Activation of chemiluminescence accompanying lipid peroxidation in monolayer liposome suspension

In the presence of rhodamine J the chemiluminescence intensity of monolayer liposomes induced by ferrous ions is increased by three orders. There is no accumulation of malonic dialdehyde. It is suggested that chemiluminescence activation is related to rhodamine J interaction with the products of lipid peroxidation whose molecules are in the excited state.     

Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.     

Light generation with Fenton's reagent. Its relationship to granulocyte chemiluminescence.

A simple chemical system consisting of FeSO4 and H2O2 (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H2O2 was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH.) and singlet oxygen (1 deltagO2) quenchers suppressed chemiluminescence of the FeSO4 + tryptophan + H2O2 system; and, deuterium oxide (2H2O) enhanced chemiluminescence of both FeSO4 reactions. These observations suggest that a radical chain reaction involving both OH. and 1 deltag O2 is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H2O2. Since iron and H2O2 are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

Bleomycin-metal interaction: ferrous iron-initiated chemiluminescence

Chemiluminescence often accompanies the spontaneous degradation of intermediates in an electronically excited state. The interaction of iron with bleomycin results in the activation of bleomycin to a reactive intermediate which can alter DNA or undergo self-inactivation. This report demonstrates that the interaction of ferrous iron with bleomycin results in chemiluminescence, that this response is iron-specific and that the presence of DNA prevents the generation of chemiluminescence. These observations suggest that the activated bleomycin intermediate may be in an electronically excited state.     

Enhanced chemiluminescence for trazodone trace analysis based on acidic permanganate oxidation in concurrent presence of rhodamine 6G

A new sensitized chemiluminescence method by acidic permanganate oxidation was developed for the sensitive determination of trazodone. A fluorescent dye as used rhodamine 6G to increase a chemiluminescence intensity. Under optimum conditions, the liner range of the calibration curve was obtained for 1–5000 nmol/L. The limit of detection was calculated from 3σ of a blank was 0.23 nmol/L. The coexistent ions and substances had no interference with the chemiluminescence measurement. The chemiluminescence spectra were measured to elucidate a possible mechanism for the system. The present method was satisfactorily used in the determination of the drugs in pharmaceutical samples and animal serums.     

Superoxide anion production by the autoxidation of cytochrome P450cam

Chemiluminescence occurs on autoxidation of oxygenated ferrous cytochrome P450_cam and is abolished by reagents that scavenge free radicals, by superoxide dismutase and singlet oxygen quenchers. We attribute the chemiluminescence to the decay of an excited singlet oxygen which arises from a superoxide anion radical precursor.     

Luminol peroxidation catalyzed by human isoferritins

The role of ferritin in catalyzing the oxidation of luminol with the production of chemiluminescence was investigated. The effect of pH was compared to its effect on K3Fe(CN)6-catalyzed oxidation and different pH optima were recorded for the two catalysts. The ferrous iron chelator, bipyridyl, enhanced the production of chemiluminescence catalyzed by FeSO4 and ferritin but had little effect on the K3Fe(CN)6-catalyzed reaction. Desferal reduced the level of chemiluminescence in the presence of FeSO4 and ferritin but was a much more effective inhibitor of chemiluminescence catalyzed by K3Fe(CN)6. The hydroxyl radical scavenger, mannitol, had little effect upon light production whereas superoxide dismutase inhibited light production. The addition of antihuman spleen ferritin completely inhibited activity. The catalytic activity of both H and L rich ferritins was affected by iron content. Activity increased until the Fe/protein ratio reached 0.04 micrograms Fe/micrograms protein and then decreased with increasing iron content. Thus activity is controlled by the iron content of the molecule and influenced by its subunit composition as is the uptake of iron into ferritin. These findings suggest that ferroxidation by ferritin is associated with the ability to generate radicals of the nitrogenous base luminol with the production of chemiluminescence. Although activity is greatest at alkaline pH there is significant activity at pH 7.4. Ferritin therefore may be able to generate free radical reactions in vivo with the acidic isoferritin being most active.     

Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.     

Chemiluminescence study on the peroxidation of linoleic acid initiated by the reaction of ferrous iron with hydrogen peroxide

Linoleic acid was used as a model system to study lipid peroxidation initiated by the reaction of ferrous iron with hydrogen peroxide. Low-level chemiluminescence of the peroxidation was measured with a high-sensitivity single-photon counter. It was found that the luminescence primarily comes from the dimol reaction of singlet oxygen and that the peak intensity of emission is a quadratic function of the concentration of either Fe2+ or H2O2, provided that the other Fenton reagent is in great excess. Under the same conditions, analysis on reaction kinetics shows a linear relationship between the maximal level of the initiator formed by the Fenton reaction and the initial concentration of Fe2+ or H2O2. This implies that the peak intensity of the chemiluminescence may be a good index of the maximal level of the initiator.     

