Oto Mestek

Oxidative stress, metabolism of ethanol and alcohol-related diseases.

Alcohol-induced oxidative stress is linked to the metabolism of ethanol. Three metabolic pathways of ethanol have been described in the human body so far. They involve the following enzymes: alcohol dehydrogenase, microsomal ethanol oxidation system (MEOS) and catalase. Each of these pathways could produce free radicals which affect the antioxidant system. Ethanol per se, hyperlactacidemia and elevated NADH increase xanthine oxidase activity, which results in the production of superoxide. Lipid peroxidation and superoxide production correlate with the amount of cytochrome P450 2E1. MEOS aggravates the oxidative stress directly as well as indirectly by impairing the defense systems. Hydroxyethyl radicals are probably involved in the alkylation of hepatic proteins. Nitric oxide (NO) is one of the key factors contributing to the vessel wall homeostasis, an important mediator of the vascular tone and neuronal transduction, and has cytotoxic effects. Stable metabolites--nitrites and nitrates--were increased in alcoholics (34.3 +/- 2.6 vs. 22.7 +/- 1.2 micromol/l, p < 0.001). High NO concentration could be discussed for its excitotoxicity and may be linked to cytotoxicity in neurons, glia and myelin. Formation of NO has been linked to an increased preference for and tolerance to alcohol in recent studies. Increased NO biosynthesis also via inducible NO synthase (NOS, chronic stimulation) may contribute to platelet and endothelial dysfunctions. Comparison of chronically ethanol-fed rats and controls demonstrates that exposure to ethanol causes a decrease in NADPH diaphorase activity (neuronal NOS) in neurons and fibers of the cerebellar cortex and superior colliculus (stratum griseum superficiale and intermedium) in rats. These changes in the highly organized structure contribute to the motor disturbances, which are associated with alcohol abuse. Antiphospholipid antibodies (APA) in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression, and they correlate significantly with the disease severity. The low-density lipoprotein (LDL) oxidation is supposed to be one of the most important pathogenic mechanisms of atherogenesis, and antibodies against oxidized LDL (oxLDL) are some kind of epiphenomenon of this process. We studied IgG oxLDL and four APA (anticardiolipin, antiphosphatidylserine, antiphosphatidylethanolamine and antiphosphatidylcholine antibodies). The IgG oxLDL (406.4 +/- 52.5 vs. 499.9 +/- 52.5 mU/ml) was not affected in alcoholic patients, but oxLDL was higher (71.6 +/- 4.1 vs. 44.2 +/- 2.7 micromol/l, p < 0.001). The prevalence of studied APA in alcoholics with mildly affected liver function was higher than in controls, but not significantly. On the contrary, changes of autoantibodies to IgG oxLDL revealed a wide range of IgG oxLDL titers in a healthy population. These parameters do not appear to be very promising for the evaluation of the risk of atherosclerosis. Free radicals increase the oxidative modification of LDL. This is one of the most important mechanisms, which increases cardiovascular risk in chronic alcoholic patients. Important enzymatic antioxidant systems - superoxide dismutase and glutathione peroxidase - are decreased in alcoholics. We did not find any changes of serum retinol and tocopherol concentrations in alcoholics, and blood and plasma selenium and copper levels were unchanged as well. Only the zinc concentration was decreased in plasma. It could be related to the impairment of the immune system in alcoholics. Measurement of these parameters in blood compartments does not seem to indicate a possible organ, e.g. liver deficiency.

Receptor for advanced glycation end products (RAGE)--soluble form (sRAGE) and gene polymorphisms in patients with breast cancer.

Receptor for advanced glycation end products (RAGE) may be involved in the pathogenesis of the cancer progression and metastasis. Pathological effects mediated via RAGE are physiologically inhibited by soluble RAGE (sRAGE), so the higher sRAGE levels may confer the patients with cancer with better outcome. The aim was to study sRAGE and RAGE gene polymorphisms in patients with breast cancer. The authors studied sRAGE and RAGE polymorphisms in 120 patients with breast cancer (subdivided based on the clinical stage, histologic grading, expression of hormonal and Her2/neu receptors) and in 92 healthy controls. Despite higher serum concentrations of AGEs, serum concentrations of sRAGE were lower in patients with breast cancer compared to healthy controls (1581 ± 777 versus 1803 ± 632 ng/mL, p < 0.05). Serum levels of sRAGE were higher in patients with advanced breast cancer (stage III), lower grade and positive estrogen receptors, and intermediate positivity of Her2/neu receptors and were also influenced genetically (Gly82Ser and 2184 AG polymorphisms of the RAGE gene). Decreased sRAGE levels in patients with breast cancer may contribute to the progression of the disease. Patients with better outcome (low grade and positive estrogen receptors) have higher sRAGE levels. Progression of the disease, may, however, increase sRAGE levels, possibly as a compensatory mechanism to counteract further progression.

