Measurement of NO in biological samples

NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Measurement of nitric oxide in biological samples1 C Csonkaa,b, T Pálic, P Bencsika,b, A Görbea,b, P Ferdinandyb,d, T Csonta,b a Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Dóm tér 9, Szeged, H-6720 Hungary, Hungary; b c Pharmahungary Group, Hajnóczy u 6, Szeged, H-6723, Hungary; Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 85, Szeged, H-6726, Hungary; d Department of Pharmacology and Pharmacotherapy, Semmelweis University, Nagyvárad tér 4, Budapest, H-1089, Hungary page head: NO detection total word counts: 11050 Corresponding author: Csaba Csonka MD PhD Cardiovascular Research Group Department of Biochemistry, University of Szeged Dóm tér 9 H-6720, Szeged, Hungary This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/bph.12832 1 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Tel: +36 62 545755, fax: +36 62 545097 e-mail: csonka.csaba@med.u-szeged.hu 2 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Summary Although the physiological regulatory function of the gasotransmitter nitric oxide (NO, a diatomic free radical) has been discovered decades ago, NO is still in the frontline research in biomedicine. NO has been implicated in a variety of physiological and pathological processes, therefore, pharmacological modulation of NO level in various tissues may have significant therapeutic value. NO is generated by the NO synthases in most of the cell types and by non-enzymatic reactions. Measurement of NO is technically difficult, due to its rapid chemical reactions with a wide range of molecules, such as e.g. free radicals, metals, thiols etc. Therefore, there are still several contradictory findings on the role of NO in different biological processes. In this review, we briefly discuss the major techniques suitable for measurement of NO (electron paramagnetic resonance, electrochemistry, fluorometry) and its derivatives in biological samples (nitrite/nitrate, NOS, cyclic GMP, nitrosothiols) and provide evidences on the advantages and disadvantages of each method. We conclude that to obtain a meaningful insight into the role of NO and NO modulator compounds in physiological or pathological processes, concomitant assessment of NO synthesis, NO content, as well as molecular targets and reaction products of NO is recommended. Key words: selection, sensitivity, specificity, detection, peroxynitrite, assay, guideline, analytical chemistry, signalling, electrode, probe, spin trap Abbreviations: BODIPY, boron-dipyrromethene; diaminofluorescein; DAMBO, DAC, phenylenediamine diamino-BODIPY; DAN, cyanine; diaminonaphthalene; DAF DAR, 3 This article is protected by copyright. All rights reserved. NO detection Accepted Article diaminorhodamine; MS #2014-BJP-0167-RCT-G.R1 Csonka et al DBNB, 3,5-dibromo-4-nitrosobenzene; diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; DETC, DEPMPO, 5- N,N-diethyl-dithiocarmabate; DMPO, 5,5-dimethyl-pyrroline-N-oxide; DTC, dithiocarbamate; ELISA, enzyme-linked immunosorbent assay; ESR, electron spin resonance spectroscopy; EPR, electron paramagnetic resonance spectroscopy; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; GC, guanylate cyclase or gas chromatography; GC-MS, gas chromatography mass spectrometry; GMP, guanosine monophosphate; HPLC, high performance liquid chromatography; MGD, N-methyl-D-glucamine dithiocarmabate; MNP, 2-methyl-2- nitrosopropane; NADPH, reduced nicotinamide adenine dinucleotide phosphate; ONOO-, peroxynitrite; PBN, alpha-phenyl-N-tert-butyl-nitrone; POBN, alpha(4-pyridyl-1-oxide)-Ntert-butyl-nitrone); carboxy-PTIO, [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1oxide]; RSNO, S-nitrosothiols; sGC, soluble guanylate cyclase; 4 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Introduction Although the physiological regulatory function of the gasotransmitter nitric oxide (NO) has been discovered decades ago, NO is still in the frontline research as shown by the continuously increasing number of annual hits for “nitric oxide” in the PubMed database. The high interest for NO in biomedical research is due to several facts: (i) NO is an ubiquitous free radial molecule found in most of the cells of all tissues intracellularly as well as in the extracellular fluids, (ii) NO is involved in a variety of physiological and pathological processes, (iii) utilization of gaseous NO and some NO donor molecules for human therapy have entered into clinical therapy and the development of further NO-related therapies are promising (see for review: (Pacher et al., 2007)). However, the chemical reactions of NO with other free radicals and various small and macromolecules raised many new questions regarding the involvement of NO in different cellular or intercellular/interorgan signalling pathways. Moreover, measurement of NO is technically difficult, due to its rapid chemical reactions with a wide range of biomolecules and its very short half-life of approximately a few seconds. Therefore, there are still contradictory findings on the role of NO in several biological processes as reviewed earlier by (Ferdinandy et al., 2003; Schulz et al., 2004). Correct application of the different NO measurement techniques are essential to gain more knowledge on NO physiology and pathology. In this review, we describe the major techniques used most frequently in the literature for measurement of NO in biological samples and provide evidences on the advantages and disadvantages of each method. 5 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Synthesis and major metabolic pathways of NO NO is a diatomic hydrophobic gas which can permeate various cellular membranes and other hydrophobic structures, and thus it has a high diffusion capacity in a physiological environment. NO can be produced in biological systems by both enzymatic and nonenzymatic reactions. In mammals, NO is biosynthesized endogenously by various isoforms of NO synthase (NOS) using the substrates L-arginine and molecular oxygen (Fig 1). NOS catalyses the oxidation of the terminal guanidino nitrogen of L-arginine to produce NO and Lcitrulline. Several cofactors are required for the reaction including NADPH, FAD, FMN, haem, tetrahydrobiopterin, and calmodulin. Insufficient availability of L-arginine and some of the cofactors (i.e. tetrahydrobiopterin), as well as S-glutathionylation of certain NOS isoforms may lead to a reduced NO formation and uncoupling of NOS resulting in superoxide production (Forstermann et al., 2011; Zweier et al., 2011). Three distinct isoforms of NOS have been described in mammals: neuronal NOS (nNOS, NOS-1), endothelial NOS (eNOS, NOS-3) and inducible NOS (iNOS, NOS-2) (Knowles et al., 1989; Mayer et al., 1989; Mulsch et al., 1989; Palacios et al., 1989; Palmer et al., 1989; Stuehr et al., 1989; Moncada et al., 1997; Forstermann et al., 2012). Moreover, existence of a putative mitochondrial NOS (mtNOS) has also been suggested (Zaobornyj et al., 2012). The constitutively expressed isoforms nNOS and eNOS are Ca2+-dependent, whereas iNOS is Ca2+-independent (Moncada et al., 1991). The biological effects of NO and the activities of NOS isoenzymes are further regulated by compartmentalisation (Villanueva et al., 2010) as well as transcriptional, posttranscriptional, and posttranslational modulations such as for instance phosphorylation, Snitrosation, interaction with modulatory proteins (e.g. calmodulin, HSP90, caveolin, etc.), dimerization, inhibition by endogenous methyl-arginines, etc. as reviewed in detail elsewhere (Zhou et al., 2009; Pautz et al., 2010; Qian et al., 2013). 