Langmuir 2003, 19, 1319-1323
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Diffusivity Study of Dihydroxy-Trihydroxy Bile Salt Systems Luciano Galantini,* Edoardo Giglio, Nicolae Viorel Pavel, and Francesco Punzo Dipartimento di Chimica, Universita` di Roma “La Sapienza”, P.le Aldo Moro 5, 00185 Roma, Italy Received July 12, 2002. In Final Form: November 13, 2002 Previously, structural units inferred from X-ray diffraction analysis of fibers, crystals, and concentrated aqueous solutions were identified as models of dihydroxy and trihydroxy bile salt micellar aggregates. Dimers and their multiples (mainly octamers and hexadecamers) or structures derived from 7/1 helices (formed by trimers) were proposed for trihydroxy salts sodium glycocholate (NaGC) and taurocholate (NaTC) or for dihydroxy salts sodium glycodeoxycholate (NaGDC) and taurodeoxycholate (NaTDC), respectively, on the basis of strong experimental evidences. However, the radii of the cylindrical structures derived from 7/1 helices were unknown. This paper deals with small-angle X-ray scattering (SAXS) measurements that have permitted to estimate the cylindrical aggregate radius of NaGDC (about 22 Å), whereas that of NaTDC is very probably about 3 Å longer. SAXS and quasi-elastic light-scattering (QELS) data have shown a linear increase of the apparent mean hydrodynamic radius (within the approximate range 40-70 Å) with the height of NaGDC and NaTDC cylindrical aggregates, suggesting that the main interparticle correlation effects, ascribed to charge, van der Waals, and volume excluded interactions, are small or compensate each other. Moreover, aqueous solutions containing one dihydroxy and one trihydroxy bile salt (NaGDC and NaGC, NaTDC and NaTC, NaGDC and NaTC, NaTDC and NaGC) and 0.8 M NaCl have been studied. QELS measurements give information on the growing processes of NaGDC and NaTDC aggregates as a function of NaGC and NaTC concentration. Experimental data and calculations performed using the above-mentioned models support that a small amount of the bile salt forming smaller size aggregates (trihydroxy salt) inhibits the growth of the bile salt forming bigger size aggregates (dihydroxy salt), giving rise to smaller size aggregates. Reasonably, this phenomenon is due to the very different and not interchangeable structures of dihydroxy and trihydroxy bile salt aggregates. It seems also that the aggregate growth is a little more inhibited when the trihydroxy molecule polar head is different from that of the aggregate molecules. Trihydroxy salt monomers and oligomers compete with dihydroxy salt trimers in the growth process and interact with their cylindrical aggregates especially by means of polar forces.
Introduction Bile salts relevant to this study are the sodium salts of 3R,12R-dihydroxy-5β-cholanoylglycine (sodium glycodeoxycholate, NaGDC), 3R,12R-dihydroxy-5β-cholanoyltaurine (sodium taurodeoxycholate, NaTDC), 3R,7R,12Rtrihydroxy-5β-cholanoylglycine (sodium glycocholate, NaGC), and 3R,7R,12R-trihydroxy-5β-cholanoyltaurine (sodium taurocholate, NaTC). They give rise to transitions [aqueous micellar solution] f [gel] f [fiber] and, sometimes, [fiber] f [crystal]. Models with well-established geometry were inferred from crystal1-4 and fiber5-7 structures, used to represent micellar aggregates, and then verified in the study of aqueous micellar solutions. Of course, fiber models should be more reliable than crystal ones, the fiber being more similar than the crystal to the aqueous micellar solution. * Corresponding author. Telephone: (+int.)-6-49913687. Fax: (+int.)-6-490631. E-mail:
[email protected]. (1) Campanelli, A. R.; Candeloro De Sanctis, S.; Giglio, E.; Scaramuzza, L. J. Lipid Res. 1987, 28, 483. (2) Campanelli, A. R.; Candeloro De Sanctis, S.; Chiessi, E.; D’Alagni, M.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1989, 93, 1536. (3) Campanelli, A. R.; Candeloro De Sanctis, S.; Galantini, L.; Giglio, E.; Scaramuzza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1991, 10, 367. (4) D’Alagni, M.; Galantini, L.; Giglio, E.; Gavuzzo, E.; Scaramuzza L. Trans. Faraday Soc. 1994, 90, 1523. (5) Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1996, 12, 1180. (6) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. Langmuir 2000, 16, 10436. (7) Bottari, E.; D’Archivio, A. A.; Festa, M. R.; Galantini, L.; Giglio, E. Langmuir 1999, 15, 2996.
