Electron spin resonance determination and superoxide dismutase activity in polymorphonuclear leukocytes in congestive heart failure.

Electron spin resonance spin trapping technique was used to measure the generation of active oxygen free radicals during the respiratory burst of phorbol myristate acetate-stimulated leukocytes, and the superoxide dismutase activity in healthy subjects and in patients with congestive heart failure. The authors also measured the concentration of peroxidation products (primarily malondialdehyde) by the thiobarbituric acid method. Experimental results showed that the electron spin resonance spectra obtained during the respiratory burst of polymorphonuclear leukocytes stimulated with phorbol myristate acetate primarily were those of the spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with the superoxide anion and to a lesser extent those of DMPO with hydroxyl radical. Compared with healthy subjects, the release of oxygen free radicals in the respiratory burst of polymorphonuclear leukocytes stimulated with phorbol myristate acetate and the concentration of thiobarbituric acid reactive product in plasma were significantly increased in patients with congestive heart failure while the activity of superoxide dismutase was markedly lower. The increased production of oxygen free radicals by polymorphonuclear leukocytes and the decreased capability of antioxidative defences might play an important role in the generation and development of cardiac failure.     

利福平和利福定对氧自由基清除作用和抗氧化作用的研究

用ESR自旋捕集技术及化学发光和紫外分光光度法研究了利福平和利福定对Fenton反应产生的羟基自由基、黄嘌呤/黄嘌呤氧化酶产生的超氧阴离子自由基的清除作用和亚油酸脂质过氧化产生的共轭二烯的抑制作用。结果发现,利福平和利福定在很低的浓度下就能对以上体系产生的氧自由基产生很强的清除作用,但对亚油酸脂质过氧化产生的共轭二烯的抑制作用很弱,说明利福平和利福定主要是一种预防性抗氧化剂。     

On the spin trapping and ESR detection of oxygen-derived radicals generated inside cells.

Recently several attempts to identify oxygen-derived radicals in whole cells by spin trapping and electron spin resonance have been reported by using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap. In the present study, the feasibility of this method is examined. Chinese hamster V79 cells and human erythrocytes served as the test systems, while OH radicals were generated by gamma radiolysis. Several spin traps were used to scavange the radicals and a distinction between exo- and endocellular ESR observable species was achieved using tri(oxalato) chromiate(III) as a line broadening agent. To distinguish between exo- and endocellular sites of radical formation, we studied the effects of high molecular weight scavengers (polyethylene glycols), which do not enter the cell. Various possible obstacles associated with trapping and detecting the radicals inside the cells were examined. The results indicate that the primary radicals react with the spin traps. However, these spin adducts decayed within the cells. Cellularly induced decay of 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl radical presented the major difficulty in detecting the endogenous radicals, and potential experimental approaches to overcome this difficulty are discussed.     

Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.

Oxygen free radicals have been demonstrated to be important mediators of postischemic reperfusion injury in a broad variety of tissues; however, the cellular source of free radical generation is still unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that bovine endothelial cells subjected to anoxia and reoxygenation become potent generators of superoxide and hydroxyl free radicals. A prominent DMPO-OH signal aN = aH = 14.9 G is observed on reoxygenation after 45 min of anoxic incubation. Quantitative measurements of this free radical generation and the time course of radical generation are performed. Both superoxide dismutase and catalase totally abolish this radical signal, suggesting that O2 is sequentially reduced from O2-. to H2O2 to OH.. Addition of ethanol resulted in trapping of the ethoxy radical, further confirming the generation of OH.. Endothelial radical generation was shown to cause cell death, as evidenced by trypan blue uptake. Radical generation was partially inhibited and partially scavenged by the xanthine oxidase inhibitor allopurinol. Marked inhibition of radical generation was observed with the potent xanthine oxidase inhibitor oxypurinol. These studies demonstrate that endothelial cells subjected to anoxia and reoxygenation, conditions observed in ischemic and reperfused tissues, generate a burst of superoxide-derived hydroxyl free radicals that in turn cause cell injury and cell death. Most of this free radical generation appears to be from the enzyme xanthine oxidase. Thus, endothelial cell free radical generation may be a central mechanism of cellular injury in postischemic tissues.     

Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages

The objectives of this investigation were (1) to determine the effects of hemoglobin on the production of reactive oxygen species by activated rat alveolar macrophages, (2) to determine a possible mechanism for these effects, and (3) to determine which part of the hemoglobin molecule is responsible for these effects. Production of reactive oxygen species by phorbol myristate acetate (PMA)-stimulated cells was assessed by measuring luminol-enhanced chemiluminescence (CL). Hemoglobin enhances PMA-stimulated CL in a dose-dependent manner. The effect is maximal at 0.5-1.0 microM hemoglobin where PMA-induced CL is increased by approximately 20-fold. Superoxide anion release from PMA-stimulated cells is not affected by hemoglobin. However, the hemoglobin-induced enhancement of PMA-stimulated CL is inhibited by superoxide dismutase, catalase, dimethylthiourea, or deferoxamine. These results suggest that hydroxyl radical may be formed from hydrogen peroxide which is derived from superoxide anion. Measurements of electron spin resonance spectra following spin trapping of radicals verify that hydroxyl radicals are produced by the cells in the presence of PMA and hemoglobin. The hemoglobin effects appear to require iron in a protoporphyrin complex, because hemin stimulates PMA-induced CL, whereas neither ferrous nor ferric iron has any effect. These findings taken together suggest that hemoglobin can act as a biological Fenton reagent to enhance the production of reactive oxygen species from alveolar macrophages and potentially contribute to lung damage during leakage of blood into the alveolar spaces.     

Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique.

Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA-stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy-radical generation.     

The cation radicals of free base and zinc bacteriochlorin, bacteriochlorophyll, and bacteriopheophytin

One-electron oxidation of zinc tetraphenylbacteriochlorin and its metal-free base yielded stable cation radicals. Electron spin resonance hyperfine splittings were assigned by selective deuterations. These results indicate that the protons of the saturated rings of the bacteriochlorins carry large spin densities, in accord with molecular orbital calculations. Comparison in vitro of the optical spectra of bacteriochlorins and their cation radicals with those of bacteriochlorophyll show close correspondence and suggest that the electron spin resonance data from the former may also prove a guide to the biological molecule. The surprising similarity in properties between the radicals of free base and zinc bacteriochlorins is maintained in the chlorophylls: cation radicals of bacteriopheophytin and methyl pheophorbide (the free bases of bacteriochlorophyll and methyl chlorophyllide, respectively) exhibit electron spin resonance properties similar to those of their magnesium-containing derivatives. The possibility that metal-free chlorophylls participate in photosynthesis is discussed.     

Ribonucleotide reductase in melanoma tissue

Summary The characteristic EPR doublet of tyrosine radicals of the growth-regulating enzyme ribonucleotide reductase was detected in human melanoma tissue grown in nude mice. This was possible through the use of an amelanotic melanoma that does not exhibit disturbing EPR signals from melanin. The content of tyrosine radicals is higher in young tumor tissues than in older ones. The clinically applied antimelanotic drug, 4-hydroxyanisole, inhibits ribonucleotide reductase in Ehrlich ascites tumor cells as demonstrated by a pronounced quenching of tyrosine radicals (IC₅₀=5 M). In amelanotic melanoma tissue tyrosine radicals of the enzyme are also quenched by 4-hydroxyanisole in concentrations down to 50 M. Thus, the inactivation of ribonucleotide reductase, which provides deoxyribonucleotides for DNA synthesis, may be a hitherto unexpected mechanism for the antitumor action of 4-hydroxyanisole.Abbreviations EPR electron paramagnetic resonance - RR ribonucleotide reductase - 4-HA 4-hydroxyanisole     

Superoxide radical scavenging activity of idebenone in vitro studied by ESR spin trapping method and direct ESR measurement at liquid nitrogen temperature