Trace Elements in Hemodialysis and Continuous Ambulatory Peritoneal Dialysis Patients

Alterations in blood and tissue concentrations of trace elements in patients with chronic renal failure have been extensively investigated. Selenium, zinc and copper are elements which play an important role in biological systems as components of proteins, enzymes and antioxidants. The concentrations of selenium, zinc and copper were determined in the plasma, erythrocytes and whole blood of patients on regular hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) treatment using the method of inductively coupled plasma mass spectrometry (ICP-MS). Analysis of isotopes 77Se, 66Zn and 65Cu was performed. Methodology presents the major limitation to valid studies on trace element levels in biological materials. One of the widely used contemporary techniques is ICP-MS. It is the most sensitive one and has a high dynamic range. The selenium concentration in the studied compartments (plasma 46.1 ± 3.0 vs. 78.0 ± 3.4 μg/l, p < 0.001; erythrocytes 90.4 ± 6.5 vs. 134.2 ± 7.6 μg/l, p < 0.01; whole blood 67.3 ± 3.1 vs. 106.4 ± 3.4 μg/l, p < 0.001) was significantly lower in HD patients compared to healthy controls. The same result was observed in plasma (63.2 ± 5.8 vs. 78.0 ± 3.4 μg/l, p < 0.05) and whole blood (82.7 ± 7.4 vs. 106.4 ± 3.4 μg/l, p < 0.01) from CAPD patients, but the selenium level of erythrocytes in CAPD patients was the same as in the control group (126.0 ± 8.8 vs. 134.2 ± 7.6 μg/l). The cooper content of erythrocytes was lower in HD patients than in controls (0.55 ± 0.02 vs. 0.66 ± 0.01 mg/l, p < 0.01) and CAPD groups (0.55 ± 0.02 vs. 0.68 ± 0.02 mg/l, p < 0.001). There were no differences in copper content in plasma (HD 1.02 ± 0.06; CAPD 1.11 ± 0.09; controls 1.02 ± 0.05 mg/l) and whole blood (HD 0.87 ± 0.04; CAPD 0.90 ± 0.05; controls 0.85 ± 0.02 mg/l) in HD and CAPD patients and healthy controls. The zinc concentration was increased in the whole blood of CAPD patients (6.68 ± 0.36 vs. 5.52 ± 0.11 mg/l, p < 0.001) and erythrocytes of HD (12.30 ± 0.23 vs. 10.11 ± 0.42 mg/l, p < 0.001), and CAPD groups (13.71 ± 0.56 vs. 10.11 ± 0.42 mg/l, p < 0.001) compared to controls. However, the plasma zinc concentration was lower in HD patients compared to blood donors (0.69 ± 0.03 vs. 0.92 ± 0.03 mg/l, p < 0.001) and CAPD patients (0.69 ± 0.03 vs. 0.95 ± 0.04 mg/l, p < 0.001). We did not find a significant increase in trace elements in whole blood after HD. These results suggest differences between plasma, erythrocytes and whole blood concentrations of the studied trace elements. The levels of trace elements are altered by HD and CAPD. A modern precise method with high accuracy, ICP-MS, which was used in our study, eliminated analytical errors and possible interferences.

Trace Elements in End-Stage Renal Disease

For human beings trace elements are essential nutrients with a gamut of functions. They are for instance indispensable components of many enzymes, so they have some regulatory functions and they may affect immune reactions and free radical generation. Altered blood levels of different trace elements have been described in patients with advanced renal failure and especially in those treated by different kinds of renal replacement therapy. Altered renal function may result in impaired renal excretion of trace elements and their accumulation or depletion in the body. The dialysate concentrate and water used for preparing the dialysate may be an important source of the accumulation or depletion of trace elements in dialyzed patients. The gain or loss of trace elements during dialysis depends on the gradient between the ultrafiltrable fraction of a particular element in serum and its concentration in the dialysis fluid, and also on the type and permeability of the dialysis membrane. There are some methodological problems concerning the handling and storing of blood samples and measurement techniques leading to the rather inconsistent results of different studies concerning trace elements in renal disease. Geographical variations and environmental contamination of soil and water and different dietary habits may significantly influence trace elements in these patients. The abnormalities of trace elements are primarily the result of uremia, and they may be further modified and sometimes greatly exacerbated by the dialysis procedure.

Fractionation of phosphorus and trace elements species in soybean flour and common white bean seeds by size exclusion chromatography-inductively coupled plasma mass spectrometry.

