Spin Trapping Of Nitric Oxide Research Articles

Dynamics of nitrergic system activation in the rat brain provoked by experimentally induced seizures

Epilepsy is a pathophysiological condition displaying a highly diverse phenotype. Consequently, comprehending the mechanisms underlying seizures necessitates moving beyond a simplistic model focused on the imbalance between the classical excitatory and inhibitory neurotransmitter systems. Nitric oxide (NO), a nonclassical and multifunctional gaseous neurotransmitter, has the potential to exert a profound influence on epileptic reactivity. Unfortunately, numerous studies have not provided clear answers about its involvement in the pathophysiology of epilepsy. The objective of our study was to delineate the temporal dynamics of alterations in nitrergic system activation after experimentally induced seizures. Seizures were induced in 2-month-old male Wistar rats by an administration of pilocarpine. Over a 6-hour observation period, seizure behaviour intensity was continuously evaluated using a modified Racine scale. At intervals of 6, 12, 24, 48, or 96 h post-chemoconvulsant administration, NO spin trapping was conducted with ferrous-diethyldithiocarbamate complexes (Fe(DETC)2). Electron paramagnetic resonance (EPR) spectroscopy was employed to quantify mononitrosyl iron complexes (NO-Fe(DETC)2) in the brain. The temporal kinetic of NO release after seizures revealed a rise in NO synthesis during the initial 12 h. Subsequently, a sharp decline occurred, returning to baseline 96 h after pilocarpine injection. Notably, our research suggests that the level of NO synthesis does not interfere with the severity of the epileptic seizures that occur. In light of this, we propose that the nitrergic system is quickly activated in the epileptic brain as a compensatory mechanism of the central nervous system. However, under usual conditions, this activation is insufficient to effectively attenuate seizures.

Spin Trapping of Nitric Oxide by Hemoglobin and Ferrous Diethyldithiocarbamate in Model Tumors Differing in Vascularization.

Animal tumors serve as reasonable models for human cancers. Both human and animal tumors often reveal triplet EPR signals of nitrosylhemoglobin (HbNO) as an effect of nitric oxide formation in tumor tissue, where NO is complexed by Hb. In search of factors determining the appearance of nitrosylhemoglobin (HbNO) in solid tumors, we compared the intensities of electron paramagnetic resonance (EPR) signals of various iron-nitrosyl complexes detectable in tumor tissues, in the presence and absence of excess exogenous iron(II) and diethyldithiocarbamate (DETC). Three types of murine tumors, namely, L5178Y lymphoma, amelanotic Cloudman S91 melanoma, and Ehrlich carcinoma (EC) growing in DBA/2 or Swiss mice, were used. The results were analyzed in the context of vascularization determined histochemically using antibodies to CD31. Strong HbNO EPR signals were found in melanoma, i.e., in the tumor with a vast amount of a hemorrhagic necrosis core. Strong Fe(DETC)2NO signals could be induced in poorly vascularized EC. In L5178Y, there was a correlation between both types of signals, and in addition, Fe(RS)2(NO)2 signals of non-heme iron-nitrosyl complexes could be detected. We postulate that HbNO EPR signals appear during active destruction of well-vascularized tumor tissue due to hemorrhagic necrosis. The presence of iron-nitrosyl complexes in tumor tissue is biologically meaningful and defines the evolution of complicated tumor-host interactions.

Role of Nitric Oxide in Hydroxylamine Oxidation by Ammonia-Oxidizing Bacteria.

An important role of nitric oxide (NO) as either a free intermediate in the NH3 oxidation pathway or a potential oxidant for NH3 or NH2OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO2- production, whereas N. europaea grown under O2 limitation produced NO equivalent to up to 40% of NO2- production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O2 limitation abolished N2O production. These results indicate different turnover rates of NO during NH3 oxidation under O2-replete and O2-limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH3 saturation and adaptation to O2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N2O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O2-limited growth conditions.

Acute ischemic stroke induces magnetic resonance susceptibility signs dominated by endothelial nitric oxide synthase activation.

Acute ischemic stroke induces deoxyhemoglobin accumulation around the ischemic region while activating endothelial nitric oxide synthase (eNOS) coupling and the subsequent release of nitric oxide (NO). Because deoxyhemoglobin is a natural NO spin trap, its interplay with NO could be prominent during acute stroke. Its interaction with NO has been shown to induce overt paramagnetic signals in vitro; our goal was to investigate whether this interplay can be detected using MRI. To verify the in vivo image effects using the deoxyhemoglobin-NO interaction during acute stroke, eNOS states were manipulated in an animal model of acute ischemia, and the susceptibility signals, cerebral perfusion, and infarction were assessed noninvasively via MR susceptibility weighted imaging (SWI). Occlusion of the right middle cerebral artery increased eNOS coupling and susceptibility signals in the ischemic cortex while abolishing regional cerebral blood flow. Pharmacological eNOS blockage led to weakened susceptibility signals in the ischemic cortex as well as worsened tissue survival. Consistently, abolishment of eNOS coupling through genetic editing reduced the regional susceptibility signals in the ischemic cortex, causing large infarcts. Upregulation of eNOS during acute ischemia sustains tissue viability through the interaction between NO and deoxyhemoglobin. This interplay can be traced in vivo using SWI and can be considered a sensitive marker revealing the delicate oxygenation status of the ischemic tissue, therefore, guiding the management of acute stroke in clinical settings.

