The Oxidative Status of Valinomycin-Resistant Normal and Thalassemic Red Cells (RBCs).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3191-3191
Author(s):  
Johnny Amer ◽  
Zipora Etzion ◽  
Robert M. Bookchin ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Oxidant Stress ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Normal Blood ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Before And After ◽  
Mercury Orange

Abstract Normal high-K+, low-Na+ RBCs, suspended in low-K+ media and permeabilized to K+ with valinomycin, become dehydrated from net loss of KCl and water. A very small fraction of light, normal RBC and larger fractions of light, sickle cell anemia (SCA) and beta-thalassemia RBC were found to be “valinomycin-resistant” (val-res) due to their Na+/K+ gradient dissipation (PNAS2000;97: 8050; BLOOD2000;96:24b). In thalassemia and SCA, although the primary lesions involve the globin genes, the major damage to the RBC membranes is mediated by oxidative stress. We previously showed (Cytometry2004;60:73) that thalassemic RBC have higher reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels than normal RBC before or after in vitro oxidant stress (treatment with hydrogen peroxide). Here, we examined the oxidative status of val-res RBC from normal and beta-thalassemia major blood. RBC suspended in a plasma-like buffer containing 15 mM KCl and 10 mM valinomycin for 45 min were then layered on arabinogalactone (Larex) with density δ=1.091, and spun at 15,000 g for 30 min. Val-res cells were identified as the low density (δ<1.091 g/ml) RBCs recovered from the interphase layer. The percent val-res RBC in beta-thalassemic samples (n = 10), was 84-fold higher (4.2 ± 0.4% (mean ± SD), range 2.5 to 6.0%) than in normal samples (0.05 ± 0.06%, range 0.02 to 0.1%) (n =10). To determine the oxidative status of the RBC, the cells were washed with PBS and stained for intracellular contents of ROS and GSH, using 2′-7′-dichlorofluoresein and Mercury Orange, respectively. RBC were analyzed by flow cytometry, using gating based on size and granularity. The Mean Fluorescence Channel (MFC) for each fluorochrome was computed. The results showed that valinomycin treatment, per se, did not affect ROS and GSH contents: MFC of the stained un-fractionated RBC was similar before and after treatment with valinomycin, indicating that large changes in MCHC had little or no effect on these measurements. In addition, the unfractionated RBC had ROS and GSH values comparable to those of the high density (val-sensitive) RBC which were recovered from the pellet of valinomycin-treated RBC following Larex fractionation. Measurements on six normal and six beta-thalassemic blood samples indicated that in each case val-res RBC had higher ROS (3.5-10 fold) and lower GSH (2.5-8 fold) levels than the unfractionated RBC or the val-sensitive RBC of the same sample. Compared with val-res cells from normal blood, thalassemic val-res RBC had higher capacity to produce ROS (1.7-fold) and had a lower GSH level (1.5-fold) compared with normal val-res RBC. These results confirm that, as with SCA, beta-thalassemia blood contains a higher percent of val-res RBC than normal blood. They show, further, that (i) both normal and thalassemic val-res RBC have higher oxidative status than other cells (val-sensitive) in the same sample; and that (ii) thalassemic val-res RBC have higher oxidative status than val-res RBC in normal blood. The present results are consistent with the possibility that oxidative stress may contribute to the generation of val-res RBCs, but do not establish a cause-effect relationship. Further studies will be needed to elucidate the origin and significance of these cells.

scholarly journals Use of low temperatures for glutathione histochemical stain.

1983 ◽  
Vol 31 (7) ◽  
pp. 975-976 ◽  
Author(s):  
P Chieco ◽  
P J Boor
Keyword(s):  
Low Temperature ◽  
Standard Method ◽  
Low Temperatures ◽  
Frozen Sections ◽  
Mercury Orange ◽  
Fresh Frozen ◽  
In Cells

We performed glutathione (GSH) staining at a low temperature to prevent GSH release from the section and, hence, improve morphology. Fresh frozen sections of liver, lung, kidney, heart, and stomach were incubated for GSH activity in an ice bath (2-5 degrees C) for 5-10 min. Low temperature incubation prevented GSH diffusion out of cells and minimized migration of granules into vessels and outside of tissue. Incubation at low temperature generally reduced the intensity of the stain compared to the standard method. We conclude that low temperature incubation improves GSH localization in cells, probably by regulating the rate, formation, and the size of GSH-mercury orange complexes.

scholarly journals Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death

2017 ◽  
Vol 43 (2) ◽  
pp. 431-444 ◽  
Author(s):  
Mohamed Jemaà ◽  
Myriam Fezai ◽  
Rosi Bissinger ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Tyrosine Kinases ◽  
Annexin V ◽  
Research Area ◽  
Beta Thalassemia ◽  
Phosphatidylserine Exposure ◽  
Mercury Orange ◽  
P21 Activated Kinase ◽  
Clinical Conditions

