Solubilization of aromatics in aqueous bile salts. I. benzene and alkylbenzenes in sodium cholate: 1H NMR study

Solubilization of Aromatics in Aqueous Bile Salts I. Benzene and Alkylbenzenes in Sodium Cholate: 1H NMR Study

were also analytical grade reagents and were used without further purification. D20 for spectroscopy was used as such. All N M R samples were from a stock solution prepared by weighing 409 mg of cholic acid in 10 ml of 0.1 M sodium phosphate in D20. The cholic acid dissolved readily and the solution formed was alkaline enough (pH 9.5 uncorrected for isotopic effects) to assure that all the acid was as its sodium salt. All solubilizates were added by 10-t~l microsyringe directly to the N M R tube and all samples were sonicated for 5 min before the measurements were done in order to equilibrate the system. All measurements were performed twice in samples prepared separately. The N M R spectra were recorded at 25°C on a JEOL FX-60 N M R spectrometer. The spectral width was 600 Hz, flip angle 45 °, and pulse repetition time 5 s in every run. The number of data points was 16K and 500 scans were accumulated for every spectrum. TMS (2%) in CC14 in sealed 1-mm-diameter capillary tube was used as an external standard.

INTRODUCTION Bile salts and their conjugates are shown to be very important biological surfactants through the formation of comicellar aggregates with lecithin, cholesterol, and other lipidic substances (1). As a model system, micellar solubilization can provide considerable insight into the nature of the interactions of small molecules with other lipid assemblies such as bilayers and biological membranes, which are responsible for the binding and/or uptake of the smaller molecules, their transport, and their chemical and metabolic reactivities (2). Bile salts are specially interesting in that respect due to their structural resemblance and biosynthetical connection to cholesterol. To measure the proton N M R chemical shifts is a wellestablished technique for the solubilization studies using the aromatic solubilizate-induced ring current shifts (socalled ASIS-effects) as an experimental evidence for comicellar aggregation (3). Micellization and solubilization in bile salts have been studied extensively by many different methods including N M R spectroscopy (4-19). In spite of the enormous work done with aqueous biliary lipid systems (e.g., Small (1), Mazer et al. (20, 21), and Holzbach et al. (22)) much less interest has been directed to the solubilization of aromatic compounds in aqueous bile salts (6, 10, 15, 16, 23-29) and no systematic approaches are available, for example, in respect to the effect of the various molecular properties of the solubilizate vs the rate and the extent of the solubilization. For that reason it was concluded that the ~H N M R chemical shift changes induced by the well-known ASIS-effects could be used as a simple and convenient method for solubilization studies in the aqueous bile salts. EXPERIMENTAL Cholic acid ( 3 a , 7 a , 1 2 a - t r i h y d r o x y - 5 f l - c h o l a n i c acid), from Merck AG, was recrystallized twice from aqueous ethanol and dried in v a c u o for several days. The melting point measured in open capillary tube was 197-198°C (lit. value 199°C; p. 23 in Ref. (1)) and TLC on silica gel G performed according to Gatmaitan et al. (30) did not show any impurity spots. Sodium phosphate, Na3PO4.12 H20, used as buffer was analytical grade reagent and was used as such. All organic compounds

RESULTS AND DISCUSSION According to the results published by Mukerjee and Cardinal (29) the association of sodium cholate is a complexed pattern including the formation of eholate dimers and one or more highe r oligomers. On the other hand, based on the articles given by Ekwall et al. (23c), Small et al. (31, 32), and Mazer et al. (33) trihydroxy bile salt micelles are, however, resistant to change in the counterion concentration, temperature, and added urea. Furthermore, Fontell (25) has reported that the association of sodium cholate is complete at 0.1 M aqueous solution. For these reasons 0.1 M sodium cholate buffered with 0.1 M sodium phosphate was chosen as a model surfactant system in this solubilization study. In Fig. 1 is described the structures of four common bile acids and the notation o f the steroid carbon skeleton. In Fig. 2 is shown the ~H N M R spectrum of 0.1 M sodium cholate in 0.1 M sodium phosphate/D20 solution and the effect of the amount of the added benzene. The assignation of the signals is done according to Small et al. (4) and Barnes and Geckle (19). Although the latter were able to assign all the proton signals in the 400MHz spectrum of cholic acid, the present 60-MHz spectrum did resolve only the two angular methyls 18 and 19 and the side-chain methyl 21 as well as the

