Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction.

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1ß and nuclear translocation of the NF-κß transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.

Separation/preconcentration of Cr(VI) with a Modified Single-drop Microextraction Device and Determination by GFAAS.

We have developed a chromium speciation and preconcentration method with the use of the graphite furnace atomic absorption spectrometry (GFAAS) technique. This method is based on single-drop microextraction (SDME) technique. Nowadays the microextractions have become popular, because low amount of organic solvent needs to be used for the separation. The sample was introduced into the extraction cell with a single chloroform droplet. For the separation and enrichment of chromium species, an ion-pair forming compound was used. After the extraction, the chromium content of the droplet was determined by GFAAS. The analytical sensitivity of the standard SDME technique was improved by increasing the volume of organic phase and by sample recirculation. Because of the increased contact area and the developed extraction device, the stability of droplet was markedly increased. As an application we have determined the r(VI) content of sea water by the GFAAS technique using these separation/enrichment methods. Under the optimized extraction conditions, the linear range, detection limit (S/N = 3) and precision (RSD, n = 3) for Cr(VI) were 0.14 - 5.00 µg/L, 0.042 µg/L, and ≤ 3.0%, respectively. The advantages of this method are the following: cost efficiency, the high enrichment of chromium species and easy usage with the GFAAS technique. Therefore the concentration of the chromium species can be determined at the ng/L level.

The antagonistic Metschnikowia andauensis produces extracellular enzymes and pulcherrimin, whose production can be promoted by the culture factors.

Biological control against microbial infections has a great potential as an alternative approach instead of fungicidal chemicals, which can cause environmental pollution. The pigment producer Metschnikowia andauensis belongs to the antagonistic yeasts, but details of the mechanism by which it inhibits growth of other microbes are less known. Our results confirmed its antagonistic capacity on other yeast species isolated from fruits or flowers and demonstrated that the antagonistic capacity was well correlated with the size of the red pigmented zone. We have isolated and characterized its red pigment, which proved to be the iron chelating pulcherrimin. Its production was possible even in the presence of 0.05 mg/ml copper sulphate, which is widely used in organic vineyards because of its antimicrobial properties. Production and localisation of the pulcherrimin strongly depended on composition of the media and other culture factors. Glucose, galactose, disaccharides and the presence of pectin or certain amino acids clearly promoted pigment production. Higher temperatures and iron concentration decreased the diameter of red pigmented zones. The effect of pH on pigment production varied depending of whether it was tested in liquid or solid media. In addition, our results suggest that other mechanisms besides the iron depletion of the culture media may contribute to the antagonistic capacity of M. andauensis.

Zinc Inhibits HIF-Prolyl Hydroxylase Inhibitor-Aggravated VSMC Calcification Induced by High Phosphate.

Vascular calcification is a life-threatening clinical condition in chronic kidney disease (CKD) and is associated with reduced zinc serum levels. Anemia is another frequent complication of CKD. Hypoxia-inducible factor (HIF) stabilizers, also known as HIF prolyl hydroxylase inhibitors (PHI), are promising candidates to treat CKD-associated anemia by increasing erythropoietin synthesis. Recent evidence suggests that HIFs play a pivotal role in vascular calcification. Our study explored feasible impacts of HIF PHI on phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs) and tested whether zinc might inhibit this mineralization process. Treatment of VSMCs with PHI aggravated Pi-induced calcium deposition and Pi uptake. PHI promoted Pi-induced loss of smooth muscle cell markers (ACTA-2, MYH11, SM22α) and enhanced osteochondrogenic gene expression (Msx-2, BMP-2, Sp7) triggering osteochondrogenic phenotypic switch of VSMCs. These effects of PHI paralleled with increased pyruvate dehydrogenase kinase 4 (PDK4) expression, decreased Runx2 Ser451 phosphorylation, and reduced cell viability. Zinc inhibited Pi-induced mineralization of VSMCs in a dose-dependent manner and also attenuated the pro-calcification effect of PHI in Pi-induced mineralization. Zinc inhibited osteochondrogenic phenotypic switch of VSMCs reflected by lowering Pi uptake, decreasing the expressions of Msx-2, BMP-2, and Sp7 as well as the loss of smooth muscle cell-specific markers. Zinc preserved phosphorylation state of Runx2 Ser451, decreased PDK4 level, and restored cell viability. PHI alone reduced the expression of smooth muscle markers without inducing mineralization, which was also inhibited by zinc. In addition, we observed a significantly lower serum zinc level in CKD as well as in patients undergoing carotid endarterectomy compared to healthy individuals. Conclusion - PHI promoted the loss of smooth muscle markers and augmented Pi-induced osteochondrogenic phenotypic switch leading to VSMCs calcification. This mineralization process was attenuated by zinc. Enhanced vascular calcification is a potential risk factor during PHI therapy in CKD which necessitates the strict follow up of vascular calcification and zinc supplementation.

