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

1. Introduction

Chemiluminescence is defined as the emission of light as result of a chemical reaction. The reactants or intermediates are chemically activated via oxidation into an electronically excited state and alternatively release light by two distinct mechanisms, which are defined as direct and indirect chemiluminescence (Figure 1). In direct chemiluminescence, the chemiluminescent molecules (A) are oxidized to an unstable excited intermediates (C*) that then return to the ground state (C) by releasing energy in form of photons. In indirect chemiluminescence, instead of directly decaying, the excited intermediates (D*) transfer the energy through an optical process to the surrounding fluorophores (E), which become then excited (E*) and release energy by light emission. This phenomenon is called chemiluminescence resonance energy transfer of light.

Ozone-based chemiluminescence detection (CLD) for nitric oxide (NO) species is well recognized as a highly sensitive and specific approach to quantify gaseous NO. The detector quantifies the light produced by the reaction of NO with ozone in the gas phase [1]. The reaction produces excited nitrogen dioxide (NO₂^*) (Reaction 1), which then emits photons when it returns to the ground state (Reaction 2). The emitted light is then amplified by a photomultiplier tube and detected.

Typically, NO is generated in a reaction chamber purged with an inert carrier gas (N₂ or Argon), which carries the generated NO along the tubing connecting the chamber to the CLD (which often is referred to as an NO analyzer). The chamber contains a reductive or oxidative solution for acidic or neutral conditions and with/without high temperatures (e.g., 60 degrees). The settings of the reaction chamber depend on the type of analyte and the type of NO derivatives that one may want to quantify. After leaving the reaction chamber, the carrier gas together with NO is then purged into a “NaOH trap”, consisting of a solution of NaOH (1 N), which prevents high-temperature acid vapor to enter and damage the NO analyzer (Figure 2) [2,3].

2. NO and its biologically relevant derivatives

^•NO, is a moderately-reactive uncharged free radical, which attracted lots of attention in late 1980s for its biological properties. It was found to be the endothelium-derived relaxing factor (EDRF), which lifted the veil of the mystery how the endothelial cells are involved in the vasodilatory effect of smooth muscle cells in the vasculature [3,4,5]. The nature of EDRF as NO was indeed revealed by using CLD [3,6]. The Nobel Prize in Physiology or Medicine for 1998 was awarded jointly to Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad for their discoveries concerning “nitric oxide as a signalling molecule in the cardiovascular system”.

By reacting with other radicals and molecules produced in biological environment, ^•NO leads to formation of reactive nitrogen species and more stable metabolites like nitrite and nitrate, which are on turn involved in the oxidation or nitrosation of biomolecules and have biological effects on their own [7,8]. These downstream species include dinitrogen trioxide (N₂O₃), nitrosothiols and nitrosamines (often abbreviated as RSNO and RNNO respectively), intracellular dinitrosyl iron complexes (DNICs), nitroxyl (HNO), ^•NO₂, peroxynitrite (ONOO⁻), nitrite, and nitrate (Figure 3, adapted from Möller et al.) [9]. Together with reactive oxygen and sulfur species are part of the reactive species interactome [10].

N₂O₃, is an intermediate in the autoxidation of ^•NO, and is derived from the reaction of ^•NO with ^•NO₂ (Reaction 3 and 4). N₂O₃ can be then hydrolyzed to two molecules of nitrite or rapidly nitrosated thiols and amines leading to RSNO, RNNO and nitrite (Reaction 5 and 6) [8,11,12,13,14] .

Instead of direct nitrosation of thiols by N₂O₃, RSNO was also proposed to being formed by reacting RS^- with ^•NO₂ to generate RS^•, which then reacts with ^•NO to obtain RSNO (Reaction 7 and 8) [15].

In addition, ^•NO can target also Fe-Heme and bind to iron from or partially from the chelatable iron pool together with ligands such as glutathione to form DNICs [16].

NO⁻ is formed by one electron reduction of ^•NO, which is found only as protonated form, HNO [9]. HNO can react with oxygen leading to formation of ONOO⁻, but it has low relevance under biological conditions due to relatively slow reaction rate (Reaction 9) [17,18].