273

Journal of Colloid and Interface Science, Vol. 105, No. 1, May 1985

0021-9797/85 $3.00 Copyright© 1985by AcademicPress,Inc. All fightsof reproductionin any form reserved.

274

NOTES

t-w

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OH

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H

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DEOXYCHOLIC ACID

OH

H

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CHOLIC ACID

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LITHOCHOLIC

ACID

CHENODEOXYCHOLIC

ACID

FIG. 1. Structures and notation for four human bile acids.

methine protons epimeric to the hydroxyls in the positions 3, 7, and 12. The other protons of the steroid skeleton gave rise only to an unresolved broad pattern and it was impractical in this study. In Fig. 3 are plotted the chemical shifts of the angular methyls of sodium cholate as a function of the added sohibilizate for benzene, toluene, ethylbenzene, n-propylbenzene, and n-butylbenzene. The other alkylbenzenes studied were p-xylene and tert-butylbenzene. Cyclohexane was used as a nonaromatic reference compound.

BENZENE

The chemical shifts of the angular methyls depend, as can be seen in Fig. 3, at the beginning quite strongly on the amount of the solubilizate added, but stay nearly constant after that. Adding more solubilizate leads to the formation of the separate organic phase, which tends to ascend as droplets and later as a layer on the surface of the aqueous solution. As a general rule the effect experienced by methyl 19 was much greater than by the other angular methyl 18. On the other hand, the maximum shift is achieved at lower molar ratios in the case of higher homologs in series of alkylbenzenes. The side-chain methyl 21 did not show remarkable sensitivity to any solubilizate tested in this study. In the case of cyclohexane both angular methyls experienced roughly the same effect, ca. 2 Hz, in the magnitude, differing by this way from all aromatics studied. The inflection points in the chemical shift vs molar ratio curves (see Fig. 3), which cannot be estimated easily by extrapolating, are calculated as a side of a triangle whose height is the maximum change and its area is obtained by integrating the chemical shift at every point subtracted with its final value from zero to the saturation molar ratio. In cases where the estimation of the inflection points by extrapolating was possible (ethylbenzene and higher homologs) the integral method gave the same results as the extrapolation. In Table I are collected the calculated inflection point values for the solubilizate/cholate saturation molar ratios. As can be seen, a general feature is the diminishing trend in the inflection point molar ratios as a function o f increasing molar volume of the solubilizate. Markina et al. (24)

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FIG. 2. The effect of the amount of the added benzene on the 60-MHz IH N M R spectrum of 0.1 M sodium cholate in D20 buffered with 0.1 M sodium phosphate. Journal of Colloid and Interface Science, Vol. 105,No. I, May 1985

NOTES

275

55-~ Hz

540

BENZENE TOLUENE

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FIG. 3. The dependence of the ~H NMR chemical shifts of the angular methyls 18 and 19 of 0.1 M sodium cholate in D20 buffered with 0.1 M sodium phosphate on the added solubilizate: benzene, toluene, ethylbenzene, n-propylbenzene, and n-butylbenzene. have also reported this effect: in 0.1 M sodium cholate for p-xylene, benzene, cyclohexane, tetradecane, and dodecane the solubilization limit molar ratios were 0.33, 0.38, 0.32, 0.04, and 0.08, respectively. According to their study cyclohexane is solubilized nearly to the same extent as benzene and is thus suitable for reference use. In the present study there exists a very great difference when compared with the value obtained by Markina et al. in the case of benzene. However, cyclohexane-induced effect gave the value in agreement with the previous data. The other literature data for quantitative estimation

