Targeting the urine and plasma determinants of thromboxane A2 metabolism in detection of aspirin effectiveness.

Detection of reduced aspirin effectiveness has gained significant importance since clinical consequences of aspirin resistance were reported. Nevertheless, due to differentiated molecular basis of aspirin resistance, the conflicting choice of referential method for detection of acetylsalicylic acid ineffectiveness has become troublesome. This study, using a rat model of antiplatelet therapy, examines the aptitude of selected TXB2 metabolism-based methods in the detection of acetylsalicylic acid effectiveness. We hypothesized that ex-vivo whole blood spontaneous TXB2 generation assay could be, contrary to basal TXB2 and urine 11-dTXB2, a novel surrogate measure for impaired acetylsalicylic acid-dependent inhibition of thromboxane synthesis. To address this hypothesis, we evaluated the sensitivity of TXB2 generation assay in hirudinized whole blood to detect acetylsalicylic acid-mediated inhibition of cyclooxygenase activity in healthy rats and diabetic rats treated with acetylsalicylic acid. In diabetic and control animals, both acetylsalicylic acid drenches in the dose-independent manner contributed to significant attenuation of basal plasma TXB2 and urinary 11-dTXB2 formation. Urinary concentrations of 11-dTXB2 were, contrary to basal TXB2, significantly higher, regardless of acetylsalicylic acid dose, among all diabetic groups, compared with corresponding control groups. Determination of TXB2 generation in whole blood enabled sensitive detection of dose-related acetylsalicylic acid effect in both groups, as well as increased TXB2 formation in diabetes. We showed for the first time that evaluation of spontaneous generation of TXB2 in hirudinized whole blood enables, contrary to basal plasma TXB2 and urine 11-dTXB2 concentrations, to sensitively determine the acetylsalicylic acid effect in healthy and diabetic subjects.

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes.

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c(50%)=258+/-24 microM HOCl) and bimane-S-glutathione (B-SG, c(50%)=125+/-16 microM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH](oxidized)=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg(2+)-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c(50%)=478+/-36 microM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.

Hypochlorous acid-induced oxidative stress in Chinese hamster B14 cells: viability, DNA and protein damage and the protective action of melatonin.

This study provides further evidence for the toxicity of hypochlorous acid (HOCl) in mammalian cells. Using the Chinese hamster B14 cell line, a significant decrease in cell viability was demonstrated after exposure to 100-200 microM HOCl for 1 h. Loss of viability was accompanied by a slight increase in DNA damage as shown by the Comet assay and by oxidation of cellular thiols. Exposure of B14 cells, erythrocyte membranes and human serum albumin to HOCl resulted in an extensive protein carbonyl accumulation. Thus, the cytotoxicity of HOCl may be due to both protein damage (carbonyl formation and oxidation of protein thiol groups) and DNA damage. The well-known antioxidant melatonin interacted with the oxidant and significantly protected cells during HOCl exposure, diminishing its cytotoxic effects and reducing protein carbonyl generation.

Hypochlorous acid-induced membrane pore formation in red blood cells.

The hyperproduction of hypochlorous acid (HOCl), an extremely toxic biological oxidant generated by neutrophils and monocytes, is involved in the pathogenesis of many diseases. In these studies, we attempted to determine the membrane and cellular events associated with HOCl-induced erythrocyte impairment and haemolysis. In vitro human erythrocyte exposure to HOCl (0.1-1.0 mM) resulted in rapid oxidation of reduced glutathione, an increase in cell osmotic fragility and the formation of transient membrane pores. The process of glutathione oxidation depended on the [oxidant]/[cell number] ratio. The HOCl-induced haemolysis observed was apparently mediated by pore formation and altered membrane electrolyte permeability. The estimated pore radius was approximately 0.7 nm and the average number per cell was 0.01. The rate constant of HOCl-produced haemolysis depended on pH. There were significant differences in haemolysis of HOCl-treated erythrocytes which had maximal stability at pH 7.2-7.3.

The mechanism of Zn-phthalocyanine photosensitized lysis of human erythrocytes.