ONOO⁻ is formed also by the reaction between ^•NO and O₂^•− (Reaction 10), which can undergo one or two electrons reduction yielding ^•NO₂ and NO₂⁻ respectively [19,20]. ONOO⁻ can be also isomerized to NO₃⁻ in the presence of metmyoglobin or methemoglobin as catalysts (Reaction 11) [21,22].

Nitrate can be derived by the reaction between ^•NO and oxyhemoglobin (Reaction 12) [23]. Under certain conditions such as in the presence of oral bacteria or the enzyme xanthine oxidoreductase, nitrate can be reduced back to nitrite, which is an important source of nitrite for further reduction to ^•NO in the body [24,25,26,27,28,29].

Nitrite can be reduced to ^•NO by proteins such as deoxyhemoglobin, deoxymyoglobin and other globins under hypoxic conditions and these mechanisms contribute to many processes such as vasodilation, neurotransmission, immune response and other physiological signallings (Reaction 13) [30,31].

NO was also described to react with deoxyhemoglobin to form nitosylhemoglobin, where NO forms a complex with the iron heme. This was described as a transporter of NO bioactivity and as an intermediate for the formation of s-nitrosohemoglobin as well as an intermediate formed during nitrite reduction into NO [30,32,33,34].

3. Measurement of NO metabolites by chemiluminescence

The NO metabolites need to be converted back to NO in the reaction chamber by reduction (nitrate, nitrite, RSNO, RNNO) or oxidation reactions (NO-Heme or DNIC).

3.1. Reduction of nitrite, RSNO and RNNO by tri-iodide

The tri-iodide-based reductive reagent consists of potassium iodide (KI) and iodine (I₂) in glacial acetic acid. This mixture is very widely used for reducing nitrite to NO in biological samples (Reaction 14) [2,35]. In addition to nitrite, tri-iodide assay can also be applied for the detection of other biological NO metabolites such as nitrosated (S- or N-nitroso) products (RXNO) (Reaction 15 to 17). Sample preparation for detection of S-nitrosated products (RSNO) requires a pre-treatment of the samples with a stabilization solution containing N-ethylmaleimide (NEM) that prevents artificial S-nitrosation of free thiols. Treatments with NEM have some drawbacks as NEM is often contaminated by nitrite and the contamination may vary from one lot number to another. It is therefore necessary to check contamination before adding NEM to the samples.

3.2. Measurement of nitrate by vanadium chloride

In comparison to nitrite and other species, nitrate is present as much higher concentration in biological specimens and makes the major part of total NO species. To be reduced it requires a stronger reductive reagent as compared to triiodide or an enzymatic reaction catalyzed by a bacterial nitrate reductase. Chemical reduction of nitrate is carried out by vanadium chloride (VCl₃) prepared in hydrochloric acid (HCl) in a final concentration of 0.1 mol/L of VCl₃ in 2 mol/L HCl (Reaction 18) [36,37]. It is worth mentioning that VCl₃/HCl reagent will not just reduce nitrate but also all those metabolites able to be reduced by the tri-iodide method. To obtain the accurate nitrate level, therefore, the results from the tri-iodide assay need to be subtracted from the observed signals in VCl₃/HCl method.

Some common reductive and oxidative solutions for the detection of NO metabolites have been listed in Table 1.

4. Multi-level analytical approaches for comprehensive analysis of NO metabolites

4.1. Chemiluminescence coupled with chromatography or mass spectrometry (MS)

CLD, compared to other optical techniques, needs no external light source, has low background signal and, therefore, has a high signal-to-noise ratio and thus is very sensitive [43,44]. For continuously monitoring gaseous pollutants in the air, a chemiluminescence nitrogen detector was developed in 1970 based on the reaction of NO with ozone [1]. Coupling CLD with analytical separative techniques like gas chromatography (GC) or high-performance liquid chromatography (HPLC) allow to combine such a highly sensitive detection technique with powerful separation methods. The selective elution of the chromatography column can separate the target analytes from unwanted interferants or impurities in the sample matrix, which improves the accuracy of CLD and the sensitivity even further [45,46].

