Evaluation of total plasma nitric oxide concentrations in broilers infused intravenously with sodium nitrite, lipopolysaccharide, aminoguanidine, and sodium nitroprusside

Nitric oxide (NO) is a potent vasodilator that is synthesized by constitutive and inducible isoforms of the enzyme NO synthase (eNOS and iNOS, respectively). The half-life of NO averages only 3 to 4 s in biological fluids, where it is rapidly converted to the stable oxidation products nitrite (NO2-) and nitrate (NO3-). Our objectives were to use 2 commercial kits to measure total plasma NO, as NO2- + NO3-, and to assess plasma NO values during experimental protocols designed to influence NO accumulation in the plasma. One kit employed copper-coated cadmium as a catalyst for reducing NO3- to NO2-; the second kit employed the enzyme NO3- reductase for the same purpose. Both then employed Griess reagent for the colorimetric determination of NO2- as a measure of total plasma NO. Broilers in Experiment 1 were infused i.v. with solutions containing increasing concentrations of sodium NO2-. Broilers in Experiment 2 were injected with 1 mg of lipopolysaccharide (LPS), which is known to stimulate iNOS activity. Both commercial kits successfully detected increases in total plasma NO attributable to ongoing i.v. NO2- infusion or to increased iNOS expression at 5 h after the LPS injection. In Experiment 3, we compared the total plasma NO responses to LPS in the presence and absence of aminoguanidine (AG), a selective inhibitor of iNOS. The AG significantly attenuated the LPS-mediated increase in total plasma NO at 5 h post-injection. In Experiment 4, broilers were infused with sodium nitroprusside (SNP), an exogenous NO donor molecule that previously had been shown to lower the pulmonary arterial pressure in broilers. The SNP infusion did substantially reduce the pulmonary arterial pressure, but an increase in total plasma NO was not detected during the SNP infusion. Overall, NO accumulation in the plasma was successfully detected after sustained infusion of NaNO2 and administration of LPS for 5 h, but biologically effective levels of NO released from SNP were not detected. Therefore, total plasma NO concentrations (assayed as NO2- + NO3-) qualitatively reflect whole-body NO synthesis, but biologically relevant quantities of NO may be produced at levels that cannot be detected by colorimetric assays.