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A sensitive electrochemical method for quantitative hydroperoxide determination
C Y O'Gara1, K R Maddipati, L J Marnett
1Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
Chemical Research in Toxicology
|September 1, 1989
Summary
This study presents a sensitive and selective assay for measuring hydroperoxides using glutathione peroxidase and HPLC. The method quantifies total hydroperoxides in biological samples, including human plasma.
Area of Science:
- Biochemistry
- Analytical Chemistry
Background:
- Hydroperoxides are key biomarkers of oxidative stress.
- Accurate quantification of hydroperoxides is crucial for understanding various biological processes and diseases.
Purpose of the Study:
- To develop a general, sensitive, and selective assay for hydroperoxide determination.
- To enable the measurement of total hydroperoxide concentration in complex biological mixtures.
Main Methods:
- Assay based on glutathione (GSH) reduction of hydroperoxides catalyzed by GSH peroxidase.
- Separation of oxidized glutathione (GSSG) from GSH using High-Performance Liquid Chromatography (HPLC).
- Quantification of GSSG using a coulometric detector.
Main Results:
- The assay is capable of measuring picomole amounts of GSSG, correlating to hydroperoxide concentration.
- GSH peroxidase exhibits broad substrate specificity, allowing for total hydroperoxide determination.
- The method was successfully applied to measure hydroperoxide levels in human plasma.
Conclusions:
- A robust and versatile assay for hydroperoxide quantification has been established.
- The method is applicable to various biological samples, including plasma, and can analyze peroxidized phospholipids.
- This assay provides a valuable tool for oxidative stress research.