4.1.1. Gas chromatography

The chemiluminescence detector for NO species coupled with GC was applied for the detection of atmospheric NO, ammonia, amines, and some nitrogen-containing compounds [1,47,48]. A well-established GC-CLD system requires attention for many critical aspects on both GC and CLD. For GC, the sample injection (split/splitless, which decides a certain ratio of sample entering the GC system), the column (such as type of stationary phase, film thickness, column inner diameter, and length of the column), GC-CLD interface (with a transfer line where analytes travel from GC to the CLD) [48].

Since GC is an analytical technique for the quantitative analysis of volatile compounds, the volatility of the analytes, therefore, is important for their vaporization to enter the gaseous mobile phase and then be transported for further separation on the column. The introduction of the sample in GC might also lead to the failure of the chemiluminescence reaction of NO with ozone. For samples with liquid matrices (like waste water, plasma or biological tissues), the injected samples need to be introduced at a proper flow rate for the combustion at the injection port of GC, which eliminates the risk of water reaching CLD [48]. For higher limits of detection, CLD was also coupled to two-dimensional gas chromatography (GCxGC), with orthogonality for the separation between two applied GC columns, for quantitative analysis of nitrogen-containing compounds in microalgae-based bio-oils, food samples, or urban aerosol samples [49,50,51].

In GC-CLD, GC introduces the chance for further separation on the GC column, however, also brings the requirement for the volatility of the samples. For those complex samples, especially biological tissues or cells, the development of sample preparation method and instrumental parameters as mentioned before for GC-CLD might be time-consuming and costly, which requires more careful consideration and more prudent handling.

4.1.2. Liquid chromatography

Instead of GC, attempts also have been made to couple the CLD for NO species with HPLC, which allows the direct introduction of non-volatile samples in the liquid matrices without vaporization [45,52]. HPLC-CLD was preliminarily applied in the analysis of nitrated polycyclic aromatic hydrocarbons, environmental combustion pollutants, which was reported to encounter the pressure variation issue in the reaction chamber. Incomplete conversion of the mobile phase to the gaseous state before the chemiluminescence reaction leads to reducing the sensitivity, which becomes worse as increasing aqueous content in the mobile phase [53].

To overcome this, a dewatering chamber equipped with drying membrane can be placed between the furnace for the oxidation and the reaction chamber for ozone chemiluminescence, where water is continuously removed from gas streams (including NO) after the oxidation [54]. The modified HPLC-CLD system in the ion chromatography mode was validated for the detection limit of ammonium nitrogen down to 5 ng in wastewater and also the capability of profiling nitrate of 80 ng and nitrite of 160 ng in a nitrite-nitrate mixture [54]. The application of HPLC-CLD for the nitrogen-specific detection of commercially synthesized peptides, including crude peptide mixtures, further demonstrated its superiority over simultaneous ultraviolet detection in peptide profiling [45,55]. On the other hand, HPLC-CLD system was also applied for quantifying the nucleotides and nucleosides standard mixture based on their nitrogen content [52].

An automated system for the analysis of nitrite and nitrate was successfully attempted in 1982, which was then commercialized and developed as ENO-10/ENO-20 and ENO-30 NOx analyzer (Eicom Corporation) [56,57,58]. Such a system was constructed to obtain high sensitivity by coupling HPLC with a diazotization reaction method (Griess reaction). In briefing, nitrite and nitrate are firstly separated on the reverse phase HPLC column, followed by flowing through a copper-loaded reduction column (where the nitrate was reduced to nitrite). Two resolved eluents then react with Griess reagent to form the azo dye compound and detected by spectrophotometer [56,58].

4.1.3. Mass spectrometry

Mass spectrometry is a powerful detection technique based on determination of mass-to-charge ratio (m/z) of target ions and has been applied for the qualitative analysis and quantitation of NO-derived species such as nitrite and nitrate by coupling with chromatography as GC-MS or LC-MS [59,60]. Instead of sole MS detection, the CLD was coupled for on-line quantitative analysis to LC-MS or GC-MS. For the sample class with a known formula, the quantitation with CLD can be calibrated with external standard, which means there is no need for preparing primary standards for the detection of each single analyte (known class but with unknown concentration) then enables high-throughput analysis. Hence, the combination of LC-MS/CLD show the advantage of CLD for quantitation in high-throughput analysis and also brings the power of MS in qualification. An example of this application is the analysis of 24 selected nitrogen-containing compounds [61].

