Nitrosylhemoglobin, an unequivocal index of nitric oxide release from nitroaspirin: in vitro and in vivo studies in the rat by ESR spectroscopy.

Electron spin resonance (ESR) spectroscopy was applied for the unequivocal detection/quantitation of nitric oxide (NO) as nitrosylhemoglobin (HbFe(II)NO) released from nitroaspirin, benzoic acid,2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX-4016; NO-ASA), the lead of a new class of nonsteroidal anti-inflammatory drugs. In both in vitro and in vivo experiments, the paramagnetic complex was detected at 100 K in the venous blood of the rat (microwave power, 20 mW) and characterized by a three-line hyperfine structure with coupling constants (A(x) and A(z)) of 17 G at g(x)=2.066 and g(z)=2.009. The kinetics of NO release from the drug were first determined in vitro by incubating rat blood with 1 mM NO-ASA and confirmed by the two-line hyperfine structure obtained with the labeled compound ((15)N-NO-ASA). In in vivo studies, the hematic levels of HbFe(II)NO were determined after oral (p.o.) and intraperitoneal (i.p.) administration of the drug (100 and 200 mg kg(-1)). In p.o. treated animals, the complex was detectable at 1 h post-dosing and its formation was maximal at 4-6 h, where the antithrombotic activity peaks. In i.p. treated animals, HbFe(II)NO complex peaks at the second hour to decline thereafter: in these animals, the ESR technique was applied to also detect nitrosylmyoglobin as an index of NO diffusion/compartmentalization in myocardial tissue. The results of this study emphasize the great potentiality of ESR spectroscopy for the study of the release, the metabolic fate and distribution of NO from nitrovasodilators.