Micellar Concentration

m m m pj m nnnnnnnnn uuuuuuuuu

Figure 3-10. Some liquid crystalline states, with various degrees of dimensional order. Polar groups are represented by dots, the hydrocarbon chains by wriggles or lines, and water by blank areas.

The three-dimensional cubic phase unit cell formed by egg lecithin at low hydration is centered cubic. The structure consists of rigid rods of finite length, all identical and crystallographically equivalent, joined three-by-three to form two interwoven three-dimensional networks. The hydrocarbon chains are fluid.

Liquid crystals with two-dimensional (2D) order may be rectangular or hexagonal. The 2D centered rectangular phase of the anhydrous Na soaps has the loci of the polar groups arranged in infinitely long ribbons of finite width. The two hexagonal phases have the loci of the polar groups arranged in infinitely long rods packed hexagonally. Hexagonal II is the water-in-oil type observed in low water-phospholipid systems and in the anhydrous soaps of divalent cations. Hexagonal I is the oil-in-water type observed in lipid-water systems containing monovalent soaps, lysolecithin, or aliphatic detergents.

Lameller systems are generally one-dimensional. However, if the chains are packed in a 2D hexagonal array, 2D order is perpendicular to the chain axis. These lamellar systems, called "gels," are ordered. Lamellae may be fully or partially interdigitated layers or bilayers. Transitions between interdigitated layers and bilayers change the lamellar thickness greatly, and such transitions may have a biological role.

(From Small, D. M. and Zoeller, R. A. [1991] Lipids. In: Encyclopedia of Human Biology, Vol. 4, pp. 725-748. Academic Press, Inc., Orlando, Florida).

Liquid crystals that have no long-range periodic order are ordered liquids (nematic and cholesteric) but have been called liquid crystals because of their birefringence. They have no true x-ray diffraction and show only x-ray scattering that corresponds to their molecular length. These observations indicate that many rod-shaped lipids lie side by side but not in strict layers like the lamellar liquid crystals. The particular lipids that exhibit this kind of liquid crystalline behavior are generally steryl esters and, in particular, choles-teryl esters.

The Liquid State—Melts, Solutions and Suspensions

The general properties of liquids, including fluidity, cohesiveness, relative incompressibil-

ity, and rapid molecular motions, are characteristic of melted lipids. Because only a 10% to 20% change in volume occurs when a lipid melts from a solid, some short-range order must remain in the liquid. It has been shown that many molten lipids form nonideal liquids consisting of clusters of a few hundred molecules, aligned along their long axes, separated by more disordered molecules. These clusters may act as nucleating centers for the formation of more ordered liquids or liquid crystals (Small, 1986). In mixtures of different lipids, some domain separation of individual lipids may occur in the liquid state.

Fluidity, which is defined as the reciprocal of viscosity, and diffusivity in liquid and liquid crystal systems are directly proportional to the free volume of the molecule (i.e., that volume above the minimum volume at zero fluidity). The free volume is a function of the temperature, and thus fluidity increases with temperature (Small, 1986).

The solubility of different types of lipids in aqueous systems is quite variable, extending from virtually insoluble (large hydrocarbons, triacylglycerols) to very soluble (soaps and detergents). The solubility of fatty acids is quite low (see Table 3-2). The solubility is less at temperatures below the MP than above it, and at any given temperature solubility decreases as the length of the hydrocarbon chain increases (i.e., the longer the chain or the larger the hydrophobic moiety, the lower the solubility). Some of the more polar lipids (e.g., K and Na soaps; detergents such as sodium dodecyl sulfate, commonly called SDS; and bile salts) form optically clear aqueous solutions in which the apparent solubility may be as high as 60 g/100 g of solution. These lipids, in fact, have a low monomer solubility but spontaneously form small aggregates of molecules called micelles when their true (monomer) solubility is exceeded. These solutions are called micellar solutions.

