Liquid crystals in living and dying systems

Liquid Crystals in Living and Dying Systems~ DONALD M. SMALL Boston University Medical School, Boston, Massachusetts 02118

Received September 20, 1976; accepted October 14, 1976 I. INTRODUCTION Liquid crystals may be defined as states of matter having characteristics of both liquids and crystalline solids, in that they have some degree of order but also some degree of fluidity. Interest in liquid crystals has expanded greatly in industry, particularly in the field of display systems and temperature sensors. However, little attention has been paid to the liquid crystalline nature of certain arrays of molecules in biological systems and even less to the accumulation of liquid crystalline deposits in human disease states. In healthy living systems, a liquid crystalline order of molecules occurs in membraneous structures such as myelin, the rods and cones of the eye, chloroplasts, certain other cellular organelles, and serum lipoproteins. While this paper will be directed particularly toward the liquid crystal systems primarily involving lipids, I should point out that there are a large number of protein systems which might be classified as liquid crystalline, for instance the major components of muscle are arranged in ordered liquid crystalline-like arrays which allow movement to occur when the appropriate energy is supplied. I will first review the physical properties of Iipids of biological importance, the types of liquid crystals which they form, and the types of interactions between various cIasses of lipids. I will then progress to a discussion of liquid crystals in living systems concentrating specifically on lipoproteins and upon the plasma membrane as a prototype of liquid crystals in 1 Supported b y H L 18623 and GM 00176 from the National Institutes of Health.

all living systems. Finally, I will show that liquid crystals often accumulate in specific human diseases. In such diseases, the delicate metabolic balance of a certain lipid species is disturbed, often by the lack of a specific catabolic enzyme, leading to the accumulation of a particular lipid species and disruption of cellular function by the accumulation. Certain lipid species while occurring in trace amounts in normal tissue, become quite toxic when accumulated in larger amounts. However, other more innocuous lipid species cause problems only because of mass effects which compromise the cell and the organ in which the lipids accumulate. II. GENERAL CONSIDERATIONS--LIQUID CRYSTALLINE STATES A. T H E R M O T R O P I C

MESOHORPHISH

When a given molecule is heated it may not melt directly into an isotropic liquid, but instead passes through intermediate states, called mesophases or liquid crystals, which are characterized by less order than the crystal, but more order than the liquid. Such molecules undergo thermotropic mesomorphism. The chemical characteristics necessary to produce liquid crystalline states have been reviewed by Gray and Winsor, and by Brown and others (1-4). The physical properties of liquid crystals have been reviewed by de Gennes (5) and McMillan (6). In general, the molecules are somewhat longer than they are wide and have a polar or aromatic part somewhere along the length of the molecule. The shape and polar-polar or aromatic interaction permits the molecules to

581 Copyright ~ 1977 by Academic Press, Inc. All rightsof reproductloa In any form reserved,

Journal of Colloid and Interface .Science, Vol. 58, No. 3, March 1, 1977

ISSN 0021-9797

582

DONALD M. SMALL

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align in partially ordered arrays. Several structural types of thermotropic liquid crystals have been described by X-ray diffraction: three-dimensional cubic, two-dimensional rectangular, tetragonal, or hexagonal symmetry

as well as those with one-dimensional order (7-11). These structures are characteristic of molecules with a polar group at one end of the molecule. Liquid crystals with long-range order in the direction of the long axis of the molecule are called smectic, layered, or lamellar liquid crystals. These states may be formed by molecules above the melting point of the crystals (stable states) or on undercooling (metastable states). If the molecule contains a long aliphatic chain, the transition from crystal to the smectic state is often related to the partial or total melting of the aliphatic chain (7-11). A number of different kinds of layered states are possible (Figs. la-i, 2a), including those composed of single or double layers of molecules, those with molecules normal or tilted or to the plane of the layer, those with frozen or melte(t aliphatic chains, and those with twisted structure (Fig. 2). In the nematic state the molecules are aligned side by side but not in specific layers (Fig. 1). This state has particular optical properties but because the molecules have no long-range order, X-ray analysis does not give much information about the intermolecular structures. A special type of nematic phase is the cholesteric phase where each molecule is slightly displaced in relation to the next giving rise to a helical arrangement of the molecules (Figs. 2b, c). Since this state was first described in cholesterol esters, it has been called the cholesteric liquid crystalline phase. It might be more simply called the twisted nematic phase since the addition of a molecule of the righthanded pitch to one of the left-handed pitch will produce an untwisted nematic phase. B. LYOTROPIC LIQUID CRYSTALS

Lyotropic liquid crystals are defined as liquid crystals forming in the presence of a solvent. A solvent penetrates a crystal liquifying a part of the molecule, thus, producing a liquid crystal state. Although both polar and nonpolar solvents may produce liquid crystals, I will confine my discussion to water as the solvent.

Journal of Colloid and Interface Scicnca, Vol. 58, No. 3, March 1, 1977

LIQUID CRYSTALS a

585 c

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Fro. 2. Twisted liquid crystalline structures. (a) twisted smectic, (b) and (c) twisted nematic (cholesteric) structure.

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J~'IG. 3. Structural models associated with thermal transitions in synthetic lecithins. Dimyristoyl lecithin. Above DSC trace showing two transitions (see arrows), the low-enthalpy transition at 11 °C and a large one at 23°C. d is the interlamellar repeat, dz the bilayer thickness, and 0 the angle of tilt. a and b are the cell parameters for the two-dimensional lattice P j . Below l l ° C the structure is L J , between the two transitions (11-23°C) the structure is YJ, and above 23°C the structure is L~. (Taken from ReL (13).)

Molecules that form liquid crystals with water must have a polar part which can attract water. Lyotropic liquid crystalline phases include the "gel phase" and the "Iamellar liquid crystalline phase" which have been termed L~ and L.,

respectively, by Luzatti (7, 8). The fl term s refers to the aliphatic chains being packed in a stiff, nearly crystalline hexagonal array, and 1p, is used if the chains are tilted with respect to the [)lane of the bilayer.

Journal of Colloid and Interface Science, VoL 58, No, 3, March 1, 1977

584

DONALD M. SMALL TABLE I Classification of Biologically Active Liplds Class

Nonpolar

Surface properties a Will not spread to form monolayer

Bulk propertiesb Insoluble

Examples Long-chain, saturated or unsaturated, branched or unbranched, aliphatic hydrocarbons with or without aromatic groups, e.g., dodecane, oetadecane, hexadecane, paraffin oil, phytane, pristane, carotene, lycopene, gadusene, squalene. Large aromatic hydrocarbons, e.g., cholestane, benzpyrenes, coprostane, benzphenantrocenes. Esters and ethers in which both components are large hydrophobic lipids, e.g., sterol esters of long-chain fatty acids, waxes of long-chain fatty acids and long chained normal monoalcohols, ethers of long chained alcohols sterol ethers, long chained triethers of glycerol.

