Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase.

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.

In vivo on-line detection of no distribution in endotoxin-treated mice by l-band ESR.

The report describes a method for tracing nitric oxide (NO) distribution in endotoxin-treated mice using in vivo low-frequency L-band (1.1 GHz) electron spin resonance spectroscopy (ESR) in combination with extracellular nitric oxide trapping complex consisting of N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). An ESR signal characteristic of the MGD-Fe-NO complex was found in the upper abdomen (liver region), lower abdomen and head region of ICR mice. The origin of NO from the L-arginine-NO synthase (NOS) pathway was confirmed using the NOS inhibitor N(G)-monomethyl-L-arginine (NMMA) and isotopic tracing experiments with 15N-labelled L-arginine. Experiments with mice lacking inducible NOS (iNOS) and matched wild type animals were performed using the NO trapping agent diethyldithiocarbamate (DETC). These experiments demonstrated that endotoxin-induced NO generation in the liver tissue of mice occurs via the iNOS isoform of NOS. The described in vivo ESR technique using a "whole body" resonator allows in vivo on-line detection of endogenous NO in mice.

Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity.

The potential anti-radical properties and cytoprotective effects of Mg-gluconate were studied. When microsomal membranes were peroxidized by a *O2- driven, Fe-catalyzed oxy-radical system (R* = dihydroxyfumarate + Fe2+), Mg-gluconate inhibited lipid peroxidation (TBARS formation) in a concentration-dependent manner with IC50 being 2.3 mM. For the entire range of .25-2 mM, MgSO4 or MgCl2 were < or = 20% effective compared to Mg-gluconate. When cultured bovine aortic endothelial cells were incubated with the R* for 50 min. at 37 degrees C, 56% loss of total glutathione occurred. Pre-treatment (10 min.) of the cells with 0.25-4 mM Mg-gluconate before R* exposure significantly (p<0.05) prevented the GSH loss to varying degrees; the EC50 was 1.1 mM. In separate experiments, with 30 min. of free radical incubation of endothelial monolayers (approximately 65% confluent), cell survival/proliferation determined by the tetrazolium salt MTT assay, decreased to 38% of control at 24 hrs; Mg-gluconate concentration-dependently attenuated the lost cell survival with EC50 of approximately 1.3 mM. For comparison, the effects provided by MgSO4 or MgCl2 were significantly lower and were < or = 1/3 as potent as that produced by Mg-gluconate. In a Fenton-reaction system consisting of Fe(II)+ H2O2, Mg-gluconate but not other Mg-salts, significantly inhibited the formation of OH radicals as determined by the ESR DMPO-OH signal intensity. Mg-gluconate also dose-dependently inhibited the 'Fe-catalyzed' deoxyribose degradation suggesting that Mg-gluconate could displace Fe from 'catalytic sites' of oxidative damage. These data suggest that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities.

Effect of septic shock on nitrate, free amino acids, and urea in murine plasma and urine.

OBJECTIVES: Concentration changes of free amino acids, urea and nitrate in plasma and urine were studied for the murine model of septic shock. METHODS: After administration of a bolus dose of bacterial lipopolysaccharide (LPS), concentrations of amino acids and urea in plasma, and urea and nitrate in urine were determined. RESULTS: For individual amino acids four different trends were observed: (1) no change ( e.g., taurine, histidine, phenylalanine, hydroxproline); (2) continuous increase (e.g., aspartate and glutamate); (3) continuous decrease (e.g., threonine, serine, asparagine, proline, methionine, tyrosine); and (4) decrease during the first 4 hours, but return to normal at 8 hours after the LPS treatment (e.g., all the other amino acids). The ratio of phenylalanine to tyrosine was increased to about 2x. In plasma, urea concentration was increased about 3x, but in urine it decreased about 4x. Nitrate levels were increased 3x in urine. CONCLUSION: These early changes in the concentrations of amino acids as well as in the urea and nitrate may be useful as sensitive markers for the early and rapid diagnosis of septic shock.

Iron attenuates nitric oxide level and iNOS expression in endotoxin-treated mice.

The effect of exogenous Fe-citrate complex (Fe doses of 120 and 240 micromol/kg) on nitric oxide (NO) production in vivo has been studied in blood and liver tissue of endotoxin-treated mice. Fe-citrate complex was administered to mice subcutaneously at the same time with intravenous injection of Escherichia coli lipopolysaccharide (LPS). Iron-dependent decrease in NO2-/NO3- and nitrosyl hemoglobin levels in blood of animals was detected at 6 h after LPS administration, suggesting systemic attenuation of NO generation. NO production in the liver tissue of LPS-treated mice was decreased after Fe administration judging from the amount of mononitrosyl-iron complexes formed in the tissue by diethyldithiocarbamate. The iNOS protein determination in the liver tissue of LPS-treated mice demonstrated iron-dependent inhibition of iNOS expression. We have found previously that exogenous iron does not affect systemic NO level when it is given at 6 h after LPS injection, i.e. after iNOS expression. This is a first report demonstrating iron-dependent iNOS down-regulation in endotoxin-treated mice.

