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 journalArticle

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 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
Volume312
Issue number2
DOIs
Publication statusPublished - Jan 15 2003

Fingerprint

High performance liquid chromatography
Cations
Erythrocytes
High Pressure Liquid Chromatography
Oxidative stress
Hemoglobins
Oxidative Stress
Glutathione
Blood
High pressure liquid chromatography
Glutathione Disulfide
Dithiothreitol
Molecular mass
glutathionyl hemoglobin
Healthy Volunteers
Antioxidants
Biomarkers
Clinical Trials
Availability
Monitoring

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography. / Pastore, Anna; Mozzi, Alessia Francesca; Tozzi, Giulia; Gaeta, Laura Maria; Federici, Giorgio; Bertini, Enrico; Russo, Anna L.; Mannucci, Liliana; Piemonte, Fiorella.

In: Analytical Biochemistry, Vol. 312, No. 2, 15.01.2003, p. 85-90.

Research output: Contribution to journalArticle

Pastore, Anna ; Mozzi, Alessia Francesca ; Tozzi, Giulia ; Gaeta, Laura Maria ; Federici, Giorgio ; Bertini, Enrico ; Russo, Anna L. ; Mannucci, Liliana ; Piemonte, Fiorella. / Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography. In: Analytical Biochemistry. 2003 ; Vol. 312, No. 2. pp. 85-90.
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