An improved method to measure nitrate/nitrite with an NO-selective electrochemical sensor.

Nitric oxide (NO) produced from NO synthase(s) (NOS) is an important cell signaling molecule in physiology and pathophysiology. It remains challenging, however, to measure NO accurately and reproducibly in many cell types producing relatively low levels of NO from the enzymes such as endothelial NO synthase (eNOS). In the present study, we describe a very sensitive and convenient analytical method that affords measurement of 1 to 2 nM concentration of NO(x) (nitrite plus nitrate) in culture media. In the present study, we used an ultra-sensitive NO-selective electrochemical sensor (AmiNO700) in combination with a highly efficient nitrate conversion method, which coupled the nitrate reductase step with the glucose-6-phosphate dehydrogenase system. An aliquot of conditioned culture media was first treated with nitrate reductase, NADPH, glucose-6-phosphate dehydrogenase and glucose-6-phosphate to convert nitrate to nitrite quantitatively. The nitrite (that is present originally plus the reduced nitrate) was then reduced to equimolar NO in an acidic iodide bath while NO was being detected by the sensor. With this analytical method, we can quantitatively and reliably measure basal and stimulated NO release from cultured endothelial cells. We believe this improved assay should be useful in measuring a wide range of NO levels, especially the low but physiologically relevant levels, in many cell types.     

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of -arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [¹⁵N]nitrite and [¹⁵N]nitrate were used as internal standards. Endogenous nitrite and [¹⁵N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [¹⁵N]nitrate they were reduced to nitrite and [¹⁵N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [¹⁵N]nitrite ([¹⁵N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Role of brain nitric oxide in the thermoregulation of broiler chicks

Nitric oxide (NO) plays a key role in body temperature (Tb) regulation of mammals, acting on the brain to stimulate heat loss. Regarding birds, the putative participation of NO in the maintenance of Tb in thermoneutrality or during heat stress and the site of its action (periphery or brain) is unknown. Thus, we tested if NO participates in the maintenance of chicks' Tb in those conditions. We investigated the effect of intramuscular (im; 25, 50, 100 mg/kg) or intracerebroventricular (icv; 22.5, 45, 90, 180 µg/animal) injections of the non selective NO synthase inhibitor L-NAME on Tb of 5-day-old chicks at thermoneutral zone (TNZ; 31–32 °C) and under heat stress (37 °C for 5–6 h). We also verified plasma and diencephalic nitrite/nitrate levels in non-injected chicks under both conditions. At TNZ, 100 mg/kg (im) or 45, 90, 180 µg (icv) of L-NAME decreased Tb. A significant correlation between Tb and diencephalic, but not plasma, nitrite/nitrate levels was observed. Heat stress-induced hyperthermia was inhibited by all tested doses of L-NAME (im and icv). Tb was correlated neither with plasma nor with diencephalic nitrite/nitrate levels during heat stress. These results indicate the involvement of brain NO in the maintenance of Tb of chicks, an opposite action of that observed in mammals, and may modulate hyperthermia.     

A new method to measure nitrate/nitrite with a NO-sensitive electrode

There are different methods to measure the unstable moleculenitric oxide (NO). We will describe a new sensitive method to measure NO by reconversion of nitrate/nitrite to NO, which will bedetermined with an amperometric Clark-type electrode. Nitrate andnitrite are the degradation products of NO. First, nitrate isenzymatically converted to nitrite with the use of the nitrate reductase. Second, nitrite is reduced to equimolar NO concentrations byan acidic iodide solution. The detection limit of the electrode in anaqueous solution was 2 nmol/l NO (meaning the threshold was dependingon the volume added: 500 µl of a 0.2 µmol/l nitrite solution addedto a 10-ml bath). This method provides the ability to assess basal andagonist-stimulated NO releases of different biological models. Wemeasured basal and carbachol-stimulated NO release of nativeendothelial cells from porcine coronary arteries and porcine aorticendothelial cell cultures. Moreover, it was possible to measure thenitrate/nitrite concentration in the coronary effluent of a guinea pigheart. In conclusion, we present a valid, highly sensitive new methodof measuring nitrite/NO in biological systems with a commerciallyavailable electrode.