Activation of the neutrophil and loss of plasma glutathione during Mg-deficiency--modulation by nitric oxide synthase inhibition.

Sprague-Dawley rats (200 g) were fed either a Mg-deficient or Mg-sufficient diet for 3 weeks. An enriched neutrophil fraction (> 85%) was isolated from the blood by sodium metrizoate/dextran gradient centrifugation. Using the superoxide dismutase. (SOD)-inhibitable cytochrome c reduction assay, the basal activity of neutrophils isolated from the Mg-deficient rats were found elevated 5 fold after two weeks, and up to approximately 7 fold after three weeks on the diet. Upon challenge by phorbol myristate acetate (PMA), unlike the Mg-sufficient cells, the Mg-deficient cells exhibited no significant activation. Treatment of the Mg-deficient rats with the nitric oxide (NO)-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) in the drinking water, significantly attenuated the basal superoxide producing activity of the neutrophils and partially restored its response to PMA challenge. In association with the neutrophil activation. Mg-deficiency resulted in 70% decrease in plasma glutathione and 220% increase in Fe-promoted, thiobarbituric acid reactive substance (TBARS) levels; both changes were significantly attenuated by L-NAME treatment. The results suggest that neutrophils from Mg-deficient rats are activated endogenously to generate oxy-radicals which might directly mediate the in vivo peroxidative indices during Mg-deficiency. Furthermore, the neutrophil activity was lowered by NO-synthase inhibition suggesting that NO overproduction during Mg-deficiency participates in the neutrophil activation process.

Iron potentiates nitric oxide scavenging by dithiocarbamates in tissue of septic shock mice.

Vanin and co-workers (Kubrina et al., Biochim. Biophys. Acta 1176 (1993) 240-244; Mikoyan et al., Biochim. Biophys. Acta 1269 (1995) 19-24) reported that short term (30 min) iron (Fe) exposure potentiates nitric oxide (NO) production in tissues of septic shock mice, based on increased formation of NO complex by diethyldithiocarbamate (DETC). We have reexamined the effect of Fe administration in mice treated with Escherichia coli lipopolysaccharide (LPS) and have not found any changes in nitrosylhemoglobin (HbNO) or (NOs- + NO3-) levels in blood 30 min after Fe-citrate complex injection. However, Fe-citrate promotes NO complex formation by iron-dependent NO traps: DETC, pyrrolidinedithiocarbamate (PDTC) and N-methyl-D-glucamine dithiocarbamate (MGD), when given simultaneously at 6 h after LPS. Rather than potentiation of NO production, our data support that short-term iron treatment (30 min) enhances in vivo spin trapping ability of dithiocarbamate.

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 (aN 12.5 G and giso = 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 perpendicular = 2.04 and g parallel = 2.02 at 77 K) with a triplet hyperfine structure at g perpendicular. Dithiocarbamate-Fe-NO complexes detected by both trapping agents were abolished by the .NO synthase inhibitor, NG-nitro-L-arginine methyl ester. Quantitatively, both trapping procedures provided 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.

Enhanced NO production during Mg deficiency and its role in mediating red blood cell glutathione loss.

The effect of dietary Mg deficiency on nitric oxide (NO) production and its role in mediating oxidative depletion of red blood cell (RBC) glutathione in rats were investigated. Male Sprague-Dawley rats were placed on Mg-deficient or Mg-sufficient diets for up to 3 wk. Plasma nitrate plus nitrite levels, determined by the Escherichia coli reductase/Griess reagent procedures, increased 1.7-fold during the 1st wk and increased 2- to 2.4-fold during the 2nd and 3rd wk on the Mg-deficient diet. In association, substantial losses (approximately 50%) of RBC glutathione occurred during the 2nd and 3rd wk. Administration of the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in drinking water (0.5 mg/ml) effectively blunted the increases in plasma nitrate/nitrite during Mg deficiency. Concomitantly, losses of RBC glutathione exhibited by Mg-deficient rats were significantly attenuated. Packed RBCs, obtained from Mg-deficient but not from Mg-sufficient animals, displayed a prominent nitrosyl hemoglobin signal detected by electron spin resonance spectroscopy; the signals of the samples from the L-NAME-treated Mg-deficient rats were greatly reduced. With isolated RBCs, losses of the glutathione could be induced directly by peroxynitrite or 3-morpholinosydnonimine, which generates NO + .O2-, but not by NO (from sodium nitroprusside) alone, in a concentration-dependent manner. The results clearly indicate that NO overproduction occurs and participates in RBC glutathione loss during Mg deficiency. Because neutrophil activation also occurs, we suggest that NO might interact with superoxide anions to form peroxynitrite, which then directly oxidizes RBC glutathione.

Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy.

We describe here a spin-trapping method combined with X-band electron paramagnetic resonance (EPR) spectroscopy for ex vivo measurement of nitric oxide (.NO) levels in the urine of both normal and lipopolysaccharide (LPS)-induced shock mice. Normal or LPS-treated mice were injected subcutaneously with a metal-chelator complex, N-methyl-D-glucamine dithiocarbamate-ferrous iron, [(MGD)2/Fe], which binds to .NO and forms a water-soluble [(MGD)2/Fe-NO] complex. At 2 h after injection of the [(MGD)2/Fe] complex, a three-line EPR signal characteristic of the [(MGD)2/Fe-NO] complex was detected in the urine of either normal or LPS-treated mice. It is estimated that the concentrations of the [(MGD)2/Fe-NO] complex in normal and LPS-treated mouse urine were 1.3 and 35 microM, respectively. This 25-fold increase in .NO levels in the LPS-treated mouse urine provides the direct evidence that LPS challenge induces the overproduction of .NO in mice. Administration of N-monomethyl-L-arginine (NMMA; 50 mg/kg) inhibited the ex vivo signal intensities of the [(MGD)2/Fe-NO] complex in the urine of either normal or LPS-treated mouse urine. Furthermore, after injection of 15N-arginine (10 mg per mouse), a composite EPR spectrum, consisting of a three-line spectrum of the [(MGD)2/Fe-14NO] complex and a two-line spectrum of the [(MGD)2/Fe-15NO] complex, was detected in the urine. These isotopic tracer experiments further confirm that the detected .NO levels in the mouse urine are produced via the arginine-nitric oxide pathway. This ex vivo spin-trapping method should readily be adapted to experiments on larger animals and provide a noninvasive way of measuring both constitutive and inducible .NO synthase activities in living animals under physiological as well as pathophysiological conditions where .NO is overproduced.

[Europium-labelled Staphylococcus aureus protein A as a reagent for determining specific antibodies]. / Belok A Staphylococcus aureus, mechennyi evropiem, kak reagent dlia opredeleniia spetsificheskikh antitel.

In this work the conditions of labeling protein A with europium ions were studied and the conjugates obtained in this study were compared with traditional peroxidase conjugates currently used in immunochemistry. The conjugates of protein A with Eu3+ chelate were obtained with the use of cyclic dianhydride of diethylenetriaminepentaacetic acid (DADETPA). Conjugation methods with the use of DADETPA was shown to permit obtaining high-quality conjugates with europium chelates. Europium-labeled protein A ensured the sensitivity of the determination of adsorbed IgG at a level of 2 ng/ml and the dynamic analytical range within 3-1,000 ng/ml, which essentially exceeded similar characteristics of peroxidase conjugates with protein A. Europium-labeled protein A was used for the detection of antibodies to Francisella tularensis in the sera of humans immunized against tularemia. The sensitivity of this assay exceeded that of the enzyme immunoassay 10- to 40-fold. A conclusion was made on the possibility of using europium labelled protein A for the determination of specific antibodies to F.tularensis. This preparation may be useful in the determination of specific antibodies in low-immune sera.

In vivo pharmacokinetics of nitroxides in mice.

The in vivo reduction kinetics of twenty different nitroxide compounds in mice has been investigated by using an electron paramagnetic resonance (EPR) spectrometer, equipped with an S-band loop-gap resonator, operating at 3.5 GHz. The diameter of the resonator (4 mm) fits well the tip of the mouse tail, thus allowing the direct detection of in vivo free radicals in the blood circulation. After intravenous injection, the nitroxide signal in the circulation of the mouse tail was followed with time; no anesthetic agent was used. For the pyrrolidine nitroxides (five-member rings) with different functional groups, the t1/2 values followed the order: = O > COO- > OH > CONH2 approximately CH2NH2 > NH2. A different trend was obtained for the piperidine nitroxides (six-member rings): COO- > CH2NH2 > OH approximately NH2 > CONH2 > = O. The most striking observation was that while the t1/2 value of the cabonyl pyrrolidine is the longest among all the nitroxides tested for this property, that of the carbonyl piperidine is the shortest. Comparison of the in vivo decay kinetics of six pairs of pyrrolidine nitroxides and piperidine nitroxides with same functional groups showed that the t1/2 values of the former are 2 to 28 times longer than the latter. It is concluded here that the pyrrolidine nitroxides are more resistant to cellular metabolism in vivo when compared to the piperidine nitroxides.