As a matter of fact, [aqueous micellar solution] f [gel] f [fiber] transitions could take place with moderate structural changes because the gel is obtained by concentrating the aqueous micellar solution and the fiber is drawn from the gel by means of the weak force of gravity. NaTDC and NaGDC fibers are formed by 7/1 clockwise helices5 stabilized by strong polar interactions involving especially the cations as deduced mainly from electrolytic conductance and dielectric measurements.6,8 A trimer, constituted by three NaTDC or NaGDC molecules related by a 3-fold rotation axis, is the repetitive unit of the 7/1 helix. In accordance with the above model, on one hand NaTDC micellar aggregation numbers, obtained from electromotive force (EMF) measurements as a function of ionic strength, pH, and bile salt concentration, are generally multiples of three,9 on the other NaGDC10 and NaTDC9 show always the presence of trimers, even at the highest ionic strengths. Moreover, the phase behavior of the NaTDC- and NaTC-water systems was lately studied as a function of concentration and temperature with emphasis on concentrated regions beyond the isotropic solution phase.11,12 It was assumed that, beyond the micellar solution, anisotropic liquid crystals, probably of the reverse type, were formed. A phase diagram with a liquid crystalline region of hexagonal-like structure was (8) Bonincontro, A.; D’Archivio, A. A.; Galantini, L.; Giglio, E.; Punzo, F. J. Phys. Chem. B 1999, 103, 4986. (9) Bottari, E.; Festa, M. R. Langmuir 1996, 12, 1777. (10) Bottari, E.; Festa, M. R. Monatsh. Chem. 1993, 124, 1119. (11) Edlund, H.; Khan, A.; La Mesa, C. Langmuir 1998, 14, 3691. (12) Marques, E. F.; Edlund, H.; La Mesa, C.; Khan, A. Langmuir 2000, 16, 5186.
10.1021/la026232o CCC: $25.00 © 2003 American Chemical Society Published on Web 01/24/2003
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reported on the basis of optical textures as well as of 2H NMR and small-angle X-ray scattering (SAXS) data.11,12 More recently, SAXS and wide-angle X-ray measurements were accomplished on NaTDC concentrated aqueous samples and fibrils that grow from these samples.13 Experiments strongly supported that the NaTDC aggregates are helices formed by trimers both in concentrated aqueous samples and in fibrils. Fibrils are composed by the same 7/1 helices of the fiber, whereas concentrated aqueous samples contain helices with a larger cross section and a shorter identity period along the helical axis than the 7/1 helices. Passing from the fiber to the isotropic solution phase the helical radius seems to increase, according to previous SAXS measurements,14 and the trimer repeat along the helical axis decreases, so that the trimer path from solid to liquid phase resembles the opening of an umbrella.13 Very probably NaGDC, that shows structural properties similar to those of NaTDC, behaves likewise. NaGC and NaTC trihydroxy salts behave in a different way than dihydroxy ones (NaGDC and NaTDC). Their crystals and fibers are formed by very stable dimers and octamers (assembly of four dimers)3,4,6,7 that are present in NaGC15 and NaTC7,16 electrolyte aqueous solutions as confirmed by EMF measurements. Micellar aggregation numbers are always multiples of two, and octamers and hexadecamers (very probably formed by two octamers) prevail in the sample with the highest concentration and ionic strength. As a matter of fact, NaTC SAXS patterns12 can be better interpreted when its monoclinic crystal a and c unit cell constants but a greater β angle are assumed.4,13 This suggests that the NaTC and, very likely, NaGC paths from the crystal and fiber to the isotropic solution phase should occur with negligible structural changes because of the dimer and octamer stiffness, at variance with the NaTDC and, very probably, NaGDC trimer behavior. The biggest NaGC and NaTC aggregates could be formed by piling octamers as in the crystal and fiber structures. Bile salt micellar aggregates play a pivotal role in hepatobiliary equilibria and allow the transport of relevant biomedical compounds. However, bile salt properties, such as forming interaction complexes with important biological compounds (for instance bilirubin-IXR), depend on the size and structure of their micellar aggregates.17-19 Since several bile salts are together present in biological