Relationship between serum nickel and homocysteine concentration in hemodialysis patients.

Severe hyperhomocysteinemia (HHC) is associated with atherosclerosis. In hemodialysis (HD) patients, one of the main causes of death is cardiovascular disease. In animals, trace elements such as cobalt, copper, iron, and nickel ameliorated vitamin B(12) deficiency-induced HHC. However, correlations between plasma total homocysteine (tHcy) and trace elements in HD patients have not been investigated. Therefore, tHcy, folate, vitamin B(12), trace elements (cobalt, copper, iron, and nickel), and some laboratory parameters such as serum total protein, albumin, transferrin, ferritin, C-reactive protein (CRP), and interleukin-6 concentrations were determined in 122 hemodialysis patients. When patients were divided into groups according to their tHcy, we found no significant differences in concentrations of cobalt, copper, and total protein, while nickel was higher, and folate, vitamin B(12), and iron were lower in patients with lower than higher tHcy. In univariate regression analysis, tHcy negatively correlated with concentrations of folate (r = -0.302, p < 0.006), vitamin B(12) (r = -0.347, p < 0.0001), nickel (r = -0.289, p < 0.006), and CRP (r = -0.230, p < 0.02) and positively with serum albumin (r = 0.316, p < 0.0004) and hemoglobin (r = 0.329, p < 0.0001) values. No relationship between tHcy and serum concentrations of cobalt, copper, iron, or other laboratory parameters was found in HD patients. The effect of cobalt and nickel on homocysteine production was assessed in human peripheral mononuclear cells (PBMCs). Nickel but not cobalt at concentrations found in HD patients significantly inhibited homocysteine, cysteine, and S-adenosylhomocysteine production in human PBMCs. These results suggest that nickel might also be involved in the regulation of the methionine-folate cycle in humans, as was demonstrated in animal experiments.

Elemental Analysis of Whole and Protein Separated Blood Serum of Patients with Systemic Lupus Erythematosus and Sjögren's Syndrome.

Systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) are systemic autoimmune diseases with complex symptoms and pathogenesis that are still not completely understood. Several studies showed that the trace element homeostasis and also the levels of antioxidant plasma proteins are changed in autoimmune disorders; however, these results are controversial. In this study, the potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), and iron (Fe) concentrations of the serum and proteins-immunoglobulin G (IgG), transferrin (Trf), albumin (Alb), and ceruloplasmin (Cp)-separated from serum samples by affinity chromatography were determined in patients with SLE and SS. Ca and K levels were found to be decreased in the case of both disorders compared to the control group, and the competitive antagonism of Cu and Zn was also observed: elevated Cu concentration together with a lower Zn concentration was measured in the sera of patients with autoimmune diseases. After fractionation, the trace element concentration of protein containing fractions altered to that of the control group. In case of the autoimmune disorders, the highest Cu concentration was determined in the Alb-containing protein fractions while the Zn level decreased in the Alb and increased in the Cp as well as in the IgG- and Trf-containing fractions compared to the healthy samples. Changes have also been found in the level and distribution of K and Ca.

The effect of a fireworks event on the amount and elemental concentration of deposited dust collected in the city of Debrecen, Hungary.

