Metabolic Fate Of Nitric Oxide Research Articles

Nitric Oxide Metabolism in Adults With Cyanotic Congenital Heart Disease

Endothelial-derived nitric oxide (NO) diffuses abluminally to regulate blood flow by activating soluble guanylate cyclase in medial smooth muscle. However, a significant fraction of NO diffuses luminally, where the extremely high reaction rate with red blood cell hemoglobin (Hb) effectively reduces luminal concentration to zero. The erythrocytosis of cyanotic congenital heart disease has potentially opposing effects, namely, a reduction in medial smooth muscle NO bioavailability because of the increase in luminal consumption of the molecule and, conversely, an increase in the elaboration of NO in response to the high endothelial shear stress of the erythrocytotic perfusate. NO metabolism in cyanotic congenital heart disease is unknown. Accordingly, this study aimed to establish the metabolic fate of NO and to determine the degree to which its levels are altered. Blood samples from 25 nonfasting patients with cyanotic congenital heart disease and 25 nonfasting normal controls were collected in Vacutainer tubes containing citrate dextrose and in separate Vacutainer tubes containing a solution that specifically preserves S-nitrosated Hb. Total NO species, plasma S-nitrosated proteins, iron nitrosyl Hb, and S-nitrosated Hb were quantified using chemiluminescence. In conclusion, a significant increase in plasma concentrations of NO metabolites and a modest increase in iron nitrosyl Hb levels were found, suggesting increased luminal consumption caused by erythrocytosis and further suggesting that hypoxemia might activate nonoxidative NO metabolic pathways and enhance tissue oxygen delivery.

Circulating and excretory nitrite and nitrate as indicators of nitric oxide synthesis in humans: methods of analysis

Nitric oxide (NO) is produced in various cells from l-arginine by the catalytical action of NO synthases (NOS). The main metabolic fate of NO includes oxidation to nitrate by oxyhemoglobin in red blood cells and autoxidation to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in plasma, serum, and urine can be used to assess NO synthesis in humans. Circulating nitrite reflects constitutive endothelial NOS activity. Excretory nitrate indicates systemic NO production. Nitrite and nitrate can be measured in plasma, serum, and urine of humans by various analytical methods which are based on different analytical principles. These methods include colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis, and mass spectrometry. The present article gives an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human plasma, serum, and urine in the framework of clinical studies and discusses their significance.

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.

Analysis of the L-Arginine / Nitric Oxide Pathway: The Unique Role of Mass Spectrometry

Nitric oxide (NO) is a gaseous radical molecule. In human organism NO is produced in various cells from Larginine by the catalytical action of NO synthases (NOS). The L-arginine/NO pathway powerfully contributes to maintain multiple physiological functions, including vascular tone, platelet function and neurotransmission. The metabolic fate of NO is very complex due to the participation of numerous compounds resulting from the ability of NO to react practically with any biomolecule to produce biologically active metabolites (e.g. S-nitrosothiols) and biologically inactive metabolites (e.g. nitrate). The concentration in biological fluids and tissues of members of the L-arginine / NO family is of particular interest, as it may characterize the status of this pathway in health and disease as well as to monitor the progress of pharmacological interventions. Thus, measurement of the NO metabolites nitrate and nitrite is suitable to assess NO synthesis in vivo. On the other hand, measurement of the circulating NOS inhibitor asymmetric dimethylarginine (ADMA) was found to reliably identify pathological conditions associated with NO-related endothelial dysfunction. Among the various analytical methods currently available for the analysis of the L-arginine / NO family, mass spectrometry (MS)-based approaches such as gas chromatography-mass spectrometry (e.g. GC-MS / MS) and liquid chromatographymass spectrometry (e.g. LC-MS / MS) emerged indispensable analytical tools for the reliable quantitative analysis of the whole NO family. The present article discusses the currently available analytical methods especially emphasizing the importance of the MS technology to the NO field of research.

232 In vitro and in vivo antitumor efficacy of NCX-4040, a nitric oxide-releasing non-steroidal anti-inflammatory drug, in combination with antineoplastic drugs on human colon cancer lines

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) has been studied in eight healthy male Caucasian subjects following p.o. administration of 1600 mg (single dose), by monitoring at different times in plasma the bioactive storage forms of NO, S-nitrosothiols (RSNO) and its oxidation products (NOx). Plasma levels of NOx and RSNO and urinary levels of NOx were determined by an ozone-based chemiluminescent assay using a sensitive Nitric Oxide Analyzer (LOQ: 10 pmol NO injected). In parallel plasma samples were analyzed by a newly developed LC–MS/MS method for analysis of NCX 4015, the metabolite bearing the nitrate ester function. Using MS/MS with multiple reaction monitoring (MRM) in negative ion mode for NCX 4015 and the internal standard (NCX 4015-13C-D2) it was possible to detect with sufficient accuracy and precision the metabolite in plasma with a quantification limit of 78.1 ng ml−1. Concentration versus time profile of plasma NCX 4015 gave a Cmax value of 161.94±47.4 ng ml−1 and a tmax 4.5±1 h. The results indicate that both NOx and RSNO (these last for the first time determined in vivo in man following oral administration of a NO-donor drug) are effective plasma markers of NO release in vivo, the latter being an earlier indicator of NO distribution (tmax 2.0±0.6 h versus 5.4±1.2 h).

