Reactive oxygen species and nitric oxide effect on the steroid hormone binding with serum albumin.

The interaction of a series of steroid hormones (DHEA, progesterone, testosterone, estradiol) with serum albumin immobilized on porous silica particles and the effect of reactive oxygen species and nitric oxide on this interaction were studied using a biochromatographic approach. The determination of enthalpy and entropy changes of this binding indicated that van der Waals interactions and hydrogen bonds predominated the hormone association with albumin. Reactive oxygen species (H(2)O(2) and OH*) increased the hormone binding affinity to albumin. On the other hand, this binding was decreased with the presence of NO*. This variation was due to conformational changes in the binding region explained by the oxidation of some residues such as free thiol and arginine. The thermodynamic analysis showed that free radical affects the van der Waals forces and/or a hydrogen bond of the hormone binding with albumin. These results explained the role of reactive oxygen species and nitric oxide in the hormone free fraction level in the blood.

Magnesium effect on the acetylcholinesterase inhibition mechanism: a molecular chromatographic approach.

The acetylcholinesterase enzyme (AChE) was immobilized on a chromatographic support to study the effect of magnesium on the binding mechanism of five AChE inhibitors (donepezil, tacrine, galanthamine, physostigmine and huperzine). The determination of the enthalpy and entropy changes of this binding at different magnesium concentration values suggested that van der Waals interactions and hydrogen bonds predominated the donepezil and tacrine association to AChE. As well, hydrophobic and electrostatic forces seemed to be the major interactions controlling the huperzine, galanthamine and physostigmine association with AChE. In addition, it appeared that magnesium cation increased the binding affinity of galanthamine and physostigmine to the active site gorge of AChE. A comparison of the inhibitors hydrophobicity to their relative bound percentage with AChE showed an affinity enhanced with the increase in the molecule hydrophobicity and confirmed that the hydrophobic forces played an important role in the AChEI-AChE binding process. This novel biochromatographic column could be useful to find a specific inhibitor for this enzyme and so open new perspectives to be investigated.

Magnesium effect on testosterone-SHBG association studied by a novel molecular chromatography approach.

A biochromatographic approach is developed to measure for the first time thermodynamic data and magnesium (Mg(2+)) effect for the binding of testosterone (TT) to sex hormone-binding globulin (SHBG) in a wide temperature range. For this, the SHBG was immobilized on a chromatographic support. It was established that this novel SHBG column was stable during an extended period of time. The affinity of TT to SHBG is high and changes slightly with the Mg(2+) concentration because the number of Mg(2+) linked to binding is low. The determination of the testosterone retention with the steroid hormone at different Mg(2+) concentrations and temperatures demonstrated that the Mg(2+) binding heat effect associated with this Mg(2+) release or uptake during this binding was in magnitude around 17kJ/mol corresponding to the model describing the electrostatic attraction that occurs between the negatively charged non specific areas of SHBG and the positively charged of magnesium. At all the magnesium concentrations studied, the DeltaH values were negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong TT-SHBG hydrogen bond networks. As well, the DeltaS values were all positive due to hydrophobic forces in the testosterone-SHBG complex formation. In addition our results suggest that adaptive conformational transitions contribute to the specific testosterone-SHBG complex formation. As well, in the biological Mg(2+) concentration domain, it was clearly demonstrated that there was an uncompetitive inhibition of Mg(2+) on TT-SHBG binding which led an enhancement of bioavailable TT. Our work indicated that our biochromatographic approach could soon become very attractive for study other SHBG-steroid (or phytoestrogen) binding.

Biochromatographic framework for analyzing magnesium chloride salt dependence on nor-NOHA binding to arginase enzyme.

