Effect of Cholesterol and Other Additives on Viscosity, Self-Diffusion

It has been empirically known that cholesterol largely increases the viscosity of oleic acid. To clarify the mechanism of the effect of cholesterol on...
0 downloads 0 Views 185KB Size
Download PDF
740

J. Phys. Chem. B 2007, 111, 740-747

Effect of Cholesterol and Other Additives on Viscosity, Self-Diffusion Coefficient, and Intramolecular Movements of Oleic Acid Makio Iwahashi,*,† Atsushi Umehara,† Kenichiro Wakisaka,† Yasutoshi Kasahara,† Hideyuki Minami,† Hideyo Matsuzawa,† Hideyuki Shinzawa,‡ Yukihiro Ozaki,‡ and Masao Suzuki§ School of Science, Kitasato UniVersity, Sagamihara, Kanagawa 228-8555, Japan, School of Science and Technology, Kwansei-gakuin UniVersity, Gakuen, Sanda Hyougo 669-1337, Japan, and Research Institute of Biological Materials, Keihanna Plaza, Seika, Soraku, Kyoto 619-0237, Japan ReceiVed: March 29, 2006; In Final Form: NoVember 11, 2006

It has been empirically known that cholesterol largely increases the viscosity of oleic acid. To clarify the mechanism of the effect of cholesterol on the intermolecular and the intramolecular (segmental) movements of oleic acid in the liquid state, we measured density, viscosity, IR, 1H NMR chemical shift, self-diffusion coefficient, and 13C NMR spin-lattice relaxation time for the liquid samples of oleic acid containing a small amount of cholesterol. Furthermore, the above measurements were also carried out for the samples of oleic acid containing a small amount of cholestanol, cholestane, cholesteryl oleate, ethanol, or benzene. Cholesterol, possessing one OH group and one double bond in its molecular structure, largely increased the viscosity and reduced the self-diffusion and the intramolecular movement of oleic acid. Cholestanol, possessing one OH group but not a double bond, and cholesteryl oleate, not possessing an OH group, also reduced the selfdiffusion and the intramolecular movement; cholestane, not possessing an OH group, slightly reduced the self-diffusion and the intramolecular movements. In contrast with these sterols, ethanol and benzene reduced the viscosity and increased the self-diffusion and the intramolecular movements. In addition, cholesterol and ethanol, both having one OH group, promoted the upfield shift of the 1H NMR signal of the carboxyl group of oleic acid; IR difference spectra for the cholesterol/oleic acid system quite resemble those for the ethanol/ oleic acid system. These results suggest that oleic acid makes a complex with cholesterol as well as with ethanol. On the basis of the formation of the complex, we have revealed the role and the functional mechanism of cholesterol to the intermolecular and the intramolecular movements of oleic acid in the liquid state.

Introduction Biomembranes have many kinds of structures but their fundamental structures are composed of phospholipid bilayers; the major natural phospholipids in the biomembranes contain cis-unsaturated fatty acids as a building part.1 The large steric hindrance of the cis-unsaturated acyl groups of the fatty acids most likely disturbs the close packing of hydrocarbon chains of phospholipids and keeps the mobility of biomembranes high, which activates the function of biomembranes even at low temperatures. The biomembranes are also composed of cholesterol and many kinds of proteins; they form a confirmed structure. In such constructing materials of biomembranes, cholesterol also controls the fluidity of the biomembranes and acts an important role in the metabolism of a living cell.2 The functions of cholesterol in membranes are most likely due to the interaction between cholesterol and fatty acid moiety in the phospholipids.3 It is well-known that the human population whose diet is rich in saturated acylglycerols exhibits increased blood cholesterol level, and hence has a higher risk of heart disease.4 Indeed, saturated fatty acids have been found to increase the levels of * To whom all correspondence should be addressed. Phone: 81-42-7789273. Fax: 81-42-788-9369. E-mail: [email protected]. † Kitasato University. ‡ Kwansei-gakuin University. § Research Institute of Biological Materials.

cholesterol in blood, especially cholesterol bound to low-density lipoproteins (LDL).5 Interestingly, monounsaturated acids, such as oleic acid, have the opposite effect, i.e., they decrease the levels of LDL-bound cholesterol.6 Furthermore, it has been empirically known that cholesterol largely increases the viscosity of oleic acid. Thus, it is interesting and important to clarify the mechanism of the interaction between oleic acid and cholesterol. Many useful purposes of oleic acid and cholesterol have been the subjects of individual research. For example, the crystallization, transformation mechanisms, and the crystal structures of R, β, and γ polymorphs of oleic acid have been precisely studied as well as the thermodynamic properties such as melting point, solubility, and heat of solution7-11 since the sufficient supplies of pure samples of oleic acid (purity >99.9%) have become available. The pressure effect on the polymorphs of oleic acid was also carried out.12 With respect to oleic acid in the liquid state, most of oleic acid molecules exist as dimers in melt and also even in nonpolar solvent such as CCl4.13 The dimers form a kind of cluster resembling a smectic liquid crystal in the liquid state.14-17 The existence of the clusters determines the physical properties of oleic acid such as viscosity, self-diffusion coefficient, and intramolecular (segmental) movements.15 Physicochemical properties and crystal structures of cholesterol also have been investigated.18-20 Furthermore, liquid crystals and gallstones of cholesterol were also precisely

