A rapid HPLC method for the quantification of GSH and GSSG in ocular lens

Siqi Liu, Naseem Ansari, Changsen Wang, Lifei Wang, Satish Srivastava

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)726-732
Number of pages7
JournalCurrent Eye Research
Volume15
Issue number7
StatePublished - 1996

Fingerprint

Crystalline Lens
Glutathione Disulfide
High Pressure Liquid Chromatography
Lenses
Glutathione
Centrifugation
Methanol
Limit of Detection
Epithelium
Molecular Weight

Keywords

  • High-performance liquid chromotography (HPLC)
  • Lens
  • Oxidized glutathione (GSSG)
  • Reduced glutathione (GSH)

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Liu, S., Ansari, N., Wang, C., Wang, L., & Srivastava, S. (1996). A rapid HPLC method for the quantification of GSH and GSSG in ocular lens. Current Eye Research, 15(7), 726-732.

A rapid HPLC method for the quantification of GSH and GSSG in ocular lens. / Liu, Siqi; Ansari, Naseem; Wang, Changsen; Wang, Lifei; Srivastava, Satish.

In: Current Eye Research, Vol. 15, No. 7, 1996, p. 726-732.

Research output: Contribution to journalArticle

Liu, S, Ansari, N, Wang, C, Wang, L & Srivastava, S 1996, 'A rapid HPLC method for the quantification of GSH and GSSG in ocular lens', Current Eye Research, vol. 15, no. 7, pp. 726-732.
Liu, Siqi ; Ansari, Naseem ; Wang, Changsen ; Wang, Lifei ; Srivastava, Satish. / A rapid HPLC method for the quantification of GSH and GSSG in ocular lens. In: Current Eye Research. 1996 ; Vol. 15, No. 7. pp. 726-732.
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T1 - A rapid HPLC method for the quantification of GSH and GSSG in ocular lens

AU - Liu, Siqi

AU - Ansari, Naseem

AU - Wang, Changsen

AU - Wang, Lifei

AU - Srivastava, Satish

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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