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.     

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.     

Critical evaluation of the in vivo nitrate reductase assay for detection of two nitrate pools in wheat leaves

The usefulness of the nitrate-free in vivo nitrate reductase assay for the study of nitrate pools in wheat leaves was investigated. Leaf sections from 7-day-old wheat seedlings, exposed 24 h before harvest to 1.5, 3.0 or 5.0 m M KNO₃ were used. After 2 to 4 h of incubation nitrite production ceased, reaching a plateau. The time required to reach the plateau and the level of the plateau increased with increasing endogenous nitrate content. At nitrite plateau the amount of nitrate left in the tissue was independent of the original nitrate content in the tissue. Addition of nitrate at plateau caused resumed nitrite production. It is concluded that nitrate was the limiting factor in nitrite production.
Oxygen inhibited nitrate reduction and stimulated further assimilation of nitrite. A considerable initial leakage of nitrate from tissue to the assay medium, followed by a slower continuous leakage, was observed throughout the incubation. N₂-flushing or inclusion of Triton X-100 in the assay medium increased nitrite production by making more nitrate available for reduction. These treatments also increased the leakage of nitrate. At plateau levels the amount of nitrate left in the tissue was dependent on the oxygen tension in the assay medium. Under low oxygen tension nearly all nitrate in the tissue was available for reduction. Nitrite production at plateau is not a useful index for a metabolic nitrate pool and nitrate left in the tissue is not a useful index for a nitrate storage pool because both parameters are highly dependent on the oxygen tension in the assay medium. Further, in view of the considerable leakage, the nitrate-free in vivo nitrate reductase assay cannot be used to detect two separate nitrate pools in wheat leaves.     

Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis

Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.     

Nark is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli

Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest-lived radioactive isotope of nitrogen, ¹³N-nitrate (half-life = 9.96 min) and the nitrite-sensitive fluorophore N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.     

Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of Escherichia coli: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite

The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using Escherichia coli membranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 microM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 microM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 microM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator.     

Inorganic nitrate is a possible source for systemic generation of nitric oxide

Nitrate and nitrite have been considered stable inactive end products of nitric oxide (NO). While several recent studies now imply that nitrite can be reduced to bioactive NO again, the more stable anion nitrate is still considered to be biologically inert. Nitrate is concentrated in saliva, where a part of it is reduced to nitrite by bacterial nitrate reductases. We tested if ingestion of inorganic nitrate would affect the salivary and systemic levels of nitrite and S-nitrosothiols, both considered to be circulating storage pools for NO. Levels of nitrate, nitrite, and S-nitrosothiols were measured in plasma, saliva, and urine before and after ingestion of sodium nitrate (10 mg/kg). Nitrate levels increased greatly in saliva, plasma, and urine after the nitrate load. Salivary S-nitrosothiols also increased, but plasma levels remained unchanged. A 4-fold increase in plasma nitrite was observed after nitrate ingestion. If, however, the test persons avoided swallowing after the nitrate load, the increase in plasma nitrite was prevented, thereby illustrating its salivary origin. We show that nitrate is a substrate for systemic generation of nitrite. There are several pathways to further reduce this nitrite to NO. These results challenge the dogma that nitrate is biologically inert and instead suggest that a complete reverse pathway for generation of NO from nitrate exists.     

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.     

Endogenous nitrate loss as an assay for nitrate reduction in vivo

An in vivo assay method for nitrate reduction is proposed, based on the use of endogenous nitrate rather than on the accumulation of nitrite. Loss of endogenous nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaves. Leaf sections were incubated in the dark in a gaseous environment of air or N₂. Nitrate disappeared under both conditions, the highest loss being observed in tissue under anaerobiosis. Nitrite accumulated only in leaf sections under anaerobiosis, but the amount of nitrite accumulated was much lower than the amount of nitrate lost. A comparative study of the capacity of barley leaf sections to use endogenous nitrate and accumulate nitrite showed that both activities were dependent on temperature in a manner characteristic of enzymatic reactions. Disappearance of endogenous nitrate increased with increasing levels of nitrate in the tissue.     

Measurement of serum nitrite/nitrate concentrations using high-performance liquid chromatography

Previous studies have reported increased serum concentrations of nitrite/nitrate – the degradation products of nitric oxide – in Plasmodium vivax malaria and uncomplicated Plasmodium falciparum malaria. In all these studies, however, nitrite/nitrate has been measured spectrometrically using Griess reagent which carries major disadvantages in the determination of serum nitrite/nitrate. The method does not allow an exact differentiation of nitrite and biogenic amines that are physiologically present in plasma. In the present study we introduce high-performance liquid chromatography as a new, accurate and cost effective method for determination of serum nitrite/nitrate levels. Significantly increased nitrate concentrations were found in malaria patients and serum values remained above normal levels for at least 21 days. It could be shown that our HPLC method is a sensitive and cost-effective method for direct determination of nitrite/nitrate in serum samples, which is not influenced by the presence of biogenic amines.     

The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash

Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10 mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.     

Effect of Sodium Nitrite and Sodium Nitrate on Botulinal Toxin Production and Nitrosamine Formation in Wieners

Wieners were formulated and processed approximating commercial conditions as closely as possible. Twenty-four batches of product were made with the addition of six levels of sodium nitrite (0, 50, 100, 150, 200, and 300 mug/g), four levels of sodium nitrate (0, 50, 150, and 450 mug/g), and two levels of Clostridium botulinum (0 and 620 spores/g). After formulation, processing, and vacuum packaging, portions of each batch were incubated at 27 C or held for 21 days at 7 C followed by incubation at 27 C for 56 days. The latter storage condition approximated distribution of product through commercial channels and potential temperature abuse at the consumer level. Samples were analyzed for botulinal toxin, nitrite, and nitrate levels after 3, 7, 14, 21, 28, and 56 days of incubation. When nitrite was not added, toxic samples were detected after 14 days of incubation at 27 C. At the lowest level of nitrite added (50 mug/g), no toxic samples were observed until 56 days of incubation. Higher levels of nitrite completely inhibited toxin production throughout the incubation period. Nine uninoculated samples, representing various levels and combinations of nitrite and nitrate, were evaluated organoleptically. The flavor quality of wieners made with nitrite was judged significantly higher (P = 0.05) than of wieners made without nitrite. The nine samples were negative for 14 volatile nitrosamines at a sensitivity level of 10 ng/g. The results indicated that nitrite effectively inhibited botulinal toxin formation at commercially employed levels in wieners and that detectable quantities of nitrosamines were not produced during preparation and processing of the product for consumption.     

Production of hydrogen peroxide and nitric oxide following introduction of nitrate and nitrite into wheat leaf apoplast

Infiltration of wheat (Triticum aestivum L.) seedling leaves with excess of nitrate, nitrite, or the NO donor sodium nitroprusside leads to increase both in content of hydroperoxide and activity of peroxidase and decrease in superoxide dismutase (SOD) activity in the leaf apoplast. Polymorphism of extracellular peroxidases and the presence of Cu/Zn-SOD have been shown in apoplast. Using an ESR assay, a considerable increase in the level of NO following infiltration of leaf tissues with nitrite has been demonstrated. These data suggest development of both oxidative and nitrosative stresses in leaves exposed to high levels of nitrate or nitrite. A possible interplay of NO and reactive oxygen species in plant cells is discussed.     

Modulation of cerebellar and hepatic nitric oxide synthase by exogenous arginine and endotoxin.

We have studied in mice the effect of treatment with exogenous arginine and/or LPS by monitoring serum nitrite/nitrate levels and by investigating the response of cerebellar and liver nitric oxide synthase (NOS). We measured NOS activity in cerebellar extracts while changes in iNOS mRNA were followed in the liver since direct assay of NOS activity proved unreliable with this tissue. In fact, liver and cerebellum extracts were both very active in converting arginine into a citrulline-like metabolite, but only cerebellum conversion was dependent on addition of NADPH and inhibitable by N(G)-methyl-l-arginine. Treatment with LPS, on its own, increased serum nitrite/nitrate levels at 5 and 20 h after injection, while treatment with LPS and arginine produced nitrite/nitrate levels in the serum even greater at 5 h, but significantly lower at 20 h. Liver iNOS mRNA levels were markedly increased by LPS, and this effect was significantly decreased when mice were also given exogenous arginine. A stimulatory effect of LPS was also found on NOS activity in the cerebellum, where a very small stimulation may have also been caused by arginine feeding. These findings indicate that LPS stimulates NOS expression/activity both in the cerebellum and in the liver and suggest a complex pattern of modulation of iNOS by arginine, with NO being first produced in excess and then downregulating iNOS expression.     

Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function

Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.     

Analysis of nitrite/nitrate in biological fluids: denitrification of 2-nitropropane in F344 rats

2-Nitropropane (2-NP), a rat hepatocarcinogen, is denitrified to nitrite and acetone by rat liver microsomes; the denitrification rate is increased using microsomes from phenobarbital (PB)-pretreated rats. To obtain evidence that denitrification of 2-NP also occurs in vivo, we attempted to determine nitrite and nitrate levels in blood sera and urines of 2-NP-treated (1.5 mmol/kg, ip, once) rats with and without PB pretreatment (80 mg/kg, ip, once daily, 3 days), using enzymatic reduction followed by the standard Griess reaction. However, due to various interfering factors, including pigment from methemoglobinemia, we found the assay had to be modified as follows: (a) reduction of nitrate to nitrite was accomplished using NADPH and nitrate reductase, (b) excess NADPH, proteins, and interfering pigments were precipitated using zinc acetate and Na(2)CO(3), and (c) the Griess reagents were prepared in 3 N HCl rather than 5% H(3)PO(4). With these modifications it became possible to show that 2-NP is indeed metabolized to nitrite in vivo and that the metabolism is increased by PB pretreatment. Two hours after 2-NP administration, rat blood serum nitrate plus nitrite levels were approximately 1600 microM (PB-pretreated) and 940 microM (vehicle-pretreated controls). The PB-pretreated and control rats, respectively, excreted 250 and 120 micromol nitrate/nitrite in the 24-h urine post 2-NP treatment. The modifications described make the method more specific, reproducible, and more widely applicable.     

Characterization of the assimilatory and dissimilatory nitrate-reducing systems in Rhodobacter: a comparative study

Abstract: Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 was found to be a cytosolic NADH-dependent enzyme whereas nitrite reductase was detected both in the periplasm and in the cytosol. In addition to nitrate and nitrite, organic nitro compounds were also able to induce nitrate reductase which was repressed in carbon-starved cells or under conditions producing low intracellular nitrate levels. By contrast, nitrate reductase from Rhodobacter sphaeroides DSM 158 was located in the periplasm, did not use NADH as electron donor in vitro and was induced independently from the carbon/nitrogen balance of the cells. Production of nitrogenous gases was never detected in either strain cultured anaerobically with nitrate or nitrite.     

Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer

Mass spectrometry-based approaches are the reference techniques for the determination of nitrite and nitrate in plasma and serum. However, due to their simplicity and rapidity, assays based on the Griess reaction or HPLC are generally used in clinical studies, but they generate diverging values for nitrite/nitrate concentration. In this study, particular attention is paid to the optimization of the deproteinization procedure for plasma and serum samples prior to nitrite/nitrate analysis by an enzymatic batch Griess assay, HPLC and GC-MS. A method is reported to verify completeness of deproteinization and to correct for nonspecific contribution to the absorbance of the diazo dye at 540 nm. With the application of such optimized procedures, we were able to significantly improve the correlation between Griess and HPLC method or the GC-MS technique for nitrite+nitrate concentrations in human serum and plasma. Despite remaining potentially interfering pre-analytical and analytical factors, the procedures reported in the present study may be helpful in a critical evaluation of limits and possibilities of the enzymatic batch Griess assay as a large-scale method for nitrite/nitrate determination in human serum in clinical studies.     

Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite

Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle, but little is known about NO production by commensal bacteria. We used a chemiluminescence assay to explore if human faeces and different representative gut bacteria are able to generate NO. Bacteria were incubated anaerobically in gas-tight bags, with or without nitrate or nitrite in the growth medium. In addition, luminal NO levels were measured in vivo in the intestines in germ-free and conventional rats, and in rats mono-associated with lactobacilli. We show that human faeces can generate NO after nitrate or nitrite supplementation. Lactobacilli and bifidobacteria generated much NO from nitrite, but only a few of the tested strains produced NO from nitrate and at much lower levels. In contrast, Escherichia coli, Bacteroides thetaiotaomicron, and Clostridium difficile did not produce significant amounts of NO either with nitrate or nitrite. NO generation in the gut lumen was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-associated with lactobacilli. We conclude that NO can be generated by the anaerobic gut flora in the presence of nitrate or nitrite. Future studies will reveal its biological significance in regulation of gastrointestinal integrity.