Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection.
A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.
Mourad
T
T
Laboratoire de Biochimie C, Hôpital Edouard Herriot, 5 place d'Arsonval, Lyon cedex 03, 69437, France.
Min
K L
KL
Steghens
J P
JP
D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004798:Enzymes; D019803:Glutathione Disulfide / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D009249:NADP / Q000737:chemistry; D011786:Quality Control; D011806:Quinolinium Compounds / Q000737:chemistry; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014020:Tissue Extracts / Q000737:chemistry
D004798:Enzymes; D011806:Quinolinium Compounds; D013438:Sulfhydryl Compounds; D014020:Tissue Extracts; D009249:NADP; D004228:Dithionitrobenzoic Acid; D019803:Glutathione Disulfide