A rapid HPLC method for the quantification of GSH and GSSG in ocular lens.
Purpose. To develop a rapid and accurate method for the quantification of reduced glutathione (GSH) and oxidized glutathione (GSSG) using micro-quantities of ocular lens. Methods. The epithelium, cortex and nucleus of the lens were separated and also the whole lens was homogenized in 3% metaphosphoric acid. The homogenate was ultrafiltered by centrifugation at 10,000 g in an Amicon microconcentrator, molecular weight cut off 3,000 g. The method does not require prior derivatization of the glutathiones. The filtrate was analyzed on a Microsorb-MV by a high performance liquid chromatography (HPLC) column using an isocratic solvent system (3% methanol and 10 mM potassium phosphate, pH 3.0) and detection at 200 nm.


RESULTS
The GSH and GSSG were eluted from the HPLC column at retention times 5 and 10 min, respectively. The detection limit was 100 pmoles applied to the column. The recovery of GSH and GSSG added to the tissue samples was 97-100%.


CONCLUSIONS
A fast and sensitive HPLC-method for the quantification of picomole quantities of GSH and GSSG in ocular lens, which does not require prior derivatization, has been developed.
Liu
S
S
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX,77555-0647 USA.
Ansari
N H
NH
Wang
C
C
Wang
L
L
Srivastava
S K
SK
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004848:Epithelium / Q000737:chemistry; D005069:Evaluation Studies as Topic; D005978:Glutathione / Q000031:analogs & derivatives* / Q000032:analysis*; D019803:Glutathione Disulfide; D066298:In Vitro Techniques; D007904:Lens Cortex, Crystalline / Q000737:chemistry; D007907:Lens Nucleus, Crystalline / Q000737:chemistry; D007908:Lens, Crystalline / Q000737:chemistry*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
D005978:Glutathione; D019803:Glutathione Disulfide