of solubilization molar ratio are based on t3C NMR measurements by Leibfritz and Roberts (6). They reported that sodium deoxycholate can take up aromatic hydrocarbons, p-xylene and 2-methylnaphtalene, in about 1:2 solubilizate/surfactant molar ratio. They did not observe, however, any effect due to the molar volume of the solubilizate on the extent of the solubilization, but the rate of molecular tumbling was reduced in the case of bicyelic compound. On the other hand, Fung and Thomas (10) have found, by using the integrated proton intensities, the values for solubilization molar ratios: 0.5 and 0.1 for Journal of Colloid and Interface Science, Vol. 105,No. 1, May 1985

276

NOTES TABLE I

Estimated Solubilization Limits for Benzene, Toluene, Ethylbenzene, p-Xylene, n-Propylbenzene, n-Butylbenzene, and tert-Butylbenzene in 0.1 M Sodium Cholate/D20 Buffered by 0.1 M Sodium Phosphate Based on the IH NMR Chemical Shift Changes of the Angular Methyls of Sodium Cholate Solubilizationlimits Methyl 19 Compound Benzene Toluene Ethylbenzene p-Xylene n-Propylbenzene n-Butylbenzene tert-Butylbenzene

Methyl 18

Molecular weight

Molarvol. (ml)

Densitya

Volume, ~1

Molarratio

Volume,~1

Molarratio

78.12 92.15 106.17 106.17 120.20 134.22 134.22

89.1 106.3 122.5 123.3 139.4 156.1 154.8

0.877 0.867 0.867 0.861 0.862 0.860 0.867

6.2 2.4 2.3 1.8 1.7 1.2 1.3

1.4 0.5 0.4 0.3 0.2 0.2 0.2

7.4 3.3 2.4 2.5 2.0 1.2 1.5

1.7 0.6 0.4 0.4 0.3 0.2 0.2

a Values taken from Handbook of Chemistry and Physics 56th Edition (R. C. Weast, Ed.), CRC Press, Boca Raton, Fla., 1975.

benzene and naphtalene, respectively. The diminishing extent in the solubilization limits with increasing molar volume observed in this study as well as by other authors is consistent with the theory for the influence of t h e Laplacian pressure inside the micelles proposed in the work of Mukerjee (2). The difference in the behavior between the angular methyls must be explained by the structural characterization of the mixed micelles. According to Small (4, 31, 32) the initial stage of the bile salt aggregation process involves the formation of globular primary micelles, in which the constituent molecules have their hydrophobic surfaces facing the interior of the micelle and their hydrophilic surfaces in contact with the solvent and in which it is possible to fit up to 10 bile salt molecules together using Stuart-Briegleb space-filling models so that the hydrophobic parts are in contact. In the case of lecithin-bile salt-mixed micelles those aggregates are disk-like in shape consisting of lecithin bilayers surrounded by a girdle of bile salt molecules. Mazer et aL (33-35) have proposed a more detailed double-disk model for the aqueous biliary lipid-mixed micelle including bile salt, lecithin, and cholesterol. In this model there can exist cholate dimers inside the disks, too. Anyhow, in all these models the methyl 19 is closer to the hydrophobic center of the dimer than the methyl 18 because the hydrophilic carboxylate tails are directed to the surrounding aqueous solution. It is now expected that the hydrophobic aromatic solubilizate will be able to penetrate into the hydrophobic interior of primary micelle and tend to solubilize closer to the methyl 19 than 18. Due to steric effects the motion of the solubilizate is restricted to some extent and this causes an anisotropic shift in the angular methyl 19 differing from the effect induced by cyclohexane. On the other hand, more polar aromatic compounds such as o-dimethoxybenzene (verJournal of Colloid and Interface Science, Vol. 105,No. 1, May 1985

