Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography

A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 mM beta-cyclodextrin, 5 mM 2-hydroxypropyl-beta-cyclodextrin, 50 mM SDS and sodium borate-dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C(18)-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 microM. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.     

Noninvasive methods to determine the critical micelle concentration of some bile acid salts

In this work the critical micelle concentrations (cmc) of four bile salts, sodium cholate, sodium glycocholate, sodium deoxycholate, and sodium glycodeoxycholate, are determined and presented. Three independent noninvasive methodologies (potentiometry, derivative spectrophotometry, and light scattering) were used for cmc determination, at 25 degrees C with ionic strength adjusted to 0.10 M with NaCl. Spectrophotometric and potentiometric studies of some bile salts were also executed at various ionic strength values, thus allowing the influence of the ionic strength on the cmc value of the bile salt to be assessed. A critical comparison of the cmc values obtained with data collected from the literature is presented. Furthermore, this work makes an evaluation of the conceptual bases of different methodologies commonly used for cmc determination, since variations in the results obtained can be related mainly to different intrinsic features of the methods used (such as sensitivity or the need to include tracers or probes) or to the operational cmc definition applied. The undoubted definition of the experimental bile salt concentration that corresponds to cmc (operational cmc) is essential since in the case of these amphiphiles the formation of micelles is not as abrupt as in the case of ordinary association colloids. The biphasic nature of their aggregation leads to a "round-shaped" variation of the experimental parameters under analysis, which makes difficult the evaluation of the cmc values and can be responsible for the different results obtained.     

Rapid determination of logarithmic partition coefficients between n-octanol and water using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) was evaluated as a rapid screening tool for the determination of logarithmic partition coefficients between n-octanol-water (logP_ow). The technique is performed by electrochromatographing a mixture of standards of known log P_ow. The logarithmic capacity factor of each standard was plotted against its log P_ow to form a linear calibration curve for a given set of chromatographic conditions. The log P_ow of an unknown is calculated by using its chromatographically determined capacity factor and extracting the log P_ow value from the calibration curve. The method was evaluated with a set of model compounds with known log P_ow. The accuracy of the method was examined and found to be within the limits required for screening purposes. The correlation of log P_ow values determined using HPLC and MECC for some novel compounds was examined. This technique allows the screening of log P_ow at a rate of four samples per hour with minimal sample requirements (<μg) and with extremely small solvent waste generated.     

The effect of tyrosine conjugation on the critical micellar concentration of free and glycine-conjugated bile salts

We investigated the effect of conjugation with the aromatic amino acid tyrosine on the critical micellar concentration (CMC) of bile salts. The CMC values were determined by surface tension and by dye solubilization. The surface tension measurement employed the Du Nouy ring detachment method and the dye solubilization measurement utilized a water-insoluble dye, 1-O-tolylazo-2-naphthol. We compared the CMC values of the sodium salts of cholyltyrosine (cholylTyr), deoxycholyltyrosine (deoxycholylTyr), deoxycholylglycyltyrosine (deoxycholylGlyTyr) chenodeoxycholyltyrosine (chenodeoxycholylTyr), chenodeoxycholylglycyltyrosine (chenodeoxycholylGlyTyr), cholyldiglycyltyrosine (cholylGlyGlyTyr) and cholylglycyltyrosine (cholylGlyTyr) with their respective glycine conjugated bile salts. Both techniques of CMC determination indicated that tyrosine conjugation to free and glycine-conjugated bile salts reduced their CMC significantly.     

Synthesis and haemolytic activity of novel salts made of nicotine alkaloids and bile acids

A series of novel salts made of nicotine alkaloids and bile acids were synthesized and their haemolytic activity was examined in vitro using human erythrocytes. All compounds were characterized by spectroscopic methods. The novel salts show membrane-perturbing properties inducing the erythrocyte shape alterations and haemolysis in dose-dependent manner. Nicotine decreases the membrane interacting potential of bile acids in the novel compounds. The presence of sulfur or selenium atom in the nicotine molecule affects the haemolytic activity of its novel salts depending on the hydrophobicity of bile acids.     

