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Erythrocyte Lysis (erythrocyte + lysis)
Selected AbstractsThiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detectionELECTROPHORESIS, Issue 10 2005Angelo Zinellu Abstract Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4°C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5-iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 57 cm × 75 ,m ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N -methyl- D -glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and ,-glutamylcysteine (GluCys) were baseline-resolved in , 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro-oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers. [source] An optimized method to separate reticulocytes from peripheral blood for molecular analysisINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2009R. PETRUZZELLI Summary A method based on immunomagnetic sorting of reticulocytes from peripheral blood was set up and combined to a commercial extraction kit for the isolation of total RNA from whole blood. This procedure resulted in high-quality RNA samples suitable for molecular analysis. We used this procedure to analyse erythroid-specific transcripts, starting from peripheral blood samples, to search for differently expressed mRNAs in patients with hereditary persistence of foetal haemoglobin. After erythrocyte lysis, CD15+and CD45+ peripheral cells were negatively sorted to remove leucocyte populations that could have affected the subsequent screening procedure. The cell sorting and RNA extraction procedure was completed within 1,2 h of erythrocyte lysis, which represents a consistent saving of time compared with other procedures. Moreover, it produced 1 ,g of total RNA per ml of blood samples, which is sufficient for molecular analysis. Therefore, our method is a reliable and efficient tool to isolate RNA from specific cell subpopulations poorly represented in peripheral blood, particularly when accurate detection and characterization of highly unstable and poorly expressed molecules is required. [source] Increased vigabatrin entry into the brain by polysorbate 80 and sodium caprateJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 2 2001D. Dimitrijevic The effects of a non-ionic surfactant, polysorbate 80, and the sodium salt of the saturated fatty acid, sodium caprate (C10), as potential brain absorption enhancers for vigabatrin were studied. Vigabatrin is an enzyme-activated irreversible inhibitor of ,-aminobutyric acid (GABA) transaminase that increases brain and cerebrospinal GABA concentrations in animals and man. Before intravenous administration, a range of concentrations of the surfactants were tested using erythrocyte lysis or the red blood cell lysis test to establish the non-toxic concentration range. Vigabatrin was dissolved in 0.1% polysorbate 80 and 0.1% sodium caprate and administered intravenously in doses of 4 mL kg,1 to male Wistar rats (230,250 g; n = 3). Rats were killed 2 h after drug and surfactant administration and the brains were immediately removed and homogenized in 0.4m perchloric acid. Selected ion monitoring electrospray mass spectrometry was used to determine the concentration of vigabatrin and GABA directly from the perchloric acid extract of the rat brain. This method was developed to increase the speed and efficiency of the analysis by removing the need for complex extraction and derivatization procedures while retaining the specificity of the mass spectrometer as a detector. The stability of both vigabatrin and GABA in perchloric acid was established by monitoring their pseudo molecular ions in standard solutions at timed intervals over 24 h. Although the detection level for vigabatrin and GABA was at least 50 pg, only GABA was detected in rat brain. Vigabatrin caused a small increase in whole brain GABA. However, GABA levels were higher in the samples with vigabatrin + enhancer than in the samples where vigabatrin alone was administered. One-way analysis of variance indicated a significant effect of the surfactants on GABA levels (F (5,17) = 11.86, P < 0.01) and vigabatrin absorption was presumed. The rectal temperature of the rats is lowered by the presence of vigabatrin in the brain. Vigabatrin alone decreased rectal temperature by 6%. When given with either polysorbate 80 or sodium caprate, the extent of temperature lowering was significantly greater (P < 0.001). There was no significant difference after 2 h between polysorbate 80 + vigabatrin, and sodium caprate + vigabatrin. [source] CTLA-4 expression in T cells of patients with atopic dermatitisPEDIATRIC ALLERGY AND IMMUNOLOGY, Issue 5 2005Sung Yon Choi Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is a surface molecule of activated T cells with sequence homologous to CD28, and may act as a negative regulator of T-cell activation. In murine animal models, cross-linkage of CTLA-4 molecules on the cell surface results in decreased T-cell proliferation, accompanied by increased interleukin (IL)-2 production and apotosis. To clarify the activation of peripheral blood T cells, we studied the CTLA-4 expression in 32 patients with atopic dermatitis who visited our institution, and 19 normal children who visited for pre-operative laboratory examination were used as normal controls. Whole blood was obtained from all subjects and stained with anti-CD3, anti-CD4, anti-CD8 monoclonal antibodies (mAb). After erythrocyte lysis with lysing solution, the cells were stained with anti-CTLA-4 mAb, and stained cells were analysed by fluorescence-activated cell sorter (FACScan) flow cytometer. Intracellular expression of CTLA-4 was significantly upregulated in peripheral blood CD3+ T cells (36.8%), CD4+ T cells (21.7%) and CD8+ T cells (18.7%) of patients with atopic dermatitis, compared with normal control (18.3%, 9.7%, 9.8%; respectively). Furthermore, CTLA-4-positive CD3+ T cells in patients with severe atopic dermatitis were significantly higher compared with milder group (42.8% vs. 32.2%). However, no significant difference was obtained in CD4+ and CD8+ T cells. Mean percentage of T cells expressing CTLA-4 in patients with atopic dermatitis was higher than the control group. These observations suggest the possibility that the disease activity can be correlated with the CTLA-4 level. [source] Protective effects of N-acetyl- L -cysteine against acute carbon tetrachloride hepatotoxicity in ratsCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2008Yu. Z. Maksimchik Abstract In recent years, N-acetyl- L -cysteine (NAC) has been widely investigated as a potentially useful protective and antioxidative agent to be applied in many pathological states. The aim of the present work was further evaluation of the mechanisms of the NAC protective effect under carbon tetrachloride-induced acute liver injuries in rats. The rat treatment with CCl4 (4,g/kg, intragastrically) caused pronounced hepatolysis observed as an increase in blood plasma bilirubin levels and hepatic enzyme activities, which agreed with numerous previous observations. The rat intoxication was accompanied by an enhancement of membrane lipid peroxidation (1.4-fold) and protein oxidative damage (protein carbonyl group and mixed protein-glutathione disulphide formations) in the rat liver. The levels of nitric oxide in blood plasma and liver tissue significantly increased (5.3- and 1.5-fold, respectively) as blood plasma triacylglycerols decreased (1.6-fold). The NAC administration to control and intoxicated animals (three times at doses of 150,mg/kg) elevated low-molecular-weight thiols in the liver. The NAC administration under CCl4 -induced intoxication prevented oxidative damage of liver cells, decreased membrane lipid peroxidation, protein carbonyls and mixed protein-glutathione disulphides formation, and partially normalized plasma triacylglycerols. At the same time the NAC treatment of intoxicated animals did not produce a marked decrease of the elevated levels of blood plasma ALT and AST activities and bilirubin. The in vitro exposure of human red blood cells to NAC increased the cellular low-molecular-weight thiol levels and retarded tert -butylhydroperoxide-induced cellular thiol depletion and membrane lipid peroxidation as well as effectively inhibited hypochlorous acid-induced erythrocyte lysis. Thus, NAC can replenish non-protein cellular thiols and protect membrane lipids and proteins due to its direct radical-scavenging properties, but it did not attenuate hepatotoxicity in the acute rat CCl4 -intoxication model. Copyright © 2007 John Wiley & Sons, Ltd. [source] | |||||||||||||