid hydrocarbon state largely excluding water. The polar head groups face the water.? The center or core of such a micelle contains the hydrocarbon chains in a hydrophobic environment. This area of the micelle possesses solvent properties for the hydrocarbon (hydrophobic) parts of other types of Reiss-Husson and Luzzati [45] used x-ray scattering technics to study the micellar shape of aliphatic detergents in micellar solutions. They showed that the shape of the micelles of certain detergents depends on the solute concentration. Spherical micelles form with sodium lauryl suffate, potassium or sodium myristate, palmitate and stearate and with the cationic detergent cetyl-trimethyl ammonium bromide above their respective CMCs. These, however, undergo transformation to rods at concentrations of amphiphile above 10 ~m/lO0 ml. The micelles formed by sodium oteate were shown to be rod-shaped at all concentrations, whereas those of cetyl trimethyl ammonium chloride were spherical at all concentrations. This study [45] and Madame Reiss-Husson's doctoral dissertation [46] should be consulted for further details. t Typical spherical soap or ionic detergent micelles in water [41] bind about 50 per cent of their counterions (Li', Na ~, K', Rb', Cs*, etc. in the case of anionic detergents or CI, Br-, I-, etc. in the case of cationic detergents), thus giving the micelle a net charge. These counterions are bound rather tightly to the surface of the micelle. The Agtt of ionic micelles usually lies between 50 and 100. The radius of these micelles is approximately the length of the constituent lipid molecule (15 to 20 A). Added counterion increases the amount of counterion bound to the micelle and this decreases the micelles' net charge. Added counterion also lowers the CMC and increases the Ag# of ionic detergents. Increases of temperature from O~ may initially lower the CMC (a minimum is usually found around room temperature), however, further increases of temperature elevate the CMC and reduce the Ag#. Further details, can be found in other studies [41, 47,48]. The CMCs of nonionic detergents are also depressed by added neutral salt, but less so than with anionic detergents. The CMC of solutions of nonionic detergents falls sharply with increases of temperature. The Ag# increases correspondingly with temperature elevation until the detergent precipitates from solution. This phenomenon is analogous to a phase separation and occurs at a temperature specific for each detergent. The temperature of precipitation is called the "cloud point." For further discussion see previous reports [41,49-52].

595

CHARACTERISTICS

OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

lipids. Thus, other insoluble lipids may be incorporated into the micelle to form "mixed micelles." This process is called solubilization and occurs only above the CMC of the mixture. The proposed model for the bile salt micelle is shown in the inset in Figure 3 [53]. The common dihydroxy and trihydroxy bile salts are steroids possessing a rigid cyclopentenophenathrene nucleus, on one side of which are clustered two or three hydroxyl groups and on the other side two methyl groups. Protruding from one end of the steroid nucleus is a short-branched aliphatic chain terminating in a strong hydrophilic group. These molecules, therefore, contain a hydrophobic side, a hydrophilic side and a short hydrophilic tail. They do not possess the clear-cut polarity that exists in long-chain aliphatic detergents. Nevertheless, they aggregate to form small micelles above their CMCs and they possess striking ability to solubilize certain classes of lipids, particularly the class II swelling amphiphiles of biologic interest. The structure and properties of these micelles stand in striking contrast to the structure and properties of spherical micelles of typical detergents. Systematic studies [40,53-56] have shown that the type of bile salt, the pH, the temperature and the counterion concentration can all appreciably affect the CMCs, the size (Ag,~s) and most probably the structure of pure bile salt micelles. Ag#s of bile salt micelles probably do not vary appreciably with the concentration of bile salt [53]. In water at 20~ the bile salts, free or conjugated, both dihydroxy and trihydroxy, form very small aggregates, usually dimers, trimers or tetramers [53]. Unlike the dimers formed by some aliphatic detergent molecules [48] which form at low concentrations below the CMC, bile salts form dimers over a wide range of concentration, well above the CMC. As the counterion concentration is increased, a marked difference manifests itself between the trihydroxy and dihydroxy bile salts. The trihydroxy bile salt micelles increase from dimers in water up to a maximum of about 7 to 9 molecules per micelle in 1.0 M sodium chloride [53]. On the other hand, the counterion concentration has a much more marked effect on the size of the dihydroxy bile salt micelles, even at relatively low salt concentration--for instance, sodium glycodeoxycholate, which forms dimers in water, forms large micelles with an aggregation number of about 63 in 0.5 M sodium chloride [53]. The marked differences in micelle size between dihydroxy and trihydroxy bile salts in response to added counterion is also reflected in the CMCs of dihydroxy and trihydroxy bile salts [56]. The CMCs of taurine conjugated bile salts were studied in increasing concentrations of sodium chloride. The CMC of taurocholate at 10~ is 3.1 mM in water with no added counterion. As the counterion concentration is increased to 0.7 M sodium chloride, the CMC remains constant. On the other hand, the CMC of taurodeoxycholate at 10~ is 1.8 mM