Soluble species of phosphorus, sulfur, selenium and eight metals (Mn, Fe, Co, Ni, Cu, Zn, Mo and Cd) in soybean flour and common white bean seeds were investigated by size exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). Samples were extracted by 0.02 mol l(-1) Tris-HCI buffer solution (pH 7.5). Fractionation of sample extracts by preparative scale SEC was accomplished using a Fractogel EMD BioSEC column (600 x 16 mm) and 0.02 mol l(-1) Tris-HCl buffer solution (pH 7.5) as mobile phase (flow rate: 2 ml min(-1)). A 2-ml sample was injected. Contents of elements in chromatographic fractions were determined by AAS, ICP-AES and ICP-MS. The elution profiles of P, Fe, Co, Ni, Cu, Zn and Mo in both samples were similar. Main species of Co, Ni, Cu, Zn and Mo were found in the low molecular weight region (2-5 kDa), whereas Fe is predominantly bound to high molecular weight compounds (180 kDa). The dominant phosphorus fraction was detected in the medium molecular weight region (10-30 kDa) and the other fraction in the low molecular weight region. Isotachophoretic analysis of chromatographic fractions revealed that the main phosphorus compound in the medium molecular weight region is phytic acid. SEC on Superdex 75 and Superdex Peptide columns (300 x 10 mm) was performed in on-line hyphenation with ICP-MS. The same mobile phase was used with a flow rate of 0.5 ml min(-1); volume of injected sample was 200 microl. Element specific chromatograms were obtained by continuous nebulization of effluent into ICP-mass spectrometer measuring intensities of 47(PO)+ and 48(SO)+ oxide ions and 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se, 95Mo and 114Cd nuclides. Chromatographic profiles of elements are generally analogous to those obtained with a Fractogel column, but better chromatographic resolution of separated species was achieved so that slight differences between samples were revealed. Estimated molecular weights of major phosphorus species in soybean flour and common white bean seed extracts are 6 and 3.6 kDa, respectively, whereas those of minor phosphorus species in both samples are 0.7 kDa. Traces of phosphorus were also detected in the high molecular weight region (130 kDa). Chromatograms of P, Ni, Cu, Zn and Mo compounds in both extracts are similar but not identical. Molecular weights of major Cu and Zn species are approximately 1 and 0.4 kDa for soybean flour and white bean seeds, respectively. In cases of Mn, Fe, Co and Se, the element profiles of soybean flour and white bean seed extracts are significantly different.

Soluble receptor for advanced glycation end-products (sRAGE) and polymorphisms of RAGE and glyoxalase I genes in patients with pancreas cancer.

OBJECTIVES The receptor for advanced glycation end-products (RAGE) takes part in the pathogenesis of many diseases, including diabetes mellitus and cancer. AGE-precursors are detoxified by glyoxalase (GLO). sRAGE, soluble RAGE, is an inhibitor of pathological effects mediated via RAGE. The aim was to study sRAGE and polymorphisms of RAGE (AGER) and GLO genes in patients with pancreas cancer (PC). DESIGN AND METHODS The studied group consisted of 51 patients with PC (34 with impaired glucose tolerance-IGT, 17 without IGT), 34 type 2 DM and 154 controls. For genetic analysis, the number of patients was increased to 170. Serum sRAGE was measured by ELISA and all polymorphisms (RAGE -429T/C, -374T/A, 2184A/G, Gly82Ser and GLO A419C) were determined by PCR-RFLP and confirmed by sequencing. RESULTS Soluble RAGE is decreased in patients with PC compared to patients with DM and controls (975+/-532 vs. 1416+/-868 vs. 1723+/-643pg/mL, p<0.001). Patients with PC and IGT have lower sRAGE levels compared to patients with PC without IGT (886+/-470 vs. 1153+/-616pg/mL, p<0.05). No relationship of sRAGE to the stage was found. We did not show any difference in allelic and genotype frequencies in all RAGE and GLO polymorphisms among the studied groups. CONCLUSION This is the first study demonstrating decreased sRAGE in patients with pancreas cancer. Its levels are even lower than in diabetics and are lowest in patients with PC and IGT. Our study supports the role of glucose metabolism disorder in cancerogenesis. Further studies are clearly warranted, especially with respect to potential preventive and therapeutic implications.

Quantification of copper and zinc species fractions in legume seeds extracts by SEC/ICP-MS: validation and uncertainty estimation.

