Detection and quantification of nitric oxide–derived oxidants in biological systems

Abstract

The free radical nitric oxide (NO^•) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NO^•-derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO^•, and we discuss the approaches currently available for the analysis of NO^•, nitrogen dioxide (NO₂^•), dinitrogen trioxide (N₂O₃), nitroxyl (HNO), and peroxynitrite (ONOO⁻/ONOOH), as well as peroxynitrite-derived hydroxyl (HO^•) and carbonate anion (CO₃^•−) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO₂⁻), nitrate (NO₃⁻), nitrosyl–metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state–of–the–art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NO^•-derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involved.

Introduction

Soon after the discovery of nitric oxide (NO^•) as a physiological mediator in the vascular, nervous, and immune systems, it became evident that this moderately-reactive free radical can give rise to secondary species, many of which are oxidizing, nitrosating, or nitrating agents toward biomolecules (1–4).

The species formed downstream from NO^• (i.e. NO^•-derived oxidants) include nitrogen dioxide (NO₂^•), dinitrogen trioxide (N₂O₃), nitroxyl (HNO), and peroxynitrite (ONOO⁻/ONOOH), as well as hydroxyl (HO^•) and carbonate anion (CO₃^•−) radicals formed from peroxynitrite (Fig. 1). These species are short-lived (half-lives are typically in the millisecond to microsecond range) and are frequently referred to in general as “reactive nitrogen species.” However, this term should be used with caution because in a similar way as “reactive oxygen species,” with which NO^•-derived species are usually grouped, this term may give the inaccurate idea that there exists only one ill-defined species that has a particular type of reactivity and targets all biomolecules (5, 6). In contrast, the different species have unique reactivities and, depending on the particular properties of each, they may lead to oxidation, nitrosation, or nitration. As a further argument against the use of the term “reactive nitrogen species,” some of the species formed downstream from NO^• (e.g. CO₃^•−) do not contain nitrogen. Finally, researchers in the nitrogen fixation field might argue that the reactive nitrogen species are those formed in the activation of nitrogen in the nitrogenase-catalyzed process of ammonia formation. Thus, in line with proposals in the free radical research field (5, 6), we suggest that the name of the identified species should be used whenever possible. When the species that are being referred to are unknown, we suggest using the term “NO^•-derived oxidants.”

Figure 1.

Nitric oxide and its biologically relevant derivatives. Nitric oxide can give rise to several species. Reaction with superoxide (O₂^•−) generates peroxynitrite (ONOO⁻); with oxyhemoglobin (HbO₂), nitrate (NO₃⁻); with oxygen (O₂), nitrogen dioxide (NO₂^•); with strong one-electron reductants, nitroxyl (HNO); with liganded iron(II) (Fe(II)L₂), dinitrosyl iron complexes (DNICs); with thiyl radical (RS^•), S-nitrosothiol (RSNO); and with NO₂^•, dinitrogen trioxide (N₂O₃). Many of these products are reactive and yield further products. Peroxynitrite at neutral pH will protonate and generate NO₃⁻, as well as NO₂^• and hydroxyl radicals (HO^•) in 30% yield. In the presence of carbon dioxide (CO₂), peroxynitrite will generate NO₃⁻, as well as NO₂^• and carbonate anion radical (CO₃^•−) in 33% yield. In the presence of reductants, peroxynitrite will be reduced to nitrite (NO₂⁻) or NO₂^•. Nitrogen dioxide can react with tyrosyl radicals (Tyr^•) to generate 3-nitrotyrosine (NO₂–Tyr) or with a reductant to form NO₂⁻. Dinitrogen trioxide can be rapidly hydrolyzed to NO₂⁻, it can be formed by NO₂⁻ in acidic pH, and it can react with thiols (RSH) to generate RSNO. In this figure, stoichiometries are not always strict, and protons are sometimes omitted for simplicity.

The preferential targets of NO^•-derived oxidants in biological systems are typically located in close proximity (in the micrometer distance range) and determined by a combination of factors, including kinetic aspects of rate constants multiplied by target concentration, compartmentalization, and membrane permeability. Some of the NO^•-derived oxidants are good one-electron oxidants that start oxygen-dependent chain reactions in both aqueous and lipidic compartments, which may amplify the effects (1, 7).

In many cases, the formation of NO^•-derived oxidants is linked to the presence of partially-reduced oxygen species, as exemplified by peroxynitrite, which is formed from the reaction of NO^• with the superoxide radical (O₂^•−). Thus, the formation of NO^•-derived oxidants is frequently related to inflammation, in which increased formation of NO^• through the inducible nitric-oxide synthase converges with increased formation of O₂^•− and other oxidants. In fact, the high reactivity of some of the species derived from NO^• make them part of the weaponry that immune cells use in their battles against microorganisms (8). In addition to their cell-damaging activity, they can have signaling roles. The recognition that hydrogen peroxide (H₂O₂) can act as second messenger (9) and that signaling actions can be extended to species derived from NO^• (10) have expanded the traditional view of oxidative stress as a misbalance between oxidant formation and antioxidant action to include the view of a disruption in regulatory pathways.

Because of their high reactivity, the species derived from NO^• have relatively short half-lives that impede their detection in biological systems through direct spectroscopic techniques. Thus, the analytical strategies used to demonstrate the formation of a certain species in a particular biological context are based either on the measurement of downstream stable products or on the use of probes that react with the species. Because these strategies are not always specific for a certain species, the modulation of the formation or decay pathways of precursors and products provides complementary evidence. For example, the modulation of NO^• and O₂^•− formation, which are the precursors of peroxynitrite, should accompany the results obtained through the detection of the stable product 3-nitrotyrosine or through the use of peroxynitrite probes. The modulation of NO^• formation can be carried out using nitric oxide synthase inhibitors, among other strategies.

In the following sections, we briefly describe the reactive species derived from NO^• in a biological milieu and NO^• itself, as well as some of the stable end products (Fig. 1). We examine methodologies used for their detection and quantification, focusing on strategies aimed at assessing their involvement in biological processes.

Nitric oxide

The discovery of nitric oxide, a free radical, as an endogenously generated effector molecule, was a paradigm shift in biological signaling. Nitric oxide (NO^•, IUPAC names nitrogen monoxide, oxidonitrogen(^•), or oxoazanyl) is a diatomic free radical produced in animals mainly by the enzymes nitric oxide synthases (NOS)³. Nitric oxide has a low dipole moment (0.159 D (11)), so it has weak intermolecular interactions and it is a gas at 1 atm and 25 °C. It is only sparingly soluble in water (1.94 ± 0.03 mm (12)) but is about 10 times more soluble in organic solvents (13). The partition coefficient in membrane models and human low-density lipoprotein is 4.4–3.4 at 25 °C (14). The diffusion through cell membranes is very rapid (14–17). The permeability coefficients of lipid membranes to NO^• range from 18 to 73 cm s⁻¹ (15, 17), similar to that of an equally thick layer of water.

Unlike several other free radical species, NO^• is not a one-electron oxidant (E⁰′ (NO^•, H⁺/HNO) ∼−0.55 V at pH 7) (18, 19). It does not abstract hydrogen atoms, and it does not add to unsaturated bonds. Importantly, NO^• does not react directly with thiols (RSH). Among the main targets of NO^• in biological systems are metal centers. Coordination to the ferrous heme in soluble guanylate cyclase is responsible for many physiological effects of NO^• (20–23). Reaction with oxyhemoglobin to form NO₃⁻ is an important sink of NO^• (24, 25). Other relevant targets of NO^• are other free radical species, in particular O₂^•− (1). Nitric oxide can also react with oxygen, and this is analyzed in the next section on autoxidation.

The effects of NO^• are exerted either via direct reactions with biological targets or indirectly via NO^•-derived oxidants. Dysregulation of NO^• homeostasis has been linked to neurodegeneration, cardiovascular disease, cancer, and inflammation. Therefore, the detection and quantification of NO^• and its derived oxidants in vitro and in vivo are relevant to understanding the molecular bases of physiological as well as pathological processes.

Nitric oxide autoxidation

The reaction of NO^• with oxygen (O₂), termed autoxidation, is a complex process that gives different products and intermediates relevant to the detection of NO^• and several of its derived species. This process is considered to be too slow to be of relevance under most physiological conditions. In the first step, two molecules of NO^• and one molecule of O₂ give two molecules of NO₂^• (Equation 1). Next, NO₂^• reacts with NO^• reversibly to give N₂O₃ (Equation 2) (26, 27). In water, in the absence of other targets, N₂O₃ is subsequently hydrolyzed to two molecules of NO₂⁻ (Equation 3).

$$2\hspace{0.17em}{\text{NO}}^{•}+{\text{O}}_2\to 2\hspace{0.17em}{\text{NO}}_2^{•}$$ (Eq. 1)

$${\text{NO}}_2^{•}+{\text{NO}}^{•}\rightleftharpoons {\text{N}}_2{\text{O}}_3$$ (Eq. 2)

$${\text{N}}_2{\text{O}}_3+{\text{H}}_2\text{O}\to 2\hspace{0.17em}{{{\displaystyle \text{NO}}}_2}^{-}+2\hspace{0.17em}{\text{H}}^{+}$$ (Eq. 3)

The rate of decomposition of NO^• is second order in NO^• and first order in O₂ concentrations, and in water the final stoichiometry is four molecules of NO^• per O₂, so that the rate equation is expressed as in Equation 4.

$$\frac{-\text{d}\left[{\text{NO}}^{•}\right]}{\text{d}t}=4\text{k}{\left[{\text{NO}}^{•}\right]}^2\left[{\text{O}}_2\right]$$ (Eq. 4)

The limiting reaction in the autoxidation of NO^• is the reaction with O₂ (Equation 1); trapping subsequent products has no effect on the overall rate (27). The rate constant of the process is third order (k ∼2 × 10⁶ m⁻² s⁻¹ (27, 28)). In aqueous solutions, the forward reaction of N₂O₃ formation (Equation 2) is very fast (k = 1.1 × 10⁹ m⁻¹ s⁻¹), and the dissociation has a rate constant k = 8 × 10⁴ s⁻¹ (29). The hydrolysis of N₂O₃ is also rapid in water and can be further accelerated by salts such as phosphate and bicarbonate (27, 30, 31).

The autoxidation of NO^• is slow in vivo because the rate of NO^• decay is second order in NO^• concentration, which is expected to be in the nanomolar range under normal conditions. This reaction can be accelerated in hydrophobic environments such as lipid membranes, lipoproteins, and proteins (32–34) due to the increase in concentration of both NO^• and O₂ because of their hydrophobicity (14, 35). This so-called “lens effect” may be of relevance where NO^• concentrations are significantly increased, especially in sites of inflammation.

Detection of nitric oxide

Nitric oxide is difficult to measure in vivo because of its short half-life (typically in the range of 0.1–10 s), reactivity, and low steady-state concentration (i.e. nanomolar to micromolar). Nonetheless, several strategies have been developed to measure NO^• or its derived species in vitro or in vivo that involve the use of absorbance, fluorescence, electron paramagnetic resonance (EPR), and electrochemistry (Fig. 2). Furthermore, NO^• can be measured by chemiluminescence, a methodology that can be adapted to also measure other species. These methods are described in the following sections.

Figure 2.

