Bile Salt Aggregates Research Articles

Equilibrium binding model of bile salt-mediated chiral micellar electrokinetic capillary chromatography.

Optimum concentration of bile salts in chiral separations depends on both the aggregation properties of the surfactant and the stability of the analyte-micelle complexes. An equilibrium model is proposed in which these two effects are treated separately. First the aggregation constants should be determined under the experimental conditions of the chiral MEKC analysis. With these data, the equilibrium concentrations of bile salt aggregates can be calculated at any total surfactant concentration. Using the Offord equation to approximate the mobilities of the enantiomer-bile salt complexes, a model function has been derived to fit the experimental mobilities. The method yields the binding constants of the enantiomers to each aggregate present. Those species are assumed to be important in the chiral recognition process, which have significantly different stability constants for the enantiomers. The method is demonstrated by the chiral separation of R- and S-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate with sodium taurodeoxycholate. Based on the calculated binding constants, tetrameric aggregates are assumed to be the discriminating species, while no significant difference in enantiomer binding to dimers was found.

Spectral properties and ionization behavior of retinoids, II.: Bile salt solutions

Purpose: The spectral properties and ionization behavior of retinoic acid (RA) and three structurally related arotinoids, MTTO, TTNPB, and TTNN, have been determined in simple micellar and mixed micellar solutions to provide a better understanding of the intestinal absorption of retinoids. Methods: Spectrophotometric and pH measurements have been made to determine the ionization constant in bile salt solutions. The fluorescent intensity and polarization of TTNN was determined. The extent of solubilization of retinoic acid in 10 mM NaTC/10 mM egg PC solutions was determined as a function of pH. Results: The rank order of wavelengths of maximum absorption, λ max, was as follows: aqueous solution>dihydroxy bile salt>trihydroxy bile salt>ethanol>mixed micelles. The interaction as reflected in the λ max of arotinoids with the bile salts at low pH depended on the mixing order, but this was not the case for retinoic acid. The rank order of the observed negative logarithm of the ionization constants, p K a obs , was aqueous solution>mixed micelles>dihydroxy bile salt>trihydroxy bile salt which reflects both the polarity as well as the electrostatic charge of the aggregates. The calculated electrostatic and dielectric effects on the ionization constant were comparable in bile salt micelles suggesting that TTNN is repositioned with a change in the ionic strength and that the size distribution of bile salt simple micelles appears to be perturbed by the presence of TTNN. In addition, the size of the bile salt-TTNN aggregate was independent of ionic strength and the type of bile salt. The solubilization of retinoic acid in bile salt/egg PC mixed micelle does not follow the expected dependence of pH indicating the presence of specific interactions. Conclusions: Retinoic acid and its derivatives exist almost exclusively in aggregated forms with RA forming a distinct type of aggregate in comparison to the arotinoids. The values of the p K a obs of the retinoids ranged from near 5 in simple bile salt aggregates to >7 in self-associated aggregates and mixed micelles which reflects the sensitivity of the ionization to the environment. This sensitivity has direct implications for the extent solubilized in the intestine and thereby complicates efforts at understanding the mechanism of the oral absorption of retinoids.

Bile acids as stationary phase in liquid chromatography

Bile acids chemically bonded onto aminopropylsilica have been employed as stationary phases in liquid chromatography. Bile acid aggregates were dynamically formed around molecules chemically anchored on the supports when the eluent contained bile salts. The bile salt aggregates achieved the separation of 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate enantiomers and dansyl amino acids.

Interaction of iron(II) with bile salts

Iron(II) ions react with small aggregates of cbolate, glycocholate, cbenodeoxycholate, and deoxycholate to form soluble and colloidal compounds. Taurocbolate under conditions used does not react with the Fe 2+ ion. Small aggregates of dibydroxy bile salts (predominating in the premicellar region, at concentrations of the bile salt above 1 mmol dm −3) have a larger affinity for Fe 2+ compared to those formed from cbolate anions. In their interactions with small aggregates of cbolate anions, the Fe 2+ ion shows an affinity comparable to that of Cu 2+ and Cd 2+ and somewhat larger than that of Zn 2+. Small aggregates of cbolate show a higher ability to mask Fe 2+ than those of taurocbolate and glycocholate. Interaction of glycocbolic acid anions with Fe 2+ ions is sufficient to prevent iron(II) precipitation.

