The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection

John Richie, Calvin A. Lang

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Abstract

A determination of glutathione, cysteine, and their disulfides using HPLC and dual electrochemical detection (HPLC-DEC) was described previously but was not validated in biological tissues for these and other important thiols and disulfides (SH/SS). Thus, our objectives were to develop this method to quantify simultaneously reduced and oxidized glutathione, cysteine, cystine, and other SH/SS in various tissues, including human blood and plasma, rat liver and hippocampus, mosquito, and spinach leaf. Optimal conditions were determined for sample processing and analysis using metaphosphoric acid and HPLC-DEC. Authentic standards of 10 common SH/SS compounds were resolved and eluted within 15 min, and all standard curves were linear from 5 to 1600 pmol. Validation was based on the following: First, tissue sample sizes were proportional to peak areas over an eightfold range. Second, recovery of SH/SS added to samples before processing was 96-101%. Finally, the results were equivalent and correlated highly with values for total SH by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) assay (r2 = 0.996) and for total glutathione by DTNB-GSSG reductase assay (r2 = 0.998). The life span of the Au/Hg electrode was limited to 200-500 samples based on the lineal range of standard curves. On the basis of these results, we believe that this method will fill a long-time need for the simultaneous determination of SH/SS in biological tissues.

Original languageEnglish (US)
Pages (from-to)9-15
Number of pages7
JournalAnalytical Biochemistry
Volume163
Issue number1
DOIs
StatePublished - May 15 1987

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High performance liquid chromatography
Sulfhydryl Compounds
Disulfides
Glutathione
Cysts
High Pressure Liquid Chromatography
Tissue
Dithionitrobenzoic Acid
Glutathione Disulfide
Assays
Nitrobenzoates
Spinacia oleracea
Cystine
Processing
Culicidae
Liver
Sample Size
Cysteine
Rats
Hippocampus

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "A determination of glutathione, cysteine, and their disulfides using HPLC and dual electrochemical detection (HPLC-DEC) was described previously but was not validated in biological tissues for these and other important thiols and disulfides (SH/SS). Thus, our objectives were to develop this method to quantify simultaneously reduced and oxidized glutathione, cysteine, cystine, and other SH/SS in various tissues, including human blood and plasma, rat liver and hippocampus, mosquito, and spinach leaf. Optimal conditions were determined for sample processing and analysis using metaphosphoric acid and HPLC-DEC. Authentic standards of 10 common SH/SS compounds were resolved and eluted within 15 min, and all standard curves were linear from 5 to 1600 pmol. Validation was based on the following: First, tissue sample sizes were proportional to peak areas over an eightfold range. Second, recovery of SH/SS added to samples before processing was 96-101{\%}. Finally, the results were equivalent and correlated highly with values for total SH by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) assay (r2 = 0.996) and for total glutathione by DTNB-GSSG reductase assay (r2 = 0.998). The life span of the Au/Hg electrode was limited to 200-500 samples based on the lineal range of standard curves. On the basis of these results, we believe that this method will fill a long-time need for the simultaneous determination of SH/SS in biological tissues.",
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AB - A determination of glutathione, cysteine, and their disulfides using HPLC and dual electrochemical detection (HPLC-DEC) was described previously but was not validated in biological tissues for these and other important thiols and disulfides (SH/SS). Thus, our objectives were to develop this method to quantify simultaneously reduced and oxidized glutathione, cysteine, cystine, and other SH/SS in various tissues, including human blood and plasma, rat liver and hippocampus, mosquito, and spinach leaf. Optimal conditions were determined for sample processing and analysis using metaphosphoric acid and HPLC-DEC. Authentic standards of 10 common SH/SS compounds were resolved and eluted within 15 min, and all standard curves were linear from 5 to 1600 pmol. Validation was based on the following: First, tissue sample sizes were proportional to peak areas over an eightfold range. Second, recovery of SH/SS added to samples before processing was 96-101%. Finally, the results were equivalent and correlated highly with values for total SH by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) assay (r2 = 0.996) and for total glutathione by DTNB-GSSG reductase assay (r2 = 0.998). The life span of the Au/Hg electrode was limited to 200-500 samples based on the lineal range of standard curves. On the basis of these results, we believe that this method will fill a long-time need for the simultaneous determination of SH/SS in biological tissues.

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