atrole) induce roughly the same effect (10 Hz) to both angular methyls differing from those in alkylbenzenes. This phenomenon may be linked to the different solubilization sites inside the micelles compared with alkylbenzenes. Some preliminary experiments with polycyclic solid aromatics such as naphtalene, biphenyl, anthracene, phenanthrene, and triphenylmethane show also a different kind of behavior. The maximum shifts experienced by the methyl 19 were much smaller than in the case of monoaromatics, being ca. 2 Hz, and the methyl 18 remained unchanged. The dilution of the benzene/cholate system at the fixed molar ratio of 1.5 by the buffer solution diminished the observed effects linearly as a function of the cholate (or solubilizate) concentration. At 0.01 M the effect was negligible. This behavior implies that there exists a dynamic equilibrium between the different micellar species or between the micellar and aqueous phase or that there are some changes in the distribution of micellar species and/or in the capacity for solubilization due to other reasons. These effects are, however, difficult to differentiate by IH NMR at 60 MHz, because there exist, according to Barnes and Geckle (19), small concentration effects for pure cholate solutions, too. As a whole the chemical shift changes form an interesting alternative to a study of the solubilization behavior of aromatic compounds. For quantitative purposes much work is needed. Unfortunately there exist very little reference data measured by other methods. However, the results obtained by the inflection point method are roughly in agreement with the theory for the Laplacian pressure effect and for higher homologs in series of alkylbenzenes the results are reasonable. The most promising aspect is the possibility to determine the solubilization sites depending on the structure of the solub'flizate. The possibilities of the high-field measurements can give

NOTES more detailed information accompanied with the concentration changes and the solubilization. Other magnetic nuclei such as 31p and ~gF with the greater dispersion of the NMR chemical shift can give also more accurate insight into these phenomena. These experiments are now in progress. ACKNOWLEDGMENTS Fruitful discussions with Professor I. Danielsson and Dr. J. B. Rosenholm, Abo Akademi, are gratefully acknowledged. REFERENCES 1. Small, D. M., in "The Bile Acids" (P. P. Nair and D. Kritchevsky, Eds.), p. 249. Plenum, New York, 1971. 2. Mukerjee, P., Pure, AppL Chem. 52, 1317 (1980). 3. Eriksson, J. C., and Gillberg, G., Acta Chem. Scand 20, 2019 (1966). 4. Small, D. M., Penkett, S. A., and Chapman, D., Biochem. Biophys. Acta 176, 178 (1969). 5. Martis, L., Hall, N. A., and Thakkar, A. L., J. Pharm. Sci. 61, 1757 (1972). 6. Leibfritz, D., and Roberts, J. D., J. Amer. Chem. Soc. 95, 4996 (1973). 7. Fung, B. M., and Peden, M. C., Biochem. Biophys. Acta 437, 273 (1976). 8. Arvidson, G., Fontell, K., Johansson, L. B.-A., Lindblom, G., Ulmius, J., and Wennerstr6m, H., Ber. Bunsenges. Phys. Chem. 82, 977 (1978). 9. Smith, W. B., J. Phys. Chem. 82, 234 (1978). 10. Fung, B. M., and Thomas, L., Jr., Chem. Phys. Lipids 25, 141 (1979). 11. Castellino, F. J., and Violand, B. N., Arch. Biochem. Biophys. 193, 543 (1979). 12. Lichtenberg, D., and Zilberman, Y., J. Magn. Reson. 34, 491 (1979). 13. Lichtenberg, D., Zilberman, Y., Greenzaid, P., and Zamir, S., Biochemistry 18, 3517 (1979). 14. Ranajit, P., Mathew, M. K., Narayanan, R., and Balaram, P., Chem. Phys. Lipids 25, 345 (1979). 15. Barnard, G. D., thesis for Ph.D., available by Univ. Microfilms, Int. Order No. 8021813. Texas Christian University, 1980. 16. Smith, W. B., and Barnard, G. D., Canad. J. Chem. 59, 1602 (1981). 17. Murata, Y., Sugihara, G., Fukushima, K., and Tanaka, M., J. Phys. Chem. 86, 4690 (1982). 18. Miyazaki, S., Yamahira, T., Morimoto, Y., and Nadai, T., Int. J. Pharm. 8, 303 (1981). 19. Barnes, S., and Geckle, J. M., J. LipidRes. 23, 161 (1982). 20. Mazer, N. A., Kwasnick, R. F., Carey, M. C., and Benedek, G. B., in "Micellization, Solubilization, and Microemulsions" (K. L. Mittal, Ed.), Vol. 1, p. 383. Plenum, New York, 1977.