Analysis of the Solubilization of Steroids by Bile Salt Micelles

Bile salt mixed micelles play an important role in the emulsification, solubilization, and absorption of cholesterol, fats, and lipid-soluble vitamins. Studies have also revealed the importance of wetting and solubilization of drugs by the bile salts; however, the capacity and specificity of bile salt simple and mixed micelles for solubilization are still undefined. Thus, in this study the aqueous solubility and the extent of solubilization in bile salt micellar solutions of a series of steroids were determined. The melting point and enthalpy of fusion were measured to determine the ideal mole fraction solubility of each steroid. From the ideal mole fraction solubility and the observed solubility of each steroid, the activity coefficient of each steroid in solution was calculated with respect to the supercooled pure liquid as the standard state. In aqueous solution, the activity coefficients decreased with increasing number of hydroxyl groups on the steroids. This trend also occurred in the bile salt solutions, although the values of the activity coefficients were smaller by about three orders of magnitude. The stereochemical position of the hydroxyl groups influenced the rank order of the activity coefficients in the aqueous and micellar solutions. Incorporation of a fluoro or methyl group resulted in an increase in the activity coefficient, whereas aromatization of the A ring of the steroid frame markedly decreased the activity coefficient. The results for the aqueous solubility are consistent with the expected interaction of the functional groups with the polar environment. The relatively small activity coefficients observed with the micellar solutions result from the nonpolar environment of the aggregates. However, the similarity of the relationship between the activity coefficient and the number of hydroxyl groups in the aqueous and micellar solutions indicates the importance of polar interactions for solubilization. These results may provide new insight into the solubilization of steroids by bile salt micellar solutions and may provide a basis for predicting solubilization of other compounds by bile salt aggregates.

Reactions of Small Aggregates of Taurine Conjugates of Dihydroxy Bile Salts with Divalent Transition Metal Ions

Formation of slightly soluble complexes between the small aggregates (dimers to tetramers) of bile salts and divalent cations, Cu2+, Cd2+, and Fe2+(Me2+), have been studied using polarography to determine concentration of free aquo ions. Polarography has been proved to be useful for studying heterogeneous equilibria. Whereas taurocholate does not form such complexes (possibly because it does not form small aggregates), taurodeoxycholate, taurochenodeoxycholate, and tauroursodeoxycholate (all bearing two OH groups) participate in formation of such complexes. Nevertheless, these complexes are much weaker than those formed with parent, unconjugated bile salts. These differences can be due to differences in stacking as well as to exchange of COO−for the less complex-forming SO3−. The stability of complexes were characterized by values of pKi, obtained from shifts of half-wave potentials with concentrations of bile salt in excess. The values of pKj, corresponding to equilibria between Me2+in solution and solids, which can be obtained from decrease of limiting currents with concentration of bile salt, are less suitable for comparison, as it is often not possible to reach sufficiently high concentrations of bile salts. Comparison of pKiwith pKjindicates that for some bile salts–Me2+combinations the same complex predominates in solution and in the solid; for others the composition of these complexes differ. For Cu2+and Cd2+, tauroursodeoxycholate forms the most stable complexes. For all three bile salts studied, Cu2+formed the least stable complexes. Possibility and consequences of formation of complexes of bile salts with Me2+ions in bile should be kept in mind.

Ionization and solubilization of 4 alkyl benzoic acids and 4 alkyl anilines in sodium taurodeoxycholate solutions.

The aqueous solubility and the extent of solubilization and ionization constant in sodium taurodeoxycholate (NaTDC) solutions of a series of benzoic acid and aniline derivatives were measured as a basis to characterize and thereby help predict the nature of the interaction of drugs with bile aggregates. The aqueous solubility and the solubilization of two series of compounds, 4-alkyl benzoic acids and 4-alkyl anilines, was measured as a function of NaTDC in 0 and 150 mM NaCl. The ionization constants were determined in water and in 50 mM NaTDC at sodium chloride concentration of 0, 75 and 150 mM by spectrophotometric titration. The diffusion coefficients of NaTDC and the solutes were measured by pulsed-field gradient spin echo NMR spectroscopy. The aqueous solubilities decreased with increasing alkyl chain length in both series, and the aniline derivatives had larger solubilities than the benzoic acid derivatives. The number of moles of solute solubilized per mole of bile salt ranged from 0.17 to 0.31 for the benzoic acid derivatives and from 1.3 to 3.0 for the aniline derivatives. The pKa values of the benzoic acid derivatives in the presence of NaTDC were higher relative to the controls, and the difference in the pKa (delta pKa,obs) increased with increasing chain length. With the aniline derivatives, the pKa values were also shifted to higher values in NaTDC relative to the control but only in the absence of salt. The presence of the solute caused a decrease in the diffusion coefficient of NaTDC, and the diffusion coefficients of the solutes decreased with increasing alkyl chain length. With the hexyl derivative, the diffusion coefficient of the solute was smaller than the diffusion coefficient of the bile salt. The chemical shift of the protons attached to carbon 18 and 19 of the salt were decreased to a greater extent in the presence of the solutes than the protons attached to carbon 26. Both the solubilization and ionization behavior of solutes were affected by the presence of bile salt aggregates. The surface potential and effective polarity of NaTDC aggregates were found to be dependent on the alkyl chain length for these two homologous series of solutes. The solubilization ratio was largely independent of alkyl chain length, but the unitary partition coefficient was dependent on both alkyl chain length as well as ionization state. The derivatives reduced the diffusivity of the micelles suggesting the formation of larger size aggregates and the solutes (hexyl derivatives) appear to favor association with the larger sized aggregates. The phenyl ring of the solutes appears to be oriented parallel to the plane of the steroid frame with preferential positioning near the hydrophobic rings.

Modelling micellar aggregation of bile salts

Modelling micellar aggregation of bile salts

Effect of Divalent Transition Metal Ions on the Aggregation of Trihydroxy Bile Salts

The interaction between anions of trihydroxy bile acids (BS−) (cholate, glycocholate, taurocholate, ursocholate, and hyocholate) and divalent transition metal ions (Cu2+, Cd2+, and Zn2+) was followed using polarographic reduction of the metal ion. For solutions containing 0.1 mMMe2+no interaction with BS−was observed at concentrations below 1 mM.At [BS−] between 1 and 10 mMslightly soluble complexes are formed, attributed to an interaction of Me2+with small aggregates of BS−. At concentrations above 10 mMBS−large (“micelle-like”) aggregates are formed, bearing free COO−groups which increase their solubility. Exact concentration limits characterizing individual ranges depend on the nature of both BS−and Me2+. Sequences of overall stability constants, solubilities, and ranges of formation of large aggregates indicate a similar stacking of hydroxy bile acid anions in both small and large aggregates. The stability of complexes with small aggregates increases in the sequence Cd2+< Fe2+< Pb2+< Zn2+< Cu2+, and their solubility in the sequence Fe2+< Cu2+< Cd2+< Zn2+. The sequence of the concentrations at which large aggregates are formed in the presence of Me2+ions resembles the sequence of CMC values obtained in the presence of Na+ions.

Dynamics of Probe Complexation to Bile Salt Aggregates

Excited triplet naphthalene and xanthone were employed as probe molecules to study the dynamics of incorporation into sodium cholate, taurocholate, and deoxycholate aggregates. The association and dissociation rate constants and the quenching rate constants for the incorporated probes were recovered from the dependence of the observed triplet decay rate constants on quencher concentrations (nitrite and cupric ions). Triplet naphthalene was incorporated into a site offering a high degree of protection from quenchers and from which dissociation was relatively slow. Triplet xanthone was incorporated into a less protected site from which dissociation was an order of magnitude faster than from the naphthalene site. We propose that naphthalene was incorporated into the hydrophobic site of primary aggregates, and xanthone was located in the region containing the hydroxyl groups in the secondary aggregates.

Probing Bile Salt Aggregates by Fluorescence Quenching

Abstract— The combination of steady‐state and time‐resolved quenching experiments was employed to study the aggregation behavior of sodium cholate at concentrations below 50 mAf. Naphthalene, anthracene and pyrene were used as fluorescent probe molecules, and protection by the aggregates from aqueous quenchers, as well as the onset of aggregation at low sodium cholate concentrations, was dependent on the shape of the probes. Protection from aqueous quenchers was inferred by comparing the efficiency for dynamic quenching in the absence and presence of sodium cholate and was best for naphthalene followed by pyrene and anthracene. Static quenching was observed, suggesting that probe molecules are located in an aggregate environment that also contains iodide. The incorporation of pyrene at low sodium cholate concentrations, as well as the small degree of static quenching observed, suggest that the shape complementarity, i.e. hydrophobic surface and packing, between pyrene and sodium cholate is optimum for aggregate formation.

Aggregation Behavior of Bile Salts in Aqueous Solution†‡

Freezing point depression, ∆Tlk, and pNa are measured and analyzed for aqueous solutions of trihydroxy (NaTC) and dihydroxy (NaDC and NaTDC) bile salts. The results show the existence of break points in the plot of ∆Tlk vs molality at 0.018, 0.013, and 0.007 m, respectively, in good agreement with previous published critical micelle concentration values. Above the break point bile salts form aggregates with average aggregation numbers of 2.59 ± 0.12 (NaTC), 5.82 ± 0.04 (NaDC), and 5.42 ± 0.47 (NaTDC). Fractions of bound counterions are also deduced, being close to 0.3 for the three bile salts studied. This indicates that only one counterion is bound for every three monomers in the aggregate. The different structural models published for the bile salt aggregates are discussed.