Many social celebrations in urban areas are followed by fireworks show. The organic and inorganic pollutants emitted during detonations are expected to affect the ambient air quality of these celebration sites. The environmental aspects of fireworks events are usually investigated by analyzing the concentration and composition of airborne particulate matter, while there is limited information regarding the effect of fireworks on the elemental concentration of deposited dust. In this study, foliage dust samples were collected in the city of Debrecen (Hungary) before and after the fireworks show, organized on the 20th of August for the celebration of a historical event. Leaf samples (Tilia tomentosa) were collected around the location of the area of festivities. The sampling sites were further divided into five areas: city center (center), Southeast (SE), Southwest (SW), Northeast (NE), and Northwest (NW). We found that the amount of deposited dust particles increased significantly after the fireworks show compared to the background; we also found significant differences in the amount of dust deposition between the different locations of the city. A statistically higher level of Ca, Mg, and Sr was detected in samples collected after the display compared to those collected during the previous days, while the concentration of other studied elements were not statistically different from the background level. Our study confirmed previous findings that the relatively high altitude of detonations allows chemicals to disperse in the fine and ultrafine aerosol fractions; thus, the emitted pollutants by fireworks shows do not increase the level of elements as markedly in deposited dust as in the inhalable fraction.

Atomization of cadmium compounds under reactive and inert high-temperature environment with stationary sample introduction.

Atomization of cadmium compounds (acetate, chloride, nitrate, perchlorate, sulfate, formate, propionate) was studied using flame atomic absorption spectrometry. Our goal was to study processes influencing atomization separately, the focus was on the contribution of thermal properties of substances to atomization. For this purpose new techniques and equipment have been developed, such as a special separated three-slot burner, quartz flame furnace, and an electrically heated thermospectrometer. According to quartz flame furnace and thermospectrometric measurements, cadmium salts do not atomize below 600 °C in an inert atmosphere. We found that in the thermospectrometer the atomization of cadmium compounds follows at least two different reaction courses. At lower temperatures (650-700 °C) a slower mechanism is dominant at higher regions of the furnace, while at 800 °C a faster mechanism demanding less residence time in the furnace becomes dominant. Under inert atmosphere the degree of atomization strongly depends on the thermal properties of substances.

Apoptogenic and necrogenic effects of mercuric acetate on the chromatin structure of K562 human erythroleukemia cells.

Time lapse video photography was used to follow the movement of individual cells after in vitro treatment with Hg(II) acetate. Cellular changes of mercuric ions were characterized by their properties of causing reduced cellular mobility (10-50microM), and complete lack of cellular movement at higher concentrations (100-1000microM). Results show that after mercury treatment at subtoxic levels (1microM): (a) chromatin changes were the earliest signs of cytotoxicity, (b) two major parts in nuclear material of K562 erythroleukemia cells could be distinguished, highly condensed supercoiled and decondensed veil-like chromatin, (c) decondensed chromosomes were rejected as clustered puffs and (d) often the nuclear material was broken down to apoptotic bodies. Nuclear changes caused by Hg(II) acetate in the concentration range between 10 and 50microM were characterized by apoptosis seen as broken nuclei and apoptotic bodies. High concentration of Hg(2+) ions (100microM) initiated necrotic nuclear changes, with enlarged leaky or opened nuclei.

A novel approach in dispersive liquid-liquid microextraction based on the use of an auxiliary solvent for adjustment of density UV-VIS spectrophotometric and graphite furnace atomic absorption spectrometric determination of gold based on ion pair formation.

This paper presents a novel approach to dispersive liquid-liquid microextraction (DLLME), based on the use of an auxiliary solvent for the adjustment of density. The procedure utilises a solvent system consisting of a dispersive solvent, an extraction solvent and an auxiliary solvent, which allows for the use of solvents having a density lower than that of water as an extraction solvent while preserving simple phase separation by centrifugation. The suggested approach could be an alternative to procedures described in the literature in recent months and which have been devoted to solving the same problem. The efficiency of the suggested approach is demonstrated through the determination of gold based on the formation of the ion pair [Au(CN)(2)](-) anion with Astra Phloxine (R) reagent and its extraction using the DLLME procedure with subsequent UV-VIS spectrophotometric and graphite furnace atomic absorption spectrometric detection. The optimum conditions were found to be: pH 3; 0.8 mmol L(-1) K(4)[Fe(CN)(6)]; 0.12 mmol L(-1) R; dispersive solvent, methanol; extraction solvent, toluene; auxiliary solvent, tetrachloromethane. The calibration plots were linear in the ranges 0.39-4.7 mg L(-1) and 0.5-39.4 µg L(-1) for UV-VIS and GFAAS detection, respectively; thus enables the application of the developed method in two ranges differing from one from another by three orders of magnitude. The presented approach can be applied to the development of DLLME procedures for the determination of other compounds extractable by organic solvents with a density lower than that of water.