10.1021/jp0619538 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/10/2007

Effects of Additives on Oleic Acid

J. Phys. Chem. B, Vol. 111, No. 4, 2007 741

studied.21 However, the direct interaction between oleic acid and cholesterol molecules especially in the liquid state has scarcely been studied. In the present study, through the measurements of density, viscosity, 1H and 13C NMR, and IR, we have studied the effect of cholesterol or other additives such as cholestanol, cholestane, cholesteryl oleate, benzene, and ethanol on the physical properties of oleic acid and then revealed the functional mechanism of cholesterol on the intermolecular and the intramolecular movements of oleic acid in the liquid state. Experimental Section Materials. Samples of oleic acid (cis-9-octadecenoic acid; >99.9% pure), cholesterol (5-cholesten-3β-ol; 99.3% pure), and cholesteryl oleate (99.3% pure) were obtained from the Research Institute of Biological Materials (Kyoto, Japan). Samples of cholestanol (5R-cholestan-3β-ol; >95% pure) and cholestane (5R-cholestane; >98% pure) were purchased from Sigma Co. These samples were used without further purification. Samples of ethanol (>99.7% pure) and benzene (>99.7% pure) purchased from Aldrich Co. were dried over 5A molecular sieves and distilled. Carbon disulfide-free carbon tetrachloride (CCl4; 99.9% pure) purchased from Dojin Co. for the IR measurement was also dried over 5A molecular sieves and distilled. All the distillations were carried out under an atmosphere of dried nitrogen. Samples for the measurements of 13C NMR spinlattice relaxation time T1 were prepared under an atmosphere of nitrogen, using a glovebox to prevent the absorption of oxygen, which would make the T1 shorter. Density and Viscosity Measurements. Density, F, and viscosity, η, for the oleic acid samples in the liquid state with or without the additives in the temperature range (30-80) ( 0.01 °C were measured on a vibration-type densimeter (Anton Paar Model DMA 58) and with two Ubbelohde viscometers (Shibata Co. Ltd.) having different diameters, respectively. Degassed pure water was used for calibrating the densimeter and the viscometers. The obtained density data were used for the calculation of the viscosity of the samples. All experiments were carried out under the condition of the mole fraction of cholesterol (xcholesterol) below 0.15, because of its relatively low solubility in oleic acid at room temperature. NMR Measurements. The self-diffusion coefficient, D, of oleic acid in liquid samples with or without the additives was determined by means of the pulsed-field gradient NMR method.22 All the measurements were made on protons at 399.65 MHz in the temperature range (30-80) ( 0.5 °C on an NMR spectrometer (Japan Electron Optics Laboratory (JEOL) Model EX400). The 1H NMR chemical shift of the carboxyl group of oleic acid in the samples with or without the additives was measured on the same NMR spectrometer, using tetramethylsilane (TMS) enclosed in an inner tube as a standard, in the temperature range (30-120) ( 0.5 °C. The 13C NMR spin-lattice relaxation time, T1, for evaluating the intramolecular movements of oleic acid in the samples with or without the additives was obtained by the inversion recovery method22 employing a 180°-τ-90°pulse sequence, using the same NMR spectrometer in the temperature range (40-80) ( 0.5 °C. IR Spectrum Measurements. IR spectra of oleic acid in CCl4 solutions with or without cholesterol or ethanol were measured at a resolution of 2 cm-1 on a Bio-Rad FT135 spectrophotometer with a cell having CaF2 windows. All the IR measurements were carried out with a light path length of 0.1 mm in a thermostated room controlled within (0.05 °C. Throughout the IR measurements, the temperature in the cell room was kept at 26 ( 0.1 °C.

Figure 1. Effect of cholesterol on the viscosity of oleic acid in the liquid state under various constant temperatures: (9) 30 °C; (O) 40 °C; (2) 50 °C; (]) 60 °C; (B) 70 °C; (0) 80 °C.

Figure 2. Effect of cholesterol on the self-diffusion coefficient of oleic acid in the liquid state under various constant temperatures: (9) 30 °C; (O) 40 °C; (2) 50 °C; (]) 60 °C; (B) 70 °C; (0) 80 °C.

Results and Discussion Effects of Cholesterol on the Physical Properties of Oleic Acid. Figure 1 shows the relationship between the viscosity, η, of oleic acid/cholesterol mixtures and the molar fraction of cholesterol, xcholesterol, at various constant temperatures (30-80 °C). The η value increases with increasing xcholesterol and largely decreases with increasing temperature. Obviously, cholesterol increases the viscosity of oleic acid. Figure 2 shows the relationship between the self-diffusion coefficient, D, of oleic acid and xcholesterol in the oleic acid/ cholesterol mixtures in the liquid state at various constant temperatures (30-80 °C). Experimental errors were within (8%. The D value decreases with increasing xcholesterol and largely increases with increasing temperature. Therefore, the self-diffusion coefficient as the microscopic property seems to inversely reflect the viscosity as the macroscopic property. To clarify the effect of cholesterol on the intramolecular (segmental) movements of oleic acid, we measured the 13C NMR spin-lattice relaxation time T1 for each carbon atom in the oleic acid molecule under the conditions of several cholesterol compositions. Experimental errors were within (5%. Reciprocal effective correlation time, 1/τc for the rotational movement of the each carbon atom in the oleic acid molecule in the liquid mixture, was calculated from the obtained T1 value by assuming that T1 of a protonated carbon is overwhelmingly dominated by dipole-dipole interaction with the attached

742 J. Phys. Chem. B, Vol. 111, No. 4, 2007

Iwahashi et al.

