Methods to detect nitric oxide and its metabolites in biological samples

Abstract

Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. NO is involved in many physiological processes including regulation of blood pressure, immune response, and neural communication. Therefore its accurate detection and quantification are critical to understanding health and disease. Due to the extremely short physiological half-life of this gaseous free radical, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of NO metabolites in biological samples provides valuable information with regard to in vivo NO production, bioavailability, and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The methods described in this review is not an exhaustive or comprehensive discussion of all methods available for the detection of NO but rather a description of the most commonly used and practical methods which allow accurate and sensitive quantification of NO products/metabolites in multiple biological matrices under normal physiological conditions.

Figure 1. Multiple pathways for the formation of 3-nitrotyrosine

3-nitrotyrosine may be generated by peroxynitrite (ONOO-), NO₂ (from autoxidation of NO), nitroxyl (HNO) in the presence of oxygen, NO₂ produced by heme or hemoprotein catalyzed, H₂O₂-dependent oxidation of nitrite $({\mathrm{NO}}_2^{-})$ , nitryl chloride (ClNO₂) produced by the interaction between $({\mathrm{NO}}_2^{-})$ and HOCl and NO2 generated from acidified ${\mathrm{NO}}_2^{-}$.

Figure 2. The Griess Reaction

The nitrosating agent dintrogen trioxide (N₂O₃) generated from acidified nitrite (or from the autoxidation of NO) reacts with sulfanilamide to yield a diazonium derivative. This reactive intermediate will interact with N-1-naphthylethelene diamine to yield a colored diazo product that absorbs strongly at 540 nm.

Figure 3

(Top) Schematic of ENO-20 method of detection for nitrite and nitrate. (bottom) Standard chromotogram of 10 pmol nitrite and nitrate injected into ENO-20 (100μl of 100nM solution of nitrite and nitrate). Sensitivity of 1nM for each anion with 100μl injection volume. No interference with protein or colored species.

Figure 4. Fluorometric Detection of Nitrite or NO Using Diaminonaphthalene

The nitrosating agent N₂O₃ generated from acidified nitrite or from the autoxidation of NO reacts with diaminonaphthlene (DAN) to yield the highly fluorescent product naphthotriazole (NAT).

Figure 5. Fluorometric Detection of NO Using Diaminofluroscein-2 diacetate

DAF-2 diacetate diffuses into cells where esterases hydrolyze the diacetate residues thereby trapping DAF-2 within the intracellular space. NO-derived nitrosating agents (NO_x) nitrosate DAF-2 to yield its highly fluorescent product DAF-2 triazole.

Figure 6. Detection of S-nitrosothiols (RSNO) by the Saville Reaction

Liberation of the nitrosating agent nitrosonium (NO⁺) by interaction of RSNO with Hg⁺² in the presence of the Griess reagents at pH 7.4 results in the formation of a diazo product that absorbs strongly at 540 nm.

Figure 7. Fluorometric Detection of RSNO

Liberation of NO⁺ by interaction of RSNO with Hg⁺² in the presence of the diaminonaphthlene (DAN) at pH 7.4 results in the formation of the fluorometric derivative naphthotriazole (NAT).

Figure 8

(Panel A) Chemiluminescence detection of nitrite, RSNO, RNNO in reductive denitrosation assay by sample preincubation with group specific chemical reagents. Subtraction of peak areas allow detection of nitrite and RSNOs. (Panel B) Chemiluminescent detection of nitrosyl heme species using oxidative denitrosation solution of ferricyanide. This method is specific for NO-heme products with no cross reactivity with RSNOs (GSNO or SNO-albumin), or RNNO (NO-pyrrolidine and N-nitroso-albumin).