277

21. Mazer, N. A., Carey, M. C., and Benedek, G. B., in "Solution Behavior of Surfactants" Theor. Appl. Aspects (Proc. Int. Symp., 1980)." (K. L. Mittal and E. J. Fendler, Eds.), p. 595. Plenum, New York, 1982. 22. Holzbach, R. T., Oh, S. Y., McDonnell, M. E., and Jamieson, A. M., in "Micellization, Solubilization, and Microemulsions'" (K. L. Mittal, Ed.), Vol. 1, p. 403. Plenum, New York, 1977. 23. (a) Ekwall, P., Acta Acad. Abo. Ser. B 17, 8 (1951); (b) Ekwall, P., "Intern. Colloq. Biochem. Probl. Lipiden, Briissels, 1953," p. 103. Koninkl. Vlaamse Acad. van Belgie, Wetenschappen, Letteren, Schone Kunsten, Klasse Wetenschappen. Briissels, 1954; (c) Ekwall, P., Fontell, K., and Sten, A., "Proceedings, 2nd International Congress Surface Activity," Vol. 1, p. 357. London, 1957. 24. (a) Markina, Z. N., Tsikurina, N. N., and Rehbinder, P. A., Dokl. Acad. Nauk 172, 1376 (1967); (b) Tsikurina, N. N., Markina, Z. N., Chirova, G. A., and Rehbinder, P. A., Kolloidn. Zh. 30, 296 (1968), English translation, p. 219. 25. Fontell, K., Kolloid. Z. Z. Polym. 250, 333 (1972). 26. Fisher, L., and Oakenfull, D., Aust. J. Chem. 32, 31 (1979). 27. Thomas, D. C., and Christian, S. D., J. Colloid. Interface Sci. 82, 430 (1981). 28. Christian, S. D., Smith, L. S., Bushong, D. S., and Tucker, E. E., J. Colloid Interface Sci. 89, 514 (1982). 29. Mukerjee, P., and Cardinal, J. R., J. Pharm. Sci. 65, 882 (1976). 30. Gatmaitan, O. G., Yotsuyanagi, T., and Higuchi, W. J., J. Colloid Interface Sci. 61, 499 (1977). 31. Small, D. M., Adv. Chem. Ser. 84, 31 (1968). 32. Carey, M. C., and Small, D. M., Arch. Int. Med. 130, 506 (1972). 33. Mazer, N. A., Carey, M. C., Kwasnick, R. F., and Benedek, G, B., Biochemistry 18, 3064 (1979). 34. Carey, M. C., Montet, J.-C., Philips, M. C., Amstrong, M. J., and Mazer, N. A., Biochemistry 20, 3637 (1981). 35. Mazer, N. A., and Carey, M. C., Biochemistry 22, 426 (1983). 36. Cardinal, J. R., and Mukerjee, P., J. Phys. Chem. 82, 1614 (1978). 37. Mukerjee, P., and Cardinal, J. R., J. Phys. Chem. 82, 1620 (1978). ERKKI KOLEHMA1NEN Department of Chemistry University of Kuopio P.O. Box 6 SF-70211 Kuopio 21 Finland Received November 21, 1983 Journalof Colloidand InterfaceScience,Vol. 105,No. 1, May 1985