596

in water and progressively falls to 0.9 mM with increasing counterion concentration. Thus, in the case of the trihydroxy bile salt the CMC and Ag#s are resistant to the concentration of counterion present, whereas with the dihydroxy bile salt the Ag$ increases and the CMC decreases as the counterion concentration is increased [56]. Compared to aliphatic detergents (see f, page 595) bile salt micelles bind little or no counterion in water. In other words, bile salt micelles are highly charged compared to aliphatic ionic micelles [56]. The effect of pH on the Ag#s of bile salts show striking differences between the dihydroxy and trihydroxy species [40,53]. Decreasing the pH of trihydroxy bile salt solutions (even to the point of precipitation [40] of sodium cholate (pH =, 6.5) or sodium glycocholate (pH = 4)), has little effect on the Ag~ of these bile salts. On the other hand, the free and glycine-conjugated deoxycholates form huge micel~es (Ag~ > 1,000) as the pH is lowered towards their pKa's [40]. The effect of temperature on bile salt micelles is complex. In general, however, the CMC falls between 10~ and 20~ remains at a minimum level between 20~ and 30~ and rises after 40~ [56]. At all temperatures and counterion concentrations the CMCs of taurocholate are about double those of taurodeoxycholate [40,56]. The structure of the pure bile salt micelle is now fairly clearly established [53-56]. Apparent micellar weights [53], nuclear; magnetic resonance studies [54], CMC and thermodynamic data [56] and experiments using space-filling molecular models indicate that pure bile salt micelles are of two types which, for convenience, we will call " p r i m a r y " and "secondary" micelles, respectively [53]. Bile salt molecules probably associate into primary micelles by "hydrophobic interactions," that is, molecules are " b o n d e d " back-to-back by association of their bulky aromatic hydrocarbon moieties in Van der Waal's or apolar interactions. Experiments using molecular models indicate that about 8 bile salts can be fitted together in this fashion leaving no appreciable space on the inside. Micelles of Ages greater than 10 are called " s e c o n d a r y " micelles. These are formed, as indicated earlier, only, by the dihydroxy bile salts in high counterion (sodium chloride) concentrations. These larger secondary micelles are probably composed of clusters of primary micelles hydrogen bonded one to the other through their hydroxyl groups. These micelles are not as asymmetric as one might imagine. Viscosity and light scattering data indicate that they are very close to being spherical (see Small's report [40] for a detailed review of the physical chemistry of the bile salts). MIXED MICELLAR SOLUTIONS

The nonpolar lipids and the polar lipids of classes I and II can be solubilized by solutions of class Ill lipids. This phenomenon has traditionally been called

The American Journal of Medicine

CHARACTERISTICS OF MIXED MICELLAR S O L U T I O N S - - C A R E Y ,

SMALL

This principle defines, in a strict sense, the phenomenon of micellar solubilization. It means that each micelle in the mixed micellar solution must contain at least 1 molecule of solubilizate ( n o n p o l a r lipids or polar class I or II lipids). To know how many moleCules of solubi[izate are in each mice~le one needs the following information: (1) The molar ratio' of solubilizate to d e t e rg e n t (class III lipid) at saturation of the micellar solution with solubilizate " s a t u r a ti o n ratio"; (2) the CMC of the saturated micellar solution; (3) the micellar weight of the saturated micelle and (4) evidence that the population of micelles is of uniform size (monodisperse); (5) the solubility of the solubilizat'e in water.* From this information one may calculate both the number of detergent molecules and solubilizate molecules in each micelle. From the shape (spherical, rod-like or disc-like, etc.) one may make an educated guess concerning the molecular arrangem e n t within the micelle. Data concerning various types of mixed micelles is given in Tables I through

III and our concepts of the structure of some of the mixed micelles is depicted in Figure 5. Although m a n y of the mixed micellar solutions obey the principle of 1 (or more) molecule(s) of s o l u b i l i zate per micelle, some systems do not. In these cases there is far less than 1 molecule of solubilizate per mlcelle. Although it is clear t h a t increased solubility of the solubilizate occurs above the CMC, it is much less than would be expected if each micelle contained 1 molecule of solubilizate. For instance, one may calculate from the data of Norman [57] that a b o u t 3 molecules of 20 m e t h y l c h o l a n t h r e n e , a five ringed nonpolar unsaturated hydrocarbon, are solubilized by 1,000 bile salt molecules. Since both the CMC and micelle size are probably the same as t h a t of the pure detergent, one can estimate t h a t there are less than 0.1 molecules of solubilizate per micelle. Further, in the case of n-decane (see Table I) [58], about 8,000 molecules of the bile salt sodium taurocholate are needed to " s o l u b i l i z e " 1 molecule of the solubilizate. Since the CMC is unchanged and the aggregation number r e m a i n s a t only 2 the principle of 1 molecule of solubilizate per micelle is clearly not followed. Thus, in a strict sense this p h e n o m e n o n should not be called " m i c e l l a r s o l u b i l i z a t i o n . " t Many nonpolar molecules and some polar molecules, such as cholesterol and its esters, whose HHB is v e r y hydrophobic appear to be " s o l u b i l i z e d " in this way. In the following section mixed micellar solutions will be classified into four types (A-D) according to type of solubilizate.

~' In general, the solubility of lipid solubilizate in water may be considered negligible. t It should b,e noted that several laboratories have used large molecules such as dyes, fluorescent steroids and antifungal agents to calculate the CMCs of bile salts [5,57,59-62]. In these cases the reported CMCs are much higher than values obtained by other methods [40,55,56]. Since the principle of 1 molecule of solubili. zate per micelle appears not to be followed, the "solubilization" of these solubilizates is perhaps not directly related to micelle formation and may not even reflect the true CMC. The fact that solubilization of these substances is very small and only occurs well above the CMC (found by other methods) suggests that solubilizati0n in these cases may not be due to micelles per se, but to some other factors such as change in water structure brought about by the presence of detergent molecules, etc. Small water-soluble polar molecules can theoretically adsorb to the polar face of both aliphatic and bile salt micelles, as has been suggested [41]. These "mixed" micelles are a poorly defined category. The adsorbed molecules cannot be considered to be true solubilizates as outlined earlier. Several studies (summarized in [41]) have been carried out to estimate what effect such substances have on the CMC and micellar weights of detergents. Some examples of these additives are u n s a t u r a t e d aromatic hydrocarbons such as benzene and its derivatives, toluene and phenol; aromatic dyes, very short-chain mono-, di- and trihydric alcohols such as methanol,

