SIMULTANEOUS DETERMINATION OF MICELLAR DISSOCIATION CONCENTRATION AND CRITICAL MICELLAR CONCENTRATION OF BILE SALTS BY pH MEASUREMENTS

This paper demonstrates a method of using a pH meter to determine the micellar dissociation concentration (mdc) and the. critical micellar concentration (cmc) of eight bile salt samples: sodium cholate (NaC), sodium deoxycholate (NaDC), sodium glycocholate (NaGC), sodium glycodeoxycholate (NaGDC), sodium glyco-chenodeoxycholate (NaGCDC), sodium taurocholate (NaTC), sodium taurodeoxycholate (NaTDC) and sodium taurochenodeoxycholate (NaTCDC). The experiments were performed by diluting the bile salt solutions with freshly distilled water and following the pH changes with a hydrogen ion electrode at 25°C. One break appears in most of the pH-concentration plots of the bile salt solutions, signifying mdc. However, two breaks appear for NaC and NaDC samples, signifying mdc and cmc. The mdc and cmc values are in good agreement with values determined by surface tension and turbidity methods and with data reported in the literature. The method described in this paper is quick, simple and requiring no sample purification. It is the only method which can be used to determine mdc and cmc simultaneously.     

Bile salt aggregates in the gas phase: an electrospray ionization mass spectrometric study

Helical and ordered structures have previously been identified by X-ray diffraction analysis in crystals and fibers of bile salts, and proposed as models of the micellar aggregates formed by trimeric or dimeric units of dihydroxy and trihydroxy salts, respectively. These models were supported by the results of studies of micellar bile salt solutions performed with different experimental techniques. The study has now been extended to the gas phase by utilizing electrospray ionization mass spectrometry (ESIMS) to investigate the formation and the composition of aggregates stabilized by noncovalent interactions, including polar (ion-ion, ion-dipole, dipole-dipole, hydrogen bonding etc.) and apolar (van der Waals and repulsive) interactions. The positive and negative ESIMS spectra of sodium glycodeoxycholate (NaGDC), taurodeoxycholate (NaTDC), glycocholate (NaGC), and taurocholate (NaTC) aqueous solutions, recorded under different experimental conditions, show in the first place that aggregates analogous to those present in micellar solutions do also exist in the gas phase. Furthermore, consistently with the condensed-phase model, the positive-ion spectra show that the trimers are the most stable oligomers among the aggregates of dihydroxy salts (NaGDC and NaTDC) whilst the dimers are the most stable among the aggregates of trihydroxy salts (NaGC and NaTC). Moreover, the binding energy of the constituent glycocholate salt units in most gaseous oligomers exceeds that of the corresponding taurocholate units. The ESIMS evidence has been confirmed by vapor-pressure measurements performed on NaGC and NaTC crystals and NaGDC and NaTDC fibers, the results of which show that the evaporation enthalpy of glycocholate exceeds that of taurocholate by some 50 kJ mol(-1).     

H＋对水溶液中脱氧胆酸钠聚集体的影响

运用pH滴定、傅立叶变换红外光谱、紫外可见光谱、激光光散射谱、ICP和元素分析等方法研究了H＋的加入对水溶液中脱氧胆酸钠（NaDC）聚集体的影响.结果表明,浓度大于cmc的NaDC水溶液具有一定的缓冲能力,NaDC浓度越高,缓冲能力越大;随溶液中H＋浓度的增加,首先形成质子化胶团,质子化胶团通过酸盐结构的氢键作用使NaDC初级胶团长大,形成较大的高级胶团,甚至形成凝胶体,最终形成HnNam(DC)n＋m沉淀.     