Measurement of Bile Salt Aggregation Equilibria Using Kinetic Dialysis and Spreadsheet Modeling

Bile salts initially self-associate into small aggregates such as dimers and trimers before forming micelles at higher concentrations. The very size of these small aggregates hampers quantitation of their formation. We have devised a method using kinetic dialysis measurements to quantitatively examine aggregation of bile salt at concentrations below 10 mM. Data for rate of dialysis versus concentration were fitted to hypothetical models of aggregation using a personal computer spreadsheet. For sodium taurocholate these data best fit a model of initial dimer and trimer formation with stepwise association constants of 100 and 160, respectively. Addition of larger aggregates to the model did not improve the fit. For sodium taurodeoxycholate the data best fit a model which included not only dimers and trimers, but also tetramers or larger aggregates up to 10-mers with stepwise association constants of 40, 400, and 1700 for dimers, trimers, and tetramers, respectively. These data agree reasonably well with existing literature and suggest that kinetic dialysis with spreadsheet modeling is a useful technique for the study of bile salt aggregation at relatively low concentrations.

Interaction between bile salts and beta-adrenoceptor antagonists.

The interactions between beta-adrenoceptor antagonists and bile salts were investigated by microcalorimetry. Nadolol and oxprenolol interactions with dihydroxy salts could be described by a 1:1 interaction model with the thermodynamic parameters indicating that the drugs were incorporated within the bile salt aggregates. This weak interaction was primarily hydrophobic although electrostatic attraction also played a role. Atenolol and metoprolol did not interact with the dihydroxy salts. None of the compounds interacted with trihydroxy bile salts or with salts below their aggregation concentration. Phase separation resulted when propranolol and alprenolol were present in dihydroxy salt solutions above a certain concentration with the interaction being of a hydrophobic and electrostatic nature. The implications of these results on in-vivo drug absorption are discussed.

The Role of Reaction Conditions in the Interaction of Cadmium(II) Ions with Cholate Anions

At "submicellar" concentrations of cholate anions reaction with cadmium(II) ions produces slightly soluble species involving small aggregates of bile salts. At large excess of cholate more soluble large aggregates ("micelles") are formed. Nature and concentration of an added neutral salt, initial concentration of Cd2+, and buffering do not affect qualitatively the pattern of observed processes. Quantitative changes due to variations of reaction conditions result in the studied processes occurring at different concentrations of the bile salts. Composition of the reaction mixture affects the concentration above which precipitation occurs, the solubility of the compounds formed, and the composition and stability of the complexes formed, as well as the concentration above which large aggregates are formed. Standard conditions, involving 1 × 10-4M metal(II) ion, 0.15 M KNO3, bile salt concentrations varying from 5 × 10-4 to 8 × 10-2M, and polarographic determination of the free metal ion concentration, proved to be most informative.

Proton magnetic resonance studies of the aggregation of taurine-conjugated bile salts.

The concentration dependence of the 500 MHz 1H-NMR spectra of taurocholate, taurochenodeoxycholate, taurodeoxycholate, and the monosulfate esters of taurochenodeoxycholate has been examined at 0.154 M NaCl in D2O. The resonances of the C18, C19, and C21 methyl groups and the C23 methylene group are differentially broadened with respect to the C25 and C26 methylene and C7 (or C12) methine groups with increasing bile salt concentration for each of the bile salts studied. These data confirm hydrophobic association and indicate that the side chain contributes to the hydrophobic surface of the bile salt. The chemical shift difference of the anisochronous C23 methylene protons is different in monomer and aggregate form. The C25 methylene protons are isochronous in monomeric form but anisochronous in aggregate form. The concentration dependence of the observed chemical shifts has been analyzed to estimate the critical concentration associated with the onset of these changes. The conformer population about the C22-C23 bond changes before the anisochronicity of the C25 methylene protons develops. This indicates that the C23 methylene group is affected by the initial stages of self-association, whereas specific motional constraints about the N-C25 bond in the taurine moiety are only induced in large primary micelles. The difference in the chemical shift of the C25 methylene protons depends on the structure of the bile salt. The relative magnitude of the shift differences is not altered by the presence of phosphatidylcholine. The data suggest that in primary micelles or mixed micelles the taurine moiety conforms to segregate the hydrophilic groups of the bile salt and effects greater van der Waals' contact between the hydrophobic surfaces.

