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Mark E Hinsdale - One of the best experts on this subject based on the ideXlab platform.

  • simplified Hemoglobin Chain detection by capillary electrophoresis
    Electrophoresis, 2005
    Co-Authors: Zak K Shihabi, Mark E Hinsdale
    Abstract:

    Hemoglobin (Hb) Chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb Chains also after sample extraction. We describe a CE method for analysis of Hb Chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the Chains were denatured and separated from the heme during electrophoresis. The common variants of the β-Chains, such as βS, βC, and βE, are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the Hemoglobinopathies.

  • simplified Hemoglobin Chain detection by capillary electrophoresis
    Electrophoresis, 2005
    Co-Authors: Zak K Shihabi, Mark E Hinsdale
    Abstract:

    Hemoglobin (Hb) Chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb Chains also after sample extraction. We describe a CE method for analysis of Hb Chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the Chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-Chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the Hemoglobinopathies.

Zak K Shihabi - One of the best experts on this subject based on the ideXlab platform.

  • simplified Hemoglobin Chain detection by capillary electrophoresis
    Electrophoresis, 2005
    Co-Authors: Zak K Shihabi, Mark E Hinsdale
    Abstract:

    Hemoglobin (Hb) Chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb Chains also after sample extraction. We describe a CE method for analysis of Hb Chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the Chains were denatured and separated from the heme during electrophoresis. The common variants of the β-Chains, such as βS, βC, and βE, are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the Hemoglobinopathies.

  • simplified Hemoglobin Chain detection by capillary electrophoresis
    Electrophoresis, 2005
    Co-Authors: Zak K Shihabi, Mark E Hinsdale
    Abstract:

    Hemoglobin (Hb) Chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb Chains also after sample extraction. We describe a CE method for analysis of Hb Chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the Chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-Chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the Hemoglobinopathies.

Bertram H Lubin - One of the best experts on this subject based on the ideXlab platform.

  • effect of excess alpha Hemoglobin Chains on cellular and membrane oxidation in model beta thalassemic erythrocytes
    Journal of Clinical Investigation, 1993
    Co-Authors: Mark D. Scott, J J M Van Den Berg, Tanya Repka, P Rouyerfessard, R P Hebbel, Y Beuzard, Bertram H Lubin
    Abstract:

    Abstract While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-Hemoglobin Chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-Hemoglobin Chains, purified alpha-Hemoglobin Chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of metHemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-Hemoglobin Chain autoxidation and appearance of membrane-bound globin. In addition, alpha-Hemoglobin Chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-Hemoglobin Chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-Hemoglobin Chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-Hemoglobin Chains. In summary, entrapment of purified alpha-Hemoglobin Chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-Hemoglobin Chains can be examined.

  • effect of excess alpha Hemoglobin Chains on cellular and membrane oxidation in model beta thalassemic erythrocytes
    Journal of Clinical Investigation, 1993
    Co-Authors: Mark D. Scott, Tanya Repka, P Rouyerfessard, R P Hebbel, Y Beuzard, J J M Van Den Berg, Bertram H Lubin
    Abstract:

    While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-Hemoglobin Chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-Hemoglobin Chains, purified alpha-Hemoglobin Chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of metHemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-Hemoglobin Chain autoxidation and appearance of membrane-bound globin. In addition, alpha-Hemoglobin Chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-Hemoglobin Chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-Hemoglobin Chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-Hemoglobin Chains. In summary, entrapment of purified alpha-Hemoglobin Chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-Hemoglobin Chains can be examined.

Arnulf Pekrun - One of the best experts on this subject based on the ideXlab platform.