From crystal to micelle: A new approach to the micellar structure

The structure of sodium and rubidium deoxycholate micellar aggregates in aqueous solutions was found to be helical and to be stabilized mainly by polar interactions. Astonishingly, the lateral surface of the helix is covered by nonpolar groups and the interior part is filled with cations surrounded by water molecules, as in the case of an inverted micelle. This helical model was inferred from the crystal structures of sodium and rubidium deoxycholates and proved by spectroscopic and diffractometric experimental data. The strategy of the approach to the determination of the micellar structure and the comparison with another model, previously proposed for the bile salt micelles, are reported. On the basis of some results obtained for sodium tauro- and glyco-deoxycholates, micellar models are suggested which could account for the biological function of these important conjugated bile salts.     

On the growth and shape of sodium taurodeoxycholate micellar aggregates: a spin-label and quasielastic light scattering investigation

Electron spin resonance (ESR) and quasielastic laser scattering (QELS) measurements have been carried out on sodium taurodeoxycholate (NaTDC) micellar aqueous solutions. Computer simulation of the ESR line shape has been used to quantitatively analyze the rotational dynamics of the cholestan-spin label (CSL) dissolved by the NaTDC micellar aggregates as a function of temperature and NaCl concentration. The local reorientation of CSL has been accounted for motionally-averaged g- and A-tensors assuming fast oscillation around the spin-probe long molecular axis. The overall Brownian tumbling of CSL-micelle complexes has been modeled by an axially symmetric rotational tensor. Good agreement with experimental spectra is obtained. Best-fit rotational parameters and QELS data suggest that, in the circumstance of large aggregation, NaTDC micelles have cylindrical shape and micellar growth occurs along the cylinder axis.     

Effect of submicellar concentrations of conjugated and unconjugated bile salts on the lipid bilayer membrane

The interaction of submicellar concentrations of various physiologically important unconjugated [sodium deoxycholate (NaDC), sodium cholate (NaC)] and conjugated [sodium glycodeoxycholate (NaGDC), sodium glycocholate (NaGC), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC)] bile salts with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles in solid gel (SG) and liquid crystalline (LC) phases was investigated using the excited-state prototropism of 1-naphthol. Steady-state and time-resolved fluorescence of the two excited-state prototropic forms of 1-naphthol indicate that submicellar bile salt concentration induces hydration of the lipid bilayer membrane into the core region. This hydration effect is a general phenomenon of the bile salts studied. The bilayer hydration efficiency of the bile salt follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC for both DPPC and DMPC vesicles in their SG and LC phases.     

Bile salt-phospholipid aggregation at submicellar concentrations

The aggregation behavior of the bile salts taurodeoxycholate (NaTDC) and sodium cholate (NaC), are followed at concentrations below critical micelle concentrations (CMCs) using the environment sensitive, fluorescent-labeled phospholipid, 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C₆-HPC). A buffer solution containing NBD-C₆-HPC is titrated with increasing NaC or NaTDC and the fluorescence changes followed. Both bile salts induced fluorescence changes below their critical micelle concentration indicating the presence of a bile salt–phospholipid aggregate. A critical control experiment using 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoic acid (NBD-X) shows that the bile salts are interacting with the longer, C16 hydrocarbon tail, not the NBD probe. The fluorescence curves were fitted to the Hill equation as a model for cooperative aggregation. The cooperativity model provides a minimum estimate for the number of bile salts to give maximal fluorescence. This number was calculated for NaC and NaTDC to have a minimum value of 2. A small aggregation number supports the existence of primary micellar aggregates at submicellar concentrations for bile salt–phospholipid aqueous solutions.     

Micellar bile salt mobile phases for the liquid chromatographic separation of routine compounds and optical, geometrical, and structural isomers

Aqueous solutions of bile salts, i.e. sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium taurocholate (NaTC), are characterized and evaluated as reversed-phase liquid chromatographic (RPLC) mobile phases. The separation of the ASTM-recommended RPLC test mix in addition to more than 50 other compounds on a C18 column demonstrates the viability of these bile salts as HPLC mobile phases. The Armstrong-Nome theory was applied and found to adequately describe the partitioning behavior of solutes eluted with these bile salts at low surfactant concentrations. The effect of alcohol additives on chromatographic retention and efficiency was also assessed. Not only are the bile salt molecules rigid and chiral, but they form helical micellar aggregates as well. Consequently, many isomeric compounds can be easily resolved with this mobile phase additive. The base-line resolution of some binaphthyl-type enantiomers with a standard C18 column and the bile salt micellar mobile phases is also demonstrated. In addition, these bile salt mobile phases may be preferable to conventional hydroorganic mobile phase systems for the separation of many classes of routine compounds. A brief prospectus on the future utilization of bile salts in liquid chromatography is presented.     