     

Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia

Prolonged exposure to arsenic results in peripheral and cardiovascular manifestations, as does impaired production of endothelial nitric oxide (NO). In vitro studies have indicated that endothelial cells undergo damage by arsenic. However, no information has been available on the relationship between NO synthesis and chronic arsenic poisoning in humans. The present study was designed to reveal this question. The subjects were 33 habitants who continued to drink well water containing high concentrations of inorganic arsenic (mean value = 0.41 microg/ml) for about 18 years in Inner Mongolia, China, and 10 other people who lived in this area but exposed to minimal concentrations of arsenic (mean value = 0.02 microg/ml) were employed as controls. Mean blood concentration of total arsenic was six times higher in exposed subjects than controls; 42.1 vs. 7.3 ng/ml, p <.001. Mean serum concentration of nitrite/nitrate, stable metabolites of endogenous NO, was lower in arsenic-exposed subjects than in controls: 24.7 vs. 51.6 microM, p<.001. In total samples, an inverse correlation with serum nitrite/nitrate levels was strong for blood inorganic arsenic (r = -0.52, p <.001) and less strong for its metabolites, monomethyl arsenic (r = -0.45, p<.005) and dimethyl arsenic (r = -0.37, p<.05). Furthermore, serum nitrite/nitrate concentration was significantly correlated with nonprotein sulfhydryl level in whole blood (r = 0.58, p<.001). In an in vitro study, we demonstrated that inorganic arsenite or arsenate suppresses the activity of endothelial NO synthase in human umbilical vein endothelial cells. These results suggest that long-term exposure to arsenic by drinking well water possibly reduces NO production in endothelial cells, resulting in a decrease in reduced nitrite/nitrate concentrations. Peripheral vascular disorders caused by arsenic may be attributable in part to impairment of NO production in vivo.     

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.     

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia-reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS-/-) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS-/- mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.     

The Role of Preventing Nitric Oxide Deficiency in the Antihypertensive Effect of Adaptation to Hypoxia

Shortage of endothelial nitric oxide (NO) manifested as decreased daily urinary excretion of nitrate and nitrite as well as attenuated endothelium-dependent relaxation of conduit and resistance vessels progresses with age-related increase of blood pressure (BP) in stroke-prone spontaneously hypertensive rats (SHRSP). Simultaneous NO-dependent suppression of vascular contractions is, apparently, due to the inducible NO synthase activity in vascular smooth muscle specific for spontaneously hypertensive rat. The adaptation of rats to hypobaric hypoxia initiated at early hypertensive stage (at the age of 5–6 weeks) decelerates hypertension progress. The antihypertensive effect of the adaptation was accompanied by stimulation of endothelial NO synthesis and prevention of impaired NO-dependent response in isolated blood vessels. Nitric oxide stores were formed in the vascular wall of SHRSP and WKY rats at the same time. The obtained data indicate that the correction of endothelial NO deficiency plays a significant role in the antihypertensive effect of adaptation to hypoxia.     

Cytokine treatment increases arginine metabolism and uptake in bovine pulmonary arterial endothelial cells

L-Arginine (L-Arg) is metabolized to nitric oxide (NO) by NO synthase (NOS) or to urea by arginase (AR). L-Arg is transported into bovine pulmonary arterial endothelial cells (BPAECs) by cationic amino acid transporter-2 (CAT-2). We hypothesized that cytokine treatment would increase L-Arg metabolism and increase CAT-2 mRNA expression. BPAECs were incubated for 24 h in medium (control) or medium with lipopolysaccharide and tumor necrosis factor-alpha (L-T). L-T increased nitrite production (3.1 +/- 0.4 nmol/24 h vs. 1.8 +/- 0.1 nmol/24 h for control; P < 0.01) and urea production (83.5 +/- 29.5 nmol/24 h vs. 17.8 +/- 8.6 nmol/24 h for control; P < 0.05). L-T-treated BPAECs had greater endothelial and inducible NOS mRNA expression compared with control cells. Increasing the medium L-Arg concentration resulted in increased nitrite and urea production in both the control and the L-T-treated BPAECs. L-T treatment resulted in measurable CAT-2 mRNA. L-T increased L-[(3)H]Arg uptake (5.78 +/- 0.41 pmol vs. 4.45 +/- 0.10 pmol for control; P < 0.05). In summary, L-T treatment increased L-Arg metabolism to both NO and urea in BPAECs and resulted in increased levels of CAT-2 mRNA. This suggests that induction of NOS and/or AR is linked to induction of CAT-2 in BPAECs and may represent a mechanism for maintaining L-Arg availability to NOS and/or AR.     