Figure 3. Effect of cholesterol on the intramolecular movements (1/ τc) of the different carbon atoms in the oleic acid molecule in the oleic acid/cholesterol mixture at 40 °C: the mole fraction of cholesterol (xcholesterol) ) 0 (]), 0.05 (0), 0.1 (2), 0.15 (O).

protons. Namely, 1/τc, which is a semiquantitative measure of the segmental (mostly rotational) movement of the molecule, is expressed as23 2 2 2 1 Nh γC γH ) T1 τc r 6

(1)

CH

where h is Planck’s constant and γC and γH are the gyromagnetic ratio of 13C and 1H, respectively; rCH is the C-H distance, usually about 0.109 nm, and N is the number of hydrogen atoms directly bound to carbon atoms. The 1/τc of the carbon atoms at various positions of the oleic acid molecule in its sample with or without cholesterol obviously increased with increasing temperature. Figure 3 shows the relationships between 1/τc and the position of the carbon atoms in the oleic acid molecule at xcholesterol ) 0, 0.05, 0.10, and 0.15 at 40 °C, as a typical example. As can be seen from the open diamonds at xcholesterol ) 0, the rotational movements for the carbon atoms at the second position attached to the carboxyl group and at the double-bonded position are considerably restricted, while those for other carbon atoms increase toward the end of the hydrocarbon chain. That is, the methyl group positioned at the end of the acid molecule moves around most vigorously. This is because, in the liquid state, two oleic acid molecules make a cyclic dimer with the hydrogen bonding between their carboxyl groups and therefore the carbon atom at the second position is just near the center of gravity of the dimer.14,15,24 Interestingly, the rotational movements of the carbon atoms at any positions decrease with increasing xcholesterol. Namely, cholesterol evidentially reduces not only the intermolecular movement such as self-diffusion but also the intramolecular movements of the oleic acid molecule. To clarify the reason for the depression effect of cholesterol on the intermolecular and the intramolecular movements of oleic acid, we measured the chemical shift, δ, of the 1H NMR signal of the carboxyl group of oleic acid in the oleic acid samples with or without cholesterol. Figure 4 shows the relationship between δ and temperature under the condition of various cholesterol concentrations. The proton signal of the carboxyl group of oleic acid in the pure sample of oleic acid (open diamond) exhibits an upfield shift with increasing temperature. The upfield shift generally means the rupture of the hydrogen bonding of the cyclic dimer composed of two carboxyl groups. Namely the temperature

Figure 4. Temperature dependence of the 1H NMR chemical shift of the carboxyl group of oleic acid with or without cholesterol: the mole fraction of cholesterol (xcholesterol) ) 0 (]), 0.05 (0), 0.1 (2), 0.15 (O).

rising causes a rupture of the cyclic dimer of oleic acid, even though the degree of the rupture is very low: it has been revealed that only ca. 1% of the oleic acid dimers dissociate into monomers in the melt at 60 °C and that ca. 2% of the dimers dissociate at 80 °C. 13) All the oleic acid samples containing cholesterol also exhibit the upfield shift of the proton signal of the carboxyl group with increasing temperature. Interestingly, at any constant temperatures cholesterol promotes the upfield shift of the proton signal of the carboxyl group of oleic acid: the upfield shift increases as xcholesterol increases. Namely, cholesterol appears to accelerate the rupture of the hydrogen bonding between the carboxyl groups of the oleic acid cyclic dimer. The rupture of the cyclic dimer, which means the increase in the concentration of the fatty acid monomer, should have decreased the viscosity and then increased the self-diffusion coefficient of oleic acid. In practice, however, as shown in Figures 1-3, quite opposite results were obtained both in the intramolecular and in the intermolecular movements of oleic acid when cholesterol was added to the oleic acid sample. To elucidate more precisely the role of cholesterol in the hydrogen bonding of oleic acid, we measured IR spectra of oleic acid in CCl4 solutions with or without cholesterol and also the IR spectrum of cholesterol itself in CCl4 solution. Figure 5 shows the IR spectra over the 3000-3800 cm-1 region for the samples of 0.2 mol dm-3 oleic acid (A), 0.2 mol dm-3 cholesterol (B), and 0.2 mol dm-3 oleic acid/0.2 mol dm-3 cholesterol mixture (C) in CCl4 solutions, respectively, at 26 ( 0.1 °C. Figure 6 also shows the IR spectra over the 15001900 cm-1 region, for the same A, B, and C samples. The contributions of CCl4 to these absorption spectra have been subtracted from these original spectrum data. In spectrum A of oleic acid in Figure 5, a small but relatively sharp band is observed at 3528 cm-1; this band is assigned to the free OH-stretching mode for oleic acid monomer.25,26 The sum of the tails of the large and broad absorption bands due to the stretching vibration of OH groups of the carboxylic acid dimer and to the stretching vibrations of CH groups of methyl and methylene groups exists below 3300 cm-1. Taking notice of spectrum B of cholesterol in CCl4, it has a relatively sharp 3622 cm-1 band, which is due to the free OHstretching mode for monomeric cholesterol, and a broadband centered at about 3340 cm-1 due to the hydrogen-bonded OH