The main pitfall of LC-MS/CLD techniques is the tailing of the peak due to clogging of the splitter and the nebulizer, which can be overcome by the adjustment of flow rate, tubing length, and the replacement by a variable flow splitter [61]. Nevertheless, LC-MS/CLD still provides the chance for a relatively comprehensive view of both quantitative and qualitative analysis. LC-MS/CLD was applied for the identification and quantitation of in vivo metabolites in complex biological matrixes such as bile, urine and plasma [62].

In GC-MS/CLD, a T-splitter was placed after the trapping chamber containing NaOH solution, which splits the NO generated by chemiluminescence reaction into CLD and mass spectrometer [63]. With GC-MS/CLD, total NO can be quantified routinely with the detection of CLD, furthermore, the ¹⁴NO and isotope labelled ¹⁵NO can be differentiated according to m/z by MS. As an example, an application of this technique is the measurement of nitrite reductase activity in a macrophage cell line lysate. J774.2 macrophage cell lysate was treated with ¹⁵NO₃⁻, which was then reduced to ¹⁵NO₂⁻ by nitrate reductase activity. Both original ¹⁴NO₂⁻ and produced ¹⁵NO₂⁻ were reduced with chemiluminescence assay to ¹⁴NO and ¹⁵NO respectively, followed by MS analysis to distinguish them. In this case, GC-MS/CLD holds the detection limit of NO-related products around 10 nM in a 100-μL sample, which was demonstrated to be beneficial for the study of nitrate reductase activity or related enzymatic pathways [63].

4.2. Coupled with microdialysis

Microdialysis was developed in 1972 based on the concept of collecting samples from small interstitial tissue with a dialysis bag, which then dramatically improved in collecting efficiency by changing the surface area of the dialysis membrane [64]. A special microdialysis probe with three-layer membrane was designed to collect NO in vivo from blood or brain tissues of rats and rabbits, which is then coupled with chemiluminescence-based detection. The integration of such microdialysis sampling system with CLD was proposed for the real-time monitoring of changes in NO concentration in vivo, which provide hints to trace variations in physiological states such as body temperature and evaluate the impact of NO donors on the concentration of NO in blood and tissues [65].

4.3. Coupled with flow injection analysis

Flow injection analysis (FIA) involves the injection of samples into a continuous carrier flow, a liquid stream, which then is mixed with other reagent flows for the reaction before reaching the detector [66]. Aqueous ammonia, nitrite and nitrate were quantified by using a flow injection system equipped with ozone-based NO chemiluminescence detector [67,68]. In this system, an aqueous sample stream was mixed with various reducing reagents (KI for nitrite only and titanium chloride for total nitrate and nitrite) continuously. Moreover, high-temperature combustion was preceded before the generated NO reached the detector to remove aqueous content. The detection limits of 10 nM for nitrite and nitrate were demonstrated by the performance study with standards. Another FIA/chemiluminescence system with and without pre-selective membrane was constructed for the monitoring of peroxynitrite in biological samples based on peroxynitrite-induced luminol chemiluminescence [69]. With this setup, detection limits were 10 and 100 pM for the calibrations with and without membrane respectively, which was proposed to be beneficial for in vivo monitoring of peroxynitrite as combined with microdialysis. A FIA/HPLC system was applied for reproducible determination of plasma nitrite level based on Griess reaction, which enables high-throughput measurements as clinical routine with a detection limit of 10 nM [70]. It helps indicating the correction of plasma nitrite level with endothelial dysfunction and cardiovascular risk factors (such as elevated blood pressure, hypercholesterolemia and etc.).

5. Advantages and drawbacks of chemiluminescence for measuring NO species

5.1. Advantages

Chemiluminescence is currently the most used technique for the quantification of NO [71] because of its high sensitivity, since it allows to detect pM concentration of NO [72], its selectivity, a wide dynamic linear range usually from 0.5 ppb to 5600 ppm NO [71], its ability for real-time monitoring of NO [73], and its commercial availability.