On-line concentration of neutral analytes for micellar electrokinetic chromatography: VI. Stacking using reverse migrating micelles and a water plug

Utility of a second enhanced field zone (water zone) is investigated for the on-line concentration of neutral analytes in micellar electrokinetic chromatography. Micellar solutions of sodium dodecyl sulfate prepared in acidic phosphate buffers are used as separation and sample solutions. Prior to long hydrodynamic injection of samples prepared in a low conductivity matrix, a long water plug is hydrodynamically injected to provide a second enhanced field zone. Practical and some fundamental considerations are presented. The technique is selective towards hydrophobic analytes. Notable detector response improvements (>100-fold) for several analytes are observed experimentally.     

Bile salts of fishes collected on the Zaire River Expedition (1974-5): their chemical nature and its possible significance

Bile salts from 21 fish species caught during the Zaïre River Expedition (1974–5) have received partial or complete chemical analysis. Conclusions from the results are as follow. (1) Polypterus bile salts are biochemically advanced, but with easily detectable features regarded as relicts from ancestral stock. The bile acids include haemulcholic acid, previously found only in a marine teleost and an unidentified acid also found in a mormyrid. (2) This mormyrid has advanced bile salts, consisting almost entirely of taurine conjugates of haemulcholic acid (present in Polypterus) , and two other C₂₄ acids. (3) Three Alestes and three Distichodus species have no more than traces of primitive bile salts; their bile acids suggest a degree of activity of intestinal microorganisms during the enterohepatic circulation not previously noticed in fishes. (4) The predatory Hydrocynus has bile acids of an omnivorous rather than a carnivorous type. (5) The Barbus species have primitive bile salts closely similar to those of Cyprinidae previously examined but containing also (in two species) a newly-discovered bile alcohol sulphate: in contrast, a Varicorhinus does not have this new substance and has a chief bile salt found so far only as a minor constituent in the Cyprinidae. (6) Two Labeo species are different from all other cyprinids examined: diey both have a new major bile alcohol sulphate. (7) Four Siluriformes show evidence of enterohepatic changes that might be characteristic of some tropical freshwater fish; three have taurine-conjugated haemulcholic acid. (8) Synodontis species are remarkable in having a high proportion of unconjugated bile acid ions and this finding was confirmed with fresh material.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

Determination of a cardiac antiarrhythmic,tricyclic antipsychotics and antidepressants in human and animal urine by micellar electrokinetic capillary chromatography using a bile salt

A micellar electrokinetic capillary chromatographic method based on the use of sodium taurodeoxycholate has been developed to detect and quantitate a total of 26 tricyclic drugs. Detection limits in urine down to 4 ng/ml have been obtained. The method uses a simple liquid-liquid extraction and recovery of analytes followed by ultraviolet detection.     

Effect of lignocaine on blood lipid in relation to partition coefficient and biological activity.

To correlate lipophilicity of lignocaine with changes in lipid composition of blood as a result of in vitro incubation with the drug, phosphorus content and fatty acid compositions of blood lipids before and after lignocaine treatment have been compared with those of a standard phospholipid, lecithin, under similar conditions of drug treatment. The change in fatty acid constituents has been correlated with the biological activity (both therapeutic and toxic) of lignocaine.     

Bile acid structure-activity relationship: evaluation of bile acid lipophilicity using 1-octanol/water partition coefficient and reverse phase HPLC