Synthesis of Nitric Oxide Donors Derived from Piloty's Acid and Study of Their Effects on Dopamine Secretion from PC12 Cells.

This study investigated the mechanisms and kinetics of nitric oxide (NO) generation by derivatives of Piloty’s acid (NO-donors) under physiological conditions. In order to qualitatively and quantitatively measure NO release, electron paramagnetic resonance (EPR) was carried out with NO spin trapping. In addition, voltammetric techniques, including cyclic voltammetry and constant potential amperometry, were used to confirm NO release from Piloty’s acid and its derivatives. The resulting data showed that Piloty’s acid derivatives are able to release NO under physiological conditions. In particular, electron-withdrawing substituents favoured NO generation, while electron-donor groups reduced NO generation. In vitro microdialysis, performed on PC12 cell cultures, was used to evaluate the dynamical secretion of dopamine induced by the Piloty’s acid derivatives. Although all the studied molecules were able to induce DA secretion from PC12, only those with a slow release of NO have not determined an autoxidation of DA itself. These results confirm that the time-course of NO-donors decomposition and the amount of NO released play a key role in dopamine secretion and auto-oxidation. This information could drive the synthesis or the selection of compounds to use as potential drugs for the therapy of Parkinson’s disease (PD).

111 - Early Administration of an NO Spin Trap Shortens the Survival and Reduces NO Production in Septic- Shocked Rat

The role of nitric oxide (NO) in shock depends on its evolution. It seems that an early NO increment compensates for systemic vasoconstriction whereas a late massive production participates in vasoplegia. NO spin traps are used to form short life time adducts with NO, and evidence for NO by electron paramagnetic resonance. The goal of this study was to evaluate the early administration of sodium diethyldithiocarbamate (DETC, an NO spin trap) on survival and NO production in septic shock. Septic shock was induced in Male Wistar rats through the iv administration of lipopolysaccharide (LPS, 5 mg/Kg). To assess their survival the animals were divided into two groups (n=6) which were treated at exactly the same time as the LPS with either: 1) Isotonic saline solution (ISS, 600 µl ip, and 600 µl sc) or 2) DETC (500 mg/Kg, diluted in 600 µl of ISS, ip) and iron citrate complex (iron sulfate and sodium citrate diluted in 600 µl of ISS, sc). Survival times were recorded and differences evaluated by the Mantel Cox Test. Two more groups (the same treatments as above) were used to measure NO2/NO3 ions (indirect NO measurement, modified Griess reaction) in arterial blood samples (1ml, replaced with 3ml of Hartman solution) before (time 0) and after (1, 2 and 6 h) LPS. The animals were sacrificed by anesthesia overdose after the last blood sample was taken. Differences were evaluated by two way ANOVA testing. Survival was significantly shorter (p=0.01) and NO2/NO3 concentration was significantly smaller (p This study was supported by CONACYT 216184 and 225115 Grants.

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

Identification of Free Nitric Oxide Radicals in Rat Bone Marrow: Implications for Progenitor Cell Mobilization in Hypertension

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

Endothelial NOS (NOS3) impairs myocardial function in developing sepsis

Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3−/− and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160 % of baseline at 10 h after sepsis induction followed by a decline to 63 % of baseline after 18 h in wildtype mice. CO was unaltered in septic NOS3−/− mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6 h post CLP. At 12 h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3−/− mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3−/− mice diminished this survival benefit. Plasma NOx- and local myocardial NOx- and NO levels (via NO spin trapping) demonstrated enhanced NOx- and bioactive NO levels in septic wildtype as compared to NOS3−/− mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-013-0330-8) contains supplementary material, which is available to authorized users.

Cardiomyocyte-restricted overexpression of extracellular superoxide dismutase increases nitric oxide bioavailability and reduces infarct size after ischemia/reperfusion

Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO−). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-012-0305-1) contains supplementary material, which is available to authorized users.

