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It is shown that the inhalation of gaseous nitric oxide (gNO) or sprayed aqueous solutions of binuclear dinitrosyl iron complexes with glutathione or N-acetyl-L-cysteine by animals or humans provokes no perceptible hypotensive effects. Potentially, these procedures may be useful in COVID-19 treatment. The NO level in complexes with hemoglobin in blood decreases as the gNO concentration in the gas flow produced by the Plazon system increases from 100 to 2100 ppm, so that at 2000 ppm more than one-half of the gas can be incorporated into dinitrosyl complexes formed in tissues of the lungs and respiratory tract. Thus, the effect of gNO inhalation may be similar to that observed after administration of solutions of dinitrosyl iron complexes, namely, to the presence of dinitrosyl iron complexes with thiol-containing ligands in lung and airway tissues. With regard to the hypothesis posited earlier that these complexes can suppress coronavirus replication as donors of nitrosonium cations (Biophysics 65, 818, 2020), it is not inconceivable that administration of gNO or chemically synthesized dinitrosyl iron complexes with thiol-containing ligands may help treat COVID-19. In tests on the authors of this paper as volunteers, the tolerance concentration of gNO inhaled within 15 min was approximately 2000 ppm. In tests on rats that inhaled sprayed aqueous solutions of dinitrosyl iron complexes, their tolerance dose was approximately 0.4 mmol/kg body weight.

—The effect of injected intravenously dinitrosyl iron complexes with glutathione ligand on the structural and conformational characteristics of albumin, which is a primary ligand of these complexes in plasma, and on molecules of the lipid phase in the erythrocyte membrane, which constitute the microenvironment of dinitrosyl iron complexes in erythrocytes, was analyzed. The analysis of EPR spectra of 5- and 16-DOXYL stearic acid spin probes in plasma and erythrocyte suspensions before and after injection of dinitrosyl iron complexes with glutathione was performed in the experiments. It has been found that injection of these complexes into an organism does not affect the molecular motion in albumin hydrophobic regions in plasma and erythrocyte membranes, while at the same time they stimulate incorporation of charged spin labels into the erythrocyte membrane. Apparently, this was due to a change in the charge on the outer surface of the erythrocytes.

—The objective of this work was to study the biological effect of dinitrosyl iron complexes (DNICs) with the glutathione ligand (GSH−DNICs) as a stabilized form of nitric oxide in a rat model of nitric oxide hyperproduction induced by inflammation. Administration of GSH−DNICs in endotoxin shock did not increase the total nitric oxide levels in rat organs, but exerted a protective effect by suppressing nitric oxide hyperproduction in the liver and led to an accumulation of the complexes with protein ligands in the kidney.

The objective of the present research was to study transformations of various physiological NO forms in an isolated rat heart, perfused with the medium containing dinitrosyl iron complexes with glutathione ligand (DNIC-GH). We showed that such aerobic perfusion resulted in accumulation of mostly diamagnetic NO physiological forms (S-nitrosothiols) in myocardial tissue. They were transformed into protein-bound mononuclear dinitrosyl iron complexes during subsequent total ischemia. Meantime, DNIC-GH injection on the onset of ischemia resulted in changes in the state of mitochondrial respiratory chain, characterized by the increase in myocardial concentration of flavosemiquinones.

Treatment of catechol, pyrogallol, DOPA, dopamine, norepinephrine, and natural polyhydroxy-1,4-naphthoquinone echinochrome with solution of potassium superoxide (KO2) in the presence of CaCl2 leads to the formation of water-insoluble dark pigments with stable paramagnetic properties (“calcium melanins”). In control experiments in the same procedure without Ca2+, the pigments were not formed. EPR spectra of the calcium melanins had little difference from each other and from known melanins in shape, line width, and the g factor about 2.004. Addition of EDTA water solution to dried paramagnetic pigments leads to their fast dissolving and disappearing of EPR signal. Formation of similar polymers is also observed during autoxidation of o-diphenols in Ca2+-containing alkaline buffer solution, however, this process takes a few days instead of few seconds in the presence of KO2. Thus, calcium (and other divalent cation M2+) can be considered as a key structural element in formation of M2+-catecholate paramagnetic polymer. We assume the existence of two types of paramagnetic centers in melanin-like polymer: M2+-stabilized o-semiquinone radical or bi-radical complex containing o-semiquinone and superoxide anion radicals, stabilized by M2+.