6 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article It has been clarified that NOS-independent reduction of dietary or endogenous sources of nitrate and nitrite are important contributors of production of NO in mammalian tissues (Lundberg et al., 2008; Rassaf et al., 2014) (Fig 1). Nitrate found in significant amounts in certain vegetables (e.g. lettuce, spinach, and beetroot, etc.), needs to be initially reduced to nitrite by nitrate reductase enzymes of bacteria of the gastrointestinal tract. Nitrite - also found in certain dietary sources - can be reduced to NO by several pathways and conditions including low pH, ascorbic acid, haemoglobin, myoglobin, polyphenols, and xanthine oxidoreductase (Lundberg et al., 2008; Rassaf et al., 2014). The formation of NO by these pathways may become especially important during hypoxia when pH becomes acidic and oxygen-dependent NOS activities are limited. NO has a short biological half-life (estimated to be a few seconds) due to its rapid reaction with a variety of molecules (Fig 1). Although the chemistry of NO is well-established in a test tube, the exact biochemistry of NO is still far from clear (Hill et al., 2010). The biologically relevant reactions of NO has been reviewed elsewhere in more details (Gow, 2006; Bryan et al., 2007; Habib et al., 2011; Tennyson et al., 2011). The major pathway for the metabolism of NO is its oxidation to nitrite and nitrate eventually followed by their urinary excretion. NO in the presence of molecular oxygen is oxidized to nitrogen-dioxide (NO2), which by reacting with another NO molecule forms N2O3, an intermedier that participates in nitrosation reactions. N2O3 may be decomposed to nitrite and a one electron reduction of NO2 may also lead to nitrite formation. Nitrite has a half-life of a few minutes in the circulation as it can be further oxidized to the more stable nitrate by certain oxyhaemoproteins such as oxygenated haemoglobin or myoglobin. Alternatively, NO may directly react with oxyhaemoproteins to form nitrate (Bryan et al., 2007). In addition to its reaction with oxygen, NO rapidly reacts with superoxide to yield peroxynitrite, a short-lived oxidant, nitrating, and nitrosating agent (Pacher et al., 2007; Radi, 2013). 7 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Important molecular targets of NO are transition metal ions. NO binds to transition metal ions to form nitrosyl-metal ion complexes. The nitrosyl-Fe2+ adduct - such as in haem - is particularly stable, as the binding of the nitrosyl ligand to Fe2+ is very strong. Relevant examples for proteins in which the formation of nitrosyl-metal complexes affect biological function include soluble guanylate cyclase (sGC, see later), haemoglobin, cytochromes, etc (Toledo et al., 2012). NO - following oxidation to N2O3 - plays an important role in the formation of S-nitrosothiols (RSNOs) via S-nitrosation of small molecular weight thiols and thiol-containing proteins (Broniowska et al., 2012). This type of reaction is often incorrectly referred to as “S-nitrosylation” (i.e. direct addition of NO to a reactant) in the literature (for more details in NO chemistry and terminology see a recent review by (Heinrich et al., 2013). Nevertheless, NO may also react directly with thiyl radicals formed after oxidation of thiols to produce RSNOs. Enzyme-dependent and -independent S-nitrosation, transnitrosation and denitrosation are potential post-translational modifications that may regulate biological function of several proteins (Lima et al., 2010; Stamler et al., 2010; Gould et al., 2013; Maron et al., 2013). RSNOs may play an important role in endogenous transport and storage of NO as well as in NO-related cell signalling. 8 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Analytical tools for the assessment of NO: what to consider before selection? In general, NO measurement techniques can be classified as direct (the target of measurement is NO itself) and indirect methods. Most of the NO measurement techniques in the literature are indirect ones, i.e. measurement of NO synthase activities, activation of molecular targets of NO such as guanylate cyclase-derived cyclic GMP, or products of reactions of NO, such as S-nitrosothiols or nitrite/nitrate. The major analytical tools for the detection of these analytes are spectroscopic or electrochemical methods. The spectroscopic methods include colorimetric, fluorometry, luminometry, and electron spin resonance spectroscopy. These analytical tools have been extensively reviewed previously (Hetrick et al., 2009; Coneski et al., 2012). The sensitivity and specificity of these techniques for NO varies a lot, and they cannot provide insight into the in situ NO levels in biological systems. Direct NO measurement techniques that are more specific for NO such as e.g. electron-spin resonance spectroscopy after in vivo or ex vivo spin-trapping or NO specific biosensors are less frequently used. Choosing the most appropriate method for measurement of NO in biological systems is not easy. Therefore, we suggest consideration of 2 additional aspects before planning experiments. First, for the measurement of NO, a variety of different commercial products are available on the market, most of which can be purchased in ready to use formats. However, these kits and/or instruments that operate with different background principles are developed for various scientific, industrial, and environmental application purposes. Second, the specificity and cross reactivity of the NO sensors with NO derivatives (e.g. reactive nitrogen species such as ONOO-) and other non-NO related molecules (e.g. reactive oxygen species) still remain a major challenge in NO research (Rodriguez-Rodriguez et al., 2012; Woolley et al., 2013). This is mainly due to the small size of the NO molecule and its complex chemical 9 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article nature in biological systems. Therefore, before scientific use, careful consideration of various aspects including background assay principle, specificity, sensitivity, advantages, disadvantages, and financials (see Table 1) is strongly recommended to choose the most proper analytical technique(s) which fits best to the study objectives. As an aid to find the most appropriate method(s) for measurement of NO in a particular study, a detailed questionnaire can be constructed and answered as suggested by Wardman (Wardman, 2007). Moreover, we suggest the use of NO donors for positive control and inhibitors of NO formation as negative controls to complete the data obtained either by direct or indirect assays to understand the exact role of NO in biological systems. In the present review we focus on the most important possibilities to determine NO or its derivatives in biological matrices. We discuss background assay principles, specificities, sensitivities, advantages/disadvantages and possible limitations. 