Polar Class I Spread to form stable insoluble nonswelling monolayer amphiphilic lipids

Insoluble or solubiIity very low

Triglycerides, diglycerides, long chained protonated fatty acids, long chained normal alcohols, long chained normal amines, long chained aldehydes, phytols, retinols, vitamin A, vitamin K, vitamin E, cholesterol, desmosterol, sitosterol, vitamin D, unionized phosphatidic acid, sterol esters of very short chain acids, Waxes in which either acid or alcohol moiety is less than 4 carbon atoms long (e.g., methyl oleate). Phosphatidyl choline, phosphatidyl ethanolamlne, phosphatidyl inositol, sphingomyelin, eardiolipin, plasmalogens, ionized phosphatidic acid, eerebrosides, phosphatidyl serine, monoglycerides, "acid-soaps," alpha hydroxy fatty acids, monoethers of glycerol, mixtures of phospholipids and glycolipids extracted from ceil membranes or cellular organelles (glycolipids and plant solfolipids).

Class I I insoluble swelling amphiphilie Iipids

Spread to form stable monolayer

Insoluble but swells in water to form lyotropic liquid crystals

Class I I I A soluble amphiphiles with lyotropic mesomorphism

Spreads but forms unstable monolayer due to solubility in aqueous substrate

Soluble; form micenes Sodium and potassium salts of long chained fatty acids, many of the ordinary anionic, cationic, above a CMC. At and nonionic detergents, lysolecithin, palmotyI low water conceneoenzyme A and other long chained thioesters trations forms liquid crystals. of coenzyme A, gangliosides, sulfo cerebrosides.

Class I I I B soluble amphiphiles, no lyotropic mesomorphism

Spreads but forms unstable monolayer due to solubility in aqueous substrate

Forms micelles but not liquid crystals

Conjugated and free bile salts, sulfated bile alcohols, sodium suit of fusidic acid, rosin soaps, saponins, sodium salt of Phenanthrene sulfonie acid. penicillins, phenothiazines.

a At air-water interface. b In aqueous systems.

the a refers to the chains being in a melted or The transition temperature from/3 to a chains more liquid-like state. In hydrated synthetic (frozen to liquid-like) is influenced by the lecithins, Janiak et al. (13) has shown that the amount of water present (the more water the L~I phase changes to a lamellar phase with a lower the transition), by the number of carbons periodic ripple ( P f ) and at higher temperatures in the fatty acid chains of the lipid (the more to the L~ phase (Fig. 3). The transition between the higher the transition), and by the number the Lot and the P0' is a low-energy first-order of double bonds and other chain substitutents, transition while the transition from Pa' to L~ is most of which decrease the transition temperaa high-energy first-order transition. Thus, the t u r e . Thus, lyotropic systems also undergo major energy change involves chain melting. thermotropic transitions. Finally, there are a Journal of Colloid and Interface Science, Vol. 58, No. 3, March i, 1977

LIQUID CRYSTALS WB,TER

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Fro. 4. The three-component system phospholipid (PL)-cholesterol ester (CE)-cholesterol (C) at constant water content. The tetrahedron at the upper left shows the position of the section containing the four-component system with 70% water by weight. This section is shown enlarged below, and is dealt with as a three-component system PL-CE-C (see text). Zone I, a lamellar liquid-crystalline phase containing varying amounts of C and CE (shown diagrammatically in the upper right); Zone II, a phase of CE containing from 0% when cholesteryl ester is crystalline (lower dashed fine) to almost 8% when the ester is in a liquid state (upper dashed line); Zone III, a two-phase zone consisting of the lamellar liquld-crystalline phase and the oily liquid phase of CE; Zone IV, an invariant zone of three phases, the ]amellar liquid-crystalline phase saturated with C and CE, the oily CE phase saturated with C, and C crystals. (Taken in part from Ref. (19).) n u m b e r of other liquid crystalline structures which occur with specific lipid classes, such as the cubic phase of certain phospholipids, the orthorhombic of phospholipids and soaps, and the hexagonal phases of phospholipids, soaps, and detergents. M a n y of these phases have been carefully described in the laboratory of Luzatti (7, 8) and presumably even other phases are possible as suggested by Mabis (14). III. C L A S S I F I C A T I O N O F B I O L O G I C A L L Y A C T I V E LIPIDS

Biologically active lipids have been classified according to their surface and bulk properties ((15, 16) ; Table I). Nonpolar molecules are defined as molecules which do not spread at an air-water interface and are insoluble in the

bulk. From the point of view of m a m m a l i a n pathobiology the most important molecules of the nonpolar class are the cholesterol esters such as cholesteryl oleate and cholesteryl linoleate (17). P o l a r lipids fall into three general classes; (I) those which form stable monolayers but are virtually insoluble in the bulk (nonswelling amphiphiles); (II) those which undergo lyotropic liquid crystal formation, b u t are quite insoluble in the bulk (swelling amphiphiles); and ( I I I ) those which form unstable monolayers, have a high solubility in the aqueous phase, and form micelles (soluble amphiphiles). I m p o r t a n t members of these classes include cholesterol and triglyceride (Class I), phospholipids and cerebrosides (Class II), and typical detergents, soaps, sulfocerebrosides,

Journal of Colloid and Interface Science, VoL 58, No. 3, Maxch 1, 1977

586

DONALD M. SMALL /

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FIO. 5. Lecithin-Iysolecithin-water ternary-phase diagram at 52°C. The structures of the lamellar, hexagonal, and micellar phases are indicated by the insets. The lecithin molecules have been drawn in darker characters so that they will stand out. The zones of hexagonal phase and micellar phase formed by lysolecithin are small, showing that these phases become saturated with small amounts of lecithin. Lecithin in excess of about 1 molecule to 10 of lysolecithin separates as a lamellar liquid crystal (Reproduced from Ref. (15, Fig. 11), with permission of the Journal of the American Oil Chemical Society.)

and gangliosides (Class III). This classification is not rigorous, thus, the classification of a given lipid may depend on the outside influences such as temperature, pH, ionic strength, etc. For instance, certain ionic lipids may fall in Class III when fully ionized, but revert to Class II or I when they become partly ionized or unionized. IV. T H E I N T E R A C T I O N S B E T W E E N LIPID SPECIES

