DETERMINATION AND CHARACTERIZATION OF NITRIC-OXIDE GENERATION IN MICEBY IN-VIVO L-BAND EPR SPECTROSCOPY

The authors have shown direct, real-time, in vivo measurement of nitri c oxide (NO) in mice by using the water soluble metal chelator complex , N-methyl-D-glucamine dithiocarbamate (MGD), and Fe(II) as monitored by EPR at L-band. The three-line EPR spectrum from the product [(MGD)( 2)-Fe(II)-NO] was observed noninvasively in lipopolysaccharide (LPS)-t reated mice. The spectrum was markedly suppressed by the administratio n, before LPS injection, of phenyl N-tert-butyl nitrone (PBN), an inhi bitor of the expression of induced nitric oxide synthase (iNOS). When N-15-arginine was administered to LPS-treated mice, a diagnostic EPR s pectrum was observed, consisting of both three- and two-line EPR signa ls, due to (MGD)(2)-Fe(II)-(NO)-N-14 and (MGD)(2)-Fe(II)-(NO)-N-15, re spectively. The results strongly suggested that the NO detected in the se experiments was synthesized by iNOS. In vivo EPR measurements of [( MGD)(2)-Fe(II)-NO] at several regions in the body (from the head to th e tail) indicated that the NO was generated mostly in the upper abdome n near the liver. These observations were confirmed by ex vivo EPR mea surements on isolated organs where higher NO levels were detected in v ivo in the liver and kidney. The spectroscopic results, combined with the pharmacokinetic data, support the model that NO detected in LPS-tr eated mice was produced mainly in the liver, and that it did not refle ct NO-adduct complex accumulated in the liver via the blood circulatio n.