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.     

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.     

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.     

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.     

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).     

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.     

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.     

Osmotic and activity coefficients of aqueous bile salt solutions at 25, 37, and 45°C

Osmotic coefficients measured by vapor pressure osmometry are reported for aqueous bile salt solutions at 25, 37, and 45°C. Solute activities decrease rapidly with increasing concentration due to premicellar association and micelle formation. In all cases, activities of the dihydroxy bile salts are lower than those of the trihydroxy bile salts. Osmotic coefficients and activity coefficients increase with increasing temperature. It is suggested that hydrophobic forces contribute substantially to the stability of primary bile salt aggregates and micelles.     

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.     

Spin and fluorescence label studies on bile salt micelles

The interior structure of micelles formed by bile salts, which differ in the number and location of the hydroxyl groups attached to the steroid nucleus, was studied by the spin label and fluorescence label methods. The results show that the interior structure of micelles formed by bile salts possessing two hydroxyl groups is more rigid than that of micelles formed by trihydroxy bile salts regardless of the terminal hydrophilic group. Even in the case of dihydroxy bile salts possessing two hydroxyl groups in the same location, the interior structures of their micelles are different from each other depending on the orientation of their hydroxyl groups. It is considered that hydroxyl groups as well as the terminal hydrophilic group play an important role in the micellar formation of bile salts.     

Measurement of bilirubin partition coefficients in bile salt micelle/aqueous buffer solutions by micellar electrokinetic chromatography

The partition coefficients for the distribution of bilirubin between aqueous phosphateborate buffer and cholic, taurocholic, taurodeoxycholic, and taurochenodeoxycholic micelles have been measured by micellar electrokinetic chromatography at pH 8.5. Determination of the partition coefficients required that the critical micelle concentration and partial specific volumes be determined for each bile salt. Critical micelle concentrations were slightly higher for the trihydroxy bile salts. Partial specific volumes of the bile salt micelles differed very little from each other, and for each bile salt they were constant over the concentration range studied, which was typically from slightly above the critical micelle concentration to 35 mM. Capacity factors were corrected for the effects of applied voltage by extrapolation of the capacity factor to zero applied volts. The free solution mobility of bilirubin, determined in the absence of bile salt, was also corrected for the effects of applied voltage. Plots of extrapolated capacity factor versus phase ratio yield the partition coefficient as the slope of a linear fit to the data. Partition coefficients for bilirubin were significantly higher for dihydroxy bile salts than for trihydroxy bile salts.     

水溶性壳聚糖降血脂作用的化学机理(Ⅰ)──水溶性壳聚糖及其衍生物对胆酸盐的结合能力研究

通过壳聚糖氧化裂解,制备了分子量为8000的水溶性壳聚糖,并通过烷基化反应合成了二乙氨乙基壳聚糖、二甲氨基(1-甲基)乙基壳聚糖及二乙基甲基铵乙基壳聚糖.在体外测定了水溶性壳聚糖及其衍生物对胆酸盐(牛磺胆酸钠和甘氨胆酸钠)的结合能力及其影响因素.结果表明,水溶性壳聚糖结合胆酸盐的能力主要取决于其阳离子化程度.修饰后的壳聚糖结合胆酸盐的能力增强,说明引入更多的胺基或铵基有利于对胆酸盐的结合.     

The influence of the ionic medium on the behaviour of sodium glycocholate and cholate aqueous solutions

In the framework of research on the sodium bile salts, solutions of sodium cholate and glycocholate had been studied only at three ionic medium concentrations. In this paper, experiments in two other concentrations of ionic medium are carried out both to verify the behaviour dependence on the ionic medium and to apply the results of this paper to a future investigation on solutions containing contemporary sodium cholate and glycocholate. To this aim, solubility, protonation constants, lead (II) salts solubility products at 25 degrees C and in 0.300 and 0.750 mol dm(-3) N(CH3)4Cl were determined. The formed species in solutions containing lead (II) together with the selected bile salts and the behaviour of the sodium salts in micellar and premicellar solutions were investigated, too. The obtained results were compared with those obtained at the other ionic medium concentrations.     

