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).     

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.     

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.     

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.     

Temperature dependence of the interaction of cholate and deoxycholate with fluid model membranes and their solubilization into mixed micelles

The interaction of bile salts with model membranes composed of soybean phosphatidylcholine (SPC) and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using high sensitivity isothermal titration calorimetry (ITC). The partitioning and incorporation of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) from an aqueous phase (pure water or 0.1 M NaCl) into fluid bilayer vesicles was studied as a function of temperature and ionic strength. The thermodynamic parameters of partitioning were determined with a model taking electrostatic interactions into account. In addition, the solubilization of SPC and POPC vesicles with NaC and NaDC as a function of temperature was also studied by ITC and the phase diagrams for the vesicle to micelle transition at two different temperatures were established. Unsaturated phospholipids require higher amounts of detergent to be transformed into micelles compared to saturated phospholipids. In addition, the width of the coexistence region of mixed micelles and mixed vesicles is larger for phosphatidylcholines with unsaturated chains. A comparison of NaDC with NaC shows the higher solubilization effectiveness of NaDC in agreement with its lower cmc. Furthermore, increasing the ionic strength decreases the amount of bile salt necessary for the formation of mixed micelles, which is also expected from the decrease of the cmc with ionic strength due to the shielding of the charges of the bile salts.     

Solubilization of negatively charged DPPC/DPPG liposomes by bile salts

The interactions of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) in 0.1 M NaCl (pH 7.4) with membranes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) and mixtures of DPPC and DPPG at molar ratios of 3:1 and 1:1 were studied by means of high-sensitivity isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). The partition coefficients and the transfer enthalpies for the incorporation of bile salt molecules into the phospholipid membranes were determined by ITC. The vesicle-to-micelle transition was investigated by ITC, DLS, and DSC. The phase boundaries for the saturation of the vesicles and their complete solubilization established by ITC were in general agreement with DLS data, but systematic differences could be seen due to the difference in detected physical quantities. Electrostatic repulsion effects between the negatively charged bile salt molecules and the negatively charged membrane surfaces are not limiting factors for the vesicle-to-micelle transition. The membrane packing constraints of the phospholipid molecules and the associated spontaneous curvature of the vesicles play the dominant role. DPPG vesicles are transformed by the bile salts into mixed micelles more easily or similarly compared to DPPC vesicles. The saturation of mixed DPPC/DPPG vesicles requires less bile salt, but to induce the solubilization of the liposomes, significantly higher amounts of bile salt are needed compared to the concentrations required for the solubilization of the pure phospholipid systems. The different solubilization behavior of DPPC/DPPG liposomes compared to the pure liposomes could be due to a specific "extraction" of DPPG into the mixed micelles in the coexistence region.     

Spectroscopic studies on the interaction between acridine orange and bile salts

The structure of sodium deoxycholate (NaDC) micellar aggregates has been previously reported to be helical, and two helical models have been proposed for the micellar aggregates of sodium taurodeoxycholate (NaTDC). Here we report NMR and UV-VIS studies on the interaction between acridine orange (AO) and NaDC or NaTDC aqueous micellar solutions. AO is known to aggregate in aqueous solutions. The addition of NaDC or NaTDC causes the breaking of the AO aggregates, although the binding geometry of the two bile salts with AO seems to be slightly different. The cationic dye interacts mainly with the C₁₈ and C,9 methyl groups of the bile salt molecules. This result agrees with one of the two NaTDC helical models and with some of its possible aggregates, and confirms again the helical structure attributed to the NaDC micellar aggregates within the limits of the experimental conditions tested by us.Devoted to Professor Giovannai Battista Marini Bettolo Marconi on the occasion of his 75th birthday.     

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.     

凝胶扩散体系中磷酸钙的沉淀图形

０引言生物矿化可分为正常矿化和异常矿化。胆结石是一种生物异常矿化，是世界性的常见病和多发病，形成机理尚不十分清楚。从结石的形成过程来看，不仅结石的组成与其形成机理有关，而且结石的组成分布和剖面图形同样与之有密切关系。人们经常可以观察到，一类结石的剖面具有周期环状图形，且棕色和乳白色环相间。其中白色环的主要成份为胆固醇；棕色环的主要成份为胆红素钙络合物，同时棕色环中还常常含有少量的磷酸钙和碳酸钙等无机难溶盐。由于结石的形成是一个长期的过程，研究表明在结石生长的过程中胆汁的组成不发生规律性的周期变化…     

Mixed Micellization of Dodecyldimethylamine Oxide with Bile Salts in Aqueous Solutions

Micellar behavior of dodecyldimethylamine oxide (DMDAO) with bile salts [sodium deoxycholate (NaDC) and sodium cholate (NaC)] with and without NaCl was studied by surface tension. Interaction parameters of the mixed micelles were estimated using Rubingh's theory. Strong synergism observed for each mixed system, which is a common feature shown by anionic-cationic mixtures. The mixed solutions remained clear even at equimolar ratio. Different behavior of the two bile salts is explained on the basis of their orientation in cationic micelles.     

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.     