Effects of Additives on Oleic Acid

Figure 5. IR spectra over the 3100-3800 cm-1 region of 0.2 mol dm-3 oleic acid (A), 0.2 mol dm-3 cholesterol (B), and 0.2 mol dm-3 oleic acid/0.2 mol dm-3 cholesterol mixture (C) in CCl4 solutions at 26 ( 0.1 °C. The peak intensities of the 3622 cm-1 bands in the B and C curves are 0.084 and 0.053, respectively.

Figure 6. IR spectra over the 1500-1900 cm-1 region of 0.2 mol dm-3 oleic acid (A), 0.2 mol dm-3 cholesterol (B), and 0.2 mol dm-3 oleic acid/0.2 mol dm-3 cholesterol mixture (C) in CCl4 solutions at 26 ( 0.1 °C. Similar results were also obtained for several lower concentration samples having absorbance less than unity for the 1710 cm-1 band.

group of the self-associated multimer.27-29 The broadband has a shoulder at about 3470 cm-1 due to the hydrogen-bonded OH group of the self-associated dimer.27 This spectrum profile is quite similar to those for ordinary alcohols.30 It is worth noting that spectrum C for the sample of oleic acid/cholesterol mixture in Figure 5 is not merely the addition of the spectra for the samples of oleic acid (A) and of cholesterol (B). In fact, with respect to the 3622 cm-1 band due to the free OH vibration of cholesterol, the peak intensity for cholesterol (B) is higher than that for the oleic acid/cholesterol mixture (C). Namely, in the mixture the number of free OH moieties of cholesterol is reduced by oleic acid. In the 1500-1900 cm-1 wavenumber region shown in Figure 6, cholesterol (B) does not exhibit any absorption. Taking notice of the sharp 1710 cm-1 band due to the carbonyl group of dimeric oleic acid,31 the peak intensity for the sample including only oleic acid in CCl4 (A) is higher than that for the sample of oleic acid/cholesterol mixture (C). Namely the number of bonded carboxyl groups of the oleic acid dimer is also reduced by cholesterol. On the other hand, concerning the small and broad 1760 cm-1 band due to the free carbonyl group of the

J. Phys. Chem. B, Vol. 111, No. 4, 2007 743 oleic acid monomer,31-33 the band height of spectrum A and that of spectrum C are almost equal. Similar results were also obtained for several lower concentration samples having absorbance less than unity for the 1710 cm-1 band. This suggests that, although the addition of cholesterol to the oleic acid samples ruptures the bonded carboxyl group of the oleic acid dimer, the number of free carboxyl groups remains almost constant: The free carboxyl groups newly produced from the dissociation of the oleic acid dimer are thought to be immediately consumed by cholesterol. Thus, the upfield chemical shift of 1H of the carboxyl group of oleic acid accompanied by adding cholesterol to the oleic acid samples (Figure 4) arises merely from the promotive dissociation of the oleic acid dimer: the decrease in the concentration of the oleic acid dimer. To clarify more precisely the hydrogen bonding between oleic acid and cholesterol we subtracted both spectra due to oleic acid (A) and cholesterol (B) from the spectrum of the oleic acid/ cholesterol mixture (C). This subtraction eliminates the contributions of the C-H vibrations of the hydrocarbon chain of oleic acid and the steroid ring of cholesterol from the observed spectrum for the sample of the oleic acid/cholesterol mixture and indicates only the change in hydrogen bonding on mixing oleic acid and cholesterol. The obtained difference IR spectra over 3100-3800 and 1500-1900 cm-1 regions are shown in Figures7, parts a and b, respectively. In Figure 7a the 3622 cm-1 difference band assigned to the free OH group of cholesterol becomes minus. This means that the free OH group of cholesterol is consumed by oleic acid molecules when mixed with oleic acid. On the other hand, the 3528 cm-1 band assigned to the free carboxyl group of oleic acid seems to slightly increase. However, Mercler et al. 29 reported that cholesterol and fatty acid ester in CCl4 solutions are bonded with each other by the OHsOdC type hydrogen bond between the OH group of cholesterol and the carbonyl group of the ester and that the absorption band for the OHsOdC type hydrogen bond also appears at 3530 cm-1. Thus, the slight increase in the 3528 cm-1 band height would likely arise from not an increase in the free carboxyl group of oleic acid but the formation of the OHsOdC type hydrogen bonding. Interestingly, two large, broad bands newly appear at 3430 and 3200 cm-1 in the difference IR spectrum in Figure 7a. The two bands are thought to result from the hydrogen bonding between the OH group of cholesterol and the OH group in the carboxyl group of oleic acid. On the other hand, the difference spectrum over the 15001900 cm-1 region (Figure 7b) indicates that the cholesterol in the oleic acid in CCl4 samples reduces the 1710 cm-1 band due to the bonded carbonyl group of oleic acid dimer but increases the 1723 cm-1 band due to the bonding between the OH group of cholesterol and the carbonyl group of oleic acid (OH-OdC band).33 Furthermore, cholesterol increases the 1690 cm-1 band, which is presumably due to the dimeric carbonyl groups influenced by the OH group of cholesterol since cholesterol has no absorption band over the 1500-1900 cm-1 region. Similar results were also obtained for the several lower concentration samples having absorbance less than unity for the 1710 cm-1 band. These results suggest that, on adding cholesterol to oleic acid, the oleic acid dimers promotively dissociate into oleic acid monomers, which rapidly combine with cholesterol through the two kinds of bonding to the OH group of cholesterol. Consequently, not only by the hydrogen bonding between the OH group of the cholesterol and the carbonyl group of oleic acid but also by the new hydrogen bonding between the OH

