Spin Trapping of Superoxide from Glass Adherent Polymorphonuclear Leukocytes Induced by N-Formylmethionyl-Leucyl-Phenylalanine

Dahinden et al. reported that N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide release from polymorphonuclear leukocytes (PMNs) lasted more than 60 min when the cells were allowed to attach to a petri dish before induction. In contrast, it lasted only for 2.5 min when cells were in suspension (J. Clin. Invest. 72: 113-121, 1983). In spite of this report, the effect of cell adhesion has been ignored in most spin trapping studies of superoxide release from PMNs. This study shows that most PMNs in a quartz flat electron paramagnetic resonance (EPR) cuvette which was placed horizontally adhered to the wall within 3 min. In contrast, if the cuvette was placed vertically, only 20-30% of the cells became adherent in 30 min. We performed spin trapping studies using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, and monitored the effect of cell adhesion on superoxide generation. When spin trapping was conducted on PMNs in suspension, the EPR signal of superoxide adduct (DMPO-OOH) was undetectable after stimulation with fMLP. However, PMNs which were allowed to adhere to the cuvette after stimulation generated superoxide for hours. Moreover, when PMNs were allowed to adhere prior to the stimulation, the magnitude of superoxide release was augmented three-to fourfold. Unlike fMLP, phorbol myristate acetate (PMA), which has been most commonly used in spin trapping studies, induced superoxide release which was not influenced by cell adhesion. We emphasize the importance of specifying the cell-adhesion-state in spin trapping studies.     

电子自旋共振-自由基捕获技术在生物学中的应用

本文结合作者实验室的工作,简要回顾了电子自旋共振-自由基捕获技术在生物领域的应用,包括新型自由基捕获探针的分子设计与合成,以及该技术在细胞、植物体系中的应用实例,并结合该技术的研究现状初步讨论了它的未来发展前景。     

用自旋捕集技术研究硒化合物对活性氧自由基的清除作用

亚硒酸钠,硒代甲硫氨酸和硒代硫酸软骨素对黄嘌呤氧化酶体系和人多形核白细胞（ＰＭＮ）呼吸暴发时产生的超氧阴离子（Ｏ）自由基;和Ｆｅｎｔｏｎ反应生成的羟自由基（·ＯＨ）有明显的清除作用,并抑制ＰＭＮ呼吸暴发时对氧的消耗。实验结果说明不同硒化合物不仅能够清除细胞体系产生的活性氧自由基,而且对化学反应生成的活性氧自由基也有清除作用。     

Spin Trapping of Inorganic Radicals

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals.

Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (·N_j· ·CN, ·SCN) are discussed in more detail.     

Direct Observation of Spin-Trapped Carbon Dioxide Radicals in Hepatocytes Exposed to Carbon Tetrachloride

Carbon dioxide radical adducts of the spin trapping agent, α-phenyl N-t-butyl nitrone (PBN), have been observed to occur in the urine and bile of rats exposed to carbon tetrachloride as well as in perfusates of liver in which the perfusion medium contained carbon tetrachloride (Connor er al., J. Biol. Chem., 261, 4542, (1986)). The carbon dioxide adduct was proven to be derived from CCI, by use of 13-C-labelled compound. These adducts were not observed in the liver itself suggesting that they might be rapidly secreted from the liver. However, using isolated hepatocytes, we have demonstrated that the carbon dioxide radical adduct can be observed directly in the liver cells as it is formed. Since this water-soluble adduct cannot be extracted by non-aqueous solvents such as chloroform or toluene, its formation in liver in vivo or in perfused livers was not detected. Lowering the oxygen tension in the system diminished the intensity of production of the carbon dioxide adduct, consistent with the adduct being produced as a result of ·OOCCl₃ generation. It is not clear the extent to which this adduct is formed as a result of the ·CO₂ radical or is produced by metabolic oxidation of the trichloromethyl radical adduct of PBN per se to the carbon dioxide radical adduct. The intensity of the signal of the carbon dioxide radical adduct suggests that adduct conversion may be the route of formation since it seems unlikely that a sufficient amount of the halocarbon could be metabolized to ·COCl or ·CO₂ radicals to generate a signal of the magnitude involved. The ·CO₂ adduct is readily observed in intact hepatocytes, but the ·CCl₃ adduct is not (although we know the ·CCl₃ adduct has been produced in these cells), indicating that the ·CO₂ adduct is present in considerable abundance compared to the ·CCl₃ adduct.     