10 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Direct methods to estimate NO Electron paramagnetic resonance spectroscopy Since NO is a free radical, hence of paramagnetic nature, electron paramagnetic resonance spectroscopy (EPR; also referred to as electron spin resonance – ESR) is considered to be the most appropriate tool for the direct detection of NO. The main advantage of EPR compared to other NO detection techniques is that it only detects paramagnetic molecules, and the EPR spectrum is a unique fingerprint of the chemical and electronic structure around the unpaired electron. EPR measures the transitions induced between the Zeeman levels of a paramagnetic molecule via its interaction with a static magnetic field and an oscillating electromagnetic field, most commonly in the X microwave frequency band, around 9-9.5 GHz. To take an EPR spectrum, the microwave frequency is held constant and the static magnetic field is swept (despite its name) across the desired range. The equation for resonance absorption is ΔE=h =gβB, in which h is Planck’s constant, is the frequency of the microwaves, β is the Bohr magneton a physical constant, B is the external magnetic field, and g is the g-value, or the Zeeman splitting factor (Weil et al., 2007). The g-value is a characteristics of the paramagnetic molecule (in case of NO in liquid g=2.035, (Hogg, 2010)). A further specific property of EPR spectra originates from the coupling of the electron spin with the surrounding nuclear magnetic spins, measured by the so called hyperfine coupling constant, A, in mT. The paramagnetic NO can be measured directly by EPR irrespective of the optical appearance of the sample, however, due to the rapid relaxation of its excited electron spin state to the ground state (Maples et al., 1991) and the high reactivity of NO, spin-trapping techniques have been developed. Spin traps are compounds that interact with the less stable 11 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article radicals, producing a more stable adduct, which can be then detected by EPR (Janzen, 1984; Tosaki et al., 1996; Berliner et al., 2004; Villamena et al., 2004). As natural spin traps, the iron centre of haemoglobin and other haem-proteins may interact with NO with high affinity forming nitrosyl derivates, which are paramagnetic and exhibit a characteristic EPR spectrum (Greenberg et al., 1990; Henry et al., 1991; Eriksson, 1994; Katayama et al., 2001). The spectra of the haemoglobin-NO is sensitive to many factors such as tertiary and quaternary structures of the protein, concentration of O2, pH, degree of hydration, temperature, etc. (Sanches, 1988; Hall et al., 1996). The detection limit of haemoglobin-NO have been reported to be ~200 nM but basal level of NO in blood appear to be below this limit (Piknova et al., 2005). There are several classes of chemical spin traps for the detection of NO like the (i) nitroxide spin traps (Arroyo et al., 1991a; Arroyo et al., 1991b) including nitrones (e.g. DMPO, 5,5-dimethyl-pyrroline-N-oxide and its related spin trap, DEPMPO, 5- diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; PBN, alpha-phenyl-N-tert-butyl-nitrone; and POBN, alpha(4-pyridyl-1-oxide)-N-tert-butyl-nitrone) and nitroso compounds (e.g. MNP, 2-methyl-2-nitrosopropane and DBNBs, 3,5-dibromo-4-nitrosobenzene), the (ii) NO cheletropic traps (NOCTs, double carbon-centered biradical equivalents, Korth et al., 1992), and (iii) the nitronyl nitroxides (e.g. carboxy-PTIO [2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxide] (Katayama et al., 2001; Hawkins et al., 2014). However, the abovementioned spin traps have several limitations like instability in certain pH and low sensitivity or selectivity (Katayama et al., 2001; Hawkins et al., 2014). The efficiency of NO spin trapping can be sharply increased by using various transition metal complexes (see, e.g. (Archer, 1993; Henry et al., 1993). Thus, the most popular and most extensively used NO spin traps are the iron (II) dithiocarbamate (DTC) type of complexes (Kleschyov et al., 2007; Hong et al., 2009; Hogg, 2010). Iron complexed with 12 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article N,N-diethyl-dithiocarmabate (DETC, (Mordvintcev et al., 1991; Mülsch et al., 1992)) is hydrophobic, whereas iron complexed with N-methyl-D-glucamine dithiocarmabate (MGD, (Komarov et al., 1993)) is hydrophilic, hence these iron complexes trap NO in different environments. One of the major advantages of using these iron complexes as spin traps is that they react with NO extremely rapidly, i.e. NO bind to dithiocarbamate complexes with a rate constant of 1-5×108 M-1s-1 (Hogg, 2010). Fe2+(DTC)2 are suitable spin traps for NO in vivo and in real-time measurements because the rate constant of the formation of NO-Fe2+(DTC)2 adduct is much larger than that with other NO spin traps (Vanin et al., 2000; Nagano et al., 2002), and also because of the high solubility of NO in membranes (Nedeianu et al., 2004). Whereas DETC is soluble in aqueous media, its ferrous and the mononitrosyl ferrous complexes precipitate at neutral pH (Csont et al., 2003). Therefore, when injected in animals, it is recommended to inject Fe2+ separately from DETC. The reagents are inexpensive and commercially available and typically used in vivo (100 mM DETC, 20 mM Fe(II), usually at a ratio of 1 iron:5 ligand (Berliner et al., 2004)). The Fe2+(DETC)2 is distributed throughout the body, as observed by the detection of the NO-Fe2+(DETC)2 complexes in different organs in vitro and in vivo in animal models (see, e.g. (Csont et al., 1998; Fejer et al., 2005); see for review: (Hong et al., 2009)), while Fe2+(MGD)2 is suitable for ex vivo spin trapping in different tissue samples and cell cultures (Csonka et al., 1999; Radak et al., 1999; Csont et al., 2010), including human tissue samples (Radak et al., 1999). NO interacts with high affinity with these Fe2+(DTC)2 complexes forming stable nitrosyl iron-dithiocarbamate complexes (according to Vanin et al, the predominant binding of iron to dithiocarbamate ligands takes place only after its binding to