Estimation of hydroxyl free radical levels in vivo based on liquid chromatography with electrochemical detection

Abstract

When hydroxyl free radical (HO•)- is produced in vivo , it can readily react with many extremely important biomolecules, including DNA, lipids, proteins, carbohydrates, and a variety of low molecular weight species. This chapter discusses the estimation of hydroxyl free radical levels in vivo . Of all the approaches in literature that claim to measure HO•- production, two are showing great promise. One approach, electron spin resonance (ESR) detection, captures HO• by reacting it with a suitable spin trap such as phenyltert-butylnitrone or 5,5-dimethylpyrroline N-oxide. Not only is the lifetime of HO• extended, but the HO•-spin trap adduct still remains paramagnetic and can thus be measured by ESR. A second approach, aromatic hydroxylation, is mechanistically similar to the spin-trap ESR-based methods discussed above, but it does not use “classical” spin trap agents, nor do the products formed have to be paramagnetic. Thus, referring to aromatic hydroxylation as a spin-trap method is quite erroneous. Aromatic hydroxylation uses the formation of products from the reaction between HO• and a suitable aromatic species as a marker of HO• production.