Validation of a method to directly and specifically measure nitrite in biological matrices

The bioactivity of nitric oxide (NO) is influenced by chemical species generated through reactions with proteins, lipids, metals, and its conversion to nitrite and nitrate. A better understanding of the functions played by each of these species could be achieved by developing selective assays able of distinguishing nitrite from other NO species. Nagababu and Rifkind developed a method using acetic and ascorbic acids to measure nitrite-derived NO in plasma. Here, we adapted, optimized, and validated this method to assay nitrite in tissues. The method yielded linear measurements over 1-300 pmol of nitrite and was validated for tissue preserved in a nitrite stabilization solution composed of potassium ferricyanide, N-ethylmaleimide and NP-40. When samples were processed with chloroform, but not with methanol, ethanol, acetic acid or acetonitrile, reliable and reproducible nitrite measurements in up to 20 sample replicates were obtained. The method's accuracy in tissue was ≈ 90% and in plasma 99.9%. In mice, during basal conditions, brain, heart, lung, liver, spleen and kidney cortex had similar nitrite levels. In addition, nitrite tissue levels were similar regardless of when organs were processed: immediately upon collection, kept in stabilization solution for later analysis or frozen and later processed. After ip nitrite injections, rapidly changing nitrite concentrations in tissue and plasma could be measured and were shown to change in significantly distinct patterns. This validated method could be valuable for investigations of nitrite biology in conditions such as sickle cell disease, cardiovascular disease, and diabetes, where nitrite is thought to play a role.