This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (<0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r >0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for >1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.

HPLC WITH O-PHTHALALDEHYDE PRECOLUMN DERIVATIZATION TO MEASURE TOTAL, OXIDIZED, AND PROTEIN-BOUND GLUTATHIONE IN BLOOD, PLASMA, AND TISSUE / R. PARONI, E. DEVECCHI, G. CIGHETTI, C. ARCELLONI, I. FERMO, A. GROSSI, P.A. BONINI. - In: CLINICAL CHEMISTRY. - ISSN 0009-9147. - 41:3(1995 Mar), pp. 448-454.

HPLC WITH O-PHTHALALDEHYDE PRECOLUMN DERIVATIZATION TO MEASURE TOTAL, OXIDIZED, AND PROTEIN-BOUND GLUTATHIONE IN BLOOD, PLASMA, AND TISSUE

R. PARONI
Primo
;
G. CIGHETTI;A. GROSSI
Penultimo
;
1995

Abstract

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (<0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r >0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for >1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.
chromatography, reversed-phase; fluorometry; sample handling
Settore BIO/10 - Biochimica
Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/181070
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