• S D Dumitrescu, A T Meszaros, S Puchner, A Weidinger, J Kaszaki, M Boros, H Redl, and A V Kozlov.
• 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria, 2Institute of Surgical Research, University of Szeged, Szeged, Hungary.
• Shock. 2015 Oct 1;44 Suppl 2:11-2.

IntroductionNitric oxide (NO) is a potent signaling molecule maintaining numerous physiological and pathophysiological processes. Because of its short lifespan, the detection of NO by electron paramagnetic resonance spectroscopy (EPR) in frozen tissues requires stabilization by specific exogenous NO-traps. Such NO-traps are unfeasible in clinical settings, and are restricted to experimental models only.MethodsWe developed an EPR-based quantification method based on the reaction of NO with endogenous NO-traps such as hemoglobin and iron. To stimulate the NO production and formation of NO-Hb and NO-Fe complexes in liver and blood, rats were injected with various doses of LPS (0-8 mg/kg). The levels of NO-Hb and NO-Fe complexes were determined by EPR at liquid nitrogen temperature, and compared to NO-Hb/NO-Fe levels in calibration samples. In additional experiments, an exogenous NO-trap DETC-Fe was used to confirm NO production.ResultsNO-Hb/NO-Fe complexes were detected in liver tissue upon LPS treatment, whereas in blood-free liver samples only NO-Fe was detected. In contrast, only NO-Hb complexes were detected in blood upon LPS treatment. Ex-vivo synthetized NO-Hb and NO-Fe were used for calibration. The calibrations were optimal if the concentrations of NO-traps were varied upon an excess of NO. We determined the concentration of Fe-NO and Hb-NO in liver in the range of 4.8-98.8 μM and 15.3-105.5 μM respectively, upon LPS treatment.ConclusionOur methodological approach enables quantification of NO production in vivo without introduction of NO-traps and allocates NO-generation either to extra- or intracellular compartments of the liver. This method can be effectively applied for analysis of human biopsies.

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