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.     

The use of Bond-Elut for the estimation of serum bile pigments bonded covalently to albumin

A simple and precise method has been devised for the quantitation of biliprotein (delta-bilirubin or albumin bound bilirubin) in serum. In the presence of caffeine/benzoate, Bond-Elut (C8, 200 mg) extracts unconjugated and conjugated bilirubin but not pigments that are covalently bonded to albumin which pass through the column and can be quantitated by a standard diazo method. Following elution from the Bond-Elut column with methanol-acetonitrile (50:50, v/v) unconjugated and conjugated bilirubin can be quantitated either as total pigments or individually by HPLC.     

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).     

Study of biologically relevant physical-chemical properties of bile salts by reverse-phase liquid chromatography

Summary The study of chromatographic behaviour of bile salts with reverse-phase HPLC columns (C18 and C8) and buffered water — methanol eluent systems indicated that the octanol-water partition coefficient and the micellar cholesterol-solubilizing capacity are linearly correlated with the chromatographic mobilities if eluents with pH>7 and 0.154 M saline concentration are used (correlation coefficients 0.98 and 0.86 respectively). The critical micellar concentration was poorly correlated with the chromatographic mobility under the various conditions tested. The pKa values of bile salts were determined by the changes of mobility with the pH of eluent. Values in the range 4.92–5.06 and 4.27–4.47 were found respectively for unconjugated and glycineconjugated bile salt, in agreement with previous estimates. Values in the range 0.74–1.02 represent the first direct evaluation for taurine-conjugates.     

High-performance liquid chromatographic separation of bilirubin conjugates: the effects of change in molarity and pH

A fast, sensitive high-performance liquid chromatographic method has been developed for the separation and quantitation of biliary bile pigments; this utilizes a C18 reversed-phase column with two solvents, a buffer and an organic solvent, which were changed in a linear gradient from a polar to a less polar combination. Nine glycosidic conjugates of bilirubin as well as unconjugated bilirubin and a suitable internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution; the species eluted in a polar to less polar fashion. Increasing the molarity of the solvent decreased the binding of non-glucuronide pigments to the column, with a decrease in their retention times, whereas for bilirubin monoglucuronide they increased. Decrease in pH, similarly, preferentially increased bilirubin monoglucuronide retention times.     

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.     

游离胆红素与固体碳酸钙的相互作用

应用红外光谱的方法研究了溶液中游离胆红素与固体碳酸钙的相互作用,探讨了色素型胆结石的形成机理.研究结果表明,溶液中游离的胆红素可以与固体碳酸钙作用,游离胆红素通过与固体碳酸钙表面的钙作用生成难溶性的胆红素钙络合物.牛磺胆酸钠、胆酸钠和脱氧胆酸钠的加入能阻止游离胆红素与碳酸钙的作用,其中加入牛磺胆酸钠后,游离胆红素几乎不与碳酸钙作用.     

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.     

The Separation of Conjugated and Unconjugated Bilirubin in Bile by High-Performance Liquid Chromatography

Abstract

A fast and simple method was developed for the separation of unconjugated bilirubin and its mono- and di-glucuronide conjugates from bile by high-performance liquid chromatography (HPLC). Unconjugated bilirubin was separated on a reversed-phase column using acetonitrile-water (70:30 v/v) as the mobile phase, while the conjugates were separated on a μ-Bondapak-carbohydrate column employing acetonitrile-water (90:10 v/v) as the eluent. The application of this method was demonstrated by the analysis of the bile pigments in rat bile.     

胆酸盐在酸性介质中自聚集作用的共振瑞利散射光谱及其分析应用研究

研究了牛磺胆酸钠、甘牛胆酸钠、胆酸钠和脱氧胆酸钠等四种胆酸盐在酸性介质中的聚集作用对共振瑞利散射光谱的影响及其分析应用. 结果表明: 在一定浓度的HCl, H₂SO₄或HNO₃溶液中, 四种胆酸盐均能自聚集形成粒径增大的聚集体. 该聚集体能导致溶液RRS显著增强, 并产生了新的RRS光谱. 不同胆酸盐在同种介质中的反应产物具有相似的光谱特征, 最大RRS波长分别位于349 (HNO₃介质), 359 (H₂SO₄介质)和369 nm (HCl介质). 在一定范围内, 胆酸盐的浓度与散射强度(ΔI_RRS)成正比. 对于不同的体系其检出限在12.0～21.8 ng/mL之间. 方法灵敏度高, 选择性较好, 而且十分简便快速. 可用于市售蛇胆川贝液和血清样品中胆酸盐的测定.     