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.     

Micellar electrokinetic chromatography of tri aza aromatic ligand compounds of iron (II): influence of bile salt type on enantiomeric separation.

Micellar electrokinetic chromatography is used with a variety of bile salt micelles to separate the enantiomers of bis(8-((pyridine-2-methylene)amino)quinoline)iron(II) hexafluorophosphate, Fe(PMAQ)2(PF6)2; bis(8-((pyridine-2-methylene)amino)lepidine iron(II) hexafluorophosphate, Fe(PMAL)2(PF6)2; and bis(1-(2-pyridinyl)ethylidine)-8-aminoquinoline iron(II) hexafluorophosphate, Fe(PEAQ)2(PF6)2. The influence of ten different bile salts on the resolution of each pair of enantiomers is investigated. Significant changes in resolution are seen depending upon the bile salt used. The dihydroxy bile salts are superior to the trihydroxy bile salts in terms of resolution, and the taurine or glycine conjugated bile salts yield better results than the unconjugated bile salts. Resolution for most enantiomers is maximized in a buffer solution containing 10-15% acetone and employing either taurochenodeoxycholic or glycochenodeoxycholic acid as the bile salt. Evidence for the separation of the corresponding Fe(III) complexes is presented.     

Composition of aqueous solutions containing sodium glycocholate and glycodeoxycholate

Composition and existence range of aggregates formed by sodium glycocholate and glycodeoxycholate contemporary present in aqueous micellar and premicellar solutions were investigated. Solubility measurements of lead (II) glycocholate and glycodeoxycholate give analytical concentration of lead (II) and glycocholate and glycodeoxycholate, respectively. Electromotive force measurements provide the free concentration of hydrogen, sodium and lead (II) ions. Experimental data obtained at 25 degrees C and at three different concentrations of N(CH3)4Cl, used as a constant ionic medium, can be explained by assuming the presence of aggregates of different composition depending on the reagent and ionic medium concentrations. Next to two species containing only glycocholate or glycodeoxycholate, the presence of aggregates formed with the contemporary participation of both bile anions in different ratios was assumed. Species with the hydrogen ion participation are not present in appreciable quantity in the investigated concentration range. As expected, the size of aggregates increases by increasing reagent and ionic medium concentration. Most of the species can be explained with a "core + link" mechanism, where all the glycocholate aggregation numbers are even, while those of glycodeoxycholate are always multiple of three. Analogy and difference with aggregates formed by the two bile anions separately are discussed.     

Bile salt-induced disintegration of egg phosphatidylcholine liposomes: a kinetic study based on turbidity changes

The disintegration kinetics of egg phosphatidylcholine small unilamellar liposomes in various bile salts (nine species) were investigated by monitoring turbidity changes with a stopped-flow apparatus. The pseudo-first-order rate constants obtained as a function of bile salt concentration (up to 25 mM) were analyzed based on a two-step model in which a penetration-saturation step of bile salt into the bilayer and a lamellar-micellar transition step were assumed for the disintegration mechanism of the bilayer. The order of the rate of the penetration-saturation step, which is assumed to be a measure of the disintegration ability, was as follows: SCDOC greater than SDOC greater than STCDOC greater than STDOC greater than STC greater than SC greater than SGCDOC greater than SGDOC greater than SGC. The results indicated that (1) the dihydroxy bile salts have a greater disintegration ability than the corresponding trihydroxy bile salts, (2) the chenodeoxy bile salts have a greater ability than the corresponding deoxy-bile salts regardless of non-conjugated or conjugated form, (3) the taurine conjugates always have a greater ability than the glycine conjugates. The penetration-saturation rate of the bile salts against the lipid bilayer depends considerably on the chemical nature of each bile salt, varying by a factor of about 10(5). In the conjugated bile salts alone, they were in a narrower range of a factor of 10(3). The physical integrity of liposomes can hardly be maintained in the bile salt-rich intestinal tract but the resulting mixed micelles may contribute substantially to solubilization and enhanced delivery of drugs.     

