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Assays for oxidative stress and antioxidant status: applications to research into the biological effectiveness of polyphenols^1,2,3,4

¹ From the Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo

ABSTRACT

Oxidative stress is a factor in many human diseases, as either cause or effect. A convenient biomarker of oxidative stress is the extent of oxidation of bases in DNA (although measures of lipid or protein oxidation may be equally informative). 8-Oxo-7,8-dihydroguanine or the corresponding nucleoside is most often measured, either chromatographically (gas chromatography-mass spectrometry, HPLC with electrochemical detection, or HPLC-tandem mass spectrometry) or enzymically, with the use of the enzyme formamidopyrimidine DNA glycosylase to convert 8-oxo-7,8-dihydroguanine to DNA breaks, which are detected with alkaline elution, alkaline unwinding, or the comet assay. Estimates of background levels of 8-oxo-7,8-dihydroguanine in normal human cells vary 1000-fold, depending on the technique used. Gas chromatography-mass spectrometry is particularly prone to oxidation of samples during derivatization, whereas HPLC suffers from this artifact to a lesser degree. In a recent interlaboratory study that measured the same samples of human cells, median values obtained with HPLC with electrochemical detection and with formamidopyrimidine DNA glycosylase differed by 10-fold. There are still questions regarding the actual level of damage, but it is probably approximately one 8-oxo-7,8-dihydroguanine residue per 10⁶ guanines. Assays for antioxidant protection against oxidative damage generally depend on measurements of decreases in a marker of oxidation. Potential dietary antioxidants can be screened with in vitro antioxidant assays or tested in cell culture systems. The best test, however, is in humans. The total antioxidant capacity of plasma is generally insensitive to dietary supplementation with antioxidants or antioxidant-rich foods. An increase in the resistance of lymphocyte DNA to oxidation in vitro is commonly seen, however, and a decrease in endogenous oxidation of DNA may be detected, especially after prolonged supplementation.

Key Words: Oxidative DNA damage • antioxidant assays • 8-oxoguanine • validation study

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