Our group demonstrated recently that arginase I inhibition reduces endothelial dysfunction and blood pressure rising in spontaneously hypertensive rats [C. Demougeot, A. Prigent-Tessier, C. Marie, A. Berthelot, J. Hypertens. 23 (2005) 971; C. Demougeot, A. Prigent-Tessier, T. Bagnost, C. Andre, Y. Guillaume, M. Bouhaddi, C. Marie, A. Berthelot, Life Sci. 80 (2007) 1128]. This discovery opens interesting perspectives in the development of new drugs against hypertension. As well, in a previous paper [T. Bagnost, Y.C. Guillaume, M. Thomassin, J.F. Robert, A. Berthelot, A. Xicluna, C. Andre, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 856 (2007) 113], a novel biochromatographic column was developed in our laboratory for studying the binding of N(omega)-hydroxy-nor-l-arginine (nor-NOHA), an arginase inhibitor, with this enzyme. In this manuscript, using this novel biochromatographic concept, the effect of magnesium chloride on the nor-NOHA/arginase binding was analyzed for the first time. This study demonstrated that the salt ions interacted with arginase and played a great role in the nor-NOHA/arginase association. For a salt concentration (x) in the medium less than 3mM, the nor-NOHA/arginase binding decreased with x due to a decrease of the charge-charge interactions between nor-NOHA and its arginase binding site. Above 3mM of salt in the medium, the affinity of nor-NOHA to arginase increased slightly with x because the net number of ions (n) (Mg(2+) or Cl(-)) released or bound upon complex formation is low. As well, it was clearly demonstrated, that above 3 mM the n value depend on the salt concentration in the bulk solvent and was approximately nil for x=12 mM. This dependence was due to a gradual and conformational change of the arginase enzyme which around 12 mM adopted a less flexible structure; its binding site was thus less accessible to nor-NOHA and nor-NOHA-arginase association decreased slightly.

In vitro antimicrobial activity of the leaf extract of Harungana madagascariensis Lam. Ex Poir. (Hypericaceae) against strains causing otitis externa in dogs and cats.

Otitis externa in dogs and cats is always caused by a combination of yeasts and bacteria, among which the most important are Malassezia pachydermatis, Staphylococcus intermedius and Pseudomonas species. These organisms often develop resistance to classical antimicrobial agents. Therefore, the aim of this study was to investigate the antimicrobial activities of an ethyl acetate leaf extract of Harungana madagascariensis against the organisms cited, to carry out the phytochemical investigation of this extract and to determine its bioactive chemical class using dilution techniques, the bioautography method and the standard phytochemical method described by Harborne (1973). Phytochemical analysis revealed the presence of saponins, tannins, flavonoids, alkaloids and anthracenic derivatives. The bioassay showed that the antimicrobial properties may be attributed to astilbin, a flavanone derivative identified on the basis of its spectroscopic data. The results suggest that the extract could be used in an antimicrobial preparation effective against the whole range of organisms incriminated in otitis externa in dogs and cats, with a minimal inhibitory concentration (MIC) of 250 microg/ml.

A stepwise stoichiometric representation to confirm the dependence of pesticide/humic acid interactions on salt concentration and to test the performance of a silica bonded humic acid column.

In a previous paper (André et al., in press), a novel chromatographic column was developed in our laboratory for studying the binding of pesticides with humic acid (HA), the main organic component in soil. It was demonstrated that this column supported a low fraction of organic modifier in the aqueous mobile phase (<0.25 (v/v)). To overcome this limitation for a practical use, a column in which the stationary phase was based on silica gel with chemically bonded humic acid was created. It was shown that this novel HA column supported a higher methanol fraction (<0.55 (v/v)). As well, the dependence of pesticide/humic acid interactions on salt (sodium chloride) concentration has been expressed in terms of a stepwise stoichiometric representation, which leads to a specific equation for the partition of the added salt between the pesticide molecule, the HA, and the pesticide/HA complex. Based on this novel equation, the dependence of the pesticide/humic acid association on the salt concentration can be formulated via a relation similar to the one of Tanford. In addition, for the first time, the calculation of the affinity energy distribution for different values of the salt concentration in the mobile phase confirmed the existence of several types of binding sites on the HA macromolecule.

Construction and evaluation of a humic acid column: implication for pesticide risk assessment.

