S Liu1, N H Ansari, C Wang, L Wang, S K Srivastava. 1. Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX,77555-0647 USA.
Abstract
UNLABELLED: 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.
UNLABELLED: 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.