Determination of oxidized, reduced and protein-bound glutathione in eye lenses by high-performance liquid chromatography and electrochemical detection.
A method for the detection of oxidized, reduced and protein-bound glutathione in eye lenses has been developed. Homogenized lens samples are deproteinated with acetonitrile and perchloric acid. Protein-bound glutathione is reduced by 1,4-dithiothreitol. Separation of the different forms of glutathione and dithiothreitol is performed by ion-pair reversed-phase high-performance liquid chromatography with sodium octylsulphate as the ion-pairing agent. The compounds are detected amperometrically using on-line-generated bromine, which oxidizes thiols and disulphides. In this way two samples can be analysed in triplicate in a single day. The lower detection limits are 80 and 48 nmol per gram wet lens for reduced and oxidized glutathione, respectively. The amounts of free reduced and protein-bound glutathione in calf lenses, determined with this method, are 6.8 +/- 0.4 and 0.96 +/- 0.03 mumol per gram wet lens, respectively. That of oxidized glutathione is less than 0.048 mumol per gram wet lens.
Ozcimder
M
M
Department of General and Analytical Chemistry, Free University, Amsterdam, Netherlands.
Louter
A J
AJ
Lingeman
H
H
Voogt
W H
WH
Frei
R W
RW
Bloemendal
M
M
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000378:metabolism*; D004229:Dithiothreitol / Q000737:chemistry; D004563:Electrochemistry; D005978:Glutathione / Q000378:metabolism*; D007908:Lens, Crystalline / Q000737:chemistry*; D010084:Oxidation-Reduction
D003459:Crystallins; D005978:Glutathione; D004229:Dithiothreitol