A micellar solution is a thermodynami-cally stable system formed spontaneously in water above a critical concentration and temperature (Fig. 3-11). The solution contains small aggregates (micelles) whose molecules are in rapid equilibrium with a low concentration of monomers. This low concentration of monomers is, in fact, the true solubility of the lipid and is called the critical micellar concentration (CMC). Above the CMC, the excess lipid forms micelles. Micellar solutions can solubilize other less soluble lipids to form mixed micelles. Micelles are spherical structures, about 2 molecular lengths in diameter

Micelles Cmc Factor Salt Curve

of Soluble Lipid

Figure 3-11. Behavior of soluble lipids at high-water concentration as a function of temperature and concentration. The temperature is plotted on the ordinate and increasing concentrations of soluble lipid (amphiphiie) on the abscissa. Micelles occur only in region Y. In this region the amphiphiie is above a concentration indicated by the curve 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 at which the CMC and CMT curves meet is termed the Krafft point. It can be considered a triple point and indicates the CMT at the CMC. (From Small, D. M. 11986] The Physical Chemistry of Lipids from Alkanes to Phospholipids. Handbook of Lipid Research, vol. 4. Plenum Press, New York.)

of Soluble Lipid

Figure 3-11. Behavior of soluble lipids at high-water concentration as a function of temperature and concentration. The temperature is plotted on the ordinate and increasing concentrations of soluble lipid (amphiphiie) on the abscissa. Micelles occur only in region Y. In this region the amphiphiie is above a concentration indicated by the curve 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 at which the CMC and CMT curves meet is termed the Krafft point. It can be considered a triple point and indicates the CMT at the CMC. (From Small, D. M. 11986] The Physical Chemistry of Lipids from Alkanes to Phospholipids. Handbook of Lipid Research, vol. 4. Plenum Press, New York.)

in pure water, but when salt is added they often enlarge and assume cylindrical or discoid shapes. Bile salts allow formation of mixed micellar solutions in bile and in intestinal contents during fat absorption. Such solutions are necessary for the proper digestion and absorption of fat and fat-soluble vitamins.

Lipids may also be suspended in aqueous systems. Insoluble lipids such as triacylglycer-ols or cholesteryl esters can be made to form relatively stable suspensions (emulsions) of lipid in water by adding an emulsifier such as a phospholipid. For instance, emulsions containing particles with a core of triacylglyc-erol and a surface layer of an emulsifier such as PC can be formed in vitro by agitation. Plasma lipoproteins are similar lipid particles formed in vivo in intestinal enterocytes or he-patocytes and modified in plasma by enzymes and transfer factors (Small, 1992). (See Chapters 7 and 14 for further discussion of lipoproteins.)

Determinants of Lipid Melting

Lipid melting is the melt (solid to liquid ) of the aliphatic chains, illustrated, for instance, by melting butter or animal fat. It is affected by chain length, polar substitution, or double bonds and is illustrated in Figure 3-12, in which the chain-melting transition (crystal to liquid chain) is shown for lipids with increasing chain length and for a variety of molecules having different substituents. Note that as the chain length increases, the melting temperature rises. Double bonds, triple bonds, methylene branches, and halide substitutions at the end of the chain decrease the melting temperature, but polar substituents, particularly those that can form hydrogen bonds or ionic bonds, increase the melting transition. The order of increasing melting temperatures for a given chain length is as follows: 1 olefins<al-kanes<ethyl esters<normal alcohols<car-boxylic or fatty acids<triacyIglycerols<l,2 di-acylglycerols = 3 monoacylglycerols<dry PC. Interaction of water with the polar groups decreases the melting transition (see anhydrous and hydrated PC in Fig. 3-12).

Surface Behavior of Lipids at the Water Interface

Lipids accumulate at air/water or oil/water interfaces. The interaction of a specific lipid with an aqueous interface depends on the hydrophilic-lipophilic balance of the lipid (i.e., the relative strengths of the hydrocarbon and water-seeking parts). Thus, when a drop of pure lipid contacts a water surface of limited area, one of three events will occur: (1) a very lipophilic lipid, like mineral oil, cholesteryl oleate, or oleoyl palmitate (a wax ester), will simply sit on the water as an intact droplet or lens; (2) a more hydrophilic lipid, like triolein or oleoyl alcohol, will spread to form a continuous insoluble monolayer of molecules in equilibrium with the remainder of the droplet; or (3) a highly hydrophilic lipid, like K oleate or Na cholate (a bile salt), will spread to form an unstable film from which molecules desorb into the water. Lipids are found in almost all interfaces between cellular compartments. Between two aqueous compartments within a cell, a membrane bilayer is present. Between fat and aqueous compartments in the cytoplasm (e.g., a fat droplet in a fat cell) or plasma (e.g., a lipoprotein particle), a phospholipid monolayer forms the interface.