Spin Trapping of Inorganic Radicals

The use of nitrose compounds and nitrones as spin traps for the detection of short-lived inorganic radicals is discussed. To a certain degree nitrones and nitroso compounds are complementary. While nitroso compounds are superior with respect to spin trapping metal-centred radicals, nitrones form more persistent spin adducts with most small inorganic radicals.

Erroneous results may be obtained when hydrolysis and redox reactions involving the spin adducts are ignored. Spin trapping of pseudohalide radicals (·N_j· ·CN, ·SCN) are discussed in more detail.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

慢性肾衰病人血清和红细胞抗氧化能力的ESR研究

用电子自旋共振(ESR)自旋捕集技术研究了正常人和肾衰病人血清和红细胞对黄嘌呤 黄嘌呤氧化酶体系产生的氧自由基的作用.结果发现:(1)正常人血清和红细胞能够有效地清除超氧阴离子自由基(O_2~-),而肾衰病人血清和红细胞清除O_2~-的能力明显比正常人血清和红细胞低;(2)正常人血清能有效地把黄嘌呤 黄嘌呤氧化酶体系产生的O_2~-转化为·OH,病人血清在这方面与正常人血清有显著性差异.     

Oxygen free radical producing activity of polymorphonuclear leukocytes in patients with Parkinson's disease

Oxygen free radicals (OFRs) have been suggested in the pathogenesis of Parkinson's disease (PD). These free radicals exert their cytotoxic effect by peroxidation of lipid membrane resulting in the formation of malondialdehyde (MDA). Polymorphonuclear (PMN) leukocyte is one of the major sources of OFR. However, the oxygen free radical producing activity of PMN leukocytes in patients with PD is not known. We therefore studied the oxygen free radical producing activity of polymorphonuclear leukocytes and MDA levels in the serum of healthy subjects and in patients with Parkinson's disease. The oxygen free radical producing activity of PMN leukocytes in blood and the MDA content in serum were significantly higher in patients with Parkinson's disease than in healthy subjects. These results indicate a possible role of oxygen free radicals in the pathogenesis of Parkinson's disease.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Endothelial Cells as A Source of Oxygen-Free Radicals An Esr Study

Endothelial cells were subjected to anoxia/reoxygenation in order to simulate some of the free radical mechanisms occurring in ischaemialreperfusion. With ESR and spin trapping using the spin traps 5.5-dimethyl-l-pyrroline-l-oxide (DMPO) and 3,3,5,5-dimethyl-l-pyrroline-l-oxide (M₄PO), the results show that upon reoxygenation of endothelial cells, following a period of anoxia, these cells generate superoxide (0₂). Cytotoxicity of the spin traps was measured by standard trypan blue exclusion methods. Cell injury or death was measured at various times during reoxygenation by lactate dehydrogenase (LDH) release. Experiments using oxypurinol, SOD, CAT and a combination of SOD and CAT show that while oxypurinol partially prevents spin adduct formation. the combination of SOD and CAT is more effective in doing so. These results suggest that the majority of the oxygen radicals produced by endothelial cells are done so exogenously. The results also suggest that endothelial cells are not only a source of oxygen radicals but also a target.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Free radical generation during chemical depolymerization of heparin

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

Degradation of Hyaluronic Acid, Poly- and Mono-Saccharides, and Model Compounds by Hypochlorite: Evidence for Radical Intermediates and Fragmentation

