In Vivo Detection of Nitric Oxide: Combining EPR and NMR

Lawrence J. Berliner

Dept. of Chemistry and Biochemistry, University of Denver, Denver CO, USA

Nitric oxide (NO), a gaseous free radical, has been implicated recently in a number of diverse physiological processes. In order to understand the mechanism by which NO is synthesized by nitric oxide synthase (NOS) in tissues and how NO mediates various physiological responses, it is useful to be able to observe real time NO generation. We have shown direct, real-time, in-vivo measurement of nitric oxide (NO) in mice using the water soluble metal chelator complex, N-methyl-D-glucamine dithiocarbamate (MGD), and Fe(II) and the lipophilic reagent, N,N-diethyl-dithiocarbamate (DETC), as monitored by EPR at L-band. The EPR spectrum from the product [(MGD)2-Fe(II)-NO] or [(DETC)2-Fe(II)-NO] was observed noninvasively in lipopolysaccharide (LPS)-treated mice. In-vivo EPR measurements of [(MGD)2-Fe(II)-NO] at several regions in the body (from the head to the tail) indicated that the NO was generated mostly in the upper abdomen near the liver. This was confirmed by ex-vivo EPR measurements on isolated organs. In the case of [(DETC)2-Fe(II)-NO] we were successful in observing relatively high concentrations of NO trapped in excised brain tissue. Lastly, the NO-adduct detected in LPS-treated mice brains was not inducible NOS, but probably rather constitutive NOS, since it was not suppressed by the administration, prior to LPS injection, of phenyl N-tert-butyl nitrone (PBN), an inhibitor of the expression of induced nitric oxide synthase (iNOS). MRI spin trapping is defined as the ability to visualize free radicals or free radical reactions in vivo by virtue of the power of paramagnetic compounds to serve as excellent contrast agents by inducing proton relaxation enhancement from rapidly exchanging water molecules. There are additional advantages in that MRI is a highly refined, commercialized technology which offers exquisite resolution by virtue of nuclear relaxation times, diffusion rates, oxygen magnetic susceptibility effects, etc.

Email: berliner@du.edu

Address: Lawrence J. Berliner, Professor and Chair, Department of Chemistry & Biochemistry, University of Denver, 2190 E. Iliff Avenue, F. W. Olin Hall Room 202, Denver, CO 80208-2436, Phone: 303-871-2436, 2435, Fax: 303-871-2254,