Detection of nitric oxide. A, oxidation of oxyhemoglobin. Methemoglobin can be detected spectrophotometrically. B, electrochemical sensor. NO^• is oxidized to nitrosonium cation (NO⁺), which is converted to nitrite (NO₂⁻). The current is directly proportional to NO^• concentration. C, reaction of NO^• with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-yloxyl-3-oxide (PTIO) to yield NO₂^• and PTI, the conversion can be followed by EPR. D, fluorogenic probes for NO^•-derived species. The 4,5-diaminofluorescein diacetate (DAF-2–DA) is cell-membrane–permeable, and the acetyl groups are removed by intracellular esterases to yield the nonfluorescent DAF-2 that then reacts with NO^•-derived species to yield the fluorescent triazole derivative DAF-2 T. At right are shown related fluorescent probes diaminonaphthalene (DAN) and 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM). The mechanisms of triazole formation from diamino-fluorogenic probes involve two possible routes: one is the direct nitrosation by N₂O₃ to give an intermediary N-nitrosamine that then diazotizes and reacts with the second amine to yield the triazole; the other mechanism requires the oxidation of the diamino probe by NO^•-derived and other one-electron oxidants, followed by the reaction of the radical with NO^• to form the N-nitrosamine. Either of these pathways implicate NO^• but with different stoichiometries. E, ozone-based chemiluminescence. F, reaction catalyzed by nitric oxide synthase to generate NO^•. l-Arginine is first hydroxylated to N^ω-hydroxy-l-arginine with O₂ and NADPH as cosubstrates. In the second step, this intermediate is oxidized by a second O₂ and 0.5 eq of NADPH to give l-citrulline and NO^•. G, bioassays to measure downstream physiological actions of NO^•.

Oxyhemoglobin oxidation

The identification of the endothelial-derived relaxing factor as NO^• back in 1987 (36) was in part made by the change in the UV-visible spectrum of deoxyhemoglobin to form nitrosyl hemoglobin, with a corresponding shift of the Soret band from 433 to 406 nm. Nonetheless, the reaction mostly used to quantify NO^• in vitro is with oxyhemoglobin, which is stable in air.

Oxyhemoglobin (Fe(II)(Hb)O₂) is oxidized by NO^• to yield NO₃⁻ and methemoglobin (Fe(III)(Hb)), which can be measured spectrophotometrically (Fig. 2A). The absorption of the Soret band is strong; thus the sensitivity of this method is relatively high (submicromolar). The maximum absorbance changes are observed at 401 (increased by reaction with NO^•) and 421 nm (decreased), with an isosbestic point at 411 nm. If multiple wavelengths can be measured, the reaction can be followed by the absorbance difference of 401–421 nm (Δϵ_401–421 = 77 mm⁻¹ cm⁻¹) (37). If there is interference at these wavelengths, the absorbance at 577 nm can be used (Δϵ₅₇₇ = 10 mm⁻¹ cm⁻¹) or even both absorbances at 577 and 630 nm to calculate oxy- and methemoglobin concentrations before and after addition of NO^• (38). Due to the fast reaction with NO^• (k = 3.7 × 10⁷ m⁻¹ s⁻¹) (39), oxyhemoglobin is also often used as NO^• scavenger. Nitrite can also oxidize oxy- to methemoglobin but at much lower rates (although autocatalytically), so when high concentrations of NO₂⁻ are expected, like with the use of NO^• donors for long-time periods, the contribution of NO₂⁻ to hemoglobin oxidation should be considered. Peroxynitrite can also oxidize oxyhemoglobin; thus, addition of superoxide dismutase is recommended as a control to prevent peroxynitrite formation from NO^• and O₂^•− so that the methemoglobin formed can be associated with NO^•. In addition, a potential O₂^•−-dependent redox cycling of hemoglobin can be avoided.

Electrochemical sensor

Electrodes specific for NO^• are commercially available, but many research laboratories make their own. Typically, they consist of a filament made of carbon or platinum and a coating to provide specificity that either attracts NO^• (40) or excludes other oxidizable species (41). At the anode, NO^• is oxidized by one-electron to nitrosonium cation (NO⁺), which is converted to NO₂⁻ (Fig. 2B). The current generated from NO^• oxidation is directly proportional to NO^• concentration with a 10 nm detection limit (a calibration curve should be run with each experiment). The electrode is covered with a gas-permeable membrane that allows diffusion of NO^• but not NO₂⁻ or other charged species. Temperature should be kept constant considering that solubility of NO^• gas is very temperature-sensitive. Microelectrodes have been designed (<1 mm) that allow direct detection in cells in real time (42).

Electron paramagnetic resonance (EPR)

Although NO^• is a free radical, i.e. it has an unpaired electron, it is difficult to detect directly by EPR, and spin-trapping techniques have to be used. Nitronyl nitroxides (with nitrone and nitroxide functional groups) are used as NO^• probes (43). They react with NO^• to give an iminonitroxide (Fig. 2C) with a dramatic change in the EPR spectrum that can be followed in a continuous and quantitative way. For example, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-yloxyl-3-oxide (PTIO) or its water-soluble analogue carboxy-PTIO react with NO^• with second-order rates constants of 10⁴ m⁻¹ s⁻¹ and a change in the EPR spectrum from five to seven lines (44, 45).

Due to its fast reaction with NO^•, carboxy-PTIO is often used as a scavenger of NO^•; however, care should be taken because NO₂^• is a product of the reaction and has its own reactivities. In addition, biological reductants like thiols, ascorbate, or O₂^•− can nonspecifically reduce the nitroxides. Encapsulation of PTIO in liposomes has been used to avoid reduction (46).

Hydrophobic and hydrophilic nitroxides are available that allow detection of NO^• at different depths of a biological membrane. Collisions of NO^• with spin labels located in water or in membranes alter both the linewidth and the spin-lattice relaxation time that can be used to qualitatively and quantitatively measure NO^• (17, 47).

Colloid iron diethyldithiocarbamate (Fe(DETC)₂) or N-methyl-d-glucamine dithiocarbamate are reliable spin traps for NO^• detection. They form iron nitrosyl complexes with characteristic three-line EPR spectra (g_av = 2.04; a_N = 1.27 mT) at room temperature that are stable in the presence of oxygen (48). Dinitrosyl iron complexes (DNIC) with thiol-containing ligands have been detected in animal and bacterial cells by EPR. These complexes are formed in vivo in the paramagnetic (EPR-active) mononuclear as well as diamagnetic (EPR-silent) binuclear forms. The amount of NO^• can calculated from the EPR amplitude signal because the linewidth of the NO-Fe(DETC)₂ EPR spectrum may vary considerably with variations in the amount of Fe(DETC)₂ in membrane lipids and the amount of Fe(III) present (48, 49).

In addition, deoxyhemoglobin or other hemeproteins in the reduced Fe(II) form react with NO^• to form nitrosyl–heme complexes that besides having characteristic UV-visible spectra are paramagnetic and can be followed by EPR. However, because of the instability of the complexes, the EPR spectra should be run at low temperatures (77 K) (50–53).

Fluorogenic probes

Fluorogenic probes have been developed that specifically react with NO^•-derived species (i.e. N₂O₃) to yield fluorescent products, such as diaminonaphthalene (DAN) and diaminofluorescein (DAF) derivatives (Fig. 3D). The most popular of this kind is 4,5-diaminofluorescein (DAF-2), where nitrosation results in the highly-fluorescent triazole DAF-2 T (λ_exc = 495 nm, λ_em = 515 nm). The esterified diacetate derivative (DAF-2-DA) is also commercially available. It is highly membrane-permeable and detects intracellular nitrosation of the probe.

Figure 3.

Chemiluminescence detection of nitric oxide and derived species. This sensitive method allows the determination of NO^• and several related species in gas or liquid phase. The system includes a purge vessel where the sample is injected. An inert gas transports NO^• from the purge vessel to a detector where ozone (O₃) reacts with NO^• yielding NO₂^• in the excited state, which decays to the basal state emitting light. Different reagents can be used in the purge vessel to selectively convert certain analytes into NO^•. A neutral buffer is used if NO^• as such is to be measured. A triiodide/acid solution reduces most derivatives to NO^•, and thus it is useful for total quantitation. The sample can be pretreated with reagents that will trap specific species. Thus, the amount of NO₂⁻ in a complex sample is obtained by the difference between untreated sample and sample pretreated with sulfanilamide and acid to trap NO₂⁻. RSNO can be removed by pretreatment with Hg(II), sulfanilamide, and acid. DNICs are sensitive to the same treatment but differently from RSNO; they decay over time (10–12 h). The remaining signal after treatment with Hg(II), sulfanilamide, and acid derives mostly from N-nitrosamines, and to a lesser degree from nitrosyl–heme. Alternatively, the purge vessel can contain Cu(II) and cysteine, which does not reduce NO₂⁻ to NO^• but effectively reduces RSNO to NO^•, and it also releases NO^• from nitrosyl-hemoglobin and N-nitrosamines. When analyzing nitrosation of hemoglobin or other heme-proteins, carbon monoxide (CO) is added to avoid reaction of NO^• with heme-proteins.

The pH should be carefully controlled because DAF-2 T fluorescence is pH-sensitive (54). Other potential artifacts are divalent cations, in particular calcium, which was reported to significantly increase the fluorescent signal from DAF-2, as well as the incident light (55). Multiple and long exposures to excitation light, instead of causing photobleaching of the dye, potentiate the fluorescence response. Thus, minimum periods of light exposure are recommended. The use of 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) is favored because of its increased photostability, stability to pH, and reactivity toward NO^•-derived species (54). The sensitivity of DAF-FM is 1.4 times higher than that of DAF-2. This increase of sensitivity is thought to result from the higher rate of the reaction with nitrosating NO⁺ equivalents due to the electron-donating effect of the methyl group (43).

The proposed mechanism of triazole formation involves N₂O₃ reacting with an amine to form an intermediate N-nitrosamine that at neutral pH can diazotize and then react with the second amine to yield the triazole (Fig. 3D) (56, 57). Alternatively, a radical intermediate of the diamino-probe is formed by NO₂^• or other strong oxidants (e.g. radicals derived from peroxynitrite or peroxidases/H₂O₂) that then react with NO^• (Fig. 3D) (58). These probes show some specificity issues, in that the triazole is not an exclusive product of NO^•, and fluorescent products can be derived from peroxynitrite, nitroxyl (HNO), and ascorbic acid, complicating the interpretation of results (59, 60). Nonetheless, the role of NO^• can be confirmed in cells using NOS inhibitors, NO^• scavengers, and also by HPLC to isolate DAF-triazole (60–62).

Novel genetically-encoded fluorescent NO^• biosensors have been developed (63). For example, one sensor has a fusion between a fluorescent protein and a bacteria-derived NO^• domain that selectively binds NO^• via a nonheme Fe(II) center. Once NO^• binds, the domain gets closer to the fluorescent protein and quenches its emission (64).

Ozone-based chemiluminescence detection of nitric oxide and related species

The chemiluminescence detection of NO^• and several other related species presents very good sensitivity and reproducibility and has become the gold standard method for quantification (62, 65–68). The sensitivity is in the nanomolar range. The method can be used with any type of gas or liquid sample, including cell lysates and tissue homogenates (68, 69).

The sample is injected into a purge vessel containing a given reactant such as triiodide. This vessel has fritted glass at the base and is purged at a constant flow rate with nitrogen or helium gas. The NO^• that was injected (or generated) in the vessel is carried by this inert gas to the detector (65, 68). The NO^• in the carrier gas passes first through a reaction cell where ozone is constantly introduced. The reaction with ozone (O₃) generates NO₂^• in the excited state (NO₂^•*) that is then carried by the inert gas flow to the detection cell where red and near IR light emission from NO₂^•* decay to the basal state is measured (68, 70). The intensity of emission is directly proportional to the amount of NO^• (Fig. 2E).

This method is not only useful to the study of NO^• but also of other oxidation products that can be converted to NO^• through different methods, such as NO₂^•, NO₂⁻, S-nitrosothiols, nitrosyl–metal complexes, and N-nitrosamines (62, 65, 66, 68). The reactant used in the purge vessel determines what species can be quantified (Fig. 3). If buffer at neutral pH is used, only NO^• as such will give a signal (69). One of the most popular reactants for chemiluminescent detection of NO^• and its derivatives is an acidic triiodide solution (65). It consists of iodine plus iodide and acetic acid (65, 68, 71). The triiodide that forms in this solution can convert NO₂⁻, S-nitrosothiols, N-nitrosamines, and nitrosyl–metal complexes to NO^• (65, 66). In the case of a biological sample that contains a mixture of these species, several tubes are prepared that include the parent sample, then one with acidic sulfanilamide to trap NO₂⁻, and another that also includes HgCl₂ to decompose S-nitrosothiols (65, 66). The difference in the measured NO^• with the different treatments indicate how much NO₂⁻ and S-nitrosothiol were in the sample.

