Role of serum carrier proteins in the peripheral metabolism and tissue distribution of thyroid hormones in familial dysalbuminemic hyperthyroxinemia and congenital elevation of thyroxine-binding globulin.

To investigate the role of thyroxine-binding globulin (TBG) and albumin in the availability of thyroid hormones to peripheral tissues, comprehensive kinetic studies of thyroxine (T4) and triiodothyronine (T3) were carried out in eight subjects with familial dysalbuminemic hyperthyroxinemia (FDH), in four subjects with inherited TBG excess, and in 15 normals. In high-TBG subjects, the reduction of T4 and T3 plasma clearance rates (by 51% and 54%, respectively) was associated with normal daily productions; T4 and T3 distribution volumes were significantly reduced. In FDH subjects T4 clearance was less reduced (by 31%) than in high TBG; consequently T4 production rate was significantly increased (by 42%); T4 and T3 distribution volumes and T3 clearance rate were unchanged. Increased T3 peripheral production in FDH (by 24%) indicates that T4 bound to abnormal albumin is more available to tissues than T4 carried by TBG, thus suggesting an important role of albumin in T4 availability to the periphery.

Molecularly imprinted solid-phase extraction method for the high-performance liquid chromatographic analysis of fungicide pyrimethanil in wine.

A method for molecularly imprinted solid-phase extraction (MISPE) of the fungicide pyrimethanil from wine samples has been investigated. The molecular imprinted polymer was obtained by iniferter-mediated grafting on porous chloromethylated polystyrene beads, using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linker. The imprinted beads were evaluated for use as a solid-phase extraction sorbent, in order to develop the extraction protocol in aqueous standards and red wine samples. The optimised extraction protocol resulted in a reliable MISPE method suitable for HPLC analysis (stationary phase: Cromolith Performance C18 column, 100 mm x 4.6 mm; mobile phase: acetonitrile-water (3:2, v/v), flow-rate: 1.00 ml/min; detection 270 nm). It was selective for pyrimethanil and the related pyrimidinic fungicides cyprodinil and mepanipyrim, while the non-pyrimidinic fungicides benalaxyl, chlozolinate, furalaxyl, iprodione, metalaxyl, nuarimol, procymidone and vinclozolin were not extracted. Recoveries performed on a wine matrix spiked with pyrimethanil at three different concentration levels were reproducible and were in good agreement with the recoveries performed on buffer, coming out between 80 and 90% (85+/-7.0% at 0.50 microg/ml, 79+/-1.6% at 2.0 microg/ml and 87+/-5.6% at 20 microg/ml). Preconcentration and quantitative extraction of pyrimethanil from wine samples was shown to be feasible down to 0.1 microg/ml.

A novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin.

A new non competitive capillary electrophoresis immunoassay format based on a separation into a capillary modified by analyte immobilisation is described. The injection of an excess of labelled antibody off-line preincubated with the analyte allows the surface capture of the free antibody and the immunocomplex detection. It was developed using the human serum albumin (HSA) as analyte, two FITC-labelled antibodies and a HSA covalently linked capillary. Two calibration curves with good run-to-run reproducibility and LOD--respectively 14.0 nM for the FITC-polyclonal antibody and 9.0 nM for the FITC-monoclonal antibody--were achieved. The assay was applied to HSA determination in spiked samples of human urine with acceptable recoveries.

Solid-phase extraction of ochratoxin A from wine based on a binding hexapeptide prepared by combinatorial synthesis.

In this paper we describe the preparation of a hexapeptide library by combinatorial synthesis and the identification of a peptide with sequence Ser-Asn-Leu-His-Pro-Lys, which showed good affinity (K(eq)=3.4 x 10(4) M(-1)) towards the mycotoxin ochratoxin A (OTA). An immunoaffinity-like stationary phase supporting such a hexapeptide was used to develop a solid-phase extraction method for the quantification of OTA in wine samples at concentration levels down to 0.10 microg l(-1). Several different wine samples fortified with OTA at 2 and 4 microg l(-1) levels showed recovery of 94.7% and 98.4% at 2.0 and 4.0 microg l(-1), respectively, without any effect on the extraction efficiency of the matrix. The efficacy of this approach was successfully tested by comparison with an immunoaffinity extraction performed on commercial cartridges.

Antibody-like peptides as a novel purification tool for drugs design.

New pharmaceutical approaches, such as biotechnology industry, genomic and proteomic studies, require the development of new analytical and preparative tools that should allow the resolution and the characterisation of complex sets of molecule mixtures in a high-throughput mode with the isolation of a single substance from complex matrices in a high degree of purity, low costs and wide availability. In this review we discuss the design of a tailor-made peptide by focusing our attention on the bioinformatic techniques and combinatorial approaches. Then synthesis, purification and characterisation of peptides with recognition properties are discussed by considering different approaches and their fitness to drug design. Moreover, the development of affinity devices and the discussion of their characteristics to optimise the purification phase are reported. Applications of peptide ligands that bind pharmaceutical compounds (i.e. therapeutic proteins, monoclonal antibodies, hormones) are described and analytical tools mentioned and evaluated. Future perspectives, in particular alternative applications of peptide ligands and their substitution with peptidomimetic compounds, are described.

