Anna Pieniazek

Investigation of albumin properties in patients with chronic renal failure

The aim of this study was the investigation of HSA properties and its structural changes after modification induced in vivo among patients with CRF who underwent haemodialysis. Application of different fluorescent dyes allowed the investigation of different regions of albumin molecule using ANS, bis-ANS, piren, piren maleimide and fluorescein isothiocyanate. As markers of oxidative modification, the total protein thiol, carbonyls, glycosylated plasma proteins and hydroperoxide were estimated in plasma. Additionally, this study investigated plasma viscosity and total antioxidant capacity (TAC) of the plasma. Results show that haemodialysis provoked significant changes in conformational properties of plasma albumin, which resulted in the loss of its biological functions. These findings suggest that oxidative stress and glycation of proteins in plasma are developed during haemodialysis. The results depict that one of the features of uraemia is the presence of signs of oxidative stress before haemodialysis. Nevertheless, oxidative stress and glycation of proteins in plasma are exacerbated during haemodialysis and are a complex process.

Alterations in Red Blood Cells and Plasma Properties after Acute Single Bout of Exercise

The aim of this study was to investigate alterations in haemoglobin conformation and parameters related to oxidative stress in whole erythrocytes, membranes, and plasma after a single bout of exercise in a group of young untrained men. Venous blood samples from eleven healthy young untrained males (age = 22 ± 2 years, BMI = 23 ± 2.5 kg/m2) were taken from the antecubital vein before an incremental cycling exercise test, immediately after exercise, and 1 hour after exercise. Individual heart rate response to this exercise was 195 ± 12 beats/min and the maximum wattage was 292 ± 27 W. Immediately after exercise, significant increase in standard parameters (haemoglobin, haematocrit, lactate levels, and plasma volume) of blood was observed as well as plasma antioxidant capacity one hour after exercise. Reversible conformational changes in haemoglobin, measured using a maleimide spin label, were found immediately following exercise. The concentration of ascorbic acid inside erythrocytes significantly decreased after exercise. A significant decline in membrane thiols was observed one hour after exercise, but simultaneously an increase in plasma thiols immediately after and 1 h after exercise was also observed. This study shows that a single bout of exercise can lead to mobilization of defensive antioxidant systems in blood against oxidative stress in young untrained men.

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.

Investigation of oxidative stress parameters in different lifespan erythrocyte fractions in young untrained men after acute exercise

What is the central question of this study? What is the influence of a single bout of exercise on the properties of erythrocyte fractions at different ages? What is the main finding and its importance? A single bout of exercise in untrained men induced oxidative stress in erythrocytes and had an influence on antioxidant defense in these cells. Old erythrocytes were more sensitive to oxidative damage than young and middle‐aged cells. Higher levels of glutathione in old erythrocyte fractions did not protect them against oxidative stress. It seems that exercise may promote the removal of old erythrocytes from the circulation.

Inhibition of glutamate receptors reduces the homocysteine-induced whole blood platelet aggregation but does not affect superoxide anion generation or platelet membrane fluidization

Abstract Homocysteine (Hcy) is an excitotoxic amino acid. It is potentially possible to prevent Hcy-induced toxicity, including haemostatic impairments, by antagonizing glutaminergic receptors. Using impedance aggregometry with arachidonate and collagen as platelet agonists, we tested whether the blockade of platelet NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors with their inhibitors: MK-801 (dizocilpine hydrogen maleate, [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), CNQX (7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile) and UBP-302 (2-{[3-[(2S)-2-amino-2-carboxyethyl]-2,6-dioxo-3,6-dihydropyrimidin 1(2H)-yl]methyl}benzoic acid) may hamper Hcy-dependent platelet aggregation. All the tested compounds significantly inhibited Hcy-augmented aggregation of blood platelets stimulated either with arachidonate or collagen. Hcy stimulated the generation of superoxide anion in whole blood samples in a concentration-dependent manner; however, this process appeared as independent on ionotropic glutamate receptors, as well as on NADPH oxidase and protein kinase C, and was not apparently associated with the extent of either arachidonate- or collagen-dependent platelet aggregation. Moreover, Hcy acted as a significant fluidizer of surface (more hydrophilic) and inner (more hydrophobic) regions of platelet membrane lipid bilayer, when used at the concentration range from 10 to 50 µmol/l. However, this effect was independent on the Hcy action through glutamate ionotropic receptors, since there was no effects of MK-801, CNQX or UBP-302 on Hcy-mediated membrane fluidization. In conclusion, Hcy-induced changes in whole blood platelet aggregation are mediated through the ionotopic excitotoxic receptors, although the detailed mechanisms underlying such interactions remain to be elucidated.

Changes in lymphocyte properties after employment of the combination of carbamylation and oxidative stress, an in vitro study.

