Estimate of the digestibility, assimilability and intestinal permeability of butyltins occurring in wine.

An estimate of the digestibility and assimilability of butyltins occurring in contaminated wines (Port, red and white) was obtained by means of in vitro studies of gastrointestinal digestion. The influence of the wine matrix on the intestinal permeability was explored by studying the accumulation of butyltins in Caco-2 monolayers either when these species are dissolved in buffer only or in the dialysates of digested wines. Some important information about the fate of the butyltin compounds ingested from contaminated wines could be achieved. Only a very small fraction of the ingested DBT and TBT, the two most toxic forms, appear to be able to reach the epithelium as judged by the small dialyzable fraction found (<2%). This is probably independent from the food/drink matrix introducing these contaminants, since the influence of the involved enzymes appear to be dominant, especially for DBT and TBT. Additionally, the intestinal permeability of the three butyltins was also very low, the wine matrix possibly having a hindrance effect in a few cases.

Elemental levels in tree-bark and epiphytic-lichen transplants at a mixed environment in mainland Portugal, and comparisons with an in situ lichen.

Samples of Platanus hybrida Brot. bark and Flavoparmelia caperata (L.) Hale thalli, from a clean area in northern Portugal (Baião), were transplanted into an exposure location at the south-western Atlantic coast, impacted by urban-industrial emissions (Sines), for a 10-month long experiment. Bark pieces were confined into nylon bags (2-mm mesh), and lichen thalli kept with their bark substrate (Pinus pinaster (Ait.) Sol.). Every two months, a double set of transplants (one for bark, one for lichens) was brought back into the laboratory, together with native samples of Evernia prunastri (L.) Ach. Following suitable cleansing and preparation procedures, field samples were put through INAA for elemental assessment. The results indicate that, regardless of signal magnitude, (1) concentrations in bark and lichen transplants are significantly correlated with atmospheric deposition for an appreciable number of elements; (2) there are a number of significant correlations between transplanted and native samples, and again between the latter and the deposition; and (3) the elements with biological patterns that follow the deposition in either transplanted or native samples (Co, V) are the very ones whose bioaccumulation seems to benefit from an alternation of wet-dry periods, which fits the precipitation record of the test site during the exposure term.

Estimation of the human intestinal permeability of butyltin species using the Caco-2 cell line model.

The main objective of the work was the setup of the Caco-2 human intestinal cell-line model for the study of the intestinal permeation of monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT). The study was focused in gathering information on (a) the relative permeability of butyltins, (b) their possible permeation routes (paracellular/transcellular) and (c) the eventual interactions between the different butyltins when occurring as a mixture. The presence of basolateral serum protein greatly influenced the permeability, causing a large net clearance, but the apparent permeability (Papp) values were comparable to that of phenolred, suggesting a low in vivo permeability of the butyltins. The found permeability pattern correlates well with the general in vivo toxicity pattern (trialkyltin>dialkyltin>>monoalkyltin). The accumulation pattern (DBT>TBT>MBT) was different from that of permeability and may be an important element regarding the elucidation of some specific strong toxic effects caused by the dialkyltins in several species. The transport of MBT and DBT was found to be dependent on the paracellular route status. An interaction between the butyltin compounds in a mixture was found for the accumulation results (the accumulation was significantly higher for the three compounds when in a mixture). A set of useful information about the butyltin accumulation and transport by the epithelial Caco-2 cell line was, thus, achieved, constituting a starting point for future research on the permeability of butyltins from contaminated food and beverages.

Pb and Cu speciation and bioavailability in port wine.

Information about the speciation of Pb and Cu in different types of Port wines (white, single-year and blended aged red, and young red wines) was gathered to estimate their respective bioavailabilities to man. For this purpose, wines were subjected to in vitro simulated gastrointestinal digestion, and the following properties were studied in the wines and its gastric and intestinal digests: (1) average conditional stability constant (K(av)) of the Cu complexes (by potentiometry), of the strongest Pb complexes (those inert to cathodic voltammetry, K'(av)), and of the respective ligand concentrations (CC or CC(inert)); (2) the distribution of the metal among the different groups of compounds of different molecular weights and/or polarities in the different bands separated by reverse phase high-performance liquid chromatography; (3) the total metal concentration present in the wines and the respective fractions present in the soluble and in the dialyzable fractions of the digest (an estimation of the assimilable fraction). The study showed that the complexing affinity for Pb (expressed by either CC(inert) or K'(av)) of white and very aged red Port wines was lower than for the remainder of the wines. For Cu, the strength of the ligands in the white wines was lower (

Assessment of the Pb and Cu in vitro availability in wines by means of speciation procedures.