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.     

胆红素与胆汁酸盐作用时的构象变化

利用PPP-SCF-CI-DV量子化学程序拟合胆红素以及胆汁酸盐-胆红素水溶液的UV和CD谱, 借以研究胆红素及其与胆汁酸盐复合物的构象性质。发现胆红素与胆汁酸盐作用时, 两个双吡咯生色团的扭角由104°变成112°, 仍保持形成分子内氢键的状态, 其UV及CD谱两个吸收峰间隔的增宽, 主要是胆红素的一侧双吡咯生色团与胆汁酸盐呈疏水性结合的原故; 二羟基和三羟基胆汁酸盐所形成的二元复合物园二色谱的差别, 是胆红素的对映选择性结合造成的。     

Structure of sodium glycodeoxycholate micellar aggregates from small-angle X-ray scattering and light-scattering techniques

Small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) measurements were accomplished on sodium glycodeoxycholate (NaGDC) aqueous electrolyte solutions as a function of NaGDC and NaCl concentrations with the aim to determine with satisfactory approximation the NaGDC micellar aggregate structure at a gross molecular level, assuming monodispersity. Different conditions of interparticle interactions by varying ionic strength (NaCl concentration from 0 to 0.70 M) and NaGDC concentration (from 0.02 to 0.10 M) were studied. Smeared scattering intensities and electron pair distance distribution functions, radii of gyration, and aggregate heights are in satisfactory agreement with the corresponding functions calculated using a 2(1) helix as model. It is formed by trimers, each one composed by three NaGDC and nine H2O molecules related by a 3-fold rotation axis, and can be described by a hollow cylinder, probably filled by water molecules, characterized by a conventional radius of 23.7 A and a trimer repeat along the helical axis of 3.6 A. The helix is considerably inhomogeneous since the volume of the cylinder occupied by NaGDC molecules is less than one-third of the total. On the other hand, calculations performed with the average radial electronic density of the helix without water molecules or totally filled by water molecules (a NaGDC/H2O molecular ratio of about 1/50) or by using a three-shell average radial electronic density, independently evaluated on absolute scale, do not show significant differences, thus supporting the helical model. The aggregate size increases for all the samples by increasing either the NaCl or NaGDC concentration. The NaGDC low concentration (0.02 M) samples with NaCl within the range 0.30-0.70 M are characterized by short cylindrical aggregates that do not give rise to sensible interference effects. This assertion is supported by the satisfactory fit between the observed apparent mean hydrodynamic radii and the calculated ones by means of the method of Ortega and Garcia de la Torre (J. Chem. Phys. 2003, 119, 9914), valid for rods with a length-to-diameter ratio > or = 0.1 in dilute solution (noninteracting rods). The NaGDC moderate concentration (0.10 M) samples with NaCl within the range 0.20-0.60 M are characterized by cylindrical aggregates that, in the presence of repulsive Coulombic interactions progressively more and more screened, produce interference effects, due to the hard-body repulsion and attractive forces, but the agreement between observed and calculated SAXS data is satisfactory. The results of the low and moderate NaGDC concentration samples seem to indicate that the aggregation number increase, produced by adding 0.10 M NaCl, is constant within an ionic strength range and occurs by the addition of oligomers formed by trimers. The samples with a variable NaGDC concentration (0.02-0.10 M) at a fixed and high NaCl concentration (0.6 M) contain cylindrical aggregates that give rise to an attractive term effect prevailing on the hard-body repulsive one. The same situation seems to occur in the NaGDC moderate concentration samples.