ethylene glycol and glycerols; and molecules containing pyranose or furanose rings, such as sugars. The CMC of detergents [41,47] may be elevated or depressed by these additives depending on several factors, such as the concentration of the additive, its molecular structure, its mode of incorporation in or on the micelle and its electrical charge. The separate effects of these molecules are difficult to identify experimentally, due to the fact that many o~f these water-soluble molecules profoundly influence the structure of water, the di electric of water, "hydrophobic interactions" and the monomer solubili.ty of the detergent. For further details, Emerson's thesis [47] should be consulted. Nuclear magnetic resonance (NMR) technics have recently been applied to the solubilization of aromatic hydrocarbons by ionic micelles [63,64]. In one study [63] persuasive evidence was presented to show that the highly polarizable hydrocarbon, benzene, is predominantly solubilized at the micelle-water interface, whereas cyclohexane (a saturated cyclic hydrocarbon) is solubilized within the hydrocarbon core of the micelles and does not exchange significantly with the aqueous phase. Nakagawa and Tori [64], also using NMR spectroscopy, measured the time-averaged exchange rate of benzene between micelles of sodium dodecyl sulphate and the intermicellar aqueous zone. The exchange rate was evaluated to be 10-' second or less for a micelle "saturated" with benzene. The interactions between water-soluble dyes and detergents (including bile salts) are discussed elsewhere [41,56,65,66].

" m i c e l l a r s o l u b i l i z a t i o n . " These solutions may be called mixed micellar solutions. Mixed micellar solutions may also be formed by mixtures of class III lipids of different types. We will, therefore, define a mixed micelle as any micelle made up of more than one lipid-like chemical species. Empirically, at least one of the chemical species must be able to form micelles alone in aqueous solutions (i.e., must be a class III lipid).

I. Micellar Solubilization. The Principle of "1 (or More) Molecule(s) of Solubilizate per Micelle."

Volume 49, November 1970

597

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

TABLE I

Type A Mixed Micelles (Detergent and Nonpolar Lipid) S01ubilizate Aliphatic Detergent(Lipid ClassIliA) Meth0xy0xyethylenedecylether in water + n-Decane[68}

Molar saturationratio

Bile Salt(Lipid ClassIIIB) Na Taur0ch01ateor Glyc0cholatein water + n-DecaneI53,56,58]

--

0.2-0.3

--

Micellar characteristics

No n-decane

No n-decane

CMC Shape

0.001 mM

Micellesaturated with n-decane 0.00068 mM

Spherical

Spherical

Size-micellar weight Ag# of detergent molecules Number of nonpolar molecules per micelle

43,100 83

88,500

0

158 46

0.00013

3.1 mM Roughly spherical

Micelle "Saturated" with n-decane* 3.3 mM Roughly spherical

850 2

900 2

0

Far less than one

NOTE: CMC -----critical micellar concentration. Ag# = aggregation number. * Mechanism of solubilization unknown (see footnote t, P. 597). II. Classification of Mixed Micelles. Mixed micelles may be formed by a detergent of either polar lipid class I l i a (aliphatic) or polar lipid class I-liB (aromatic) and A. Nonpolar lipids B. Class I nonswelling amphiphiles C. Class II swelling amphiphiles D. Other chemical species of class III amphiphiles. In the succeeding section the properties of each type of mixed micelle formed by a class I l i A lipid will be compared and contrasted with an example of each type of mixed micelle formed by a class IIIB lipid. Since there are many types of class IliA lipids (the anionic, cationic, nonionic zwitterionic and ionicnonionic a~iphatic detergents) some differences may exist between them in respect to formation of mixed micelles with other lipids. We will, therefore, confine our discussion to mixed micelles formed with common aliphatic detergents, such as sodium dodecyl sulfate (anionic) or polyoxyethylene ethers (nonionic) which have been extensively studied.* Bile salts will be discussed exclusively as examples of class IIIB lipids although they are not unique in this subclass of lipids.t

A major consideration in solubilization, be it industrial (soaps or detergents) or physiologic, is the solubilization ratio of insoluble lipid to soluble lipid. Or, in other words, how much insoluble lipid can be solubilized by a g i v e n amount of soluble micelieforming lipid? Since it is believed that a micellar phase is necessary for the absorption of fat from the alimentary tract [2,3,5,6,17], absorption can be accounted for by the ability of bile salt to hold large amounts of insoluble lipids in mixed micellar solution in gut luminal contents (i.e., micellar solutions of bile salts with insoluble biologic lipids have high saturation ratios). This characteristic of mixed micellar systems will dominate our discussion of both aliphatic and bile salt mixed micelles in the succeeding section. We must stress at this point that mixed micelles formed by aliphatic detergents (class I l i A lipids) are not only of industr!a! interest, but also may have physiologic application. In clinical situations in which bile cannot enter the intestine, such as in complete biliary obstruction or total biliary fistula, as much as 80 per cent of ingested fat can still be absorbed. The absorption can be accounted for by virtue of the fact t h a t even at the slightly acidic pH

r In general, the greater the number of molecules in a micelle, the greater the capacity of the micelle to solubilize any class of lipid. Nonionic detergents possess very low CMCs (CMCs approximately 100 times lower than that of an analogous ionic detergent) [41,49]. As a general rule, the Ag# of a micelle (ionic or nonionic) varies inversely with the CMC. Therefore, the Ag#s of nonionic micelles are correspondingly much larger than the Ag#s of ionic micelles, and solubilization capacities of nonionic micelles for other insoluble lipids are accordingly greater than that of ionic micelles [51]. Theoretically, the effect of increases of temperature on the solubilizaLion capacities of both types of micelles should vary, depending on whether increase in temperature decreases

the Ag# (ionic micelles) or increases the Ag# (nonionic micelles). However, in practice, anomalous results are nearly always found due to a true increase in the solubility of the solubilizate in the intermicellar aqueous phase and in the hydrocarbon cores of the micelles as the temperature is increased [41,50,51]. Increases in temperature thus result in an increased saturation ratio irrespective of the species of micelle-forming lipid. t We have recently shown [67] that the steroid antibiotic, sodium fusidate, which is derived from the fungus Fusidium coccineum, possesses micellar properties similar to trihydroxy bile salts, with respect to Ag#s, CMCs, response to added counterion and solubilizing capacity for insoluble swelling amphiphiles (class II lipids).

