HPLC-electrochemical detection of tocopherol products as indicators of reactive nitrogen intermediates

The nitric oxide (NO) free radical serves diverse functions in mammalian physiology, facilitating processes that range from vasodilation to neurotransmission to host-pathogen defense. Despite the fascinating biochemical utility of this small diatomic gas, NO and its derived oxidation or reduction products [reactive nitrogen species (RNSs) or reactive nitrogen intermediates (RNIs)] can be deleterious to cells and tissues under conditions of pathophysiology. Recent years have witnessed a tremendous and continuing scientific interest in RNI, both as targets for pharmacotherapy and as biomarkers for disease. Accordingly, methods have been developed to quantify RNI in real time under controlled experimental conditions. Such methods usually employ either electron paramagnetic resonance (EPR) spin traps (see ) or electrochemical sensors (Cserey and Gratzl, 2001). Nonetheless, the transient nature of NO and RNIs often precludes their routine assessment in animal experiments or human clinical studies where tissue must be archived and stored at a later time. To circumvent these limitations, methods have been invented to detect and quantify stably nitrated products of RNI reaction with ambient biomolecules. This chapter describes the theory and methodology for detection of lipid-phase tocopherol nitration products, especially 5-nitro-gamma-tocopherol (5-NO2-gammaT) by high-performance liquid chromatography with electrochemical detection (HPLC-ECD).