The general interactions between lipids of different class in aqueous systems were first outlined by Dervichian (18) and later described in a number of phase diagrams (15). The interaction between phospholipids, cholesterol, and cholesterol esters in 700-/0 water (19) are depicted in Fig. 4. The Zone 1 on the right-hand side consists of a single phase made up of varying amounts of phospholipid and cholesterol and small amounts (less than 2o-/0) of choles-

tero! ester. The dimensions of this phase will vary depending upon its composition as well as on the specific phospholipid chosen. On the left-hand side there is a single phase (Zone II) consisting of cholesterol ester into which is incorporated a few percent of free cholesterol. This phase can exist, depending upon the temperature, as a crystalline phase which excludes all cholesterol (lower dashed line), or as a smectic liquid crystalline phase, or as a cholesteric liquid crystalline phase or finally, as an isotropic oil. The absolute amount of cholesterol solubilized in these phases varies from zero when the ester is crystalline up to almost 8% in a liquid phase at body temperature. Mixtures falling in Zone III, that is, between Zones I and II, will separate into two phases along tie lines connecting the composition with appropriate points on the phase boundaries. Mixtures falling in Zone IV contain excess

Journal of Colloid and Interface Science, Vol. 58, No. 3, March 1, 1977

LIQUID CRYSTALS

cholesterol which, at equilibrium, precipitate as cholesterol monohydrate. The relationship between Class II and Class I I I molecules in water is illustrated in Fig. 5 showing the interrelation of lecithin, a swelling amphiphile, with lysolecithin, a biological detergent with a low critical micellar concentration (15). At low lecithin concentrations, the detergent molecules are able to incorporate a small amount of lecithin into a hexagonal liquid crystalline phase and with excess water and transorm this hexagonal liquid crystalline phase into a micellar phase. However, it takes quite a large amount of lysolecithin, perhaps 20 molecules to one of lecithin to form a mixed micellar solution. As the amount of lecithin is increased in the phase diagram, the lysolecithin is incorporated into the lamellar liquid crystalline phase. Since the polar groups are not charged, the lamellar phase does not swell excessively. However, with a more charged detergent the liquid crystalline phase might swell up to as high as 95% water with very large spacings between the sheets of lamellar liquid crystal. Thus, the interaction of detergent-like molecules and other amphiphiles depends upon : (1) the weight ratio between the two molecules, (2) the presence of charge, etc. Large ratios of detergent completely solubilize insoluble amphiphilic lipids but smaller ratios are incorporated into lyotropic liquid crystalline phases. V. LIQUID CRYSTALS IN LIVING SYSTEMS A. MEMBRANES

A single plasma membrane or an intracellular organelle membrane such as mitochondrial membrane or nuclear membrane cannot strictly be considered liquid crystalline. They do, however, have the characteristics of certain liquid crystalline systems in that at least some of the lipids are in a fluid-like state and at least part of the membrane has a bilayer-like configuration. This has been established in a large number of biological membranes ranging from plasma membranes to mitochondrial membranes, Figure 6 shows plots of the compositions

587

of plasma membranes from a large nmnber of species and of intracellular organelle membranes. The composition indicates that there is very little cholesterol ester (and for that matter triglyceride) and that the predominant lipids making up virtually all of the membrane systems are free cholesterol and phospholipids. Only the golgi apparatus, the sites of packaging cholesterol esters into lipoproteins, has an appreciable quantity of cholesterol ester. Membranes also contain varying amounts of proteins (20). Figure 7 presents Singer and Nicholsons' (21) concept of the fluid mosaic membrane. It illustrates proteins, the large potato-like masses, floating in a sea of bilayer. Some of the proteins penetrate through the membrane and some merely penetrate part way. This interesting concept gives no indication of the differences in lipid species which are actually present in the membrane. While phospholipids and free cholesterol are the major membrane lipids, there are a large number of other species of lipids which are present in small amounts in most membranes and certain other components present in relatively large amounts in specific membranes. These other membrane lipids include, for instance, all of the glycosphingolipids from gangliosides to ceramides, other sterols, aliphatic and aromatic alcohols, minor quantities of fatty acids, mono, di, and triglycerides, and cholesterol ester. Thus, while the lipid composition is made up of about 90% phospholipid and free cholesterol, other species, nonpolar and polar lipid, are present in small amounts within membranes. This point is illustrated in a schematic view of the membrane drawn to illustrate the heterogeneity of lipid molecules and potential states of molecules existing in a hypothetical plasma membrane, in Fig. 8. It is probably realistic to suggest that most lipids are in a more or less fluid state; therefore, the continuous phase of the membrane, if one were looking down on the surface, would be "fluid." Those areas rich in cholesterol are somewhat less fluid than those areas relatively free of cholesterol. However, it is also quite possible that parts of the membrane are relatively solid with the chains existing in the

Journal of Colloid and Interface Science, VoI. 58, No. ,3, March 1, 1977

588

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Fzo. 6. Composition of cellular membranes plotted on triangular coordinates as in Fig. 4. Plasma membranes from different ceUs contain varying amounts of cholesterol. However, in mammals most plasma membranes e.g., red blood cells (rbc), white blood cells (lymphocytes, ]y; leucocytes; lu), myelin (my), intestinal microvillous membranes (mvm), and liver cell membranes (P) contain appredable cholesterol. Lower forms such as mycoplasma (M.laid) and most bacteria contain little or no cholesterol. On the other hand, intracellular organelle membranes (lower triangle) contain less cholesterol than plasma membranes. Only the golgi complex (GC) contains appreciable cholesteryl ester. GM, golgimembranes; mit, mitochondria; CP, chloroplasts; RRo and RRi, retinal receptor cells outer and inner segments; mic, microsomes. Data taken from: (1) Nelson, G. J. (1972), Lipid composition and Journal of Colloid a~d Interface Science, Vol. 58, No. 3, NI&rch 1, 1977

LIQUID CRYSTALS

form. These may form ridges extending across the bilayer which may be barriers to lateral diffusion of molecules within the more fluid parts of the membrane. They may occur around protein molecules as boundary lipid, and in highly ordered areas of the membrane, they may be the predominant species. Furthermore, there may be islands on one side or the other of the membrane which have a/3 chain configuration due to the lateral phase separations or to the interaction of the polar groups of certain lipids with proteins or cations such as calcium and magnesium (22). Thus, while lipids and some proteins may undergo diffusion within a membrane, it is quite possible that one-half of the membrane might be able to move independent of the other half, that is, the liquid monolayer of the membrane flowing past the solid part and vice versa. Furthermore, if a change in the surface pressure occurs at one part of the membrane, for instance, by removal of a specific lipid by biochemical reaction, a pressure gradient exists in the membrane and channels of flow may exist within that pressure gradient. Therefore, lipids are shown flowing, not just diffusing. The important feature of this concept is that the membrane is made up of a large number of lipids, mostly swelling amphiphile phospholipids which carry a host of minor compounds. These include certain other lipids which may be in more solid form with the chains in the ~ form, some nonpolar and polar nonswellifig lipids which are solubilized and carried along in the membrane and finally, some soluble lipids such as the gangliosides, carried in the membrane in trace amounts, probably for purposes of recognition. As will be shown later, it is this heterogeneity of lipid composition which can give rise to abnormal tiquid crystalline deposits occurring in certain metabolic disorders.

