Nitric oxide (NO) is a diffusible, transient, reactive molecule that has physiological effects in the picomolar-to-micromolar range. Acting through soluble guanylate cyclase activation, NO is an important physiological regulator of the cardiovascular, nervous, and immunological systems. NO is bio-available by two routes. It can be endogenously generated by constitutive or induced enzymes like Nitric Oxide Synthase, or it can be orally ingested as nitrates / nitrites for rapid uptake into circulation and subsequent conversion.
The reactive nature of nitric oxide allows it to act as a cytotoxic factor when released during an immune response by cells such as macrophages. The reactivity also allows NO to be easily converted to a toxic radical that can produce nitrosative damage to cells, organelles and molecules such as DNA. Nitrosylation however can be a regulated post-translational modification in cell signaling. The balance and dynamics of the regulatory/damage facets of NO are major forces in mitochondrial signaling and dysfunction. NO is linked not only to coronary heart disease, endothelial dysfunctions, erectile dysfunction, and neurological disorders, but also diabetes, chronic periodontitis, autism, cancer, and assorted age-related diseases.
With a half-life in vivo of a few seconds or less, the physical properties of Nitric Oxide make it challenging for direct detection methods. However, colorimetric methods can be applied to measure its stable break-down products nitrate (NO3-) and nitrite (NO2-).
ICT’s Nitric Oxide Colorimetric Detection Kit has two assay options. The first option is designed to quantitatively measure endogenous Nitrite. In the second option, Nitrate is converted to Nitrite using Nitrate Reductase and Total Nitric Oxide is measured. To obtain the Nitrate concentration, endogenous Nitrite is subtracted from the Total Nitric Oxide value. The kit has been designed to work in a variety of samples. Sample types validated include: water, buffers, serum, plasma, urine, saliva, and tissue culture medium (TCM). Please read the complete kit insert before performing this assay. Both Nitrate and Nitrite standards are provided to generate standard curves for the assay and all samples should be read off the appropriate standard curve. For
Nitrite detection, samples are mixed with the Color Reagents A and B and incubated at room temperature for 5 minutes. The colored product is read at 540 – 570 nm. The concentration of Nitrite in the sample is calculated, after making a suitable correction for any dilution of the sample, using software available with most plate readers.
Total Nitric Oxide content is measured after the sample is incubated with Nitrate Reductase and NADH. The reductase in combination with NADH reduces Nitrate to Nitrite. After a 20 minute incubation at room temperature, Color Reagents A and B are added and incubated at room temperature for 5 minutes. The colored product is read and calculated as with the Nitrite determination above. The concentration of Nitrate in the sample is calculated by subtracting the measured Nitrite concentration from the Total Nitric Oxide concentration for the sample.
This kit uses Nitrate and Nitrite Standard solutions calibrated to the US National Institute for Science and Technology Standard Reference Materials and ISO/IEC standards. This kit is for research use only. Not for use in diagnostic procedures.