The radical scavenging activity of oxidized and reduced idebenone (ID-O and ID-H, respectively) against superoxide radical (O2(-*) was studied in vitro using two methods: (1) O2(-*) radicals were generated enzymatically in a hypoxanthine (HPX)-xanthine oxidase (XOD) system and detected by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trapping. Superoxide dismutase and other scavengers added to this system competed to various extents with DMPO to trap O2(-*) radicals, resulting in a decrease of the ESR signal intensity of the DMPO-OOH spin adduct. ID-O reacted about 12-fold quicker (k = 4.48 x 10(4) M(-1)s(-1)) with the O2(-*) radicals than ID-H (k = 3.62 x 10(3) M(-1)s(-1)) x (2) O2(-*) radicals were generated chemically in potassium superoxide (KO2)-crown ether system. Quinoid compounds reacted with the O2(-*)radicals to form semiquinone radicals that could be observed by ESR. At liquid nitrogen temperature (-196 degrees C), the ESR signal of O2(-*) radicals could be observed directly, thus allowing us to estimate the scavenging activity of ID-O and ID-H. These experiments also revealed that ID-O possesses an O2(-*) radical scavenging activity, whereas ID-H reacts quantitatively much slower. Analyzing various quinone compounds, it has been established that the O2(-*) radical scavenging process is a reversible, most probably oscillating, monovalent electron transfer from superoxide to the quinone, and that the O2(-*) radical scavenging activity depends on the redox potential, i.e., on the actual state of oxidation of the quinones.     

Mitochondrial sources of H2O2 generation play a key role in flow-mediated dilation in human coronary resistance arteries

We previously showed that hydrogen peroxide (H2O2) contributes to flow-induced dilation in human coronary resistance arteries (HCRAs); however, the source of this H2O2 is not known. We hypothesized that the H2O2 is derived from superoxide (O2*-) generated by mitochondrial respiration. HCRAs were dissected from right atrial appendages obtained from patients during cardiac surgery and cannulated with micropipettes. H2O2-derived radicals and O2*- were detected by electron spin resonance (ESR) using BMPO as the spin trap and by histofluorescence using hydroethidine (HE, 5 micromol/L) and dichlorodihydrofluorescein (DCFH, 5 micromol/L). Diameter changes to increases in pressure gradients (20 and 100 cm H2O) were examined in the absence and the presence of rotenone (1 micromol/L), myxothiazol (100 nmol/L), cyanide (1 micromol/L), mitochondrial complex I, III, and IV inhibitors, respectively, and apocynin (3 mmol/L), a NADPH oxidase inhibitor. At a pressure gradient of 100 cm H2O, ubisemiquinone and hydroxyl radicals were detected from effluents of vessels. Including superoxide dismutase and catalase in the perfusate reduced the ESR signals. Relative ethidium and DCFH fluorescence intensities in HCRAs exposed to flow were enhanced (1.45+/-0.15 and 1.57+/-0.12, respectively compared with no-flow) and were inhibited by rotenone (0.87+/-0.17 and 0.95+/-0.07). Videomicroscopic studies showed that rotenone and myxothiazol blocked flow-induced dilation (% max. dilation at 100 cm H2O: rotenone, 74+/-3% versus 3+/-13%; myxothiazol, 67+/-3% versus 28+/-4%; P<0.05). Neither cyanide nor apocynin altered flow-induced dilation. These results suggest that shear stress induced H2O2 formation, and flow-induced dilation is derived from O2*- originating from mitochondrial respiration.     

Microbial glycolipids: possible virulence factors that scavenge oxygen radicals.

Two important pathogens of developing countries, Mycobacterium leprae, the etiologic agent of leprosy, and Leishmania donovani, the protozoal parasite that causes kalaazar, persist in the human host primarily in mononuclear phagocytes. The mechanisms by which they survive in these otherwise highly cytocidal cells are presently unknown. Since the best understood cytocidal mechanism of these cells is the oxygen-dependent system that provides lethal oxidants including the superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2), we sought specific microbial products of these organisms that might enable them to elude oxidative cytocidal mechanisms. Phenolic glycolipid I of M. leprae and lipophosphoglycan of L. donovani are unique cell-wall-associated glycolipids produced in large amounts by the organisms. In this study, phenolic glycolipid I derivatives and lipophosphoglycan were examined for their ability to scavenge potentially cytocidal oxygen metabolites in vitro. Electron spin resonance and spin-trapping indicate that phenolic glycolipid I derivatives and lipophosphoglycan are highly effective in scavenging hydroxyl radicals and superoxide anions. The results suggest that complex glycolipids and carbohydrates of intracellular pathogens that can scavenge oxygen radicals may contribute to their pathogenicity and virulence.     