NO, since nitrosylated iron manifests much higher affinity for these ligands that for any non-thiol compounds (Vanin et al., 2009)). At ambient temperature the solution of NO-Fe2+(DTC)2 is characterized by the isotropic EPR signal at a g-value of 2.035 and triplet hyperfine structure (not shown) and a 13 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article spectrum with axial symmetry in the frozen state (Fig 2A). The triplet (three-line) splitting originates from the hyperfine interaction of an unpaired electron with the 14N nucleus of the NO ligand (solution spectra are characterised in (Nedeianu et al., 2002)). DETC penetrates the cell wall and binds not only the free intracellular Fe2+ but also endogenous Cu2+ forming a Cu2+(DETC)2 complex, characterised by a four-line EPR spectrum that overlaps with the NO- Fe2+(DTC)2 spectrum at low temperature (Suzuki et al., 1997); see also Fig 2B. Specimens in a quartz tube with a small sample volume (<100 L) are measurable in an X-band EPR spectrometer. However, larger biological samples (e.g., tissues, organs, and live animals) cannot be measured with a conventional X-band spectrometer due to the high dielectric loss of water at such frequencies and the small size of the EPR cavity resonator. EPR spectroscopy at lower frequencies, the L-band (0.4–1.6 GHz) or S-band (1.6–4 GHz), can be used for in vivo EPR imaging (Nagano et al., 2002; Fujii et al., 2004). Since the NO- Fe2+(DTC)2 complexes are still reactive free radicals, freezing of the samples until and during measurement is required. Measurements are done typically below 200 K. The detection limit of NO by the DTC-based spin trapping EPR method is 0.05 nM in biological samples (Mülsch et al., 1992; Khoo et al., 2004). We found the detection limit for NO released from an NO donor to be 0.4 nM in solution under anaerobic conditions (Nedeianu et al., 2004). However, precise and absolute quantification of EPR detection of NO in biologic samples is not reliable due to an uneven distribution of the spin-trap complex Fe2+(DTC)2 in the aqueous and the lipid phase of biological samples (Ferdinandy et al., 1997). In addition, since the concentration of the Fe2+(DTC)2 complexes can’t be predicted from the concentration of Fe2+ and DTC administered, and since the trapping efficiency of the spin trap in the given environment is not known, absolute quantitation of NO is not a realistic objective in practice. Hence, in the vast majority of studies, relative changes in the NO level are reported as NO signal intensity in arbitrary units. 14 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Typical EPR spectra are shown in Fig 2 for NO trapped with Fe2+(DETC)2 (traces A, C, and D), together with the most common compromising signal Cu2+(DETC)2 (trace B), and the spectra of control samples. The EPR spectrum is proportional with the first-derivative of the microwave absorption (y-axis, measured in arbitrary units) as function of the magnetic field (B, x-axis). The second integral of the EPR spectrum (the area under the absorption spectrum) is proportional to the number of spins present in the active volume of the resonator. Therefore, in this case the second integrals can be used for relative quantitation of NO. In (Csont et al., 1998) for instance, analysis of NO content was performed with double integration of all spectra, after subtracting the background signal of Cu 2+(DETC)2. However, in most studies the background signal also changes significantly from sample to sample, even if the same test tube is used for the sample and the background signals. Since the second integral approach is very sensitive to the baseline, an alternative approach is followed: the use of a positive control, which is the same tissue but loaded with an NO donor and the spin trap. A positive control yields an NO triplet that has much higher intensity than the background signal, hence it can be analysed and fitted easily (this technique was used in, e.g., (Fejer et al., 2005)). A fit of a simulated NO signal to the positive control is also shown in Fig 2 trace A, solid line. A simple approach to evaluate NO spectra is to measure the peak-to-peak amplitude of the +1 and/or the -1 NO peak and compare them with that of a positive control signal (Radak et al., 1999; Csont et al., 2010). In summary, EPR measurement of NO spin-trapped with Fe2+(DTC)2 provides a very sensitive and one of the most specific method for direct detection of NO both in vivo and in vitro. The EPR spectrum is characteristic for the nitrosyl-adduct and it can be recorded under various conditions, however, the limitation of this method is that complex evaluation of the results requires significant expertise. 15 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Electrochemical assays using microelectrodes specific for NO There are a lot of different electrochemical assays available for NO measurement. They all integrate the advantages of electrochemistry: small size, continuous (often in situ) and fast measurements, no chemical contamination of the samples (thus samples can be used for other assays). However, large differences may occur among the different electrodes in selectivity. Selectivity is controlled by the voltage applied between the electrodes (~860 mV) and the NO-selective layer around or on the surface of the electrode. The majority of electrodes on the market dedicated to measure NO uses micro ion electrodes measuring simply nitrite and/or nitrate. For the advantage and disadvantage of nitrite and nitrate measurement versus direct NO measurement, see next sections. Comparing with the classical Griess spectrophotometric assay which is the gold standard of nitrite/nitrate determination, use of electrodes has several advantages: (i) requires smaller amount of samples; (ii) wide range of samples can be used including turbid, opaque or even non liquid samples e.g. cell cultures, tissues and tissue homogenates; (iii) per sample cost savings over Griess, (iv) avoid interferences during measurements from other components e.g. NADPH and antioxidants (v) minimize sample preparation steps. To date, among the several electrochemical techniques that have been shown to be useful for the direct measurement of NO, amperometric detection of NO is the most popular technique sensitive enough to detect relevant concentrations of NO in real-time and in vivo (Serpe et al., 2007; Davies et al., 2008; Yap et al., 2013). Generally, this technique involves applying a fixed (poise) voltage potential to a working electrode, vs. a reference electrode, and monitoring the redox current produced by the oxidation of NO (Serpe et al., 2007). This technique has proven to be very useful for NO detection due to its fast response time of less than a few seconds, and its acceptable sensitivity. In amperometry, both electrodes are 16 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article