598

The American Journal of Medicine

CHARACTERISTIGS

TABLE II

OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

Type B Mixed Micelles (Detergent and Insoluble Nonswelling Polar Lipid)

Solubilizate Aliphatic Detergent(Lipid ClassILIA) Methyoxypolyoxyethylenedecyl ether in water + n-Decanol [41,68] Molar saturation ratio

=-

Micellar characteristics

No n-decanol

CMC Shape Size-micellar weight Ag# of detergent molecules Number of solubilizate molecules per micelle Other

Bile Salt (Lipid ClassIII B) Na deoxycholatein water + n-Decanol [53,56,58,69,70]

0.55

-No n-decanol

39

0.001 mM

Micelle saturated with n-decanol 0.00034 mM

3.1 mM

Spherical 43,100 83

Spherical 213,000 351

Spherical 850 2

Micelle saturated with n-decanol Not studied but probably low Probably spherical Probably large ?

0

192

0

Probably much greater than 1

...

Capacity of saturated micelle to solubilize n-decane increased [41]

Elimination of the property of solubilizing nonpolar hydrocarbons such as n-decane at saturation [70]

* Decreases markedly as chain length of alcohol increases. of the gut lumen (pH 6.0 to 6.5) some soap micelles and perhaps lysolecithin micelles can form. These molecules, which are typical aliphatic (class IliA) amphiphiles, can incorporate small amounts of other lipolytic products such as monoglycerides and protonated fatty acids into their micelles. However, solubilization of lipids by these micelles is not nearly as efficient as by an organism's own specialized detergents (i.e., the bile salts), as will be shown in the following sections. The saturation ratios, CMCs and apparent micelle shape and size are given both for the aliphatic detergents and bile salts before and after saturation with different insoluble lipids in Tables I through III. A suggested structure for each type of mixed micelle is schematically shown in Figure 5. Type A. Mixed micelles with nonpolar lipids: Table I compares the properties of a nonionic detergent, methyoxyoctaoxyethylene decyl ether-ndecane, mixed miceiles [68] and bile salt-n-decane mixed micelles [58]. In the case of the aliphatic detergent there is considerable evidence that the nonpolar solubilizate is solubilized in the hydrophobic hydrocarbon core of the micelle as illustrated in Figure 5 (top left) [41,63]. The nonionic detergent micelle (Table I) can solubilize 1 molecule of ndecane for every 3 detergent molecules. The saturation ratios are somewhat smaller in the case of ionic aliphatic detergents, due to their smaller Ag#s [51]. The slight fall in CMC, =30 per cent, below that of the pure detergent occurs pari passu with an increase in Ag#. With any given detergent the saturation ratio decreases with an increase in the chain length of the nonpolar compound. Further, in general,

Volume 49, November 1970

a crystalline nonpolar lipid is less well solubilized than a liquid nonpolar lipid. The mechanism of n-decane solubilization by bile salts is unknown (refer to section "Mixed Micellar Solutions," I and footnotet, page 597). However, it is clear that nonpolar molecules are very poorly "solubilized" by the bile salts (Table I).

Type B. Mixed micelles with class I polar lipids (nonswelling amphiphiles): The mode of solubilization of insoluble nonswetling amphiphiles in aliphatic detergent micelles is shown on the left in Figure 5. These lipids, such as medium- or long-chain alcohols or protonated fatty acids, intercalate themselves between the detergent molecules of the micelle. They become oriented with their polar groups towards the water environment and their hydrocarbon " t a i l s " toward the center of the micelle and thus become solubilized. As indicated by the example given in the left hand column in Table II, the CMC falls compared with the CMC of the pure detergent. The Ag# goes up significantly and is accounted for, not only by the incorporation of solubilizate molecules, but also by an increase in Ag# of the nonionic agents themselves. Ionic detergent micelles show similar features in response to the addition of an insoluble nonswelling amphiphile [41]. The CMC and Ag# vary inversely. That is, as more solubilizate is incorporated into the micelle the Ag# increases and the CMC falls. The saturation ratios for class I polar solubilizates in ionic and nonionic detergents are greater than the values obtained when nonpolar hydrocarbons of similar chain length are solubilized. As the CMC falls and the Ag# increases, the ability of these mixed micelles

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CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

TABLE III

Type C Mixed Micelles (Detergent and Insoluble Swelling Polar Lipid) Solubilizate Bile Salt (Lipid ClassIIIB) Na glyc0ch01ateor Na taur0cholatein 0.15 NaCI + Lecithin [40,53,87,88,89,90]

Aliphatic Detergent(Lipid ClassIliA) Lysolecithinin water + Lecithin [20,84,85,86J

Micellar characteristics

No lecithin

CMC

0.195 mM

Shape

Probably spherical 92,400

Size-micellar weight Ag# of detergent molecules Number of solubilizate molecules per micelle Other

2.0

--

0.05-0.10

--

Molar saturation ratio

Micelle saturated with lecithin Not studied, but probably very low Asymmetrical 1,500,000

No lecithin

Micelle saturated with lecithin

3.1 mM

0.2 mM

Spherical

Almost spherical

180

2,630

2,800 5

125,000 62

0

280

0

125 Shows an increased capacity to solubilize class I polar lipids, e.g., cholesterol, when

swollen with lecithin

to solubilize nonpolar hydrocarbons such as decane increases [41]. The bile salts, as shown in the right hand column of Table II, are excellent solubilizers of m e d i u m - c h a i n alcohols such as n-decanol and also of medium-chain carboxylic (heptylic, nonylic, decanoic) acids [69-72]. In the case of nodecanol, Fontell [69] showed t h a t deoxycholate-n-decanol micelles were large but probably still spherical. The solubility of nonpolar hydrocarbons such as decane in bile salt micelles swollen with insoluble nonswelling a m p h i p h i l e s is not increased, in contrast to type B mixed micelles formed with aliphatic detergents. When the chain length of the solubilizate is increased, solubility in bile salt micelles decreases markedly. For instance, the solubilization ratio for n-decanol in sodium deoxycholate is a b o u t 3 [69,70] or for decanoic acid in sodium taurocholate about 2 [71]. When the chain length is increased to 18 carbon

