Comparison of spectrophotometric and biological assays for nitric oxide (NO) and endothelium-derived relaxing factor (EDRF): Nonspecificity of the diazotization reaction for NO and failure to detect EDRF

Endothelium-derived relaxing factor has been tentatively identified as nitric oxide (NO) partially on the basis of chemical assays. In the present study, saline solutions that were either bubbled continuously for 30 min with NO (NO/X) or prepared using 25 ml of NO/ml (NO/25) produced equivalent relaxations of segments of rabbit aorta which had the endothelium removed. NO solutions prepared using 0.1 ml of NO/ml (NO/0.1), and 3 mM sodium nitrite (NO₂^-) were significantly (P < .05) less potent vasodilators than NO/X and NO/25 (order of potency: NO/X = NO/25 > NO/0.1 > NO₂^-). A novel automated method was developed to monitor nitrogen oxides using continuous-flow spectrophotometric detection (diazotization reaction). The absorbance readings for the solutions were NO/X > NO/25 = NO/0.1 = NO₂^-. Argon purging of NO/X, NO/25 and NO/0.1 solutions significantly (P < .05) reduced (44-100%) the bioactivity of these solutions in inverse proportion to the initial volume of NO used in their preparation. In contrast, the absorbance values were unchanged, indicating that the chemical assay was not correlated with the bioassay. Varying the duration of NO gassing (1-30 min) significantly (P < .05) increased the absorbance values, while having no effect on the vascular relaxations, elicited by the solutions. The diazotization assay did not detect nitrogen oxides released from cultured endothelial cells by bradykinin, ATP, or A23187, whereas the bioassay readily detected endothelium-derived relaxing factor release. These data indicate that the vasoactive properties and chemical composition of NO solutions depend on the preparative technique. In addition, the diazotization reaction lacks the specificity necessary to distinguish NO from other related substances.