fluids, the properties of each bile salt can be influenced by the others. The aim of this paper is to study as first step two bile salt systems and to establish the role of a bile salt on the growing process of another one. Aqueous electrolyte (0.8 M NaCl) solutions containing NaGDC-NaGC, NaTDC-NaTC, NaGDC-NaTC, and NaTDC-NaGC have been investigated by quasi-elastic light-scattering (QELS) measurements. The high NaCl concentration ensures greater changes in the apparent mean hydrodynamic radius (Rh) of a bile salt when a second bile salt is added. Calculations based on the above-mentioned bile salt (13) Galantini, L.; Giglio, E.; La Mesa, C.; Pavel, N. V.; Punzo, F. Langmuir 2002, 18, 2812. (14) Matsuoka, H.; Kratohvil, J. P.; Ise, N. J. Colloid Interface Sci. 1987, 118, 387. (15) Bottari, E.; Festa, M. R.; Franco, M. Langmuir 2002, 18, 2337. (16) Bottari, E.; Festa, M. R.; Franco, M. Analyst 1999, 124, 887. (17) D’Alagni, M.; D’Archivio, A. A.; Giglio, E.; Scaramuzza, L. J. Phys. Chem. 1994, 98, 343. (18) D’Alagni, M.; D’Archivio, A. A.; Galantini, L.; Giglio, E. Langmuir 1997, 13, 5811. (19) Bonincontro, A.; Briganti, G.; D’Archivio, A. A.; Galantini, L.; Giglio, E. J. Phys. Chem. B 1997, 101, 10303.
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models have been accomplished to clarify the growth mechanism. Experimental Section Materials. NaGDC, NaTDC, NaGC, and NaTC (Sigma) were used as received. Commercial powders were heated at 50 °C under a pressure of 10-2 Torr for 1 day before preparing the aqueous electrolyte solutions. NaCl (Merck, suprapur) was used. QELS Measurements. A Brookhaven instrument constituted by a BI-2030AT digital correlator with 136 channels and a BI200SM goniometer was used. The light source was a Uniphase solid-state laser system model 4601 operating at 532 nm. Dust was eliminated by means of a Brookhaven ultrafiltration unit (BIUU1) for flow-through cells, the volume of the flow cell being about 1.0 cm3. Nuclepore filters with a pore size of 0.1 µm were used. Samples were placed in the cell for at least 30 min prior to measurement to allow for thermal equilibration. Their temperature was kept constant at 25 °C within 0.5 °C by a circulating water bath. Measurements were repeated after 24 and 48 h to be sure that data were reproducible. The timedependent light scattering correlation function was analyzed at a fixed angle of 90°. Apparent diffusion coefficients did not depend on the exchanged wave vector in the range 30-150° in our experimental conditions. Scattering decays were analyzed by means of cumulant expansion up to second order because higher order contributions did not improve the statistics. SAXS Measurements. SAXS measurements were carried out on NaGDC aqueous solutions (0.1 M) containing NaCl from 0 to 0.7 M (increment of 0.1 M) by using a Kratky compact system equipped with a NaI scintillation counter and a Ni-filtered Cu KR radiation (λ ) 1.5418 Å). Measurements were done in a temperature-controlled room at 25 ( 1 °C. Scattering curves were recorded within the range 0.012 e k e 0.5 Å-1 (k ) 4π sin θ/λ) and the scattering intensity was put on an absolute scale. Experimental data were handled by the method of the indirect Fourier transformation.20,21
Results and Discussion Geometry of Dihydroxy and Trihydroxy Bile Salt Aggregates. Models with well-defined geometry are necessary for NaGDC, NaTDC, NaGC, and NaTC aggregates in order to decide about their growing mechanism in a dihydroxy-trihydroxy bile salt system (see QELS results below). Very probably, NaTDC isotropic solution phase contains cylindrical aggregates with a trimer repeat of 3.6 Å and a radius longer than the fiber radius (10.2 Å) and that inferred from concentrated (40.0 wt %) aqueous samples (15.85 Å).13 NaGDC has a fiber structure almost equal to that of NaTDC5 and shows structural properties similar to those of NaTDC. Therefore, NaGDC isotropic solution phase should contain cylindrical aggregates very similar to those of NaTDC. This hypothesis has been confirmed by means of SAXS measurements accomplished on NaGDC aqueous solutions (0.1 M) containing NaCl from 0 to 0.7 M (increment of 0.1 M). The scattered intensity I(k) and the electron pair distance distribution function p(r) can be calculated for a model of known geometry and compared with the observed ones.22 The p(r) function strongly depends on the shape and size of the scattering particles and vanishes at the maximum electron pair distance (DM) within the particles. A very good agreement has been obtained both for I(k) and p(r) using as model 21 helices formed by trimers constituted by three NaGDC molecules related by a 3-fold rotation axis, coinciding with the 21 helical axis. The trimer repeat along the helical axis is 3.6 Å as in NaTDC concentrated aqueous samples13 and the helical radius is 21.7 Å, much longer than that (20) Glatter, O. J. Appl. Crystallogr. 