EPR Detection of Metals and Nitrosyl-Heme 211 25 In Vivo Detection of Transition Metals and Nitrosyl-Heme Complexes Using Ex Vivo Electron Paramagnetic Resonance Spectroscopy David M. Hall and Garry R. Buettner 1. Introduction Cellular redox environment is a critical determinant of stress-induced cellular responses and the progression of disease (1). Under normal (nonstress) conditions, the cell maintains a strong reducing environment that favors reductive over highly compartmentalized oxidative biochemistry. Exposure of cellular macromolecules to reactive oxygen (ROS) and reactive nitrogen species (RNS) is tightly controlled. Environmental stress can shift the redox balance away from reductive biochemistry however, promoting transition metal activation, and nonprogrammed oxidative and/or nitrosative reactions. Metal- catalyzed oxidative and nitrosative stress have been implicated in the etiology of numerous clinical disorders including inflammation, ischemia-reperfusion injury, rheumatoid arthritis, and aging (1). While the oxygen- and nitrogen-centered radicals themselves are labile and difficult to directly measure in biological systems without the aid of pharmacologic spin-trapping agents, endogenous biomolecules that have been modified by ROS/RNS can be relatively long-lived, allowing us to detect their presence using electron paramagnetic resonance spectroscopy (EPR). In this chapter, we present a technique for characterizing biomarkers of ROS/RNS production and metal activation in whole blood and intact tissues. With this method we have quantitated temporal changes in semiquinone radical (a biomarker of mitochondrial stress), ceruloplasmin (an acute phase ferroxidase antioxidant protein), transferrin, and nitrosyl-heme production in vivo, by sam- 211 From: Methods in Molecular Biology, vol. 196: Oxidants and Antioxidants: Ultrastructure and Molecular Biology Protocols Edited by: D. Armstrong © Humana Press Inc., Totowa, NJ