589

There are specific well-known membrane-like structures which are ordered within in concentric lamellae or stacked one on top of another, which can be specifically described as liquid crystalline. These include the rods and cones of the eye, the myelin sheaths of nerves and white matter in the brain, perhaps the'lamellar bodies of dipahnitoyl lecithin of the lung. B. SERUM[ I~IPOPROTEINS

A second kind of liquidcrystalwhich can occur normally in humans occurs within a small

particle called low-density lipoprotein. This particle has been shown by Deckelbaum el al. to undergo a transition from a smectic to a less ordered state at about body temperature (2325). The postulated structure is given in Fig. 9. Below a specific temperature (the transition temperature), the lipoprotein exists as a shell of phospholipid-free cholesterol and protein surrounding a core of cholesterol esters and a small amount of triglyceride. The core is arranged in a smectic layered structure. While the exact position of the molecules is not known, a rough estimate of the layered structure is shown in Fig. 9 (24). If the lipoprotein is heated, it undergoes a transition with an enthalpy AH, equal to that for the extracted esters of the lipoprotein going from smectic to the cholesteric phase (AH = 0.7 calories per gram of ester), and the sharp fringe seen by X ray at 36 .A, due to the ester is seen only below the transition. These and other facts strongly indicate the presence of a smectic-like liquid crystalline phase in low-density lipoprotein. Since body temperature varies from approximately 38°C in the core of the body to as low as 26°C in the skin, it is quite probable that the low-density lipoproteins in humans are periodically undergoing transitions as they pass from the center of the body to the

metabolism of erythrocytes, Chap. 7 in "Blood Lipids and Lipoproteins: Quanfitation, Composition, and Metabolism" (Gary J. Nelson, Ed.), Interscience; (2) Korn, E. D. (1966) Science 153, 1491-1498, (3) Zambrano, E., et al. (1975), Biochim. Biophys. Acta 380, 357-369; (4) Robins, S. J. (1971), Biochim. Biophys. Acta 233, 550-561 ; (5) Sj6strand, F. S. (1969), Morphological aspects of lipoprotein structures, Chap. 3 in "Structure and Functional Aspects of Lipoproteins in Living Systems" (E. Tria and A. M. Scanu, Eds.), Academic Press, New York. Journal of Colloid and Interface Science. Vol. 58, No. 3, March 1, 1977

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Fro. 7. The lipid-globular protein mosaic model with a lipid matrix (the fluid mosaic model) ; schematic three-dimensional and cross-sectional views. The solid bodies with stippled surfaces represent the globular integral proteins, which at long range are randomly distributed in the plane of the membrane. At short range, some may form specific aggregates, as shown (By permission from Singer and Nicholson, Ref. (21).)

periphery. The pathological significance of this transition has not yet been established. VI. LIQUID CRYSTALS I N PATHOLOGICAL CONDITIONS

Many, but not all, of the diseases which accumulate liquid crystals involve a specific block in the catabolism of a particular lipid

species. Under normal circumstances, when all of the appropriate metabolic apparatus is present in the system, all lipids are turned over within the cell and remain at relatively fixed concentrations. The synthesis or intake of lipids from lipoproteins guarantees an adequate content of the appropriate lipids within the cell and the breakdown, principally by enzymes

FIG. 8. Lipid heterogeneity in membranes. Schematic diagram of a membrane to show differences in lipids within the membrane. Proteins are similar to those shown in Singer's model (Fig. 7). Arrows indicate possible directions of movement of liplds in the plane of the membrane. Unidirectional lipid flow may occur down pressure gradients on either or both sides of the membrane. Movement of one monolayer in opposition to the other is also possible. (a) Gangliosides (b) cholesterol (c) a patch of lipid with stiff chains on outer face of bilayer (d) a patch of stiff-chained lipids on the inner half of the bilayer (e) a line of stiff-chained lipids creating a boundary within the membrane (f) a small patch of stiff-chained lipids (g) the cyto-skeletal system. Journal of Colloid and Interface Science,

Vol. 58, No. 3, March 1, 1977

LIQUID CRYSTALS

591

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triglyceride A. Below the transition in intact LDL, at 10°, the cholesterol esters are arranged in 4 concentric layers with a periodicity of about 36 ~ in a smectic-like state. B. Above the transition, at 45 °, this layered arrangement is lost, but the organization of cholesterol ester molecules is not totally random, and some degree of motional restriction persists. In both A and B, cholesterol esters are the dominant lipid in the particle core. All LDL triglycerides and about 15% of LDL free cholesterol are dissolved in this core. Around the neutral lipid core are the polar lipids of LDL, phospholipids, and most of the free cholesterol. Since these two lipid classes cannot cover all the surface of the neutral lipid core, we suggest that some aproprotein is adjacent to neutral lipid. Thus, the outer shell is made up of apoprotein and polar lipids mutually interacting with each other at the surface as well as with the neutral lipids in the LDL particle core. The water of hydration of the LDL particle also forms part of this outer shell. (Taken from Ref. (24).) within the lysosomes (the digestive organ of the cell) and b y specific removal mechanisms carried out b y certain lipoproteins, control the level of lipid within the celt. When a specific catabolic enzyme is lacking, the lipid which would be broken down b y that enzyme accumulates within the cell. I t first accumulates in the sites of the cell in which it is prevalent or being synthesized. Then, as the cell recognized t h a t excess of this lipid is present, intracellular lysosomes engulf the excess lipid and a t t e m p t

to break it down. However, since the specific enz~vne is deficient the lipid accumulates. Thus, the cell can be affected in at least two ways, one b y a specific malfunction of the organdies of the celt b y an excess of the specific lipid, or second b y massive engorgement within lysosomes of the uncatabolized lipid species. I t is obvious that a n y of the different kinds of lipid species depicted in the model m e m b r a n e in Fig. 8 m a y accumulate if the enzyme necessary to catabolize t h a t particular lipid is lacking in a

