Detection of dinitrosyl iron complexes by ozone-based chemiluminescence

Dinitrosyl iron complexes (DNICs) are important intermediates in the metabolism of nitric oxide (NO). They have been considered to be NO storage adducts able to release NO, scavengers of excess NO during inflammatory hypotensive shock, and mediators of apoptosis in cancer cells, among many other functions. Currently, all studies of DNICs in biological matrices use electron paramagnetic resonance (EPR) for both detection and quantification. EPR is limited, however, by its ability to detect only paramagnetic mononuclear DNICs even though EPR-silent binuclear are likely to be prevalent. Furthermore, physiological concentrations of mononuclear DNICs are usually lower than the EPR detection limit (1 μM). We have thus developed a chemiluminescence-based method for the selective detection of both DNIC forms at physiological, pathophysiological, and pharmacologic conditions. We have also demonstrated the use of the new method in detecting DNIC formation in the presence of nitrite and nitrosothiols within biological fluids and tissue. This new method, which can be used alone or in tandem with EPR, has the potential to offer insight about the involvement of DNICs in many NO-dependent pathways. •Use of different reagents enables selective quantification of specific classes of NOx species in a biological matrix.•O3-based chemiluminescence with ferricyanide in PBS detects both bi- and mononuclear DNICs selectively.•As low as 25 pmol NO released from DNICs can be detected, which is ≥ 10-fold more sensitive than currently available methods.•Glutathione-liganded DNICs rapidly convert to high molecular weight DNICs in plasma.