Non-purged voltammetry explored with AGNES.

The reduction of oxygen increases pH in the surroundings of an electrode. A theoretical model estimates the steady-state pH profile from the surface of the electrode up to the bulk solution. A very simple formula predicts that, in non-deaerated solutions, for bulk pH-values between 4.0 and 10.0, the corresponding surface pH could be as high as 10.3, regardless of the thickness of the diffusion layer and composition of the sample (except if it has a buffering capacity). For bulk pH lower than 3.0 or higher than 10, pH increases are negligible. Less steep pH-profiles are obtained using buffers (such as MOPS 0.01 M or MES 0.01 M). The change in surface pH modifies the local speciation and hinders the standard interpretation of voltammetric responses. The electroanalytical technique Absence of Gradients and Nernstian Equilibrium Stripping (AGNES), implemented with Screen Printed Electrodes (SPE), provides experimental insights into this phenomenon. AGNES probes the free metal concentration at the electrode surface, from which the surface pH can be estimated for systems of known composition. These estimations agree with the theoretical model for the assayed systems. Additionally, the quantification of the bulk free Zn(2+) and Cd(2+) concentrations with specific modifications of AGNES for non-purged synthetic solutions is discussed. In general, more accurate determinations of the bulk free metal concentrations in non-purged solutions are expected: (i) when the calibration is performed in a medium where the pH increase induces similar changes in the surface free metal concentration and in the sample solution and (ii) when the system is more buffered.

Direct determination of free metal concentration by implementing stripping chronopotentiometry as the second stage of AGNES.

The electroanalytical technique Absence of Gradients and Nernstian Equilibrium Stripping (AGNES) has been extended by applying stripping chronopotentiometry (SCP) as the re-oxidation stage in the determination of the free concentration of Zn(2+), Cd(2+) and Pb(2+). This new approach, called AGNES-SCP, has been implemented with screen-printed electrodes (SPE) and the standard Hanging Mercury Drop Electrode (HMDE). Clear advantages of this variant have been shown: (i) the easy resolution of the peaks of different metals present in mixtures and (ii) the sparing of blanks. A rigorous computation of the faradaic charge along the SCP stage takes into account the contribution of other oxidants, which can be efficiently measured at the end of the deposition stage of AGNES. The free Cd concentration determined in an oxalate solution at pH 6 with an HMDE as the working electrode agreed well with values obtained with a Cd Ion Selective Electrode. The free metal concentration measured using an SPE for the system Cd and nitrilotriacetic acid (NTA) at pH = 4.8 also conformed well with Visual MINTEQ results.

Experimental verification of the metal flux enhancement in a mixture of two metal complexes: the Cd/NTA/glycine and Cd/NTA/citric acid systems.

Rigorous computation of the metal flux crossing a limiting surface of a system that contains a mixture of 1 : 1 metal complexes under steady-state planar diffusion in a finite domain and under excess of ligand conditions predicts, for some cases, an enhancement of the metal flux with respect to that expected in a system with independent complexes. Indeed, the coupling of the dissociation kinetics of both complexes can yield higher metal fluxes than expected with important environmental implications. By using the voltammetric techniques AGNES and stripping chronopotentiometry, this paper provides experimental evidence of this enhancement for two systems: Cd/NTA/glycine and Cd/NTA/citric acid. The flux measured in both cases is in good agreement with the flux computed for the global system, exhibiting maximum enhancement ratios above 20%. Theoretical discussion of the flux enhancement factors and of the conditions for this enhancement are also provided.

Molecularly imprinted polymer for analysis of zidovudine and stavudine in human serum by liquid chromatography-mass spectrometry.

A molecularly imprinted polymer (MIP) using zidovudine (AZT) as template and methacrylic acid as monomer was prepared. The synthesis of the MIP was performed in acetonitrile. The synthesized material was then tested for the solid-phase extraction of AZT from different media (pure organic solvents and hydro-organic mixtures). An optimised procedure was developed for the selective extraction of AZT with a recovery of 96% using the MIP and only 3% on a non-imprinted polymer used as control polymer. A specific capacity of 0.2 micromol g(-1) was determined. The specificity of the MIP was evaluated by studying the retention behaviour of two others nucleoside analogues. The feasibility of the MIP to selectively extract AZT and stavudine (d4T) from human serum was also demonstrated with recoveries of 80 and 85% respectively. The lower limit of quantification (LLOQ) and the lower limits of detection (LLOD) for AZT were 5.10(-7) and 10(-7) M respectively.

Quantification of zidovudine and its monophosphate in cell extracts by on-line solid-phase extraction coupled to liquid chromatography.

A simple and rapid analytical method for the simultaneous quantification of zidovudine (AZT) and its monophosphate (AZTMP) in cell extracts has been developed using high-performance liquid chromatography (HPLC) with on-line solid-phase extraction and 2-aminoethyl-3'-azido-2',3'-dideoxythymidin-5'-yl phosphodiester sodium salt as internal standard (IS). The cell extract samples were directly injected on a short reversed-phase precolumn using an aqueous buffer containing an ion-pairing reagent as a mobile phase. Under these conditions, the analytes were retained on the precolumn whereas the proteins were discarded. The analytes were then transferred onto the analytical column by increasing the strength of the eluent. The calibration curve was linear over a concentration range of 0.5-100 microg/ml. Inter- and intra-day accuracy and precision results satisfied the accepted criteria for bioanalytical validation. This method was used to study the decomposition pathway of a model pronucleotide in an in vitro approach.