The speciation of Pb and Cu in white and red table wines was investigated, in order to estimate their respective bioavailability to man. For this purpose, wines were subjected to in vitro gastrointestinal digestion, and the following properties were studied in the wines and in their gastric and intestinal digests: (1) the average conditional stability constant (Kav) of the strongest complexes (those inert to cathodic voltammetry) and of the respective ligand concentration (CCinert); (2) the distribution of the metal among the different bands of reverse phase high performance liquid chromatography (RP-HPLC) (groups of compounds of different molecular weight and/or polarity); (3) the total metal concentration and metal present in the soluble and in the dialyzable fractions of the digest. The CCinert of the red wines and the respective digests were much greater than those of the white wines and their digests. The conditional stability constants of the strongest soluble complexes after the digestion ranged between 5.9 and 6.1 for Pb. These parameters could not be determined for Cu. After the digestion the dialyzable metal fraction (a relative index of the metal potentially available for interaction with the inner biologic ligands) was only 16% of the total Pb in red wine, 62% in white Verde and 75% in white wine. For Cu the dialyzable metal fraction was 45% of the total metal in red wine, 64% in white Verde and 98% in white wine.

The influence of Cu concentration on ethanolic fermentation by Saccharomyces cerevisiae.

In the present work, the influence of Cu concentration on alcoholic fermentation by Saccharomyces cerevisiae was studied in white grape musts and in YNB medium containing glucose. In the YNB medium, the yield of ethanol, relative to the control, doubled in the presence of 0.50 and 1.0 mM Cu. As for production of ethanol from musts, only minor effects were observed at different Cu concentrations, which indicates that Cu levels do not effect changes in fermentation, and, therefore, are below any toxic level regarding the yeast performance.

Role of polyphenols in copper complexation in red wines.

Four monovarietal red wines, Tinta Roriz, Tinta Barroca, Touriga Nacional, and Touriga Francesa, were studied for their complexometric properties toward copper. For the purpose, ion selective electrode potentiometric titrations were carried out, and the data were treated by two methods suitable for heterogeneous ligands: the Scatchard plot, which provided the complexation capacity (CC(total) = maximum number of moles of sites per liter), and the differential equilibrium function (DEF), which provided the conditional stability constants of the sites effectively involved in the metal complexation (K(DEF)) at a given titration point. The different wines displayed similar properties: CC(total) ranged from 4.5x10(-3) (Tinta Barroca) to 4.9x10(-3) M (Touriga Francesa), and log K(DEF) values were between 4.2 and 6.5. The range of log(|Cu|(bound)/CC(total)) = log theta embraced in the titration was between -0.2 and -1.9. With regard to the natural levels of copper, from 4.4x10(-7) (Tinta Roriz) to 1.3x10(-6) M (Touriga Nacional), and ligands (CC(total)) in the wines, it can be expected that copper will be strongly complexed in the studied wines (log K(DEF) > 6). Studies carried out, in parallel, for solutions of isolated seed tannins and skin polyphenols (mainly anthocyans) showed that their contribution to the CC(total) of the wines was <40% for tannins and <2% for skin polyphenols. However, skin polyphenols were shown to be much stronger copper ligands (log K(DEF) = 6.7, log theta = -1.5) than tannins (log K(DEF) = 4.5, log theta = -1.5).

Atmospheric metal pollution (Cr, Cu, Fe, Mn, Ni, Pb and Zn) in Oporto city derived from results for low-volume aerosol samplers and for the moss Sphagnum auriculatum bioindicator.

A low-volume aerosol sampler with filters and bags of Sphagnum auriculatum were exposed, in parallel, to the atmosphere of Oporto city for approx. 2 months in 1994, during a dry weather period. The levels of Cr, Cu, Fe, Mn, Ni, Pb and Zn in the moss (weekly samples) and in the filters (daily samples) were determined by atomic absorption spectrophotometry and the results were compared. For all the heavy metals, the rate of metal uptake by moss was significantly correlated with the metal concentration in atmospheric aerosols. The results indicated that moss bags of S. auriculatum can provide a quantitative estimation of the concentration of different heavy metals in urban atmospheres, when specific calibration by mechanic monitoring, at the same sampling point, is performed during a first stage of biomonitoring. The mean aerosol metal concentrations found in the Oporto atmosphere were similar to those observed in other urban atmospheres in different countries. The relative order of the mean metal concentrations was Fe (1.8 micrograms/m3) > Zn > Pb > Cu > Cr > Mn > Ni (20 ng/m3). The aerosol Pb levels were monitored at different sampling points over various periods of time between 1991 and 1997. The mean Pb levels were < or = 0.5 microgram/m3 and approximately constant at each sample point up to January 1996. After that date it decreased by approx. 50%, in consequence of the reduction of the Pb concentration in leaded gasoline.

Copper(II) complexation properties and surfactant activity of 3-[N, N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid and N-(2-Hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid pH buffers which may affect trace metal speciation in in vitro studies.