1974, 7, 147. (21) Glatter, O. J. Appl. Crystallogr. 1977, 10, 415. (22) Glatter, O. Acta Phys. Austriaca 1980, 52, 243.
Study of Dihydroxy-Trihydroxy Bile Salt Systems
of the 7/1 helix in the NaGDC fiber (10.1 Å). The NaGDC anion has a nearly fully extended side chain and its long axis makes an angle of about 60° with the helical axis, which has the carboxylate group as the closest one. The SAXS study of NaTDC aqueous solutions (0.1 M) containing NaCl from 0 to 0.8 M (increment of 0.1 M) is in progress. On the basis of preliminary data, cylindrical aggregates similar to those of NaGDC are present. Also, the NaTDC trimer repeat is consistent with the value of 3.6 Å, but the cylindrical radius is not yet determined. However, it seems to be longer than that of NaGDC (21.7 Å) because the NaTDC radius of gyration of the cross section (average value 14.3 Å) is greater than that of NaGDC (average value 12.2 Å). Reasonably, the NaTDC longer radius should be mainly due to the greater length of its side chain, which in the fully extended conformation is about 3 Å longer. Therefore, a rough value of 25 Å can be assumed. This value is supported by sedimentation and diffusion experiments carried out on NaTDC aqueous micellar solutions.23 Sedimentation measurements in media of different densities, obtained by addition of NaCl, NaBr, and NaI (concentrations 0.1, 0.5, 1.0, and 2.0 M), indicated a high degree of hydration. Provided that the variations in the media do not change the aggregate structure, the micellar aggregate density and degree of hydration were 1.084 g cm-3 and about 2 mL water/g NaTDC, respectively, in 0.5 M salt,23 namely, under conditions which are the closest to those of our SAXS samples. These values are in good agreement with those of 1.087 g cm-3 and 2.0 mL water/g NaTDC calculated for a cylindrical aggregate assuming molecular volumes of 658 and 30 Å3 for NaTDC (specific volume 0.76 cm3 g-1) and H2O, respectively, together with a trimer volume of 7069 Å3 (volume of a disk with a height of 3.6 Å and a radius of 25 Å). The micellar aggregate density increases with increasing NaCl, NaBr, or NaI concentration (and, hence, ionic strength) up to 1.205 g cm-3 in 2.0 M salt, thus suggesting a decreased hydration.23 In this case, an explanation is that the aggregate cross-sectional area decreases together with its radius (about 16.4 Å for a density of 1.205 g cm-3), keeping constant the trimer repeat of 3.6 Å. The corresponding structure could be very close to that observed for a 40.0 wt % NaTDC aqueous sample which has a trimer repeat and radius of 3.6 and 15.85 Å, respectively,13 whereas the trimer motion could resemble the opening of an umbrella passing from high to low ionic strengths. Alternatively, the density can increase by means of an inclusion of inorganic salt ions into micellar aggregates, keeping constant the trimer repeat and radius. This effect was invoked to explain the specific conductance measurements of NaTDC, NaGDC, NaTC, NaGC, and sodium deoxycholate aqueous solutions (0.1 M) containing NaCl within the concentration range 0-0.8 M.6 It must be stressed that this explanation should be valid for NaTDC and NaGDC samples with NaCl up to 0.8 (NaTDC) and 0.7 (NaGDC) M concentration at least, on the basis of the SAXS data, which indicate a constant cylindrical radius. Models of NaGC and NaTC aggregates are based on their octamer geometry (see QELS results below). An octamer can be approximately represented as a disk with a height of about 8 Å (NaGC and NaTC) and a radius of about 18 (NaGC)3,6 and 19 (NaTC) Å.4,7 QELS Study of Dihydroxy-Trihydroxy Bile Salt Systems. All the systems are aqueous solutions containing 0.8 M NaCl to minimize charge interactions. Experimental (23) Laurent, T. C.; Persson, H. Biochim. Biophys. Acta 1965, 106, 616.