encased within a protective Faraday-shielded stainless steel sleeve. The tip of sleeve is covered with a NO-selective membrane and the sleeve itself contains electrolyte (Davies et al., 2008). NO diffuses across the gas permeable/NO-selective membrane (e.g. nitrocellulose, chroloprene, silicon, teflon, graphene, Nafion, cellulose acetate and polycarbazole) and ultimately is oxidized at the working electrode surface producing a redox current (Serpe et al., 2007; Wang et al., 2009). The selectivity of the membranes to NO is still a question of debate, and after NO diffuses through the membrane, there is no more qualitative control on the analytical signal. The amount of NO oxidized is proportional to the current flow between the working and reference electrodes, which is measured by an NO meter. The redox current generated by the oxidation of NO in biological systems is extremely small, typically in the range of 1–10 pA corresponding to an approximately 10-8-10-9 M concentration range (Table 1). In addition to amperometry, voltammetry techniques may provide an alternative electrochemical approach to detect NO, however, they are less frequently used. These techniques typically employ a classical 3-electrode configuration consisting of a working electrode, reference electrode, and a counter electrode. Besides the aforementioned classic Clark-type electrodes where selectivity is insured by a passive NO-selective membrane or layer around the electrode, a new generation of NO electrodes have been recently developed (Wang et al., 2009; Bedioui et al., 2010; Jiang et al., 2013; Yap et al., 2013). Instead of membranes believed to ensure NO-selectivity, a NOspecific active scavenger layer is electropolymerized onto the surface of a glassy carbon electrode which can chemically react directly with NO. Among these materials, electropolymerized film of metalloporphyrins has been used most extensively (Diab et al., 2003; Li et al., 2009). Electric signal forms only after the direct chemical link between NO and the electropolymerized layers of the electrode, thus electrocatalytic oxidation of NO 17 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article enhance NO specificity of the electrode significantly. Parallel with advancements of nanotechnology e.g. (Santos et al., 2013), there is a great progress in the development of these electrodes recently, representing a great promise in future technology suitable for direct NO measurements in biological samples. Even if the electrochemical techniques offer great promise for the measurement of NO production, some disadvantages should be considered. The tip of the electrode is used to detect the signal, therefore, location of the electrode is critical as small changes in its position can dramatically influence spatial information. Theoretically, this problem can be eliminated by using ultramicroelectrode sensor arrays in which each electrode, or groups of electrodes, are individually addressable, and used for mapping the analyte to achieve spatio–temporal analysis (Quinton et al., 2011; Griveau et al., 2013). As NO dissolves well in lipids, distance of the electrode from different membranes may also influence signal intensity. The diameter of electrodes which varies in the m-mm range also influences the sensitivity of the electrode since the surface of the electrode (determined by diameter and length) is proportional to the NO signal detected. To get reliable results using electrochemical assays, preparation of standard solutions to calibrate the electrode is also a critical step in the measurement, and all the following options are rather problematic: (i) application of gaseous NO to prepare a standard solution (concentration can be calculated from the dissolving coefficient, and deoxygenation of the standard is important to prevent oxidation of NO); (ii) known concentration of NO donors, or (iii) NO produced by chemical reactions (Serpe et al., 2007). Temperature may affect sensitivity of the electrode by influencing the partial pressure of dissolved NO, the permeability of the coatings, and the conductivities of various sensor components. Therefore, a careful temperature control is recommended during calibration and experiments. External electric noise sources (e.g. magnetic stirrers, fluorescent lights, MRI machines, electric motors, computers, pumps and other electric instruments) may couple into 18 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article the sensor signal path electromagnetically and impose undesirable signals in the output record. Because NO signal intensity is in the pA range, it is important to ground and shield the system properly (Serpe et al., 2007). 19 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Fluorometry Fluorescence assays are sensitive techniques for detection of NO. The assay principle is that NO and/or its oxidative derivatives react with a nonfluorescent compound forming a fluorescent product. Fluorescent compounds (called frequently probes or dyes) greatly vary in their specificity for NO and applicability in biological systems. In order to detect the spatial and temporal dimensions of NO generation and accumulation in living cells, the fluorescent compounds must pass several strict criteria. The probe should be small, cell or membrane permeable, non-toxic, water soluble, photostable, have excitation/emission wavelengths in the visible range to minimize damage of biological samples, well-separated excitation/emission wavelengths, possess a large extinction coefficient and quantum yield, have a large signal-to- noise ratio and linear response. In addition, it is highly desirable that the molecular probe be selective for NO among competing reactive nitrogen species. If oxidized forms of NO such as nitrite and nitrate, and also reactive oxygen species, such as superoxide, hydrogen peroxide, peroxynitrite do not react with the probes to give a fluorescent product, than under physiological conditions fluorescent dye is not formed in the absence of NO. Once the fluorescent dye is formed, it can be detected by any instrument suitable for detection of fluorescence, including flow cytometers, microscopes, fluorescent microplate readers and fluorometers. Fluorescence microscopy can provide 2D or even 3D imaging, and high spatialtemporal resolution, therefore, biologically seems to be the most informative detection technique using fluorescence probes despite limited quantitations. There are two main classes of different fluorescent probes specifically designed for NO measurement: organic-based and metal-based sensors. In both instances, the goal is to alter the fluorescent properties of the probe by specific reaction with NO. Organic probes employ fluorophores with pendant functional groups that serve to quench their fluorescence 20 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article until restored by a