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 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.
Pastore
Anna
A
Laboratory of Biochemistry, Children's Hospital and Research Institute Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome, Italy.
Mozzi
Alessia Francesca
AF
Tozzi
Giulia
G
Gaeta
Laura Maria
LM
Federici
Giorgio
G
Bertini
Enrico
E
Lo Russo
Anna
A
Mannucci
Liliana
L
Piemonte
Fiorella
F
D002412:Cations / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry*; D005260:Female; D005978:Glutathione / Q000032:analysis*; D006441:Hemoglobin A / Q000032:analysis; D006454:Hemoglobins / Q000032:analysis*; D006801:Humans; D008297:Male; D010084:Oxidation-Reduction
D002412:Cations; D006454:Hemoglobins; C404464:glutathionyl hemoglobin; D006441:Hemoglobin A; C020622:hemoglobin B; D005978:Glutathione