Degradation of hyaluronic acid by oxidants such as HO^· and HOCl/ClO⁻ is believed to be important in the progression of rheumatoid arthritis. While reaction of hyaluronic acid with HO^· has been investigated extensively, reaction with HOCl/ClO⁻ is less well defined. Thus, little is known about the site(s) of HOCl/ClO⁻ attack, the intermediates formed, or the mechanism(s) of polymer degradation. In this study reaction of HOCl/ClO⁻ with amides, sugars, polysaccharides, and hyaluronic acid has been monitored by UV-visible (220–340 nm) and EPR spectroscopy. UV-visible experiments have shown that HOCl/ClO⁻ reacts preferentially with N-acetyl groups. This reaction is believed to give rise to transient chloramide (R—NCl—C(O)—R′) species, which decompose rapidly to give radicals via either homolysis (to produce N· and Cl·) or heterolysis (one-electron reduction, to give N· and Cl⁻) of the N—Cl bond. The nature of the radicals formed has been investigated by EPR spin trapping. Reaction of HOCl/ClO⁻ with hyaluronic acid, chondroitin sulphates A and C, N-acetyl sugars, and amides gave novel, carbon-centered, spin adducts, the formation of which is consistent with selective initial attack at the N-acetyl group. Thus, reaction with hyaluronic acid and chondroitin sulphate A, appears to be localized at the N-acetylglucosamine sugar rings. These carbon-centered radicals are suggested to arise from rapid rearrangement of initial nitrogen-centered radicals, formed from the N-acetyl chloramide, by reactions analogous to those observed with alkoxyl radicals. The detection of increasing yields of low-molecular-weight radical adducts from hyaluronic acid and chondroitin sulphate A with increasing HOCl/ClO⁻ concentrations suggests that formation of the initial nitrogen-centered species on the N-acetylglucosamine rings, and the carbon-centered radicals derived from them, brings about polymer fragmentation.     

The haemolytic reactions of 1-acetyl-2-phenylhydrazine and hydrazine: A spin trapping study

Free radical production from the reaction of hydrazine and 1-acetyl-2-phenylhydrazine (AcPhHZ) with oxyhaemoglobin and with human red blood cells, has been observed by the electron spin resonance technique of spin trapping. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the free radical intermediates detected depended on the hydrazine derivative, oxyhaemoglobin and the oxyhaem/hydrazine derivative concentration ratio.

The reaction of hydrazine with oxyhaemoglobin in the presence of DMPO gave a nitroxide which was identified as a reduced dimer of DMPO. Whereas hydrazine-treated red blood cells, in the presence of DMPO, gave a nitroxide spin adduct which was identified as the hydroxyl radical spin adduct of DMPO, 5,5-dimethyl-1-pyrrolidino-1-oxyl (DMPO-OH).

The reaction of AcPhHZ with oxyhaemoglobin, in the presence of DMPO, gave DMPO-OH, the phenyl radical spin adduct of DMPO, 5,5-dimethyl-2-phenylpyrrolidino-1-oxyl (DMPO-Ph) and an oxidised derivative of DMPO, 5,5-dimethyl-2-pyrrolidone-1-oxyl (DMPOX). The amounts of DMPO-Ph, DMPO-OH and DMPOX observed depended on the 1-acetyl-2-phenyl-hydrazine/oxyhaemoglobin concentration ratio; DMPOX replaced DMPO-OH as the concentration of AcPhHZ was decreased. DMPOX production has been previously associated with the production of highly oxidised haem iron-oxygen intermediates. AcPhHZ treated red blood cells gave DMPO-Ph and DMPO-OH spin adducts in the presence of DMPO.

DMPO had little to no effect on the rate of oxygen consumption by oxyhaemoglobin with hydrazine and AcPhHZ. Moreover, the rate of oxyhaemoglobin oxidation induced by hydrazine, was not decreased by DMPO whereas the rate of oxyhaemoglobin oxidation induced by AcPhHZ was decreased approx. 40% by DMPO. DMPO (10 mM) gave a small decrease in haemolysis and lipid peroxidation induced by 1 mM hydrazine and AcPhHZ in a 1% suspension of red blood cells.