Measurement of reactive species

Abstract This chapter describes the fundamental principles behind the detection and measurement of reactive species (RS) in vivo, as well as the quantification of the damage that they can do to biomolecules (oxidative damage). The use of electron spin resonance (ESR) is described in detail, with a particular focus on spin traps such as PBN, DMPO and DEPMPO, TEMP, TEMPD, and POBN, and the technique of immuno-spin trapping. The detection of hydroxyl radicals by spin trapping, aromatic hydroxylation, deoxyribose degradation, and other techniques is presented, as is the detection of superoxide and nitric oxide by ESR and other techniques, both direct and indirect (e.g. Griess reaction). Peroxynitrite measurement is described, as is that of singlet O2 (using fluorescent probes, spin traps, scavengers and quenchers, monomol and dimol light emission, and deuterium oxide). The measurement of cellular redox state and peroxide levels is explained in detail, especially the use of roGFPs, cpYFP, Amplex red, and Hyper or boronate-based H2O2 detectors. Essential points to be considered in the use of DCFHDA and other ‘ROS probes’ (luminol, lucigenin, L-012, HPF, APF, dihydrorhodamine, dihydroethidium) are presented in detail. Biomarkers of oxidative damage to bilirubin (biopyrrins) and urate (allantoin) are presented. The techniques used to measure oxidative damage to DNA (strand breaks, 8-hydroxyguanine, formamidopyrimidines), lipids (peroxides, conjugated dienes, loss of PUFAs, TBA test, aldehydes, isofurans, isoprostanes), and proteins (carbonyls, oxidation products of specific amino acid residues) are discussed. Finally, the value and limitations of ‘total antioxidant capacity’ determinations in food and body fluids are presented.