Small-angle neutron scattering study of sodium cholate and sodium deoxycholate interacting micelles in aqueous medium

Small angle neutron scattering (SANS) measurements of D₂O solutions (0.1 M) of sodium cholate (NaC) and sodium deoxycholate (NaDC) were carried out atT= 298 K. Under compositions very much above the critical micelle concentration (CMC), the bile salt micelle size growths were monitored by adopting Hayter-Penfold type analysis of the scattering data. NaC and NaDC solutions show presence of correlation peaks atQ = 0.12 and 0.1 ?_-1 respectively. Monodisperse ellipsoids of the micelles produce best fits. For NaC and NaDC systems, aggregation number (9.0, 16.0), fraction of the free counterions per micelle (0.79, 0.62), semi-minor (8.0 ?) and semi-major axes (18.4, 31.7 ?) values for the micelles were deduced. Extent of micellar growth was studied using ESR correlation time measurements on a suitable probe incorporating NaC and NaDC micelles. The growth parameter (axial ratio) values were found to be 2.3 and 4.0 for NaC and NaDC systems respectively. The values agree with those of SANS.     

Micellization of bile salts in aqueous medium: a fluorescence study

Owing to the physiological importance of the micellization process of bile salts, the critical micelle concentration (CMC) becomes a fundamental parameter in the evaluation of their biological activities. The present study suggests fluorescence probing, using 1,6-diphenylhexatriene (DPH), as a simple, convenient, sensitive and economic method for monitoring the micellization process of bile salts in aqueous medium. Three independent parameters: fluorescence intensity, anisotropy and lifetime of DPH have been employed successfully for determining the CMC of two bile salts, sodium deoxycholate (NaDC) and sodium cholate (NaC), in aqueous medium. The CMC values reported by all the above three parameters of DPH are found to be same and it is 16 mM for NaC and 6 mM for NaDC at 25 degrees C in unbuffered solution. The effect of temperature and ionic strength on the micellization process has also been investigated employing DPH as a fluorescent probe. Increasing temperature leads to the formation of fluffier micelles with less rigid interior for both NaC and NaDC. The micelle core of NaC is less perturbed by the presence of NaCl whereas in case of NaDC, the aggregates provide DPH a more nonpolar and rigid environment in presence of NaCl than that in absence of salt.     

Aqueous sodium dehydrocholate-sodium deoxycholate mixtures at low concentration

The behavior of the sodium dehydrocholate (NaDHC)-sodium deoxycholate (NaDC) mixed system was studied by a battery of methods that examine effects caused by the different components of the system: monomers, micelles, and both components. The behavior of the mixed micellar system was studied by the application of Rubingh's model. The obtained results show that micellar interaction was repulsive when the aggregates were rich in NaDHC. The gradual inclusion of NaDC in micelles led to a structural transformation in the aggregates and the interaction became attractive. The bile salts' behavior in mixed monolayers at the air-solution interface was also investigated. Mixed monolayers are monotonically rich in NaDC, giving a stable and compact adsorbed layer. Results have shown that the interaction in both micelles and monolayer is not ideal and such behavior is assumed to be due to a structural factor in their hydrocarbon backbone.     

