Nitric oxide production by endothelial cells: comparison of three methods of quantification

Vascular endothelial cells have been found to produce a relaxant mediator, identified as nitric oxide (NO) and implicated in numerous physiological functions. Subsequently, there has been an intensive search for accurate and specific detection methods to measure biological NO production. In the present study, we compared three approaches to evaluate NO production, based respectively on the Griess reaction (that quantifies nitrites and nitrates after their reduction), on the hemoglobin reaction (that quantifies oxyhemoglobin to methemoglobin transformation by NO), and on the electrochemical NO detection with a porphyrinic micro-probe. Comparison was made both under standard conditions and biological conditions, through calibration curves and measurements of histamine-induced NO production by cultured human endothelial cells and its modulation by L-arginine and N(omega)-monomethyl-L-arginine. We demonstrated that these three methods differ in terms of sensitivity and selectivity. The hemoglobin reaction and nitrate measurements suffer from a lack of specificity. Nitrite determination by the Griess reaction was hardly suitable for kinetic studies but it remains useful for the evaluation of basal NO production. The electrochemical technique, although it does not allow measurement of basal NO production, is the only one to exhibit great sensitivity and specificity and to allow instantaneous and non destructive measurements. This study brings up the potential hazards and pitfalls that may be associated with the various methods.