Two independent methods have been developed and compared to determine the lipophilicity of a representative series of naturally occurring bile acids (BA) in relation to their structure. The BA included cholic acid (CA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), hyodeoxycholic acid (HDCA), ursocholic acid (UCA), hyocholic acid (HCA), as well as their glycine and taurine amidates. Lipophilicity was determined using a 1-octanol/water shake-flask procedure and the experiments were performed at different pH and ionic strengths and at initial BA concentrations below their critical micellar concentrations (CMC) and the water solubility of the protonated form. The experimental data show that both the protonated (HA) and ionized (A-) forms of BA can distribute in 1-octanol, and consequently a partition coefficient for HA (logP' HA) and for A- (logP' A-) must be defined. An equation to predict a weighted apparent distribution coefficient (D) value as a function of pH and pKa has been developed and fits well with the experimental data. Differences between logP for protonated and ionized species for unconjugated BA were in the order of 1 log unit, which increased to 2 for glycine-amidate BA. The partition coefficient of the A- form increased with Na+ concentration and total ionic strength, suggesting an ion-pair mechanism for its partition into 1-octanol. Lipophilicity was also assessed using reverse phase chromatography (C-18-HPLC), and a capacity factor (K') for ionized species was determined. Despite a broad correlation with the logP data, some BA behaved differently. The logP values showed that the order of lipophilicity was DCA greater than CDCA greater than UDCA greater than HDCA greater than HCA greater than CA greater than UCA for both the protonated and ionized unconjugated and glycine-amidate BA, while the K' data showed an inversion for some BA, i.e., DCA greater than CDCA greater than CA greater than HCA greater than UDCA greater than HDCA greater than UCA. The logP data fitted well with other indirect measurements of BA monomeric lipophilicity such as albumin binding or accessible total hydrophobic surface area data calculated by energy minimization and molecular computer graphics. Differences between unconjugated and amidated BA are consistent with the presence of an amide bond and a lower pKa when pH dependence was studied. Capacity factors, on the other hand, were related to properties of BA micelles such as cholesterol-solubilizing capacity and membrane disruption, reflecting the BA detergency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of an extracellular factor that stimulates bile salt hydrolase activity in Lactobacillus sp. strain 100–100

Bile salt hydrolase activity in Lactobacillus sp. strain 100-100 is strictly intracellular. The strain produces an extracellular factor that stimulates the intracellular hydrolase activity. The factor is inducible by conjugated bile salts, has an apparent molecular mass over 12 kDa but less than 25 kDa, is stable in air, and resistant to pronase and heat. It is partially extractable into organic solvents and inactivated by a sulphydryl group inhibitor. We postulate that the factor functions by a novel mechanism to facilitate entry of conjugated bile salts into the bacterial cells.     

Effect of aspirin on blood-lipid interaction and lipid peroxidation phenomena in relation to partition coefficient and biological activity

Considering the lipophilicity of aspirin (log P = -1.15), a significant contributor to its action mechanism, interaction of the drug with the whole lipids of goat blood have been investigated using phospholipid binding and lipid peroxidation phenomena as the parameters under investigation. The lipid content change along with the peroxidation induced by aspirin and its suppression with ascorbic acid had been quantitatively measured. Significant loss in phospholipid was observed after incubation of whole blood with aspirin in varying periods of time. This may be ascribed to binding affinity of aspirin with lipid constituents in blood, which may have potential role in its therapeutic effect. Lipid peroxidation induction potential of aspirin caused significant extent of peroxidation. Ascorbic acid, an antioxidant could significantly reduce aspirin induced lipid peroxidation.     

Enantioseparation of ofloxacin and its four related substances with ligand exchange–micellar electrokinetic chromatography using copper(II)‐L‐isoleucine complex as chiral selector

A ligand‐exchange micellar electrokinetic capillary electrophoresis system with copper(II)‐L‐isoleucine complexes as the chiral selector incorporated in micelles of sodium dodecyl sulfate (SDS) was developed for the enantioseparation of ofloxacin and its four related substances (impurities A, C, E, and F). The effects of important parameters affecting separation such as buffer pH, SDS concentration, chiral selector concentration, and organic additive were investigated in detail. Under optimum experimental conditions, enantioseparation of ofloxacin, impurities A, C, E, and F enantiomers was accomplished with resolutions of 4.28, 2.83, 3.40, 3.58, and 2.46, respectively. Further, simultaneous separation of impurities A, C, E, and F enantiomers was achieved using 10 mmol/L NH₄OAc as the running buffer containing 4 mmol/L copper sulfate,20 mmol/L L‐isoleucine, 20 mmol/L SDS, and 5% methanol at pH 8.5. To the best of our knowledge, the simultaneous enantioseparation of four impurities of ofloxacin has not been reported previously.     