Additional methods for more selective chemical reduction of S-nitrosothiols include copper-based assays where the reactant in the purge vessel consists of a buffer at neutral pH and Cu(II) plus an excess of cysteine (66, 71). In this case the copper is reduced to Cu(I) by cysteine, which then reduces S-nitrosothiols to NO^• and thiol. The use of Hg(II) is recommended to discriminate the signal from N-nitrosamines (66). For applications in blood, a modification of the method that includes carbon monoxide has been developed that prevents capture of NO^• by hemoglobin (72).

Nitric oxide synthase activity

The NOS enzymes catalyze the oxidation of arginine to NO^• and stoichiometric amounts of citrulline (Fig. 2F) (73). Therefore, the rate of NO^• formation can be estimated from the rate of citrulline formation from arginine and saturating concentrations of NOS cofactors (NADPH, FAD, FMN, tetrahydrobiopterin, and calcium/calmodulin). Radiolabeled arginine is used, and the reaction is stopped with EDTA, which binds calcium and inactivates the enzyme. The radiolabeled citrulline product is separated from arginine by cation-exchange chromatography (cationic arginine is retarded and zwitterionic citrulline is eluted) and measured in a liquid scintillation counter (74). Because citrulline in the cell could be derived from non-NOS pathways, controls should be performed with addition of a NOS inhibitor as well as omission of NADPH.

There are commercially available kits to follow NOS activity indirectly, by measuring the time course of NO₂⁻ formation spectrophotometrically using the Griess reaction described below.

Bioassays for nitric oxide

The production of NO^• in mammalian cells can be detected indirectly by measuring its biological activities like vasodilation, platelet aggregation, and guanylate cyclase activation (Fig. 2G).

Cyclic GMP

In the cellular context, cyclic GMP (cGMP) is formed not only by guanylate cyclases stimulated by NO^• (NO-GC or soluble GC) but also by the membrane natriuretic peptide receptor-coupled guanylate cyclases (GC-A and GC-B). Therefore, to measure levels of cGMP as an indirect measurement of NO^•, controls with inhibitors of NOS should be included. The different methods used to determine cGMP have been recently reviewed in Ref. 75 and include radiolabeled [α-³²P]GTP (76), a cGMP antibody in a commercially available enzyme-linked immunosorbent assay (ELISA), and fluorescence-based cGMP indicators (77).

Vessel relaxation

The seminal studies that introduced NO^• to the biological scenario as a critical regulator of blood flow were related to its identification as an endothelium-derived relaxing factor (36, 67). This function is explained by the formation of NO^• from endothelial NOS, subsequent diffusion to the underlying smooth muscle, and activation of soluble guanylate cyclase, which initiates a signaling cascade that ultimately leads to vasodilation and increased blood flow. Thus, a method amply used by physiologists and pharmacologists to detect production of NO^• consists of measuring tension in isolated vascular preparations treated with agonist and antagonists of NO^•-dependent signaling (67, 78).

Inhibition of platelet aggregation

A way to test NO^• production is to follow inhibition of platelet aggregation after activation. It is a very simple, inexpensive method first described in 1962 (79). Washed human platelets are equilibrated at 37 °C in a turbidometric platelet aggregometer in the absence and presence of a system that produces NO^•. An activator like thrombin is added to induce aggregation, and turbidimetry is followed with time (80).

Nitrogen dioxide

Nitrogen dioxide (NO₂^•) is a reddish-brown free radical gas that forms part of air pollution. In biological systems, there are different endogenous sources of NO₂^•. These include: (a) NO^• autoxidation (see section above); (b) NO₂⁻ oxidation, a reaction that is catalyzed by different heme-dependent peroxidases (Equations 5–7) and Cu,Zn-superoxide dismutase (81–83) or performed nonenzymatically by strong one-electron oxidants such as CO₃^•−, HO^• or peroxyl radicals (ROO^•) (84–86); and (c) homolysis of the peroxo bond of peroxynitrous acid (ONOOH) or of short-lived adducts formed from the reaction of peroxynitrite (ONOO⁻) with carbon dioxide (CO₂), with carbonyl-containing compounds, or with metal centers (87–89).

$$\text{peroxidase-heme}+{\text{H}}_2{\text{O}}_2\to \text{peroxidase-compound}\hspace{0.17em}\text{I}+{\text{H}}_2\text{O}$$ (Eq. 5)

$$\text{peroxidase-compound}\hspace{0.17em}\text{I}+{\text{NO}}_2^{-}\to \text{peroxidase-compound}\hspace{0.17em}\text{II}+{\text{NO}}_2^{•}$$ (Eq. 6)

$$\text{peroxidase-compound}\hspace{0.17em}\text{II}+{\text{NO}}_2^{-}\to \text{peroxidase-heme}+{\text{NO}}_2^{•}$$ (Eq. 7)

The solubility of NO₂^• in water is low. The reported Henry coefficient (∼1.4 × 10⁻² m atm⁻¹ at 20 °C) (90, 91) is quite uncertain due to its rapid (k = 4.5 × 10⁸ m⁻¹ s⁻¹ (92)) dimerization to dinitrogen tetroxide (N₂O₄). The latter has ∼100-fold increased solubility in water (90) and rapidly hydrolyzes to NO₂⁻ and NO₃⁻ (k = 1000 s⁻¹) (90, 93). However, under most physiological conditions where NO₂^• concentrations are low (<1 μm), dimerization, which is a reversible process with a K_eq = 7 × 10⁴ m⁻¹, is outcompeted by bimolecular reactions of NO₂^• with different targets, some of which are far more concentrated (93–95). The partition coefficients in organic solvents indicate that NO₂^• is slightly hydrophobic, although less than NO^•, which suggests a minor “lens effect” for NO₂^• reaction kinetics in membranes or other hydrophobic biological systems (94, 96).

The reduction potential of the NO₂^•/NO₂⁻ pair is 0.99 V at pH 7 (97). Thus, it is a good one-electron oxidant. According to kinetic considerations, NO₂^• is predicted to react mostly with thiol-containing molecules (k_RS− ∼10⁸ m⁻¹ s⁻¹) (98) and ascorbate (k = 1.8–3.5 × 10⁷ m⁻¹ s⁻¹ at pH 7.4) (99), whereas urate (k = 2 × 10⁷ m⁻¹ s⁻¹ at pH 7.4) is a main target in plasma (98). In hydrophobic media, NO₂^• can initiate lipid peroxidation (100). Furthermore, it can add to alkene double bonds in a fast and reversible process to form nitroalkyl radicals, which eventually undergo cis-trans–isomerization (101) or form nitro-derivatives. The biological formation, characteristics, and relevance of nitrated fatty acids has been reviewed (93, 102–105). Finally, NO₂^• reacts at diffusion-controlled rates with other radical species, such as tyrosyl radicals in proteins, to form 3-nitrotyrosine (see section on 3-nitrotyrosine below). The reversible reaction with NO^• leads to the formation of the nitrosating species N₂O₃ (Equation 2).

Detection of nitrogen dioxide

The UV-visible absorption spectrum of NO₂^• shows a broad band peak at ∼400 nm with an absorption coefficient of 200 m⁻¹ cm⁻¹ in aqueous solution (26). The low absorption coefficient, the low stability of the radical, and the need to make corrections for N₂O₄ and NO₂⁻ absorption limit the technique. Nitrogen dioxide is frequently detected and quantified by chemiluminescence methods, in which the intensity of the emitted light is proportional to the concentration of NO₂^•. Some of these methods rely on the reaction of NO^• with ozone (see section above on ozone-based chemiluminescence detection of nitric oxide and related species) and therefore require that NO₂^• be reduced to NO^• using particular catalytic converters. The latter are usually nonspecific due to reduction of other nitrogen-containing compounds (106, 107). Nitrogen dioxide can also be converted to NO^• photolytically using UV-LED irradiation (108). In addition, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) in alkaline solution reacts with NO₂^• giving rise to intense chemiluminescence, although other one-electron oxidants can also lead to light emission (109).

Detection of NO₂^• in cells and tissues requires different methodologies. Because NO₂^• is a strong one-electron oxidant, it can react with typical redox probes such as 2′,7′-dichlorodihydrofluorescein (DCFH₂). In experimental designs, addition of NO₂⁻ may be useful to convert other one-electron oxidants to NO₂^•.

One strategy depends on the ability of NO₂^• to rapidly combine with free or protein tyrosyl radicals to form 3-nitrotyrosine (110). This is analyzed in the section below on 3-nitrotyrosine. Furthermore, nitration of green fluorescent protein (GFP) leads to a decrease in its intrinsic fluorescence and was used to evaluate NO₂^• formation. Although the decrease in fluorescence intensity is not specific for nitration, it can be utilized in combination with pharmacological modulation of NO^• levels to indicate NO₂^• formation (59).

Finally, because of the radical nature of NO₂^•, EPR has been utilized either by direct detection of NO₂^• in salt matrices and low temperatures or by using spin traps such as nitromethane at alkaline pH or nitrone compounds (93, 111–113). Nitroso spin traps do not trap NO₂^• (113).

Due to the short half-life of NO₂^• in aqueous solutions even in the absence of other targets, as a result of dimerization and hydrolysis of N₂O₄, the study of the kinetics of NO₂^• reactions requires the use of very fast methodologies that allow measurements to be made in the microsecond time scale, such as pulse radiolysis. Furthermore, the low absorption coefficient of NO₂^• limits its direct detection so that product monitoring or competition kinetics need to be used. Competition with 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) oxidation to ABTS⁺ is frequently employed due to the high extinction coefficient of the latter radical at 414 nm (3.6 × 10⁴ m⁻¹ cm⁻¹) (98, 114).

Dinitrogen trioxide and its detection

Dinitrogen trioxide (N₂O₃) can be formed from NO₂^• reaction with NO^• (26, 27) and is considered an important intermediate in the autoxidation of NO^• (27) (see section on autoxidation above). It can also be formed from NO₂⁻ at acidic pH, with an equilibrium constant of 3 × 10⁻³ m⁻¹ (Equation 8) (115).

$$2\hspace{0.17em}{\text{HNO}}_2\rightleftharpoons {\text{N}}_2{\text{O}}_3+{\text{H}}_2\text{O}$$ (Eq. 8)

As discussed in the section on autoxidation, N₂O₃ is rapidly hydrolyzed to NO₂⁻ (Equation 3), and this reaction is accelerated by certain salts, such as phosphate and bicarbonate (27, 30, 31). Therefore, the first approach to measure the production of N₂O₃ is by monitoring NO₂⁻ (see section below on nitrite and nitrate).

N₂O₃ is considered an important nitrosating species in vitro because it reacts rapidly with thiolates and amines to give the corresponding nitrosated species (116, 117), but its role in biological nitrosation is uncertain because of the slow kinetics of NO^• autoxidation (116, 118). Because both NO₂^• and NO^• are slightly hydrophobic (94, 96), it was suggested that N₂O₃ formation should be accelerated in hydrophobic regions. However, the nitrosation of thiols buried in the hydrophobic regions actually decreases because the dissociation to the more reactive thiolate is disfavored (119). Anyway, the formation of S-nitrosothiols is not specific to N₂O₃, and other mechanisms may be more relevant (116, 118), so that S-nitrosothiols are not necessarily good indicators of N₂O₃ formation (see section below on S-nitrosothiols).

Besides measuring NO₂⁻ and S-nitrosothiols, another method to detect N₂O₃ is to use fluorogenic probes such as diaminonaphthalene (DAN) and diaminofluorescein (DAF), which were discussed in the section on detection of nitric oxide and suffer from the same issues as S-nitrosothiols.

Nitrosyl–metal complexes and their detection

Intracellular dinitrosyl iron complexes (DNICs) are formed from NO^•, a ligand such as GSH, and loosely bound iron, also called labile or chelatable iron pool (120). Recent studies have shown that the exposure of cells to NO^•, either exogenous or endogenous, leads to the formation of more DNICs than S-nitrosothiols, in a 4:1 ratio (69). DNICs have been proposed to be relevant precursors in the nitrosation of thiols (121).