atoms the saturation ratios for both the alcohol and acid fall to less than 0.2 [58,73,74]. Bile salts are even poorer solubilizers of steroid alcohols such as cholesterol, sitosterol, lanosterol and vitamin D.* T y p e C. M i x e d m i c e l l e s with class II p o l a r l i p i d s (swelling amphiphiles): Detailed information is scarce on mixed micelle formation with insoluble swelling a m p h i p h i l e s and aliphatic detergents. Some data are available on lysolecithin-lecithin mixed micelles [84,85], and a phase diagram lecithin-lysolecithin-water has been published [20] which shows t h a t the saturation ratio is low. The features of this micelle are summarized in the left hand column of T a b l e III and the structure of the micelle is suggested schematically in Figure 5. The saturation ratio is low; depending on published figures, it requires between 10 [84] and 20 [20] molecules of lysolecithin to solubilize 1 molecule of lecithin. At this saturation the viscosity of the solution is high and the structure of the micelle

8, The poor capacity of bile salt solutions, whether free or conjugated, to solubilize the biologically important monohydric steroid alcohol cholesterol, an insoluble nonswelling amphiphile, is well known [10,13,20,75-83]. The solubilization maximum of cholesterol varies somewhat from individual bile salt to individual bile salt, varying from 20 molecules of sodium deoxycholate to 1 of cholesterol to 130 sodium taurocholate molecules to 1 of cholesterol in solutions of the respective bile salt well above the CMC [83]. The studies carried out to determine the Ag#s of micelles of sodium taurocholate and sodium taurodeoxycholate saturated with cholesterol by ultracentrifugation technics have shown negligible enlargement of the micelles by t h e cholesterol [40]. These results probably mean that even though some of the micelles

might contain a cholesterol molecule, each micelle cannot contain a molecule of cholesterol at the same time. Thus, the principle of 1 (or more) molecule(s) per micelle is not obeyed. It has been suggested, on the basis of diffusion coefficients of cholesterol in bile salt micelles, that two types of micelle are present, one small (Ag# ---- 5 or 6) and one large (Ag# about 30). The large is supposed to contain a cholesterol molecule [82]. However, no polydispersity could be found using ultracentrifugal technics

600

[40].

The real mechanism of cholesterol solubilization in bile salt solutions is not known. Specific data concerning the CMCs of this system are not available, but owing to the very low saturation ratios, the CMCs probably do not vary much from the CMCs of the pure bile salts.

The American Journal of Medicine

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

TYPE

SOLUBILIZATE

SOLUBILIZER ALIPHATIC DETERGENTS

A

BILE SALTS

NON-POLAR

LIPIDS

(e,g. n-decaneor n-dodecone)

POLAR LIPIDS

B

cL,ssl INSOLUBLE NONSWELLING A MPHIPHILES

~'~1~

(e.g n-dodecanol)

INSOLUBLE AMPHIPHILES e.g. phospholipids such as lecithin

D

CLASS Trr

~ ? ~

SOLUBLE AMPHIPHILES (e.g No soaps of

Iong-chamfalty acids) LONGITUDINALSECTION CROSSSECTION

Figure 5, Classification of mixed micelles. Solubilized molecules are shown with heavy lines. The capitals A.D in the far left column give the four major types of mixed micefles classified according to the aqueous interactions of the solubilizates shown in the second column from the left. The classes in this column, therefore, are identical with the classes of lipids given in Figure 3. The center column gives a pictorial representation of the structure of each type of mixed micelle when an aliphatic detergent is the solubilizer and a lipid typical of each class is the solubitizate. It is to be noted that the structure of the aliphatic mixed micelle of type C departs considerably from sphericity. The two columns on the right of the figure give both longitudinal and cross.sectional views of the suggested structure of bile salt mixed micelles. B, bile salt-octadecanol; C, bile salt-lecithin; D, bile salt-aliphatic detergent. The exact portion of the aliphatic chains (true bilayer or partially overlapping bilayer) is not known. The structure of bile salt-nonpolar micelles, if they exist, is not known (see text and footnote1, page 597). There is good evidence that the swollen bile salt micelle of type C is the most likely structure of the mixed micelles in bile. The text should be consulted for details.

is highly asymmetric. The micellar w e i g h t is about fifteen times greater than the micellar weight of pure lysolecithin. The most likely explanation for the asymmetric micelle shape is that of an oblate ellipsoid (Figure 5). The lecithin molecules would occupy the narrow transverse section of the micelle as a bilamellar leaflet. The axis of rotation of the ellipsoid would be parallel to the axis of the lecithin molecules. The lysolecithin molecules would be oriented in a fashion similar to the spherical micelle and would shield the hydrophobic parts of the lecithin from contact with water. Other class II polar lipids, such as glyceryl-l-monooleate (monoolein), are also poorly solubilized by aliphatic detergents. Hofmann's [5] published figures for the saturation ratios of sodium oleyl taurate-monoolein and sodium lauryl sulfatemonoolein micelles are 0.3 and 0.4, respectively. Bile salts, on the other hand, possess an enormous capacity to solubilize insoluble swelling amphiphiles (class II lipids). This fact is of paramount importance to their physiologic action. Some of the characteristics of bile salt-lecithin micelles are shown in Table III.

