Basic Principles of Reactivity in Free Radical Chemistry

This review is concerned with an overall survey of reactivity in free radical chemistry. A concise classification is given of elementary reaction steps which can be combined in different ways to account for overall chemical transformations: radical forming reactions, radical transformations, and radical destroying reactions. From this is derived the concept of the chain reaction which leads on to an up-to-date theory for understanding reactivity in free radical processes. Finally, a few aspects of autoxidation are discussed.     

A Novel Interleukin Stimulating Free Radical Production by Granulocytes

Polymorphonuclear granulocytes (PMN) are potent producers of free oxygen-derived radicals. Since other granulocyte functions are affected by interleukins, we investigated whether free-radical production can be initiated by a similar mediator. For estimation of free radical production, SOD-inhibitable lucigenin-dependent chemiluminescence and SOD-inhibitable cytochrome C reduction were used. As a source of interleukins, serum-free 24 h culture supernatants of human mononuclear cells (MNC) stimulated with bacterial lipopolysaccharide were prepared. Addition of such supernatants to PMN caused stimulation of sod-inhibitable chemiluminescence and superoxide production. Studies with separated MNC showed that monocytes were the cellular source of the activity. Biochemically, this activity of the supernatants was due to a heat-labile glycoprotein with a MW of approx. 60KDa. This mediator, termed granulocyte chemiluminescence inducer (GCI), appears to be distinct from interleukin 1 (a and j?) and interferon (a and y). In conclusion we describe a novel monokine, granulocyte chemiluminescence inducer (GCI), which initiates granulocyte free radical production. This interaction of monocytes and granulocytes may also in vivo constitute a new and potent pathway leading to stimulation of free oxygen production by granulocytes.     

A Novel Interleukin Stimulating Free Radical Production by Granulocytes

Polymorphonuclear granulocytes (PMN) are potent producers of free oxygen-derived radicals. Since other granulocyte functions are affected by interleukins, we investigated whether free-radical production can be initiated by a similar mediator. For estimation of free radical production, SOD-inhibitable lucigenin-dependent chemiluminescence and SOD-inhibitable cytochrome C reduction were used. As a source of interleukins, serum-free 24 h culture supernatants of human mononuclear cells (MNC) stimulated with bacterial lipopolysaccharide were prepared. Addition of such supernatants to PMN caused stimulation of sod-inhibitable chemiluminescence and superoxide production. Studies with separated MNC showed that monocytes were the cellular source of the activity. Biochemically, this activity of the supernatants was due to a heat-labile glycoprotein with a MW of approx. 60KDa. This mediator, termed granulocyte chemiluminescence inducer (GCI), appears to be distinct from interleukin 1 (a and j?) and interferon (a and y). In conclusion we describe a novel monokine, granulocyte chemiluminescence inducer (GCI), which initiates granulocyte free radical production. This interaction of monocytes and granulocytes may also in vivo constitute a new and potent pathway leading to stimulation of free oxygen production by granulocytes.     

Free Radical Scavenging Properties of Sulfinpyrazone

Sulfinpyrazone, a potent uricosuric drug, was tested in vitro for its scavenging action against oxygen free radicals. In this study, sulfinpyrazone was able to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with IC 50 value of 29.82 &#119 g/ml compared to butylated hydroxytoluene (BHT, IC 50 value=20.15 &#119 g/ml) and Trolox (IC 50 value=16.01 &#119 g/ml). It was able to scavenge superoxide anion with IC 50 value of 27.72 &#119 g/ml compared to Trolox (IC 50 value=22.08 &#119 g/ml) and ascorbic acid (IC 50 value=14.65 &#119 g/ml). The hydroxyl radical scavenging activity of sulfinpyrazone is in a concentration-dependent fashion. In the range of concentrations used, sulfinpyrazone was not a scavenger toward H 2 O 2 . However, the intracellular H 2 O 2 -induced 2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence in HL-60 cells was significantly reduced by sulfinpyrazone during 30-60 min of incubation. Finally, phorbol-12-myristate-13-acetate induced-lucigenin chemiluminescence in whole blood was markedly inhibited by sulfinpyrazone. Our results suggest a new direction for the pharmacological actions of sulfinpyrazone in free radical scavenging properties.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Characterization of Free Radical Defense System in High Glucose Cultured HeLa-tat Cells: Consequences for Glucose-induced Cytotoxicity

HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1 has a decreased antioxidant potential. In this work, we used this model to investigate the effect of a high glucose level (20 mM) on the glucose induced cytotoxicity and on the antioxidant system. In comparison to cell culture under control medium, HeLa-wild cell cultured under 20 mM glucose did not exhibit necrosis or apoptosis, contrary to HeLa-tat cell presenting a significant increase in necrotic or apoptotic state. Moreover after 48 h culture under high glucose level the HeLa-tat proliferation rate was not higher than the one of HeLa-wild cells. In HeLa-wild cell high glucose level resulted in an induction of glutathione reductase activity in opposition to HeLa-tat cells where no change was observed. High glucose level resulted in 20% increase in GSSG/GSH ratio in HeLa-wild cells and 38% increase in HeLa-tat cells. Moreover, high glucose level resulted in a dramatic cytosolic thiol decrease and an important lipid peroxidation in HeLa-tat cells. No significant change of these two parameters was observed in HeLa-wild cells. In both cell lines, high glucose resulted in an increase of total SOD activity, as a consequence of the increase in Cu,Zn-SOD activity. High glucose did not result in an increase of Mn-SOD activity in both cell lines. As a consequence of tat tranfection Mn-SOD activity was 50% lower in HeLa-tat cells in comparison to HeLa-wild cells. This work emphasizes the importance of the antioxidant system in the glucose induced cytotoxicity.     

Free Radical Participation in Bacterial Bioluminescence

The metastable intermediate II produced on reaction of bacterial luciferase with reduced flavin mononucleotide and O₂, reacts with any of several stable free radicals to produce bioluminescence. The bioluminescence spectrum is very similar to that from the well-studied intermediate II and aldehyde reaction, and the number of photons per luciferase molecule reacted is at least 40% of the aldehyde reaction.     

Free radical scavenging properties of sulfinpyrazone

Sulfinpyrazone, a potent uricosuric drug, was tested in vitro for its scavenging action against oxygen free radicals. In this study, sulfinpyrazone was able to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with IC 50 value of 29.82 μg/ml compared to butylated hydroxytoluene (BHT, IC 50 value=20.15 μg/ml) and Trolox (IC 50 value=16.01 μg/ml). It was able to scavenge superoxide anion with IC 50 value of 27.72 μg/ml compared to Trolox (IC 50 value=22.08 μg/ml) and ascorbic acid (IC 50 value=14.65 μg/ml). The hydroxyl radical scavenging activity of sulfinpyrazone is in a concentration-dependent fashion. In the range of concentrations used, sulfinpyrazone was not a scavenger toward H 2 O 2 . However, the intracellular H 2 O 2 -induced 2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence in HL-60 cells was significantly reduced by sulfinpyrazone during 30-60 min of incubation. Finally, phorbol-12-myristate-13-acetate induced-lucigenin chemiluminescence in whole blood was markedly inhibited by sulfinpyrazone. Our results suggest a new direction for the pharmacological actions of sulfinpyrazone in free radical scavenging properties.     