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.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

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.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Involvement of vascular endothelial nitric oxide synthase in development of experimental diabetic nephropathy in rats

Endothelial nitric-oxide synthase (eNOS) acts as a common pathogenic pathway in diabetic nephropathy (DN). However, its functional consequences are still not fully understood. Caveolin, a membrane protein, inhibits the eNOS by making caveolin-eNOS complex, and its expression is upregulated during diabetes mellitus (DM). This study was designed to determine the role of caveolin in eNOS-mediated NO synthesis and release in DN. DM in rat was induced by feeding of high-fat diet (HFD) for 2 weeks, followed by single dose of streptozotocin (STZ) (35 mg/kg, ip) further followed by HFD for further 8 weeks. Serum nitrite/nitrate ratio was measured to determine the plasma level of NO. Diabetic rat, after 6 weeks of STZ, developed elevated level of BUN, protein in urine, urinary output, serum creatinine, serum cholesterol, kidney weight, kidney weight/body weight, and renal cortical collagen content, while serum nitrite/nitrate concentration was significantly decreased as compared to normal control group. Treatment with sodium nitrite (NO donor), L: -arginine (NO precursor), daidzein (caveolin inhibitor), and combination of L: -arginine and daidzein for 2 weeks markedly attenuated these changes and increased serum nitrite/nitrate ratio. However, treatment with L-NAME, a eNOS inhibitor, significantly attenuated the L: -arginine-, daidzein-, or combination of L: -arginine and daidzein-induced ameliorative effects in DN. The finding of this study suggests that caveolin plays a vital role in the eNOS-mediated decrease in renal level of NO, which may be responsible for the development of DN in rats.     

Concomitant presence of N-nitroso and S-nitroso proteins in human plasma

Nitric oxide (NO)-mediated nitrosation reactions are involved in cell signaling and pathology. Recent efforts have focused on elucidating the role of S-nitrosothiols (RSNO) in different biological systems, including human plasma, where they are believed to represent a transport and buffer system that controls intercellular NO exchange. Although RSNOs have been implicated in cardiovascular disease processes, it is yet unclear what their true physiological concentration is, whether a change in plasma concentration is causally related to the underlying pathology or purely epiphenomenological, and to what extent other nitrosyl adducts may be formed under the same conditions. Therefore, using gas phase chemiluminescence and liquid chromatography we sought to quantify the basal plasma levels of NO-related metabolites in 18 healthy volunteers. We find that in addition to the oxidative products of NO metabolism, nitrite (0.20 +/- 0.02 micromol/l nitrite) and nitrate (14.4 +/- 1.7 micromol/l), on average human plasma contains an approximately 5-fold higher concentration of N-nitroso species (32.3 +/- 5.0 nmol/l) than RSNOs (7.2 +/- 1.1 nmol/l). Both N- and S-nitroso moieties appear to be associated with the albumin fraction. This is the first report on the constitutive presence of a high-molecular-weight N-nitroso compound in the human circulation, raising the question as to its origin and potential physiological role. Our findings may not only have important implications for the transport of NO in vivo, but also for cardiovascular disease diagnostics and the risk assessment of nitrosamine-related carcinogenesis in man.     