Solvent effect on testosterone-antitestosterone interaction.

The inhibition of the binding between testosterone and antitestosterone antiserum caused by organic solvents was studied at pH 7.4, 298 K. Inhibition curves were obtained at variable ranges of molar fractions for the following solvents: methanol (range 0-0.4), ethanol (0-0.317), 1-propanol (0-0.082), 2-propan-ol (0-0.260), t-butanol (0-0.223), ethylenglycol (0-0.189), 2-methoxyethanol (0.036), 2-butoxyethanol (0-0.063), 1,4-dioxan (0-0.124), tetrahydrofuran (0-0.238) and acetonitrile (0-0.392). Steroid-antibody binding decreases with increasing molar fraction of solvent in the reaction mixture for all but tetrahydrofuran and acetonitrile, which enhance binding at low molar fraction then cause a sharp inhibition. Molar fraction of solvent that causes a 50% binding inhibition is uncorrelated to some solvent properties (i.e. dielectric constant, polarity index, dipole moment) but is inversely correlated to the molecular mass of the solvent. The correlation becomes better by taking into account the length of the solvent molecule, or the Randic molecular connectivity index, suggesting that binding inhibition could be related to the length of the solvent molecules that displace water around the steroid molecule. However, the increase of binding observed at low molar fraction with tetrahydrofuran and acetonitrile, together with very different shapes of inhibition curves suggest that a molecular mechanism based on the differential solvation of the steroid by solvent and water molecules must be taken into account to explain adequately the solvent effect on testosterone-antitestosterone interaction.

Immunochemical methods for environmental monitoring.

Immunochemical methods for environmental analysis must be taken into consideration more for their ability to expand the potential of analytical measures rather than for substituting current methodologies. Moreover, the full potential of these methods has yet to be realized. Indeed, the terms and concepts of immunology are new to most analytical chemists, even if environmental science has always been an interdisciplinary field. On the other hand, the clinical development of immunoassays means that much experience has been gained in the analysis of blood, urine, and tissue samples. The immunochemical analysis of samples from soils, ground water, waste chemicals, poses new challenges in sample preparation that have yet to be extensively studied, and in the future there may be immunoassays better suited for the particular problems associated with environmental monitoring.

Chromatographic characterization of molecularly imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid.

Two polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared by utilising the technique of the non-covalent molecular imprinting polymerisation in an aqueous medium. The polymers obtained were packed in HPLC columns and the effects of the mobile phase composition on the retention of the imprinting molecule and the selectivity of the stationary phases towards several analogous structures were studied by liquid chromatography. The columns showed a good level of selectivity towards the template and strictly related molecules. It was found that the molecular recognition mechanism acting on the columns was dependent on a combination of ion pair and hydrophobic interactions.

Kinetics of the reaction between testosterone and antitestosterone antiserum.

In order to characterize from a kinetic viewpoint the antibody population mainly involved in the binding of testosterone by its homologous antiserum, the kinetics of the association reaction between [1,2,6,7-3H]-testosterone and rabbit antiserum anti-testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin (Ab R2603-1) was followed at pH 7.4 and at constant ionic strength, at temperatures ranging from 2 degrees C to 37 degrees C and at concentration near to work conditions for testosterone radioimmunoassay; dextran coated charcoal suspension was used for the bound/free separation. In the examined concentration range, the observed kinetics trends can be explained by assuming the existence of two classes of antibody binding sites, Ab1 and Ab2. The kinetics of the dissociation reaction of the testosterone-antibody complex was also followed after the addition of a large excess of unlabeled testosterone. At 22.0 degrees C, association and dissociation rate constants are 2.1.10(7) s-1M-1 and 3.7.10(-3) s-1, respectively, for the Ab1 class of antibody binding sites, and 3.6.10(6) s-1M-1 and 7.0.10(-4) s-1 for the Ab2 class. Equilibrium constants obtained from kinetic data were very similar for both classes of antibody binding sites and in good agreement with the equilibrium values obtained from linear Scatchard plot. The order of magnitude of the second order rate constants and the high activation enthalpy for the forward and reverse reaction suggest a mechanism more complex than a simple second order.

Application of an ELISA to the determination of benalaxyl in red wines.

The applicability of an ELISA for detection and quantification of benalaxyl in red wine samples is described. The study of the influence of this matrix on the reliability of the assay indicates that red wine samples require a rapid and simple cleanup step before ELISA assay. Recovery and precision of the method were evaluated by spiking red wine samples with benalaxyl in the 0.5-24 ng/mL range. Benalaxyl can be determined with good accuracy and precision up to 0. 5 ng/mL in starting red wine samples (detection limit of 0.13 ng/mL). No false negative or positive results were obtained. Authentic red wine samples were analyzed by ELISA and by RP-HPLC. The amounts of benalaxyl found by ELISA were in good agreement with RP-HPLC analysis.