Disadvantages of the some zwitterionic pH buffers are (i) that they can interact with metal ions as well as protons, and (ii) that they may have a surfactant effect in chemical or in vitro biological or biochemical studies. This has to be taken into account when a buffer is selected. Here, the copper-complexing capacity and the surfactant activity of three compounds, 3-[N,N-bis (2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid) (HEPPSO), and piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), were investigated. Global stability constants (log betaabc) of copper(II)-buffer complexes were determined, at 25 degrees C, 0.5 M ionic strength, and at 0.8 mM buffer concentration, by pH and pCu ion-selective electrode measurements. Here, betaabc corresponds to the equilibrium: aCu2+ + bL- + cH+ left and right arrow (CuaLbHc)(2a-b+c); HL = DIPSO or HEPPSO; c: +1 = proton; -1 = hydroxide. Using SUPERQUAD constants were calculated, giving: DIPSO: log beta110 = 5.02, log beta120 = 8.99, log beta130 = 13.0, log beta140 = 16.3, log beta14-1 = 9.26, log beta14-2 = 0.645, log beta150 = 20.5, log beta160 = 24.3, log beta16-1 = 16.1, log beta16-2 = 8.98; HEPPSO: log beta110 = 4.29, log beta120 = 8.35, log beta130 = 12.1, log beta140 = 15.9, log beta150 = 19.6, log beta160 = 23.4, log beta16-1 = 14.9. The pKa values were determined at higher buffer concentrations, giving a value 7.33 for DIPSO and 7.84 for HEPPSO at 2.0 mM buffer concentration. Effects of buffer concentration on stability and acidity constants were investigated and compared with measurements using voltammetric and potentiometric stripping analysis, confirming no copper(II) complexation by Pipes. Surfactant activities were determined using alternating current polarography, confirming marked surface activity of 10 mM of DIPSO or HEPPSO.

Electrochemical evidence of surfactant activity of the hepes pH buffer which may have implications on trace metal availability to cultures in vitro.

Hydrogen ion buffers are required for many different types of in vitro biological and chemical studies, but they may not be inert enough, thus interfering with the system under study. N-Hydroxyethylpiperazine-N'-ethanesulfonic acid (Hepes), a zwitterionic buffer in the 7.0-8.0 pH range, has been used in different biological studies. In this work, surfactant activity of Hepes and the buffer's capability to bind copper(II) were investigated through different electrochemical techniques: ion-selective electrode potentiometry, potentiometric stripping analysis, differential pulse anodic stripping voltammetry, normal pulse anodic stripping voltammetry, and alternating current polarography. Hepes concentrations between 0.1 (0.24 g liter-1) and 25 mmol liter-1 (6.0 g liter-1) were studied at pH 8.0 (and at pH 6.5 for comparison). At pH 8.0 Hepes displayed surfactant activity, particularly when magnitude of Hepes > 10 mmol liter-1 (2.4 g liter-1). The ability of Hepes to bind copper(II) was detected when Hepes was in large excess (magnitude of Hepes/magnitude of Cu between 10,000 and 2000). Eventual implications in the bioavailability of copper(II) are also discussed.

Application of potentiometric stripping analysis for speciation of copper complexes with a non-adsorbable ligand on a mercury electrode.

Potentiometric stripping analysis (PSA) was used for determination of conditional stability constants (delta') of copper(II) complexes. Glycine was used as a model of a non-adsorbable ligand on the mercury electrode that forms well defined 1:1 and 1:2 copper(II)-glycine complexes, which are labile within the time scale of the analytical technique. The calculations were performed by the DeFord-Hume method, which was applied to the shifts in peak potential (dt/dE vs. E) provoked by the presence of different concentration of the ligand in the metal solution. For comparison purposes, the study was also carried out by differential pulse anodic stripping voltammetry (DPASV). The results obtained by PSA both at pH 6.0, logbeta'(1) = 5.0 +/- 0.2 and logbeta'(2) = 7.6 + 0.2, and at pH 6.5, logbeta'(1) = 5.7 +/- 0.6 and logbeta'(2) = 8.5 +/- 0.4 (standard deviations are given), were in agreement with those obtained by DPASV and from the literature, which indicates that PSA is suitable for this type of study.

Acid-base ionization equilibria of 1-(2-carbamylethyl) imidazole and its 2-methyl and 2-ethyl derivatives.

The acid and base ionization constants of 1-(2-carbamylethyl)-2-alkylimidazoles, as well as the acid ionization constants of acrylamide and acetamide, have been determined by potentiometry. Concentration constants at 10.0, 25.0 and 40.0 degrees and I = 0.1M (KNO(3)) were measured. From plots of log K vs. 1/T the thermodynamic parameters (DeltaH and DeltaS) of the ionization reactions were calculated.

Simultaneous determination of the acid and basic ionization constants of imidazole.

The acid and base ionization constants of imidazole (LH) have been determined simultaneously by potentiometry. Concentration constants were obtained at 10.0, 15.0, 25.0 and 40.0 degrees with I = 0.1M (KNO(3)), the values (and standard deviations) at 25.0 degrees being pK(a1)(LH(2)(+)) = 7.002 +/- 0.006 and pK(a2)(LH(2)(+)) = 12.588 +/- 0.004. The following values were obtained for the corresponding thermodynamic parameters: DeltaH(1) =38 and DeltaH(2) = 38 kJ mole ; DeltaS(1) = -8 and DeltaS(2)= -112 J.mole(-1).K(-1).