specific reaction with NO. Metal-based probes take advantage of the high reactivity of NO with transition metals to form metal-nitrosyl complexes (McQuade et al., 2010b). The first fluorescent probe developed for the measurement of NO was diaminonaphthalene (DAN) (Ji et al., 1988). However, Ji and colleagues suggested that chemical nitrosation of DAN requires O2, therefore, DAN directly measures oxidative derivatives of NO (N2O3). Later Misko et al developed an assay using DAN to detect nitrite in biological samples (Misko et al., 1993). Another popular organic-based fluorescein probe, diaminofluorescein (DAF), was developed more than 15 years ago (Kojima et al., 1998a; Kojima et al., 1998b). DAF is essentially nonfluorescent until it reacts with NO (or more precisely N2O3) to form a fluorescent benzotriazole. Beside specificity, cell membrane permeability was improved by developing its diacetate derivative which passively diffuses across cellular membranes. Once inside cells, it is deacetylated by intracellular esterases to become membrane-impermeable. These probes have been extensively used for estimation of NO production in a variety of biological samples. However, it turned out that DAF-2 can be enzymatically converted into a variety of highly fluorescent derivatives both intra- and extracellularly, of which only a minor part appeared NO-dependent (Roychowdhury et al., 2002). Thus, DAF-fluorescence does not necessarily indicate NO·production, therefore, it is not generally accepted as a NO-specific probe. The presence or absence of oxygen (i.e. in ischemia or reperfusion) can also influence fluorescence through formation of N2O3 (Rumer et al., 2012). Despite these limitations, further applications of DAF were developed, i.e. a high-throughput technique based on DAF-fluorescence capable of detecting RSNOs (Doctor et al., 2005). Other frequently used organic NO fluorophores are o-diaminorhodamines (DARs; typically DAR-2 and DAR-4M AM), o-diamino-BODIPYs (DAMBOs; typically DAMBO and DAMBO-CO2Et), and o-phenylenediamine cyanines (DACs) (Nagano, 2009; 21 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Zhang et al., 2014). The currently used organic fluorescent probes are reviewed in (Nagano, 2009; Nagano, 2010; Woolley et al., 2013). Since organic probes are not suitable for direct detection of NO, metal-based probes were also developed. As transition metals can reversibly form bonds with NO, many researchers have focused on metal–ligand constructs where the ligand contains a fluorophore. (McQuade et al., 2010a). To date, NO sensing has been accomplished by using a wide array of metal ions, including Co(II), Fe(II), Ru(II), Rh(II), and Cu(II) (Hong et al., 2009; Hu et al., 2011). One of the most promising of metal-based sensors for direct, cellular detection of NO is a copper-based fluorescent probe CuFL (Lim et al., 2006; McQuade et al., 2010b). Although, the use of fluorescent techniques is a promising tool to detect NO, it has several disadvantages. The optimal dilution buffer and working concentration of the fluorescent dye, and optimal loading concentration, time and temperature are needed to be determined empirically, which can vary among different labs. Moreover, quantitation of NO is nearly impossible. It is desirable to use the lowest dye concentration yielding fluorescence signals with adequate signal-to-noise ratios. Moreover, close excitation/emission wavelengths, cytotoxicity, strong autofluorescence, small extinction coefficient and insufficient solubility in neutral buffers, pH-dependency of the fluorescence intensity can be mentioned as further disadvantages of this method. Some further limitations still exist to affect sensitive imaging of NO, such as poor photostability (e.g. DAFs, DACs, and transition metal complex probes), small changes of fluorescence quantum yield after reaction with NO (e.g. DACs and transition metal complex probes), fast leakage from cells (e.g. DAFs and DARs), and possible fluorescence interference from biological matrix (e.g. DAFs, DAMBOs, and DARs) (Zhang et al., 2014). A great deal of improvement has been made to fluorescent NO probes in recent years, mostly in the area of sensitivity and selectivity e.g. (Nagano, 2010; Rodriguez-Rodriguez et 22 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article al., 2012; Woolley et al., 2013; Alam et al., 2014; Zhang et al., 2014). Although these improvements have allowed for more accurate measurements of NO, the specificity of fluorophores for NO is still a very important issue and needs to be further validated in biological systems. 23 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Indirect methods to estimate NO Determination of nitrate/nitrite Due to its short half-life, the direct measurement of NO is extremely challenging in a complex biological environment. As NO is rapidly metabolized to nitrite and nitrate in the presence of oxygen (Fig 1), the determination of both nitrite and nitrate (termed NOx) is commonly used to estimate total NO production. A number of analytical techniques have been developed to determine nitrite and nitrate in biological samples including the Griess colorimetric assay, fluorometry, flow or sequential injection analysis with visible absorbance, chemiluminescence, and electrochemical detection (Moorcroft et al., 2001; Bryan et al., 2007). Chromatographic methods including GC–MS, capillary electrophoresis, and HPLC have also been developed using a variety of detection systems. A major general disadvantage of nitrite/nitrate determination to estimate tissue NO level is that dietary intake of nitrite (e.g. cured meat) and nitrate (e.g. vegetables) are rather significant which markedly influences plasma NOx level (Rassaf et al., 2014). In addition, environmental conditions such as nitrite/nitrate pollution in chemical reagents, cell culture media, and even plastic labware may interfere with nitrite/nitrate analysis. Nitrite and nitrate can be measured from a variety of biological fluids. A relatively simple, long-known colorimetric assay based on the Griess reaction is probably used most extensively for assaying NOx. The Griess reaction – also called diazotization assay - is based on the conversion of nitrite to a purple-coloured azo-dye that can be spectrophotometrically assayed at a wavelength of ~540 nm. The most widely used reagents required for the reaction are sulphanilamide and N-naphthyl-ethylenediamine (Moorcroft et al., 2001; Sun et al., 2003; Bryan et al., 2007; Hetrick et al., 2009). Advantages of this method include a strong literature 24 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article background, numerous commercially available reagent kits, and wide availability of infrastructure. However, prior to this reaction nitrate needs to be reduced either chemically or enzymatically to nitrite in order to determine NOx. Chemical reduction is not specific for the nitrate-nitrite conversion and nitrite may be reduced further to NO thereby leading to underestimation of nitrite (Sun et al., 2003). Enzymatic reduction of nitrate by nitrate reductase requires NADPH, however, excess NADPH interferes with the subsequent Griess reaction and thus limits sensitivity of the assay. Moreover, the high protein content of cell lysates and plasma may interfere with nitrate reduction or the Griess reaction, therefore deproteinization of samples is highly recommended. Since the detection limit is ~0.5 µM of nitrite/nitrate, this assay is inadequate to measure physiological amounts of NO (Sun et al., 2003; Hetrick et al., 2009). The other most widely used approach for the analysis of nitrite and nitrate is based on electrochemical detection. For general advantages and disadvantages of the electrochemical approach see previous sections. 