Differential effect of buffer on the spin trapping of nitric oxide by iron chelates.

Nitric oxide synthase (NOS) generates nitric oxide (NO*) by the oxidation of l-arginine. Spin trapping in combination with electron paramagnetic resonance (EPR) spectroscopy using ferro-chelates is considered one of the best methods to detect NO* in real time and at its site of generation. The spin trapping of NO* from isolated NOS I oxidation of L-arginine by ferro-N-dithiocarboxysarcosine (Fe(DTCS)2) and ferro-N-methyl-d-glucamide dithiocarbamate (Fe(MGD)2) in different buffers was investigated. We detected NO-Fe(DTCS)2, a nitrosyl complex, resulting from the reaction of NO* and Fe(DTCS)2, in phosphate buffer. However, Hepes and Tris buffers did not allow formation of NO-Fe(DTCS)2. Instead, both of these buffers reacted with Fe2+, generating sparingly soluble complexes in the absence of molecular oxygen. Fe(DTCS)2 and Fe(MGD)2 were found to inhibit, to a small degree, NOS I activity with a greater effect observed with Fe(MGD)2. In contrast, Fe(MGD)2 was more efficient at spin trapping NO* from the lipopolysaccharide-activated macrophage cell line RAW264.7 than was Fe(DTCS)2. Data suggested that Fe(DTCS)2 and Fe(MGD)2 are efficient at spin trapping NO* but their maximal efficiency may be affected by experimental conditions.     

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.     

Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD.

The objective of this study was to elucidate the nitric oxide-forming reactions of the iron-N-methyl-D-glucamine dithiocarbamate (Fe-MGD) complex from the nitrogen-containing compound hydroxyurea. The Fe2+(MGD)2 complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. The reaction of Fe2+(MGD)2 with NO yields the resultant NO-Fe2+(DETC)2 complex, which has a characteristic triplet EPR signal. It is widely believed that only NO reacts with Fe2+(MGD)2 to form the NO-Fe2+(MGD)2 complex. In this report, the mechanism leading to the formation of NO-Fe2+(MGD)2 was investigated using oxygen-uptake studies in conjunction with the EPR spin-trapping technique. We found that the air oxidation of Fe2+(MGD)2 complex results in the formation of the Fe3+(MGD)3 complex, presumably concomitantly with superoxide (O3*-). Dismutation of superoxide forms hydrogen peroxide, which can subsequently reduce Fe3+(MGD)3 back to Fe2+(MGD)2. The addition of NO to the Fe3+(MGD)3 complex resulted in the formation of the NO-Fe2+(MGD)2 complex. Hydroxyurea is not considered to be a spontaneous NO donor, but has to be oxidized in order to form NO. We present data showing that in the presence of oxygen, Fe2+(MGD)2 can oxidize hydroxyurea to yield the stable NO-Fe2+(MGD)2 complex. These results imply that hydroxyurea can be oxidized by reactive oxygen species that are formed from the air oxidation of the Fe2+(MGD)2 complex. Formation of the NO-Fe2+(MGD)2 complex in this case could erroneously be interpreted as spontaneous formation of NO from hydroxyurea. The chemistry of the Fe2+(MGD)2 complexes in aerobic conditions must be taken into account in order to avoid erroneous conclusions. In addition, the use of these complexes may contribute to the overall oxidative stress of the system under investigation.     

Electron paramagnetic resonance imaging of nitric oxide organ distribution in lipopolysuccaride treated mice