In this paper, humic acid (HA), known to play a large role in the binding and transport of pesticides in soil, was immobilized on a chromatographic support. Then, the association of some herbicides and rodenticides with the main soil component HA was examined using this novel chromatographic column. It appeared that HA has a lower affinity for neutral than for charged pesticides. Moreover, the influence of various parameters was investigated on the pesticide retention in order to providevaluable information about both the binding mechanism and the utilization conditions of the HA column. For all the pesticides studied, a change was clearly vizualized in the HA-pesticide association mechanism at a critical value of the Na+ concentration in the bulk solvent, x(c), equal to 0.6 M. Around this value, the HA structure balanced between a flexible linear conformation for x < x(c) and a random coil form for x > x(c). This work confirmed the conformation change on HA immobilized on silica. As well, only for the charged pesticides, it was clearly pointed out that below a Na+ concentration equal to 0.3 M, the pesticide binding to HA decreased when the salt concentration was enhanced due to an ion pair formation and a competition effect between the sodium cation and pesticide to bind to the HA molecule. Furthermore, it was established that the HA column was stable during an extended period of time, indicating that the HA column could soon become very attractive to determine the risk assessment of pesticides.

Zinc-human serum albumin association: testimony of two binding sites.

The zinc cation (Zn(2+)) binding to human serum albumin (HSA) was studied using a non-equilibrium approach in order to prove two HSA binding sites. The effect of the bulk solvent pH and column temperature T on this binding and the corresponding thermodynamic data were also investigated. It appeared that the association process can be divided into two pH value ranges due to a predominant Zn(2+) interaction with either HSA site I or site II. It was also demonstrated that the Zn(2+) affinity for the site II was weakly affected by modifying the mobile phase pH whereas for the site I, the affinity constant increased strongly with increasing the pH of the bulk solvent.

Analysis of the progesterone displacement of its human serum albumin binding site by beta-estradiol using biochromatographic approaches: effect of two salt modifiers.

The mechanisms of (i) the binding of two sex-hormones (i.e. progesterone and beta-estradiol) to human serum albumin (HSA) and (ii) the progesterone displacement of its HSA binding cavity by beta-estradiol were studied by biochromatography using three different methods. In the first time, zonal elution method was used to prove the direct competition effect between the two sex-hormone. In the second time, the competition effect between beta-estradiol and progesterone to bound on the same HSA site was analysed by the competitive bi-Langmuir approach. Finally, the thermodynamic data of these two binding processes were studied. The Gibbs free energy value (Delta(approximately)G degrees) of the displacement equilibrium was negative demonstrating that beta-estradiol displaced progesterone of its HSA binding cavity. Moreover, the effect of two chloride modifiers (i.e. Na(+), Mg(2+)) on these two binding processes were analysed. Results showed that in the salt biological concentration ranges, the Mg(2+) cation enhanced strongly the bioavailable progesterone, whereas the Na(+) cation interacted slowly on the progesterone displacement of its HSA binding site by beta-estradiol. This study showed that it must be useful to carry out more in vivo test on the magnesium supplementation effect for women who suffer from estrogen dominance syndrome.

Phloroglucinol: novel synthesis and role of the magnesium cation on its binding with human serum albumin (HSA) using a biochromatographic approach based on Langmuir isotherms.

In this paper, a new and efficient method for synthesis of phloroglucinol with an overall yield of 60% was described. As well, the phloroglucinol association on an immobilized human serum albumin (HSA) column was analyzed in biochromatography by the determination of its Langmuir distribution isotherms. The role of the magnesium cation Mg2+ on the phloroglucinol-HSA binding process was as well analyzed. The results showed that in the Mg2+ concentration range (0.7-2 mM) (including its biological concentration range, i.e. 0.75-0.90 mM), increasing the Mg2+ concentration increased the fraction of free phloroglucinol (not linked with HSA) and thus its biological effect.

Triazinic herbicide determination by gas chromatography-mass spectrometry in breast milk.