Analysis of creatinine, vanilmandelic acid, homovanillic acid and uric acid in urine by micellar electrokinetic chromatography

A simultaneous determination of vanilmandelic acid, homovanillic acid, creatinine and uric acid using capillary electrophoresis was investigated. The optimum conditions of buffer concentration, pH and surfactant concentration were studied, and high resolution was obtained using a 30 mM phosphate buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate. The detection was by UV absorbance at 245 nm and the column was a fused-silica capillary of 67 cm×75 μm I.D.. The determination of these metabolites in human urine was completed within 15 min without any interferences.     

Specific optical rotation is a versatile tool for the identification of critical micelle concentration and micellar growth of tartaric acid–based diastereomeric amphiphiles

Four novel tartaric acid–based diastereomeric chiral amphiphiles, two being enantiomers of the other two, have been synthesized and investigated using chiroptical spectroscopic methods, along with tensiometry and dynamic light scattering experiments. We found that an inflection point in specific optical rotation (SOR) values at ~0.32 mM corresponds to the critical micelle concentration (CMC). The increase in magnitude of SOR values beyond CMC corresponds to the growth of aggregates. For enantiomers, oppositely signed SOR values were observed, ruling out the possibility for the presence of aggregation size mediated artefacts. SOR values did not exhibit concentration dependence for a chiral tartaric acid based non‐aggregating analogue further establishing the absence of artefacts or anomalous interaction of tartaric acid based head group with solvent. Electronic circular dichroism spectra showed no significant changes in band positions or intensities with concentration. Due to the requirement for higher concentrations (~200 mM) needed to obtain vibrational circular dichroism spectra, these measurements are not found to be useful for studying concentration dependent properties of chiral amphiphiles.     

The influence of mixed salts on the capacity of HIC adsorbers: A predictive correlation to the surface tension and the aggregation temperature

Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in downstream processing of biopharmaceuticals. During HIC, salts are the governing additives contributing to binding strength, binding capacity, and protein solubility in the liquid phase. A relatively recent approach to increase the dynamic binding capacity (DBC) of HIC adsorbers is the use of salt mixtures. By mixing chaotropic with kosmotropic salts, the DBC can strongly be influenced. For salt mixtures with a higher proportion of chaotropic than kosmotropic salt, higher DBCs were achieved compared with single salt approaches. By measuring the surface tensions of the protein salt solutions, the cavity theory—proposed by Melander and Horváth—that higher surface tensions lead to higher DBCs, was found to be invalid for salt mixtures. Aggregation temperatures of lysozyme in the salt mixtures, as a degree of hydrophobic forces, were correlated to the DBCs. Measuring the aggregation temperatures has proven to be a fast analytical methodology to estimate the hydrophobic interactions and thus can be used as a measure for an increase or decrease in the DBCs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:346–354, 2016     

Octanol–water partition of nonzwitterionic peptides: Predictive power of a molecular size-based model

A remarkably simple, molecular size-based model developed to predict octanol–water partition coefficients for organic compounds is tested on a set of 188 neutral peptides with available experimental partition data. Despite using only two parameters, it gives a promising correlation (r² = 0.914; σ = 0.455, F = 1978.0), and predictions are in a realistic range even for larger peptides (cyclosporin, melanotan, sandostatin) where common, overparametrized fragment methods become quite unreliable. Ion-pair partitioning and the extraction constant formalism is briefly reviewed to describe the sigmoidal lipophilicity profile of ionizable, nonzwitterionic peptides. It seems possible to extend the present model to estimate apparent partition coefficients measured around neutral pH and physiological conditions for monoionic peptides; however, as no standard conditions are yet defined and only relatively small number of experimental data are available, the situation here is more complex. Proteins 30:86–99, 1998. © 1998 Wiley-Liss, Inc.