Dinitrosyl iron complexes (DNICs) have been traced in rat blood and organs after intravenous infusion of Oxacom. It is shown that the active principle (DNIC with glutathione) is rapidly distributed through the organism and deposited in blood and organs as protein-bound DNICs. The specific levels of DNIC in the main body organs are comparable, whereas its apparent lifetimes relate as blood < heart = lung < liver < kidney. Spin trapping assays indicate that protein-bound DNICs are a major but not the only form of NO deposition; the next largest depot is most probably formed by S-nitrosothiols. The gradual release of NO from such pools ensures the smooth and prolonged hypotensive effect of Oxacom.

Injection of dinitrosyl iron complexes with glutathione at the onset of 40-min regional myocardial ischemia in rat was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After introducing the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that in postischemic reperfusion, the rate of decrease in the content of these complexes in the ischemic area increases, which reflects effective scavenging of short-lived reactive oxygen species by the dinitrosyl iron complexes.

The effects of a complex of dinitrosyl iron with glutathione (DNIC-GS) lyophilized on dextran, its hydrolysis products (glutathione, nitrosoglutathione, dextran), as well as nitric oxide released from the drug on the energy metabolism and functional recovery of isolated perfused rat heart subjected to global ischemia and reperfusion have been studied. Infusion of 100 nM DNIC-GS after ischemia substantially enhanced the recovery of coronary flow, cardiac contractile and pump functions during reperfusion, with simultaneous preservation of myocardial high-energy phosphates and cell membrane integrity. It was shown by EPR that these effects were associated with transfer of Fe+(NO+)2 groups from DNIC-GS to thiol-containing proteins of cardiomyocytes and coronary vessels. Combined infusion of 100 nM DNIC-GS and 25 μM 2-(phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, after ischemia profoundly reduced the metabolic and functional recovery of reperfused hearts. After postischemic administration of an equivalent amount of DNIC-GS hydrolysate (completely decomposed complex), most of the indices did not differ from those in control or were significantly lower. Thus, inclusion of Fe+(NO+)2 groups into myocardial tissue and spontaneous release of nitric oxide trigger the protective mechanisms in the ischemic heart.

Changes in nitric oxide concentration in the rat myocardium in situ during temporary occlusion of the anterior descending coronary artery and subsequent reperfusion were monitored by microdialysis in the risk zone and a normal zone, using an NO trap (complex of ferrous ions with N-methyl-D-glucamine dithiocarbamate, Fe3+-MGD). The amplitude of the EPR signal of the reduced adduct NO-Fe2+-MGD in the samples from the risk zone increased during the 40-min occlusion and remained higher than the initial or the current control values during 60-min postischemic reperfusion, indicating substantial NO production. By the end of reperfusion, the infarct size was 47 ± 3% of the risk area. The contents of ATP, creatine phosphate, and total creatine in the risk zone decreased to respectively 44 ± 4, 51 ± 5, and 60 ± 3% of the initial values, whereas the level of lactate was six times the initial. The normal zone of the left ventricle showed no changes in NO or energy metabolite levels throughout the experiment. Thus, intense nitric oxide production in acute regional ischemia and reperfusion is associated with disturbance of energy metabolism, cell membrane damage, and death of cardiomyocytes.

The interaction between the glutathione-containing dinitrosyl iron complexes and the superoxide radical generated in mitochondria and in the xanthine-xanthine oxidase system was studied. Both superoxide and hydroxyl radicals proved to be involved in destruction of dinitrosyl iron complexes. However, the iron within dinitrosyl complexes is unlikely to catalyze decomposition of hydrogen peroxide yielding hydroxyl radical. It was found that iron dinitrosyl complexes with various anion ligands efficiently inhibited the formation of probucol phenoxyl radical in the hemin-H2O2 system, different components of these complexes being involved in the antioxidant action.

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6,-tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls.

