Nitrite/ Nitrate Determination Assay

Nitric oxide is an important molecular messenger in the vascular and nervous systems. It has multiple physiological roles, such as vasorelaxation or neuronal signaling, but it also has other complex pathophysiological effects. It is synthesized by the three isoforms of the nitric oxide synthases (eNOS, nNOS and iNOS) from L-arginine in the endothelial cells, neurons, macrophages, etc. and in biological systems it is decomposed to nitrite and nitrate.

The overproduction of nitric oxide may lead to oxidative and nitrosative stress. It has been demonstrated that they enhance the development of a variety of diseases, as well as the ageing process.

Regarding nitrosative stress, high levels of iNOS have been found in various inflammatory diseases such as arthritis and obesity, and increased levels of NO have been also associated to other cardiovascular diseases.

G-Biosciences’ Nitrate/Nitrite Determination Kit is recommended for the determination of nitrite and nitrate, which is a method for the detection of nitric oxide formation.

The assay described here measures the nitrite and nitrate anions. Firstly, the nitrate is reduced to nitrite catalyzed by the nitrate reductase with cofactors and specific compounds to eliminate interferences.

The detection is based on the final product detection (diazonium compound, λ_max= 540 nm) obtained after nitrite reaction in several steps with sulfanilamide. Nitrite only determinations can then be made in a parallel assay where the samples were not reduced before the colorimetric assay. The nitrate levels are determined by the subtraction of nitrite levels from the total.