It is important to underline that the ozone-based chemiluminescence can be used not only for the detection of NO, but also for nitrite, nitrate, S-nitrosothiols, nitrosyl-metal complex and N-nitrosamines [2,33,38,74,75,76,77,78].

Moreover, CLD can be easily coupled with other analytical techniques like GC or LC for better accuracy and sensitivity in the quantification of respectively volatile and non-volatile molecules [1,45], or with LC-MS and GC-MS which allow the differentiation and detection of labelled ¹⁵NO according to the m/z ratio [63].

5.2. Pitfalls

As mentioned before, the ozone-based chemiluminescence detector can detect pM concentration of nitrite, therefore it is important to avoid contamination coming from the water used for the preparation of reagents, standards, samples and cleaning procedures. For this reason, every step that involves the use of water, must be done with distilled water, filtered by a Milli-Q system which was demonstrated to contain the lowest level of nitrite among the other sources of water [79]. Contaminations is not the only reason why the samples preparation is a critical step. In fact, since NO has a short half-life and nitrite in the blood rapidly reacts with oxyhemoglobin to form nitrate, it is essential that the sample preparation is carried on in conditions that allow to preserve the endogenous NO metabolites. This could require working quickly and many steps during both sample collecting and processing [80]. Regarding the sensitivity of the ozone-based chemiluminescence, it is necessary to mention that different machines have different sensitivities, therefore for comparing data, the measurements need to be done with the same machine [79]. In fact, the reproducibility and sensitivity of the measurement is affected from the ozone gas stream, which is difficult to keep it stable, and from the carrier gas flow rate [81].

Moreover, the chemiluminescence methods required very specific and relatively expensive equipment, time-consuming detection procedure, a frequent equipment maintenance, as well as the knowledge of the chemical reagents used, the reactions that occur during the measurement and the specific operating system for data analysis [71,72]. Concerning these last points, for example, the measurement of S-nitrosothiols, which are released slower than nitrite in tri-iodide, could lead to hard data analysis because of a large width of the peaks [79].

Taking into account the advantages and pitfalls of this method, if the proper precautions are taken to avoid contamination, interference in measurement and errors in data analysis, ozone-based chemiluminescence can be considered one of the best techniques for the determination of NO, mainly due to its high sensitivity, selectivity and possibility of coupling with other analytical techniques.

6. Alternatives for the detection of nitric oxide species

Chemiluminescence detection is one of the most widely used techniques for the determination of NO and NO derivatives, but undoubtedly, many other methods are currently in use. In this section we give a quick overview of the alternatives that can be used, which are also summarized in Table 2.

6.1. Electrochemical sensors

The detection of NO by using electrochemical sensors exploits the phenomena of electrooxidation [82,83] or electroreduction which occur on a metal surface working as an electrode. This method allows a direct NO measurement with high sensitivity and selectivity [82]. One example of electrochemical sensor is the ion-selective electrode (ISE) that can be used specifically for nitrate detection (NO₃⁻ - ISE) [84].

6.2. Fluorescence

Fluorescent probes allow an indirect detection of NO via the formation of a fluorescent molecule. The most used fluorescent agents are the 2,3-diaminonaphthalene (DAN) which reacts with nitrite under acidic conditions to produce the fluorescent deprotonated naphthotriazol [85], and the diaminofluoresceins (DAFs) which of NO can be oxidize forming a fluorescent derivative triazolofluorescein [86,87].

6.3. Electron paramagnetic resonance (EPR)

EPR is a specific method to indirectly detect NO formation in cells or tissues using spin traps like Hb, nitronylnitroxides and iron-dithiocarbamate complex [88,89,90]. Moreover, NO-derived produced in biological system like nitrosylhemoglobin (HbNO) and dinitrosyl iron complex can be only defined by EPR spectrum.

6.4. Mass spectrometry

A membrane inlet mass spectrometry (MIMS) was used for measuring NO in aqueous solution (detection limit of 10 nM and linear response to 50 μM). This technique uses a semipermeable membrane that allow to introduce selectively small molecules like NO into the MS [91].