The recent development of electron paramagnetic resonance (EPR) permits its application for in vivo studies of nitric oxide (NO). In this study, we tried to obtain 3D EPR images of endogenous NO in the abdominal organs of lipopolysuccaride (LPS) treated mice. Male ICR mice, each weighing about 30 g, received 10 mg/kg of LPS intraperitoneally. Six hours later, a spin trapping reagent comprised of iron and an N-dithiocarboxy sarcosine complex (Fe(DTCS)₂, Fe 200 mM, DTCS/Fe = 3) were injected subcutaneously. Two hours after this treatment, the mice were fixed in a plastic holder and set in the EPR system, equipped with a loop-gap resonator and a 1 GHz microwave. NO was detected as an NO-Fe(DTCS)₂ complex, which had a characteristic 3-line EPR spectrum. NO-Fe(DTCS)₂ complexes in organ homogenates were also measured using a conventional X-band EPR system. NO-Fe(DTCS)₂ spectra were obtained in the upper abdominal area of LPS treated mice at 8 h after the LPS injection. 3D EPR tiled and stereoscopic images of the NO distribution in the hepatic and renal areas were obtained at the same time. The NO-Fe(DTCS)₂ distribution in abdominal organs was confirmed in each organ homogenate using conventional X-band EPR. This is the first known EPR image of NO in live mice kidneys.     

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron-dithiocarbamate complexes

The free radical, nitric oxide ( radicalNO), is responsible for a myriad of physiological functions. The ability to verify and study radicalNO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of radicalNO at relevant cellular sites. With this in mind, several iron-chelates (Fe2+(L)2) have been developed, which have provided a useful tool for the study and identification of radicalNO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of radicalNO detection is dependent on the Fe2+(L)2 complex. The development of more efficient and stable Fe2+(L)2 chelates may help to better understand the role of radicalNO in vivo. In this paper, we present data comparing several proline derived iron-dithiocarbamate complexes with the more commonly used spin traps for radicalNO, Fe2+-di(N-methyl-D-glutamine-dithiocarbamate) (Fe2+(MGD)2) and Fe2+-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2). We evaluate the apparent rate constant (kapp) for the reaction of radicalNO with these Fe2+(L)2complexes and the stability of the corresponding Fe2+(NO)(L)2 in presence of NOS I.     

EPR spectroscopy of common nitric oxide - spin trap complexes

Two commonly used hydrophobic and hydrophilic spin traps for NO, namely Fe2+(DETC)(2)and Fe2+(MGD)(2), respectively, were analyzed via EPR spectroscopy. EPR spectra of trapped NO, together with field position standards, were recorded both in the frozen state and at room temperature. We present a detailed characterization of the EPR spectra of the above paramagnetic NO complexes, concerning g-value, hyperfine splitting and linewidths. This study also provides spectroscopic data required to develop a quantitative and sensitive detection system for nitric oxide both in hydrophobic and hydrophilic aqueous media.     

X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine)

Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of (.)NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)(2) were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of (.)NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of (.)NO, but also the redox properties of liver and lung might determine the formation of complexes of (.)NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for (.)NO-metry at room temperature using Fe(DTCS)(2) as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of (.)NO production in a whole organ, such as murine liver.     

Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems

While the Fe(2+)-dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe(2+) complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe(3+) complexes. Following exposure to NO, diamagnetic NO-Fe(3+) complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO-Fe(3+)-MGD and lipid soluble NO-Fe(2+)-DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO-Fe(2+)-MGD complexes with yield of up to 50% and the balance is converted to Fe(3+)-MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO-Fe(3+)-MGD into NO-Fe(2+)-MGD (60-90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe(2+)-MGD. With isotope labeling of the NO-Fe(3+)-MGD with (57)Fe, it was shown that these complexes donate NO to Fe(2+)-MGD. NO-Fe(3+)-MGD complexes were also formed by reversible oxidation of NO-Fe(2+)-MGD in air. The stability of NO-Fe(3+)-MGD and NO-Fe(2+)-MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron-dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.     