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

scholarly journals A flow cytometric assay for intracellular nonprotein thiols using mercury orange

Cytometry ◽  
1988 ◽  
Vol 9 (6) ◽  
pp. 529-532 ◽  
Author(s):  
Jose Enrique O'Connor ◽  
Bruce F. Kimler ◽  
Michael C. Morgan ◽  
Kathryn J. Tempas
Keyword(s):  
Flow Cytometric Assay ◽  
Flow Cytometric ◽  
Mercury Orange ◽  
Nonprotein Thiols

Oxidative Stress of RBC in a Murine Model of Beta-Thalassemia Can Be Reversed by Treatment with Antioxidants.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3643-3643
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Eliezer Rachmilewitz ◽  
Ella Guy ◽  
Stefano Rivella
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Mouse Model ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Beta Thalassemia ◽  
Light Scatter ◽  
Intracellular Ros ◽  
Mercury Orange ◽  
Dichlorofluorescin Diacetate

Abstract Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia and sickle cell anemia. The oxidative status within RBC is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, such as reduced glutathione (GSH). Using flow cytometric methods, we previously showed that RBC obtained from patients with thalassemia (Amer et al. Eur J Haematol70:84,2003; Cytometry60:73,2004) or sickle cell anemia (Amer et al. Blood, 104:972a,2004) exhibit oxidative stress. In the present study, we assessed the extent of RBC oxidative stress and the effects of antioxidant administration, using the thalassemic mouse model Th3/+. This model closely mimics the phenotype observed in patients affected by beta-thalassemia intermedia, such as low hemoglobin levels (7 to 9 gr/dL), splenomegaly and iron overload. In addition, the mature RBC have a shorter life-span and are characterized by anisocytosis, poikilocytosis and hypochromatism. RBC were derived from normal and thalassemic mice before and 4 hours after i.p. injection of the antioxidants N-acetyl cysteine (NAC), vitamin C (Vit. C) or tocotrinol - a mixture of vitamin E derivatives, at a dose of 150 mg/kg. Intracellular ROS was determined in dichlorofluorescin diacetate-stained RBC following stimulation with 2 mM H2O2; GSH content was assessed in RBC stained with mercury orange. Cells were analyzed by flow cytometry: RBC were gated according to size (forward light scatter) and granularity (side light scatter), their fluorescence was measured and the Mean Fluorescence Channel (MFC) was calculated. Fig. 1 shows the average MFC of ROS and GSH of normal and thalassemic mice treated or not treated with anti-oxidants (N=6 in each group). The results show a significantly higher (2.6-fold) production of ROS and lower (three-fold) levels of GSH in RBC from the thalassemic mice versus those in RBC from normal mice. Administration of antioxidants decreased the ROS of normal and thalassemic RBC by 1.4-fold and 2.6-fold, respectively, whereas GSH levels were significantly increased both in the normal (2.7 fold) and in the thalassemic (9.4-fold) RBC. The results show that the RBC of thalassemic mice are under oxidative stress that could be ameliorated by a short antioxidant treatment. Hence, this mouse model recapitulates the oxidative stress found in thalassemic patients and can serve as a model for studying the effects of antioxidant therapy. The flow cytometry methodology used is helpful in following up the results of the treatment and in evaluating its efficacy in reducing oxidative stress. Figure Figure

scholarly journals Enhanced Suicidal Erythrocyte Death Contributing to Anemia in the Elderly

2015 ◽  
Vol 36 (2) ◽  
pp. 773-783 ◽  
Author(s):  
Adrian Lupescu ◽  
Rosi Bissinger ◽  
Tobias Goebel ◽  
Madhuri S. Salker ◽  
Kousi Alzoubi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Statistical Significance ◽  
Annexin V ◽  
The Elderly ◽  
Elderly Individuals ◽  
Erythrocyte Surface ◽  
Mercury Orange ◽  
Orange Fluorescence ◽  
Underlying Mechanisms

Background/Aims: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca2+-concentration ([Ca2+]i), oxidative stress and ceramide. The present study explored whether eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. Methods: Blood was drawn from healthy young (n=11, age 31.3±1.7 years) and elderly (n=16, age 88.6±0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. Results: Elderly individuals suffered from severe anemia (hemoglobin 10.5±0.3 g/100 ml) despite enhanced number of reticulocytes (2.3±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5±0.2%) than in the young volunteers (1.3±0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca2+]i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. Conclusions: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

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