Fluorescent probe studies of metal salt effects on bile salt aggregation

Fluorescent probe studies of metal salt effects on bile salt aggregation

Novel chiral separation techniques based on surfactants

Aqueous solutions of three different general types of chiral organized surfactant media are characterized and evaluated as mobile phases in micellar liquid chromatography (MLC), or as the transport vehicle in liquid membrane (LM) based separations. The surfactant systems examined included bile salts (e.g., sodium cholate, sodium deoxylcholate, sodium taurocholate), mixed surfactant systems containing a chiral surfactant additive (e.g., sodium dodecyl sulfate with octyl glucopyranoside or digitonin), and a chiral quaternary ammonium ionene polyelectrolyte. The preliminary data indicate that all of these surfactant organized assemblies can function as either chiral mobile phase additives or the active component of liquid membranes for the resolution of optical isomers, in addition to separating other isomeric and routine compounds. Specifically, the bile salt aggregates are useful in the separation of substituted binaphthyl enantiomers, whereas mixed micellar systems such as sodium dodecyl sulfate/chiral nonionic surfactant media appear to be effective in the separation of some derivatized amino acid optical isomers and anomeric saccharides. The ionene media can function as the enantioselective discriminating agent in the liquid membrane based separation of enantiomeric dansyl amino acids. Our preliminary results on these systems are summarized in this paper. The salient features, advantages, limitations, and potential applications of these new chiral surfactant-mediated separation techniques are discussed.

The association of sodium cholate and sodium deoxycholate in solutions of aromatic alcohols

Equivalent conductivities (Λ eq) of sodium cholate (SC) and sodium deoxycholate (SDOC) in water and in solutions containing aromatic alcohols were measured at 25°C. The measurements of the solubilization of alcohols by SC and SDOC aggregates in aqueous solutions have also been performed at the same temperature. It has been found that Λ eq versus √ C plots of SC and SDOC in water and in solutions of aromatic alcohols do not show a sharp decrease in Λ eq over a narrow range of concentration, as usually found in systems undergoing micelle formation. Instead, bile salts exhibit non micellar association characteristics in the range of concentrations used. Equilibrium constants and partition coefficients of aromatic alcohols in solutions of SC are lower than those of the same alcohols in solutions of SDOC. These results show that SDOC forms larger aggregates than SC. Transfer free energies of alcohols from water to bile salt aggregates and methylene group contribution to transfer free energy provide the evidence that aromatic alcohols are solubilized in the interior of bile salt aggregates as well as the interference.

The influence of bile salt structure on self-association in aqueous solutions.

The relationship between chemical structure and the concentration at which self-association occurs in water or in 0.15 M Na+ ion was examined for more than 50 bile salts and bile salt analogues varying in substituents on the steroid nucleus or in the structure of the side chain. Nuclear substituents varied in type (alpha- or beta-hydroxy, or oxo group) and number (1, 2, or 3); side chain structure varied in the nature of the ionic group (unconjugated, glycine- or taurine-conjugated, or zwitterion) or length of the side chain (5-, 4-, or 3-carbon atoms). The midpoint of the concentration range over which aggregation occurred was called the critical micellar concentration (CMC), even though bile salt aggregation is known to be more gradual than that of most typical ionic detergents. CMC values were obtained by surface tension measurements using an improved maximum bubble-pressure method, as well as by dye solubilization. Results obtained by the two methods agreed well. The CMC values varied from about 1 to greater than 250 mM. For a given bile salt, the addition of a hydroxy or oxo group increased the CMC; and for a given number of substituents, the changing of a hydroxy group to an oxo group increased the CMC values as well. The orientation of hydroxy substituents also influenced the CMC values: the changing of a hydroxy substituent from an alpha- to a beta-configuration increased the CMC values, as bile salts possessing alpha- and beta-hydroxy substituents had higher CMC values than bile salts with only alpha-hydroxy substituents. Inspection of space-filling models suggested the hypothesis that the greater contiguous hydrophobic area of the molecule, the lower the CMC value. The CMC value also increased exponentially as the side chain was shortened from C5 to C4 to C3. Conjugation of the side chain carboxylic group with glycine or taurine, although increasing the length of the side chain, caused little change in the CMC values. The addition of Na+ ion to a total concentration of 0.15 M lowered the CMC in a predictable manner for all anionic bile salts. The results indicate that the concentration at which bile salt aggregation occurs varies widely and is determined not only by the number, type, and orientation of nuclear substituents, but also by side chain structure.