Diastereoisomeric resolution of a pronucleotide using solid phase extraction and high performance liquid chromatography: application to a stereoselective decomposition kinetic in cell extracts.

A stereospecific HPLC methodology has been developed for the diastereoisomeric resolution of a mononucleotide prodrug in cell extracts. This method involves the use of solid phase extraction on a C18 cartridge. Diastereoisomers and internal standard resolutions were performed on a cellulose based chiral column (Chiralcel OD-H) used in the normal phase mode. The method was validated in terms of specificity, recovery, linearity (diasteroisomers mixture concentration: 3-60 micromol L(-1)), precision and accuracy and detection limit (1.67 and 1.33 micromol L(-1) for first and second eluted diastereoisomer). This method was applied to the determination of the apparent rate constants of disappearance and half-lives of each stereoisomers. This permits to conclude to the stereoselectivity of the enzymatic activity involved in the decomposition pathway of 2.

Column selection and method development for the separation of nucleoside phosphotriester diastereoisomers, new potential anti-viral drugs. Application to cellular extract analysis.

Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases used in normal and reversed-phase modes were developed for the diastereoisomeric separation of mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine (AZT). The resolutions were performed with two silica-based celluloses using normal and reversed-phase methodologies: Tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H and Chiracel OD-RH) and Tris-methylbenzoate (Chiralcel OJ and OJ-R). Two amyloses phases, Tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and Tris-(S)-1-phenylethylcarbamate (Chiralpak AS), were used in normal-phase mode. Additionally, we developed separation using two stationary phases with immobilized cyclodextrins in reversed-phase and polar-organic modes. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols, acetonitrile or water in the mobile phase were also tested for the different separation modes. An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. The different columns gave complementary results in term of resolution. Limits of detection and quantification were 0.12-0.20 and 0.40-0.67 microm, respectively. This analytical method was applied in a preliminary study for the pronucleotide 2 quantification in cellular extract.

[Oxidative metabolism of HIV-infected macrophages: the role of glutathione and a pharmacologic approach]. / Métabolisme oxydatif dans les macrophages infectés par le VIH: rôle du glutathion et approche pharmacologique.

Oxidative stress and glutathione deficiency seem to play a major role in the pathogenesis of HIV infection, as suggested by the increased survival of HIV-infected patients treated with N-acetylcysteine, a prodrug of glutathione. However, beneficial effects of GSH-replenishing drugs are restricted in vivo by the high concentrations needed to obtain biological effects and their low bioavailability. In this study, we evaluated the antiretroviral and antioxidant activities of new more lipophilic GSH-replenishing molecules, in macrophages infected in vitro with HIV-1. In these experimental conditions, a prodrug of N-acetylcystéine and beta-mercaptoethylamine, I-152 demonstrated a potent anti-HIV activity, increased intracellular GSH level, and decreased TNF-alpha production. Altogether, these results suggest that I-152 could be beneficial as adjuvant therapy of antiretrovirals in HIV-infected patients, especially in those with damages to the central nervous system or with mitochondrial damages associated with highly active antiretroviral therapy.

NAC/MEA conjugate: a new potent antioxidant which increases the GSH level in various cell lines.

I-152 is a prodrug of NAC and MEA with potent pro-GSH effects in human macrophages, astrocytes and lymphocytes. This molecule could be of interest in HIV infection in respect to its antioxidant and anti-HIV activities, but also in other diseases to counteract oxidative stress, that is, inflammation, cardiovascular diseases, and neurodegenerative diseases.

Voltammetric analysis of heterogeneity in metal ion binding by humics.

The complexation of Cd, Pb, and Cu by fulvic acids at a fixed pH and ionic strength is studied by means of different voltammetric techniques at any metal-to-ligand ratio. When using Reverse Pulse Polarography (RPP) the complex species are electrochemically labile and not subject to significant electrodic adsorption. RPP titrations of fulvic acid with metal ions are interpreted on the basis of a recently proposed analytical expression for limiting currents valid for fully labile heterogeneous complexation. The voltammetric data are transformed into the corresponding binding curve, i.e., the fraction of occupied sites vs free metal concentration. Finally, the competition between metal ions and protons in their interaction with the fulvic binding sites as well as the concomitant polyelectrolytic effects are analyzed in terms of the NICCA-Donnan model. The results show that voltammetric techniques can be applied to the studies of heterogeneous complex systems in a broad range of metal-to-ligand ratios.

Multivariate analysis of superficial scald susceptibility on Granny Smith apples dipped with different postharvest treatments.