744 J. Phys. Chem. B, Vol. 111, No. 4, 2007

Iwahashi et al.

Figure 8. Effect of various additives on the viscosity of oleic acid at 40 °C: (B) cholestane; ([) ethanol; (0) benzene; (O) cholesterol.

Figure 7. (a) A difference spectrum in the 3100-3800 cm-1 region for the oleic acid/cholesterol system (0.2 mol dm-3 oleic acid/0.2 mol dm-3 cholesterol mixture (C), 0.2 mol dm-3 oleic acid (A), 0.2 mol dm-3 cholesterol (B)). The band due to the free OH group largely decreases. The OH-OdC band appears near 3530 cm-1; the two new bands due to hydrogen bonds between the OH of cholesterol and the OH of the carboxyl group of oleic acid appear around 3200 and 3430 cm-1. (b) A difference spectrum in the 1500-1900 cm-1 region for the oleic acid/cholesterol system (0.2 mol dm-3 oleic acid/0.2 mol dm-3 cholesterol mixture (C), 0.2 mol dm-3 oleic acid (A), 0.2 mol dm-3 cholesterol (B)). The 1710 cm-1 band due to the bonded carbonyl group of the oleic acid dimer largely decreases, while the 1723 cm-1 band due to the OH-OdC band increases. Similar results were also obtained for the several lower concentration samples having absorbance less than unity for the 1710 cm-1 band.

group of cholesterol and the OH groups of the carboxyl group of oleic acid, a type of complex is produce; this complex reduces the intermolecular and the intramolecular movements of the oleic acid in the liquid state. To determine more clearly the effect of cholesterol on the various physical properties of oleic acid, we investigated the influence of the other additives such as cholestane, cholestanol, cholesteryl oleate, benzene, and ethanol on the viscosity, the self-diffusion coefficient, and the intramolecular movement of oleic acid. Effect of Other Additives on the Physical Properties of Oleic Acid. Figure 8 shows the concentration dependence of various additives on the viscosity of oleic acid at 40 °C. Cholestane (filled circle) increases the viscosity, whereas ethanol (filled diamond) and benzene (open square) decrease the viscosity. For a comparison, the results of cholesterol (open circles) are also plotted in the same figure. Cholesterol largely decreases the viscosity. A similar tendency was observed at other constant temperatures (30, 50, and 60 °C).

Figure 9. Effect of various additives on the self-diffusion coefficient of oleic acid at 40 °C: (9) cholestanol; (B) cholestane; ([) ethanol; (0) benzene; (O) cholesterol.

Figure 9 shows the concentration dependence of the additives on the self-diffusion coefficient, D, of oleic acid at 40 °C. Cholestanol (filled square), not having any unsaturated bonds, decreases the D value of oleic acid with an increase in its concentration as much as cholesterol having an unsaturated bond. Thus, the unsaturated bond of sterols seems to scarcely influence the physical properties of oleic acid. Cholestane slightly decreases the D value. Contrary to the above sterols, ethanol and benzene increase the D value. A similar tendency was observed at other constant temperatures (30, 50, and 60 °C). Namely the additives having a steroid ring have a tendency to reduce the intermolecular movements of oleic acid, whereas the other additives such as ethanol and benzene increase the intermolecular movements. Figure 10 shows the effect of the other additives such as cholestane, cholesteryl oleate, ethanol, and benzene on the reciprocal of the effective correlation time, 1/τc, for the rotational movement of the segments in the oleic acid molecule at 40 °C. Triangles show the results for the samples of oleic acid without any additives. For a comparison, the results of cholesterol are also plotted in the same figure. Cholestane and cholesteryl oleate decrease the rotational movements of oleic acid, whereas benzene and ethanol increase the movements. It should be noticed that cholesteryl oleate decreases the movement as largely as cholesterol does. A similar

Effects of Additives on Oleic Acid

Figure 10. Effect of various 0.1 mol dm-3 additives on the intramolecular movements of oleic acid at 40 °C: (4) without additives; (B) cholestane; (2) cholesteryl oleate; (0) benzene; ([) ethanol; (O) cholesterol.