6.5. UV-visible spectrophotometry for NO determination

Spectrometric detection of NO is based on the change of absorption spectrum when oxyhemoglobin is oxidized to methemoglobin in the presence of NO in aqueous solution [92].

6.6. Griess assay

Griess assay is a colorimetric method used for the determination of nitrite. For the nitrate, a reduction from nitrate to nitrite is needed before the assay. This method is based on the formation of a red-violet colored azo compound (λ_max ≈ 540 nm) by the reaction of nitrite with the amino group of sulfanilic acid to form a diazonium cation, which then couples to α-naphthylamine [93].

Table 2. Main techniques applied for the determination of NO and NO derivatives.

Method	Common applications	Applications	References
Electrochemical sensors	Direct NO detection via electrooxidation or electroreduction;	Real-time NO quantification in biological system; NO detection in tissues and cells	[82,83]
Fluorescence	Indirect detection of NO via formation of a fluorescent molecule	Detection of NO in cells and tissues	[85,86,87]
Electron paramagnetic resonance (EPR)	Indirect detection of NO; Direct detection of HbNO and dinitrosyl iron complex	NO and HbNO detection in cells and tissues	[88,89,90]
Mass spectrometry	Detection of NO via multiple ion detection (MID)	NO detection in aqueous solution	[91]
UV-visible spectrophotometry	Indirect detection of NO via oxyhemoglobin oxidation	NO formation in cells and tissues	[92,94]
Griess assay	Determination of nitrite and nitrate via formation of an azo dye in acidic condition	Determination of nitrite level in biological system	[93,95]

Table 2. Main techniques applied for the determination of NO and NO derivatives.

Method	Common applications	Applications	References
Electrochemical sensors	Direct NO detection via electrooxidation or electroreduction;	Real-time NO quantification in biological system; NO detection in tissues and cells	[82,83]
Fluorescence	Indirect detection of NO via formation of a fluorescent molecule	Detection of NO in cells and tissues	[85,86,87]
Electron paramagnetic resonance (EPR)	Indirect detection of NO; Direct detection of HbNO and dinitrosyl iron complex	NO and HbNO detection in cells and tissues	[88,89,90]
Mass spectrometry	Detection of NO via multiple ion detection (MID)	NO detection in aqueous solution	[91]
UV-visible spectrophotometry	Indirect detection of NO via oxyhemoglobin oxidation	NO formation in cells and tissues	[92,94]
Griess assay	Determination of nitrite and nitrate via formation of an azo dye in acidic condition	Determination of nitrite level in biological system	[93,95]

7. Practical considerations

7.1. General concerns about choosing chemiluminescence as a detection tool for NO species

Many techniques are capable of detecting NO species in the biological samples. However, it is necessary to choose the technique depends on the target. For example, as the main players in the nitrate-nitrite-NO/nitrosothiols biological pathway, nitrite and nitrate can be measured by the Griess assay diazotiation of nitrite (nitrate requires reduction to nitrite first) or by ozone-based chemiluminescence detection of NO [96,97,98,99]. The Griess assay with colorimetric determination of the diazotization product is simple, fast and also affordable (chemicals and UV-vis spectrophotometer are relatively low costs as compared to other instruments). As mentioned before in chapter 5, ozone-based chemiluminescence depends on the availability of the NO analyzer for the direct measurement of gaseous NO or those derived from the reduction of nitrite and nitrate. The detection procedures by using ozone-based chemiluminescence also requires more experience to avoid contamination and for carrying out the reactions, but it is superior for sensitivity.

As reported by many researchers, Griess assay has a limit of detection around 1 μM [99,100,101,102]. In contrast, ozone-based chemiluminescence is able to quantify NO at the range of nM, which is much more sensitive as compared with the Griess assay [103]. Moreover, the direct quantification allows ozone-based chemiluminescence analyzer to measure even exhaled NO [104,105].