Volume 49, November 1970

A maximum of 2 moles of lecithin can be solubilized by 1 mole of bite salt in a micellar solution [13,87]. The most rational way that this phenomenon can be explained is in terms of a bile salt-lecithin micelle consisting of a cylinder of bilamellar lecithin molecules surrounded on its perimeter by a cylinder of bile salts [20,40,54], as shown schematically in Figure 5. In other words, if bile salts are added to a suspension of the lamellar liquid crystals of lecithin ( " m y e l i n figures") in an excess of water (see Figure 3 inset of class II polar lipids), the bile salt molecules can readily chop up the extended bilayer configuration of the myelin figures into small disc-like portions of the bilayer. The bile salts " b o n d " hydrophobically onto the exposed hydrocarbon chains of the lecithin molecules. In this manner the micelle presents a universal hydrophilic surface to the aqueous milieu both at its ends and on its sides (previous studies [14,20,40,54,87-90] should be consulted for the experimental evidence for this model). The CMC of this system falls steeply as more and more lecithin is added [88]. The value reaches a plateau at about

601

CHARACTERISTICS OF MIXED MICELLAR SOLUTIONS--CAREY,

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0.2mM (in the case of taurocholate-lecithin micelles). Concomitantly the micellar weight increases enormously(Table III). Bile salts solubilize monoglycerides with similar efficiency. Hofmann [5] found that the saturation ratios for glyceryl-l-monooleate dissolved in conjugated bile salt solutions varied from 1.4 to 1.7, four or five times greater than the capacities of the long chain detergents for the same lipid. The micelle is appreciably swollen by the addition of even small amounts of monoolein [40]. As the number of molecules of monoolein increases in the mixed micelle, a proportionately smaller increase in the number of bile salt molecules in the micelle occurs [40]. Both bile salt-lecithin and bile salt.monoglyceride micelles possess a capacity greater than pu re bile salt micelles to solubilize cholesterol and fatty acids respectively (see section on "Bile and Complex Mixed Micelles").

increased. Benzonana [91] has shown that the presence of 1 molecule of sodium oleate for 5 molecules of sodium deoxycholate results in a decrease in the CMC from about 3.0 to 2,0 mM. In a study on mixtures of bile salts and Tweens (nonionic detergents) by light scattering [92], it was found that mixed micelles of intermediate size are formed at all mixing ratios. The Ag# of the mixed micelles varied directly with the nature and amount of the nonionic detergent. Mixtures of different bile salts also form mixed micellar solutions whose properties vary with the ratios and types of bile salts employed.*

Type D. Mixed micelles--binary detergent mixtures (class III, polar lipids): Binary mixtures of soaps or

gallbladder bile is about 84 per cent water, 11.5 per cent bile salts, 3 per cent lecithin, 0.5 per cent cholesterol and 1 per cent of other components (bile pigments, proteins and inorganic ions and cations) [93,94]. Neglecting the last components, which are present in small quantities and are soluble in water, bile can be considered an aqueous mixture of three very different lipid species. The interactions between bile salts, lecithin and cholesterol in water have been studied in vitro and expressed as phase diagrams [10,12-14]. Certain ratios of these lipids in an excess of water formed a clear micellar solution. This micellar solution was able to solubilize a maximum of 4 per cent cholesterol, and it was suggested that cholesterol can be solubilized and transported in bile in high concentrations in this complex three-component micelle. In other words, the bile salt micelle swollen

typical detergents, class I l i A amphiphiles, are miscible in all proportions. The micelle structure probably varies continuously from that of one pure component to that of the other. A reasonable guess of the structure of a micelle in a micellar solution con. taining 50 per cent sodium dodecyl sulfate and 50 per cent sodium oleate is indicated schematically in Figure 5. The individual molecules are both present in the micelle and are oriented without distinction of type with their hydrophobic tails towards the center of the micelle and their polar groups on the surface. Both are present as individual molecules outside the micelle. Thus, both contribute to the measured CMC. Theoretically, the species with the lower CMC has a greater concentration in the micelle and a conversely lower concentration as monomer than its over-all concentration in the total solution, i.e., the species with the lower CMC should partition favorably into the micelle. As a corollary, the CMC of the mixture lies somewhere between the CMCs of the components and nearly always closer to the CMC of the more hydrophobic component than would be expected by ideal mixing. Owing to the huge difference in the CMCs of ionic and nonionic detergents, the CMCs of their binary mixtures lie much closer to the CMC of the nonionic component at practically all mixing ratios. It has also been clearly established [41] that the degree of dissociation of the ionic detergent is increased as the proportion of the nonionic material is increased. Bile salts and soaps also form mixed micelles. For example, all proportions of sodium cholate and sodium oleate form a micellar solution when an excess of water is present [20]. The characteristics of sodium taurocholate-potassium oleate mixed micelles have been studied by ultracentrifugation technics [40]. The bile salt micelle becomes larger and larger as the weight ratio of potassium oleate is

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COMPLEX NATURALLY OCCURRING MIXED MICELLES I. Natural Bile and Bile Salt-Lecithin-Cholesterol Micelles. The average composition of normal human

" The CMCs and Ag#s of bile salt mixtures have been studied in this laboratory [40,56]. With respect to this type of micelle the CMC behaves in a fashion analogous to other detergent mixtures [41]; the CMCs of a binary mixture (dihydroxy and trihydroxy) lie closer to the CMC of the more hydrophobic dihydroxy bile salt than ideal mixing would suggest. Normal human bile and intestinal luminal contents contain a mixture of six conjugated bile salts, the glycine and taurine conjugates of cholic, deoxycholic and chenodeoxycholic acids. The Ag#: of an equimolar mixture of conjugated di- and trihydroxy bile salts in physiologic concentration of salt (0.15 M sodium chloride) at 20~ is approximately 15 [56]. Although not studied at 37~ extrapolation based on the temperature effects on pure bile salt micelles [53] leads one to predict that the Ag~: might be slightly less, say 10 to 12 at body temperature. Bile salt molecules, therefore, would be expected to exist in the ileum of man in mixed aggregates containing approximately this number of monomers. Hofmann [5] found that the approximate CMC of such a mixture was 2 mM with "solubilized" azobenzene. Our studies [56] showed that at 37~ in 0.15 M sodium chloride the CMC of an equimolar mixture of taurine conjugated bile salts is 1.6 mM.