Fractions of Cu and Zn species in legume samples (common white bean, pea, chick pea and lentil seeds and defatted soybean flour) were analysed by on-line hyphenation of size exclusion chromatography and inductively coupled plasma-mass spectrometry. Samples were extracted by 0.02 mol l(-1) Tris-HCl buffer solution, pH 7.5. The extraction efficiency lay in the region 60-90 and 60-80% for Cu and Zn, respectively. Quantification of elements in the individual chromatographic fractions was carried out by isotope dilution (ID) and external calibration (EC) techniques. For ID analysis the chromatographic effluent was mixed with the flow of (65)Cu and (68)Zn isotope enriched solution and the isotope ratio values (63)Cu/(65)Cu and ((64)Zn+(66)Zn)/(68)Zn were measured. In the case of EC technique calibration solutions of elements were injected to the flow of mobile phase by the second injector. Prior entering detector the effluent was mixed with the flow of internal standard solution (In, 50 mug l(-1)). Both methods have similar precision, however the behaviour of both studied elements was not the same. The chromatographic analysis itself was the main source of variability in the case of Cu. For Zn species analysis, the extraction process and the manipulation with the extract, played the significant role too. It was probably caused by lower stability of the present zinc chelates. The total amounts of Zn found in all chromatographic fractions represented 85-95% of Zn in sampled extract whereas those of Cu approached 100%. In case of small peaks the results of ID and EC were not the same. The EC results were lower then ID results. The great deal of results uncertainty accounts for the precision.

Comparison of the Suitability of Various Atomic Spectroscopic Techinques for the Determination of Selenium in Human Whole Blood

A method was developed for the determination of selenium in human blood by ICP-MS. The procedure involves sample dilution with Triton X-100 and HNO 3 (1 ml of whole blood+1 ml of 1% Triton X-100+1.5 ml of 0.14 mol l -1 HNO 3 +0.2 ml of 5 mg l -1 In solution+water to 10 ml) and determination using the 77 Se line. Standard samples were modified with a solution containing NaCl, Fe, Ca, Br, S, and K. No corrections were made for interferences from polyatomic ions. The applicability of the ICP-MS, ICP-OES, ETAAS and HGAAS techniques to the determination of Se in human blood was compared. Limits of detection (3 s ) of these methods were 6, 76, 14 and 8 µg l -1 Se, respectively. The ICP-MS method was found to be well suited to routine analysis.

A419C (E111A) Polymorphism of the Glyoxalase I Gene and Vascular Complications in Chronic Hemodialysis Patients

Advanced glycation end products (AGEs) take part in the pathogenesis of vascular, diabetic, and uremic complications. Their precursors are detoxified by the glyoxalase system. Our aim was to study A419C (E111A) single nucleotide polymorphism (SNP) of the glyoxalase I gene in hemodialysis (HD) patients. A419C SNP, several laboratory parameters including soluble receptor for AGEs (sRAGE), and clinical data were studied in 214 HD patients and 89 controls. Allelic and genotypic frequencies did not differ between HD patients and controls. A419C SNP was significantly linked with serum sRAGE, which sensitively reflects the AGE burden of the organism (3986 ± 1638 pg/mL in the CC variant versus 3277 ± 1398 pg/mL in the AC variant and 3297 ± 1445 pg/mL in the AA variant, P < 0.01). In the CC variant, significantly higher prevalence of cardiovascular disease and peripheral vascular disease was found, while the prevalence of hypertension, diabetes mellitus, and dyslipidemia did not differ between genotypes. In summary, in this study we demonstrate for the first time the association of A419C polymorphism of the glyoxalase I gene with sRAGE levels and show the genetic predisposition to vascular complications in HD patients.

Application of size-exclusion chromatography-inductively coupled plasma mass spectrometry for fractionation of element species in seeds of legumes.

Fractionation of soluble species of P, Mn, Fe, Co, Ni, Cu, Zn, Se and Mo in pea and lentil seeds was made by on-line hyphenation of size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry. Seed samples were extracted with 0.02 mol l(-1) Tris-HCl buffer solution, pH 7.5. SEC was performed on Superdex 75 and Superdex Peptide columns (300 x 10 mm) with the same buffer solution as the mobile phase. Monitoring of oxide ion 47(PO)+ was used for detection of phosphorus compounds. Other elements were detected as ions of 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se and 95Mo nuclides. Elements in individual elution zones were quantified using external calibration. Complete chromatographic recoveries of elements were found in cases of phosphorus, nickel and copper. Substantial parts of manganese and zinc, as well as traces of cobalt, selenium and molybdenum are retained on the column. Injection of EDTA solution removes these elements from the column. Chromatographic profiles of pea and lentil samples are very similar for all elements except Mo. Main element species in the high-molecular-mass region (approx. 190,000 rel. mol. mass unit) were detected in case of Fe. Low-molecular-mass species (<2000 rel. mol. mass unit) as major element forms are typical for Cu and Zn.