Detailed methods for the quantification of nitric oxide in aqueous solutions using either an oxygen monitor or EPR

The interest in nitric oxide has grown with the discovery that it has many biological functions. This has heightened the need for methods to quantify nitric oxide. Here we report two separate methods for the quantification of aqueous stock solutions of nitric oxide. The first is a new method based on the reaction of nitric oxide with oxygen in liquid phase (*NO + O2 + 2H2O --> 4HNO2); an oxygen monitor is used to measure the consumption of oxygen by nitric oxide. This method offers the advantages of being both simple and direct. The presence of nitrite or nitrate, frequent contaminants in nitric oxide stock solutions, does not interfere with the quantification of nitric oxide. Measuring the disappearance of dissolved oxygen, a reactant, in the presence of known amounts of nitric oxide has provided verification of the 4:1 stoichiometry of the reaction. The second method uses electron paramagnetic resonance spectroscopy (EPR) and the nitric oxide trap [Fe2+-(MGD)2], (MGD = N-methyl-D-glucamine dithiocarbamate). The nitrosyl complex is stable and easily quantitated as a room temperature aqueous solution. These two methods are validated with Sievers 280 Nitric Oxide Analyzer and cross-checked with standards using UV-Vis spectroscopy. The practical lower limits for measuring the concentration of nitric oxide using the oxygen monitor approach and EPR are approximately 3 microM and 500 nM, respectively. Both methods provide straightforward approaches for the standardization of nitric oxide in solution.     

Nitric oxide-forming reaction between the iron-N-methyl-D-glucamine dithiocarbamate complex and nitrite

The objective of this study was to elucidate the origin of the nitric oxide-forming reactions from nitrite in the presence of the iron-N-methyl-D-glucamine dithiocarbamate complex ((MGD)(2)Fe(2+)). The (MGD)(2)Fe(2+) complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. Although it is widely believed that only NO can react with (MGD)(2)Fe(2+) complex to form the (MGD)(2)Fe(2+).NO complex, a recent article reported that the (MGD)(2)Fe(2+) complex can react not only with NO, but also with nitrite to produce the characteristic triplet EPR signal of (MGD)(2)Fe(2+).NO (Hiramoto, K., Tomiyama, S., and Kikugawa, K. (1997) Free Radical Res. 27, 505-509). However, no detailed reaction mechanisms were given. Alternatively, nitrite is considered to be a spontaneous NO donor, especially at acidic pH values (Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Arch Biochem. Biophys. 357, 1-7). However, its production of nitric oxide at physiological pH is unclear. In this report, we demonstrate that the (MGD)(2)Fe(2+) complex and nitrite reacted to form NO as follows: 1) (MGD)(2)Fe(2).NO complex was produced at pH 7.4; 2) concomitantly, the (MGD)(3)Fe(3+) complex, which is the oxidized form of (MGD)(2)Fe(2+), was formed; 3) the rate of formation of the (MGD)(2)Fe(2+).NO complex was a function of the concentration of [Fe(2+)](2), [MGD], [H(+)] and [nitrite].     

Spin trapping of vascular nitric oxide using colloid Fe(II)-diethyldithiocarbamate

Currently available EPR spin-trapping techniques are not sensitive enough for quantification of basal vascular nitric oxide (NO) production from isolated vessels. Here we demonstrate that this goal can be achieved by the use of colloid Fe(DETC)(2). Rabbit aortic or venous strips incubated with 250 microM colloid Fe(DETC)(2) exhibited a linear increase in tissue-associated NO-Fe(DETC)(2) EPR signal during 1 h. Removal of endothelium or addition of 3 mM N(G)-nitro-l-arginine methyl ester (L-NAME) inhibited the signal. The basal NO production was estimated as 5.9 +/- 0.5 and 8.3 +/- 2.1 pmol/min/cm(2) in thoracic aorta and vena cava, respectively. Adding sodium nitrite (10 microM) or xanthine/xanthine oxidase in the incubation medium did not modify the intensity of the basal NO-Fe(DETC)(2) EPR signal. Reducing agents were not required with this method and superoxide dismutase activity was unchanged by the Fe(DETC)(2) complex. We conclude that colloid Fe(DETC)(2) may be a useful tool for direct detection of low amounts of NO in vascular tissue.     