Journal of Colloid and Interface Science, V o l . 58, N o . 3, M a r c h 1, 1977

592

DONALD M. SMALL

density lipoprotein and when cellular stores of cholesterol begin to fall, the receptor takes up the lipoprotein. The lipoprotein is taken into the lysosome, and the cholesterol esters are hydrolyzed to free cholesterol which then moves out of the lysosome and resupplies the cell with cholesterol. This free cholesterol then decreases the number of receptor sites and shuts off the cholesterol synthesis within the cell (which was NE I trying to compensate for the previous low level) to achieve a new steady state. Any excessive cholesterol ingested may be removed by a parCE I \\PL ticular tissue fluid carrier, probably the serum high-density lipoprotein, which circulates Fie,. 10. Lipid composition of tissues in cholesterol ester storage disease, Tangier disease, and control. through the extracellular tissues. Thus, a Points 1 and 2 are liver tissues from patients with beautiful balance is maintained between upcholesterol ester storage disease (39). Points 3-9 are take, synthesis, utilization, and removal. The tissues from patients with Tangier disease: 3, lymph liver probably also takes up low-density lipotissue (43); 4, tonsil tissue (43); 5, spleen tissue protein but this organ is also responsible for re(46, 47); 6 and 7, tonsil tissue (43); 8, spleen tissue (43); and 9, liver tissue (47). Points 10-16 are tissues moving the large amounts of cholesterol arrivfrom control patients : 10, liver (39) ; 11, lymph (39, 43) ; ing via the chylomicron remnants (39), that is, 12, spleen (43); 13, tonsil (43); 14, spleen (43); 15, the lipoproteins formed in the intestine which splenic artery (47); and 16, spleen (47). The point contain ingested cholesterol. Normally, the (x) (47) in Zone II is lipids isolated from droplets found liver seems to have an obligate step taking the in the spleen tissue from a patient with Tangier disease. cholesterol-rich remnants from the blood. In the liver cells lysosomes exist to break down the given cell population. Furthermore, the characingested cholesterol, which in turn regulates teristics of the lipid deposits will depend upon the cholesterol synthesis within the liver cell, and physical properties of the lipid which is accumuperhaps controls the excretion of cholesterol lating, and its interaction with other adjacent into the bile either as free cholesterol or as bile lipids. Some lipids merely accumulate as relaacid, the major metabolic product of cholestively harmless droplets within the cell while terol. In a very rare disease called cholesterol others affect specific functions such as nervous ester storage disease (39, 41) it was discovered conduction enzymatic action, membrane perthat the lysosomal enzyme for the breakdown meability, etc. of cholesterol ester is deficient throughout the tissue (41). These patients therefore take up A. D I S E A S E S A C C I Y M U L A T I N G cholesterol to some extent in the peripheral tisN O N P O L A R LIPIDS sue but much more in the liver and cannot Cholesterol ester storage disease. Cellular me- break down the ester that is retained within the tabolism of cholesterol is complex, but the lysosome of the liver cell; thus it accumulates tissue-culture work of Bailey (26) and Rothblat in large amounts. For illustration, the composi(27) and recently that of Brown and Goldstein tion of liver cells is shown in Fig. 10 on the on fibroblasts (28-30) have done much to eluci- ternary phase diagram. Normal liver (point date the metabolism of cholesterol in periph- 10) has very little cholesterol ester although eral tissue. The work of Dietschy and others there is more than the pure membrane frac(36, 37) has helped us to understand choles- tion due to the presence of small amounts terol metabolism in the liver. In general, many of cholesterol ester-rich lipoprotein ill the liver. peripheral tissue cells have a receptor for low- However, the liver from patients with cholesc

Journal of Colloid and Interface Science, Vol. 58, No. 3, March I, 1977

LIQUID CRYSTALS

a

b

593

c

FIG. 11. Individual spleen foam cells from a patient with Tangier disease. Spaces in cells are nuclei. Same field of 400)< : top, ordinary light; middle, crossed polars 20°C; bottom, crossed polars 48°C. At 48°C, droplets in cell on left (a) are still birefringent, those in the middle cell (b) are nearly melted and those in the cell on right (c), are completely melted. Birefringent droplets are smectic liquid crystal phases

of cholesteryl esters. Different melting temperatures of droplets in different cells indicate a different cholesteryl ester mixture in each cell (from Ref. (47)). terol ester storage disease contain nearly 90% of their total lipid as cholesterol ester. Such patients m a y carry up to a kilogram of cholesterol ester in their liver. Since the ester is not a particularly toxic substance as it exists

within the Iiver, it seems to create damage within the liver cells only b y its mass effect. I n 1967 the author examined a sample of unfixed liver from patient T H of Dr. Schiff (41). The cells were full of smectic liquid crystals

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DONALD M. SMALL

FIG. 12. Smear of center of a human utherosclerotic plaque. Crossed polars 90X. Crystals are cholesteryl monohydrate and birefringentdroplets are cholesterylester phase in the smecticliquid crystalline phase. which melted sharply at 35°C. The birefringent droplets were isolated and shown to be 980-/o cholesterol ester. Thus the deposits in this patient were almost pure cholesterol ester capable of forming smectic liquid crystals at temperatures very close to body temperature. Tangier disease. Tangier disease is a rare, inherited disorder discovered in the population of Tangier Island, and discussed in great detail by D. S. Frederickson and associates at the National Institutes of Health (42, 45). These patients lack the ability to synthesize adequate quantities of high-density apoprotein; therefore they have about a 600-fold decrease in the major apoprotein of high-density lipoprotein and consequently extremely low levels in the serum. Thus, they lack one of the major acceptor systems for cholesterol in the tissue. Patients with Tangier disease were recognized as having very large tonsils full of cholesterol esters (42). Furthermore, a number of other organs containing reticuloendothelial cells, those cells which scavenge debris within the body, were found to contain large quantities of

cholesterol ester. Figure 10 shows that the composition of the lipids (42-47), taken from a number of organs in the patient with Tangier disease, is extremely high in cholesterol ester. However, many tissues did not appear to have massive amounts of esters and only the reticuloendothelial system appears to accumulate the lipids. Thus it has been suggested that highdensity lipoproteins are particularly necessary in removing cholesterol from those specific cells, the macrophages of the reticuloendothelial system, which scavenge the cholesterol containing remnants of lipoproteins and cell membranes which have lost their use in the body. These patients, while sometimes having enormous spleens and tonsils full of cholesterol ester, rarely develop serious symptoms and apparently live a relatively normal life. Figure 11 shows live cells taken from the spleen of a patient with Tangier disease. Over 80% of all of the cells are birefringent at 37 ° and have a uniaxial positive sign of birefringence within each droplet (46, 47). The cells as well as the droplets isolated from tissue give a smectic A

LIQUID CRYSTALS

X-ray diffraction pattern at 37°C with the major narrow angle fringe at 36 A (46, 47). Thus most of the lipids are in the smectic liquid crystalline phase at body temperature. The chemical composition of the esters from such cells is very rich in oleic acid and their mean transition temperature from smecfic to isotropic is at about 41°C which is similar to the temperature for the smectic-cholesteric transition of cholesterol oleate 07). The composition of the droplets isolated from the cell is plotted on Fig. 10 (47) and shows that they are almost pure cholesterol ester. Atherosclerosis. The etiology of atherosclerosis is not so simple. This widespread disease may have several etiologic factors, several relating to abnormalities in the metabolism of the serum lipoproteins (48-50). The lesions are often small but occur in strategic places such as the coronary vessels or arteries going to the brain and therefore they cause problems by restricting the blood flow to vital organs. Figure 12 shows a smear from an atherosclerotic plaque of a patient who had died of atherosclerosis. This plaque is typical of advanced lesion and is loaded with crystals of cholesterol monohydrate and also with optically positive birefringent droplets. These isolated droplets contain 940/0 cholesterol ester and 6% free cholesterol, that is, they are the cholesterol ester phase saturated with cholesterol. Plaques also contain many droplets which are isotropic. The lack of birefringence is probably due to high concentrations of cholesterol linoleate and other more unsaturated esters as well as the presence of small amounts of triglyceride in the cholesterol ester phase. All these low-melting constituents (17) lower the transition temperature of the higher melting esters. Atherosclerosis apparently starts at a very early age with excessive cholesterol beginning to accumulate in the membranes of the arterial intima (19). Thus as the membrane becomes saturated with cholesterol it begins to change its characteristics and to accumulate cholesterol esters. These esters appear to be accumulated within cells both in lysosomes and also in droplets not bounded by lysosomal