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Figure 1. Experimental Rh values of 80 mM NaGDC or NaTDC aqueous solutions containing 0.8 M NaCl as a function of NaGC or NaTC concentration. Average standard deviation is (0.3 Å.
Figure 2. Experimental Rh values of 0.8 M NaCl aqueous solutions containing NaGDC + NaGC (100 mM) vs NaGDC concentration, black circles; NaGDC, black squares; NaGC (its concentration is given by 100 - NaGDC concentration), open diamonds. Average standard deviation is ( 0.3 Å. Open triangles represent Rh values calculated by means of models.
Rh values of NaGDC or NaTDC (80 mM) with NaGC or NaTC (0-150 mM) are shown in Figure 1. They considerably decrease by increasing the NaGC or NaTC concentration. Remarkable changes occur at very small NaGC or NaTC concentrations (for instance, NaGDC or NaTDC Rh values decrease of 14 or 5 Å, respectively, when the NaGC or NaTC concentration is 5 mM). Among the possible hypotheses, two are the most reasonable to explain the Rh decreasing. The first one is that each aggregate is composed by only one bile salt and, therefore, Rh values decrease because those of NaGC or NaTC are much smaller than the NaGDC or NaTDC ones (Figures 2 and 3). The experimental Rh for a system of noninteracting particles and when the light-scattering form factor is in the limit where approximates unity24,25 is given by
) Σn (n2 Xn)/ Σn [(n2 Xn)/(Rh)n] where n is the aggregation number of a micellar aggregate, Xn is its mole fraction, and (Rh)n is its hydrodynamic radius. The second one is that Rh decreases because the NaGDC or NaTDC growth is inhibited when NaGC or NaTC (24) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.; Benedek, G. B. Biochemistry 1979, 18, 3064. (25) Schurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1983, 87, 308.
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Figure 3. Experimental Rh values of 0.8 M NaCl aqueous solutions containing NaTDC + NaTC (100 mM) vs NaTDC concentration, black circles; NaTDC, black squares; NaTC (its concentration is given by 100 - NaTDC concentration), open diamonds. Average standard deviation is (0.3 Å. Open triangles represent Rh values calculated by means of models. Table 1. Some Relevant Data of NaGDC (A) and NaTDC (B) Samples C (mM) CNaCl (M) Rh (Å) DM (Å) hSAXS (Å) h (Å) 1A 2A 3A 4B 5B 6B 7A 8A 9A 10A 11A 12B 13B 14B 15B
100 100 100 100 100 100 20 40 60 80 100 20 40 60 80
0.5 0.6 0.7 0.6 0.7 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8
42 56 67 43 52 64 42 53 66 77 88 33 41 48 55
110 150 180 122 139 170
101 144 175 111 130 163
104 143 174 106 132 165 104 134 171 202 233 77 101 120 140
n 87 119 145 88 110 137 87 112 142 168 194 64 84 100 117
monomers or oligomers interact with NaGDC or NaTDC micellar aggregates. The Rh values of aqueous solutions containing 0.8 M NaCl and NaGDC or NaGC or NaGDC + NaGC are reported in Figure 2. Those of NaGDC remarkably decrease by adding NaGC. The NaGDC + NaGC samples keep constant the global concentration (0.1 M). The corresponding data for NaTDC and NaTC are shown in Figure 3. The first hypothesis can be roughly tested by means of the above-mentioned relationship, assuming that each bile salt is monodisperse in all the samples and that its Rh is equal both in the one- and in the two-bile-salt systems having the same concentration of the bile salt. Experimental (Rh)n values can be used for NaGDC and NaGC or NaTDC and NaTC to calculate . Moreover, also the n values are needed. These can be estimated for our models as follows. A linear relationship has been observed between experimental Rh values of the six NaGDC and NaTDC samples at the highest ionic strengths studied by SAXS (to minimize charge interactions that can strongly affect QELS and SAXS experimental data) and their aggregate cylindrical heights (hSAXS, see Table 1). The hSAXS values are inferred from the DM ones by fixing the cylindrical radius at 21.7 Å (NaGDC) and 25 Å (NaTDC). The equation is hSAXS ) 2.81 Rh - 14.49 (regression value 0.9951) and has been used to calculate the heights h of samples 1-15 (Table 1) from their experimental Rh values (fifth column of Table 1), although two slightly different linear rela-