Plasma nitrite reserve and endothelial function in the human forearm circulation

Attenuation of endothelium-derived nitric oxide (NO) synthesis is a hallmark of endothelial dysfunction. Early detection of this disorder may have therapeutic and prognostic implications. Plasma nitrite mirrors acute and chronic changes in endothelial NO-synthase activity. We hypothesized that local plasma nitrite concentration increases during reactive hyperemia of the forearm, reflecting endothelial function. In healthy subjects (n = 11) plasma nitrite and nitrate were determined at baseline and during reactive hyperemia of the forearm using reductive gas-phase chemiluminescence and flow-injection analysis, respectively. Endothelium-dependent dilation of the brachial artery was measured as flow-mediated dilation (FMD) using high-resolution ultrasound. Results were compared to patients with endothelial dysfunction as defined by reduced FMD (n = 11). Reactive hyperemia of the forearm increased local plasma nitrite concentration from 68 +/- 5 to 126 +/- 13 nmol/L (p < 0.01), whereas in endothelial dysfunction nitrite remained unaffected (116 +/- 12 to 104 +/- 10 nmol/L; n.s.), corresponding to nitrite reserves of 94 +/- 21 and -8 +/- 4%. This was accompanied by a significantly greater increase in brachial artery diameter (FMD: 8.5 +/- 0.4% vs 2.9 +/- 0.5%, for healthy subjects and endothelial dysfunction, respectively; p < 0.001). This observation suggests that nitrite changes reflect endothelial function. Assessment of local plasma nitrite during reactive hyperemia may open new avenues in the diagnosis of vascular function.     

Redox thiol status plays a central role in the mobilization and metabolism of nitric oxide in human red blood cells

We assessed the redox thiol status influence on nitric oxide (NO) metabolism and efflux in erythrocytes stimulated with acetylcholinesterase substrate (acetylcholine, ACh) and inhibitor (velnacrine maleate, VM). Erythrocyte suspensions from healthy donors were incubated with increasing concentrations of dithiothreitol (1-50 μM), in the presence and absence of acetylcholine/velnacrine (10 μM). Levels of NO, nitrite/nitrate, S-nitrosohemoglobin, peroxynitrite and S-nitrosoglutathione were determined by spectrofluorimetric and spectrophotometric methods.Dithiothreitol significantly mobilized NO toward nitrite/nitrate and S-nitrosoglutathione, and decreased the amount of NO efflux. Both ACh/VM induce changes on the levels of erythrocyte nitrite/nitrate dependent on the DTT concentration. Higher levels of peroxynitrite and S-nitrosoglutathione were seen with velnacrine in presence of DTT 1 and 50 μM.We concluded that dithiothreitol-induced activation of erythrocyte thiol status decreases NO efflux and allows greater intracellular NO mobilization onto different derivative molecules, both in the absence and presence of acetylcholinesterase substrate and inhibitor.     

Thiols enhance NO formation from nitrate photolysis

Nitrate is generally considered an inert oxidative breakdown product of nitric oxide (NO). Whereas it has been shown that limited amounts of NO are produced during the photolysis of nitrate in aqueous solution, the photochemistry of nitrate in biological matrices such as plasma is unknown. We hypothesized that thiols, which are ubiquitously present in biological systems, may significantly enhance NO-quantum yields from nitrate photolysis. Exposure of fresh human plasma to high-intensity UV-light resulted in NO-formation (19 +/- 3 nmol/l/min) as measured by gas phase chemiluminescence, and this signal was almost completely abolished by the removal of plasma N-oxides (2 +/- 1 nmol/l/min). Reconstitution of NOx-depleted plasma samples with a physiological concentration of nitrate, but not nitrite, restored photolytic NO-generation to values comparable to na?ve plasma. Addition of the thiol-reducing agent, dithiothreitol or the sulfhydryl-bearing amino acid, L-cysteine increased NO-formation above control levels. Thiol-blockade by either N-ethylmaleimide (NEM) or mercuric chloride (HgCl2) reduced basal NO formation from 19 +/- 3 to 7 +/- 2 and 4 +/- 1 nmol/l/min, respectively. Exposure of plasma to UV-light increased NO-adduct concentrations from 18 +/- 5 to 1662 +/- 658 nmol/l. Collectively, our results show that thiols facilitate photolytic conversion of nitrate to NO and NO-adducts such as S-nitrosothiols. This may lead to substantial overestimation of the latter when photolysis-based methodologies are used for their determination. Whether this novel reaction channel also has in vivo relevance remains to be investigated.