Melanin photoprotection in the human retinal pigment epithelium and its correlation with light-induced cell apoptosis

Time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to study melanin free radicals in human retinal pigment epithelium (RPE) cells and tyrosine-derived synthetic melanin. TREPR signal traces from RPE cells reveal in vivo light-induced melanin free radical photochemistry in more detail than previously known. Electron spin polarization reflecting a non-Boltzmann population within the energy levels of the spin system is observed in RPE cells as the result of the triplet state photoproduction and subsequent disappearance of free radicals in the melanin polymer. In a set of RPE cells cultured from individual sources, differences in optical absorption, continuous wave EPR spectra, and TREPR signals were correlated with apoptosis assays performed by flow cytometry. Continuous wave EPR spectra of RPE cells and TREPR of acidified synthetic melanin suggest that increased melanin aggregation provides an increase in photoprotection in the RPE cells that are relatively less susceptible to blue light-induced apoptosis.     

UV causation of melanoma in Xiphophorus is dominated by melanin photosensitized oxidant production

Controversy continues both as to which wavelengths of sunlight cause melanoma and the mechanisms by which these different wavelengths act. Direct absorption of UVB by DNA is central in albino animal models, but melanin-pigmented models have shown major contributions by wavelengths longer than UVB that are thought to be mediated by photosensitized oxidant production. The only model for which the action spectrum of melanoma causation is known is a genetically melanoma-susceptible specific cross of Xiphophorus fish. We used electron paramagnetic resonance to quantitatively detect the UV induction of reactive melanin radicals in situ in the melanin-containing cells in the skin of this model and derived the action spectrum for melanin-photosensitized oxidant production (Phi(ox)). This action spectrum was identical to that for melanoma induction (Phi(mel)). These results confirm the hypothesis that melanin-photosensitized radical production is the major causative step of melanoma in this model and demonstrate that the wavelengths and mechanisms of melanoma causation in different models are dependent on the presence of melanin. This approach should be applicable to humans, thus providing an accurate surrogate for Phi(mel) for prevention studies.     

Nuclear coupling of 33S and the nature of free radicals in irradiated crystals of N-acetyl-L-cysteine.

Hyperfine structure due to 33S in its natural abundance of 0.76% has been measured in the electron spin resonance of free radicals produced by x-irradiation of single crystals of N-acetyl-L-cysteine at 77 K. These measurements proved that the radicals produced at 77 K with principal g values of 1.990, 2.006, and 2.214 are monosulfide radicals with the 3p unpaired electron density of 0.70 on the S. They are believed to be negatively charged molecules RCH2S-H or neutral RCH2SH2 radicals in which 90% of the spin density of the captured electron is concentrated in a d-p hybrid orbital on the S. As the temperature is raised to 300 K, these, as well as the carbon-centered radicals produced at the lower temperature, are mostly converted to neutral disulfide radicals RCH2SS like those observed in irradiated cystine.     

On the State of the Iron and the Nature of the Ligand in Oxyhemoglobin

The structure of the oxygenated heme group of oxyhemoglobin may be formulated as [Hb(Heme d(1/2) (5)).OO(-)]. The heme iron atom is formally ferric, and the ligand is bound superoxide anion. When deoxyhemoglobin combines reversibly with oxygen a partial transfer of an electron occurs from the ferrous iron atom to the oxygen molecule. By surrendering its electron the iron atom has become ferric; in accepting an electron the ligated oxygen molecule has become a new species, the bound superoxide anion (.OO(-)).The configuration of the heme iron atom is deduced from comparison of the optical spectrum in the visible region of oxyhemoglobin with that of alkaline ferric hemoglobin whose configuration is established by electron paramagnetic resonance spectroscopy. The configuration of both species is low spin ferric heme iron (Heme d(1/2) (5)). The configuration of the ligated oxygen molecule of oxyhemoglobin is not accessible to study by magnetic or optical probes. However it may be known by analogy with the configuration of the ligated oxygen molecule of reversibly oxygenated cobalt complexes whose structure has been proved by both electron paramagnetic resonance and x-ray diffraction analysis. It is bound superoxide anion (.OO(-)). Other physical studies bearing on the structure of the oxygenated heme group are discussed. Reasons are given for believing that the proposed formulation of the oxyhemoglobin structure is consistent with the known stability of oxyhemoglobin.     