The choice of the technique needs to be based on the detection range and the sample type. If the target sample is complex, coupled technique such as HPLC-CLD or GC-CLD can be one of the solutions instead of sole chemiluminescence detection. As mentioned above, such coupling system with chromatography provides additional separation before chemiluminescence detection, which reduces the matrix effect and the chance of under-or over-estimation. Furthermore, MS can bring highly accurate qualitative analysis even possibility of isotopic tracing with ¹⁵N-labelled substrate for studies of enzymatic pathway and metabolomics. The GC/MS-CLD approach developed by Cornelius et al. provided the detection limits from 10 to 30 pmol for ¹⁴NO₂⁻ and ¹⁵NO₂⁻ as well as the capability of studying enzymatic reduction of nitrate reductase in murine macrophage J774 cell lysates.

7.2. Technical details on the procedure

7.2.1. Carefully choose calibrants and a range of calibration concentrations

As all semiquantitative and quantitative analytical techniques the calibrants should be prepared in a matrix as close as possible as the samples, which means the interference from other compounds in the sample also needs to be included in the calibrants. The classes of target analyte (nitrite, nitrate, etc.) and their estimated level in the samples need to be confirmed to define the calibration range. The detected concentrations of samples need to be located in the linear range of the calibration, otherwise, samples need to be diluted.

7.2.2. Interference from other components in the matrix, non-objective reaction

Chemiluminescence depends on the reaction of NO with ozone. However, some species such as ethylenic hydrocarbons, sulfur compounds, and carbonyls can react with ozone and produce chemiluminescence signals, which may lead to interference for accurate detection [106]. Some substituted ethylenes can react with ozone and produce strong chemiluminescence signals at higher or lower pressure in the analyzer. Sulfur compounds, such as hydrogen sulfide or dimethyl sulfide, can produce intensive signals once they react with ozone. Carbonyls can be introduced from experiment treatment, such as treatment with CO, which interferes with the detection by reacting with ozone. The disturbance of interferants like ethylenic hydrocarbons and sulfur to NO/ozone reaction can be eliminated by increasing detection wavelength to that > 600 nm (440 to 470 nm for ethylenic hydrocarbons, <400 nm for sulfur). However, even some of the interference can be reduced or overcome by methods such as adjusting the wavelength, pressure of the analyzer, or filtering, the artificial contamination needs to be avoided or carefully evaluated before starting.

7.3. Notes for ozone-based chemiluminescence detectiion in biological samples

Some critical points for technical procedures of ozone-based chemiluminescence in biological samples have been summarized in Table 3.

8. Successful application in different fields

The determination of NO and its metabolites has already been used for a long time in different fields. Already fifty years ago CLD was used for identifying and understanding the composition of air to detect the presence of nitrogen species and air pollution [107]. CLD of nitrogen species NOx in the air to the determine air pollution as well as the composition of exhausted air from motors and cars was one of the first applications of CLD of NO species and is still used until today [108,109]. Furthermore, the concentration of NO and NO_x is measured in lakes, oceans and rivers as well as drinking water to investigate the pollution of nitrate by increasing use of fertilizer in agriculture [110,111,112].

The analysis of NO species in the gas phase is not only used for environmental monitoring but also used for biomedical and clinical research and diagnosis. In 1991 it was shown that endogenous NO is present in exhaled air of different animal species and humans and can be measured by CLD [113]. Following-up studies could find differences in exhaled NO in asthmatic patients compared to healthy individuals [114]. CLD was also applied to detect NO released by vasoactive drugs in exhaled air in pigs [115]. In this interesting a pioneering study the authors concluded that the measurement of exhaled NO could be a possible indicator of pulmonary endothelial dysfunction or hypertension. Measurement of exhaled NO is still used as a reproducible noninvasive indicator for inflammation of the airways [116,117].

These studies mainly focused on the measurement of NO in exhaled air, but the measurement of NO metabolites in plasma, urine, or tissues is used in various medical fields to investigate the role of endogenous NO production or the effect of intake of nitrate in the body [118,119,120,121]. NO produced in the body is immediately metabolized. Thus, the production of NO by NO synthase (NOS) is measured by NO metabolites [122,123]. Nitrite concentration in plasma was proposed as an indicator of endothelial dysfunction [70,124]. Global eNOS KO mice show decreased circulating nitrite and nitrate metabolites [122,123,125,126]. Recently we found that mice lacking eNOS in the endothelial cells show a decrease in circulating nitrite and nitrate in plasma. However, reactivation of eNOS expression only in endothelial cell in global eNOS KO mice do not recover the nitrite levels in plasma of the mice. This indicate that the levels of nitrite in plasma are not only dependent on eNOS activity in the endothelium. Another finding of this study was that also eNOS expressed in RBCs contribute to the circulating nitrite levels.[97]

In addition, the activity of nNOS and iNOS may also contribute to overall NO metabolites [123]. Recently it has been shown that decreased NO and sulfide availability as detected by CLD is a hallmark of COVID-19 [127].