Figure 11. Effect of various additives on the chemical shift of the 1H NMR of the carboxyl group of oleic acid at 30 °C: (O) cholesterol; (B) cholestane; (2) cholesteryl oleate; (0) benzene; ([) ethanol.

tendency was observed at other temperatures (30, 50, and 60 °C). Namely the additives having a steroid ring reduce the intramolecular movements of oleic acid, whereas the other additives such as ethanol and benzene increase the intramolecular movements. Figure 11 shows the effect of the additives on the chemical shift of 1H NMR for the carboxyl group of oleic acid at 30 °C. Benzene, cholestane, and cholesteryl oleate, not possessing the OH group, show little contribution to the chemical shift, whereas cholesterol and ethanol, possessing the OH group, cause considerable upfield shift with increasing concentrations. Similar results were obtained at 50 °C. The OH group of cholesterol or ethanol is thought to rupture the cyclic hydrogen bonding between the carboxyl groups of oleic acid and then newly bind with the free carboxyl group and/or the carbonyl group of oleic acid. This produces a new complex between oleic acid and cholesterol or ethanol. The oleic acid/cholesterol complex most likely resembles the cholesterol oleate in its structure because cholesteryl oleate depresses the intramolecular movement of oleic acid as much as cholesterol does (Figure 10). On the other hand, oleic acid and ethanol also form a complex fundamentally resembling the oleic acid/cholesterol complex. This is because the difference-IR spectra in the regions

J. Phys. Chem. B, Vol. 111, No. 4, 2007 745 of 3000-3800 and 1500-1800 cm-1 for the oleic acid/ethanol system were very similar with those for the oleic acid/cholesterol system shown in Figure 7a,b. It has been reported that in protic and dipolar aprotic solvents the fatty acid dimers are broken up to form 1:1 or 1:2 hydrogenbonded complexes with the solvents.34 This has been demonstrated by a variety of techniques in water,35 alcohols,36 amides,37,38 sulfoxdes,39 acetonitrile,40 and acetone.36 The predominant formation of heteroassociated species in such solvents is thought to be sometimes due to a simple mass action effect, and sometimes to an inherent instability of the dimer. With the appearance of the heteroassciation, these protic solvents give lower dimerization constants for the fatty acids than the completely nonpolar solvents do.35 Now we know that the oleic acid/cholesterol complex reduces the intermolecular and the intramolecular movements of oleic acid, while the oleic acid/ethanol complex increases those movements, although both complexes have a similar structure. Then why does the oleic acid/cholesterol complex reduce the intermolecular and the intramolecular movements of oleic acid? On the other hand, why does the oleic acid/ethanol complex increase the intermolecular and the intramolecular movements of oleic acid? The fatty acids such as oleic acid, cis-6-octadecenoic, cis11-octadecenoic, trans-9-octadecenoic (elaidic), and octadecanoic (stearic) acids aggregate and form clusters possessing the structure of a quasismectic liquid crystal in their pure liquid states.14,15 Namely, the dimerized acid molecules arrange longitudinally and alternately to make an interdigitated structure in the clusters, which determine the properties of the liquids of the fatty acids.15 The complex formed between cholesterol and oleic acid, resembling cholesteryl oleate in its structure, most likely easily penetrates into the clusters composed of oleic acid dimers as schematically shown in Figure 12 and improve the packing of the dimers inside the clusters by the attractive interaction between their hydrophobic moieties of cholesterol and oleic acid molecules. As a result, the viscosity of oleic acid increases and then the self-diffusion coefficient and the intramolecular movements of oleic acid decrease with an increase in the concentration of cholesterol. At the air-water interface the condensation of assembly of oleic acid molecules by cholesterol molecules has been reported:3 Miscibility and interactions between the components of the oleic acid/cholesterol system were studied based on the analysis of surface pressure-area isotherms completed with Brewster angle microscopy images. Oleic acid was found to form a miscible, but nonideal mixed monolayer with cholesterol in the monolayers: Negative deviations from ideality were observed in all the surface pressure-area plots. This suggests that, in the mixed monolayers, oleic acid and cholesterol have strong interactions and good miscibility at all molar ratios. This reflects close-packing arrangements between the bulky cholesterol molecule and the hydrocarbon chain of oleic acid. The molecular arrangement in the oleic acid cluster containing cholesterol molecules resembles that in the above mixed monolayer composed of oleic acid and cholesterol at the airwater interface. That is to say, as shown in Figure 12, the schematic drawing of the oleic acid cluster including the oleic acid/cholesterol complexes shows that, at the cross section A-B of the cluster, the OH group of cholesterol and the COOH group of oleic acid also take similar orientation as that in the monolayer at the air-water interface:3 The molecular situation at the cross section resembles that in the mixed monolayer composed of

