Eduardo Martins

Accumulation of calcium and loss of potassium in the hippocampus following transient cerebral ischemia: a proton microprobe study

This study explored (a) whether postischemic accumulation of calcium in hippocampal neurons precedes or occurs pari passu with light microscopical signs of delayed neuronal necrosis, and (b) whether calcium initially accumulates in dendritic domains, presumed to have a high density of agonist-operated calcium channels. Transient ischemia of 10-min duration was induced in rats, and the animals were studied after 1, 2, 3, and 4 days of recovery. We measured total calcium and potassium contents in the stratum oriens, pyramidale, radiatum, and moleculare of the CA1 and CA3 sectors, using particle induced x-ray emission (PIXE) in the proton microprobe mode. The results showed significant accumulation of calcium and loss of potassium after 3 and 4 days of recovery in the CA1 sector, which developed neuronal necrosis, but not in the CA3 sector, which showed only occasional damage. In a few animals, calcium accumulation (and loss of potassium) was observed with no or only mild visible damage, but in the majority of animals the accumulation of calcium correlated to signs of neuronal necrosis. Since calcium accumulation was similar in all strata examined, the results failed to reveal preferential accumulation in dendritic or somal regions. Based on our results and those of Dux et al., we emphasize the possibility that delayed neuronal death is, at least in part, caused by increased calcium cycling of plasma membranes and gradual calcium overload of mitochondria.

Cadmium(II), zinc(II), and copper(II) ions binding to bovine serum albumin. A 113Cd NMR study

Abstract The interaction between bovine serum albumin and the metals Cd(II), Zn(II) and Cu(II) was studied by 113 Cd NMR. A 113 Cd titration of the protein revealed two strong Cd(II) binding sites. Competition between Zn(II) (or Cu(II)) and Cd(II) for the strong sites indicates that there is a common strong binding site for Cu(II), Zn(II) and Cd(II), with higher affinity for Cu(II) and Zn(II) than for Cd(II). Competition between serum albumin and NTA for Cd(II) did not allow conclusive evaluation of the intrinsic binding constants, probably due to the formation of ternary complexes NTACd(II)protein. Chemical shifts are in good agreement with histidyl residue involvement in one site, and probably oxygen ligands in a second site. The site responsible for strong binding of Zn(II) contains two histidyl residues, but not thiol groups.

Solute transfer in on-line analytical flow-through dialyzers

Abstract Mass transfer in infinite parallel-plate dialyzers with co-flow between sample and detector streams is discussed for three different theoretical models. Analytical solutions with coupled diffusion and membrane transfer equations were obtained for plug flow in both channels. The finite-difference approximation method was used to obtain numerical solutions for a laminar-flow regime. Results obtained with a mixing-cup model under steady-state conditions were also included. With the dimensions typical for analytical dialyzers, there were only small differences between the laminar-flow and plug-flow models. The mixing-cup model predicted higher fluxes through the membrane than the other two models, particularly when the channel heights were increased. The theoretical results were compared with experimental results for dialysis of zinc(II) ions and the flow dependence agreed reasonably well with theory provided that the hydrostatic pressures were equal on both sides, and that stresses which could result in membrane bulging were kept low.

Accumulation of calcium in substantia nigra lesions induced by status epilepticus. A microprobe analysis.

Status epilepticus of sufficient duration (greater than 30 min) causes a unique lesion of substantia nigra pars reticulata (SNPR), and of globus pallidus (GP). This lesion, which encompasses a pan-necrotic destruction of neurons and glial cells seems to develop during ongoing seizures. We decided to investigate if the lesion is accompanied by net calcium accumulation. Seizures of 20 and 60 min duration were induced by the administration of flurothyl, and the tissue was frozen in situ either at the end of the seizure periods, or after recovery periods of 15 or 120 min. The total calcium and potassium contents of caudoputamen, neocortex, GP and SNPR were measured using particle induced X-ray emission (PIXE) in the microprobe mode. Seizures of 20 min duration did not cause net accumulation of calcium. When the duration of seizures was extended to 60 min the results varied depending on the location. In caudoputamen, which does not incur neuronal damage, no calcium accumulation was observed. In GP and SNPR, such a rise was unequivocally demonstrated, with calcium content increasing to about 150% of controls. The increase in calcium correlated to a decrease in potassium content. It is concluded that epileptic cell death occurs pari passu with accumulation of calcium although it cannot be stated that this accumulation is the cause of the cell death. It is speculated that seizures increase the permeability of the blood-brain barrier to calcium, and that enhanced blood to tissue transfer increases the calcium load of metabolically strained cells.

