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Hypericin is a photosensitizer compound used in the photodynamic therapy (PDT). PDT is an alternative cancer treatment strategy whose function is dependent on the photosensitizers accumulating selectively in tumor cells and following visible or infra-red light induced activation lead to the apoptosis/necrosis of the tumor cells via the formation of reactive oxygen species. Thus, the cellular redox balance is essential for the efficacy of PDT. Among the protective enzyme systems glutathione S-transferases (GST, E.C.2.5.1.18) function in detoxification, protection against oxidative stress and intracellular transport of molecules. It is known that isoenzymes of GST and especially GST-pi is increased in cancer cells and it plays very important functions in the development of resistance to anticancer drugs. Since photosensitizers are used intravenously, it is important to elucidate the effects of photosensitizers on the erythrocyte enzymes. The aim of the present study was to investigate the impact of hypericin on human erythrocyte GST-pi (heGST-pi). Purification yield of 71% and purification fold of 2550 were achieved by using conventional chromatographic methods. The specific activity of the enzyme is found as 51 U/mg protein. Hypericin inhibited heGST-pi in a dose dependent manner and inhibition was biphasic. Noncompetitive type of inhibition was observed with both substrates, GSH and CDNB. The inhibitory constant (K i ) values obtained from Lineweaver-Burk, Dixon, secondary plots; slope and y-intercept versus 1/S (substrate) and from non-linear regression analysis were in good correlation: K i (GSH) was calculated as 0.19 ± 0.01 μM and K i (CDNB) as 0.26 ± 0.03 μM.

Purpose: Elevated red blood cell distribution width (RDW) has been associated with atrial fibrillation (AF) in cross-sectional and prospective studies. In this study, we aim to evaluate the relation of preablation RDW levels to late AF recurrence following cryoablation., Methods: A total of 299 patients with symptomatic paroxysmal or persistent AF despite ≥ 1 antiarrhythmic drug(s) who were scheduled for cryoballoon-based AF ablation were enrolled in this prospective study., Results: A total of 299 patients (55.40 ± 10.60 years, 49.20 % male) were involved and followed up at a median time of 24 (6-44) months. Patients with late AF recurrence had higher RDW levels (14.30 ± 0.93 vs. 13.52 ± 0.93 %, p < 0.001). Multivariate Cox proportional hazard regression analysis showed that RDW level was an independent predictor for late AF recurrence (HR 1.88, 95 % CI 1.41-2.50, p < 0.001) along with left atrial (LA) diameter (HR 3.09, 95 % CI 1.81-5.27, p < 0.001), duration of AF (HR 1.04, 95 % CI 1.01-1.07, p = 0.02), and early AF recurrence (HR 6.39, 95 % CI 3.41-11.97, p < 0.001). A cut-off level of 13.75 % for RDW predicted late AF recurrence following cryoballoon-based pulmonary vein isolation (PVI) with a sensitivity and specificity of 78.00 and 70.00 %, respectively., Conclusion: These findings suggest that elevated RDW may be a predictor of late recurrence following cryoballoon-based AF ablation. Further studies are needed to establish its exact pathophysiologic and prognostic roles.

Toxic oxygen metabolites may contribute to the development of tissue damage, and play a role in the pathogenesis of malignancies, some acute and chronic pulmonary diseases, and in cell damage by radiomimetic agents, which can be seen in patients with ataxia-telangiectasia (A-T). Oxidative stress resulting from increased free radical production and/or defects in antioxidant defences is also involved in neurodegenerative disorders. Thus, oxidative stress could account for several aspects of the pleiotropic phenotype of A-T patients. The aim of this study was to determine the activities of the enzymes involved in cellular antioxidant metabolism in A-T patients to see if there is any defect which may result in constant oxidative stress. Superoxide dismutase (SOD) and catalase activities of erythrocytes, in contrast to lymphocytes, were found to be significantly higher in patients than in healthy controls. Our results may be another indication for the presence of constant oxidative stress in A-T patients as suggested previously.

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.

The first step in the separation of adenine nucleotides from different types of tissues or cells is deproteinization. Several sample preparation methods successfully used for a number of tissues or cells failed to work with erythrocytes. Use of strong acids or bases for deproteinization resulted in a low yield due to the hydrolysis of adenine nucleotides. Moreover, the neutralization of these acids or bases increased the ionic strength, resulting in broad and overlapping peaks. In neutral salt precipitation methods, saturated salts caused clogging of the capillaries. A new deproteinization procedure method was developed. The samples were deproteinized by heating of erythrocytes in boiling distilled water at 95 degrees C for 5 min. The denatured proteins were removed by centrifugation and membrane filtration. The adenine nucleotides were then separated using a polyacrylamide coated capillary. Depending on the type, diameter, length of the capillary and the voltage applied, an average of 16.50 min was sufficient for the separation of adenine nucleotides. All adenine nucleotides were clearly resolved and gave very sharp peaks. The amount of each adenine nucleotide was calculated from the areas under the peaks and AEC values were calculated using the integrator software. The AEC value of 0.91+/-0.04 (n=10) obtained for healthy persons was in good agreement with the literature value of 0.85-0.95. These reported method for sample preparation and capillary electrophoresis is simple, fast and inexpensive compared to the previously reported sample preparation, HPLC and enzymatic methods for the determination of AEC.

Human erythrocyte glutathione disulfide reductase was purified using serially connected 2',5'-ADP-Sepharose 4B affinity and anion-exchange columns. About 11,000-fold purification was achieved with 90% yield. The specific activity of the final preparation was 140 units per milligram of protein. The purified enzyme gave a single band on both native and SDS-PAGE with a subunit mass of 58 kDa. Its pH optimum was 7.20. The Michaelis constants determined at pH 7.4, 37 degrees C, fell within the range of previously reported values [K(m)(NADPH) = 18 microM, at 30-200 microM NADPH; K(m)(GSSG) = 72 microM, at 40-1000 microM glutathione disulfide, both at saturating concentrations of the second substrate]. The affinity eluent NADPH and its oxidized form NADP+ were successfully removed from the enzyme on the ion-exchange column. The purification method developed is very useful when the enzyme source material is scarce (e.g., in preparations from human tissues) and may find further application in the purification of other NAD(P)H-dependent enzymes which might be inactivated by their affinity eluent(s).

Effect of chronic halothane exposure on hepatic and erythrocyte lipid peroxidation and erythrocyte osmotic fragility and morphology were determined in rats exposed to 0.4% halothane, 8 h per day for 40 days. Hepatic lipid peroxidation was increased in the halothane-treated group compared to controls. Lipid peroxidation was not increased by halothane exposure in erythrocytes without hydrogen peroxide, but after peroxide supplementation lipid peroxidation increased more in the erythrocytes of halothane-exposed rats than in control rats. We have observed significant morphological changes in erythrocytes from halothane-treated rats. In addition, erythrocytes of halothane-treated rats were more fragile in saline solutions compared to those of controls. Our results suggest that chronic halothane exposure is not only hepatotoxic but also affects erythrocyte membrane structure and stability.

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 degrees C were 2.14 and 2.74 M-1 min-1 in the absence and presence of 50 mM borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent pK alpha of 8.1-8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 +/- 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 mM ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 +/- 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 +/- 1.3 per subunit.