Determination of Nitric Oxide and Its Metabolites in Biological Tissues Using Ozone-Based Chemiluminescence Detection: A State-of-the-Art Review

Abstract

Ozone-based chemiluminescence detection (CLD) has been widely applied for determining nitric oxide (^•NO) and its derived species in many different fields, such as environmental monitoring and biomedical research. In humans and animals, CLD has been applied to determine exhaled ^•NO and ^•NO metabolites in plasma and tissues. The main advantages of CLD are high sensitivity and selectivity for quantitative analysis in a wide dynamic range. Combining CLD with analytical separation techniques like chromatography allows for the analytes to be quantified with less disturbance from matrix components or impurities. Sampling techniques like microdialysis and flow injection analysis may be coupled to CLD with the possibility of real-time monitoring of ^•NO. However, details and precautions in experimental practice need to be addressed and clarified to avoid wrong estimations. Therefore, using CLD as a detection tool requires a deep understanding of the sample preparation procedure and chemical reactions used for liberating ^•NO from its derived species. In this review, we discuss the advantages and pitfalls of CLD for determining ^•NO species, list the different applications and combinations with other analytical techniques, and provide general practical notes for sample preparation. These guidelines are designed to assist researchers in comprehending CLD data and in selecting the most appropriate method for measuring ^•NO species.

Keywords: chemiluminescence detection; clinical studies; vascular function; •NO metabolites.

Figure 1

Direct and indirect chemiluminescence. Green: basal state; yellow: excited state. (I) Direct chemiluminescence, A: chemiluminescent molecule, B: oxidant, C*: excited state of intermediate, C: ground state of intermediate. (II) Indirect chemiluminescence, D*: excited state of intermediate, D: ground state of intermediate, E: ground state of fluorophore, E*: excited state of fluorophore.

Figure 2

Apparatus for ozone-based chemiluminescence. A: supply gas, N₂, B: supply gas fine control for purging, C: waste outlet, D: injection port of sample, E: heating circulation, F: cooling circulation, G: NaOH trap, H: gas filter; in the CLD: the emission hv is collected by the I: optical filter and then converted by the J: photomultiplier tube (PMT) to amplify the signal in mV.

Figure 3

The reactions of ^•NO to form its derived species. Nitric oxide (^•NO) can react with O₂^•− to form peroxynitrite (ONOO−), which then can yield nitrate (NO₃⁻) or be reduced to NO₂⁻ and ^•NO₂. NO₃⁻ can be also derived from the reaction between ^•NO and oxyhemoglobin (HbO₂). Nitrosyl (HNO) can be formed by reduction of ^•NO. ^•NO can bind to iron (Fe(II)) to form dinitrosyl iron complexes (DNICs). ^•NO can also react with nitrogen dioxide (^•NO₂), with N₂O₃ as intermediate, to generate nitrite (NO₂⁻) and nitrosated product (RXNO).

Figure 4

Schematic representation of HPLC-CLD with a pyrolysis oven, as described in Ref. [61]. The eluents from an HPLC column were transformed into the gaseous state within the pyrolysis oven. This system showed some sensitivity issue in the presence of water in aqueous matrixes.

Figure 5

Schematic representation of the HPLC-CLD coupled with a high-temperature oven and a dewatering chamber, as described in Ref. [62]. The dewatering chamber, positioned post the high-temperature oven, serves to remove the aqueous content in the sample flow, thereby enhancing the sensitivity of CLD detection.

Figure 6

Schematic representation of LC-MS/CLD, as described in Ref. [67], featuring a flow splitter positioned after LC. The flow splitter divides eluents from LC into two streams, with one stream directed toward CLD for quantitative analysis and the other directed toward MS for qualitative analysis.

Figure 7

Schematic representation of GC-MS/CLD, as described in Ref. [69], featuring a T-splitter positioned after the NaOH solution chamber. The T-splitter divides the eluents into two streams, with one stream directed toward CLD for quantitative analysis and the other directed toward GC oven followed by MS detection.

Figure 8

Schematic representation of FIA-CLD, as described in Ref. [73]. An aqueous sample is mixed with KI or the mixture (KI + TiCl₂) before reaching the reaction coil. After high-temperature combustion in the ^•NO converter, the ^•NO products are detected using CLD.