tionships (Rh vs hSAXS) with better regression values (0.9991 and 0.9990) fit the 1-3 NaGDC or 4-6 NaTDC samples. Aggregation numbers can be obtained from the heights because n ) (3/3.6) h. The equation is valid within the Rh approximate range 40-70 Å (Table 1) but not for SAXS samples at lower ionic strengths, which have Rh < 40 Å and are very probably affected by intermicellar correlation effects ascribed to charge interactions. Three samples lie outside this range. One is the sample 12 with a Rh value (33 Å) which approaches that of the cylindrical radius (25 Å), giving rise to an aggregate of spheroidal shape. The height of the corresponding cylindrical aggregate has been calculated to be 77 Å by putting the volume of a sphere with a radius of 33 Å equal to that of a cylinder with a radius of 25 Å. The other two are samples 10 and 11 with Rh values of 77 and 88 Å, for which the equation has been considered to be roughly valid on the basis of the following reasons. The linear increase of Rh with h seems to indicate that the main interparticle correlation effects at high ionic strength, because of van der Waals26 and volume-excluded interactions, are negligible because they either are small or compensate each other. On the other hand, linear relationships are observed between experimental Rh values of NaGDC samples 7-11 or NaTDC samples 6 and 12-15 and their concentrations C (mM). The equations are Rh ) 0.58 C + 30.44 (NaGDC, regression value 0.9992) and Rh ) 0.39 C + 24.67 (NaTDC, regression value 0.9989). Thus, the Rh linear dependence on h and C implies that of h on C. The equations are h ) 1.63 C + 71.0 (NaGDC, regression value 0.9995) and h ) 1.08 C + 56.1 (NaTDC, regression value 0.9988). Samples 10 and 11 fit the first equation and, probably, that of Rh versus hSAXS as samples 7-9 do. If this is true and monodispersity is assumed, each concentration increase of 20 mM within the range 20-100 mM causes an n average increase of about 27 (9 trimers) for NaGDC and 18 (6 trimers) for NaTDC (Table 1), in agreement with a growth occurring by a stepwise incorporation of trimers in the aggregates. As far as NaGC and NaTC aqueous solutions containing 0.8 M NaCl are concerned, their experimental Rh values are approximately constant within the concentration range 20-100 mM (Figures 2 and 3). Their average values of 16 (NaGC) and 17 (NaTC) Å have been used in the calculations. These values can satisfactorily compare with those calculated for spheroidal hexadecamers, formed by piling two octamers, having a height of about 16 Å and a radius of about 18 (NaGC)3,6 and 19 (NaTC) Å.4,7 Therefore, an n value of 16 has been always assumed for the NaGC and NaTC aggregates. The results of calculations are shown as open triangles in Figures 2 and 3. The bad agreement with the experimental data (black circles) allows us to discard the first hypothesis. Furthermore, an acceptable agreement is not obtained also varying the n value of 16 for NaGC and NaTC aggregates in a wide range. Obviously, the agreement is fair only when the trihydroxy salt concentration (80 mM) is much greater than that of the dihydroxy one (20 mM), so that the value depends especially on the trihydroxy salt (Rh)n value. The second hypothesis, hence, should be the most reasonable. Micellar aggregates of both dihydroxy5,13 and trihydroxy6,7 salts grow mainly by means of strong polar interactions along hydrophilic channels containing sodium ions, water molecules, carboxylate, sulfonate, and hydroxyl groups. Ion-ion, ion-dipole, dipole-dipole, and hydrogen(26) Janich, M.; Lange, J.; Graener, H. J. Phys. Chem. B 1998, 102, 5957.