Lipid Classification Based on Physical Interaction with Water

Because most known biological systems are aqueous systems, a classification based on the behavior of lipid in water and at aqueous interfaces (Table 3-4) is given to help one predict how certain classes of lipids will assemble and distribute in cells. This classification generally applies to lipids with melted chains. Nonpolar lipids are water insoluble, do not spread at an air/water interface, have extremely limited solubility in membranes (less than 3% by weight of total membrane lipids), and will usually be found in intracellular droplets. These lipids are highly soluble in organic solvents like hexane (C6H]4) and chloroform (CHC13). All other lipids are am-phiphiles (i.e., they have affinity for both oil and water). The interaction with water (or hydrocarbons) is determined by the nature of the polar group (water-soluble) and the mass of the hydrocarbon part.

Class I polar lipids have a weak polar group compared with the hydrocarbon mass. They are virtually insoluble in water but

Lipid Melting Temperature

Figure 3-12. Effects of polar substltuents on melting of the hydrocarbon chain for a variety of lipids. The major chain-melting transition (i.e., to liquid chain) temperatures for a variety of lipids are plotted against the number of carbons in the aliphatic chain. The stronger the interactions of the polar groups with each other, the higher the melting point (MP). This is illustrated by the difference between fatty acids, which can form hydrogen bonds between the carboxyl groups, and ethyl esters of fatty acids, which cannot. The MP of the hydrogen-bonded fatty acids is 30 to 40° C higher. Note that the melting transitions increase in temperature with increasing hydrocabon chain length, even in water. The presence of water, however, lowers the chain transition when compared with the dry lipid, as shown for phosphatidylcholine (PC). DG, diacylglycerol; MG, monoacylglycerol; TG, Triacylglycerol.

Number of Carbons

Figure 3-12. Effects of polar substltuents on melting of the hydrocarbon chain for a variety of lipids. The major chain-melting transition (i.e., to liquid chain) temperatures for a variety of lipids are plotted against the number of carbons in the aliphatic chain. The stronger the interactions of the polar groups with each other, the higher the melting point (MP). This is illustrated by the difference between fatty acids, which can form hydrogen bonds between the carboxyl groups, and ethyl esters of fatty acids, which cannot. The MP of the hydrogen-bonded fatty acids is 30 to 40° C higher. Note that the melting transitions increase in temperature with increasing hydrocabon chain length, even in water. The presence of water, however, lowers the chain transition when compared with the dry lipid, as shown for phosphatidylcholine (PC). DG, diacylglycerol; MG, monoacylglycerol; TG, Triacylglycerol.

TABLE 3-4

Classification of Biologically Active Lipids

Class

Surface Properties* Bulk Properties Examples

Nonpolar

Will not spread to form monolayer

Insoluble

Polar Class I: Insoluble, nonswelling amphiphiles

Class II: Insoluble swelling amiphiphilic lipids

Spreads to form stable monolayer

Spreads to form stable monolayer

Class IIIA: Soluble amphiphiles with lyotropic mesomorphism*

Class HIB: Soluble amphiphiles, no lyotropic mesomorphism

Insoluble, or solubility very low

Insoluble but swells in water to form lyotropic liquid crystals

Spreads but forms unstable monolayer because of solubility in aqueous substrate

Spreads but forms unstable monolayer due to solubility in aqueous substrate

Soluble, forms micelles above a critical micellar concentration; at low water concentrations forms liquid crystals Forms micelles but not liquid crystals