Aggregation behavior of sodium deoxycholate and its interaction with cetyltrimethylammonium bromide in aqueous solution studied by NMR spectroscopy

In pure sodium deoxycholate (NaDC) dilute solution, the transverse relaxation times (T ₂) of nearly all proton signals of NaDC obey single-exponential decay, with exception for proton at 3-position (H3) that decays in a two-exponential manner. The two components with different mobility of H3 of NaDC indicate that the molecules are probably associated in head-to-tail pairs via hydrogen bonds in dilute solution. In the mixed NaDC/cetyltrimethylammonium bromide (CTAB) solutions, the T ₂ values of interested NaDC protons are far less than those in pure NaDC solution, especially when the concentrations of the two components are close to equal-molar. The results of self-diffusion coefficients and the chemical shifts confirm further that the strong interaction occurs between the two components in mixed solutions, especially for equal-molar condition. The arrangement of the mixed aggregates can be speculated for the cross-peaks of proton pairs occurring between NaDC and CTAB molecules in two-dimensional and rotating frame nuclear Overhauser enhancement spectroscopy spectra.     

Effect of sodium deoxycholate and sodium cholate on DPPC vesicles: A fluorescence anisotropy study with diphenylhexatriene

Effects of two bile salts, namely sodium deoxycholate (NaDC) and sodium cholate (NaC), on DPPC small unilamellar vesicles have been investigated using the steady-state fluorescence anisotropy (r _ss ) of diphenylhexatriene (DPH) as a tool. It was found that the variation of r _ss is sensitive enough to monitor different stages of interaction of bile salts with DPPC vesicles. NaDC induced significant changes in the membrane well below its CMC (6 mM). Even at 4 mM, which is still lower than the CMC, the phospholipids were completely solubilised by the NaDC micelles. The effect of NaC on DPPC vesicles, however, was much less significant, especially in the sub-micellar concentration regime. Being more hydrophilic NaC does not interact with the membrane efficiently. Complete solubilisation of phospholipids took place only when the concentration of NaC was above its CMC (16 mM). The experiments also showed that the bile salt-induced changes of vesicle structure were strongly dependent on the concentration of the bile salt and not on the molar ratio of lipid and bile salt.     

Temperature-induced chirality reversal of induced circular dichroism of premicellar aggregates of acridine orange derivatives and dodecanoyl-l-threonine in aqueous solution

Circular dichroism (CD) and absorption spectra were measured for acridine orange derivatives: 3,6-bis(dimethylamino) acridine (AO), 3,6-bis(dimethylamino)-10-methylacridinium bromide (C₁AO), and 3,6-bis(dimethylamino)-10-dodecylacridinium bromide (C₁₂AO) in aqueous dodecanoyl-l-threonine (C₁₂Thr) solutions at 30, 40, 50, and 60 °C and pH 8–9. CD spectra were not induced for AO and C₁AO in C₁₂Thr aqueous solutions. Induced circular dichroism (ICD) based on the exciton interaction was found for premicellar aggregates of C₁₂AO with C₁₂Thr, but the micellar aggregates failed to induce CD for solubilized C₁₂AO at a higher concentration than C₁₂Thr critical micelle concentration. The maximum ICD intensity was observed for 1:1.2 aggregates of C₁₂AO and C₁₂Thr. The ICD spectrum indicated positive chirality at 30 °C, but negative chirality at 50 °C. The chirality transition occurred at 40∼45 °C. The slow change in both the absorption and ICD spectra is ascribed partly to the rearrangement of dye alignment and partly to the growth of the aggregate; the system reaches final phase separation after a few days.     

Correlation between Small-Angle X-ray Scattering spectra and apparent diffusion coefficients in the study of structure and interaction of sodium taurodeoxycholate micelles

