Preparation of Standards and Measurement of Nitric Oxide, Nitroxyl, and Related Oxidation Products

Abstract

Nitric oxide (NO) is a vasodilator, neurotransmitter, and inflammatory mediator which is unstable at physiologic O2 tensions. NO′s susceptibility to oxidation accounts for its short biological half-life in vivo and greatly complicates its measurement. Measuring NO requires an understanding of the redox reactions and partitioning that determine its survival and distribution. In aqueous solutions NO rapidly partitions to the gas phase and virtually all the NO in the aqueous phase is oxidized to nitrite (NO−2). The survival of NO in oxygenated water is inversely proportional to the PO2. Nitrate (NO−3) is produced mainly in the presence of oxidizing agents, such as the oxyhemoproteins. NO is readily measured by a chemiluminescence assay in which the reaction of NO with ozone yields light in direct proportion to NO levels (detection threshold ≈5 pmol). This assay is relatively specific as few other biological compounds exist as gases and interact with ozone. The chemiluminescence assay can be modified to measure NO−2 by refluxing the sample with an acid and a reducing agent, such as potassium iodide (KI). Reduction of NO−3 to NO requires a stronger reducing agent (vanadium III). Acidification and reduction renders the chemiluminescence assay less specific for NO as not all NO−2 is derived from NO. NO−2 is a ubiquitous contaminant of H2O and is a product of bacterial denitrification enzymes. The threshold for detection of NO−2 by chemiluminescence (5 × 10−11 mol) is lower than that of conventional colorimetric assays using the Griess reagent (threshold≈10−9 mol). The chemiluminescence assay is best calibrated against NO gas (10 ppm) or saturated NO solutions. Saturated solutions of NO (≈2 mM) are prepared by bubbling deoxygenated H2O with 99.99% NO gas using a custom-designed apparatus. Since NO levels in vivo reflect a complex balance of NO synthesis, reduction–oxidation, and partitioning, a single type of assay of only one compartment is rarely adequate to characterize NO metabolism.