Long-chain, saturated or unsaturated, branched or unbranched, aliphatic hydrocarbons with or without aromatic groups (e.g., dodecane, octadecane, hexadecane, paraffin oil, phytane, pristane, carotene, lycopene, gadusene, squalene) Large aromatic hydrocarbons (e.g., cholestane, benzopyrenes, coprostane, benzophenanthrocenes) Esters and ethers in which both components are large, hydrophobic lipids (e.g., steryl esters of long-chain fatty acids, waxes of long-chain fatty acids, and long-chain normal monoalcohols, ethers of long-chain alcohols, steryl ethers, long-chain triethers of glycerol) Triglycerides, diglycerides, long-chain protonated fatty acids, long-chain normal alcohols, long-chain normal amines, long-chain aldehydes, phytols, retinols, vitamin A, vitamin K, vitamin E, cholesterol, desmosterol, sitosterol, vitamin D, un-ionized 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) Phosphatidylcholine, phosphatidylethanolamine, phosphatidyl inositol, sphingomyelin, cardiolipid, plasmalogens, ionized phosphatidic acid, cerebrosides, phosphatidylserine, monoglycerides, acid soaps, a-hydroxy fatty acids, monoethers of glycerol, mixtures of phospholipids and glycolipids extracted from cell membranes or cellular organelles (glycolipids and plant sulfolipids)

Sodium and potassium salts of long-chain fatty acids, many of the ordinary anionic, cationic, and nonionic detergents, lysolecithin, palmitoyl and oleoyl coenzyme A and other long-chain thioesters of coenzyme A, gangliosides, sulfocerebrosides

Bile salts, sulfated bile alcohols, sodium salts of fusidic acid, rosin soaps, phenanthrene sulfonic acid, penicillins, phenothiazines

*Lyotropic mesomorphism means the formation of liquid crystals on interaction with water.

From Small, D. M. (1986) The Physical Chemistry of Lipids from Alkanes to Phospholipids. Handbook of Lipid Research, vol. 4. Plenum Press, New York.

spread to form stable monolayers. That is, they have a surface solubility They have limited solubility in membranes, and they partition between membranes and intracellular lipid droplets. Cholesterol and free protonated long-chain fatty acids are good examples. The partition coefficient (Kp) of cholesterol between membranes and oils [Kp = % (w/w) in membranes/% (w/w) in oil] varies from about 6 in membranes versus cholesteryl ester oil to 22 in membranes versus triacylglycerol oil (Miller and Small, 1987). Thus on a weight basis, much more cholesterol distributes to membranes than to oils. For fatty acids such as oleic acid, the Kp between membranes and oils is about 12.

Class II lipids have more balanced polar and hydrocarbon parts and are the "membrane formers." They are insoluble in water and in oil and seek out the interface between oil and water; thus, they are good emulsifiers. When no water-oil interface is present, they form bilayers.

Class III lipids have a very strong polar moiety relative to the hydrophobic region, have measurable monomer solubility, and form micelles. They also partition into membranes and can be disruptive to membranes. They are oil insoluble, are not found in fat droplets, and are not readily soluble in hex-ane and chloroform. They are soluble in polar solvents like methanol (CH3OH) or ethanol (CH3CH2OH).

PROPERTIES OF DIETARY FATS AND OILS AND THEIR PRODUCTS: DIACYLGLYCEROLS, MONOACYLGLYCEROLS, AND FATTY ACIDS

Physical properties of dietary triacylglycerols and their hydrolysis products affect the digestion and absorption of dietary lipids and thus have important physiological implications.

