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 language | English |
|---|---|
| Pages (from-to) | 85-90 |
| Number of pages | 6 |
| Journal | Analytical Biochemistry |
| Volume | 312 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jan 15 2003 |
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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 journal › Article
}
TY - JOUR
T1 - Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography
AU - Pastore, Anna
AU - Mozzi, Alessia Francesca
AU - Tozzi, Giulia
AU - Gaeta, Laura Maria
AU - Federici, Giorgio
AU - Bertini, Enrico
AU - Russo, Anna L.
AU - Mannucci, Liliana
AU - Piemonte, Fiorella
PY - 2003/1/15
Y1 - 2003/1/15
N2 - 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.
AB - 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.
KW - Cation-exchange HPLC
KW - Glutathionyl-hemoglobin
KW - Oxidative stress
KW - Tissue redox status
UR - http://www.scopus.com/inward/record.url?scp=0037439380&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037439380&partnerID=8YFLogxK
U2 - 10.1016/S0003-2697(02)00500-6
DO - 10.1016/S0003-2697(02)00500-6
M3 - Article
C2 - 12531191
AN - SCOPUS:0037439380
VL - 312
SP - 85
EP - 90
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -