Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the L-arginine/nitric oxide area of research

In the Griess reaction, first reported by Johann Peter Griess in 1879 as a method of analysis of nitrite (NO(2)(-)), nitrite reacts under acidic conditions with sulfanilic acid (HO(3)SC(6)H(4)NH(2)) to form a diazonium cation (HO(3)SC(6)H(4)-N[triple bond]N(+)) which subsequently couples to the aromatic amine 1-naphthylamine (C(10)H(7)NH(2)) to produce a red-violet coloured (lambda(max) approximately 540 nm), water-soluble azo dye (HO(3)SC(6)H(4)-NN-C(10)H(6)NH(2)). The identification of nitrite in saliva has been the first analytical application of this diazotization reaction in 1879. For a century, the Griess reaction has been exclusively used to identify analytically bacterial infection in the urogenital tract, i.e. to identify nitrite produced by bacterial reduction of nitrate (NO(3)(-)), the major nitrogen oxide anion in human urine. Since the discovery of the l-arginine/nitric oxide (l-Arg/NO) pathway in 1987, however, the Griess reaction is the most frequently used analytical approach to quantitate the major metabolites of NO, i.e. nitrite and nitrate, in a variety of biological fluids, notably blood and urine. The Griess reaction is specific for nitrite. Analysis of nitrate by this reaction requires chemical or enzymatic reduction of nitrate to nitrite prior to the diazotization reaction. The simplicity of the Griess reaction and its easy and inexpensive analytical feasibility has attracted the attention of scientists from wide a spectrum of disciplines dedicated to the complex and challenging L-Arg/NO pathway. Today, we know dozens of assays based on the Griess reaction. In principle, every laboratory in this area uses its own Griess assay. The simplest Griess assay is performed in batch commonly as originally reported by Griess. Because of the recognition of numerous interferences in the analysis of nitrite and nitrate in biological fluids and of the desire to analyze these anions simultaneously, the Griess reaction has been repeatedly modified and automated. In recent years, the Griess reaction has been coupled to HPLC, i.e. is used for post-column derivatization of chromatographically separated nitrite and nitrate. Such a HPLC-Griess system is even commercially available. The present article gives an overview of the currently available assays of nitrite and nitrate in biological fluids based on the Griess reaction. Special emphasis is given to human plasma and urine, to quantitative aspects, as well as to particular analytical and pre-analytical factors and problems that may be associated with and affect the quantitative analysis of nitrite and nitrate in these matrices by assays based on the Griess reaction. The significance of the Griess reaction in the L-Arg/NO pathway is appraised.     

Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high-performance anion-exchange liquid chromatography for their determination

Measurements of nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO(2)(-) and NO(3)(-) in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO(2)(-)/NO(3)(-) contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO(2)(-) and NO(3)(-) in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO(2)(-) and NO(3)(-) content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO(2)(-) and NO(3)(-) in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO(2)(-) and NO(3)(-) in standards was 3 pmol. Recoveries of NO(2)(-) and NO(3)(-) from spiked plasma (1-100 microM KNO(2)/KNO(3)) and from extracted standards (1-250 microM) were approximately 100%. Intra-assay and inter-assay RSDs for NO(2)(-) and NO(3)(-) in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO(2)(-) and NO(3)(-) in various biological fluids, e.g., plasma.     

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Modification of the cadmium reduction assay for detection of nitrite production using fluorescence indicator 2,3-diaminonaphthalene.

The measurement of nitric oxide (NO) is important for characterizing the regulatory roles of NO in various biological systems. In this communication we report that cadmium (Cd) reduction of nitrate (NO(-)(3)) to nitrite (NO(-)(2)) can be quantitated by using the fluorescence indicator, 2,3-diaminonaphthalene (DAN) to detect the sum of NO(-)(3) and NO(-)(2) (NO(-)(x)) from endothelial cells. This assay is at least 10-fold more sensitive than when Cd reduction is coupled with the spectrophotometric Greiss reaction and can be used to quantitate the small amounts of NO(-)(x) generated from the constitutive form of endothelial nitric oxide synthase (eNOS). In addition various P(2) purinoceptor agonists and antagonists do not interfere the Cd reduction/DAN assay. Thus the Cd reduction/DAN assay can be used not only to characterize P(2) purinoceptor release of NO(-)(x) from cultured endothelial cells but also to quantitate NO(-)(x) levels in serum.     

Nitric oxide homeostasis in Salmonella typhimurium: roles of respiratory nitrate reductase and flavohemoglobin

Nitric oxide (NO) is generated in biological systems primarily via the activity of NO synthases and nitrate and nitrite reductases. Here we show that Salmonella enterica serovar Typhimurium (S. typhimurium) grown anaerobically with nitrate is capable of generating polarographically detectable NO after nitrite (NO(2)(-)) addition. NO accumulation is sensitive to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Neither an fnr mutant nor an fnr hmp double mutant produces NO, indicating the involvement in NO evolution from NO(2)(-) of protein(s) positively regulated by FNR. Contrary to previous findings in Escherichia coli, we demonstrate that neither the periplasmic nitrite reductase (NrfA) nor the cytoplasmic nitrite reductase (NirB) is involved in NO production in S. typhimurium. However, mutant cells lacking the membrane-bound nitrate reductase, NarGHI, and membranes derived from these cells are unable to produce NO, demonstrating that, in wild-type S. typhimurium, this enzyme is responsible for NO production. Membrane terminal oxidases cannot account for the NO levels measured. The nitrate reductase inhibitor, azide, abrogates NO evolution by Salmonella, and production of NO occurs only in the absence from the assays of nitrate; both features reveal a marked similarity between the NO-generating activities of this bacterium and plants. Unlike the situation in E. coli, an S. typhimurium hmp mutant produces NO both aerobically and anaerobically. Under aerobic conditions, when a functional flavohemoglobin is present, no NO is detectable. We propose a homeostatic mechanism in S. typhimurium, in which NO produced from NO(2)(-) by nitrate reductase derepresses Hmp expression (via FNR and NsrR) and NorV expression (via NorR) and thus limits NO toxicity.     

ReviewMethods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.     

Fluorometric measurement of nitrite/nitrate by 2,3-diaminonaphthalene

We describe a step-by-step protocol for measuring the stable products of the nitric oxide (NO) pathway: nitrite, nitrite plus nitrate and nitrate. This described protocol is easy to apply and is about 50 times more sensitive than the commonly used Griess reaction or commercially available assay kits based on the Griess reaction. It also allows the study of minimal changes in the NO pathway. With this method, it takes about 3 h to analyze the above-mentioned stable products in culture supernatants or in various body fluids, and the method has a sensitive linear range of 0.02-10.0 microM. This restricted linear range suggests that the technique is useful for studying small changes of nitrite and nitrate, rather than for routine diagnostic measurements.     

Nitric oxide and nitrite-based therapeutic opportunities in intimal hyperplasia

Vascular intimal hyperplasia (IH) limits the long term efficacy of current surgical and percutaneous therapies for atherosclerotic disease. There are extensive changes in gene expression and cell signaling in response to vascular therapies, including changes in nitric oxide (NO) signaling. NO is well recognized for its vasoregulatory properties and has been investigated as a therapeutic treatment for its vasoprotective abilities. The circulating molecules nitrite (NO(2)(-)) and nitrate (NO(3)(-)), once thought to be stable products of NO metabolism, are now recognized as important circulating reservoirs of NO and represent a complementary source of NO in contrast to the classic L-arginine-NO-synthase pathway. Here we review the background of IH, its relationship with the NO and nitrite/nitrate pathways, and current and future therapeutic opportunities for these molecules.     

Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography

Various analytical techniques have been developed to determine nitrite and nitrate, oxidation metabolites of nitric oxide (NO), in biological samples. HPLC is a widely used method to quantify these two anions in plasma, serum, urine, saliva, cerebrospinal fluid, tissue extracts, and fetal fluids, as well as meats and cell culture medium. The detection principles include UV and VIS absorbance, electrochemistry, chemiluminescence, and fluorescence. UV or VIS absorbance and electrochemistry allow simultaneous detection of nitrite and nitrate but are vulnerable to the severe interference from chloride present in biological samples. Chemiluminescence and fluorescence detection improve the assay sensitivity and are unaffected by chloride but cannot be applied to a simultaneous analysis of nitrite and nitrate. The choice of a detection method largely depends on sample type and facility availability. The recently developed fluorometric HPLC method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) and the enzymatic conversion of nitrate into nitrite, offers the advantages of easy sample preparation, simple derivatization, stable fluorescent derivatives, rapid analysis, high sensitivity and specificity, lack of interferences, and easy automation for determining nitrite and nitrate in all biological samples including cell culture medium. To ensure accurate analysis, care should be taken in sample collection, processing, and derivatization as well as preparation of reagent solutions and mobile phases, to prevent environmental contamination. HPLC methods provide a useful research tool for studying NO biochemistry, physiology and pharmacology.     

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.     

A spectrophotometric assay for nitrate in an excess of nitrite.

Miranda et al. have developed a method for simultaneous evaluation of nitrate and nitrite concentrations using reduction of nitrate by vanadium(III) combined with detection by the acidic Griess reaction [K.M. Miranda, M.G. Espey, D.A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide 5 (2001) 62-71]. The sensitivity of the nitrate assay decline if the mixture analyzed contains a large excess of nitrite relative to nitrate, for instance, in the case of oxidation products of nitric oxide (NO) in aerated solutions, or in sweat. By this reason nitrite should be removed before the nitrate assay, if [NO2-]>[NO3-]. Here we lay out an improved method allowing the above limitation to be erased, using sulfamic acid for nitrite removal. We also describe some modifications that enhance the reproducibility of the assay.     

Catalysis of intermolecular oxygen atom transfer by nitrite dehydrogenase of Nitrobacter agilis

Nitrobacter agilis, which contains a very active nitrite dehydrogenase, was studied in vivo under anaerobic conditions by the 15N NMR technique. When incubated with equimolar 15NO3- and unlabeled nitrite (or 15NO2- and unlabeled nitrate) the bacterium catalyzed an isotope exchange reaction at rates about 10% those observed in the nitrite oxidase assay. When incubated with 18O-labeled 15NO2- and 18O-labeled 15NO3-, the 18O was observed to exchange at similar rates from both species into water. Finally, when incubated with equimolar [18O]nitrate and 15NO2-, intermolecular 18O transfer was observed to result in formation of double labeled nitrate and nitrite at similar rates. 18O was transferred from nitrate to a 15N species or to water at approximately equal rates under the conditions of the experiments. It is argued that the enzyme responsible for these exchange reactions is nitrite dehydrogenase and not nitrate reductase. This work and the related experiments of DiSpirito and Hooper (DiSpirito, A.A., and Hooper, A.B. (1986) J. Biol. Chem. 261, 10534-10537) represent the first demonstrations of intermolecular oxygen atom transfer among oxotransferases. Mechanistic implications are discussed.     

Time-course of changes in plasma nitric oxide following lipopolysaccharide and turpentine injection in rats

The effect of lipopolysaccharide (LPS) and turpentine on nitric oxide (NO) production were investigated in rats. Because of short half-life of NO in biological fluids, the plasma nitrite and nitrate concentrations (two stabile metabolites of NO) were measured based on Griess reaction, which is indirect assay for NO production. Injection of LPS at an intraperitoneal dose of 50 μg/kg caused a 3,5-fold increase in plasma nitrite within 3 h and nitrite levels remained significantly elevated 6, 12, and 24 h after endotoxin treatment with LPS. However, injection of turpentine at an intramuscular dose of 20 μl/rat did not alter plasma nitrite concentration at selected times after turpentine treatment (7, 10, 14, and 24 h postinjection). These results further support the hypothesis that NO is involved in pathogenesis of febrile response due to LPS in rats. Because turpentine did not change concentration of NO in plasma, the role of NO, as mediator/modulator, in development of turpentine fever appears to be controversial and needs further experimental verification.     

Codenitrification and denitrification are dual metabolic pathways through which dinitrogen evolves from nitrate in Streptomyces antibioticus

We screened actinomycete strains for dinitrogen (N(2))-producing activity and discovered that Streptomyces antibioticus B-546 evolves N(2) and some nitrous oxide (N(2)O) from nitrate (NO(3)(-)). Most of the N(2) that evolved from the heavy isotope ([(15)N]NO(3)(-)) was (15)N(14)N, indicating that this nitrogen species consists of two atoms, one arising from NO(3)(-) and the other from different sources. This phenomenon is similar to codenitrification in fungi. The strain also evolved less, but significant, amounts of (15)N(15)N from [(15)N]NO(3)(-) in addition to (15)N(15)NO with concomitant cell growth. Prior to the production of N(2) and N(2)O, NO(3)(-) was rapidly reduced to nitrite (NO(2)(-)) accompanied by distinct cell growth, showing that the actinomycete strain is a facultative anaerobe that depends on denitrification and nitrate respiration for anoxic growth. The cell-free activities of denitrifying enzymes could be reconstituted, supporting the notion that the (15)N(15)N and (15)N(15)NO species are produced by denitrification from NO(3)(-) via NO(2)(-). We therefore demonstrated a unique system in an actinomycete that produces gaseous nitrogen (N(2) and N(2)O) through both denitrification and codenitrification. The predominance of codenitrification over denitrification along with oxygen tolerance is the key feature of nitrate metabolism in this actinomycete.     

Nitrate in exhaled breath condensate of patients with different airway diseases.

There is an increasing interest in the measurement of nitric oxide (NO.) in the airways. NO. is a free radical that reacts rapidly with reactive oxygen species in aqueous solution to form peroxynitrite which can then break down to nitrite (NO(2)(-)) and nitrate (NO(3)(-)). NO(3)(-) is considered a stable oxidative end product of NO. metabolism. The aim of this study was to assay NO(3)(-) in exhaled breath condensate (EBC) of normal nonsmoking and smoking subjects, asthmatics, patients with obstructive pulmonary disease (COPD), and patients with community-acquired pneumonia (CAP). EBC was collected using a glass condenser and samples were assayed for NO(3)(-) by ion chromatography followed by conductivity measurement. NO(3)(-) was detectable in EBC of all subjects. NO(3)(-) was elevated in smokers [median (range)] [62.5 (9.6-158.0) microM] and in asthmatics [68.0 (25.8-194.6) microM] compared to controls [9.6 (2.6-119.4) microM; p=0.003 and p=0.006, respectively], whereas NO(3)(-) was not elevated in COPD patients [24.1 (1.9-337.0 microM]. The concentration of NO(3)(-) in patients with CAP [243.4 (26.1-584.5) microM] was higher than that in controls (p=0.002) and NO(3)(-) values decreased after treatment and recovery from illness [40.0 (4.1-167.0) microM, p=0.009]. This study shows that NO(3)(-) is detectable in EBC of healthy subjects and it varies in patients with inflammatory airway diseases.     

Magnetic resonance study of the transmembrane nitrite diffusion.

Nitrite (NO(2)-), being a product of metabolism of both nitric oxide (NO(*)) and nitrate (NO(3)-), can accumulate in tissues and regenerate NO() by several mechanisms. The effect of NO(2)- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO(2)- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO(2)). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO(2) transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO(2)(-)-mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by (31)P NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO(2)- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 microM NO(2)- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NO(x) in myocardium increased from initial 7.8 to 24.7 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.     

Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method.

Nitric oxide (NO) is a signal molecule with functions such as neurotransmission, local vascular relaxation, and anti-inflammation in many physiological and pathological processes. Various factors regulate its intracellular lifetime. Due to its high reactivity in biological systems, it is transformed in the bloodstream into nitrates (NO(-)(3)) by oxyhemoglobin. The Griess reaction is a technically simple method (spectrophotometric, 540 nm) for the analysis of nitrites (NO(-)(2)) in aqueous solutions. We studied the interference of common anticoagulants in the quantification of nitrate and nitrite in plasma samples by the Griess method. We obtained rat plasma using heparin or sodium EDTA as anticoagulants, then added, or otherwise, known NO(-)(3) amounts in order to calculate their recovery. We also studied the effect of ultra-filtration performed before Griess reaction on plasma and aqueous solutions of various anticoagulants (heparin, EDTA, and also sodium citrate) to compare the recoveries of added NO(-)(3) or NO(-)(2). We used standards of NO(-)(3) or NO(-)(2) for quantification. We conclude that: (i) The bacterial nitrate reductase used to reduce NO(-)(3) to NO(-)(2) is unstable in certain storage conditions and interferes with different volumes of plasma used. (ii) The ultrafiltration (which is sometimes performed before the Griess reaction) of plasma obtained with EDTA or citrate is not recommended because it leads to overestimation of NO(minus sign)(3). In contrast, ultrafiltration is necessary when heparin is used. (iii) The absorbance at 540 nm attributed to plasma itself (basal value or background) interferes in final quantification, especially when ultrafiltration is not performed. For the quantification of plasma NO(-)(3) we recommend: sodium EDTA as anticoagulant, no ultrafiltration of plasma, and measurement of the absorbance background of each sample.     

Measurement of nitric oxide metabolites in brain microdialysates by a sensitive fluorometric high-performance liquid chromatography assay

Nitric oxide (NO), formed from arginine by a specific neuronal NO synthase, is an important neurotransmitter in various regions of the central nervous system. While intracerebral microdialysis is an elegant technique to study local extracellular neurotransmitter concentrations in vivo, NO metabolites (nitrate, nitrite (NO(x))) are difficult to study at high temporal resolution because of low tissue concentrations and small sample volumes. We developed a sensitive fluorometric high-performance liquid chromatography (HPLC)-coupled NO(x) assay adapted for the use in brain microdialysate samples. The assay includes an initial enzymatic step in which nitrate is reduced to nitrite. Nitrite is acidified to N2O3, which reacts with 2,3-diaminonaphthalene to form 1-(H)-naphthotriazole. This reaction product can be readily isolated and quantitated by HPLC with fluorometric detection. The theoretical assay sensitivity is less than 1 nM, but numerous sources of contamination must be eliminated in the sampling and assaying process to reliably monitor brain NO(x) outflow by microdialysis.     

The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts

Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.