Small-Angle X-ray Scattering (SAXS) and Dynamic Light Scattering (DLS) measurements were carried out on aqueous micellar solutions of the ionic biological detergent sodium taurodeoxycholate (NaTDC). Apparent diffusion coefficients (D(app)) and SAXS spectra of NaTDC 0.1 M solutions at different ionic strengths (0.1-0.3 M NaCl) were reported. A comparative analysis of SAXS spectra and D(app) data was performed to infer information on particle structure and interaction potential. Uniform particles with a spherical, an oblate, and a prolate symmetry were used to model the micelles in the data interpretation. A hard-core interaction shell of suitable thickness and a screened Coulomb potential of the electric double layer (EDL potential) were alternatively used to represent the long-range repulsive tail of the interaction potential. The Percus Yevick and the Rescaled Mean Spherical Approximation were applied. To compare the data of the two techniques, for each sample, a D(app) was calculated from the SAXS best-fitting geometrical parameters and interparticle structure factor of the micelles. Hence, a fitting procedure involving both the scattering and D(app) data was performed. The interpretation of SAXS spectra does not allow the discrimination between the oblate and the prolate symmetries of the aggregates. On the other hand, the comparison of calculated and experimental D(app) values indicates that the prolate ellipsoid is better suited to represent the micelle shape. Moreover, the agreement between calculated and experimental D(app) values is sensitively better at the lowest NaCl concentration when the EDL potential is used. A rodlike micellar growth and a progressive screening of the electrostatic interactions is testified by the trends of best-fitting parameters as a function of the added electrolyte.     

Photophysical and photodynamical study of fluoroquinolone drug molecule in bile salt aggregates

Photophysical properties of two widely used antibiotic fluoroquinolone drugs, namely Norfloxacin (NOR) and Ofloxacin (OFL) have been investigated in biomimicking environments formed by bile salts. Experimental results demonstrate that photophysical enhancement and fall of a particular prototropic species are sensitive to the excitation wavelength in bile salt aggregates. Excitation at shorter wavelengths reveals quenching of fluorescence of these fluoroquinolone with addition of sodium deoxycholate (NaDC), sodium taurocholate (NaTC) and sodium glycodeoxycholate (NaGDC). On the contrary, we observe a steady increase in the fluorescence intensity with a continuous redshift upon excitation at longer wavelength. The experimental results were rationalized in terms of the fact that, neutral and zwitterionic species of fluoroquinolone molecules in bile salt aggregates are selectively excited at shorter wavelength while the cationic form of fluoroquinolone molecules are excited at longer wavelength. The excess hydronium ions in the hydrophilic surface of bile salt aggregates convert the neutral species of NOR and OFL into cationic species causing an enhancement in the emission intensity. We found that NaGDC and NaTC because of the conjugate head group are more effective in converting the neutral species of fluoroquinolones into a cationic species than NaDC. The quenching order is in accordance with hydrophobicity indices of bile salt.     

Binding Study of the [Ru(NH3)5pz]2+ Complex to Bile Anion Aggregates through Kinetic Measurements

The electron transfer reaction between [Ru(NH₃)₅pz]²⁺ and [Co(C₂O₄)₃]^3? was studied in the presence of monomers and aggregates of bile salts (sodium deoxycholate, sodium taurodeoxycholate, and sodium glycocholate) at 298.2 ± 0.1 K. The results show a decreasing rate constant with the successive addition of bile salts. To rationalize the trends of the reaction rate on the [bile salts], two models were used. One of them takes into account the aggregation feature by considering a stepwise self‐association between monomers, whereas the other assumes the formation of a critical micellar concentration. Binding constants between [Ru(NH₃)₅pz]²⁺ species and deoxycholate or taurodeoxycholate aggregates were higher than that for glycocholate aggregates. These results are consistent with the way in which the monomers are added to form the bile anion aggregates.     

