Chemiluminescence quantification of NO and its derivatives in liquid samples

Nitric oxide (NO) is a ubiquitous gas with potent biological effects, including vasodilation, neuronal signaling, and antimicrobial activity. NO is a free radical and can readily react with other molecules, in particular, iron centers and oxygen. At physiological concentrations in aqueous solutions, even in the presence of oxygen, NO is reasonably stable. Under these conditions, NO is oxidized almost exclusively to nitrite (NO2-). In cell lysates and tissue extracts with iron-containing proteins, however, NO is postulated to have a very short half-life, with the major oxidation product being nitrate (NO3-). In mammalian cells, NO is generated via the action of the NO synthases (NOS), of which there are three known isotypes. NO can also be generated from the chemical decomposition of S-nitrosothiols, and there is some indication that naturally occurring S-nitrosothiols, such as S-nitrosoalbumin, may be natural reservoirs of NO in vivo. Here we describe a methodology to measure variations in NO in liquid samples using chemiluminescence. The protocols described allow us to distinguish between various products of NO chemistry, thus providing a sensitive method of measurement of NO concentration within a sample. They also allow us to distinguish between the various products that may be generated when NO reacts with molecules in complex biological samples such as cell lysates and supernatants.