Study of Dihydroxy-Trihydroxy Bile Salt Systems
bonding interactions, for instance, stabilize the aggregate structures and involve especially the hydrated cations as inferred from X-ray5-7,13 and electrolytic conductance measurements.6 An interesting question is whether trihydroxy salts interact with dihydroxy salt aggregates mainly by means of polar or nonpolar forces. Trihydroxy salt monomers and oligomers (very probably not trimers in accordance to EMF results7,15,16) must compete against the NaGDC or NaTDC trimers in the growth process. The trimer incorporation in the aggregates occurs mainly by means of strong polar interactions. Reasonably, trihydroxy salt monomers and oligomers should give rise to rather equal strong interactions with NaGDC and NaTDC aggregates to succeed in the inhibition of the growth. Therefore, polar interactions should be preferred to nonpolar ones. The bases of dihydroxy salt cylindrical aggregates seem to be the most probable, even though not the exclusive, candidates as binding sites. Monomers, dimers, and higher oligomers of trihydroxy salts, forming small-size aggregates, could interact with these bases especially by means of polar forces and interfere with the growth process. Hence, the complete or also partial neutralization of these binding sites inhibits the aggregate growth. An important point is the role of the trihydroxy salt polar head in the inhibition of the dihydroxy salt aggregate growth. To clarify this point, Rh values of aqueous solutions containing 0.8 M NaCl and NaGDC (80 mM) + NaTC (0-30 mM) or NaTDC (80 mM) + NaGC (0-30 mM) have been determined (Figure 4). In these cases, the polar heads of each bile salt of the pair are different. The trends are similar to those of Figure 1. NaGDC or NaTDC Rh values decrease of 17 or 7 Å when the NaTC or NaGC concentration is 5 mM. These values are slightly greater than those of 14 and 5 Å found for bile salt pairs with the same polar head. The small differences (2-3 Å only) could be due to the ability of a NaGC or NaTC molecule to replace a NaGDC or NaTDC molecule in a trimer. EMF measurements show that trimers are always present also at high ionic strengths,9,10 suggesting that they are the building blocks of the NaGDC and NaTDC aggregates. The replacement could occur only or chiefly when the two bile salts have the same polar head because the main interactions stabilizing the trimer are
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Figure 4. Experimental Rh values of 80 mM NaGDC or NaTDC aqueous solutions containing 0.8 M NaCl as a function of NaTC or NaGC concentration. Average standard deviation is (0.3 Å.
those between sodium ions, polar heads, and water molecules, and are the same existing in a NaGDC or NaTDC trimer. On the other hand, the 3-fold symmetry is practically preserved, because the dihydroxy and trihydroxy salt molecules with the same polar head differ only in one hydrogen atom or hydroxyl group at C7. Moreover, these atoms are not essential in stabilizing the trimer, since the atoms belonging to a molecule are very far from those of the other two molecules of the trimer. Therefore, some trihydroxy salt molecules could contribute to form trimers, which are incorporated in the aggregates, and would be subtracted from those that can be utilized to inhibit the NaGDC or NaTDC aggregate growth. Of course, the trimer formation is improbable for energy and symmetry reasons when the polar heads are different. Thus, all or almost all of the trihydroxy salt molecules can be utilized to inhibit the aggregate growth, and this gives rise to shorter aggregates and lower Rh values. Acknowledgment. This work was sponsored by Italian Ministero per l’Universita` e per la Ricerca Scientifica e Tecnologica and by Italian Consiglio Nazionale delle Ricerche. LA026232O