Electronic and stereochemical characterizations of the photoinduced intermediates of nitrosyl complexes of metal (S = 5/2)-substituted hemoproteins trapped at low temperature

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric myoglobin (NO-Fe(III)Mb) and manganese(II)-porphyrin-substituted myoglobin (NO-Mn(II)Mb) was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the electronic and structural natures of the photoinduced intermediates of these hemoprotein-ligand complexes trapped at low temperature. The photoproduct of NO-Fe(III)Mb at 5 K exhibited entirely new X-band EPR absorptions in the magnetic field strength from 0 to 0.4 tesla. The widespread absorption together with distinct, sharp zero-field absorption was consistently observed in the photoproduct of the isoelectronic NO-Mn(II)Mb. These novel ERP signals indicate a spin-coupled pair with an effective spin of S = 2 between the high spin metal center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. On the other hand, the photolyzed form of nitrosyl complexes of Fe(III)- and Mn(II)-Glycera hemoglobins, in which the distal histidine of Mb is replaced by a leucyl residue, exhibited somewhat broader EPR absorptions similar to those of the corresponding native Fe(III)- or unliganded Mn(II)-Glycera hemoglobins, respectively, indicating that the photodissociated NO molecule moved farther away from the metal center in the heme pocket. These observations show the importance of the interaction of the distal residue with the ligand in determining the nature of the photolyzed states.     

The registration of nitric oxide production in mammals using a water-soluble complex of N-methyl-D,L-glucamine dithiocarbamate with Fe3+

The level of nitric oxide production in the intact rabbit organism was studied using the water-soluble complex of Fe3+ with MGD as a selective spin trap for nitric oxide. The Fe(3+)-MGD3 complex was injected intravenously. It was shown by the EPR method that this injection resulted in the formation of paramagnetic complexes in the urine, as Cu(2+)-MGD2, and nitric oxide spin adducts: nitric oxide-Fe(2+)-MGD2 and nitric oxide-Fe(3+)-MGD2. The level of nitric oxide production was estimated by the ratio of the total amount of these adducts to the nitric oxide-Fe(2+)-MGD2 level, formed after the addition of excessive S-nitrosoglutathione. This value for intact animals was 1.33 +/- 0.13%.     

The relationship between the production of nitric oxide and the injury of cardiomyocytes caused by in vivo rat regional myocardial ischemia and reperfusion

Changes in nitric oxide concentration in rat myocardium in vivo during temporary occlusion of the anterior descending coronary artery, followed by reperfusion were studied by microdialysis assay in risk and intact areas by using an NO spin trap (complex of ferrous ions with N-methyl-D, L-glucamine dihiocarbamate, Fe3+-MGD2). The amplitude of the EPR signal of the NO spin adduct NO-Fe2+-MGD2 in the risk area increased during the 40-min occlusion and remained higher than the initial level during 60-min postischemic reperfusion, indicating a substantial nitric oxide production. The size of the infarction in the risk area by the end of reperfusion was 47 +/- 3 %, the contents of ATP, phosphocreatine, and total creatine decreased to 44 +/- 4, 51 +/- 5, and 60 +/- 3 %, correspondingly, as compared with initial values, and the level of lactate was six times higher than the initial one. In the intact area of the left ventricle, the level of nitric oxide and high-energy metabolites did not change throughout the experiment. It was shown that the intensive nitric oxide production, in acute regional ischemia and reperfusion are related to the disturbance of energy metabolism, the damage to cytoplasmic membranes, and the death of cardiomyocytes.     