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with lecithin solubilizes the cholesterol. In general, the composition of bile samples from normal human subjects falls in the predicted micellar zone of the in vitro study, i.e., normally, bile is less than saturated with cholesterol. In contrast, the composition of bile samples from patients with cholesterol gallstones, for the most part, fell either close to the line separating the micellar from the other zones, or clearly in a zone in which cholesterol crystals would be found in equilibrium with the micellar solution. This fact suggested that the physical state of bile in the human gallbladder is predicted from the model in vitro system and that other components (inorganic salts, bile pigments, proteins, etc.) apparently do not significantly influence the solubility characteristics of cholesterol in bile. Recently it has been suggested that the composition of hepatic bile from patients with cholesterol gallstones is highly supersaturated with cholesterol when compared with the model system [95]. It was suggested, therefore, that a subtle defect in hepatic metabolism resulted in abnormal bile supersaturated with cholesterol. In the gallbladder, nucleation of the supersaturated bile occurs, leading to precipitation of cholesterol--a condition which, if chronic, would give rise to cholesterol stones [96]. No systematic studies have as yet been carried out on the CMC's, shape, size or stability of artificial model solutions of bile salt-lecithin-cholesterol micelles. Therefore, we must turn our attention to the studies that have been carried out on whole human bile. Using such technics as paper electrophoresis [97103], free electrophoresis [104], ultracentrifugation [100,102,104-108], gel filtration [109-115] and recently electron microscopy [116], all investigators agree that a "macromolecular complex" containing variable amounts of bile salts, lecithin, cholesterol and perhaps protein and bilirubin occurs in human bile. It must be stressed, however, that in almost all of these experiments bile was examined by technics commonly used for studying true macromolecular colloids such as proteins.* The results of most of these studies must be interpreted with considerable caution. For instance, electron microscopy technics [116] involve dehydration of the specimen and therefore alter or destroy micelles. I sopycnic grad lent ultracentrifugation with 60 per cent w/v caesium chloride [105,106] must alter micellar size because of the huge salt concentration (see footnote f, page 595). Gel filtration technics [109-115] disrupt micelles by True macromolecules are usually large single molea r e joined by stronger bonds (covalent bonds, ionic bonds, hydrogen bonds) than those "hydrophobic forces" or "dispersion forces" which are responsible for aggregation of individual molecules into micelles. Thus, technics adequate for isolating true macromolecules usually disrupt or at least modify micelles. cules whose atoms

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trapping the bile salts in the gel lattice. This causes the lecithin and cholesterol to precipitate out as micro-liquid crystals [114,115]. Electrophoresis [97-103] involves placing the micellar solution in an electric field, which may result in separation of charged molecules from uncharged molecules. Unfortunately, in all studies in which ultracentrifugal sedimentation technics were used to measure micellar weights [102,104,107,108] the specimens of bile had to be diluted at least 1:10 in order to reduce the optical density sufficiently to make schlieren optical examination possible. This dilution may decrease the concentration of bile salt below the CMC or change saturation ratios. Further, constants needed to calculate micellar weights by ultracentrifugal methods (partial specific volume and diffusion coefficient of micelle) have been on]y crudely estimated. Errors in these constants can give rise to large errors in micellar weights. Thus, only conflicting reports regarding the size of the "micellar complexes" in bile are available [102,105,106,107,117]. Estimated micellar weights have been published for whole bile which range from 11,000 [105] to 150,000 [114]. In fact, micellar weights would be expected to vary with lecithin content like the lecithin-bile salt system [40]. Technical difficulties are also encountered in measuring the CMC of bile. Tamesue and Juniper [108] measured the CMCs of bile by surface tension and ultracentrifugal technics. The CMCs of the same specimen by these two technics corresponded reasonably well and were of the order of 0.9 to 2.2 mM (expressed as total bile salts) with a mean of 1.45 BM. The only other CMC value for human gallbladder bile found in the ]iterature is 1.5 B M reported in one specimen by Furusawa [117] who used a surface tension method. Surface tension versus concentration curves are complex and difficult to interpret when such mixed micel~es are under consideration [11], and thus these CMC values should be accepted with reservation. Until such time as model micellar systems of bile salt lecithin and cholesterol are studied systematically and until technics are available to estimate accurately the characteristics of the mixed micelles of natural bile, this subject must, perforce, remain a terra incognita. II, Micellar Phase of Intestinal Luminal Content During Digestion of a Fatty Meal. In 1964 Hofmann

and Borgstrom [118] published the results of their extensive experiments on fat digestion in man. They found that after a test meal containing corn oil, all the lipids in intestinal content are present as an emulsified oil phase coexisting simultaneously with an aqueous "micellar phase" composed chiefly of fatty acids, monoglycerides and bile salts. Their results were consistent with the hypothesis previously advanced by Bergstrom and BorgstrSm [119] that the absorption of dietary lipid takes place from a micellar solution of bile salts containing the lipolytic products