It is well known that oxidative stress and carbamylation alter macromolecule properties and functions. We evaluated the influence of sodium cyanate (NaOCN) and the combination of cyanate and hydrogen peroxide (H2O2) on nonenzymatic antioxidant capacity (NEAC), total thiols, reduced glutathione (GSH) and hydroperoxide level in mononuclear blood cells (MNCs). We also examined plasma membrane properties of MNCs using the spin labeling method in EPR spectroscopy (electron paramagnetic resonance spectroscopy). We showed that MNCs are resistant to cyanate treatment up to a concentration of 2mM (survival test). On the other hand, a significant loss of antioxidant defense of cells, e.g. NEAC upon NaOCN, H2O2 and the combination of cyanate and hydrogen peroxide was observed. Carbamylation slightly decreased GSH and the free thiol level, but H2O2 and its combination with NaOCN lead to a decrease in their amounts. A markedly higher level of hydroperoxides was only observed in the cells treated with H2O2. We found a significant decrease in lipid membrane fluidity at the depth of 12th and 16th carbon atoms of fatty acids in lymphocytes treated with cyanate or H2O2. The combination of both substances acted synergistically and induced profound changes in comparison to cyanate and hydrogen peroxide used alone.

Changes in the Conformational State of Hemoglobin in Hemodialysed Patients with Chronic Renal Failure

The aim of this study was to evaluate the properties of internal components of erythrocytes in chronic renal failure (CRF) patients undergoing hemodialysis (HD) in comparison to control subjects. For investigation of conformational state of hemoglobin and nonheme proteins (NHP) the maleimide spin label (MSL) in electron paramagnetic resonance (EPR) was applied. The studies were performed using MSL in whole cells and hemolysate as well as proteins separated by ion exchange chromatography and checked by electrophoresis. Additionally the level of –SH groups in hemolysate and isolated internal proteins of CRF erythrocytes was determined using 4,4′-dithiodipyridine. All measurements were performed before and after hemodialysis. Oxidative stress accompanying CRF/hemodialysed patients caused a significant decrease in the mobility of internal components inside erythrocytes indicated by MSL (P < 0.02). The significant decrease in mobility of spin labeled HbA1c and HbA both before and after HD (P < 0.0002) as well as in nonheme proteins before hemodialysis (P < 0.05) versus control was indicated. Decrease in mobility of internal components of erythrocytes was accompanied by loss of thiols before and after hemodialysis versus control in NHP (P < 0.05), HbA1c (P < 0.0002), and HbA (P < 0.0005). These findings showed oxidative influence of hemodialysis on hemoglobins and internal nonheme proteins in erythrocytes of CRF patients.

Indoxyl sulfate generates free radicals, decreases antioxidant defense and leads to damage to mononuclear blood cells

Indoxyl sulfate (IS) is a uremic toxin that has been associated with inflammation and oxidative stress as well as with the progression of chronic kidney disease (CKD). IS is a protein metabolite that is concentrated in the serum of CKD patients. IS is a well-known uremic toxin, but there are very few reports on the effect of IS on cells including mononuclear cells (MNCs). We hypothesized that a high concentration of IS in CKD patients may induce changes in redox balance in the in vitro cells exposed. In the present study, we investigated the effect of IS on free radical production, antioxidant capacity, and protein damage in the mononuclear blood cells. As already determined, the concentrations (0.2 or 1 mM) of IS used in this study do not affect the survival rate of MNCs. For both the concentrations of IS, there was an increase in superoxide and nitric oxide and a release of other reactive oxygen species (ROS) inside the cells, as measured using fluorescent probes. However, an increase in other ROS as indicated by H2DCF-DA was found only for 1 mM of IS. Moreover, a decrease in the non-enzymatic antioxidant capacity and an increase in the superoxide dismutase activity after incubation of the cells with IS were observed. Furthermore, we found an increase in the levels of carbonyl compounds and peroxides in the cells treated with both the concentrations of IS. The obtained results show that IS induces oxidative stress and a decrease in antioxidant defense in cells leading to lipid and protein damage.

Alterations in conformational state of albumin in plasma in chronic hemodialyzed patients

Objective In chronic hemodialyzed (CH) patients the balance between production of reactive oxygen species and antioxidant defense system is disturbed and shifted towards oxidative conditions. The properties of albumin in CH patients were studied before hemodialysis (HD) and post-HD. Methods Two oxidants were applied, organic t-butyl hydroperoxide (t-BOOH) and inorganic hydroperoxide (H2O2), for oxidation of albumin molecules. By comparison, albumin from healthy donors was also modified by both oxidants. The thiol content in albumin was determined by the Ellman method. Albumin properties were evaluated with the spin labelling technique using two covalently bound spin labels, maleimide (MSL) and iodoacetamide (ISL), and fatty acid spin probe, 16-doxylstearic acid (16-DS). Results A decrease in thiols level in HD albumin was greater than in control albumin. The t-BOOH modified the microenvironment at the binding site of MSL and ISL in control albumin molecules to a greater extent than hydrogen peroxide. Control albumin treated with t-BOOH and H2O2 showed an increase in the mobility of 16-DS. However, no changes were observed in albumin from CH patients treated with either of the oxidizing agents. Conclusion Both oxidants induced strong conformational changes in albumin from healthy volunteers, but were less effective or ineffective in modification of albumin derived from CH patients. These results show that albumin from CH patients is highly modified in vivo and is not vulnerable to oxidation in the same way as normal albumin.