胆盐与磷酸钙的相互作用

胆汁的pH条件下(pH=6～8)，应该生成无定形磷酸钙(ACP)，而在胆结石中磷酸钙通常以羟基磷灰石的形式出现.利用谱学方法研究了ACP与胆盐的作用.结果表明，胆盐以胶团的形式与ACP作用，在溶液中形成复合胶团，使其溶解度增加.不同类型胆盐与ACP的作用能力不同：脱氧胆酸钠(NaDC) > 牛磺胆酸钠(NaTC) > 胆酸钠(NaC).胆盐与ACP中结合钙的亲和能力大于结合钙的亲和能力，使ACP在胆汁的环境下容易转化为羟基磷灰石.     

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.     

“Three-in-one” Complexes Formed by Anionic Guests and Monosubstituted Cationic Alkyldiamino β-Cyclodextrin Derivatives

The effect of electrostatic interactions on the complexation of ionic guests by charged β-cyclodextrin (βCD) derivatives is reviewed. Special attention is paid to the numerous studies concerning the effect of electrostatic interactions on (i) the complexation of fluorescent and UV probes; (ii) the catalytic and chiral recognition properties of βCD derivatives; and (iii) the complexation of two bile salts (sodium cholate, NaC, and sodium deoxycholate, NaDC). The formation of three-in-one complexes between NaC and alkyldiamino βCD derivatives is also presented.     

Functionalization of silver and gold nanoparticles using amino acid conjugated bile salts with tunable longitudinal plasmon resonance

The vital bioactivities of bile salts are physiologically important molecules. The concept of using bile acids and their conjugates in nanoscience is a novel idea, which opens up fascinating prospects and gives way for various versatile properties. Here in, we report novel strategy for the synthesis of aqueous stable, silver and gold nanoparticles (Ag & AuNPs) using naturally occurring amino acid conjugated sodium salt of taurocholate (NaTC) and glycocholate (NaGC) as reducing and capping agents. The formation of nanoparticles was kinetically monitored using UV–vis spectroscopy at different time intervals. It was noticed, that the rate of reduction of AgNO₃ is much faster than the HAuCl₄ at fixed concentration of bile salts. Furthermore, the size and shape of the NPs are controlled and achieved by changing the nature of bile salts. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD) techniques for morphological studies. The interaction between nanoparticles with bile salts was investigated using FT-IR spectroscopy, cyclic voltammetry (CV) and thermogravimetric analysis (TGA).     

Supramolecular Linear Conglomerates Formed by β-Cyclodextrin Dimers and Sodium Deoxycholate

The formation of supramolecular structures by the complexation of two bile salts--sodium cholate (NaC) and sodium deoxycholate (NaDC)--with four new head-to-head g -cyclodextrin dimers was studied by NMR techniques. All dimers form 1:2 (dimer:bile salt) stoichiometry complexes with NaC. With NaDC, linear supramolecular conglomerates of an n : n stoichiometry were obtained for all dimers. ROESY spectra confirmed the presence of electrostatic interactions when a protonated amino group is present in the linking group. The dependence of the pseudo-equilibrium constants with the electrostatic interactions and steric hindrance is discussed.     

Interactions of phenothiazine drugs with bile salts: Micellization and binding studies

An evaluation of the interactions of phenothiazine tranquilizer drugs (promazine hydrochloride; PMZ and promethazine hydrochloride; PMT) with bile salts viz., sodium cholate (NaC) and sodium deoxycholate (NaDC) in aqueous medium, investigated through different physicochemical measurements is presented in this work. The mixed micellization behavior and surface properties of the phenothiazine-bile salt systems have been analyzed by conductivity and surface tension measurements. Application of different theoretical approaches to all the phenothiazine-bile salt mixtures shows a non-ideal behavior. Further, the spectroscopic techniques such as UV-visible and steady state fluorescence have been employed to study the binding of phenothiazines with bile salts. The stoichiometric ratios, binding constants (K), and free energy change (ΔG) for the phenothiazine-bile salt complexes were estimated from the Benesi-Hildebrand (B-H) double reciprocal plots obtained by using the changes in spectral intensities of phenothiazines on addition of bile salts. The results are discussed in the light of use of bile salts as promising drug delivery agents for phenothiazines and hence improve their bioavailabilty.     

Effects of bile salts on percolation and size of AOT reversed micelles

The effects of two trihydroxy bile salts, sodium taurocholate (NaTC) and 3-[(3-cholamidylpropyl)dimethylammonio]-1-propane sulfonate (CHAPS), on the size, shape and percolation temperature of reversed micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane were studied. The percolation temperature of the reversed micelles decreased upon inclusion of bile salts, indicating increased water uptake. Dynamic light scattering (DLS) measurements showed consistent enlargement of reversed micelles upon addition of the bile salts; the hydrodynamic radius increased sixfold in the presence of 10 mM CHAPS and doubled in the presence of 5 mM NaTC. Inclusion of the enzyme yeast alcohol dehydrogenase (YADH) increased the percolation temperature and distorted the spherical structure of the AOT reversed micelles. The spherical structure was restored upon addition of bile salt. These results may help to explain the increase in activity of YADH in AOT reversed micelles upon addition of bile salts.