Melting and Crystallization of Acylglycerols, Fatty Acids, and Acid-Soaps

The melting and crystallization temperatures for a variety of triacylglycerols, diacylglycer-

ols, and monoacylglycerols that are present during fat metabolism have been reported (Small, 1991). The MPs of many individual dietary triacylglycerols are above body temperature. The pure saturated triacylglycerols tristearin (MP 73.1°C) and tripalmitin (MP 66.4°C) are not well absorbed. Pancreatic lipase will not hydrolyze them, and they pass through the gut unhydrolyzed and unab-sorbed, still in solid form. Dietary fat is nevertheless well absorbed. Some food fats that are partly solid at body temperature, such as lard and tallow, are digested and absorbed. So why are the fats with high MPs such as lard well absorbed? Although foods contain some simple saturated triacylglycerols, most fat is a mixture of complex triacylglycerols (see Table 3-3), and MPs of mixed fats are very broad. Beef fat starts to melt at about 25°C and finishes melting at 55 to 60°C. At 37°C, most of the fat is melted. Therefore, it is a good substrate for intestinal and gastric lipases.

Once hydrolysis of fat or oil begins in the stomach by action of gastric lipase, fatty acids are released. Long-chain saturated fatty acids also have quite high MPs (Fig. 3-13); for instance, the MPs for myristic, palmitic, and stearic acids are 54, 63, and 70°C, respectively. In addition, they are poorly soluble. For instance, the solubility of palmitic acid at 25°C is 3X10"5 g/L (10 nmol/L) (Small, 1986). In the stomach, the pH is low (1.5 to 3), and fatty acids are protonated (RCOOH). However, at the pH of the duodenum (5.5 to 7.5), the fatty acid would be rapidly converted into hydrated acid-soaps [e.g., H+K+(RCOO-)2] (Cistola et al„ 1986; Small, 1986). Like protonated fatty acids, these compounds are almost insoluble in aqueous media, but the melting temperatures for acid-soaps are about 10°C lower than for the corresponding acids (Fig. 3-13). For instance, the melting temperatures for hydrated K acid-soaps are 43°C (myristic), 51°C (palmitic), and 61°C (stearic) (Cistola et al„ 1986). It should be noted here that alkali metal soaps of fatty acids (K+RCOO-) do not form until the pH is quite high, more than 8.5. Therefore, the form of most fatty acids at biological pH (5.5 to 8) is the acid-soap and at pH lower than 5, the protonated acid. The commonly held concept that fatty acids are biological detergents is incorrect. Sodium and

Biological Detergents

Number of Carbons

Figure 3-13. The melting point of saturated fatty acids (RCOOHj, acid-soaps [e.g., H* K* (RCOO~)2l, and potassium soaps (K*RCOO~) plotted against the number of carbon atoms in the molecule. R stands for CH3-(CH2j„-CH2-.

Number of Carbons

Figure 3-13. The melting point of saturated fatty acids (RCOOHj, acid-soaps [e.g., H* K* (RCOO~)2l, and potassium soaps (K*RCOO~) plotted against the number of carbon atoms in the molecule. R stands for CH3-(CH2j„-CH2-.

K soaps of fatty acids are detergents, but because they do not form except at high pH, they do not exist in vivo under most circumstances.

Thus when 1 mol of dietary fat is hy-drolyzed by intestinal lipases, the products are 2 mol of fatty acid and 1 mol of 2-monoacyl-glycerol. If the fatty acids released are unsaturated or medium-chain (less than 14 carbon-) saturated fatty acids, they are above their MPs and can be solubilized by bile salts and moved to the intestinal brush border membrane where they are absorbed. However, if the fatty acids released by the lipase have a high content of long-chain (18 to 24 carbon-) saturated fatty acids, which are below their MPs, they form a solid precipitate that is not solubilized well by bile acids and thus are poorly absorbed (Redgrave et al., 1988; Small, 1991). This effect of MP on solubility appears to be a factor in certain "low-calorie" structured fats.

Fatty acids may also be malabsorbed if they are hydrolyzed from glycerides in a high calcium environment. Calcium intake correlates with the fecal loss of fatty acids. The probable reason for this is that with an adequately high concentration of calcium ions, fatty acids will react to form calcium soaps, Ca2+ (RCOO-)2, which have very high MPs (>200°C), precipitate in the intestine, and pass out in the feces (Carey et al., 1983).

