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Phytochemicals and cellular redox potential: Variations in measurement of glutathione levels using three assays
Author(s) -
Marisiddaiah Raju,
Wiener Doris,
Gong Xiaoming,
Rubin Lewis P
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.27.3
Subject(s) - glutathione , chemistry , redox , biochemistry , dtnb , oxidizing agent , glutathione reductase , glutathione disulfide , chromatography , enzyme , glutathione peroxidase , organic chemistry
Purpose Glutathione (GSH) is the principal intracellular redox couple. Several methods have been devised to measure GSH tot , GSH reduc and GSH oxid (GSSG) in biological samples. However, different techniques and assay conditions can yield conflicting results. Methods We measured the GSH redox couple in normal and tumor cell lines using three methods. In some experiments, we compared the GSH couple ±lycopene or lutein. We examined two methods involving fluorimetric or luminometric detection and a spectrophotometric GR:DTNB (enzymatic) recycling method. Results The luminometric and spectrophotometric assays consistently yielded GSH red >> GSSG (the physiologically anticipated result). However, the fluorimetric assay yielded >90% of GSH tot as GSSG. An important element in this technique is use of perchloric acid (PCA) protein precipitation to prevent enzymatic degradation of GSH to GSSG. Conclusion The discrepancies among glutathione assay methods may result from use of a powerful oxidizing agent (PCA), causing artificial GSSG generation. The GSH:GSSG couple is an important target of phytochemical action, including carotenoids and flavinoids. More consistent measurements of GSH homeostasis should clarify biological effects. Grant Funding Source : NIH, USF