Nitric oxide release and distribution following oral and intraperitoneal administration of nitroaspirin (NCX 4016) in the rat

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs), has been studied in the rat following p.o. and i.p. administration of 100 mg/kg, by monitoring in plasma the bioactive storage forms of NO (S-nitrosothiols, RS-NO) and its oxidation products (nitrites/nitrates, NOx) by a chemiluminescent assay. In parallel, plasma was analyzed for unchanged drug and metabolites by reverse-phase HPLC. In orally treated rats, no unchanged drug is observed in the 0–24 h interval post-dosing, but only salicylic acid (SA), NOx and RS-NO. The time-course of SA formation parallels that of plasma NOx (plateau after 6 h). Nitrosothiols in plasma are detectable at 1 h, peak at 4 h post-administration, and decline thereafter. The results relative to i.p. administration show a more pronounced and rapid NO delivery (peak of both NOx and RS-NO at 1 h and plateau between 1 and 2 h), still coincident with the peak of SA, and the presence in plasma of NCX 4015 (a metabolite of NCX 4016 which still bears the nitrate function). In myocardial tissue from p.o. treated rats, no drug or metabolites were ever detected, and the NOx levels were always in the range of the controls. Conversely, following i.p. treatment, we observed a rapid compartmentalization within the heart of the unchanged drug, which rapidly disappears in favour of its breakdown products NCX 4015 and SA, with a concomitant rise in myocardial NOx levels up to 2 h. To check the stability of NCX 4016 in the acidic gastric milieu and to explain the different distribution of the drug following p.o. or i.p. administration, the gastric content of the orally-treated animals at different post-dosing times was analysed by HPLC. The unchanged drug was detected up to 8 h post-dosing (levels slowly decreased with time), and the only metabolite to be detected was the O-deacetylated derivative (NCX 4023), which was present in low concentrations up to 4 h post-dosing. This indicates that NCX 4016 does not undergo biotransformation in the upper part of gastrointestinal tract (no direct release of NO in this district) and that the stomach acts as a reservoir for the drug.

Reductive nitrosylation and S-nitrosation of hemoglobin in inhomogeneous nitric oxide solutions

Elucidating the reaction of nitric oxide (NO) with oxyhemoglobin [HbFe(II)O 2] is critical to understanding the metabolic fate of NO in the vasculature. At low concentrations of NO, methemoglobin [HbFe(III)] is the only detectable product from this reaction; however, locally high concentrations of NO have been demonstrated to result in some iron-nitrosylhemoglobin [HbFe(II)NO] and S-nitrosohemoglobin (SNO-Hb) formation. Reductive nitrosylation through a HbFe(III) intermediate was proposed as a viable pathway under such conditions. Here, we explore another potential mechanism based on mixed valenced Hb tetramers. The oxidation of one or two heme Fe(II) in the R-state HbFe(II)O 2 has been observed to lower the oxygen affinity of the remaining heme groups, thus creating the possibility of oxygen release and NO binding at the heme Fe(II) sites. This mixed valenced hypothesis requires an allosteric transition of the Hb tetramer. Hence, this hypothesis can account for HbFe(II)NO formation, but not SNO-Hb formation. Here, we demonstrate that cyanide attenuated the formation of SNO-Hb by 30–40% when a saturated NO bolus was added to 0.1–1.0 mM HbFe(II)O 2 solutions. In addition, HbFe(II)NO formation under such inhomogeneous conditions does not require allostericity. Therefore, we concluded that the mixed valenced theory does not play a major role under these conditions, and reductive nitrosylation accounts for a significant fraction of the HbFe(II)NO formed and approximately 30–40% of SNO-Hb. The remaining SNO-Hb is likely formed from NO oxidation products.