The most selective technique to measure DNICs is through EPR (120, 122). Mononuclear DNICs show a characteristic EPR spectrum (48, 122). EPR has several advantages such as the ability to measure signals in optically opaque samples, a good sensitivity (200 nm), and the capacity to distinguish enzymatically generated NO^• by the change in the spectrum using [¹⁵N]arginine (122).

DNICs made with GSH can also be analyzed by UV-visible spectrophotometry, provided there is a separation step such as HPLC. They show a spectrum with maximum absorbance below 200 nm and characteristic peaks at 310, 360, and 680 nm (ϵ = 9200, 7400, and 200 m⁻¹ cm⁻¹, respectively) for the diamagnetic binuclear form, or 390 nm (ϵ = 3900 m⁻¹ cm⁻¹) for the paramagnetic mononuclear form (48).

Cellular DNICs can also be quantified through ozone-based chemiluminescence, using the triiodide method. Care should be taken in quantification because the signal is time-sensitive and decays within hours, and also because DNICs are sensitive to HgCl₂, analogously to S-nitrosothiols (69). To distinguish between signals from S-nitrosothiols and DNICs, it was proposed to stabilize S-nitrosothiols in the cell lysate using a buffer containing diethylenetriaminepentaacetic acid and N-ethylmaleimide and to analyze the sample immediately after extraction and 20 h later to ensure the full decay of DNICs (69).

Formation of protein nitrosyl–metal complexes is particularly relevant in red blood cells, because NO^• can react with deoxyhemoglobin to yield nitrosyl-hemoglobin. The detection of this product predominates at low oxygen tensions (123) because deoxyhemoglobin will be more abundant and because oxyhemoglobin will rapidly decompose NO^• to NO₃⁻ (24). Nitrosyl-hemoglobin can be quantified in vitro through UV-visible spectrophotometry and shows absorption maxima at 403 and 575 nm (36). In red blood cells, nitrosyl-hemoglobin is difficult to quantify by spectrophotometry where there is a mixture of different forms of hemoglobin absorbing at the same wavelength. EPR, in contrast, is specific for nitrosyl-hemoglobin and allows its quantification in packed red blood cells. The limit of quantification was calculated to be 200 nm. Under normal conditions, the amount of nitrosyl-hemoglobin in human blood is below the detection limit. However, patients exposed to 80 ppm NO^• inhalation treatment increased its nitrosyl-hemoglobin levels up to 2 μm (124).

S-Nitrosothiols

The formation of S-nitrosothiols is undoubtedly linked to the formation of NO^• in biological systems. Nevertheless, the exact chemistry is still under debate. In fact, thiols or rather thiolates can be nitrosated by the products of NO^• autoxidation (see section above on autoxidation) in a direct mechanism by N₂O₃ or stepwise by NO₂^• and NO^• (Equation 9-11) (116, 125).

$${\text{RS}}^{-}+{\text{N}}_2{\text{O}}_3\to \text{RSNO}+{\text{NO}}_2^{-}$$ (Eq. 9)

$${\text{RS}}^{-}+{\text{NO}}_2^{•}\to {\text{RS}}^{•}+{\text{NO}}_2^{-}$$ (Eq. 10)

$${\text{RS}}^{•}+{\text{NO}}^{•}\to \text{RSNO}$$ (Eq. 11)

Although the formation of NO₂^• and N₂O₃ can be accelerated by hydrophobic regions in lipid membranes and even proteins (32–34), autoxidation is still too slow to be biologically significant (118). Furthermore, in cells, oxygen inhibits rather than increases thiol nitrosation, arguing against a significant role for NO^• autoxidation in biological thiol nitrosation (118).

Regarding the mechanisms of biological thiol nitrosation, there is evidence supporting the intermediacy of nitrosyl–iron complexes (DNICs) (69, 121), as well as the intermediacy of cytochrome c (126).

S-Nitrosothiols undergo further reactions with other thiols, such as trans-nitrosation, where the nitroso moiety is transferred regenerating the original thiol (127). For instance, the trans-nitrosation from S-nitrosoglutathione to cysteine occurs with k = 140 m⁻¹ s⁻¹ (127). Thioredoxin catalyzes both trans-nitrosation and denitrosation (128). Alcohol dehydrogenase class III catalyzes the reduction of S-nitrosoglutathione efficiently and has therefore been called S-nitrosoglutathione reductase (129).

Detection of S-nitrosothiols

Several methods have been developed to quantify total S-nitrosothiols and also to identify proteins that are nitrosated. S-Nitrosothiols show a UV spectrum with a maximum at 335 nm. The absorptivity for S-nitrosoglutathione at 335 nm is 922 m⁻¹ cm⁻¹; therefore, the sensitivity of the spectrophotometric analysis is low (above 50 μm) and depends on having a purified sample or on chromatographic or capillary electrophoresis separation (130).

Another historically important method for S-nitrosothiols is by Saville (131). In this method the S-nitrosothiol is treated with Hg(II). Tight binding to the thiolate releases NO⁺ that is rapidly hydrolyzed to NO₂⁻ (131). The released NO₂⁻ is then measured by the Griess method. This method has micromolar sensitivity (see section below on detection of nitrite).

Antibodies against S-nitrosocysteine have been used in immunohistochemical assays, Western blotting, and immunoprecipitation. However, specificity issues and the advent of biotin switch techniques that also allow mapping the modified cysteine within a protein have discouraged their use (66).

The gold standard method to quantify S-nitrosothiols is ozone-based chemiluminescence that provides nanomolar sensitivity and is appropriate for most biological applications (Fig. 3). As discussed in the section on chemiluminescence, S-nitrosothiols can be quantified using the triiodide reaction and also using copper ions and reductants (66).

It was early observed that several proteins could be S-nitrosated (132) but unbiased approaches to the S-nitrosoproteome were only possible after the introduction of the “biotin switch” method in 2001 (133). The original method involved blocking free thiols in S-nitrosated proteins with methyl methanethiosulfonate, then specifically reducing the nitrosated thiols with ascorbate, followed by reaction of these thiols with N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide. The biotinylated proteins could then be selectively captured by using the specific binding to immobilized streptavidin (133). Some issues with this method have been pointed out, namely that it is very difficult to ensure that all free thiols are effectively blocked in the first step, that ascorbate does not reduce S-nitrosothiols directly but through the intermediacy of metals in solution (134), and that no chemical trace is left to indicate that the thiol was effectively nitrosated.

Relative quantification of protein S-nitrosation can be achieved through different means, including isotope-coded affinity tags (ICAT) and stable isotope labeling by amino acids in cell culture (SILAC). Both methods are based on using a light and a heavy isotope-containing tag. In ICAT, samples to compare are processed in parallel and tagged with biotin derivatives that include either light or heavy isotope linkers, and then mixed and further processed (135). SILAC involves adding either light or heavy isotope-containing arginine and lysine to control or stimulated cell cultures (136). The cell lysates from both cell cultures can then be mixed and processed as in the biotin switch method. If the same peptide is enriched from both control and treated samples, it will elute at the same time in the LC-MS analysis, but the mass spectra will differ by a known number of Da, and the relative amounts can be calculated from the intensities in the MS peaks (136).

Other approaches to identify S-nitrosated proteins and the location of the modification include the use of organomercurial compounds to either trap or tag S-nitrosothiols, after blocking free thiols (137) or the selective reaction of S-nitrosothiols with derivatized phosphines to tag S-nitrosated peptides in one step through reductive ligation (138).

Nitrite and nitrate

Nitrite (NO₂⁻, IUPAC name dioxidonitrate(1−)) and nitrate (NO₃⁻, IUPAC name trioxidonitrate(1−)) were considered for a long time to be rather inert products of NO^• oxidation. The concentration of NO₃⁻ in plasma of fasting individuals is 20–40 μm, and it is considered to derive mostly from the reaction between NO^• and oxyhemoglobin, but also from the diet (139). The concentration of NO₂⁻ in plasma is significantly lower (50–300 nm) because there are several processes by which it can be converted to NO^• or further oxidized to NO₃⁻ (140). Nitrate is concentrated in saliva and can be converted to NO₂⁻ by bacteria in the oral cavity (139). Xanthine oxidase has also been shown to reduce NO₃⁻ to NO₂⁻, but it seems to be a minor contribution compared with the oral microbiome (140).

Nitrite can be reduced to NO^• by different proteins, including deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, and aldehyde oxidase (141). The reduction by deoxyhemoglobin is thought to be quantitatively the most important pathway for the generation of NO^• from NO₂⁻ and responsible for the NO^•-like effects of NO₂⁻ infusion in the presence of red blood cells (141). The NO₂⁻ reductase activity of deoxyhemoglobin leads to the formation of NO^• and methemoglobin (Equation 12).

$${\text{NO}}_2^{-}+\text{Fe}\left(\text{II}\right)\left(\text{Hb}\right)+{\text{H}}^{+}\to {\text{NO}}^{•}+\text{Fe}\left(\text{III}\right)\left(\text{Hb}\right)+{\text{OH}}^{-}$$ (Eq. 12)

Detection of nitrite and nitrate

There are several methods to detect NO₂⁻ in biological samples. The simplest method to measure NO₂⁻ is the Griess method, which sensitivity is in the micromolar range. The method is based on the diazotization of sulfanilamide by NO₂⁻ in acidic pH and the subsequent reaction with N-(1-naphthyl)ethylenediamine to yield an intensely pink-colored product with absorption maximum at 540 nm (Fig. 4) (142). The measurement of NO₃⁻ is usually done by first converting it to NO₂⁻, either by using vanadium chloride or the enzyme NO₃⁻ reductase (142). The analysis can readily be automated to measure NO₂⁻ and NO₃⁻ (143). Lower concentrations of NO₂⁻ (down to 20 nm) can be quantified by the formation of the fluorescent triazole derivative of DAN (see section above on fluorescent detection). In this case, the reaction of NO₂⁻ with DAN is done at acidic pH (Fig. 3), and then the fluorescence of the product is measured at alkaline pH (144).

Figure 4.

Detection of nitrite and nitrate with the Griess method. Sulfanilamide reacts with NO₂⁻ in acidic pH to yield a diazonium intermediate that subsequently reacts with N-(1-naphthyl)ethylenediamine to yield the intensely colored Griess product with absorption maximum at 540 nm. NO₃⁻ can be converted to NO₂⁻ with vanadium chloride or NO₃⁻ reductase to allow both NO₃⁻ and NO₂⁻ to be measured.

Nitrite and nitrate can also be quantified by HPLC, capillary electrophoresis, and GC-MS (145, 146). For low concentrations such as those often encountered in biological samples, the ozone-based chemiluminescence method (see section on chemiluminescence and Fig. 3) offers the required nanomolar sensitivity. In this case, the purge vessel needs to be filled with the triiodide acidic solution that converts NO₂⁻ to NO^• that is then carried to the detection cell by the carrier gas. Nitrate is measured by first reducing it chemically or enzymatically to NO₂⁻. The contribution of other species such as S-nitrosothiols to the signal is controlled by running samples treated with acidic sulfanilamide to trap all free NO₂⁻.

Nitroxyl

The product of the one-electron reduction of NO^• is HNO (nitroxyl, azanone, nitrosyl hydride, and hydrogen oxonitrate). The reduction potential of this process, E⁰′ (NO^•, H⁺/HNO) ∼−0.55 V at pH 7 (18, 19), is quite low, but high enough to make endogenous HNO formation a possibility. Biological studies are usually performed using nitroxyl donors (e.g. Angeli's salt). The ground state of HNO is a singlet in which all the electrons are spin-paired, whereas that of NO⁻ (nitroxyl anion, oxonitrate (1−)) is a triplet with two unpaired electrons (18, 19). Thus, deprotonation is spin-forbidden and slow, and the pK_a of HNO is 11.4 (147).