595 c

E

CE

L

FIG. 13. Possible progression of the arterial infima from normal child to advanced plaque. The chemical compositions of the child and adolescent are taken from Refs. (52, 53), as presented by Small and Shipley (19). The chemical data of the mature intima, the fatty streak, the intermediate lesion, and the advanced plaque are taken from (51). The lipid composition of the intima in children appears to consist of a single lamellar liquid crystalline phase, that is like cellular membranes. By adolescence, the membrane system appears to be saturated with cholesterol. The mature intima (i.e., from people 45-70) appear normal but microscopically contain a second phase of cholesterol ester. The fatty streak is an abnormal lipid deposit consisting of a large quantity of cholesteryl ester in the smectic liquid crystalline phase. The intermediate lesion (51) looks like a fatty streak but has become supersaturated with cholesterol. Finally, the advanced lesion is a necrotic lesion with many dead cells containing many cholesterol monohydrate crystals,'the smecfic and isotropic phase of cholesteryl esters=as well as a lamellar membrane phase of phospholipid.

membrane. When these lesions become visible to the maked eye they are called fatty streaks. Furthermore, the esters tend to be richer in oleate than in linoleate suggesting that some of the esters are synthesized within the cell or that polyunsaturated esters are selectively hydrolyzed. Thus the early stage of the fattystreak lesion of atherosclerosis is characterized by the deposition of cholesteryl oleate-rich smectic liquid crystals primarily within the cells. Advanced lesions of atherosclerosis called plaques, are lipid-rlch lesions, full of cholesterol crystals (see Fig. 12), which protrude into the lumen of large arteries, They also contain many

Journal of Cotloid and-Interface Science, VoL 58, No. 3, March 1, 1977

596

D O N A L D M. S M A L L POLAR I,NON-SWE LLING AMPHIPHILES

Surface

H'philic

CHOL ES~'IhB:?

!!!!!! 25°C,38 dyn/cm '38~ z, L

Bulk

illlllI Chol Monohydrate

revertsto anhydrous form at 80~C

CERAMIDE Crystalline structure unknown m,p. ) 70°C

Livinq Systems

Disease

Constituent of membranes May control membrane viscosity

Gallstone Atherosclerosis

Major intermediate in sphingnlipid metabolism Trace amounts

Farber's disease

intissue 2,SWELLINS AMPHIPHILES H'phobic LECITHI N

I H'philie

11iii,

J-o

:+-~cIA-~'-

Maior component of all membranes

LCATdeficiency

Lc~,Tc ~5°C SPHINGOMYEUN

i~ Major component of membranes

~55dynlcm

Niemonn -Pick disease

L/~,Tc=50"42"C toLQ CEREBROSIDES Glucose Cer

glu-cer Gaucher's disease

Galactose Car

Crystal or Gel Tc ~70~C to LC~

Components of nervous tissue

LIz , L/~ Tc ?

Minor component of tissue

Fobry~ disease

Minor constituents of tissue

MOachromatic leucodystrophy

Minor constituents of many tissues

GM2 Toy-Sachs disease

gal-cer -Krabbe's disease

OL=OS,OB-;

'J4"Joi 1

5, SOLUBLE

AMPHIPHILES

Miceles

H'phobic JH'philic GaI-CEREBROSIDE SULFATE I

GANGLIOSIDES GM2

- - ~ L

GMI

Low pH, stable mono(ayer High pH, unstable State probably depends on charge: monolayer ionized =micelles unionized= Lj~or LC~

P~

~°'~ "~

°4 °" L°,, "~o,,

Form unslable Form [arge monolayers micelles (Both GM2 & GM I)

,ro.fZ~ GMIGenera[

ganglosidos[s

•

FIG. 14, Physical characteristics of polar lipids accumulating in certain diseases. Surface properties at ambient temperature include: collapse pressure of monolayer (dynes/era), limiting surface area (Z_2/molecules) and film viscosity, L = liquid, C = condensed. Ceremides and cerebrosides form solid film.

Journal o[Colloid and Interface S4ence, Vol. 58, No. 3, March 1, 1977

LIQUID CRYSTALS

597

TABLE II Class

NonpoIar

Polar

Major lipid accumulating CE~

l--Nonswelliug Cholesterol Ceramide 2--Swelling Lecithin

Disease

Tissue affected

Atherosclerosis

Arterial intima

CE storage disease Tangier

Liver cells

Cholesterol gallstones Farber's

LCAT deficiency

Sphingomyelin

Neimann-Pick

Glucosyl cerebroside

Gauchers (glueosyt ceramidosis)

Galaetosyl eerebroside

Krabbe's (galactosyl ceramidosis) Fabrys

Composition of deposit

Physical state of deposit

40% Chol., 40% CE, 20% PL >90% CE

Cholesterol monohydrate, smeetic LC of CE, liquid oil, La of PL Smectic CE

RE cells of liver, spleen nodes, etc.

>90% CE

Smectic CE

Bile

Choh

Brain, kidney, etc.

?

Crystalline cholesterol monohydrate ? Crystalline ceramide

Blood and other tissues RE cells of liver, spleen, lymph nodes, kidney, lung, CNS

70% lecithin, 30% cholesterol 45 mole% sphingomyelin, 20 mole% other PL, 35 mole% cholesterol 59% glueosyl cerebroside 11% lecithin 30% cholesterol

RE cells of liver, spleen, hone marrow, lung, lymph nodes (CNS) CNS, kidney

? La 70 ~_

? La 60

Galactosyl cerebroside

? La

RE system, endothelium, kidney, nervous tissue, heart, lung

Lysosomal bodies high content of globoside 3.