Kinetics of the interaction of ferrous oxide with oxidized lipids and possibility of chiluminescent determination of hydroperoxides]

The theory shows that for lipids containing hydroperoxides of unsaturated fatty acids there exists a verge concentration of Fe2+: at concentrations of Fe2+ lower than the verge one the peroxidation process develops autocatalytically and at higher concentrations it looks pulseshaped. In the second case the amplitude of pulse concentration of peroxide radicals and the square root from the maximum value of chemiluminescence intensity are proportional to the concentration of hydroperoxides present in the system at the time of ferrous ions injection. The former two values do not depend on the concentration of inoxidized fatty acids. In the experiments of chemiluminescence of partially oxidized linoleic acid and lecithin in methanol-benzene solutions (1 : 2) a perfect correlation between the experimental data and theoretical conclusions was revealed.     

Cytochrome P450 and steroid hydroxylase activity in mouse olfactory and vomeronasal mucosa

Neopterin and 7,8-dihydroneopterin, two compounds which are secreted by activated macrophages, have been shown to interfere with radicals generated by cellular and certain chemical systems. Reduced pterins were reported to scavenge whereas aromatic pterins promoted or reduced radical mediated reactions or had no effect. However, recently it was found that high concentrations of 7, 8-dihydroneopterin enhanced luminol dependent chemiluminescence and T-cell apoptosis, suggesting an enhancement of free radical formation. In this study hydroxylation of salicylic acid was used for detection of hydroxyl radicals. It is shown that in solutions of 7,8-dihydroneopterin hydroxyl radicals were formed in the absence of any radical source. The presence of EDTA chelated iron enhanced hydroxyl radical formation. Whereas the addition of iron accelerated the hydroxylation reaction, 7,8-dihydroneopterin was responsible for the amount of hydroxylation products. In the presence of superoxide dismutase or catalase, as well as by helium purging, hydroxylation was inhibited. Our data suggest that in solutions of 7, 8-dihydroneopterin superoxide radicals are generated which are converted to hydroxyl radicals by Fenton or Haber-Weiss type reactions. While superoxide might be generated during autoxidation of ferrous iron, dihydroneopterin seems to be involved in regeneration of ferrous iron from the ferric form.     

Reduction of ferric iron by L-lactate and DL-glycerol-3-phosphate in membrane preparations from Staphylococcus aureus and interactions with the nitrate reductase system.

Membrane fractions with L-lactate dehydrogenase, sn-glycerol-3-phosphate (G3P) dehydrogenase, and nitrate reductase activities were prepared from Staphylococcus aureus wild-type and hem mutant strains. These preparations reduced ferric to ferrous iron with L-lactate or G3P as the source of reductant, using ferrozine to trap the ferrous iron. Reduction of ferric iron was insensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) with either L-lactate or G3P as reductant, but oxalate and dicumarol inhibited reduction with L-lactate as substrate. The membranes had L-lactate- and G3P-nitrate reductase activities, which were inhibited by azide and by HQNO. Reduction of ferric iron under anaerobic conditions was inhibited by nitrate with preparations from the wild-type strain. This effect of nitrate was abolished by blocking electron transport to the nitrate reductase system with azide or HQNO. Nitrate did not inhibit reduction of ferric iron in heme-depleted membranes from the hem mutant unless hemin was added to restore L-lactate- and G3P-nitrate reductase activity. We conclude that reduced components of the electron transport chain that precede cytochrome b serve as the source of reductant for ferric iron and that these components are oxidized preferentially by a functional nitrate reductase system.     

Flow-injection-chemiluminescence method for the determination of penicillin G potassium.

The degradation product of penicillin G potassium can react with potassium permanganate in acidic medium and produce chemiluminescence, which is greatly enhanced by formaldehyde. The optimum conditions for this chemiluminescent reaction were studied in detail using a flow-injection system. The experiments indicated that under optimum conditions, the chemiluminescence intensity was linearly related to the concentration of penicillin G potassium within the range 1.0 x 10(-7)-1.0 x 10(-5) g/mL, with a detection limit (3sigma) of 7 x 10(-8) g/mL. The relative standard deviation was 1.0% for 4.0 x 10(-7) g/mL penicillin G potassium solution (n = 11). This method has the advantages of simple operation, fast response and high sensitivity. The method was successfully applied to the analysis of penicillin G potassium in raw medicines.     

ADP—Fe~（2＋）启动脂质过氧化的化学发光研究

以化学发光法和雨二醛测定法为实验手段研究了ＡＤＰ—Ｆｅ２＋启动的脂质过氧化反应以及几种金属离子对该反应的影响、结果表明,当反应体系中只有ＡＤＰ－Ｆｅ２＋存在时,通过化学发光法和丙二醛测定法都可以现察到脂质过氧化反应在０—５分钟内有一“潜伏期”存在,同时在微粒体浓度保持不变的条件下,增大二价铁离子的浓度,则脂质过氧化的水平增强。如果反应体系中同时加入ＡＤＰ—Ｆｅ２＋与ＡＤＰ—Ｆｅ３＋,则反应起始时的“潜伏期”消失。当ＡＤＰ—Ｆｅ３＋、ＡＤＰ—Ａｌ３＋和ＡＤＰ－Ｐｂ２＋单独存在时本身并不启动脂质过氧化,但对ＡＤＰ－Ｆｅ２＋启动的脂质过氧化都有增强作用,并且三价铁离子对鼠肝微粒体脂质过氧化的增强作用随着ＡＤＰ－Ｆｅ２＋浓度的增大而逐渐加强。将化学发光法与雨二醛测定法的结果加以比较,发现微粒体本身对它的脂质过氧化反应过程中的发光具有猝灭作用。     