Nitroxyl can react with soft electrophiles (18). In vivo, the preferential reactions of HNO are with thiols and metal centers. For example, the reaction between HNO and GSH, which is present in millimolar concentrations inside cells, has a rate constant of 3.1 × 10⁶ m⁻¹ s⁻¹ (148). In addition, HNO can dimerize yielding nitrous oxide (N₂O) and water (k = 8 × 10⁶ m⁻¹ s⁻¹) (147). Nitroxyl can also react with oxygen to form peroxynitrite with a rate constant of 1.8–2 × 10⁴ m⁻¹ s⁻¹ at pH 7.4 (148), but this process is relatively slow under biological conditions and has low relevance. More information on the biochemistry of HNO can be found in Refs. 149–152.

Detection of nitroxyl

Nitroxyl can be detected by observing the dimerization product, N₂O, by gas chromatography (GC) (152). It can also be detected by membrane inlet MS, in which HNO traverses a membrane before reaching the mass spectrometer (153) (Fig. 5, A and B).

Figure 5.

Detection of nitroxyl. A, detection based on the HNO dimerization product N₂O by GC. B, membrane inlet MS for the detection of HNO and its decay products. C, reaction with metalloporphyrins for spectrophotometric detection. D, electrochemical sensor based on the reaction of HNO with a cobalt(III) porphyrin. E, fluorogenic probe based on the reaction of copper(II) to copper(I). F, reaction between HNO and a nitroxide TEMPO derivative to form NO^• and a hydroxylamine which, appropriately derivatized, is fluorescent. G, reaction between HNO and a 2-mercapto-2-methylpropionic acid fluorogenic derivative. H, reaction of HNO with an ester of 2-(diphenylphosphino)benzoic acid to give a benzamide phosphine oxide and an alcohol that, appropriately derivatized, is fluorescent. Some protons are omitted for simplicity.

Iron(III) porphyrins react with HNO forming nitrosyl–iron(II) porphyrins (154). The products can be detected both spectrophotometrically and by the typical three-line EPR signal of a ferrous nitrosyl complex under anaerobic conditions (155). Manganese(III) porphyrins also react with HNO leading to a large shift in the UV-visible Soret band, which can be used for colorimetric detection of HNO (Fig. 5C) (156).

Cobalt(III) porphyrins react with HNO and constitute the basis of an amperometric electrochemical sensor for HNO (Fig. 5D). In the resting state, the polarized electrode (0.8 V) contains Co(III) porphyrin. When the porphyrin reacts with HNO it forms a Co(III)–NO⁻ complex that is oxidized releasing NO^• and the Co(III) porphyrin, ready for another cycle. The current intensity is proportional to HNO, and the sensitivity is in the nanomolar range. The success of the electrode is based on the fact that HNO reacts with Co(III) and not with Co(II) porphyrins, whereas NO^• reacts with Co(II) and not with Co(III). This is an advantage of Co(III) over Fe(III) porphyrins, which react both with NO^• and HNO (149, 157, 158).

Nitroxyl can reduce Cu(II) to Cu(I) and NO^•. This is the basis of a group of fluorogenic probes in which the reduction of the metal ion is concomitant with the release of fluorescence quenching (Fig. 5E). The probes should be used with caution for the potential reduction by other reductants, as well as interference from hydrogen sulfide (H₂S), S-nitrosothiols and oxygen (159–161).

Nitroxyl reacts with stable nitroxide free radicals such as (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO) with rate constants of 10⁴–10⁵ m⁻¹ s⁻¹ forming the hydroxylamine and NO^• (Fig. 5F) (162, 163). Fluorogenic TEMPO derivatives have been prepared in which the nitroxide group quenches the fluorescence, which is released when the nitroxide is converted to the hydroxylamine (160, 164). Due to the complex chemistry and to the potential to react with other reductants and oxidants, the use of the nitroxide probes in biological systems is limited.

Nitroxyl reacts fast with thiols. The formation of GSH sulfinamide (GS(O)NH₂) from the reaction of GSH with HNO can be used as footprint for HNO. An N-hydroxysulfenamide is formed as an intermediate, and the final sulfinamide can be separated and detected by HPLC or MS (Equation 13) (165, 166).

$$\text{RSH}+\text{HNO}\to \text{RSNHOH}\to \text{RS}\left(\text{O}\right){\text{NH}}_2$$ (Eq. 13)

A probe has been developed that consists of an ester of 2-mercapto-2-methylpropionic acid and a fluorescent compound. The reaction of HNO with the thiol forms an N-hydroxysulfenamide intermediate that cyclizes releasing the fluorophore (Fig. 5G) (160, 167).

Nitroxyl reacts fast with arylphosphines to yield phosphine oxides and azaylides (Equation 14). The rate constants are in the order of 10⁶ m⁻¹ s⁻¹ (168, 169). The azaylides are indicative of the formation of HNO and can be detected by NMR and MS, although, depending on the phosphine used, they may hydrolyze to the corresponding phosphine oxide. Although arylphosphines are resistant to reductants, possible interference by S-nitrosothiols is a potential concern (170).

$$2\hspace{0.17em}{\text{R}}_3\text{P}+\text{HNO}\to {\text{R}}_3\text{P}=\text{O}+{\text{R}}_3\text{P}=\text{NH}$$ (Eq. 14)

The azaylides are nucleophilic and can react with an adjacent electrophilic group such as an ester or a carbamate. When the azaylide attacks the carbonyl, alcohol is released, and a unique amide phosphine oxide product is formed (Fig. 5H). This product, as well as the alcohol, can serve as reporters for HNO. The hydrolysis of the probe should be controlled as well as possible interference from S-nitrosothiols (160, 169–171).

Despite the progress in the development of methods to measure HNO, the potential limitations should be carefully addressed. More than one method should be used, preferentially in combination with HPLC or MS detection of HNO-specific products (160).

Peroxynitrite

Peroxynitrite (ONOO⁻) and peroxynitrous acid (ONOOH) are formed through the diffusion-controlled reaction between O₂^•− and NO^• (Equation 15). IUPAC names for ONOO⁻ and ONOOH are (dioxido)oxidonitrate(1−) and (hydridodioxido)oxidonitrogen, respectively. In this text, the term peroxynitrite is used for the mixture of ONOO⁻ and ONOOH, unless specified.

$${\text{NO}}^{•}+{{\displaystyle {\text{O}}_2}}^{•-}\to {\text{ONOO}}^{-}$$ (Eq. 15)

Peroxynitrite is a powerful one- and two-electron oxidant; the reduction potentials are E⁰′ (ONOOH, H⁺/NO₂^•, H₂O) = 1.6 V and E⁰′ (ONOOH, H⁺/NO₂⁻, H₂O) = 1.3 V (172). Peroxynitrous acid can traverse membranes through simple diffusion, whereas ONOO⁻ can use anion channels. The anion is a good nucleophile, and ONOOH can act as an electrophile. In buffer, ONOOH (pK_a 6.8, Equation 16) can decay to nitric acid (HNO₃) plus a 30% fraction of HO^• and NO₂^• radicals (Equation 17), but this process (k = 0.9 s⁻¹ at pH 7.4 and 37 °C) is relatively slow and has limited physiological significance. The most relevant biological targets for peroxynitrite are peroxiredoxins, GSH peroxidases, CO₂, and metal centers (1, 7, 86). The peroxiredoxins are thiol-dependent peroxidases that constitute the most efficient peroxynitrite scavengers known to date, with rate constants of ∼0.1–10 × 10⁷ m⁻¹ s⁻¹ and high concentrations in different cellular compartments (173). In addition, peroxynitrite can react fast (k ∼10⁴ m⁻¹ s⁻¹ at pH 7.4) (174) with CO₂, which is abundant in tissues (1.2 mm), leading to the formation of secondary radicals, CO₃^•− and NO₂^•, in 33% yield (Equation 18) (89).

$${\text{ONOO}}^{-}+{\text{H}}^{+}\rightleftharpoons \text{ONOOH}$$ (Eq. 16)

$$\text{ONOOH}\to 0.7\hspace{0.17em}{\text{HNO}}_3+0.3\hspace{0.17em}{\text{NO}}_2^{•}+0.3\hspace{0.17em}{\text{HO}}^{•}$$ (Eq. 17)

$${\text{ONOO}}^{-}+{\text{CO}}_2\to \left[{\text{ONOOCO}}_2^{-}\right]\to 0.67\hspace{0.17em}{\text{NO}}_3^{-}+0.33\hspace{0.17em}{\text{NO}}_2^{•}+0.33\hspace{0.17em}{\text{CO}}_3^{•-}$$ (Eq. 18)

The reactions with metal centers are diverse. Peroxynitrite can be reduced by one electron yielding NO₂^• as the metal center is oxidized, or by two electrons yielding NO₂⁻. In addition, some hemeproteins (e.g. methemoglobin) catalyze peroxynitrite isomerization to NO₃⁻, whereas others (e.g. Fe(III) cytochrome c) do not react at all (1, 7).

Peroxynitrite detection

Because of its short life in cells and tissues (∼1 ms) (7), peroxynitrite cannot be detected in biological samples through direct spectroscopic techniques. Nevertheless, the UV absorbance of ONOO⁻ (ϵ₃₀₂ = 1700 m⁻¹ cm⁻¹) (175) has proven to be very useful for the quantification of stock solutions in the laboratory at alkaline pH, as well as for following ONOO⁻ decay in stopped-flow kinetic experiments.

One analytical approach for the detection of peroxynitrite is the use of probes that react with peroxynitrite itself or with its downstream radicals (NO₂^•, CO₃^•−, and HO^•). Because the specificity of the probes is not always straightforward, particularly for the latter, the modulation of O₂^•− and NO^• formations, which are the precursors of peroxynitrite, should accompany the results obtained with probes. Another analytical approach to evidence the involvement of peroxynitrite in a certain biological process is the detection of nitrotyrosine, a stable product formed from the reaction of radicals derived from peroxynitrite with tyrosine residues. As in the case of the probes, confirmatory evidence is required. These approaches are described in the next sections.

A growing number of small fluorogenic organic molecules designed and synthesized to detect peroxynitrite are reported constantly, having different selectivity and sensitivity toward this oxidant. The basic common characteristic is to have weak basal fluorescence, which is largely increased upon oxidation (176–178). In terms of the reaction mechanism, fluorogenic probes can be divided in two main groups: 1) probes that react with the radicals derived from peroxynitrite and yield a fluorescent end product by a radical mechanism; and 2) probes that react directly through a nucleophilic attack by peroxynitrite anion (ONOO⁻) to a particular functional group of the electrophilic probe, releasing masked fluorescence. The probes that react directly with ONOO⁻ are potentially more straightforward, specific, and quantitative. They must react fast (>10⁵–10⁶ m⁻¹ s⁻¹) and outcompete other routes of decay. Besides, genetically-encoded fluorescent protein sensors for peroxynitrite have been described recently (179, 180). They use similar principles as some of the chemical probes that lead to direct detection of this oxidant (i.e. boronate-based compounds, see below).

Importantly, detection methods based on probes reveal only a minor fraction of total peroxynitrite, because a large proportion reacts with other targets in the cell. Moreover, the fraction trapped by the probe may vary with cell type or metabolic state according to the abundance of alternative targets (181). For a full review of chemical probes for peroxynitrite detection, see Refs. 176, 181.

Probes that react with the radicals derived from peroxynitrite

Probes frequently used for oxidant detection in biological systems are reduced dyes like 2′,7′-dichlorodihydrofluorescein (DCFH₂) and dihydrorhodamine (DHR-123). Although extensively used, they present a series of limitations and caveats (5). The general reaction mechanism is a one-electron oxidation by potent one-electron oxidants such as those derived from peroxynitrite (NO₂^•, CO₃^•−, and HO^•) (182) yielding a radical intermediate (DCF^•−), which is afterward oxidized to highly resonant moieties responsible for the increase in fluorescence emission (DCF) (Fig. 6A). These probes do not react directly with peroxynitrite (178, 183). Neither NO^• nor O₂^•− are able to oxidize either probe at significant yields; however, these radicals may react with the radical intermediate (DCF^•−) in termination reactions giving nonfluorescent products (184). In addition to peroxynitrite-derived radicals, other potent one-electron oxidants such as those produced from heme peroxidases and other metalloproteins in the presence of H₂O₂ can generate fluorescent DCF (177). Thiyl radicals (RS^•) derived from the oxidation of GSH can also oxidize DCFH₂ with a significantly high rate constant of ∼10⁷ m⁻¹ s⁻¹ at pH 7.4 (185). Moreover, DCF-dependent fluorescence can be self-amplified by redox-cycling of the one-electron oxidized dye (5).

