Simultaneous determination of reduced and oxidized glutathione in freshly isolated rat hepatocytes and cardiomyocytes by HPLC with electrochemical detection.
Glutathione and glutathione disulphide constitute an essential thiol redox system present in the cell. The balance in favour of the latter is an indication of oxidative stress. Glutathione and glutathione disulphide quantification in isolated cells may therefore be essential for the evaluation of mechanistic and comparative studies of toxic xenobiotics. In this study, a rapid and sensitive isocratic reverse-phase high-performance liquid chromatographic method using coulometric detection was implemented for the simultaneous detection of glutathione and glutathione disulphide, in freshly isolated hepatocytes and cardiomyocytes of the rat. The method implemented proved to be effective for the measurement of glutathione and glutathione disulphide in control conditions and for the detection of variations in this redox system, induced by tert-butylhydroperoxide. tert-Butylhydroperoxide is an organic peroxide, which has been used as a model molecule for inducing oxidative stress in isolated cells. A comparative study with a previously published HPLC-electrochemical detection method was performed.
Remião
F
F
ICETA/CEQUP, Toxicology Department, Faculty of Pharmacy, University of Porto, 4050/047 Porto, Portugal. remiao@mail.ff.up.pt
Carmo
H
H
Carvalho
F
F
Bastos
M L
ML
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D005978:Glutathione / Q000032:analysis*; D019803:Glutathione Disulfide / Q000032:analysis*; D022781:Hepatocytes / Q000737:chemistry* / Q000187:drug effects; D008297:Male; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D051381:Rats; D017208:Rats, Wistar; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D020122:tert-Butylhydroperoxide; D005978:Glutathione; D019803:Glutathione Disulfide