Effect of sodium salicylate, sodium oxalate, and sodium chloride on the micellization and adsorption of sodium deoxycholate in aqueous solutions

The salicylate ion increases the rate of bile flow (choleretic effect) and bile salts are known to affect the colonic absorption of oxalate. Owing to this physiological relevance of salicylate and oxalate ions, critical micelle concentration (cmc) values of sodium deoxycholate (NaDC) were determined in aqueous sodium oxalate, sodium salicylate, and sodium chloride solutions by using surface tension, fluorescence, and EMF methods. The results indicate, besides a counterion effect, the influence of coanions on the cmc. In the range from 25 to 40 °C, cmc increases almost linearly with temperature. In the temperature range from 30 to 40 °C, the counterion binding constant β of NaDC micelles has the same value (0.17±0.01) in the presence of sodium chloride and sodium salicylate. On the other hand, in sodium oxalate solution β=0.05±0.02 when oxalate concentration is less than or equal to c* and β=0.48±0.04 above c*, where c*≈0.038 mol kg(-1). EMF measurements also supported this type of counterion binding to NaDC micelles in sodium oxalate solutions. In sodium oxalate solution, at c* a change in the shape of deoxycholate micelles is expected to take place. Salicylate, oxalate, and chloride coanions have a similar effect on the adsorption of NaDC. This study reveals that the choleretic effect of salicylate is not due to the influence of salicylate ions on the micellization of NaDC.     

Thermodynamics of demicellization of mixed micelles composed of sodium oleate and bile salts

Isothermal titration calorimetry (ITC) was used to determine the critical micelle concentration (cmc) and the thermodynamic parameters associated with the demicellization of sodium oleate (NaO) and mixed micelles composed of the bile salt (BS) sodium cholate (NaC) or sodium deoxycholate (NaDC), respectively, and NaO at a molar ratio of 5:2. The influence of the ionic strength (pure water and 0.1 M NaCl at pH 7.5) as well as that of the temperature (10-70 degrees C) were analyzed. For NaO, two cmc's were detected, indicating a two-step aggregation process, whereas only one cmc was observed for the two BSs. A single aggregation mechanism is also evident for the demicellization of mixed micelles (BS/NaO 5:2). Increasing the ionic strength induces the well-known decrease of the cmc. The cmc shows a minimum at room temperature. The cmc(mix) of the mixed micelles was analyzed using models assuming an ideal or nonideal mixing behavior of both detergents. The thermodynamic parameters describing the enthalpy (deltaHdemic), entropy (deltaSdemic), and Gibbs energy change (deltaGdemic), as well as the change in heat capacity (deltaCp,demic) for demicellization, were obtained from one ITC experiment. From the temperature dependence of deltaHdemic, the change of the hydrophobic surface area of the detergents from the micellar into the aqueous phase was derived. In all cases, the deltaCp,demic values are positive. In addition, the temperature dependence of the size of the formed aggregates was studied by dynamic light scattering (DLS). DLS indicated two populations of aggregates in the mixed system, small primary micelles (0.5-2 nm), and larger aggregates with a hydrodynamic radius in the range of 50-150 nm.     

链式与面式两种不对称疏水结构表面活性剂之间的相互作用热力学

本文通过等温滴定量热法(ITC)、电导法和浊度法研究了阴离子生物表面活性剂脱氧胆酸钠(NaDC)及其与相反电荷的十二烷基三甲基溴化铵(DTAB)在水溶液中的自组装热力学.ITC结果支持了NaDC在水溶液中先生成预胶束再形成稳定胶束的分步聚集模型,由此得到了NaDC的预胶束和胶束化过程的一系列热力学参数,并讨论了它们形成的热力学机理.进一步研究了具有头-尾链式和疏水-亲水刚性面式非对称结构的DTAB/NaDC混合体系的聚集热力学行为,得到了富NaDC临界混合胶束浓度(cmcmix)、富DTAB临界胶束浓度(CM)及对应过程的转变焓.结果表明,NaDC面式结构与DTAB链式结构的对称性差异以及相反电荷的相互作用,导致混合体系有别于单一表面活性剂或头-尾链式结构的混合体系的聚集行为.混合溶液的聚集行为受控于表面活性剂浓度和摩尔分数的变化.富NaDC胶束化过程为熵驱动,而富DTAB的两种胶束形态转变过程为熵焓共同驱动的热力学机理.这些结果对于从热力学角度认识胆汁酸盐的自组装机理以及与传统的头-尾链式结构的表面活性剂相互作用机理和相行为有重要的意义.