Epr detection of endogenous nitric oxide in postischemic heart using lipid and aqueous-soluble dithiocarbamate-iron complexes

Spin-trapping techniques combined with electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (·NO) production were compared in the ischemic-reperfused myocardium for the first time, using both aqueous-soluble and lipophilic complexes of reduced iron (Fe) with dithiocarbamate derivatives. The aqueous-soluble complex of Fe and N-methyl-D-glucamine dithiocarbamate (MGD) formed MGD2-Fe-NO complex with a characteristic triplet EPR signal (a_N12.5 G and g_iso = 2.04) at room temperature, in native isolated rat hearts following 40 min global ischemia and 15 min reperfusion. Diethyldithiocarbamate (DETC) and Fe formed in ischemic-reperfused myocardium the lipophilic DETC2-Fe-NO complex exhibiting an EPR signal (g = 2.04 and g = 2.02 at 77K) with a triplet hyperfine structure at g. Dithiocarbamate-Fe-NO complexes detected by both trapping agents were abolished by the ·NO synthase inhibitor, N^G-nitro-L-arginine methyl ester. Quantitatively, both trapping procedures provi ded similar values for tissue ·NO production, which were observed primarily during ischemia. Postischemic hemodynamic recovery of the heart was not affected by the trapping procedure. (Mol Cell Biochem 175: 91–97, 1997)     

Protonation of nitrite is an obligatory stage in the generation of nitric oxide from nitrite in biological systems

The yield of nitric oxide from 1 mM sodium nitrite differs 200 times when the process was initiated by 10 mM sodium dithionite in the solution of 5 or 150 mM HEPES-buffer (pH 7.4). Dithionite acted both as a strong reductant and an agent that induced a local acidification of solutions without notable change in pH value. The amount of nitric oxide was estimated by the EPR method by measuring the incorporation of nitric oxide to water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which led to the formation of EPR-detectable mononitrosyl iron complexes with MGD (MNIC-MGD). Ten seconds after dithionite addition, the concentration of MNIC - MGD complexes reached 2 microM in 5 mM HEPES-buffer in contrast to 0.01 microM in 150 mM HEPES-buffer. The difference was suggested to be due to a higher life-time of zones with decreased pH values in a weaker weak buffer solution. The life-time was high enough to ensure the protonation of a part of nitrite. The resulting nitrous acid was decomposed to form nitric oxide. The difference in the formation of nitric oxide from nitrite was also observed in weak and strong buffer solutions in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, the ratios of nitric oxide yields in weak and strong buffer did not exceed 3-4 times. The increase in the formation of nitric oxide from nitrite was characteristic for the solutions containing both proteins. Large amounts of nitric oxide formed from nitrite was observed in mouse liver preparation subjected to freezing-thawing procedure followed by incubation in 150 mM HEPES-buffer (pH 7.4) and addition of dithionite. The proposition was made that the presence of zones with low pH value in cells and tissues can ensure the predominant operation of the acid mechanism formation of nitric oxide from nitrite. The contribution of the formation of nitric oxide from nitrite catalyzing with heme-containing proteins nitrite reductases can be minor one under these conditions.     

A new method for monitoring nitric oxide production using Teflon membrane microdialysis

The measurement of nitric oxide (NO) by electron spin resonance (ESR) is complicated by potentially toxic spin-trapping agents, which may affect the NO-producing cells per se and/or cause artifacts and systemic side effects. These problems can be addressed by preventing direct interaction between the agent and the biological system. In the present study, we utilized Teflon as a barrier between the spin trap and the living cell, since the material is permeable to gas only. Our aim was to investigate if NO could diffuse across the membrane in sufficient amounts to be trapped and quantified by ESR. We used standard microdialysis equipment and specially designed dialysis probes, or tubing, with Teflon membranes. Sodium nitroprusside was used as a NO donor and Fe-N-dithiocarboxysarcosine (Fe(DTCS)2) as a spin trap. NO readily diffuses through Teflon and could be quantified in concentrations considerably below 50 nM in a reproducible and accurate manner. In cell cultures of activated murine macrophages, NO synthesis from iNOS could be monitored and we noted a huge increase in NO concentration by superoxide dismutase. We conclude that spin trapping of NO by Fe(DTCS)2 across Teflon membranes is an attractive approach for quantifying and monitoring nitric oxide production without interfering with cell viability.     

