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

Anna Pastore, Alessia Francesca Mozzi, Giulia Tozzi, Laura Maria Gaeta, Giorgio Federici, Enrico Bertini, Anna L. Russo, Liliana Mannucci, Fiorella Piemonte

Research output: Contribution to journalArticlepeer-review


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 + HbA1C + 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.

Original languageEnglish
Pages (from-to)85-90
Number of pages6
JournalAnalytical Biochemistry
Issue number2
Publication statusPublished - Jan 15 2003


  • Cation-exchange HPLC
  • Glutathionyl-hemoglobin
  • Oxidative stress
  • Tissue redox status

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology


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