On-line dialysis of some metal ions and metal complexes

Abstract The effect of ionic strength, pH and complexing ligands on the dialysis of metal ions, particularly zinc(II), through cellulose acetate membranes, was studied under flow conditions. The dialysis factor, which depends on both the mass transfer and the membrane permeability, was found to be independence of ionic strength in the interaval 0.05–0.3 M and to increase only slightly with pH 4.6 and 7.0. Some common buffer constituents had no effect, but chloride and calcium ions affected the transfer rates. The rate of transfer of the ligands histidine, NTA and EDTA was of the same order of magnitude as that of the metal ions. The transfer rate of the Ni(II)-EDTA complex was the same as that of a mixture of Ni(II) and EDTA. Generally, addition of the chelating agents decreased the metal-ion transfer rates. Partition coefficients between the membrane polymer and the buffers were evaluated and compared with the dialysis factors and there were significant differences with regard to the range of variation, effect of concentration and pH-dependence. The time scale of uptake or release of metal ions by the polymer is much longer than that of a steady-state transfer in dialysis. Partition and dialysis may not be directly related to each other.

A flow-through cell for differential pulse anodic stripping voltammetry

Abstract A flow-through voltammetric cell with a hanging mercury drop electrode has been developed to fit the static mercury drop electrode (PAR 303). The design has resulted in a linear increase of sensitivity with flow rate and an enhancement of sensitivity by the wall-jet effect. The cell is used in a flow injection system in which samples are introduced with a Růžicka—Hansen injector. The mercury drop is held at plating potentials while the sample peak passes through the cell. Stripping is done under stopped flow conditions, to reduce noise, after the sample has been washed completely from the cell. The stripping thus takes place into the carrier electrolyte which always has a constant composition independent of sample constituents. Film-forming interfering species will, however, remain on the surface of the mercury drop. The effect of medium exchange on films produced by l -cysteine is reported. The flow-through medium exchange simplifies deaeration, speeds up analysis and reduces contamination.

Hollow fiber cartridges for removal of particulate matter from natural waters prior to matrix isolation and trace metal enrichment using an 8-quinolinol chelating ion exchanger in a flow system

Abstract Natural water samples are normally filtered through a 0.45 μm membrane filter before analysis. Such filters clog rapidly, however, when the content of humic acids is high. An alternative approach for natural water filtration is reported in this paper. Sample solutions are pumped into tubing containing a coaxial hollow fiber and a needle valve restrictor at the outlet. One end of the hollow fiber is plugged and the other is connected to a chelating ion exchange column for uptake of trace metal ions. The differential pressure between the inside and the outside of the fiber forces a filtered liquid through the fiber and into the ion exchanger. It was found that flow rates of about 2.0 ml min−1 could be obtained with a pressure difference of around 1 bar. The concentrations of copper, cadmium and cobalt ions were the same in the filtrate as in the inlet solution, indicating that the membrane is uncharged. In one configuration more than 4 I of a sample containing 20 μg ml−1 humic acids could be passed through the fiber before clogging caused unacceptable reduction of the flow rate. A hollow fiber cartridge, therefore, seems to be very useful for enrichment of ions from samples with a high solids content. The trace metals were enriched about 15 times using the described procedure.

Investigation of Ischaemic rat brains using a proton microprobe

Abstract Calcium and iron concentrations in the dorsal hippocampus from rats subjected to transient ischaemia were measured with a proton microprobe. In control animals no difference in calcium concentration could be detected in cellular and dendritic layers but in postischaemic brains increased calcium concentrations were found in both structures. The staining and fixation of the tissue slices for visual inspection of the beam spots using light microscopy are described.

Model experiments for determinations of zinc—amino acid complexes in biological fluids by polarography

Abstract The Ilkovic constant was determined for zinc and zinc—amino acid complexes by using differential pulse polarography in 0.1 M sodium chloride—sodium diethylbarbiturate buffer at pH 7.4. A single well-behaved wave was obtained, except for cysteine and cystine for which useful calibration curves could not be obtained. The non-equilibrium dialysis of the amino acid complexes was studied in a hollow-fiber dialyzer. It is concluded that the dialysis step could be used in a method intended to measure available zinc but that the polarographic method needs further modification.

Flow-through dialysis of zinc(II) from model and human sera

Dialysis of zinc(II) through cellulose-acetate membranes was studied in a flow-through dialyzer. The metal ion concentration was determined with differential pulse anodic stripping voltammetry in a flow-through cell with a detection limit of 10(-9) M zinc(II). The concentration of zinc(II) in the dialysate decreased by x750 when albumin was added in an excess corresponding to the physiological concentrations. Addition of amino acids in physiological concentrations had little or no effect on the transfer of zinc(II) through the membrane. Albumin solutions and a model serum containing inorganic salts, amino acids, lipids, and carbohydrates were compared with pooled serum. Zinc(II) was transferred faster from the latter at equal concentrations of exchangeable total zinc. Predialysis of the pooled serum decreased the zinc(II) transferring capability indicating some unknown mode of transport from the untreated serum.