? L~

Metachromatic leucodystrophy

Myelin,white matter, kidney

Swoilen La or LB ? mixed micelles

GM2

Tay Sachs

Nervous tissue. liver, lung, spleen

GM1

General gangliosidosis

Brain, liver, spleen, kidney, bone

Myelin-54% PL, 18% chol., 21% cer sulfate, 7% other lipids 30-40% GM2, rest of lipid is cholesterol PL and other glycosphingolipids High amount of GM1, varying amounts of other lipids

Globoside 3 (gaI-gal-glu-cer)

3--Soluble Cerbroside sulfate

Swollen La or Ltd, ? mixed micelles with other lipids

Swollen La or L¢, ? mixed micelles with other lipids

a Abbreviations: CE = cholesterol esters; chol. = cholesterol; PL = phospholipids; Cer = ceramide; GM = gangliosides; R E = retieuloendothelial system; La = lamellar liquid crystal with "fluid" chains; L~ = lamellar liquid crystal with "frozen" chains.

droplets and a few smectic liquid crystals at body temperature (19, 51). The mean lipid compositions of the intima are shown in Fig. 13. The young child's arterial intima is mainly phospholipid and contains only a single phase unsaturated with cholesterol or cholesterol esters (19, 52, 53). The

early lesion, the fatty streak, is extremely rich in cholesterol esters (51, 54) and many of these are in the smectic state (46, 51, 55, 56). The advanced lesion moves into the zone when three phases are present, and indeed these phases have been physically identified (19, 51) and partially isolated (56). It is this lesion which

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598

D O N A L D M. SMALL

compromises arterial blood flow and causes myocardial infarctions and strokes. Katz et al. have found an intermediate lesion which looks like a fatty streak but is supersaturated with cholesterol and has properties intermediate between plaques and normal fatty streaks (51). It is possible that certain enzymatic processes involved in the breakdown of stored cholesterol ester initiates supersaturation of free cholesterol within the cell and precipitation of cholesterol monohydrate. This solid intracellular phase initiates destruction and death of the cell which in turn gives rise to the development of the advanced necrotic plaque. B. DISEASES ACCU]~ULATING POLAR LIPIDS

A number of specific enzyme deficiencies lead to the accumulation of various polar lipids in the body (See Ref. 57, Chapters 27, 29-35 for an excellent review). Figure 14 gives a list of the various polar lipids which accumulate in certain diseases and their physical properties as far as is known. Table II lists the various diseases in which these lipids accumulate, the major organs affected, the best estimate of the composition of the accumulation, and speculation concerning the physical state of the accumulation. Each disease will be discussed by lipid class,

1. Nonswdling Amphiphilic Lipid Accumulation

of excess cholesterol such that it precipitates ill the gallbladder in the form of cholesterol monohydrate (59-65). Bile lipids form a number of lyotropic liquid crystals (59-63) which may be implicated in the gallstone formation (63). These crystals grow to form stones of almost pure cholesterol monohydrate which then can block the biliary tract and cause serious disease (65). Presumably mechanisms for cholesterol gallstone disease involve either inadequate secretion of bile salts to solubilize the cholesterol or excessive cholesterol secretion (65-66). The exact metabolic reasons for this have not yet been discovered athough obesity is a factor ill the secretion of excess cholesterol (66). An enzymatic defect in the conversion of cholesterol to bile acid has been suggested as a possibility for the decreased bile salt secretion in certain gallstone patients (67). Cholesterolosis of the gallbladder. Certain patients with gallstones occasionally develop the deposition of lipids, possibly cholesterol esters, in the wall of the gallbladder. These lipids appear to be present in the form of smectic liquid crystals (68). Dogs fed high doses of cholesterol develop similar lesions containing liquid crystals (68).

2. Swelling AmphiphiIic Lipid Accumulation Lecithin cholesterol acyl transferase deficiency.

Farbers disease. Farbers disease is a very rare disorder in which the central molecule in sphingolipid metabolism accumulates because an enzyme required for its breakdown is deficient (58). Ceramide contains two rigid chains and a small polar group. It has a melting point above 79°C and does not swell ill water at 25°C (D. M. Small, unpublished observations). The physical state and precise composition of the lesions have not been determined but one would predict that if ceramide separates as a phase that it would form a crystalline deposit. Cholesterol gallstone disease. Cholesterol gallstone disease is caused by the secretion into bile

While there has been no lysosomal enzyme discovered leading to an accumulation of lecithin within the cells, there is a specific disease involving a serum enzyme necessary for lecithin catabolism. This serum enzyme, lecithin cholesterol acyl transferase (LCAT), catalizes the transfer of a fatty acid from lecithin to free cholesterol to produce cholesterol ester and lysolecithin (69). The site of the reaction is the surface of certain serum lipoproteins. The lysolecithin is removed from the particle by albumin and the cholesterol ester is deposited within the core of the lipoprotein. In lecithin cholesterol acyl transferase deficiency normal lipoproteins cannot be formed because the

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1977

599

L I Q U I D CRYSTALS

cholesterol esters are not formed. Therefore, the serum contains lipoproteins extremely rich in lecithin and free cholesterol (70-73) in a ratio of 1:1 which forms stacks of small fragments of lecithin-cholesterol lamellar liquid crystals. Furthermore, such deposits have been found in tissues within cells as lamellar bodies. Neimann-Pick disease. Sphingomyelin is a ubiquitous sphingophospholipid which has properties rather similar to egg lecithin (74) except that its chain melting transition is higher (75). The transition from Lo to L~ is from about 32 to 42°C (75). In Neimann-Pick disease the enzyme necessary for the degradation of sphingomyelin is absent and this molecule accumulates in large amounts in many tissues (76-78). Several forms of the disease are present, among them a severe juvenile form in which the patient dies of massive accumulation of sphingomyelin in the brain and a later form in which the patient lives much longer and the sphingomyelin accumulates mainly in nonneural tissues (78). The aggregates formed as shown by electron microscopy consist of lamellar aggregates containing cholesterol, sphingomyelin and lecithin (79). Cerebrosides (Gaucher's disease and Krabbe's disease). Each of two major cerebrosides, that is, glucosyl ceramide and galactosyl ceramide, is involved in a specific disease. Specific enzymes necessary for the breakdown of one or the other of these molecules to the parent compound, ceramide, are lacking. In Gaucher's disease (57), glucosyl ceramide accumulates in massive amounts in the liver and spleen and other tissues of the body. Glucosyl ceramide has rather rigid hydrocarbon chains, has a high transition temperature of about 70°C and forms a solid monolayer at 37°C (80). The deposits contain a large quantity of the glucocerebroside and smaller amounts of free cholesterol and other phosphclipids. The tissues have been studied by X-ray diffraction by Lee el al. (81), and have been shown to be basically an L~ liquid crystal with a possible twist of the plane of the lamellae. The treatment of the tissue however, prior to the X-ray diffraction studies may have distorted the true structure.