Endotoxin challenge increases xanthine oxidase activity in cattle: effect of growth hormone and vitamin E treatment

In addition to its basic role in the metabolism of purine nucleotides, xanthine oxidoreductase (XOR) is involved in the generation of oxygen-derived free radicals and production and metabolic fate of nitric oxide (NO). Growth hormone (GH) and Vitamin E (E) have been shown previously to modify immune response to infection. Our objective was to determine in heifers the effect of endotoxin challenge (LPS; 3.0 μg/kg BW, i.v. bolus, Escherichia coli 055:B5) on xanthine oxidase (XO) activity in plasma and liver and the modification of this response by daily treatment with recombinant GH (0.1 mg/kg BW, i.m., for 12 days) or GH+E (E: mixed tocopherol, 1000 IU/heifer, i.m., for 5 days). In experiment 1, 16 heifers (348.7±6.1 kg) were assigned to control (C, daily placebo injections), GH, or GH+E treatments and were challenged with two consecutive LPS injections (LPS1 and LPS2, 48 h apart). After LPS1, plasma XO activity increased 290% ( P<0.001) at 3 h, reached peak (430%) at 24 h and returned to basal level by 48 h after LPS2. XO responses (area under the time×activity curve, AUC) were greater after LPS1 than LPS2 ( P<0.001). Total plasma XO responses to LPS (AUC, LPS1+LPS2) were augmented 55% ( P<0.05) over C with GH treatment but diminished to C responses in GH+E. There was a linear relationship ( r 2=0.605, P<0.001) between total response in plasma XO activity and plasma nitrate+nitrite concentration. In experiment 2, 24 heifers (346±6 kg) were assigned to C or GH treatments and liver biopsy samples were obtained at 0, 3, 6, and 24 h after a single LPS challenge. Hepatic XO activities increased 63.3% ( P<0.05) 6 h after single LPS challenge and remained elevated at 24 h (100.1%, P<0.01) but were not affected by GH treatment. Results indicate that LPS-induced increases in plasma XO activity could be amplified by previous GH treatment but attenuated by E administration. The data also suggest that E may be effective in controlling some mediators of immune response associated with increased production of NO via the effect on XO activity and its production of superoxide anion as well as uric acid.

Chemiluminescence and LC–MS/MS analyses for the study of nitric oxide release and distribution following oral administration of nitroaspirin (NCX 4016) in healthy volunteers

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) has been studied in eight healthy male Caucasian subjects following p.o. administration of 1600 mg (single dose), by monitoring at different times in plasma the bioactive storage forms of NO, S-nitrosothiols (RSNO) and its oxidation products (NO x ). Plasma levels of NO x and RSNO and urinary levels of NO x were determined by an ozone-based chemiluminescent assay using a sensitive Nitric Oxide Analyzer (LOQ: 10 pmol NO injected). In parallel plasma samples were analyzed by a newly developed LC–MS/MS method for analysis of NCX 4015, the metabolite bearing the nitrate ester function. Using MS/MS with multiple reaction monitoring (MRM) in negative ion mode for NCX 4015 and the internal standard (NCX 4015- 13C-D 2) it was possible to detect with sufficient accuracy and precision the metabolite in plasma with a quantification limit of 78.1 ng ml −1. Concentration versus time profile of plasma NCX 4015 gave a C max value of 161.94±47.4 ng ml −1 and a t max 4.5±1 h. The results indicate that both NO x and RSNO (these last for the first time determined in vivo in man following oral administration of a NO-donor drug) are effective plasma markers of NO release in vivo, the latter being an earlier indicator of NO distribution ( t max 2.0±0.6 h versus 5.4±1.2 h).

Abnormal metabolic fate of nitric oxide in Type I diabetes mellitus.

Reduced bioavailability of endothelium-derived nitric oxide is implicated in diabetic macrovascular and microvascular disease. In patients with diabetes, we hypothesised that protein glycosylation can alter nitric oxide binding affinity of haemoglobin and plasma proteins, hence reducing nitric oxide availability and causing an alteration in nitric oxide metabolism. Binding of nitric oxide to haemoglobin was studied across a range of glycosylation levels in vitro (HbA(1c) 5.9 to 9.8%). In clinical studies nitrate, nitrite, nitrosyl haemoglobin and plasma nitrosothiols were measured in venous blood from 23 patients with uncomplicated Type I (insulin-dependent) diabetes mellitus and 17 non-diabetic control subjects. Samples were analysed at baseline and after nitric oxide was added ex vivo. Nitric oxide-haemoglobin binding was increased at a HbA(1c) greater than 8.5% compared with 5.9% (p<0.01). Basal nitrosyl haemoglobin was higher in diabetic patients compared with the control subjects (0.59+/-0.12 micro mol/l vs 0.24+/-0.12 micro mol/l, p<0.05). Plasma nitrosothiols, and nitrite and nitrate (NOx) concentrations were similar in diabetic patients compared with the control subjects (7.64+/-0.79 micro mol/l vs 5.93+/-0.75 micro mol/l, 13.98+/-2.44 micro mol/l vs 12.44+/-2.15 micro mol/l, respectively). In blood from diabetic patients, added nitric oxide was metabolised preferentially to nitrosyl haemoglobin and plasma nitrosothiols, with a twofold increase in nitrosyl haemoglobin observed across all concentrations of nitric oxide (p<0.05). These preferential increases correlated positively with HbA(1c). Nitrosyl haemoglobin is increased in patients with Type I diabetes. Preferential metabolism to nitrosyl haemoglobin and nitrosothiols occurs after increases in nitric oxide. Our results show an accentuated association between nitric oxide and glycosylated proteins, especially deoxygenated haem. An altered metabolic fate of nitric oxide could influence microvascular regulation and tissue perfusion.