A solid-phase extraction procedure using a graphitized carbon black cartridge for extraction and cleaning of a series of five triazines (atrazine, deethylatrazine, deisopropylatrazine, ametryne and prometryne) from breast milk samples was developed. Using a chemometric methodology, the optimisation of both the analysis time and the triazinic herbicide separation by gas chromatography-mass spectrometry (GC-MS) was then carried out with only 18 experiments. Detection and quantification limits for 1ml breast milk sample were, respectively, 0.3 and 1 ppb for each studied compound. The variation coefficients were less than 5% over the concentration range from 1 to 100 ppb. The accuracy was between 98.63 and 104.62% for each triazinic herbicide. The recovery was between 58.64 and 63.22% for the concentration range from 1 to 100 ppb for each triazinic herbicide. The assay was successfully applied to the analysis of several breast milk samples.

A chromatographic approach to analyze dansyl amino acid-HP-beta-CD association using macrocyclic antibiotic as the stationary phase.

The retention mechanism for a series of D,L-dansyl amino acids in high-performance liquid chromatography is investigated using a teicoplanin stationary phase and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as the mobile phase additive (0-16mM). A theoretical treatment is developed to determine the HP-beta-CD influence on the equilibrium between the teicoplanin phase and the aqueous medium, respectively. From the experimental data, the association constants of the D,L-dansyl amino acids-HP-beta-CD inclusion complexes are determined and discussed in relation to the enantiomer structure. A thermodynamic study confirms that both the retention and complexation mechanisms are independent of the dansyl amino acid molecular structure and its absolute carbon configuration.

[Fractal art in separative sciences]. / L'art des fractales en sciences séparatives.

Fractal geometry has provided a mathematical formalism for describing complex and dynamical structures. It has been applied successfully in a variety of areas such as astronomy, economics and biology. Because of its success in such a variety of areas it is natural to develop fractal application in separative sciences.

Mobile phase viscosity and velocity dependence on protein retention using nonequilibrium chromatographic techniques.

Nonequilibrium chromatography (NEC) is an alternative chromatographic procedure for the separation of macromolecules. The retardation of a protein series is studied using a phosphate buffer as a mobile phase with various concentrations of glycerol fraction (used as a viscosity modifier) at different mobile phase velocities and a C1 column with a very low packing particle diameter as a stationary phase. It is shown that the two factors (viscosity and velocity) of the mobile phase constituted important parameters in the retention mechanism of the proteins in NEC. The retardation velocity domain is divided into two regions. For low velocity regions, the protein retention decreased with a mobile phase velocity increase. This retention is enhanced above a critical value of the mobile phase velocity. The transition between the two well-known NEC methods, slalom chromatography and hydrodynamic chromatography, is clearly visualized for the first time for the protein retention of particular values of the mobile phase velocity.

A mathematical model for hydrodynamic and size exclusion chromatography of polymers on porous particles.

When packed columns filled with porous particles are used for the separation of macromolecules, either size exclusion chromatography (SEC), hydrodynamic chromatography (HDC), or a combination of both determine the macromolecule retention mechanism. This paper develops a simple mathematical model to describe a molecular weight calibration graph, which includes both HDC and SEC. There is a transition between the HDC calibration region at higher molecular weights to an SEC region at lower molecular weights. The degree to which SEC and HDC are mixed depends on the particle diameter, the relative size of the pores, and the macromolecule size. In addition, using fractal considerations, the fractal character of the apparent selectivity between two adjacent peaks on the chromatogram is shown. This model constitutes an attractive tool to enhance the expansion of these two chromatographic techniques for the separation of biological or synthetical macromolecules.

Comparison of two hard keratinous substrates submitted to the action of a keratinase using an experimental design.

The influence of temperature, pH, keratinase concentration, substrate concentration and incubation time on the soluble proteins released by a new keratinase from Doratomyces microsporus was studied with a second-order experimental design. Only 15 or 18 spectrophotometric analyses were required to determine the optimal experimental conditions for this keratinase on nail and hoof. This study was carried out by measuring, according to Smith's method, the concentration of soluble proteins released by the enzyme on two substrates: nails and sheep hooves. Results give optimum conditions for the keratinase to release the soluble proteins: pH 8.2, keratinase concentration 0.14% (weight of keratinase lyophilisate/final volume) and substrate concentration 5% (weight of nail powder/final volume) for nails; temperature 38.8 degrees C, pH 9, substrate concentration 5% (weight of hoof powder/final volume) and a 5 h 55 min incubation time for hooves.