The phthalocyanines have recently been suggested as one of most effective possible sensitizers for photodynamic therapy and the blood viral inactivation. The further characterisation of the mechanism of human red blood cell lysis and membrane alterations upon photodynamic treatment in the presence of Zn-phthalocyanine was the aim of this study. It was found that there were (2.7+/-0.4).10(7) dye binding sites per red blood cell with the association constant equal to (1.4+/-0.3).10(4) M(-1). Two types of the photosensitized haemolysis: haemolysis during irradiation ("light" haemolysis) and post-irradiation haemolysis ("dark" haemolysis) were studied. The erythrocyte membrane hyperpolarisation, membrane fluidisation and cell swelling preceded the "light" haemolysis. The modification of the erythrocyte membrane band 3 protein by DIDS (an inhibitor of anion exchange) increased the rate of the "light" haemolysis. The rate of "dark" haemolysis was higher and that of "light" haemolysis was lower in potassium media in comparison to sodium ones. The rates of photohaemolysis depended on the erythrocyte membrane potential: a decrease of membrane potential inhibited both types of haemolysis. The cell shrinkage in the presence of sucrose (up to 15 mM) inhibited the "dark" haemolysis but significantly increased the "light" haemolysis. Oxidation of intracellular oxyHb to metHb by nitrite, which drastically decreases intracellular oxygen concentration, as well as GSH concentration, inhibited the rate of the "light" haemolysis. The results allow for the conclusion that the mechanism of photochemical ("light") haemolysis is not of a colloid-osmotical type, in contrast to the post-irradiation ("dark") haemolysis. The photochemical oxidation or denaturation of band 3 protein plays a significant role in the formation of haemolytic holes. The membrane lipid peroxidation, as well as glutathione oxidation, does not participate in the process of photosensitized haemolysis. From the inhibition of "dark" haemolysis by sucrose the apparent pore radius was estimated to be about 1.1 nm. The pores appear to be transient short-lived ones, the average pore number per cell was 0.02.

Usefulness of whole blood aggregometry and its comparison with thromboxane generation assay in monitoring acetylsalicylic acid effectiveness--a multiparametric study in rats.

BACKGROUND: There is a need for consensus concerning universal methodological criteria for detection of suboptimal response to acetylsalicylic acid (ASA) therapy. Therefore, animal models to test for ASA effectiveness remain of interest. Our objective was to verify the usefulness of multiparametric whole-blood impedance aggregometry and thromboxane A(2) generation, which are the most popular techniques used for monitoring of ASA treatment effectiveness. METHODS: Using multiparametric analysis of whole-blood impedance aggregometry, we examined which parameters of platelet aggregation or disaggregation allow for the best discrimination between ASA-treated (4 or 40 mg/kg for 60 days) and non-treated male rats. The effectiveness of ASA-mediated inhibition of platelet cyclooxygenase-1 was verified by determination of plasma thromboxane B(2) and urine 11-dehydro-thromboxane B(2), accepted as reference assays for monitoring of ASA-mediated platelet cyclooxygenase-1 inhibition. RESULTS: Two of the platelet agonists used, collagen (1 mg/L) and arachidonic acid (0.5 mmol/L), allowed discrimination of control and ASA-treated animals, whereas adenosine diphosphate (5 micromol/L) was not effective. It is noteworthy that only ASA-mediated changes in duration of the rising phase for platelet aggregation and the area under the curve for collagen-induced aggregation allowed significant discrimination between low and high ASA dose and remained correlated with the reference parameter, plasma thromboxane B(2). CONCLUSIONS: Analysis of aggregation curves, routinely based only on the amplitude and rate of platelet aggregation, may not be enough discriminative to distinguish between varying ASA doses and treatment schedules.

Low-intensity near-infrared laser radiation-induced changes of acetylcholinesterase activity of human erythrocytes.