Spin trapping of nitric oxide produced in vivo in septic-shock mice.

A nitric oxide (.NO) spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy has been employed to measure the in vivo production of .NO in lipopolysaccharide (LPS)-treated mice. The in vivo spin-trapping of .NO was performed by injecting into mice a metal-chelator complex, consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+), that binds to .NO and forms a stable, water-soluble [(MGD)2-Fe(2+)-NO] complex, and by monitoring continuously the in vivo formation of the latter complex using an S-band EPR spectrometer. At 6 h after intravenous injection of LPS, a three-line EPR spectrum of the [(MGD)2-Fe(2+)-NO] complex, was observed in the blood circulation of the mouse tail; the [(MGD)2-Fe2+] complex was injected subcutaneously 2 h before EPR measurement. No signal was detected in control groups. Administration of NG-monomethyl-L-arginine, an .NO synthase inhibitor, caused a marked reduction in the in vivo EPR signal of the [(MGD)2-Fe(2+)-NO] complex, suggesting that the .NO detected is synthesized via the arginine-nitric oxide synthase pathway. The results presented here demonstrated, for the first time, the in vivo real time measurement of .NO in the blood circulation of conscious, LPS-treated animals.

Protein A used in DELFIA for the determination of specific antibodies.

The conditions of protein A labelling with Eu chelates were studied. The conjugates obtained were compared with those from horseradish peroxidase used conventionally in immunochemical practice. Protein A-Eu conjugates were obtained by a method applied previously for antibody labelling with indium and europium chelates using the bicyclic dianhydride of diethylenetriaminepenta-acetic acid (DADTPA) with some modifications. The Eu-labeled protein A ensured a sensitivity of the IgG determination at the level of 2 ng/ml and a dynamic range of the determination from 3 to 1000 ng/ml, which significantly exceed analogous values for the protein A-peroxidase conjugates. The Eu-labeled protein A was used for the determination of antibodies to Francisella tularensis in the sera of humans vaccinated against tularemia. The assay values exceeded by 10-40-fold the results of an ELISA in sensitivity. It was deduced that the Eu-labeled protein A can be effectively used for the determination of antibodies specific to a tularemia causative agent. In particular, this compound can be useful for the determination of specific antibodies in low immune sera.

[The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]. / Opsoniziruiushchaia aktivnost' syvorotok obez'ian gamadrilov, immunizirovannykh preparatom vneshnikh membran Francisella tularensis (po dannym metoda liuminolzavisimoi khemiliuminestsentsii).

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.

[Liberation of non-penetrating spin labels from erythrocytes and liposomes after treatment with bacterial protein]. / Osvobozhdenie nepronikaiushchikh spinovykh metok iz éritrotsitov i liposom pod deistviem bakterial'nogo belka.

Liberation of non-penetrating spin labels from erythrocytes and liposomes was studied at their interaction with delta-endotoxin of Bacillus thuringiensis israelensis. The yield of non-penetrating spin labels was shown to be a highly sensitive indicator of the disturbance of the membrane barrier function.

[Computer analysis of electron paramagnetic resonance spectra of spin labels in the study of biological membranes]. / Mashinnyi analiz spektrov élektronnogo paramagnitnogo rezonansa spinovykh metok v issledovanii biologicheskikh membran.

Application of computer analysis to ESR spectra of maleimide spin labels in erythrocyte ghosts and ESR spectra of "spin sacks"--erythrocyte ghosts and liposomes containing concentrated solution of non-penetrating spin label was described. The analysis of the ESR spectra of spin labels gives exhausting information about the parameters of spin hamiltonian, peculiarities of the movement of nitroxyl radicals and their distribution between the cell and medium.

[Computer modeling of ESR spectra of the plasma in patients with Down's syndrome]. / Modelirovanie s pomoshch'iu EVM spektrov EPR plazmy krovi u bol'nykh s sindromom Dauna.

ESR technique at 77 degrees K was used for studying the blood plasma ESR signals of patients with Down syndrome and of healthy people. It was observed that the first exhibited a tendency towards a decrease of ESR signal with g = 4.3 and increase of the ratio of ceruplasmin (g = 2.05) and transferrin (g = 4.3) ESR signal amplitude. A computer simulation has been carried out by means of special mathematical program of experimental ESR spectra of the blood plasma.