Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography |
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Authors: | Pastore Anna Mozzi Alessia Francesca Tozzi Giulia Gaeta Laura Maria Federici Giorgio Bertini Enrico Lo Russo Anna Mannucci Liliana Piemonte Fiorella |
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Affiliation: | Laboratory of Biochemistry, Children's Hospital and Research Institute Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome, Italy. |
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Abstract: | Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials. |
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Keywords: | Glutathionyl-hemoglobin Cation-exchange HPLC Tissue redox status Oxidative stress |
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