Continuous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex

Abstract

Nitric oxide (NO) generation in murine macrophages was determined in real time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl- d-glucamine dithiocarbamate was utilized as the spin trap. This spin trapping compound reacts with NO in solution to form a specific room-temperature stable, mononitrosyl complex which is readily detected and identified by EPR spectroscopy. Mouse peritoneal macrophages were placed in an EPR sample-cell and activated by lipopolysaccharide and γ-interferon at 37°C, followed by an additional incubation in oxygenated medium without these activation agents. After various incubation periods, spin trap solution was infused to replace the medium in the sample cell, and the time evoluation of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes of the increase in the EPR intensity with time. In comparison to the NO (or NO 2 −) generation rate obtained under similar experimental conditions using the Griess reaction assay, the spin trapping method was found to be more sensitive, with a lowest limit of the detection of 3 pmol/min. In addition, by using the spin trapping method. NO generation from the same cells could be measured consecutively during various stages of activation, because infusion of the spin trap solution did not affect the viability of macrophages.