Reversed-phase chromatographic method for specific determination of glutathione in cultured malignant cells.
A chromatographic method for the specific determination of glutathione in malignant cell lines is described. The method is based on the ability of glutathione-S-transferase to specifically and quantitatively conjugate glutathione to 1-chloro-2,4-dinitrobenzene and chromatographic quantitation of the resultant conjugate, dinitrophenyl-S-glutathione, by reversed-phase liquid chromatography. The assay can be performed on 20,000 g supernatants of cell homogenates without acid extraction. 2-Mercaptoethanol, a sulfhydryl compound often used as a thiol-protective agent to preserve enzymatic activities of a number of enzymes, did not interfere with glutathione determination by this method. The dinitrophenyl-S-glutathione isolated from either standard glutathione samples or from cell homogenates was shown to be identical to authentic dinitrophenyl-S-glutathione using mass spectrometry. Recovery of glutathione in standard samples by the current method was identical to that determined using 5,5'-dithiobis(2-nitrobenzoic acid). Exogenous glutathione added to supernatants of cell homogenate in the presence or absence of 2-mercaptoethanol was also completely recovered.
Awasthi
S
S
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77550.
Ahmad
F
F
Sharma
R
R
Ahmad
H
H
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004228:Dithionitrobenzoic Acid; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D014407:Tumor Cells, Cultured
D004228:Dithionitrobenzoic Acid; D005982:Glutathione Transferase; D005978:Glutathione