Detection of dinitrosyl iron complexes by ozone-based chemiluminescence

Abstract

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.

Nitric oxide metabolism in the human placenta during aberrant maternal inflammation

“…2018; Mukosera et al . 2018) and other groups have reported the same in vascular tissue (Mülsch et al . 1991).…”

“…Following our previous report (Mukosera et al . 2018), we therefore hypothesized that high molecular weight DNICs are more stable than low molecular weight DNICs, resulting in their previously observed long‐lasting bioactivity (Тimoshin et al . 2015) and the long half‐life that we observe in plasma.…”

“…Different purge vessel reagents were used to determine the concentrations of specific types of NOx species formed (or lost) following recently published protocols that allow for discrimination between nitrate, nitrite, nitrosothiols and iron‐bound NO (FeNO) in the form of DNICs and haeme nitrosyls (haemeNO) (Mukosera et al . 2018). In brief, triiodide reagent was used (assay referred to as I 3 − ) to determine the presence of all NOx species (nitrite, nitrosothiols, N‐nitrosyls and FeNO) except for nitrate.…”

L-NAME releases nitric oxide and potentiates subsequent nitroglycerin-mediated vasodilation

“…Macrophage RAW 264.7 cells were cultured as reported [17]. Cells within 20 passages were incubated under normoxia (21% O 2 /5% CO 2 ) for 10 h in the presence and absence of 1 mmol/L L-NAME, 1 mmol/L L-NMMA, 1 μmol/L antimycin A, 100 μmol/L deferoxamine, 10 μmol/L resveratrol, 800 U/ml PEG-catalase, or 800 U/ml catalase.…”

“…Briefly, overall NOx concentrations were measured by direct injection of the sample into triiodide and nitrite concentrations were taken as the portion of the signal that was eliminated by prior reaction of the sample with acid sulfanilamide. In addition to measuring NO itself, the triiodide reagent facilitates detection of NO released from several types of NO adducts, including nitrite, S-nitrosothiols (SNO), and nitrosyl-iron complexes [17], but excluding nitrate.…”