Krabbe's disease is characterized by the deposition of the gatactoceramide and the morphology of the lesions is very similar to that of Gaucher's disease; however, there is no physicochemical data concerning the structure of the deposits. It is interesting to note the studies on mongalactosyl diglyceride by Shipley el al. (82). This molecule is similar to galactoceramide, but it forms an inverted hexagonal phase when hydrated with water. Ceramide trihexosidosis (Fabry's disease). Ceramide trihexoside (Globoside III) that is, the globoside with three sugars accumulates in Fabry's disease (57, 83). In this disease, the enzyme necessary to remove the last sugar is absent (84). Liquid crystalline deposits are seen in many tissues in this disease including the kidney, liver, spleen, bone marrow, etc. Ceramide trihexoside also accumulates in blood (85) cells and renal deposits appear to compromise renal function. While the exact nature of the deposit is not known, their optical properties suggest that they are lameltar liquid crystals of either L~ or L~ form.

3. Soluble Amphiphilic Lipid Accumulation Melachromatic leucodyslrophy. Cerebroside sulphate, a detergent-like molecule with a charged sulfate group on the sugar molecule, has been shown to form unstable monolayers and to form micelles in high concentrations of water (80). At lower water concentrations or at low pH it probably forms L~ and L~ forms at the appropriate temperatures. It is a minor constituent of most living tissues but accumulates in a disease called metachromatic leucodystrophy, in which the enzyme necessary for the removal of the sulfate is absent (57, 86). The disease affects myelin and other nervous tissues often causing severe symptoms in infants and early death in those afflicted with this disease. The liquid crystalline deposits appear lamellar by EM but their specific structural characteristics and composition are not yet known. Gangliosidoses (Tay Sachs GM-2 disease and generalized G-1 gangliosidosis). Gangliosides are

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600

DONALD M. SMALL

sphingolipids with 4 or more sugar-like polar groups and at least 1 scialic acid residue. In T a y Sachs disease ganglioside GM-2 with four sugars cannot be broken down and it accumulates in the brain (57, 87-92). Gangliosides have been shown to act as detergents in water systems (93-95), and also to solubilize phospholipids such as lecithin in model systems (96). I n the brain the liquid crystalline deposits appear by E M to be highly swollen lamellar aggregates (97). I t is perhaps because of the charge on the ganglioside and the large, bulky polar head group that the lamellae are separated by large amounts of fluid. This particular factor along with the disruption of other cellular functions due to excess gangliosides throughout the cell, causes the cells in the brain to accumulate not only large amounts of lipid, but also excessive amounts of water. The brains of children dying of this disease contain excess contents of water (57). General GM-1 gangliosidosis is characterized by the deposition of more complex gangliosides containing 5 or more sugar residues which cannot be broken' down (57). The characteristics of general gangliosidosis as well as the lesions are similar to those of T a y Sachs disease. I have gathered together a group of diseases in which liquid crystals have been implicated in their pathogenesis, or which accumulate specific lipid deposits having liquid crystalline characteristics. These deposits m a y behave as either thermotropic or lyotropic liquid crystals or both. I t is evident in m a n y cases that these deposits arise from the inability of certain cells t o catabolize a certain lipid species and therefore the species becomes a predominant lipid and separates from the menlbranes, either as the pure lipid or in combination with other membrane lipids. The physical properties of the accumulation determines the nature of cell dysfunction and the ultimate fate of the individual afflicted with the disease. ACKNOWLEDGMENTS The author wishes to thank D. Armitage, W. Curatolo, M. Juniak, V. Liepkalms and others of the Biophysics Institute for their helpful criticism of this manuscript.

REFERENCES 1. GRAY, G. W., "Molecular Structure and the Properties of Liquid Crystals." Academic Press, New York, 1962. 2. G~AY, G. W., ANDWINSOR,P. A. (Eds.), "Liquid Crystals and Plastic Crystals." Norwood, Chichester, England, 1974. 3. BRowN, G. H., AND SEAW,W. G., Chem. Rev. 57, 1049 (1957). 4. BROWN,G. H., DOAN'E,.[. W., ANDNERO,V. D., "A Review of the Structure and Physical Properties of Liquid Crystals." CRC Press, 1971. 5. DEGEN~CES, P. G., "The Physics of Liquid Crystals." Oxford Univ. Press, New York, 1974. 6. MCMILLAN,J., Phys. Rev. A4, 1238 (1971); A6, 936 (1972); A9, 1419 (1974). 7. TARDIEU, A., LUZZATI,V., AND RE~AN, F. C., J. Mol. Biol., 75, 711 (1973). 8. RANCK,J. L., MATEU,L., SADLER,D. M., TARDIEU, A., GULIK-KRYZWICKI,T, AND LUZATTI~V., J. Mol. Biol. 85, 249 (1974). 9. DE VRIES,A., Mol. Cryst. Liq. Cryst. 10, 31 (1970). 10. CItAV~AN, D. (Ed), "The Structure of Lipids." Methuen, London, 1965. 11. CHAPMAN, D., AND WALLACI~, D. F. H. (Eds.), "Biological Membranes," Vol. 2. Academic Press, New York, 1973. 12. SI~IrLEY, G. G., in "Biological Membranes," D. Chapman and D. F. H. Wallach (Eds.), Vol. 2, Chapt. 1. Academic Press, New York, 1973. 13. ~ANIAK,M. ~[., SMALL,D. M., ANDSalPLEY, G. G., Biochemistry 15, 4575 (1976). 14. MABIS,A. J., Acta Crystallog. 15, 1152 (1962). 15. S~tALL,D. M., J. Amer. Oil Chem. Soc. 45, 108 (1968). 16. SMALL,D. M., Fed. Proc. 29, 1320 (1970). 17. SMALL,D. M. in "Surface Chemistry of Biological Systems" (M. Blank, Ed.), p. 55. Plenum Press, New York, 1970. 18. DERVlCHIAN,D. G., Trans. Faraday Soc. 42B, 180 (1946). 19. S~LE, D. M., AND SmPLE¥, G. G., Science 185, 222 (1974). 20. GUIDO'rTI,G., Annu. Rev. Biochem. 41, 731 (1972). 21. SINGER,S. J., AND NICOLSON,G. L., Science 175, 7188 (1972). 22. PAPAHADJOPOULAS,D., MOSCARELLO,M., EYLAR~ E. H., AND ISAC, T., Biochim. Biophys. Acta 401, 317 (1975). 23. DECKELBAUM,R. J., SHIPLEY,G. G., SMALL,D. M., LEES, R. S., ANDGEOROE,P. K., Science 190, 392 (1975). 24. DECXXLBAUM,R. J., SmPLE¥, G. G., AND SMALL, D. M., J. Biol. Chem. 252, 744 (1977). 25. SEARS, B., DECKELBAUM,R. 7", ~'ANIAK,M. ~'.,

Journal of Collold and Interface Science, Vol. 58, No. 3, March 1, 1977

LIQUID CRYSTALS

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Journal of Colloid and Interface Science. Vol. 58, No. 3, March I, 1977