HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue.
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.
Paroni
R
R
Department of Laboratory Medicine, IRCCS, H. San Raffaele, Milan, Italy.
De Vecchi
E
E
Cighetti
G
G
Arcelloni
C
C
Fermo
I
I
Grossi
A
A
Bonini
P
P
D000818:Animals; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol; D004355:Drug Stability; D005033:Ethylmaleimide; D005978:Glutathione / Q000032:analysis* / Q000097:blood; D006801:Humans; D007668:Kidney / Q000737:chemistry; D010084:Oxidation-Reduction; D010472:Perchlorates; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000737:chemistry; D009764:o-Phthalaldehyde
D004220:Disulfides; D010472:Perchlorates; D011506:Proteins; D013438:Sulfhydryl Compounds; D009764:o-Phthalaldehyde; D005978:Glutathione; D005033:Ethylmaleimide; D004229:Dithiothreitol