746 J. Phys. Chem. B, Vol. 111, No. 4, 2007

Figure 12. Schematic drawing of the oleic acid cluster including the complex composed of oleic acid and cholesterol. The dotted line (AB) shows the cross section of the array of polar parts of oleic acid and cholesterol molecules. The molecular situation at the cross section resembles to that of the mixed monolayer of oleic acid and cholesterol at the air-water interface.3

oleic acid and cholesterol molecules at the air-water interface, where the polar groups such as the OH group of the cholesterol and the COOH group of oleic acid are oriented to the water surface. At the air/water interface, the van der Waals attraction between cholesterol rings and alkyl chains of unsaturated acids is responsible for the deviations from ideality observed in these systems. This reflects good matching between the bulky cholesterol molecule and the shorter hydrocarbon chains of unsaturated fatty acids. Consequently, for the liquid of the oleic acid/cholesterol mixture, the formation of the oleic acid/cholesterol complex is thought to be the origin of the depressive effect on the molecular movements of oleic acid. On the other hand, the complex composed of oleic acid and ethanol also penetrates into the cluster consisting of oleic acid dimers. However, it does not condense the arrangement of the oleic acid dimers in the cluster but disturbs the arrangement because of too small hydrophobic moiety of ethanol, even if the oleic acid/ethanol complex exists in a similar position in the cluster as the oleic acid/cholesterol complex does. This produces an increase in the intermolecular and the intramolecular movements of oleic acid. In the case of the addition of benzene to the oleic acid sample, benzene molecules penetrating into the cluster composed of oleic acid dimers would disturb the arrangement of the oleic acid dimers in the cluster and promote the intermolecular and the intramolecular movements of oleic acid. Also, another explanation is determined for the depression of the intermolecular and intramolecular movements of oleic acid by the addition of cholesterol. Namely, the hydrogen bonding in the oleic acid dimer is much stronger than that in cholesterol. In spite of this, the degree of self-association in cholesterol is higher as compared with that of oleic acid. Oleic

Iwahashi et al. acid molecules form predominantly cyclic dimers of exceptional stability, whereas cholesterol molecules may form higher associates, beyond dimers. Thus, an addition of cholesterol to oleic acid increases the viscosity and decreases the self-diffusion coefficient. This explanation seems to be possible. In fact, besides the proton signal of the COOH group in the 1H NMR chemical shift results for the samples of the oleic acid/cholesterol mixture (and also the oleic acid/ethanol mixture), a signal of the OH proton of cholesterol (or ethanol) existed at around 4 ppm. Consequently, cholesterol (or ethanol) molecules seem to coexist as their aggregates or their monomers in the liquid oleic acid. However, by this explanation only we are not able to explain the obvious decrease in the peak height of the 1710 cm-1 band due to the dimeric carbonyl groups (Figure 6) and also the decrease in the peak height of the 3622 cm-1 band due to the momomeric OH groups of cholesterol (Figure 5). In general, alcohols and dipolar aprotic solvents have higher solubility to the fatty acids having a relatively short hydrocarbon chain, such as octanoic and decanoic acids, than the nonpolar solvents do.41 Consequently, in the oleic acid/cholesterol mixture there would be oleic acid dimers, oleic acid monomers, oleic acid/cholesterol complexes, cholesterol monomers, and cholesterol aggregates. However, the oleic acid/cholesterol complexes are thought to contribute more to the properties of oleic acid in the liquid state than the cholesterol aggregates do. This is because cholesteryl oleate depresses the molecular mobility of carbon atoms of oleic acid as much as cholesterol does as shown in Figure 10. Conclusion The mechanism of the effect of cholesterol on the physicochemical properties of oleic acid in its liquid state was clarified through the measurements of density, viscosity, IR, 1H NMR chemical shift, self-diffusion coefficient, and 13C NMR spinlattice relaxation time for the oleic acid samples containing a small amount of additive such as cholesterol, cholestanol, cholestane, cholesteryl oleate, ethanol, or benzene. Oleic acid forms a complex with cholesterol as well as with ethanol. On the basis of these complex formations and the existence of the clusters composed of oleic acid dimers, we have revealed the role and the functional mechanism of cholesterol to the intermolecular and the intramolecular movements of oleic acid in the liquid state. Acknowledgment. The authors express their thanks to Mr. Kazuki Ishihara, Miss Haruna Matsuda, and Mr. Yoshihiro Tomizawa for their assistance with the IR and NMR measurements. References and Notes (1) Gennis, R. B. Biomenbranes: Molecular Structure and Function; Springer-Verlag: New York, 1989; Chapter 1. (2) Myant, N. B. The Biology of Cholesterol and Related Sterols; William Heinemann Medical Books Ltd.: London, UK, 1981; p 324 . (3) Seoane, R.; Minones, J.; Conde, O.; Minones, J., Jr.; Casas, M.; Eribarnegaray, E. J. Phys. Chem. B 2000, 104, 7735. (4) Krummel, D. In Krause’s Food, Nutrition and Diet Therapy; Mahan, K., Escott-Stump, S., Eds.; Saunders Co.: Philadelphia, PA, 1996; pp 526-528. (5) Grundy, S. M. Am. J. Clin. Nutr. 1994, 60, 9865. (6) Segura, R. In Nutricion y Dietion; Consejo General de Colegious Farmaceuticos, Vol. 2; Madrid, Spain, 1993; Chapter 17, p 583. (7) Abrahamsson, S.; Ryderstedt-Nahringbauer, I. Acta Crystallogr. 1962, 15, 1261. (8) Koyama, T.; Ikeda, K. Chem. Phys. Lipids 1980, 26, 149. (9) Sato, K.; Biostelle, R. J. Cryst. Growth 1984, 66, 44.