25 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Estimation of NO formation by measuring NOS activity Measurement of NOS activity may provide an alternative solution for investigating NO metabolism both in vivo and vitro (Fig 1). NOS activity can be determined by measuring either L-citrulline or the NO metabolites nitrite/nitrate. The classical citrulline assay is based on the conversion of 3H- or 14 C-labeled L-arginine to L-citrulline, where after removal of unreacted L-arginine with resin, radioactivity of the end-product L-citrulline is proportional with NOS activity. Radioactivity is usually measured by liquid scintillation counter. This method is suitable to distinguish Ca2+-dependent and Ca2+-independent NOS activities. It is relatively sensitive (Hevel et al., 1994) and after improvement by (Giraldez et al., 1998) it became suitable for detection of low levels of NOS activity found in certain tissues (e.g. heart). NOS enzyme activity measured in vitro conditions strongly depends on the availability of exogenously administered cofactors such as tetrahydrobiopterin and NADPH. A radiochemical HPLC-based citrulline assay was developed for the measurement of NOS activity in intact tissue samples (de Bono et al., 2007; Crabtree et al., 2009a; Crabtree et al., 2009b), thereby avoiding the influence of the measurements by exogenously administrated cofactors. Other commonly used assays for measurement of NOS activity are based on detection of nitrite/nitrate generated from NO. These assays are less specific but more simple and cost effective compared to the citrulline assay, and are also suitable for high throughput analysis. Another method to improve specificity and sensitivity of NOS activity assays based on nitrate/nitrite determination involves a GC–MS based measurement of 15 N-labeled nitrite/nitrate converted from L-[guanidino-15N]-arginine-derived 15NO (Tsikas, 2008; Shin et al., 2011). 26 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article NOS activity assays indicate the amount of enzymatically active NOS protein in a biological system under optimized in vitro conditions, however, they do not necessarily reflect in vivo NO production and actual NO concentration. Care should be taken to avoid interference by other enzymes affecting L-arginine metabolism (e.g. arginase) and nonenzymatic NO formation should be also considered. Specificity of the assays should be ensured by application of inhibitors of NOS isoforms in parallel measurements. 27 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Detection of S-nitrosothiols in biological systems Protein RSNOs derived from NO and/or its derivatives (Fig 1) play an important role in NO signalling in both physiological and pathological conditions. Therefore, measurement of RSNOs contributes to the understanding of NO biochemistry, however, it does not directly reflect actual NO levels. To date no universal direct method exists to identify protein RSNOs specifically. The most commonly used methods for the detection of S-nitrosated proteins are (i) Saville reaction (simplest and least expensive) in which mercury replaces a nitrosyl from a thiol group to form nitrite followed by Griess assay (Saville, 1958); (ii) biotin switch (suitable to detect low µM concentrations of biotinylated proteins without requiring special instrumentation) (Jaffrey et al., 2001; Forrester et al., 2009) and (iii) chemiluminesence-based assays (Basu et al., 2008). A DAF-fluorescence-based high-throughput technique measuring RSNOs in the low nanomolar range was also described (Doctor et al., 2005). Mass spectrometry detection became the gold standard for directly studying low-level RSNOs in a physiologically relevant context (Barglow et al., 2011), however, it is expensive and requires 15 N-labeled internal standards (Gow et al., 2007). The abovementioned methods suffer from lengthy preparative protocols and selectivity issues. Most recently, triarylphosphines and cyclization reactions with phosphines have been addressed to be the milestones of direct detection of RSNOs, however, these methods require further improvements (Bechtold et al., 2012). 28 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Estimation of NO formation by measuring cyclic GMP In biological systems, the most known target of NO is sGC that is responsible for the synthesis of cyclic GMP (Fig 1). The connection between NO and cyclic GMP in most of the tissues is so close that cyclic GMP is frequently used as a surrogate of the rate of NO synthesis both in vitro and in vivo (Tsikas, 2008), however, the relation of tissue NO and cGMP level in the heart tissue seems more complicated (Csont et al., 1998). There are several available methods to measure cyclic GMP in a variety of biological samples including tissue lysate, blood, urine, and culture supernatants. Radioimmunoassays are commercially available, with high specificity and sensitivity (pM range) (Steiner et al., 1972; Evgenov et al., 2004). The method is based on the competitive binding of cyclic GMP in the sample and a radioiodinated derivative of cyclic GMP (125I cyclic GMP) to a highly specific antibody. After separation of antibody bound cyclic GMP from free cyclic GMP, 125I is determined by a gamma-counter. A similar immune-detection approach applying non-radioactive reagents is used in commercially available ELISA assays with colorimetric detection of optical densities by a plate reader. Another method for cyclic GMP quantification is liquid chromatography coupled to tandem mass spectrometry (LC-MS) characterized by complicated sample preparation and high sensitivity and selectivity (Lorenzetti et al., 2007; Martens-Lobenhoffer et al., 2010). All these abovementioned methods are expensive and labour intensive. Moreover, these are endpoint assays, not suitable for real-time measurements. However, real-time monitoring of cyclic GMP based on homogenous time-resolved fluorescence has been recently described (van Mastbergen et al., 2012). In addition, NO reporter assays were also developed allowing real-time detection of NO synthesis within living cells (Wunder et al., 2005; Wunder et al., 2007). 