Importance of Conjugation of the Bile Salt on the Mechanism of Lipolysis

We aim to advance the discussion on the significance of the conjugation of bile salts (BS) in our organism. We hypothesize that conjugation influences the rate of lipolysis. Since the rate of lipolysis is a compound parameter, we compare the effect of conjugation on four surface parameters, which contribute to the rate. Since deconjugation is due to gut microbiota, we hypothesize that microbiota may affect the rate of lipolysis. A meta-analysis of literature data of critical micelle concentration, β, aggregation number, and molar solubilization ratio has been performed for the first time. In addition, critical micelle concentration (CMC), interfacial tension, and lipolysis rate measurements were performed. It was found that the unconjugated BS in mixed micelles increases the antagonism between the BS, therefore, increasing the CMC. This correlated with the effect of unconjugated BS on the solubilization capacity of mixed micelles. The collected literature information indicates that the role of the BS and its conjugation in our organism is a key factor influencing the functioning of our organism, where too high levels of unconjugated BS may lead to malabsorption of fat-soluble nutrients. The experimental lipolysis results irrevocably showed that conjugation is a significant factor influencing the rate.     

Ionic effect on the binding of bilirubin to the imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate)

A molecularly imprinted polymer (MIP) capable of detecting bilirubin was successfully synthesized. Bilirubin template was imprinted in poly(methacrylic acid-co-ethylene glycol dimethylacrylate) [poly(MAA-co-EGDMA)]. MAA and EGDMA were used as the monomer and the cross-linker, respectively. The optimal solvent conditions to maintain its stability were discussed. Solvent system based on ethylenediamine tetraacetic acid (EDTA) and ascorbic acid was compared with respect to the stability of bilirubin. pH and bilirubin concentration were both investigated for the bilirubin stability. Blue light as well as aeration was applied to inspect the regarding effects. The cross-linking effect was further confirmed by the thermogravimetric analysis (TGA). The effect of salts, such as NaCl and KCl on the binding capacity of the molecularly imprinted polymer was also discussed. Further, the rat serum and bile samples were applied and the binding of the MIPs for bilirubin was thus confirmed.     

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。     

Micellization of bile salts in a formamide solution: A gas liquid chromatography study

Sodium cholate and sodium deoxycholate dissolved in formamide were applied as stationary phases in gas chromatography. The critical micelle concentration of sodium cholate and deoxycholate in formamide was determined by surface tension measurements. The relation of retention times vs. concentration of bile salts was investigated for isomers of monoterpenes and xylenes. The enthalpy of binding of selected compounds with sodium cholate and sodium deoxycholate monomers and micelles was determined.     

Thin-layer chromatography/liquid secondary ion mass spectrometry in the determination of major bile salts in dog bile

Positive and negative ion liquid-state secondary-ion mass spectrometry (LSIMS) was applied to several bile acids and bile salts and their spectra were measured directly from the surface of silica gel thin-layer chromatograms. Such spectra were identical to the LSIMS spectra of the pure compound at the same concentration. Three-dimensional ion images were obtained of a model mixture of cholic, chenodeoxycholic and lithocholic acids in both the positive and negative ion modes. A sample of dog bile was prepared, and the bile acids extracted from it were separated by high-performance TLC; TLC/LSIMS spectra were obtained for sodium taurocholate and sodium taurochenodeoxycholate/taurodeoxycholate, the predominant bile salts present. Quantitative estimates of the analytes were obtained by monitoring the ion intensity for the sample spot and a standard spot that had been developed in parallel on the same TLC plate. The concentration of sodium taurocholate in the bile of this particular dog was found to be 38 mg/ml.     