  • post transcriptional effects of interleukin 3 interferon gamma erythropoietin and butyrate on in vitro Hemoglobin Chain synthesis in congenital hemolytic anemia
    Haematologica, 2001
    Co-Authors: Dirk Reinhardt, Regina Ridder, Wilfried Kugler, Arnulf Pekrun
    Abstract:

    BACKGROUND AND OBJECTIVES: Various agents modulate Hemoglobin synthesis. In vitro modulation of translation in Hemoglobin Chain synthesis was analysed in patients with congenital hemolytic anemia (n=32) and healthy controls (n=17). DESIGN AND METHODS: Enriched reticulocytes were co-incubated with (3)H-leucine and cytokines or butyrate. Reversed-phase chromatography enabled separation of alpha-, beta- and gamma-globin Chains. Globin Chain synthesis was calculated from measured (3)H-leucine incorporation. Transferrin, erythropoietin, interleukin-3 and interferon-gamma receptors were detected by flow cytometry. Reverse-transcription polymerase Chain reaction (RT PCR) was used to demonstrate changes of RNA stability. RESULTS AND DISCUSSION: Interleukin-3, interferon-gamma and butyrate caused a significant 2-fold increase (range 1.8-2.4; p<0.01) of the alpha- and beta-Chain synthesis in congenital hemolytic anaemias. Analysis of gamma-globin Chain synthesis revealed a lower, i.e. 1.4 fold increase (range 1.32 to 1.41; p<0.03). The absolute amount of globin synthesis was calculated to be 2.9 x 10(-12) g/reticulocyte/24h. After incubation with interleukin-3 the absolute additional synthesis of the alpha-globin Chain reached 1.31 x 10(-12) g/reticulocyte/24h, of the beta-globin Chain, 1.15 x 10(-12) g/reticulocyte/24h and of the gamma-globin Chain, 0.26 x 10(-12) g/reticulocyte/24h. Butyrate and interferon-gamma had no or even an inhibiting effect on reticulocytes from normal controls, while interleukin-3 stimulated alpha- and gamma-Chain synthesis (1.4 and 2.4 fold, respectively; p<0.03) suggesting an increase of fetal Hemoglobin (HbF). Erythropoietin showed no stimulating influence. Membrane associated interleukin-3 receptors were detected in 0.78+/-0.14%, and interferon-gamma receptors in 0.1+/-0.015% of the red cells. Erythropoietin receptors were extremely rare (0.05+/-0.015%). The expression of transferrin receptors (CD71) correlated with the extent of globin Chain stimulation. The alpha-, and beta-globin mRNA content of the reticulocytes after interleukin-3 incubation, as measured by RT-PCR, increased. INTERPRETATION AND CONCLUSIONS: Hemoglobin Chain synthesis could be modulated post-transcriptionally by interleukin-3, interferon-gamma and butyrate. Transferrin receptor and globin RNA stability might be involved in this phenomenon.

Mark D. Scott - One of the best experts on this subject based on the ideXlab platform.

  • effect of excess alpha Hemoglobin Chains on cellular and membrane oxidation in model beta thalassemic erythrocytes
    Journal of Clinical Investigation, 1993
    Co-Authors: Mark D. Scott, J J M Van Den Berg, Tanya Repka, P Rouyerfessard, R P Hebbel, Y Beuzard, Bertram H Lubin
    Abstract:

    Abstract While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-Hemoglobin Chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-Hemoglobin Chains, purified alpha-Hemoglobin Chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of metHemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-Hemoglobin Chain autoxidation and appearance of membrane-bound globin. In addition, alpha-Hemoglobin Chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-Hemoglobin Chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-Hemoglobin Chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-Hemoglobin Chains. In summary, entrapment of purified alpha-Hemoglobin Chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-Hemoglobin Chains can be examined.

  • effect of excess alpha Hemoglobin Chains on cellular and membrane oxidation in model beta thalassemic erythrocytes
    Journal of Clinical Investigation, 1993
    Co-Authors: Mark D. Scott, Tanya Repka, P Rouyerfessard, R P Hebbel, Y Beuzard, J J M Van Den Berg, Bertram H Lubin
    Abstract:

    While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-Hemoglobin Chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-Hemoglobin Chains, purified alpha-Hemoglobin Chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of metHemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-Hemoglobin Chain autoxidation and appearance of membrane-bound globin. In addition, alpha-Hemoglobin Chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-Hemoglobin Chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-Hemoglobin Chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-Hemoglobin Chains. In summary, entrapment of purified alpha-Hemoglobin Chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-Hemoglobin Chains can be examined.