CONTINUOUS MONITORING OF CELLULAR NITRIC-OXIDE GENERATION BY SPIN-TRAPPING WITH AN IRON-DITHIOCARBAMATE COMPLEX

Nitric oxide (NO) generation in murine macrophages was determined in r eal time using the electron paramagnetic resonance (EPR) spin trapping method. An iron complex of N-methyl-D-glucamine dithiocarbamate was u tilized as the spin trap. This spin trapping compound reacts with NO i n solution to form a specific room-temperature stable, mononitrosyl co mplex which is readily detected and identified by EPR spectroscopy. Mo use peritoneal macrophages were placed in an EPR sample-cell and activ ated by lipopolysaccharide and gamma-interferon at 37 degrees C, follo wed by an additional incubation in oxygenated medium without these act ivation agents. After various incubation periods, spin trap solution w as infused to replace the medium in the sample-cell, and the time-evol ution of the EPR signal of the spin adduct (NO-complex) was recorded. Rates of NO generation were calculated based upon the initial slopes o f the increase in the EPR intensity with time. In comparison to the NO (or NO2-) generation rate obtained under similar experimental conditi ons using the Griess reaction assay, the spin trapping method was foun d to be more sensitive, with a lowest limit of the detection of 3 pmol /min. Ln addition, by using the spin trapping method, NO generation fr om the same cells could be measured consecutively during various stage s of activation, because infusion of the spin trap solution did nor af fect the viability of macrophages.