Solubilization and precipitation of cholesterol in aqueous solution of bile salts and their mixtures

Solubilization of cholesterol by mixed micelles of sodium chenodeoxycholate with sodium ursodeoxycholate was investigated in carbonate-tetraborate buffer (Kolthoff) solution at pH 10 and 37°C. It was found that the mixing of the two bile salts gives a negatively synergetic effect on solubilization of cholesterol. The solubilizing power of bile salts for cholesterol was remarkably influenced with the change in mole fraction of sodium ursodeoxycholate (X _UDC).The behavior of bile salt solutions saturated with cholesterol was examined by measuring the surface tension. Two break points were observed in the curves of surface tension vs. concentration. The break points seem to correspond to a CMC in the absence of solubilized cholesterol and another CMC in the presence of solubilized cholesterol inside bile salt micelle.     

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.     

毛细管胶束电动色谱法分离测定胆红素亚组分

 用毛细管胶束电动色谱法成功地分离和定量测定了血清及胆汁中α，β，γ及δ等４种胆红素亚组分。二牛磺酸胆红素浓度在５１０μｍｏｌ／Ｌ以下时，峰面积与浓度呈良好的线性关系，二牛磺酸胆红素浓度即使为１．５μｍｏｌ／Ｌ时仍可检测出明显的定量峰。用一份２５５μｍｏｌ／Ｌ的二牛磺酸胆红素标准液进行重复性检测，第１次定量结果为２６０μｍｏｌ／Ｌ，出峰时间１６ｍｉｎ；第５０次电泳结果为２３８μｍｏｌ／Ｌ，出峰时间１６．７ｍｉｎ。     

Effects of high taurocholate load on activities of hepatic alcohol metabolizing enzymes

Membrane-associated cytotoxicity induced by hydrophobic bile salts is a major contributing factor leading to liver diseases. Administration of ursodeoxycholate reduces serum liver enzymes in chronic liver diseases but the nature of this effect is still unclear. Using alcohol metabolising enzymes as cellular markers, the hepatotoxic properties of hydrophobic bile salts and the putative hepatoprotective effect of ursodeoxycholate was examined. Two animal models of biliary retention, bile duct obstruction and choledochocaval fistula was used to investigate the effect of taurocholate on the hepatic alcohol metabolizing enzymes: cytosolic alcohol dehydrogenase, microsomal ethanol oxidizing system, catalase and aldehyde dehydrogenase before and after the infusion of taurocholic acid or tauroursodeoxycholic acid for two days period. Bile duct obstruction was found to be similar to or slightly exceeds choledochocaval fistula in the degree of retention. Following the taurocholic acid infusion, the serum alcohol dehydrogenase activity as well as microsomal ethanol oxidizing system and aldehyde dehydrogenase were greatly increased but the level of cytosolic alcohol dehydrogenase and catalase activities was found to be lower in either or both models in comparison with the control animals. However, the tauroursodeoxycholic acid infusion did not induce any significant changes in the levels of all the alcohol metabolizing enzyme activities in either or both models. These findings suggest that hydrophobic taurocholic acid (7alpha) affects the plasmalemma to allow leakage of cytosolic alcohol dehydrogenase into the blood circulation, stimulates the biosynthesis of microsomal ethanol oxidizing system and aldehyde dehydrogenase, and suppresses the biosynthesis of alcohol dehydrogenase and catalase. But in contrast, the hydrophilic tauroursodeoxycholic acid (7beta) provided hepatoprotective effect.     

Colloidal Stability and Cytotoxicity of Polydopamine-Conjugated Gold Nanorods against Prostate Cancer Cell Lines

Prostate cancer is one of the most common cancers in men. Cell invasion is an important step in the process of cancer metastasis. Herein, gold nanorods (GNRs) and polyethylene glycol (PEG)-coated GNRs were conjugated with polydopamine (PDA). The PDA-nanoconjugates demonstrated excellent colloidal stability upon lyophilization and dispersion in cell culture media with or without the addition of fetal bovine albumin (FBS), compared to unconjugated GNRs. PDA-nanoconjugates exhibited a considerable cytotoxicity against DU-145 and PC3 prostate cancer cell lines over a concentration range of 48 μg/mL–12 μg/mL, while they were biocompatible over a concentration range of 3.0 μg/mL–0.185 μg/mL. Furthermore, PDA-nanoconjugates demonstrated possible anti-invasion activity towards prostate cancer cell lines, particularly DU-145 cell line, by reducing cell migration and cell adhesion properties. The PDA-nanoconjugates could be considered a promising nano-platform toward cancer treatment by reducing the invasion activity; it could also be considered a drug delivery system for chemotherapeutic agents.