[Techniques for measurement of nitric oxide in biological systems: principles and practice]

Despite being small and simple in structure the nitric oxide free radical (NO.) is now proving to be of vital physiological significance, and it has been shown to play important roles in complex processes such as vasodilatation, inflammation, thrombosis, immunity and neurotransmission. To conduct meaningful research into the role of NO., it is necessary to accurately determine its concentration. Its direct and quantitative measurement, however, has been little discussed inspite of the abundance of studies on this compound. Generally most authors refer to indirect qualitative measurements, such as employment of NO-synthase inhibitors, measurement of cGMP or citrulline, and the detection of NO.-induced physiological effects such as vascular relaxation. The primary difficulties in the direct measurement of NO stem from its short lifetime and very low concentrations. Notwithstanding these problems, several quantitative methods for measuring NO. have been established. The most commonly used techniques are as follows: 1) UV-visible spectrophotometry of the diazotization product of the nitrite, NO-hemoglobin or methemoglobin, 2) fluorometry of the fluorescent product of the nitrite, 3) detection of chemiluminescence by its reaction with ozone or luminol/H2O2, 4) amperometric microelectrode assay, and 5) electron spin resonance spectrometry. All the aforementioned techniques have certain limitations that should be considered carefully prior to each application.