RAPID-DETERMINATION OF GLUTATHIONE STATUS IN FISH LIVER USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND ELECTROCHEMICAL DETECTION

A rapid and sensitive method for the detection of reduced (GSH), oxidi sed (GSSG) and protein-bound (PSSG) glutathione in fish liver, using r eversed-phase HPLC with electrochemical detection has been developed. Separation was carried out isocratically at room temperature using 0.0 20 M sodium phosphate, pH 2.7 as mobile phase. A series dual-channel e lectrochemical detector was used for the simultaneous determination of GSH and GSSG. PSSG was determined after reduction by 1,4-dithiothreit ol. The detection limits found for a 3:1 signal-to-noise ratio were 16 .2 and 8.1 pmol for GSH and GSSG, respectively. The results obtained d emonstrate that this method could be useful for measurement of the glu tathione redox status in fish liver and are consistent with those repo rted for other fish. The method has been applied to follow the oxidati ve stress induced in vivo by copper(II) ions in the gilthead seabream fish (Sparus aurata). At longer times after copper(II) injection, the glutathione redox status of the exposed fish returned to a more reduce d state, suggesting the existence of adaptive processes.