Figure 6.

Detection of peroxynitrite. A, mechanism of 2′,7′-dichlorodihydrofluorescein (DCFH₂) oxidation. The reduced probe (DCFH₂) is oxidized by peroxynitrite-derived radicals and other one-electron oxidants yielding a radical intermediate (DCF^•−) that is oxidized by oxygen to yield fluorescent DCF. Thin arrows show alternative reactions and redox cycles. AH⁻ stands for ascorbate. B, boronate oxidation by peroxynitrite. The major pathway (>85%, above) consists of heterolytic cleavage of the peroxyl bond leading to phenol which, appropriately derivatized, is fluorescent. The minor radical pathway (below) involves homolytic cleavage of the peroxyl bond giving NO₂^• and a phenyl-type radical that yields the nitro-derivative (201). C, structures of boronate-derived probes. CBA and CBE indicate a boronic acid or a boronic pinacolate ester attached to a coumarin scaffold, respectively (193). Fl-B (194), FlAmBE (192), and FBBE (205) are boronic pinacolate ester derivatives linked to a fluorescein scaffold with structural modifications as shown. D, genetically-encoded boronate-based GFP for the detection of peroxynitrite. Nucleophilic attack by the peroxynitrite anion to the phenylalanine boron moiety results in the formation of tyrosine and fluorescence. Modified from Ref. 176.

Luminol chemiluminescence can also be used to detect the radicals derived from peroxynitrite (NO₂^•, CO₃^•−, and HO^•) as well as other strong one-electron oxidants. The mechanism involves the one-electron oxidation of luminol to initiate the reactions that lead to light emission. Chemiluminescence is increased in the presence of CO₂ and inhibited by thiols, urate, and NO^• (176, 184, 186).

The lack of specificity of these probes constitutes an important limitation. DCFH₂, DHR, and luminol are not specific for peroxynitrite and cannot be used as unique tools. These probes may be useful to detect an overall increase in oxidant generation in cells, but they do not allow us to identify the particular species involved. Their use for the detection of peroxynitrite or any other particular oxidant is ambiguous and inconclusive and should be avoided unless complemented with other more specific methods.

Probes that react directly with peroxynitrite anion

Another mechanistic possibility for the detection of peroxynitrite involves a nucleophilic attack by the oxidant to an electrophilic functional group supported on the probe, leading to fluorescence. Different functional groups have been proposed as electrophilic centers, like activated ketones (187–189), α-ketoamides (190, 191), and boronates (boronic acids or boronic esters) (192–194).

A group of probes named HKGreen (HK is Hong Kong) numbered 1–4, in which the electrophilic center is an activated ketone, has been reported for peroxynitrite detection. Structurally, HKGreen-1 holds a trifluoro-ketone linked to a dichlorofluorescein scaffold through an aryl ether linkage (187); HKGreen-2 holds a trifluoro-ketone linked to BODIPY through phenol (188); and HKGreen-3 holds a trifluoro-ketone linked to a rhodol scaffold (189). All of them can react with peroxynitrite by a mechanism where the ketone generates a dioxirane intermediate upon reaction with the oxidant, which oxidizes the phenyl ring and releases the fluorescent molecule. The poor yields of the reactions of HKGreen-1–3 with peroxynitrite (187–189) limit their utility in biological systems.

Another possible electrophilic functional group is α-ketoamide. DCM-KA, an α-ketoamine moiety attached to dicyanomethylene-4H-pyran, was recently proposed (191). Although it selectively responds to peroxynitrite with fluorescence emission in the near-IR region (λ_em >600 nm), the probe has a slow response (90 s), although complete kinetic analyses are lacking (191).

The arrival of organoborane compounds to the redox biology field over the last decade represented a major breakthrough. Boronate-based probes are successfully applied to the assessment of peroxynitrite in biological systems (192, 194). Even though they were originally conceived for hydrogen peroxide detection (195, 196), it was later demonstrated that boronate derivatives react considerably faster with peroxynitrite than with hydrogen peroxide (192–194) (see below). These compounds are particularly promising due to the simplicity of their reaction with peroxynitrite and are arising as the most suitable probes for detecting and even quantifying this oxidant. They can also be directed to compartments (e.g. mitochondria) (197).

Structurally, boronic acids are trivalent boron-containing organic compounds that hold one alkyl or aryl substituent and two hydroxyl groups to fill the remaining valences on the boron atom (RB(OH)₂). Replacement of the hydroxyl groups of boronic acids by alkoxy or aryloxy groups gives boronic esters (RB(OR′)₂). The sp²-hybridized boron atom possesses a vacant p orbital due to the deficiency of two electrons and contains only six valence electrons, thus making boronic acids and boronic esters electrophilic centers and mild Lewis acids (198). Therefore, several nucleophiles like peroxynitrite (ONOO⁻), ionized hydrogen peroxide (HOO⁻), peroxymonocarbonate (HCO₄⁻), amino acid hydroperoxides (ROO⁻), or hypochlorite (ClO⁻) are prone to react with the electrophilic center of boronates (Equation 19). The rate constant of the reaction with ONOO⁻ is several orders of magnitude higher (k_ONOO− ∼10⁶, k_ClO− 10², k_HCO4− 170, k_ROO− ∼20, and k_H2O2 ∼1–2 m⁻¹ s⁻¹) at physiological pH (193, 194, 199, 200).

$$\text{ArB}{\left(\text{OR}\right)}_2+{\text{ONOO}}^{-}\to \text{ArOH}+{{\displaystyle {\text{NO}}_2}}^{-}+\text{HO-B}{\left(\text{OR}\right)}_2$$ (Eq. 19)

The reaction mechanism with peroxynitrite, similar to that with H₂O₂, involves a nucleophilic addition of ONOO⁻ (or HOO⁻) to the electrophilic boron atom generating an anionic quaternary intermediate that suffers a subsequent heterolytic cleavage at the peroxyl bond and hydrolysis giving phenol as the major nonradical end product (∼85–99% yield) (194, 201). The phenol, appropriately derivatized, is fluorescent. In addition, the reaction with ONOO⁻ typically involves a minor pathway where homolytic cleavage of the peroxyl bond gives radical intermediates, NO₂^• and a phenyl-type radical, that combine to form a nitrobenzene-type product. This product is a peroxynitrite footprint, because the major phenol product is common for all ROO⁻ or ClO⁻ (Fig. 6B) (201). The mechanism of oxidation of boronates by ONOO⁻ has been studied, both experimentally and theoretically, using density functional theory calculations (193, 201). Moreover, work using isotope-labeling studies is ongoing to track the origin of oxygen atoms in the oxidation products of the redox probes.

Although the high rate constant predicts preferential reaction of boronate probes with peroxynitrite versus other oxidants, confirmation of peroxynitrite involvement in a certain process should be provided by the detection of the products of the minor radicalar pathway of reaction, as well as by modulation of the formation and decay of peroxynitrite precursors.

Several boronic acids or boronic esters attached to different fluorescent scaffolds (coumarin-derivatives (202–204), fluorescein-derivatives (192, 194, 205), or BODIPY-derivatives (206, 207)) are described as fluorogenic probes, where the reaction with the oxidant releases the oxidized fluorescent product (Fig. 6C).

Fluorescein-dimethylamide boronate (FlAmBE) (192), fluorescein-boronate (Fl-B) (194), and 4-(pinacol boronate)benzyl-derivative fluorescein methyl ester (FBBE) (205) were recently reported as fluorogenic probes derived from modified fluorescein attached to a pinacol boronic ester for monitoring peroxynitrite in biological systems. Unlike Fl-B or FlAmBE, where there is a straight reaction mechanism with peroxynitrite as described above, the oxidative conversion of FBBE to the fluorescent product (fluorescein methyl ester) is a two-step reaction (205). The reaction of an oxidant (ROO⁻ or ClO⁻) toward the boronate group leading to the corresponding phenol is the first step of this process. The second step is a slow p-methide quinone elimination leading to the formation of fluorescein methyl ester. Although the rate constant for the reaction of FBBE with ONOO⁻ is high (>10⁵ m⁻¹ s⁻¹), the buildup of the fluorescent product is slow (k = 0.09 s⁻¹), due to a gradual p-quinone methide elimination (205).

An improvement of boronate probes derived from fluorescein scaffolds with respect to coumarin scaffolds is that the spectroscopic properties of the former (FlAmBE: λ_ex = 485 nm and λ_em = 535 nm (192); Fl-B: λ_ex = 492 nm and λ_em = 515 nm (194); and FBBE: λ_ex = 494 nm and λ_em = 518 nm (205)) allow their use in common laboratory equipment for cellular assays, such as flow cytometry and epi-fluorescence microscopy. In contrast, other reported probes such as coumarin-7-boronic acid (CBA) (202) and/or coumarin-7-boronic acid pinacolate ester (CBE) (λ_ex = 332 nm and λ_em = 470 nm) present limitations for those techniques. Moreover, although coumarin-derived and fluorescein-derived boronic probes react with comparable kinetic rate constants toward peroxynitrite (k ∼1 × 10⁶ m⁻¹ s⁻¹), Fl is a more efficient fluorophore than 7-hydroxycoumarin (COH), and it has a higher molar absorption coefficient (ϵ_{Fl, 490 nm} = 76,900 m⁻¹ cm⁻¹; ϵ_{COH, 323 nm} = 11,200 m⁻¹ cm⁻¹) and higher quantum yield (Φ_{F, Fl} = 0.93; Φ_{F, COH} = 0.15) (208, 209). Therefore, Fl-B is more sensitive than CBA. Recently, Fl-B enabled the detection of basal rates of peroxynitrite generation in vascular endothelial cells dependent on endothelial NOS activation, secondary to ionomycin-induced calcium ion influx (194).

Curiously, a boronate-derived probe named GSH-ABAH, supported on 4-amino-2-(benzo[d]thiazol-2-yl)phenol (ABAH) and with a thiol-reactive maleimide substituting the amino group, is proposed to simultaneously detect ONOO⁻ and biological thiols by a combination of excited state intramolecular proton transfer and photoinduced electron transfer, giving an increase in fluorescence emission when both analytes are present (210). This unusual strategy may need an extensive characterization of the reaction and independent validation under biologically-relevant conditions.

Recently, a novel genetically-encoded boronate-based GFP was reported for the detection of peroxynitrite in mammalian cells (179, 180). GFP fluorescence requires the presence of a key tyrosine residue (Tyr-66) that together with Ser-65 and Gly-67 participate in a cyclized structure inside a protein barrel that constitutes the actual fluorophore (211). Modern synthetic biology techniques allow the incorporation of unnatural amino acids during protein translation, by which Tyr-66 is substituted by phenylalanine boronic acid (p-B(OH)₂Phe), resulting in a nonfluorescent version of GFP. Upon exposure to peroxynitrite, the boronate-modified Phe is converted to Tyr generating the actual GFP fluorophore (Fig. 6D). It is interesting to point out that GFP had been previously proposed as a way to follow the formation of peroxynitrite and other nitrating species (59). However, in this early report, the critical Tyr-66 became nitrated to 3-nitrotyrosine, which resulted in a decrease of fluorescence intensity.

Because the available methodologies for peroxynitrite detection have a series of limitations, there are several practical aspects that must be taken into consideration. When studying peroxynitrite generation in biological systems, the pharmacological modulation is mandatory to confirm the identity of the radical precursors and oxidants that are being generated. For instance, the use of enzyme inhibitors is useful for unequivocally identifying peroxynitrite formation (i.e. modulation by NOS inhibitors or by suppression of O₂^•− formation). For a full discussion on practical considerations when assessing peroxynitrite in cells using fluorogenic probes, see Refs. 176, 181.