Nitric oxide reaction with red blood cells and hemoglobin under heterogeneous conditions.

Understanding the interaction of nitric oxide (NO) with red blood cells (RBCs) is vital to elucidating the metabolic fate of NO in the vasculature. Because hemoglobin (Hb) is the most abundant intraerythrocytic protein and reacts rapidly with NO, the interaction of NO with Hb has been studied extensively. We and others have shown the NO reaction with RBCs is nearly 1,000-fold slower than the reaction with cell-free Hb. Because the reaction rate of NO with cell-free Hb and RBCs is quite different, we hypothesize that different reaction products evolve under locally high NO concentrations, which can be generated by bolus NO addition or NO inhalation. Here we use electron paramagnetic resonance to show that bolus NO addition to cell-free Hb solutions results in nitrosylhemoglobin [HbFe(II)NO] formation as a minor product through a MetHb-dependent pathway. Further, the RBC is shown to be more prone to form HbFe(II)NO under this heterogeneous condition compared with an equivalent free-Hb solution. In both cases, trapping MetHb with cyanide blocked the formation of HbFe(II)NO. We conclude that the formation of HbFe(II)NO is a heterogeneous phenomenon involving three successive reactions of NO with the same heme molecule. These results were supported further by mathematically modeling NO-Hb reactions and diffusion.

Biotransformation of nitric oxide.

Previous investigations into the health effects of nitrogen oxides (NOx) have mostly been conducted with special reference to nitrogen dioxide (NO2) and its direct effects on the respiratory system, while the study of nitric oxide (NO) has been disregarded. We carried out a study on NO by exposing rats and mice to 15NO or administering 15N-nitrite and 15N-nitrate to these animals by IP injection in order to elucidate the metabolic fate of NO. The results of our study and previous findings led us to assume that the major metabolic path of inhaled NO is as follows: inhaled NO reacts with hemoglobin, forming nitrosyl-hemoglobin (NOHb), and from NOHb, nitrite (NO2-) and nitrate (NO3-) are generated. Major quantities of NO3- are discharged into the urine and a certain amount is discharged into the oral cavity through the salivary glands and transformed to NO2-. Part of this NO2- is converted to N2 gas in the stomach. Nitrate in the intestine is partly reduced to ammonia (NH3) through NO2-, reabsorbed into the body, and converted to urea. Most of the metabolites of inhaled NO are excreted rapidly from the body within 48 hr.

Biotransformation of Nitric Oxide

Previous investigations into the health effects of nitrogen oxides (NOx) have mostly been conducted with special reference to nitrogen dioxide (NO2) and its direct effects on the respiratory system, while the study of nitric oxide (NO) has been disregarded. We carried out a study on NO by exposing rats and mice to 15NO or administering 15N-nitrite and 15N-nitrate to these animals by IP injection in order to elucidate the metabolic fate of NO. The results of our study and previous findings led us to assume that the major metabolic path of inhaled NO is as follows: inhaled NO reacts with hemoglobin, forming nitrosyl-hemoglobin (NOHb), and from NOHb, nitrite (NO2-) and nitrate (NO3-) are generated. Major quantities of NO3- are discharged into the urine and a certain amount is discharged into the oral cavity through the salivary glands and transformed to NO2-. Part of this NO2- is converted to N2 gas in the stomach. Nitrate in the intestine is partly reduced to ammonia (NH3) through NO2-, reabsorbed into the body, and converted to urea. Most of the metabolites of inhaled NO are excreted rapidly from the body within 48 hr.

Metabolic fate of nitric oxide.

The metabolic pathway of inhaled NO in rats was investigated with 15NO. After 15NO exposure, the content of the 15N (atom % excess) in blood, red cells, serum, each organ tissue (perfused with saline solution) and urine were estimated. The contents of 15N in the samples of blood, serum, red cells, and urine were relatively high and those of lung, trachea, liver, kidney, and muscle were low.