Nitrite and nitrate are important chemical components in almost all living organisms. In vivo, nitrite and nitrate come from the NO oxidation pathway without excessive uptake of nitrite and nitrate.

Nitrite can react with chromogenic agent producing light red azo-compound. The content of nitrite can be calculated indirectly by measuring the OD value at 550 nm.

The supernatant for chromogenic reaction should not contain sediment, otherwise it will affect the results.

1.    Blank tube: Add a* mL of Double distilled water into a 2 mL EP tube.

Standard tube: Add a* mL of 100 μmol/L Sodium nitrite standard solution into a 2 mL EP tube.

Sample tube: Add a* mL of Sample into a 2 mL EP tube.

(a* = volume of sample = volume of standard = volume of double distilled water.)

(Reference sampling quantity: 0.2~0.4 mL for serum (plasma) and 0.1~0.2 mL for tissue or cells homogenate.)

2.    Add 0.8 mL of Reagent 1 and oscillate fully with vortex mixer.

3.    Add 0.4 mL of Reagent 2 and oscillate fully with vortex mixer.

4.    Stand the tubes at room temperature for 10 min and centrifuge at 2000 g for 10 min. (The supernatant after centrifugation must be clarified, and if there is precipitate, it must be centrifuged again.)

5.    Take 0.8 mL of supernatant into a 2 mL EP tube, add 0.4 mL of Chromogenic agent.

6.    Mix fully and stand the tubes at room temperature for 15 min. Set to zero with double distilled water and measure the OD values of each tube at 550 nm with 0.5 cm diameter cuvette.