3-Nitrotyrosine

Peroxynitrite can lead to the nitration of free and protein-bound tyrosines. The mechanism of tyrosine nitration is free radical-dependent and does not directly involve peroxynitrite. Instead, it involves a strong one-electron oxidant and NO₂^•. The initial one-electron oxidant can be CO₃^•−, NO₂^•, oxometal complexes such as Compounds I and II of hemeperoxidases, HO^•, and lipid peroxyl radicals (LOO^•), among others (Fig. 7). Tryptophan, nucleic acid bases, and polyunsaturated fatty acids can also become nitrated, but the better studied is tyrosine (1).

Figure 7.

Free radical-mediated formation of nitrotyrosine. Strong one-electron oxidants oxidize tyrosine to tyrosyl radical, which combines with NO₂^• forming 3-nitrotyrosine. Some protons are omitted for simplicity.

Due to the radical nature of the mechanism of tyrosine nitration, other sources of one-electron oxidants and NO₂^• that do not involve peroxynitrite can give rise to 3-nitrotyrosine. This is the case of hemeperoxidases in the presence of hydrogen peroxide and NO₂⁻ (82). In addition, tyrosine can also become nitrated by the reaction of tyrosyl radicals with NO^• followed by two oxidation steps (212). Nitrating species can also be generated by NO₂⁻ under acidic conditions (213). Thus, the detection of 3-nitrotyrosine alone cannot be considered a specific footprint of peroxynitrite unless in the light of additional confirmatory evidence.

Nitration modifies the properties of the tyrosine. First, the acidity of the phenolic group is increased (pK_a of 3-nitrotyrosine 7.5), generating an extra negative charge in the protein at neutral pH. This is useful for separative strategies based on isoelectric focusing of nitrated proteins (214, 215). Second, the 274 nm absorbance maximum shifts to 360 nm at acidic pH (ϵ₃₆₀ = 2790 m⁻¹ cm⁻¹) or 440 nm at alkaline pH (ϵ₄₄₀ = 4400 m⁻¹ cm⁻¹) (184). Although this red shift is useful for chromatographic separations with UV-visible detection and for the quantification of 3-nitrotyrosine standards, it can lead to photochemical decomposition in MALDI-based MS approaches with 337 nm lasers. Finally, the hydrophobicity of a 3-nitrotyrosine-containing peptide increases with respect to the unmodified one. 3-Nitrotyrosine is considered a relatively stable end product. Putative 3-nitrotyrosine denitrase and reductase activities that catalyze the removal of the nitro group or its reduction to amino in 3-nitrotyrosine-containing proteins, respectively, have been reported in mammalian tissues and in bacteria. The enzymes responsible have not yet been identified (216–218).

Tyrosine nitration is a low-yield process. For instance, peroxynitrite-dependent tyrosine nitration in phosphate buffer ranges from ∼6 to ∼18% of the peroxynitrite added in the absence or presence of bicarbonate, respectively, and much less in the presence of competing biomolecules (174, 219). In cells and tissues, a relatively small number of proteins is found significantly nitrated and in only a few tyrosine residues. This introduces challenges in the analytical strategies, because the detection of about one 3-nitrotyrosine within one million tyrosines is required (1, 4, 220–222).

Another challenge is the artifactual formation of 3-nitrotyrosine during sample processing, for example, from residual NO₂⁻ under acidic conditions. This is a major methodological concern that has contributed to the thousand-fold scattering of 3-nitrotyrosine values in biological samples (222–225). Separation of 3-nitrotyrosine from its precursors (226) or reduction to 3-aminotyrosine by sodium dithionite (227), early in sample preparation protocols, is useful in preventing artifactual nitration. In MS-based procedures, tyrosine labeled with stable isotopes can be added to the sample to control for artifactual nitration, in addition to serving as internal standard for tyrosine measurement (224, 228).

The analytical challenges increase even further if the aim is to detect a nitrated peptide or protein rather than free 3-nitrotyrosine, because there is the need to extract, separate, and enrich modified proteins and to hydrolyze them into peptides or amino acids.

Several approaches have been developed for the qualitative and quantitative assessment of free or protein-bound 3-nitrotyrosine. These methods include immunoassays, HPLC with various detection methods, and gas or liquid chromatography coupled to MS. Due to the potential for sensitivity and selectivity, strategies based on MS have become the gold standard of 3-nitrotyrosine determination and have started to yield reference values. For example, the concentration of 3-nitrotyrosine in plasma is subnanomolar (222). A thorough discussion of the method of validation applied to 3-nitrotyrosine analysis can be found in Refs. 221, 222. In the following sections, a brief description of available methods is provided.

Antibody-based methods for 3-nitrotyrosine detection

Nitration changes the immunogenicity of a protein through the generation of new epitopes. This has biomedical consequences in the triggering of immune reactions and in the generation of autoimmune responses (229, 230). In addition, this is the basis of procedures for the detection of 3-nitrotyrosine rooted on the use of antibodies. Poly- and monoclonal antibodies have been raised and are now commercially available. Immunohistochemical methods, immunoblotting techniques, and ELISAs have been developed and applied to the study of biological samples (184, 231–234). The typical readout of antibody-based methods is the difference in immunoreactivity in samples versus controls. However, procedures are subject to wide variations, particularly regarding the selectivity of the antibody, and unless validated, they lead to qualitative or at best semi-quantitative results (223). Recently, a highly-sensitive electrochemiluminescence-based ELISA was introduced for the assessment of biological samples and validated with MS (235).

Analysis of free 3-nitrotyrosine by HPLC-based methods

HPLC with UV-visible detection (236) or fluorescence detection (following derivatization with fluorogenic compounds) (237) has been employed to measure 3-nitrotyrosine. However, these approaches are not sensitive or selective enough for their application to biological samples. Reduction to 3-aminotyrosine followed by HPLC with electrochemical detection presents improved sensitivity (225), but it cannot match the selectivity provided by MS-based approaches (222, 223).

Analysis of free 3-nitrotyrosine by GC-MS, GC-MS/MS, and LC-MS/MS

Strategies based on GC for the separation of the analytes must follow the conversion of 3-nitrotyrosine to a volatile form, either by reduction to 3-aminotyrosine and/or derivatization. Particularly useful is the incorporation of fluorine atoms in the derivative through the use of fluorinated reagents in the case of MS procedures based on negative ion electron capture detection (226, 238–240). The perfluorinated derivatives undergo electron capture and dissociation to yield a single intense anion that can be monitored by selected ion monitoring (GC-MS analysis). Subsequent analysis by selected reaction monitoring after collision-induced dissociation (GC-MS/MS) increases selectivity.

The use of LC instead of GC presents the advantage that no derivatization steps are needed. Positive electrospray ionization mode is usually used. LC-MS is not selective enough, but collision-induced dissociation of 3-nitrotyrosine in LC-MS/MS analysis produces a few characteristic ions that can be used for quantification. Specifically, protonated unlabeled 3-nitrotyrosine (m/z 227) yields species with m/z 181 and m/z 90.

In both GC and LC-based MS methods, quantitative data can be obtained by comparison with the signals produced by an internal standard, which allows corrections for variability, sample losses, and matrix effects. The internal standard for MS consists of 3-nitrotyrosine labeled with the stable isotopes ¹³C or ¹⁵N. These isotopic homologues behave identically to nonlabeled 3-nitrotyrosine in extraction, chromatography and ionization steps but provide ions with increased m/z. Some of these standards are commercially available. In a typical procedure, labeled 3-nitrotyrosine is added to the samples and the calibration standards in known amounts. The area or height of the desired peak is related to that of the internal standard in the samples and is compared with the calibration data. The inclusion of labeled tyrosine in addition to labeled 3-nitrotyrosine allows the quantification of total tyrosine and the control of artifactual nitration (224, 228). For example, in a typical procedure 3-[¹³C₆]nitrotyrosine is added in a known amount to a sample as internal standard for 3-nitrotyrosine quantification; [¹³C₉¹⁵N₁]tyrosine is added as internal standard for total tyrosine quantification, and [¹³C₆]tyrosine is added to control for potential artifactual generation of 3-nitrotyrosine during sample processing (241).

Analysis of 3-nitrotyrosine in proteins

Quantitative data on the amount of 3-nitrotyrosine in a protein-containing sample can be obtained by extraction of total proteins followed by hydrolysis by chemical or enzymatic methods and, finally, analysis of free 3-nitrotyrosine by the procedures described above. The extraction step, typically with an organic solvent, is useful for NO₂⁻ elimination as well as protein precipitation. Total hydrolysis can be achieved with 6 m hydrochloric acid at 116 °C in vacuo, following NO₂⁻ removal (225, 228). Alkaline hydrolysis has also been tried (239). Alternatively, a mixture of proteolytic enzymes (Pronase) can be used, but the former is preferred, because enzymatic hydrolysis is not always complete, and the enzymes themselves can contribute to the values of tyrosine and 3-nitrotyrosine measured.

If information on a particular protein instead of a whole mixture is required, separation or enrichment steps can be included. These steps include mono- or two-dimensional electrophoresis and chromatographic or immunoaffinity procedures. A useful aspect to take into account in the design of separation steps is the increase in acidity of tyrosine residues upon nitration, which leads to a decrease in the pI of the protein (214, 215).

To reveal the sites of nitration in the proteins, peptide mapping can be performed by hydrolysis with trypsin to obtain the tryptic fragments followed by LC-MS/MS to separate the peptides and analyze them. The modified peptides are then identified by search algorithms. Importantly, the results should be confirmed through targeted MS/MS approaches, in which prior knowledge of the modification site allows for selected/multiple reaction monitoring and parallel reaction monitoring (221, 241, 242).

In MALDI-based procedures for peptide and protein analysis, 3-nitrotyrosine residues absorb the light of typical lasers (337 nm) and produce a characteristic pattern of ions in which the m/z is decreased by 16 and 30. The unique triplet signal enables to further identify the nitrated peptides (243, 244). Nevertheless, methods based on electrospray ionization present the advantages that photochemical decomposition is avoided and that the typical ion with m/z 181 can be detected and serve as a footprint for 3-nitrotyrosine (221).

A typical proteomics approach for the detection of nitrated proteins in complex samples would involve two-dimensional separation, immunoblotting, manual removal of selected spots, in-gel digestion, and MS/MS identification, with confirmation by targeted approaches (220–222, 245–247). A critical discussion of caveats in nitroproteomics can be found in Refs. 220–222.

Carbonate anion radical

Carbonate anion radical (CO₃^•−, IUPAC name trioxocarbonate(−1)) is a strong one-electron oxidant with a standard reduction potential (E⁰′ (CO₃^•−, H⁺/HCO₃⁻)) of +1.77 V (248). In biological systems, it can be generated by the homolysis of the initial adduct formed by the reaction of ONOO⁻ and CO₂, ONOOCO₂⁻. Although some debate exists in relation to the lifetime of the adduct and the yields of the CO₃^•− and NO₂^• radicals (249), it is now accepted that the adduct has a very short half-life (<1 μs) that precludes its participation in bimolecular reactions and that it homolyzes to CO₃^•− and NO₂^• in ∼33% yields. The experimental and theoretical evidence in support of the radical model has been recently reviewed (89).

Carbonate anion radical can also be formed in biological systems by the one-electron oxidation of bicarbonate or its conjugate base, carbonate anion (CO₃²⁻), by strong enough one-electron oxidants such as HO^• (E⁰′ = +2.31 V at pH 7) (250, 251). This route is expected to be of relevance inside the phagosomes of activated immune cells, where bicarbonate is considered to be a main target for HO^• (252). Compounds I and II of heme peroxidases cannot oxidize HCO₃⁻ (93). In contrast, CO₂/HCO₃⁻ enhances the peroxidatic activity of Cu,Zn-superoxide dismutase (253, 254), and although the mechanism is controversial, it leads to CO₃^•− formation through an enzyme-bound intermediate (199, 255, 256). Xanthine oxidase can also lead to CO₃^•− (257–259).