Saturated diacylglycerols also have high MPs (e.g., dipalmitin, 70.1°C; distearin, 77.2°C), which are not affected by hydration. These saturated diacylglycerols are class I polar amphiphiles (see Table 3-4) and partition mainly into the surface of fat droplets, lipoproteins, and membranes. As surface constituents of fat droplets, they might interfere with lipoly-sis of intestinal fat or of chylomicrons in the plasma. Monoacylglycerols in the hydrated state have considerably lower melting temperatures than in the dry state (about 25°C lower). However, even in water, the longer-chain saturated monoacylglycerols, such as

NUTRITION INSIGHT Structured Triglycerides

Recently the synthesis of "structured triglycerides" (Babayan, 1982) has become a popular method for reducing the caloric values of triglycerides (triacylglycerols). Structured triglycerides may be produced in mass amounts either by mixing short-chain fatty acids with a long-chain triglyceride and transesterifying or by mixing together 2 or more relatively pure triglycerides of chosen composition and then transesterifying and isolating particular classes of triglycerides. For instance, very long-chain triglycerides may be transesterified with medium-chain triglycerides to produce a series of triglycerides that have 1 very long-chain fatty acid and 2 medium-chain fatty acids (e.g., dioctanoyl, behenoyl glycerol). These triglycerides, called "caprenin," have melting characteristics (MP ~32°C) similar to those of chocolate, but because a large proportion of the very. long-chain fatty acid is malabsorbed and the medium-chain fatty acids have less caloric value, the overall caloric value per molecule is considerably (50% to 60%) less than that of cocoa butter.

Similarly, triglycerides containing 2 molecules of very short acids, such as acetate, propionate, or butyrate, and 1 molecule of a long-chain fatty acid have been synthesized, and these generally form oils. Other structured triglycerides include those made with medium-chain and essential fatty acids, such as linoleic, linolenic, EPA, and DHA. These may be useful in infant formulas and for treating patients with severe debilitation and essential fatty acid deficiencies (Jensen et al.r 1994). Procedures for synthesis and definition of the physical and biochemical characteristics of structured triglycerides have been described, and it is likely that some will become included into our food sources and others used in therapy.

2-monostearin, could be crystalline at body temperature, and these could potentially segregate into partially crystalline structures on the surfaces of fat globules in the intestine or of lipoproteins in plasma. If such monoacyl-glycerols accumulate in high enough quantities, this could slow lipolysis. Monoacylglycer-ols accumulate sufficiently in normal lipolysis for this to occur (Boyle et al., 1995).

Effects of Positions of Fatty Acids in Mixed-Chain Triacylglycerols upon Their Absorption from the Intestine

It has been known for some time that the stereospecific position of fatty acids on triacylglycerols can alter the availability of the component fatty acids for absorption from the small intestine in both rats and in humans (Small, 1991). Filer et al. (1969) gave newborn infants a formula that contained either lard, which has an abundance of palmitate in the 2 position (see Table 3-3), or randomized lard, in which palmitate was randomly distributed to all positions. Ninety-five percent of all the fatty acids was absorbed from natural lard, but only 72% was absorbed from the randomized lard. In the randomized lard, both palmitic and stearic acids were malabsorbed, 58% and 40%, respectively Similar observations were made in studies with cocoa butter and randomized cocoa butter (Kritchevsky, 1988). The principal triacylglycerols in cocoa butter are SOS, POP and SOP (O, oleoyl, P, palmitoyl, and S, stearoyl), and absorption of the saturated stearate and palmitate is somewhat low. After randomizing the fatty acids, the saturated acids appear more in the 2 position, and absorption is improved. These studies appear to indicate that if the saturated fatty acid is in the 2 position, it is more likely to be absorbed than if it is in the 1 or 3 position.

In studies in rats with OSO and OOS, as shown in Figure 3-14, about 94% of the stearate in OSO was absorbed, whereas only about 62% of the stearate in OOS was absorbed (Redgrave et al., 1988). When intestinal enzymes hydrolyze 1 mol of OSO, they produce 1 mol of 2-monostearin and 2 mol of oleic acid. The MP of hydrated 2-monostearin is close to the body temperature of rats (39°C), and it can be solubilized by bile salts and

Intestinal Absorption of X in OOX Series OSO (94%)

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