The objective of this research was to elucidate the factors effecting the permeability of cell membranes of gram-negative bacteria toward hydrophobic compounds. Ultrasound treatment, cell age, and the phase state of phospholipid membranes were considered. Spin-labeling EPR method was used to quantify the penetration and distribution of a lipophilic spin probe, 16-doxylstearic acid (16-DS), in Pseudomonas aeruginosa cell membranes. This bacterium was chosen because of its reported resistance to the action of hydrophobic antibiotics caused by the low permeability of the outer cell membrane for hydrophobic compounds. EPR spectra were collected from cell pellets and cell lysates. The overall spin probe uptake was measured in 10% SDS-cell lysates. Lysis with 0.6% SDS revealed the fraction of the probe located in membrane sites readily accessible to the surfactant. The results indicated a structural heterogeneity of P. aeruginosa membranes, with the presence of structurally "stronger" and "weaker" sites characterized by different susceptibility to the SDS treatment. The intracellular concentration of 16-DS was higher in insonated cells and increased linearly with the sonication power. EPR spectra indicated that ultrasound enhanced the penetration of the probe into the structurally stronger sites of the inner and outer cell membranes. The effect of ultrasound on the cell membranes was transient in that the initial membrane permeability was restored upon termination of the ultrasound treatment. These results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes. This resistance may be reduced by the simultaneous application of antibiotic and ultrasound. This hypothesis was confirmed in our experiments with P. aeruginosa exposed to erythromycin.

The aim of this study was to relate changes in the redox state of mitocondrial electron carriers to the 'burst' of oxyradicals in postischemic myocardium. The free radical EPR signals of control and re-oxygenated rat hearts were mainly due to coenzyme Q10, the line width was 0.81 +/- 0.02 mT, and the intensities (1.58 +/- 0.12) x 10(16) and (1.41 +/- 0.13) x 10(16) spins/g. The low-temperature spectra of oxygenated myocardium contained a predominant signal from a S3 Fe-S center and weak signals from N1b, N2, N3, N4 and S1 centers. Global ischemia caused cardinal changes in the redox state of the mitochondrial respiratory chain. The low-temperature EPR spectrum now contained intensive signals from most Fe-S centers. The amount of coenzyme Q10 semiquinones decreased during global ischemia, but the content of flavosemiquinones increased. The line width of the signal of the ischemic heart was 1.28 +/- 0.03 mT, and its intensity corresponded (3.16 +/- 0.94) x 10(16) spins/g. The spin-trapping experiments with TEMPONE-H showed that the rate of oxyradical generation in isolated cardiomyocytes essentially increased after hypoxia or on adding rotenone and antimycin A. It became equal to 4.2 +/- 0.3, 8.2 +/- 0.6 and 7.1 +/- 0.5 nmol/min mg-1 mitochondrial protein, respectively. The maximal stimulatory effect was observed in the presence of both inhibitors. The addition of superoxide dismutase, but not catalase, suppressed the formation of oxyradicals.

A new experimental approach for spin-trapping of oxygen radicals in a selected region of the heart in situ is described. This approach is based on microdialysis, and it permits the detection of oxygen radicals in conditions of local ischemia and restoration of normal blood flow. Increased hydroxyl radical generation in an ischemic area of canine myocardium, as a result of 40 min local occlusion, has been studied.

To assess the effect of a polymeric surfactant, Pluronic P-105 on the activity of electron transport chains in the mitochondria of HL-60 cells, the bioreduction rates of two membrane-localized lipophilic spin probes, 16-doxylstearic acid methyl ester (16-DSME) and 5-doxylstearic acid (5-DS), were studied. In addition, the effect of Pluronic on the bioreduction rate of the DNA-intercalating spin-labeled anthracyclin drug, Ruboxyl (Rb) was evaluated. For 16-DSME, the bioreduction kinetics was zero order with regard to the nitroxide concentration, indicating that the rate was controlled by the concentration of the reducing enzyme(s), which depends on the activity of the electron transport chains. The introduction of Pluronic at concentrations higher than 0.01% resulted in the decrease of the 16-DSME bioreduction rate. The data suggested that short-term cell incubation with Pluronic resulted in reduced activity of the electron transport chains in the mitochondria of HL-60 cells. This was corroborated by the results of an MTT assay. For 5-DS, the bioreduction kinetics was first order in the absence of Pluronic, but did not follow any simple kinetic law after a short-term cell incubation with Pluronic. For Rb, the degree of nitroxide bioreduction dropped progressively with increasing Pluronic concentration. Thus, incubating cells with polymeric surfactants modulates the intracellular energy metabolism, which can affect the rates of energy-dependent intracellular processes.

A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.