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of triglyceride digestion, i.e., fatty acids and 2-monoglycerides. Furthermore, it was later shown that in two important clinical disorders, the blind loop syndrome [120,121] and the ileectomy syndrome [122], respectively, both of which are associated with steatorrhea, the percentage of lipid in the micellar phase of jejunal content was significantly decreased pari passu with a decreased intraluminal concentration of bile salts. An aqueous micellar phase of bile salt-monoglycerides, bile salts-ionized fatty acids or bile salts-monoglycerides-fatty acids is also essential for the intestinal absorption of cholesterol [123,124] and the "fat-soluble" vitamins [125].* Monoglycerides are nonionic insoluble-swelling amphiphiles (class II polar lipids). Their liquid crystalline behavior in water [31,126] is akin to the zwitterionic phospholipid lecithin already discussed; in other words, they spontaneously swell in water to form liquid crystals. Partially ionized fatty acids "acid soaps" can be incorporated into this liquid ~;rystalline array [28]. In an excess of water a mixture of monoglycerides and partially ionized fatty acids form "mYelin figures," that is liquid crystals dispersed in an excess of water. The addition of an adequate amount of bile salt to such a system causes a rapid decrease in viscosity and turbidity, and the resultant transparent solution is micellar. Practically nothing is known concerning the micellar structure, shape, size or other characteristics of this system. Borgstrom and Feldman [127,128] attempted to measure the micellar diameters of these aggregates (bile salt-monoglyceride and bile salt-monoglyceride-cholesterol micelles) using gel filtration. Their figures of 30 to 60 A for the diameter of these micelles must be accepted with caution owing to the fallacies inherent in this method of estimation, as already mentioned. It seems to us that the most plausible structure of these micelles and the monoglyceride-fatty acid-bile salt micelle is also that of a bimolecular cylinder of monoglyceride (with or without fatty acid and/or cholesterol) molecules, the hydrophobic perimeter of which is clothed by a coat of bile salt molecules analogous to bile salt-lecithin micelle. Proof of this model must await further studies. No published data on the physical-chemical properties of the micellar phase during fat digestion are available. The problem is complex owing to the fact that the concentration of a given lipid in jejunal contents at any one moment of time represents the resultant of addition of lipid from the stomach reservoir, digestion by pancreatic lipase, and absorption of micella,r lipid by the microvillous membrane. In contradistinction to whole bile, which is a relatively static micellar system, we need to know the rate -'.' For an up to date summary of the function of bile in the alimentary canal the recent review by Hofmann [6] should be consulted.

604

constants for the equilibrium of each of the solubilized tipids in the gut between emulsion droplets, mixed micelles, extramicellar fluid (as monomers) and absorptive membranes to estimate the average micellar composition as it might exist in vivo. The usual method for the separation of intestinal content into an oil phase and micellar phase requires centrifugation at 100,000 g for at least twelve hours [118], during which time significant artifacts may be introduced as a result of this isolation procedure. These difficult problems will have to be elucidated before fat digestion and absorption are comprehensively understood at the molecular level.

SUMMARY In higher animals lipid molecules are largely waterinsoluble, but must be digested, absorbed, transported and secreted in an aqueous medium. One of the means by which these phenomena may occur is by the association of insoluble lipids with soluble lipids in small molecular aggregates called "mixed micelles." Insoluble !ipids are thus rendered "soluble." Since bile and the aqueous phase of intestinal content are mixed micellar solutions containing large amounts of insoluble lipids, we thought it worthwhile to focus on the properties of micellar solutions and the mechanisms by which insoluble lipids, especially those of biologic interest, are brought into aqueou s Solution. We have, therefore, classified lipids into soluble and insoluble species and suggested, as far as is presently possible, how soluble lipids form mixed micelles with insoluble lipids. Mixed micelles are divided into two categories, depending on whether the micelle-forming component (soluble lipid) is an aliphatic detergent (soaps, etc.) or a complex aromatic detergent (bile salts, etc.). The amounts of insoluble lipid solubilized by either of the two types of detergent varies markedly with the chemical type of insoluble lipid (nonpolar, polar class I and pola( class II). Bile salts have an extremely poor capacity to solubilize aliphatic or aromatic nonpolar lipids. In fact, it is far from certain that these types of molecules are actually solubilized in the bile salt micelle. Aliphatic detergents, on the other hand, solubilize nonpolar detergents effectively within the core of the micelle. The ability of bile salt micelles to solubilize insoluble polar molecules depends, not only on the class of the insoluble molecule, but also on the molecular configuration and the nature of the polar and nonpolar parts of the molecules. If the chain length is only 8 to 12 carbons, the molecules are very well solubilized, regardless of the nature of the polar group. If the chain is long (C~G-C2_,)the molecule is well solubilized only if the insoluble lipid possesses a strong enough polar moiety to permit water to swet] the molecules and form liquid crystals (class II polar lipids). Long chain nonswelling lipids (class I polar

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lipids) are solubilized much less effectively. Polar molecules whose nonpolar part is a bulky aromatic structure (e.g., cholesterol) rather than an aliphatic chain are very poorly dispersed and the mechanism of solubilization is unknown. Aliphatic detergents solubilize both class I and class II insoluble lipids. In general, aliphatic detergents solubilize long chain and aromatic class I lipids far more efficiently than the bile salts, whereas the converse is true for class II lipids. The marked differences in solubilizing capacity between the mixed micelles formed by aliphatic detergents and by bile salts can be explained by their different micellar structure, a structure imposed by their very different molecular configuration. The very marked ability of the natural detergent bile salt to disperse and solubilize the class II lipids such as phospholipids, monoglycerides and "acid soaps" is physiologically important as these lipids are the main insoluble lipids f o u n d in bile and in intestinal contents during fat digestion. Further, a striking change in the solvent properties of bile salts occurs after class II lipid is added to their solutions. The swollen bile salt-lecithin or bile saltmonoglyceride micelle now contains a liquid hydrocarbon region similar to an aliphatic micelle, in which

OF MIXED MICELLAR SOLUTIONS--CAREY, SMALL

appreciable quantities of class I lipids such as cholesterol and long-chain fatty acids may be solubilized. The resulting complex micelle appears to have a distinctive molecular arrangement. The probable structure is that of a small bimolecular cylinder of class II lipid (e.g., lecithin or monoglyceride) containing interdigitated class I lipids and surrounded on its perimeter by a " h y d r o p h o b i c a l l y b o n d e d " layer of bile salt molecules. This structure permits the exterior of the micelle to he covered with the watersoluble polar groups and hides the hydrocarbon parts on the interior. Although this model has been primarily derived from in vitro studies on mixed micelles, especially the bile salt-lecithin micelle, little information is available concerning the properties of the complex micelles existing in bile and in intestinal content. Until the characteristics of these micelles are known, one can only extrapolate their properties from model systems.

ACKNOWLEDGMENT Our thanks are due to the Medical Research Council of Ireland and the General Research Support Grants Committee of University Hospital, Harrison Avenue, Boston, for partial financial support to M. C. Carey.

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