Quantification of nitric oxide in biological samples by electron spin resonance spectroscopy

We describe a method for the detection of the nitric oxide radical (NO) in aqueous media, cells, and tissues in the presence of oxygen that is based on the trapping of NO by the ferrous iron-diethyldithiocarbamate complex (Fe(DETC)3). Yeast, cells, and tissues provide the ferrous iron to accumulate this complex in the hydrophobic membrane compartment upon incubation with DETC. Therefore, trapping NO generated in cells and tissues requires only preincubation with DETC, whereas in cell-free media DETC-loaded yeast must be added. Fe(DETC)3 avidly and nearly quantitatively binds NO, yielding a stable paramagnetic mononitrosyl complex (NOFe(DETC)2) exhibiting a characteristic electron spin resonance (ESR) signal with gτ = 2.035 and g| = 2.02 in the frozen state. The amount of NO trapped is calculated by calibration with a standard, and the intracellular free ferrous iron content can be determined by titration with exogenously added NO. The detection limit is 0.05 nmol NO in sample volumes of 0.7 ml, depending on the quality of the ESR instrument. The method is also suitable for on-line recording of NO formation proceeding in aqueous incubates exposed to the magnetic field and for measurement of NO formation in living organisms.