Measurement of Nitrate and Nitrite in Extracellular Fluids: A Window to Systemic Nitric Oxide Metabolism

Abstract There is a growing body of both clinical and experimental data that demonstrates that activation of the immune system, whether locally or systemically, is associated with an increased production of nitric oxide (NO) as measured by increases in plasma and/or urinary levels of nitrate (NO − 3 ) and nitrite (NO − 2 ). Because NO may mediate some of the tissue injury and dysfunction observed in certain pathophysiologic conditions associated with immune system activation (e.g., arthritis, colitis, sepsis) a simple, sensitive, and inexpensive method to measure NO − 3 and NO − 2 levels in the plasma and urine may provide an important tool for monitoring NO production and possibly disease activity in vivo . Therefore, the objective of this article is to outline the methods used in our laboratory to quantify NO − 3 and NO − 2 in extracellular fluids such as plasma, urine, and/or lymph. These same methods may also be used for other extracellular fluids including saliva, tears, sweat, and possibly bile. We describe two methods that require that NO − 3 first be reduced to NO − 2 and then NO − 2 determined spectrophotometrically by the Griess reaction. We have found that the enzymatic reduction of NO − 3 to NO − 2 using either a commercially available preparation of nitrate reductase (Aspergillus nitrate reductase; Boehringer-Mannheim) or the nitrate reductase associated with Escherichia coli which have been grown anaerobically in the presence of large amounts of nitrate to be the most satisfactory methods. Both methods are relatively inexpensive, simple, and fairly sensitive. Studies from our laboratory have determined that heparin should be removed from plasma (or lymph) samples prior to addition of the Griess reagent because we have found that acidification of dilute heparinized plasma may result in substantial precipitation of the sample.