TY - JOUR
T1 - A rapid HPLC method for the quantification of GSH and GSSG in ocular lens
AU - Liu, Siqi
AU - Ansari, Naseem H.
AU - Wang, Changsen
AU - Wang, Lifei
AU - Srivastava, Satish K.
N1 - Funding Information:
Supported in part by NIH grants: EY 01677, EY 08547, and DK 36118.
PY - 1996
Y1 - 1996
N2 - 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 Mircosorb-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 10 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.
AB - 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 Mircosorb-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 10 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.
KW - High-performance liquid chromotography (HPLC)
KW - Lens
KW - Oxidized glutathione (GSSG)
KW - Reduced glutathione (GSH)
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U2 - 10.3109/02713689609003455
DO - 10.3109/02713689609003455
M3 - Article
C2 - 8670780
AN - SCOPUS:0029781342
SN - 0271-3683
VL - 15
SP - 726
EP - 732
JO - Current Eye Research
JF - Current Eye Research
IS - 7
ER -