Measurement of plasma nitrite by chemiluminescence

Studies have demonstrated that plasma nitrite (N(O-)(2)) reflects endothelial nitric oxide (NO) production. In addition, N(O-)(2) has been shown to have biological activities associated with its reduction to NO in blood and tissues. Therefore, determination of plasma N(O-)(2) has been proposed as a prognostic marker for cardiovascular diseases. Typical concentrations of N(O-)(2) in the plasma are in the nanomolar range and determination of this N(O-)(2) poses a challenge in terms of both sensitivity and specificity. Thus, a highly sensitive, chemiluminescence method that is based on the reduction of N(O-)(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 N(O-)(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 N(O-)(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 N(O-)(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 N(O-)(2). Using the present method, plasma N(O-)(2) in fasting human subjects has been determined to be in the range of 56-210 nM (mean +/- SD = 110 +/- 36 nM; n = 8).