29 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Although determination of cyclic GMP is frequently used to describe NO production in biological systems, cyclic GMP level is modified by several other factors including activities of particulate GC and phosphodiesterases, sampling time, subcellular localisation and NO-independent regulation of sGC (Lucas et al., 2000; Bender et al., 2006; Francis et al., 2010). 30 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Conclusions A wide range of analytical methods are available for the detection of NO in biological samples, however, each method has certain advantages and limitations. Therefore, the use of direct NO detection methods as first choice should be considered. In addition, a series of methods to follow NO synthesis, NO content, molecular targets and reaction products of NO are recommended to get meaningful insights into the role of NO in a certain physiological or pathological process. Moreover, application of NO donors and NOS inhibitors to provide appropriate positive and negative controls is also recommended to overcome limitations of individual methodologies. 31 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Acknowledgement We thank partial financial support from the Hungarian National Science Fund (OTKA K101633, OTKA ANN 107803, OKTA K109737, PD106001), TÁMOP-4.2.2.A11/1/KONV-2012-0035. F.P. holds a Szentágothai Professorship of the National Excellence Program of Hungary (TÁMOP 4.2.4.A/2-11-1-2012-0001). G.A. holds a Bolyai János fellowship from the Hungarian Academy of Science. 32 This article is protected by copyright. All rights reserved. NO detection MS #2014-BJP-0167-RCT-G.R1 Csonka et al Accepted Article Author Contributions CC: conception and design, writing sections of ‘Fluorometry’, ‘Electrochemical assays using microelectrodes specific for NO’, ‘Electron paramagnetic resonance spectroscopy’, and ‘Analytical tools for the assessment of NO: what to consider before selection?’, Table 1, Fig 1; revising of MS TP: writing section of ‘Electron paramagnetic resonance spectroscopy’, Fig 2; PB: writing sections of ‘Estimation of NO formation by measuring NOS activity’ and ‘Detection of S-nitrosothiols in biological systems’; AG: writing section of ‘Estimation of NO formation by measuring cyclic GMP’ PF: writing Introduction; consultation, proof reading, editing of MS, improvement of English, TC: conception and design, writing sections of ‘Synthesis and major metabolic pathways of NO’ and ‘Determination of nitrate/nitrite’, ‘Analytical tools for the assessment of NO: what to consider before selection?’ Conclusion, and Summary, editing and revising of MS and final approval 33 This article is protected by copyright. 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EPR: Electron paramagnetic resonance spectroscopy. i(Mülsch et al., 1992; Khoo et al., 2004; Nedeianu et al., 2004); ii(Griveau et al., 2013); iii(Zhang et al., 2014), iv(Sun et al., 2003) Technique EPR Sensitivity 0.05-0.4 nMi Advantages -direct, considered as “stand alone” assay -the most specific method, due to NO specific spin traps Disadvantages -semiquantitative -expensive, not particularly common instrumentation -complex evaluation requires significant expertise -direct -continuous -real time, portable -no chemical contamination of the sample -difficult to calibrate -influenced by temperature and ambient electric noise -uncertain specificity (NO-specific membrane or layer) -sensitive for electrode tip position -direct -sensitive (detection limit in nM range) -can be 2D, 3D -uncertain specificity (NO-specific fluorescent dyes) -semiquantitative -cheap, fast -commercially available ready to use kits -indirect, indicative of NO oxidative products -only measures nitrite (nitrate should be reduced) -interfere with dietary and environmental nitrite and nitrate -measures enzymatic production of NO -sensitive -specific -indirect -nonenzymatic NO formation is not considered -measures active NOS protein under optimized vitro conditions, does not reflect in situ NO synthesis n.a. -important NO target besides cyclic GMP -represents downstream NO signalling -indirect -represents downstream NO signalling -does not reflect actual NO levels n.a. -most known cellular target of NO is sGC resulting in cGMP formation -represents downstream NO signalling -indirect, assesses effect of NO on sGC -represents downstream NO signalling -does not consider NO-independent cGMP formation n.a. -use of them completes other assays -helps to understand the role of NO -alone does not reflect NO production NO spin trapping followed by spectrometry in magnetic field Electrochemistry 0.3-10 nMii amperometry or voltammetry using NO-specific electrode Fluorometry 0.6-8 nMiii spectrometry or imaging of fluorophore-labelled NO Griess assay 500 nMiv diazotization assay measures nitrite by photometry NOS activity n.a. biochemical enzyme activity assay RSNO detection of nitrosated proteins/peptides cGMP assays measurement of cyclic GMP level NO donors and NOS inhibitors in 43 This article is protected by copyright. All rights reserved. NO detection Csonka et al. Accepted Article Figure legends Figure 1. Major metabolic pathways of NO. Figure 2. X-band EPR spectra of NO-Fe2+(DETC)2 complex in left ventricular tissue samples of rat hearts. (A): dotted line, positive control (NO donor sodium nitroprusside), with gain reduced by a factor of 10 compared to curves B-D; solid line, sum of Lorentzians fitted to the NO signal. (B): background spectrum, Cu2+(DETC)2. (C): negative control (NOS inhibitor NG-nitro-L-arginine). (D): increased NO production in nitroglycerin-tolerant animals. The solid lines on trace (D) are the fitted spectrum from trace A, but scaled down to match the +1 and -1 hyperfine lines, upper and lower solid lines, respectively, of the NO spectral component of the spectrum. The +1, 0, –1 numbers indicate the hyperfine lines of NOFe2+(DETC)2 triplet (perpendicular orientation). The +3/2, +1/2, –1/2, –3/2 numbers indicate the hyperfine lines of Cu2+(DETC)2 (perpendicular orientation). Sample temperature was 160 K, central field and scan range are 335.6 mT and 34 mT, respectively. The g=2.004 position of the g-value was determined by using a g standard. (Figure is modified with permission from (Csont et al., 1998)). 44 This article is protected by copyright. All rights reserved. NO detection Csonka et al. Accepted Article Conflict of interest: none 45 This article is protected by copyright. All rights reserved. Accepted Article NO detection Csonka et al. bph_12832_f1 46 This article is protected by copyright. All rights reserved. Accepted Article NO detection Csonka et al. bph_12832_f2 47 This article is protected by copyright. All rights reserved.