Due to the variability of circulating NO, nitrate and nitrite levels, these metabolites could not be used as a clinical biomarker until now. However, measuring these metabolites with CLD is still considered as an important approach to understand the NO-pathway in health and disease.

Regarding to nitrate and nitrite levels in the circulation, it is important to note that a main factor of modulating those levels is the intake of nitrate by diet. There are multiple studies using CLD for analyzing nitrate and nitrite levels in food and drinking water [128], which is also important due to the fact that a high intake of nitrate has been considered as harmful and cancerogenic due to the conversion of nitrate to nitrite by bacteria and further metabolism into nitrosamine [129,130]. Furthermore, sodium nitrate and potassium nitrate as well as sodium nitrite and potassium nitrite are used in the food industry especially for preservation of meat as additives [131]. It is, therefore, important to control nitrate and nitrite levels to avoid potential health risks [132]. On the other hand, it has been shown that that the supplementation of nitrate lowers the blood pressure in human and is cardioprotective and shows the importance of the intake of nitrate by dietary [120,121,133].

NO and NO metabolites are also determined in this field using chemiluminescence among other method [134,135]. NO signaling is known to play an important role in plant biology and is involved in multiple processes [136]. Similar to animals and humans, plants can use NADH and nitrite to generate NO but do not express nitric oxide synthase. The measurement of these molecules is not just important for a better insight in the signaling of plants but can also provide information about the accumulation of nitrate in leaves and vegetables of plants due to the use of fertilizer.

To conclude, chemiluminescence detection of nitric oxide is a widely used technique in multiple fields and is continuously improved and adapted to new possible usage.

9. Summary and conclusion

The observation that NO reacts with ozone, leading to the emission of light was first described and used for quantifying NO by amplifying the signal in a photomultiplier. Chemiluminescence-based NO detection allowed researchers to confirm the nature of the EDRF to be NO and afterwards to investigate NO and its derived species in biological samples in a specific, precise and direct way.

The high sensitivity and wide dynamic range make chemiluminescence detection an indispensable tool for quantitative analysis. To increase applicability and specificity of detection for nitrite and nitrate or other metabolites many scientists have combine the advantages of chemiluminescence detection with various analytical separative approaches, such as chromatography, mass spectrometry, microdialysis, and flow injection analysis. These multilevel approaches enable researchers to obtain both quantitative and qualitative profiles including isotope tracing of ¹⁴NO and ¹⁵NO for enzymatic activity or in vivo monitoring of important NO-derived species such as nitrite and peroxynitrite.

Chemiluminescence-based NO detection has been applied in various fields, including the investigation of environmental pollution related to NO_x, biomedical and clinical research on endogenous NO, the study of NO metabolites as indicators of endothelial dysfunction. It is important to note that ozone-based CLD is a highly sensitive method for the detection of NO species, but needs to be operated under controlled conditions. In fact, the system needs to be carefully calibrated, sample preparation and analysis need to be performed taking into consideration sample composition and target analytes, presence of contaminants and molecules that could interfere with the reactions in the chamber or in the detector. Other important factor to consider are sample collection and storage, and technical issues (system leakage, baseline, contamination from air, exhaustion of the ozone in the chamber etc.).

In conclusion, chemiluminescence-based NO determination is a very useful and versatile tool for the quantitative analysis of NO and its metabolites in various samples and can be applied in many fields. The power of chemiluminescence can be further enhanced when coupled with other analytical separative techniques, which deliver a more vivid picture of the status of NO metabolism in various samples. These techniques coupled with cell-specific genetic manipulation of NOS enzyme and/or pharmacological/dietetic intervention will reveal how NO metabolism is regulated in vivo.