Nuclear coupling of 33S and the nature of free radicals in irradiated crystals of cysteine hydrochloride and N-acetyl methionine.

The 33S hyperfine structure has been observed in the electron spin resonance of irradiated crystals of cysteine-HCl-H2O and of N-acetyl DL-methionine. An analysis of the results shows that in both substances the free radicals that are stable at room temperature are disulfide radicals, RCH2SS. A possible mechanism for formation of these radicals is proposed.     

Direct detection of oxygen free radicals produced by myocardial reperfusion using electron spin resonance spectroscopy

There is a growing evidence for the role of oxygen free radicals (OFR) in mediating myocardial tissue injury during myocardial ischemia and particularly during reperfusion. But almost all of the evidence was indirect, using electron spin resonance (ESR) spectroscopy, we have directly measured OFR generated in ischemic and reperfused isolated rabbit hearts. 17 hearts were rapidly frozen in liquid nitrogen after their arrest by cardioplegic solution and sampled after 150 min of sustained hypothermic global ischemia or after reperfusion. The ESR spectra obtained from experiment have directly demonstrated that OFR is produced in significant amounts in the isolated rabbit hearts during early stage of reperfusion but only small amount during ischemia. The mitochondrial electron transport chain appeared to be the main source of OFR. We found that superoxide dismutase scavenged OFR generated during reperfusion efficiently, but catalase did not. We believe that superoxide anion, not hydroxyl radical, is the main OFR which is responsible for myocardial reperfusion injury. We also found that Salvia, a traditional Chinese medicine, a very efficient OFR scavenger, had the similar effect as superoxide dismutase.     

ELECTRON SPIN RESONANCE OF IRRADIATED SINGLE CRYSTALS OF L-PHENYLALANINE-HCL

Single crystals of L-phenylalanine-HCl have been observed with electron spin resonance after irradiation with a cobalt 60 gamma-ray source. The predominant signals observed are from long-lived benzyl radicals. The results indicate that one form of radiation damage to proteins containing this amino acid is breakage of the carbon-carbon bond to release the benzyl radical from the polypeptide chain. Hyperfine structure due to proton couplings of the two methylene hydrogens and of the hydrogens on the ring of the benzyl radical is observed and analyzed to give the electron spin density on the various carbons. The spin density on the methylene carbon is found to be 0.71; that on carbon C((2)), C((4)), or C((6)) is 0.17. The sum of the spin densities on C((1)), C((3)), and C((5)) is -0.22.     

Free radicals formed by exposure of pyrimidine solids to sodium atoms: an electron spin resonance study.

Sodium atoms have been deposited on various pyrimidine powders under high vacuum at 77 degrees K and the electron spin resonance spectra of the resulting free radicals have been observed. Generally, the spectra show that the electron of the Na goes into a molecular orbital of the pyrimidine ring and the Na+ ions become attached to a carbonyl oxygen of the resulting pyrimidine anion. In 5-fluorouracil and 5-chlorouracil, however, the halogen is evidently abstracted by the Na to form NaF or NaC1 and the neutral uracil radical. Thymine shows evidence for H-addition radicals as well as the Na+-[thymine]-complex. The H source for the addition radicals may be an H-2-0 impurity in the sample, with which the Na atoms combine to release the H atoms. In addition to a resonance with g equals 2.00 from the pyrimidine anion radical, broad resonances with g greater than 2 were observed for 5-bromouracil, 5-chlorouracil and 5-iodouracil, as well as for alloxan and cytosine. These resonances, generally unstable at room temperature, are believed to arise from electrons trapped in interstitial sties or vacancies in the lattice.