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Measurement of Plasma Nitrite by Chemiluminescence

  • Enika Nagababu
  • Joseph M. Rifkind
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 610)

Abstract

Studies have demonstrated that plasma nitrite (\( {\textrm{NO}}_2^ - \)) reflects endothelial nitric oxide (NO) production. In addition, \( {\textrm{NO}}_2^ - \) has been shown to have biological activities associated with its reduction to NO in blood and tissues. Therefore, determination of plasma \( {\textrm{NO}}_2^ - \) has been proposed as a prognostic marker for cardiovascular diseases. Typical concentrations of \( {\textrm{NO}}_2^ - \) in the plasma are in the nanomolar range and determination of this \( {\textrm{NO}}_2^ - \) poses a challenge in terms of both sensitivity and specificity. Thus, a highly sensitive, chemiluminescence method that is based on the reduction of \( {\textrm{NO}}_2^ - \) by potassium iodide and iodine is being used to determine the nitrite in biological fluids. This method has the sensitivity, but also measures other nitric oxide species such as S-nitrosothiols and N-nitrosamines. We, therefore, developed an alternative method based on the reduction of \( {\textrm{NO}}_2^ - \) by ascorbic acid in strongly acidic media. As part of the methodology, glacial acetic acid and ascorbic acid are introduced into the purge vessel of the NO analyzer. Samples containing \( {\textrm{NO}}_2^ - \) are injected into the purge vessel and the chemiluminescence signals generated as a result of the formation of NO are then measured. We find that under these conditions \( {\textrm{NO}}_2^ - \) is stoichiometrically reduced to NO. Other traditional NO-generating species, such as S-nitrosothiols, N-nitrosamines, nitrated lipids, and nitrated proteins, did not interfere in the determination of plasma \( {\textrm{NO}}_2^ - \). Using the present method, plasma \( {\textrm{NO}}_2^ - \) in fasting human subjects has been determined to be in the range of 56–210 nM (mean ± SD = 110 ± 36 nM; n = 8).

Key words

Plasma nitrite nitric oxide endothelial nitric oxide synthase chemiluminescence ascorbic acid 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Enika Nagababu
    • 1
  • Joseph M. Rifkind
    • 1
  1. 1.Molecular Dynamics SectionNational Institute on Aging, National Institutes of HealthBaltimoreUSA

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