Real-Time, In-Vivo Measurement of Nitric Oxide Using Electron Paramagnetic Resonance Spectroscopic Analysis of Biliary Flow

The nitric oxide free radical (•NO) is formed by the enzymatic oxidation of arginine in a reaction catalyzed by various isoforms of the enzyme nitric oxide synthase (NOS). •NO serves myriad physiologic functions, including actions as a vasodilator. Under conditions of inflammation, macrophages (and certain other permissive cell types) synthesize copious quantities of •NO through the expression of an inducible isoform of NOS (iNOS). •NO may serve a defensive function against pathogens, acting as a microbial toxin. Combination of •NO with the superoxide radical anion (O₂ �⁻ ), also syntheisized by activated immune cells, leads to the formation of the highly reactive oxidant peroxynitrite (ONOO⁻ ). •NO and its redox congeners have received much attention as pathophysiologic agents in both acute and chronic inflammation, septic shock, cardiovascular biology, and neurodegenerative disorders. The bioanalysis of •NO is complicated by the relative instability of this species in a biological mileu, where the •NO radical can decompose through oxygen-dependent pathways or alternatively be consumed through reaction with thiol moieties and heme groups. A method to continuously monitor the •NO level in anesthetized rats, using an in vivo trapping reaction of NO by iron-dithiocarbamate complex, is shown in this chapter.