OBJECTIVE: The aim of the present study was to investigate the transformations of red blood cells produced by low-intensity infrared laser radiation (810 nm). BACKGROUND DATA: Low-intensity (the output power of a laser device in the milliwatt range) laser radiation as a local phototherapeutic modality is characterized by its ability to induce non-thermic, nondestructive photobiological processes in cells and tissues. However, the exact theory concerning the therapeutic effects of laser biostimulation has not been developed. MATERIALS AND METHODS: The suspensions of human erythrocytes in PBS (10% hematocrit) were irradiated with near-infrared (810 nm) therapy laser at different light doses (0-20 J) and light power (fluence rate; 200 or 400 mW) at 37 degrees C. As the parameters characterizing the cell structural and functional changes membrane acetylcholinesterase (AchEase) activity, the membrane potential, the level of intracellular glutathione, the level of products of membrane lipid peroxidation, and the cell osmotic stability were measured. RESULTS: It was found that near-infrared low-intensity laser radiation produced complex biphasic dose-dependent changes of the parameters of AchEase reaction in the dose-dependent manner: at smaller doses of radiation (6 J) the maximal reaction rate and Michaelis-Menten constant value decreased, and at higher radiation doses these parameters increased. No significant changes of erythrocyte stability, cellular redox state (reduced glutathione or lipid peroxidation product levels), or cell membrane electrochemical potential were observed. CONCLUSION: Low-intensity near-infrared laser radiation (810 mn) produced AchEase activity changes, reflecting the effect of light on the enzyme due to energy absorption. Protein molecule conformational transitions and enzyme activity modifications in cells have been suggested as laser radiation-induced events.

Effect of low-intensity (3.75-25 J/cm2) near-infrared (810 nm) laser radiation on red blood cell ATPase activities and membrane structure.

OBJECTIVE: The biostimulation and therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the exact mechanism of action of the laser radiation with living cells is not yet understood. The aim of the present work was to investigate the effect of laser radiation (810 nm, radiant exposure 3.75-25 J/cm(2)) on the structure of protein and lipid components of red blood cell membranes and it functional properties. The role of membrane ATPases as possible targets of laser irradiation was analyzed. BACKGROUND DATA: A variety of studies both in vivo and in vitro showed significant influence of laser irradiation on cell functional state. At the same time another group of works found no detectable effects of light exposure. Some different explanations based on the light absorption by primary endogenous chromophores (mitochondrial enzymes, cytochromes, flavins, porphyrins) have been proposed to describe biological effects of laser light. It was suggested that optimization of the structural-functional organization of the erythrocyte membrane as a result of laser irradiation may be the basis for improving the cardiac function in patients under a course of laser therapy. MATERIALS AND METHODS: Human red blood cells or isolated cell membranes were irradiated with low-intensity laser light (810 nm) at different radiant exposures (3.75-25 J/cm(2)) and light powers (fluence rate; 10-400 mW) at 37 degrees C. As the parameters characterizing the structural and functional changes of cell membranes the activities of Na(+)-, K(+)-, and Mg(2+)-ATPases, tryptophan fluorescence of membrane proteins and fluorescence of pyrene incorporated into membrane lipid bilayer were used. RESULTS: It was found that near-infrared low-intensity laser radiation changes the ATPase activities of the membrane ion pumps in the dose- and fluence rate-dependent manner. At the same time no changes of such integral parameters as cell stability, membrane lipid peroxidation level, intracellular reduced glutathione or oxyhaemoglobin level were observed. At laser power of 10 mW, an increase of the ATPase activity was observed with maximal effect at 12-15 J/cm(2) of light dose (18-26% for the total ATPase activity). At laser power of 400 mW (fluence rate significantly increased), inhibition of ATPases activities mainly due to the inhibition of Na(+)-, K(+)-ATPase was observed with maximal effect at the same light dose of 12-15 J/cm(2) (18-23% for the total ATPase activity). Fractionation of the light dose significantly changed the membrane response to laser radiation. Changes in tryptophan fluorescent parameters of erythrocyte membrane proteins and the increase in lipid bilayer fluidity measured by pyrene monomer/excimer fluorescence ratio were observed. CONCLUSIONS: Near-infrared laser light radiation (810 nm) induced long-term conformational transitions of red blood cell membrane which were related to the changes in the structural states of both erythrocyte membrane proteins and lipid bilayer and which manifested themselves as changes in fluorescent parameters of erythrocyte membranes and lipid bilayer fluidity. This resulted in the modulation of membrane functional properties: changes in the activity of membrane ion pumps and, thus, changes in membrane ion flows.