Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography

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 A₀; (c) the peak could be detected by incubating hemoglobin A₀ with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin β 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 (HbA₀ + HbA₂ + 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.