Effects of Additives on Oleic Acid (10) Suzuki, M.; Ogaki, T.; Sato, K. J. Am. Oil Chem. Soc. 1985, 62, 1600. (11) Kobayashi, M.; Kaneko, F.; Sato, K.; Suzuki, M. J. Phys. Chem. 1986 90, 6378. (12) Hiramatsu, N.; Inoue, T.; Suzuki, M.; Sato, K. Chem. Phys. Lipids 1989, 51, 47. (13) Iwahashi, M.; Suzuki, M.; Czarnecki, M. A.; Liu, Y.; Ozaki, Y. J. Chem. Soc., Faraday Trans. 1995, 91, 697. (14) Iwahashi, M.; Yamaguchi, Y.; Kato, T.; Horiuchi, T.; Sakurai, I.; Suzuki, M. J. Phys. Chem. 1991, 95, 445. (15) Iwahashi, M.; Kasahara, Y.; Matsuzawa, H.; Yagi, K.; Nomura, K.; Terauchi, H.; Ozaki, Y.; Suzuki, M. J. Phys. Chem. B 2000, 104, 6186. (16) Iwahashi, M.; Takebayashi, S.; Umehara, A.; Kasahara, Y.; Minami, H.; Matsuzawa, H.; Inoue, T.; Takahashi, H. Chem. Phys. Lipids 2004, 129, 195. (17) Iwahashi, M.; Takebayashi, S.; Taguchi, M.; Kasahara, Y.; Minami, H.; Matsuzawa, H. Chem. Phys. Lipids 2005, 133, 113-124. (18) Small, D. M. The Physical Chemistry of Lipids; Plenum Press: New York, 1986; Chapters 6 and 11. (19) Iwahashi, M.; Minami, H.; Suzuki, T.; Koyanagi, M.; Yao, H.; Ema, K.; Ashizawa, K. J. Oleo Sci. 2001, 50, 693. (20) Iwahashi, M.; Minami, H.; Yamauchi, Y.; Iimura, K.; Kato, T. J. Oleo Sci. 2001, 50, 787. (21) Boldrini, P. Physiol. Chem. Phys. 1979, 11, 303. (22) Farrar, T. C.; Becker, E. D. Pulse and Fourier Transform NMR; Academic Press: New York, 1971; Chapter 2. (23) Hertz, H. G. Nucl. Magn. Reson. Spectrosc. 1967, 3, 159. (24) Abraham, R. J.; Toftus, P. Proton and Carbon-13C NMR Spectroscopy; Heyden & Son Limited: 1978; Chapter 6. (25) Liddel, U.; Becker, E. D. Spectrochim. Acta 1957, 10, 70.

J. Phys. Chem. B, Vol. 111, No. 4, 2007 747 (26) Coburn, W. C., Jr.; Grunwald, E. J. Am. Chem. Soc. 1958, 80, 1318. (27) Parker, F. S.; Bhaskar, K. R. Biochemistry 1968, 7, 1286. (28) Harris, D. C.; Bertolucci, M. D. Symmetry and Spectroscopy-An Introduction to Vibration and Electronic Spectroscopy; Dover Publications, Inc.: New York, 1978 p 210. (29) Mercler, P.; Sandorfy, C.; Vocelle, D. J. Phys. Chem. 1983, 87, 3670. (30) Vinogradov, S. N.; Linnell, R. H. Hydrogen Bonding; van Nostrand Reinhold Co.: New York, 1971. (31) George, W. O.; Mcintyre, P. S. Infrared Spectroscopy; John Wiley & Sons: New York, 1987; p 335. (32) Colthup, N. B.; Daly, L. H.; Wiberley, S. E. Introduction to Infrared and Raman Spectroscopy; Academic Press Inc.: New York, 1990; p 291. (33) Nakanishi, K.; Solomon, P. H.; Furutachi, N. Infrared Spectroscopy; Nankoudou: Tokyo, Japan, 1978; p 129. (34) Pata, S. The chemistry of carboxylic acids and esters; IntersciencePublisher: London, UK, 1969; p 238. (35) Pimentel, G. C.; McClellan, A. C. The hydrogen bond; W. H. Freeman: San Francisco, CA, 1960; p 59. (36) Izailov, N. A.; Kremer, V. A.; Kutsyna, L. M.; Titov, E. V. Chem. Abstr. 1960, 54, 23629f. Naumova, A. S.; Smorodirov, V. S. Chem. Abstr. 1964, 61, 12693e. (37) Mandel, M.; Decroly, P. Nature 1964, 201, 290. (38) Dawson, L. R.; Vaughan, J. W.; Pruitt, M. E.; Eckstrom, H. C. J. Phys. Chem. 1962, 66, 2684. (39) Hadzi, D.; Kopilarov, N. J. Chem. Soc. A 1966, 439. (40) Reever, L. B. Trans. Faraday Soc. 1959, 55 1684. (41) Hashimoto, T. A Handbook of Oil and Fats Chemistry; Maruzen Inc.: Tokyo, Japan, 1990; Chapter 3.