Nitrite protonation as a necessary stage in the generation of nitric oxide from nitrite in biological systems

The yields of nitric oxide from 1 mM and 10 mM sodium dithionite in 5 or 150 mM solutions of HEPES buffer (pH 7.4) differed by a factor of 200. Dithionite acted as both a strong reducing agent and an agent responsible for local acidification of the solutions without significant changes in pH. The concentration of nitric oxide was estimated by electron paramagnetic resonance (EPR) by monitoring its incorporation into water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which resulted in the formation of EPR-detectable mononitrosyl complexes of iron. Ten seconds after dithionite addition, the concentration of mononitrosyl iron complexes reached 2 μM, whereas it did not become greater than 0.01 μM in 5 mM HEPES buffer. It has been suggested that this difference results from a longer lifetime of a localized decrease in pH in a weaker buffer solution. This time could be long enough for the protonation of some nitrite molecules. Nitrous acid thus formed decomposed to nitric oxide. A difference in nitric oxide formation from nitrite in weak and strong buffer solutions was also observed in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, in the weak buffer the nitric oxide yield was only three-four times greater than in the strong buffer. An increase in the nitric oxide yield from nitrite was observed in solutions containing both proteins. A significant amount of nitric oxide from nitrite was formed in mouse liver preparation subjected to freezing and thawing procedure followed by slurrying in 150 mM HEPES buffer (pH 7.4) and dithionite addition (10 mM). We suggest that the presence of zones with lowered pH values in cells and tissues may be responsible for the predominance of the acidic mechanism of nitric oxide formation from nitrite. The contribution of nitric oxide formation from nitrite catalyzed by heme-containing proteins as nitrite reductases may be minor under these conditions.     

Nitric oxide transfer from the NO-donor S-nitroso-N-acetylpenicillamine to monomers and dimers of water-soluble iron-porphyrins

The nitrosylation of two water-soluble iron-porphyrins, the anionic Fe(III)-meso-tetrakis(p-sulfonatophenyl)porphyrin (FeTPPS(4)) and the cationic Fe(III)-meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (FeTMPyP), by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was studied using optical absorption spectroscopy. The influence of ionic and non-ionic micelles on rates of nitric oxide transfer was investigated. Initially, the effect of the micelles on the pH-dependent equilibrium between monomeric and micro-oxo-dimeric species of the iron-porphyrins was examined. It is not affected in micelle-porphyrin systems with electric charges identical in sign. The non-ionic micelles of polidocanol induce a small negative pK shift. In contrast, the dimerization equilibrium of porphyrins in oppositely charged micellar phases is displaced to lower pH by approximately 2 units. Nitric oxide binding to monomers and micro-oxo-dimers was examined at pH 5.0 and 8.0, respectively. Contrary to nitrosylation by NO gas, SNAP induces reductive nitrosylation. There was no observed NO-Fe(III)porphyrin intermediate. Nitrosylation rates were obtained and compared in aqueous buffer and in micellar systems. Monomers nitrosylate much faster than micro-oxo-dimers. Oppositely charged micelles prevent nitrosylation of the iron-porphyrins or considerably enhance nitrosylation times. Nitrosylation rates are comparable to transnitrosylation rates between several S-nitrosothiols and thiol-containing proteins, suggesting biological relevance for the process.     

ESR characterization of a novel spin-trapping agent, 15N-labeled N-tert-butyl-alpha-phenylnitrone,as a nitric oxide donor

We previously found that one of the pharmacological effects of N-tert-butyl-alpha-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15N to identify its corresponding 15NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15N-PBN, the starting material, ammonium-15N chloride, was converted to 2-amino-15N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-15N, and finally reacted with benzaldehyde to give 15N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)2, as a trapping agent to measure the NO levels of 15N-PBN or 14N-PBN in vitro, the peak intensity of 15NO[Fe(MGD)2] was over 50% stronger than that of 14NO[Fe(MGD)2], and that 15NO and 14NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15NO derived from 15N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15NO from PBN and 14NO generated from cells. These results suggested that 15N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.