[Studies on iodine exchange in thermal therapy with salsobromoiodic water]. / Osservazioni sul ricambio dello iodio nella terapia termale con acque salsobromoiodiche.

We have attempted a quantitative evaluation of the iodine taken in with the thermal waters from Salsomaggiore during therapeutic bathing, inhalation (dry and damp spray), or ingestion. For this purpose to 127I we have applied the metabolic parameters obtained through a 131I inhalation test and a 125I ingestion test. Of the iodine inhaled by aerosol 45% becomes exhaled; by 24 hours 2% is in the serum and in the extra-thyroid area of iodine distribution, 16% in the thyroid, 16% in the urine. By adding the amount of iodine exhaled to that found in the metabolic cycle of iodine, we find that about 21% of the inhaled iodine is still missing. This amount is trapped in the respiratory tract from where it disappears only very gradually. At the end of the 24 hours, therefore, in the metabolic cycle of the iodine we find 34% of that inhaled, whereas we find 87% of that ingested. The level of iodine in the serum reached in thermal therapeutic inhalation, never stays at a level which might alter the functioning of a normal thyroid. The amount of inhaled iodine which is excreted with the urine is usually eliminated during the first excretions. Experimental studies suggest that the iodine taken in during bathing in the thermal-pools mainly comes from iodine released from the water through the addition of hypochlorites, and is then inhaled through breathing the air just above the water.

The "soluble sandwich" approach for immunoassays: methodological and instrumental implications.

The simultaneous presence of homogeneous and heterogeneous reactions at different binding sites of a multiepitope antigen makes the description of the kinetic parameters of the so called "one step" solid phase immunometric assays complex. The authors extended the "one step" approach to the concept of the "soluble sandwich" methodology which differs from the former by the delayed solid phase capture of the biotinylated immunocomplex to a streptavidin coated solid support. Using prolactin monoclonal IEMAs as a model, the equilibria involved in the reactions have been studied on a thermodynamic basis through a description of the kinetics of the interactions between biotinylated Mabs and solid phase streptavidin both in presence and/or in absence of the antigen and HRP-conjugated antibody. A comparative evaluation of models in which the biotinylated antibody was previously insolubilized on the streptavidin solid phase has been performed as well. The experimental work was carried out by using 125I labelled McBiot and Prolactin to trace individual interactions and peroxidase/H2O2/TMB systems to develop the enzymatic analytical signals. A new instrument/data reducing system was also optimized to expand the OD reading range provided by conventional, single wavelength colorimeters. The greater flexibility theoretically expected for the "soluble sandwich" approach and the possibility to extend the analyte working range without detrimental effects on the readability of low doses responses have been experimentally confirmed.

The complexation of mercury (II) and organomercurial compounds by 8-hydroxyquinoline-bovine serum albumin conjugates.

The complexing properties of conjugates between 8-hydroxyquinoline and bovine serum albumin (Ox-BSA) towards inorganic and organic mercury were studied. Two Ox-BSA conjugates (different substitution ratio) were prepared and their complexing properties were studied. Through the use of titration curves with mercury (II), methylmercury and ethylmercury an evaluation of the complex stoichiometry and stability was obtained, showing that Ox-BSA has good affinity for all investigated mercuric compounds and that the stability increases in the order: Hg (II) < CH3Hg+ < C2H5Hg+, whatever conjugate is considered. Complexes show a stoichiometry of 1:1 between mercury and 8-hydroxyquinoline residues, except with the high substituted conjugate and Hg2+ ion. The skill of the high substituted conjugate to bind inorganic and organic mercury in the presence of NaCl was also studied. Organic mercuric complexes do not show significant modification due to NaCl. Nevertheless, considering inorganic mercury, the number of retained metal ions per protein molecule increases if the NaCl concentration becomes higher than 0.1 M, probably because at high NaCl concentrations 1:1 complexes between mercury and 8-hydroxyquinoline are preferred to 1:2 complexes.

Functionalized biopolymers as soluble macromolecular chelating agents.

Two different conjugates of bovine serum albumin (BSA) with lysine and a derivative of imidazole have been synthesized to obtain watersoluble macromolecules with binding properties against bivalent transition metal ions. Syntheses have been carried out using the 60 aminogroups or the 99 carboxylic groups on BSA for the coupling reactions, with such molar ratios able to produce highly substituted BSA. The skill of each conjugate to bind metal ions in aqueous medium was studied through the use of titration curves with some metal ions, characterized by a good affinity for the free ligand. Both the conjugates allow us to recover a high number of metal ions per protein molecule, close to the number of ligand molecules on the BSA surface in the case of the lysine conjugate, whereas in the case of the imidazole conjugate M3L complexes are performed.