Multivariate methods were used to present the results of different postharvest treatments applied to control superficial scald disorder in Granny Smith apples. In a two-season study, the treatments applied by dips were diphenylamine (DPA), Semperfresh (a sucrose ester coating) formulated alone or with either ascorbyl palmitate or n-propyl gallate, and CaCl(2). DPA correlated negatively with scald, whereas the rest of the treatments showed no clear relationship with the disorder. By means of partial least squares, the incidence of superficial scald after cold storage was shown to correlate with quality parameters (firmness and acidity), measured at the end of storage. In contrast, ethylene production did not appear as a relevant factor in the incidence of scald.

Relationships between volatile production, fruit quality, and sensory evaluation in Granny Smith apples stored in different controlled-atmosphere treatments by means of multivariate analysis.

Aroma compounds, quality parameters, and sensory evaluation of Granny Smith apples were analyzed after 3, 5, and 7 months of cold storage in three controlled-atmosphere (CA) treatments, in which oxygen and carbon dioxide were held at 1, 2, and 3%. During poststorage ripening, the apples were kept at 20 degrees C for 1, 5, and 10 days before analytical measurements were made. The highest volatile emission was obtained after 5 months of storage in all CA treatments, reaching its highest value when a low-oxygen CA (LO) was used. Ultralow-oxygen CA (ULO) showed the highest ability to maintain apple firmness. The correlation among analytical and sensory parameters suggests that ethyl 2-methylbutyrate, 1-butanol, pentyl acetate, and tert-butyl propionate are the aroma compounds with the highest influence in the sensorial score. Concerning CA treatments, LO and ULO appear to be very valuable technologies for maintaining the sensorial quality even after 7 months of storage.

Synthesis and in vitro anti-HIV activity in human monocyte-derived macrophages of 2-oxothiazolidine-4(R)-carboxylic acid derivatives.

Oxidative stress and glutathione (GSH) deficit may play an important role in HIV infection pathogenesis, and oral administration of GSH-replenishing drugs such as N-acetylcysteine (NAC) and 2-oxothiazolidine-4(R)-carboxylic acid (OTC) may be associated with an increased survival rate of HIV-infected patients. Nevertheless, beneficial effects of these molecules are restricted in vivo by the high concentrations that are necessary to obtain biological effects, rapid extracellular metabolization, and low availability and plasma concentrations. We synthesized OTC derivatives that are more lipophilic than OTC and theoretically able to overcome these limitations and to generate, in addition to cysteine, other substrates of the gamma-glutamyl cycle. Their antiviral effects were investigated in human HIV-1/Ba-L-infected monocyte-derived macrophages. In our experimental conditions, OTC exhibited anti-HIV-1 effects and little cytotoxicity at high doses. None of the nine tested derivatives showed higher cytotoxicity than OTC, nor anti-HIV-1/Ba-L activity.

[The relationships between apoprotein levels and body mass and atherogenic indices and sports activity in Aragonese schoolchildren]. / Relaciones entre los niveles de apoproteínas con los índices de masa corporal, aterogénico y la actividad deportiva en escolares aragoneses.

OBJECTIVE: The main purpose of this study was to establish the relationship between the apoproteins normally employed in clinical practice and the Quetelet indexes, atherogenic and physical activity in order to know how these parameters are modified in relationship to life style and hence, be able to modify inadequate habits in order to slow down the atherogenic process. PATIENTS AND METHODS: In a group of 82 schoolchildren of both sexes, we assessed the relationships between the levels of apoproteins A-1 and B, and the quotient apoprotein A-1/apoprotein B with the atherogenic indexes and the body mass (Quetelet) in relationship to the practice of sports. RESULTS: The average values obtained for each parameter studied were within the established normal range, although 4.88% of the boys and 2.7% of the girls showed lower levels of apoprotein A-1 and 9.76% of the boys and 10.8% of the girls showed higher levels of apoprotein B. In addition, 19.25% of the boys and 8.1% of the girls showed a body mass index superior to 2 standard deviations for their age. The correlations found showed statistic value (p < 0.01) with the atherogenic index for both groups, whereas the Quetelet index was only significant (p < 0.05) with the apoprotein B in both groups and for the quotient apoA-1/apoB (p < 0.05) in the case of the boys. Physical activity did not show an important relationship with the rest of the analyzed parameters since only the girls showed statistical value (p < 0.05) with the quotient apo A-1/apo B and with the atherogenic index. CONCLUSIONS: These data allow us to conclude by pointing out the importance of the establishment of adequate dietetic habits, avoiding obesity as well as high levels of apo B, and the establishment of continuous and prolonged physical activity as hygiene for the healthy life in children to prevent modifications in the apoprotein profiles which may include an atherogenic risk.

A theoretical approach to describe monolayer-liposome lipid interaction.

It is known from several studies on the interaction between membrane models that mechanisms such as fusion or lipid exchange can play an important role in the process of internalization by cells of lipid vesicles and also in the physical stability of liposomes. In this paper it is shown that a simple monolayer-liposome model can be used to simulate experimentally observed interactions between lipid vesicles and cell surfaces. From experimental data, a simple theoretical model is formulated to interpret the variation with time of surface pressure as a function of liposome concentration. The congruency of the physico-chemical hypothesis and its validity are studied and correlated with results from experimental systems.