Effect of dimethylsulfoxide on hepatic HbNO formation in septic shocked rats

It is known that dimethylsulfoxide (DMSO) scavenges superoxide. Oxidative stress as well as nitric oxide (NO) have a role on the pathophysiology of septic shock. Since NO and superoxide form peroxynitrite, the goal of the study was to evaluate the effect of DMSO on NO availability in septic shock. Septic shock was produced in male Wistar (230–250 g) rats through lipopolysaccharide (LPS) administration (8mg/kg) in the absence or presence of DMSO treatment (1 ul/g, subcutaneous, one hour before LPS). 3.5 h after LPS or isotonic saline solution (control) all the animals received a NO spin trapping. 30 min later animals were sacrificed by anesthesia overdose (pentobarbital) and a liver biopsy was frozen on liquid nitrogen. The adduct hemoglobin‐NO (HbNO) was detected through electron paramagnetic resonance (EPR) in 100 mg of liver. HbNO increased significantly in the samples of shocked rats. However the higher values of HbNO were found in samples isolated from septic rats treated with DMSO. It is concluded that DMSO increased NO availability in the liver of rats subjected to septic shock. The biological significance of DMSO effect remains to be established

Reactions of the melatonin metabolite N1‐acetyl‐5‐methoxykynuramine with carbamoyl phosphate and related compounds

N-[2-(6-methoxyquinazolin-4-yl)-ethyl] acetamide (MQA) is a compound formed from the melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK). We followed MQA production in reaction systems containing various putative reaction partners, in the absence and presence of hydrogen peroxide and/or copper(II). Although MQA may be formally described as a condensation product of either N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with ammonia, or AMK with formamide, none of these combinations led to substantial quantities of MQA. However, MQA formation was observed in mixtures containing AMK, hydrogen peroxide, hydrogen carbonate and ammonia, or AMK, hydrogen peroxide, copper(II) and potentially carbamoylating agents, such as potassium cyanate or, more efficiently, carbamoyl phosphate. In the presence of hydrogen peroxide, copper(II) and carbamoyl phosphate, MQA was the major product obtained from AMK, but the omission of copper(II) mainly led to another metabolite, 3-acetamidomethyl-6-methoxycinnolinone (AMMC). This was caused by nitric oxide (NO) generated under oxidative conditions from carbamoyl phosphate, as shown by an NO spin trap. MQA formation with carbamoyl phosphate was not due to the possible decomposition product, formamide. The reaction of AMK with carbamoyl phosphate under oxidative conditions, in which inorganic phosphate and water are released and which differs from the typical process of carbamoylation via isocyanate, may be considered as a new physiological route of MQA formation.

NO spin trapping in biological systems

The most recent data on mechanisms of spin trapping of nitric oxide (NO) by iron dithiocarbamate complexes in animal and plant cells and tissues are considered. The rationale is as follows: 1 In the absence of NO in cells and tissues, iron binds primarily to compounds others than dithiocarbamate ligands, e.g., tricarbonic acids. 2. Predominant binding of iron to dithiocarbamate ligands takes place only after its binding to NO, since nitrosylated iron manifests much higher affinity for these ligands that for any non-thiol compounds. 3.Within the composition of mononitrosyl dithiocarbamate complexes, iron exists predominantly in the oxidized (Fe3+) form, i.e., these complexes are originally diamagnetic. Their subsequent single-electron reduction to the paramagnetic, EPR -detectable form is mediated by endogenous or exogenous (e.g., dithionite) reducing agents. 4.Superoxide-mediated transition of paramagnetic mononitrosyl dithiocarbamate iron complexes into EPR-silent state can be accompanied by significant reduction of EPR-detectable complexes. This defect can be overcome through the use of the so-called ABC method. 5. In contrast to hydrophobic complexes fast decomposition of water-soluble mononitrosyl iron complexes in animal organisms testifies to low efficiency of these complexes in determination of NO content in animal cells and tissues.

Nitrite–nitric oxide control of mitochondrial respiration at the frontier of anoxia

Actively respiring animal and plant tissues experience hypoxia because of mitochondrial O2 consumption. Controlling oxygen balance is a critical issue that involves in mammals hypoxia-inducible factor (HIF) mediated transcriptional regulation, cytochrome oxidase (COX) subunit adjustment and nitric oxide (NO) as a mediator in vasodilatation and oxygen homeostasis. In plants, NO, mainly derived from nitrite, is also an important signalling molecule. We describe here a mechanism by which mitochondrial respiration is adjusted to prevent a tissue to reach anoxia. During pea seed germination, the internal atmosphere was strongly hypoxic due to very active mitochondrial respiration. There was no sign of fermentation, suggesting a down-regulation of O2 consumption near anoxia. Mitochondria were found to finely regulate their surrounding O2 level through a nitrite-dependent NO production, which was ascertained using electron paramagnetic resonance (EPR) spin trapping of NO within membranes. At low O2, nitrite is reduced into NO, likely at complex III, and in turn reversibly inhibits COX, provoking a rise to a higher steady state level of oxygen. Since NO can be re-oxidized into nitrite chemically or by COX, a nitrite–NO pool is maintained, preventing mitochondrial anoxia. Such an evolutionarily conserved mechanism should have an important role for oxygen homeostasis in tissues undergoing hypoxia.

