Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis.
We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (Mr cut-off 3000 Da). The analysis was performed at a constant temperature (28 degrees C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 microM for GSH and 1.2 microM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1 +/- 2.6 and 0.45 +/- 0.15 (nmol/mg protein +/- S.D.), respectively. The ratio of GSH to GSSG was 17.8 +/- 1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification.
Muscari
C
C
Department of Biochemistry, G. Moruzzi, Center of Research on Heart Metabolism, University of Bologna, Italy.
Pappagallo
M
M
Ferrari
D
D
Giordano
E
E
Capanni
C
C
Caldarera
C M
CM
Guarnieri
C
C
D000818:Animals; D019075:Electrophoresis, Capillary; D005978:Glutathione / Q000032:analysis*; D007202:Indicators and Reagents; D008297:Male; D008928:Mitochondria / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000737:chemistry; D008930:Mitochondria, Liver / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D007202:Indicators and Reagents; D005978:Glutathione