Reevaluation of the Griess method for determining NO/NO2- in aqueous and protein-containing samples

Nitric oxide (NO) is an important intracellular and extracellular signal substance. Nitrite is one product of the oxidative metabolism of NO. The purpose of this study was to establish a simple method of determining nitrite (NO2-) to provide a means of estimating the endogenous formation of NO or NO2-. A flow injection analysis (FIA) based on the Griess reaction was developed for this purpose. Using a standard additive method, it is possible to eliminate matrix effects such as those that can occur in samples containing protein. This measuring method is suitable for measurements in effluates or protein-rich cellular supernatants. The sensitivity of the method is 2 nmol/L for samples in aqueous phases and 8 nmol/L for protein-containing phases. The two-point discrimination is 2 nmol/L. A linear correlation between nitrite and signal level can be demonstrated over a range of 0.002-5 micromol/L. Reproducibility, including sample preparation and analysis, can be specified with a coefficient of variation (C.V.) of 6.7%. Day-to-day variability for identical samples 0.8% (C.V.). This study presents examples of the application of this method (measurements in blood samples and in isolated perfused hearts) and compares them to established methods of measuring NO and NO2. We found the FIA method to be equally sensitive as NO measurement by means of oxyhemoglobin assay. The FIA method is seven times more sensitive than HPLC methods, and its design is significantly simpler. Compared to the traditional Griess method, its sensitivity is higher by a factor of 500. With its high sensitivity, high reproducibility, and its unsurpassed low susceptibility to interference, this method of analysis provides a means of reliably determining nitrite concentration as a marker of NO formation in various matrices. Therefore, it can be a valuable instrument in experimental and clinical studies to determine the physiologic and pathophysiologic relevance of NO.