Measuring Brain Tissue Oxygenation under Oxidative Stress by ESR/MR Dual Imaging System

The in vivo measurement of oxygen in tissues is of great interest because of oxygen's fundamental role in life. Many methods have been developed for such measurement, but all have been limited, especially with regard to repeated measurement, degree of invasiveness, and sensitivity. We describe electron spin resonance (ESR) oximetry with paramagnetic oxygen-sensing probe for in vivo measurement of oxygen in brain tissues by home-made ESR/MR dual imaging spectroscopy. Lithium 5, 9, 14, 18, 23, 27, 32, 36-octa-n-butoxy-2,3-naphthlocyanine (LiNc-BuO) radical was employed as the solid oxygen-sensing probe, and we confirmed its ability to report partial pressure of oxygen (pO(2)) in brain tissues of live animals under normal and pathological conditions for more than a month. pO(2) measurements could also be made repeatedly on the same animal and at the same location. The implantation site of LiNc-BuO in examined rats was verified by 0.5 T magnetic resonance (MR) imaging. Septic-shock rats were used to monitor tissue oxygenation during pathological state. A decline in pO(2) levels from severe hypotension during sepsis was detected, and generation of nitric oxide (NO) in brain tissues was confirmed by NO spin trapping. ESR oximetry using oxygen-sensing probe and NO spin-trapping can be used to monitor pO(2) change and NO production simultaneously and repeatedly at the same site in examined animals.

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 Fe 2+(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.

Evaluation of lipid‐based carrier systems and inclusion complexes of diethyldithiocarbamate–iron to trap nitric oxide in biological systems

The success of spin trapping techniques in vivo hinges on whether spin traps with high trapping efficiency and biocompatibility can be developed. Currently, two iron chelates based on the dithiocarbamate structure (hydrophilic ferro-di(N-methyl-D-glucamine-dithiocarbamate, or Fe(II)-MGD, and lipophilic ferro-di(diethyldithiocarbamate), or Fe(II)-DETC), are used for spin trapping of nitric oxide (NO) in biologic systems. However, detection efficiency is hampered by a complex redox chemistry for Fe(II)-MGD and by the insolubility of Fe(II)-DETC in water. To circumvent these problems, two new spin trap formulations based on Fe(II)-DETC were developed: a lipid-based carrier system stabilized by lecithin and inclusion complexes in hydroxypropyl-beta-cyclodextrin. The capability of these two systems to trap NO was determined and compared to the standard spin traps in vitro (in the presence of an NO donor) and in vivo (after induction of septic shock in mice). The sensitivity of the detection of NO was significantly increased (by a factor of 4) using the lipid-based carrier systems or inclusion complexes compared to the standard spin trap agents.

Ultrasound liberates nitric oxide (NO) from the caged NO compound N, N′-bis(carboxymethyl)- N, N′-dinitroso- p-phenylenediamine sodium salt

To determine whether nitric oxide (NO) can be released from a cage compound N, N′-bis(carboxymethyl)- N, N′-dinitroso- p-phenylenediamine sodium salt (BNN 5 Na), we sonicated different concentrations of BNN 5 Na solutions containing an NO spin trap, (MGD) 2Fe 2+, and then measured (MGD) 2Fe 2+–NO signal using electron paramagnetic resonance (EPR). We also investigated the role of cavitation by saturating the solutions with Ar, He or Xe gases before sonication. The result showed that ultrasound can liberate NO from caged NO compound at rates highest with Xe and lowest with He. These results suggest that high-temperature due to cavitations induced by ultrasound are capable of releasing NO from caged NO compounds. This finding also opens up to a new possibility for the use of ultrasound in a controlled release of active compound (e.g. drug) from its caged form for therapeutic purposes.

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.

Tissue oxygenation in sepsis; new insights from in vivo EPR.

Nitric oxide (NO) is a key mediator in the maldistribution of oxygen by tissue and organ dysfunction observed in sepsis. Despite this, few techniques are capable of measuring these parameters directly in vivo. We describe here several techniques that have been developed by our group to address this directly by in vivo EPR in animal models of sepsis. Oxygen-sensitive materials can be implanted or administered and report on local tissue pO2. Spin trapping of NO can simultaneously report on tissue NO content. Repeat measures of these parameters can be made directly from a defined tissue site, allowing development of new models and experiments to study the defects in tissue and organ function seen in sepsis.