The pK_a of HCO₃^· is <0, indicating that the radical only exists in its anionic form in biological systems (260, 261). Accordingly, it cannot diffuse through membranes (262), and it is confined to water-soluble compartments. It can react both by electron transfer or hydrogen abstraction mechanisms (259). It can also participate in oxygen transfer reactions, particularly when reacting with itself or with other free radicals. Furthermore, although some examples of CO₃^•− addition reactions have been proposed, the products formed are usually unstable, which makes their detection difficult.

The main intracellular targets of CO₃^•− are thiol-containing moieties, particularly GSH due to its high cellular abundance (k = 5.3 × 10⁶ m⁻¹ s⁻¹ at pH 7) (93, 263), solvent-exposed sulfur-containing residues (k = 4.5 × 10⁷ and 3.6 × 10⁷ m⁻¹ s⁻¹ at pH 7 for Cys and Met, respectively), as well as aromatic residues in proteins (k = 4.6 × 10⁷ and 7 × 10⁸ m⁻¹ s⁻¹ at pH 7 for Tyr and Trp, respectively), ascorbate (k ∼10⁹ m⁻¹ s⁻¹ at pH 11.4), guanine bases in nucleic acids (k ∼10⁸ m⁻¹ s⁻¹), and particularly in plasma, urate (k_urate estimated to be ∼10⁸ m⁻¹ s⁻¹) (86, 264). Carbonate anion radical promotes protein–protein (particularly ditryptophan) or DNA–DNA cross-links (265, 266). It also rapidly reacts with transition metal centers or organic cofactors (267–269). Interestingly, bicarbonate enhanced the susceptibility of bacterial suspensions to ionizing radiation, which implies extracellular HO^• (252). The higher toxicity caused by extracellular CO₃^•− compared with HO^• is a consequence of the higher diffusion distances of the former, derived from its lower reactivity and longer lifetime.

Detection of carbonate anion radical

The lack of specific biomarkers for CO₃^•− complicates its detection, both in vitro or in vivo. Carbonate anion radical has a characteristic light absorption spectrum with a peak at 600 nm (ϵ₆₀₀ = 1930 m⁻¹ cm⁻¹) (260, 270). This has been utilized for measuring the kinetics of the reaction of the radical with different compounds by pulse radiolysis techniques (263, 264, 268). Time-resolved resonance Raman spectroscopy has also been used to characterize CO₃^•−, which shows a strong polarized and intense band at 1062 cm⁻¹ (261). Furthermore, CO₃^•− can be directly detected by fast-flow EPR where it shows a singlet band at g = 2.0113 (89, 271). Due to the short half-life of the radical, which is in the microsecond range, direct detection requires rapid or fast flow methodologies, which are not always available or require high reactant amounts (259). Alternatively, spin-trapping EPR methodologies have also been utilized. Indeed, the reaction of CO₃^•− with DMPO yields an initial unstable adduct, which has been proposed as the O-centered radical DMPO-OCO₂^·, that then decays to DMPO-OH^• (272, 273). Carbonate anion radical can also be detected by means of chemiluminescence. Spontaneous chemiluminescence has been proposed to rely on radical dimerization to an excited species (CO₂)₂* that decays to the ground state with light emission at ∼440 nm (Equations 20 and 21) (274–277).

$${{\displaystyle 2{\text{CO}}_3}}^{•-}\to {{\displaystyle {\left({\text{CO}}_2\right)}_2}}^{*}+{\text{O}}_2$$ (Eq. 20)

$${{\displaystyle {\left({\text{CO}}_2\right)}_2}}^{*}\to 2{\text{CO}}_2+hv$$ (Eq. 21)

Additionally, the presence of bicarbonate enhanced the yield of luminol chemiluminescence caused either by hydrogen peroxide plus catalyzers or by peroxynitrite, which can be ascribed to CO₃^•− formation (186, 257). This is in accordance with CO₃^•− oxidation of aromatic and heterocyclic molecules with second-order rate constants ranging from 5 × 10⁵ to 5 × I0⁷ m⁻¹ s⁻¹ at pH 7 (263, 278).

Hydroxyl radical and its detection

The extremely reactive hydroxyl radical (HO^•) can be formed from the homolysis of peroxynitrous acid, which gives NO₂^• and HO^• in 30% yield, while the rest decays to nitric acid (Equation 16-18). This spontaneous decay has a relatively slow rate constant (0.9 s⁻¹ at pH 7.4 and 37 °C) and will be outcompeted by direct reactions with targets such as thiol or selenoproteins, metal centers, or CO₂, minimizing the relevance of peroxynitrous acid homolysis in biological HO^• generation (1). Nevertheless, it is possible that a very small amount of peroxynitrite-derived HO^• initiates oxygen-dependent chain reactions in which oxidation events are amplified, particularly in lipid-rich compartments (279). Undoubtedly, the formation of HO^• is relevant in in vitro systems in the absence of alternative targets that react directly with peroxynitrite. In fact, the detection of HO^• formation from peroxynitrite contributed to build the concept that NO^• can be the precursor of strong oxidants (280). In biological systems, HO^• is mainly generated through the Fenton reaction, i.e. through the reduction of hydrogen peroxide to HO^• and H₂O by reduced metal ions, Fe(II) or Cu(I). It can also be formed from photochemical processes (e.g. photolysis of hydrogen peroxide) and through the radiolysis of water. With a reduction potential of +2.31 V at pH 7 (250), HO^• is so oxidizing that it can react with most biomolecules. In addition, the rate constants are close to the diffusion limit, with values of 10⁹–10¹⁰ m⁻¹ s⁻¹. The mechanisms of HO^• reactions include one-electron abstraction, hydrogen atom abstraction, and addition to double bonds or aromatic rings.

The detection of HO^• can be carried out by different approaches that include laser-induced fluorescence and EPR associated with spin trapping (281). The kinetics of HO^• reactions have been studied by pulse radiolysis combined with competition kinetics. Hydroxyl radical can also be detected with the use of probes. Due to the very high reactivity of HO^• with biomolecules, very high probe loading is required, which can raise concerns. Because HO^• adds to aromatic rings, classical methods involve the hydroxylation of salicylic acid or phenylalanine (to m-tyrosine) (282). Often, the hydroxylation reaction can be followed by fluorescence. Various coumarins and many other probes have been developed recently and are reviewed in Refs. 177, 281, 283.

Some reflections on the detection of nitric oxide–derived oxidants

The fate of a certain species is determined by kinetics (i.e. rate constant multiplied by target concentration) as well as by compartmentalization. Detection methods based on probes reveal only a fraction of the total species, because a large fraction reacts with targets and remains undetected. Knowledge of the rate constants and concentrations of probe and targets can help estimate the actual amount of oxidant produced from the amount detected. For example, the rate of peroxynitrite formation that was detected in endothelial cells with a boronate probe was ∼0.01 μm s⁻¹, but it translated into a total of ∼0.1 μm s⁻¹ when the scavenging by other targets was taken into account (194).

Given the loose specificity of some of the methodologies for the detection of NO^•-derived oxidants, modulation of NO^• formation is mandatory. This can be pharmacologically achieved through the use of NOS inhibitors such as N-nitro-l-arginine methyl ester or N^G-monomethyl-l-arginine. Nitric oxide donors may be used to recapitulate the actions of NO^•-derived oxidants following NOS inhibition. Alternatively, genetically engineered cell lines or animals in which NOS are knocked out or overexpressed can be used (284). Complementary evidence can be achieved with inhibitors of the formation of other precursors. For example, the levels of the peroxynitrite precursor, O₂^•−, can be turned down with NADPH oxidase inhibition or superoxide dismutase overexpression. The role of NO^• in a certain process can also be probed using a scavenger such as oxyhemoglobin, which reacts fast with NO^• forming NO₃⁻. In this regard, the heterologous expression in mammalian cells of bacterial enzymes with strong NO^• dioxygenase activity has been proposed as a tool for NO^• depletion (285).

Finally, when a certain NO^•-derived oxidant or downstream product is detected in the context of a pathophysiological process, it is tempting to conclude that the relation is causative and that the NO^•-derived oxidant or downstream product participates in the development of the pathology. However, the relation may be only associative; the NO^•-derived oxidant or downstream product may constitute, at best, a biomarker. Further evidence involving pharmacological or genetic modulation of NO^•-dependent pathways and their impact on biological outcome is needed to reveal causality.

Concluding remarks

Three decades of research have contributed to building the concept that NO^• can exert biological effects both through the direct and usually reversible interaction with specific targets or through the generation of secondary species, many of which can oxidize, nitrosate, or nitrate biomolecules. The reactive species derived from NO^• are typically short-lived, and their preferential targets depend on kinetic and compartmentalization aspects. Their elusive character imposes technical challenges in their detection and quantification. In general terms, the strategies are based either on the detection of stable end products or on the use of synthetic probes. These methods are summarized in Table 1. Rigorous use of these strategies requires understanding of the mechanistic pathways involved. In many cases, probes are able to react with several species, and efforts are under way to expand the toolset with probes of suitable selectivity. The evidence about the role of a particular species in a certain pathophysiological process should combine data obtained through more than one analytical or immunochemical procedure as well as complementary evidence coming from the modulation of the formation or decay pathways of the species. A better identification and quantification of NO^•-derived oxidants and of their relevant molecular targets, strongly grounded in chemical and biochemical knowledge, will assist to expand rational applications of the NO^• field to biology and medicine.

Table 1.

Summary of methods for the detection of nitric oxide–derived oxidants

Method	NO•	NO2•	N2O3	NO2−	NO3−	DNICs	RSNO	HNO	ONOO−/ONOOH	CO3•−	HO•
Oxyhemoglobin oxidation, UV-visible, <1 μm	Yes, fast			Yes, but very slow					Yes, slower than NO•
NO•-selective electrode	Yes, in neutral buffer			No, but NO2− forms NO• in acid			No, only with Cu(II) and cysteine
EPR with spin traps	Yes, cPTIO (forms NO2•) or Fe(MGD)2	Yes, nitromethane and nitrone traps									Yes, DMPO and other traps
Direct EPR, 200 nm sensitivity	Nitrosyl heme with Fe(II) heme, 77 K					Yes		Nitrosyl heme with Fe(III) heme, 77 K		Fast flow
Fluorogenic diamino probes (DAN and DAF)	Not direct, but through N2O3 or oxidants and NO•		Yes, fast and direct reaction	Yes, in acidic pH
NOS activity, radiolabeled arginine	Yes, controls with NOS inhibitors and without NADPH
Ozone-based chemiluminescence, high sensitivity, 10 nm	Yes, neutral buffer in purge vessel	Reductant in purge vessel		Acidic triiodide in purge vessel		Acidic triiodide in purge vessel	Acidic triiodide in purge vessel
Luminol chemiluminescence		Yes							Not direct but through derived free radicals	Yes	Yes
DCFH2 and DHR fluorescence		Yes							Not direct but through derived free radicals	Yes	Yes
HPLC				Yes	Yes	Yes	Low Mr RSNO	Glutathione sulfinamide			m-Tyrosine or hydroxylated probes
UV spectrophotometry		Pulse radiolysis	Pulse radiolysis			Must be purified	Low sensitivity (50 μm)		Stocks and stopped-flow	Pulse radiolysis
Griess for nitrite detection (micromolar sensitivity)	No, but oxidation gives NO2−		No, but hydrolysis product is NO2−	Yes	Not direct, but after reduction to NO2−		Not direct, but with Hg(II)
Biotin-switch proteomics							Yes, provides protein identity
N2O detection								Yes
Oxidized metalloporphyrins, UV-visible or EPR								Yes, interference by reductants and NO•
Co(III)-porphyrin electrode								Yes
Cu(II)-fluorogenic probes								Yes, interference by reductants
Nitroxide probes								Yes, interference by reductants
2-Mercapto-2-methylpropionic acid fluorogenic probe								Yes, confirm specific product
Arylphosphine probes								Yes, RSNO interference, confirm specific product
Detection of 3-nitrotyrosine, several methods, MS confirmation desirable	Not direct, only in the presence of strong oxidants	Yes, product of NO2• with tyrosyl radical		Yes, in acid					Not direct but through derived free radicals	Forms tyrosyl radical	Forms tyrosyl radical
Boronic acid probes									Yes, direct and fast, H2O2 reaction very slow