Novel approach to the study of the chiral discrimination. Mechanism in a series of imidazole derivatives using HPLC.

In high performance liquid chromatography (HPLC) using poly (octadecylsiloxane) as a stationary phase, phosphate buffer as a mobile phase, a series of R-S imidazole derivatives as solutes, and beta-CD (beta-CD) and hydroxypropyl-beta-CD (HP-beta-CD) as chiral selectors, a study on the hydrophobic effect on both the solute complexation with the chiral selector and chiral discrimination mechanisms was carried out by varying the sucrose concentration c in the mobile phase and the column temperature T. An original mathematical treatment was developed to calculate the degree of complexation, rho (the percent of complexed guest solute), and the number of sucrose molecules excluded from both the uncomplexed solute-RP18 stationary phase interface (when the solute transfer occurred) and the solute-CD interface during the complexation process. This number of sucrose molecules was an image of the relative intensity of the hydrophobic effect and the inclusion degree of the solute in the chiral selector. Different Van't Hoff plot shapes of the degree of complexation and sucrose molecule number were observed with beta-CD and HP-beta-CD indicating a change in the solute inclusion and chiral discrimination.

Role of the water as a surface tension modifier for the retention of a series of dansylaminoacids on a beta-CD stationary phase.

In reversed phase liquid chromatography (RPLC), using a surface tension treatment, the retention and separation of a series of d,l-dansylaminoacids were investigated with native beta-cyclodextrin as a chiral stationary phase. The enantioselectivity thermodynamic parameters were determined from linear van't Hoff plots. An analysis of the experimental variations in the retention factor with different fractions of water in the mobile phase was performed. The number of water molecules, n, excluded from the solute beta-cyclodextrin cavity interface when the analyte transfer occurred, was determined. Using these n values, the relative degrees of compound inclusion were calculated and correlated to both the steric bulkiness of the solute and the thermodynamic data.

Column efficiency and separation of DNA fragments using slalom chromatography: hydrodynamic study and fractal considerations.

Novel equations (Guillaume Y. C.; et al. Anal. Chem. 2000, 72, 853) were developed to describe the large double-stranded DNA molecule retention in slalom chromatography (SC). These equations were applied for the first time to model both the "apparent selectivity" and the resolution between two eluted DNA fragments on a chromatogram. A study of the column efficiency corroborated the fact that slalom chromatography is not based on an adsorption or equilibrium phenomenon, but can be attributed to a hydrodynamic phenomenon. Using a combination of the dynamics of DNA fragment progression in the column and fractal considerations, it was shown that the apparent selectivity depends both on the DNA fragment sizes and mobile-phase flow rate and therefore a balance between two hydrodynamic regimes. A chromatographic response function was also used to obtain the most efficient separation conditions for a mixture of DNA fragments in a minimum analysis time. The chromatographic data confirmed that in SC the flow rate can increase or maintain the separation efficiency with an associated decrease in the analysis time. This constitutes an attractive outcome in relation to the classical chromatographic separation.

Mobile-phase-viscosity dependence on DNA separation in slalom chromatography.

Slalom chromatography (SC) is an alternative chromatographic procedure for the separation of relatively large double-stranded DNA molecules and is based on a new principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase with various concentrations of viscosity modifier (i.e. glycerol) and a C1 column as a stationary phase. The DNA molecule retention was accurately described over the glycerol concentration range using a model previously established. It was shown that the eluent viscosity increase enhanced the slalom chromatographic capacity to separate the DNA fragments. A connection between SC and 'hydrodynamic chromatography' processes was predicted to link the two processes in a global separation mechanism based on a non-equilibrium principle.