自由基、营养、天然抗氧化剂与衰老

影响衰老的因素很多,其中主要有遗传基因、饮食营养、生活方式、运动多少、心理状态、医疗条件及环境因素等。其中饮食营养是非常重要的因素,而且是可以控制的因素。研究证明,营养不良和营养过剩都会影响健康和寿命,而健康和延长寿命最有效的方法是限食(限能)。最近,美国和英国的权威杂志《Science》和《Nature》发表研究文章表明,节食,以及一些自由基清除剂,如小分子多酚类物质白藜芦醇,可以启动长寿基因SIRI1,抑制肿瘤基因p53,阻断细胞凋亡,延缓衰老和延长寿命。早在1955年,Dr. Harman发表的“衰老的自由基理论”就提出,体内产生过多自由基是引起衰老的重要因素,保持体内自由基和抗氧化剂的平衡可以延缓衰老。我们的实验还证明,天然抗氧化剂茶多酚可延长果蝇的寿命,使果蝇匀浆中的超氧化物歧化酶(superoxide dismutase,SOD)活性增加,脂质过氧化水平降低,改善高脂引起的果蝇寿命缩短、SOD活性降低以及脂质过氧化水平的升高。作者最近的实验还证明,茶多酚可以防止氧化应激引起的线虫寿命的缩短,还可以预防和治疗6-OHDA引起的大鼠帕金森氏综合症,山楂黄酮可以预防和治疗蒙古沙鼠缺血引起的中风,大豆异黄酮和尼古丁可以预防和治疗转基因线虫和小鼠老年痴呆症。饮食营养和天然抗氧化剂研究的进展将对人类健康和延缓衰老,提供新的线索并做出重大贡献。     

运动对人体自由基代谢的影响

自由基代谢普遍存在于机体各组织。正常情况下,人体自由基的产生与清除处于平衡状态。而人体自由基产生过多或机体清除自由基能力下降将给机体带来损伤。本文采用文献资料法,阐述了自由基的类型、功能、自由基产生的机制以及抗氧化系统的种类等基本理论。通过对力竭运动、耐力运动和无氧运动3种不同形式的运动对自由基代谢及抗氧化酶活性的研究得出结论:不同的运动形式及负荷方式对体内自由基代谢及抗氧化酶活性将产生不同影响;运动对自由基代谢影响具有器官与组织差异性。这些研究成果为科学指导运动训练和健身活动、快速有效的消除运动性疲劳并增强运动能力提供重要的科学理论依据。     

The Action of Nine Chelators on Iron-Dependent Radical Damage

Nine iron chelators were tested in five systems for their effects on radical-generation and conversion at chelator: iron molar ratios from 0.1 to 10. Stimulatory actions might distinguish toxic from safer chelators. Radical-generating reactions which represent different aspects of iron (ferrous and ferric) availability were studied: a) the reaction with hydrogen peroxide to hydroxylate benzoate; b) the oxidation of ascorbate; c) the reaction with hydrogen peroxide to fragment proteins; d) the reaction with hydrogen peroxide to permit amplified chemiluminescence; and e) the induction of peroxidation of mitochondrial membrane lipids. The compounds used were HBED, CP130, Desferal, EDTA, pyridine-hydrazone (CGP 43'902B), Ferrozine, CP 94 (CGP 46'700), LI (CGP 37 391) and rhodotorulic acid (CGP 45 274). Only the hexadentate compounds HBED, CP130 and Desferal were uniformly inhibitory (“protective”). The protective compounds were also apparently more stable during radical fluxes than the other chelators.     

抗氧化剂与自由基清除剂对硒性白内障形成的影响

本文观察了8种化合物(抗氧化剂及自由基清除剂)对大鼠亚硒酸钠性白内障的影响。实验分为正常对照组,亚硒酸钠组及药物对抗组。亚硒酸钠组系给13日龄大鼠皮下注射亚硒酸钠(6μmoles/kg体重),间日一次,逐次递增1μmole/kg体重,连续5次,药物对抗组则同时腹腔注射抗氧化剂或自由基清除剂,每日观察并记录白内障的发生频率及程度,实验表明,一些抗氧化剂及自由基清除剂能够有效的对抗亚硒酸钠性白内障的发生发展,其中AC1、AC3及AC3的效果尤为明显。本文的结果为探讨白内障形成机理及防治提供了实验依据。     

活性氧自由基诱发的胆红素聚合、聚集及稳定自由基的形成

 为了深入了解胆红色素类结石形成的触发机制,我们研究了活性氧自由基与胆红素的作用。结果表明:·O_2和·OH均可诱发胆红素聚合和聚集,井使胆红素转变成稳定的自由基,从而引起脂质过氧化和透明质酸分子降解。经自由基处理的胆红素溶解度减小,粒度分布趋向颗粒变大.根据以上事实,重点讨论了自由基触发的胆色素类结石的致病过程。     

Free Radical Metabolism of Alcohols by Rat Liver Microsomes

By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN.

The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP⁺ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P₄₅₀-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P₄₅₀ where they react with alkyl alcohol molecules.

The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.     

Free radicals in wheat flour change during storage in air and are influenced by the presence of ozone during the growing season

Electron paramagnetic resonance (EPR) spectra of wheat flour show components from Fe(III), Mn(II) and free radicals (FR). The metal signals were higher in the samples from the stressed plants, and reflected the higher total levels of these elements determined analytically. They remained essentially constant throughout the experiment, but the FR signal increased progressively with time over a period of 4-6 months after milling, after which it reached a maximum. The rate of increase in the FR signal during this period was considerably higher in the flour from plants that had been exposed to elevated ozone levels.     

Free Radicals in Wheat Flour Change During Storage in Air and are Influenced by the Presence of Ozone During the Growing Season

Electron paramagnetic resonance (EPR) spectra of wheat flour show components from Fe(III), Mn(II) and free radicals (FR). The metal signals were higher in the samples from the stressed plants, and reflected the higher total levels of these elements determined analytically. They remained essentially constant throughout the experiment, but the FR signal increased progressively with time over a period of 4-6 months after milling, after which it reached a maximum. The rate of increase in the FR signal during this period was considerably higher in the flour from plants that had been exposed to elevated ozone levels.     

Free Radical Scavenging by Myocardial Fatty Acid Binding Protein

Recent investigations have indicated the presence of a fatty acid binding protein (FABP) in mammalian heart. This protein binds free fatty acids and their esters with high affinity, however, its physiological role remains unknown. Since FABP constitutes a significant amount of cystolic protein, it is likely that it would be a target for free radical attack. To test this hypothesis, FABP was examined for scavenging against free radicals such as the superoxide anion (O^?₂,). hydroxyl radical (OH') and hypochlorite radical (OCl') which may be present in an ischemic reperfused heart. Our results suggest that FABP scavenges O^?₂, OH' and OCl' as indicated by the FABP inhibition of O^?₂-dependent reduction of cytochrome c, OH'-dependent hydroxybenzoic acid formation and OCl'-mediated chemiluminescence response. FABP was found to be a more potent scavenger of these free radicals compared to bovine serum albumin. Furthermore, FABP was more effective in scavenging OH' than O^?₂, and inhibited OH' mediated lipid peroxidation process. These results indicate that FABP can scavenge free radicals which may be present in an ischemic/reperfused heart and, thus, may play a significant physiological role in the heart during ischemia and reperfusion.     

胆红素自由基对大鼠肝细胞损伤作用的研究

用不同自由基源处理的胆红素与鼠肝细胞相互作用,结果表明：胆红素自由基可引起鼠肝细胞脂质过氧化,使总谷胱甘肽及ＧＳＳＧ水平明显下降,细胞损伤后,乳酸脱氢酶外漏,上述结果与自由基浓度正相关,而与其种类无关,牛血清白蛋白有明显的抑制作用。差示光谱表明：胆红素可能与细胞色素Ｐ－４５０快速形成络合物。根据以上结果,重点讨论了胆红素自由基对肝细胞损伤的化学本质及其与胆结石形成的关系。     

尼日利亚螫毛果(Cnestis ferruginca)中一种具有清除自由基的活性化合物的分离鉴定

用二苯基苦基苯肼自由基薄层实验法（DPPH-TLC-ASSAY）,从尼日利亚螫毛果（Cnestis ferruginca)植物的甲醇提取物中发现了一种具有很强的清除自由基活性的物质;用柱色谱及反相制备柱色谱（RP-LPPLC）,制备出了该活性物质的纯品;用质谱（HR ESI-MS）,核磁共振氢谱（^1H0,碳谱（^13C,DEPT)。紫外光谱及化学方法对该物质进行了结构鉴定,鉴定结果为对苯酚基-6-O-反式咖啡酸基-β-D-吡喃葡萄糖甙。