Generation of toxic phospholipid(s) during oxyhemoglobin-induced peroxidation of phosphatidylcholines

When egg yolk diacylglycerophosphocholine (PC) liposomes were incubated with human oxyhemoglobin, peroxidation of liposomal lipid was induced, as monitored by an increase of thiobarbituric acid (TBA)-reactive substances, an increase of lipid hydroperoxides and the generation of chemiluminescence in the presence of luminol. During the reaction, cytotoxic substance(s), which induced shedding of acetylcholinesterase-enriched vesicles from human erythrocytes, were produced. Formation of TBA-reactive substances and lipid hydroperoxides preceded generation of chemiluminescence, conversion of oxyhemoglobin to methemoglobin and production of the toxic substances. Either superoxide dismutase or catalase could suppress generation of chemiluminescence, but not other events. Methemoglobin or ferrous ion plus ascorbate could induce peroxidation of the liposomes without production of the cytotoxic substance(s). Synthetic PCs containing both saturated and polyunsaturated fatty acyl chains caused the production of cytotoxic products which induced shedding of vesicles from erythrocytes, whereas those containing only polyunsaturated fatty acyl chains did not, suggesting that the molecular species which can produce cytotoxic products may be phospholipids containing both saturated and polyunsaturated fatty acids. The mechanism of oxyhemoglobin-induced peroxidation of lipids will be also discussed.     

Features of intermediary steps around the 688-nm substance in the heme oxygenase reaction

The degradation of protoheme in the heme oxygenase reaction involves three oxidation steps: from protoheme to hydroxyheme, from hydroxyheme to a 688-nm substance, a protein-bound intermediate, and from the 688-nm substance to a biliverdin-iron complex. The 688-nm substance has a ferrous iron and it readily binds carbon monoxide to form a CO-complex, called the 638-nm substance (Yoshida, T., Noguchi, M., & Kikuchi, G. (1980) J. Biochem. 88, 557-563). The ferric 688-nm substance was prepared from the 638-nm substance by the addition of potassium ferricyanide together with aspiration to eliminate CO. The ferric 688-nm substance did not show any distinct absorption maximum in the red region of the absorption spectrum. The ferric 688-nm substance was readily reduced on the addition of the NADPH-cytochrome P-450 reductase system, but the ferric 688-nm substance could also be reduced spontaneously though at a very low rate. The ferrous 688-nm substance free from excess reducing agents was prepared by passing the 638-nm substance through a column of Sephadex G-25. The ferrous 688-nm substance was degraded to a biliverdin-iron complex much more rapidly in the presence of the NADPH-cytochrome P-450 reductase system than in its absence, indicating that a reducing equivalent is essential for the initiation of heme degradation even when starting from the ferrous 688-nm substance. Cyanide was found to bind to the ferrous 688-nm substance to form a stable compound; the cyanide compound formed could revert to neither the ferrous 688-nm substance nor the 638-nm substance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two phases in development of chemiluminescence during lipid peroxidation induced by Fe2+ ions

After cessation of the stirring of reagents in the egg yolk lipoprotein suspension where lipid peroxidation had been initiated by ferrous ions a drop of chemiluminescence (CL) was observed after a lag period, the duration of which increased in the course of stationary-state CL development. The CL intensity was but slightly dependent on oxygen pressure in the gas phase above incubation mixture within oxygen partial pressure limits 760-40 mm Hg. These data suggest that the CL quantum yield is growing during lipid peroxidation development as a result of the formation in the system of some compounds amplifying the CL intensity and/or development of a new CL reaction whose rate depends purely on oxygen and whose photon emitters are different.     

Formation of superoxide anion during ferrous ion-induced decomposition of linoleic acid hydroperoxide under aerobic conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.     

Peroxide induced chemiluminescence in an in vitro proline hydroxylation system.

This communication describes a hydrogen peroxide (HOOH) induced chemiluminescence (CL) in an in vitro aromatic (proline) hydroxylation system. The reactive components of the system are ascorbic (AA), ethylene diamine tetraacetic disodium salt (EDTA), ferrous sulfate, and HOOH. The CL is (1) nearly dissipated within three minutes, (2) enhanced and/or sustained by proline and polylysine to a greater degree than by alanine, (3) partially inhibited by a,a' dipyridyl, EDTA, and ethanol, (4) most dependent upon the presence of Fe2+, AA, and HOOH. The in vitro proline hydroxylation system is more effective than ground state oxygen in terms of the CL produced and the percent of hydroxyproline formed.     

Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification

Human polymorphonuclear neutrophilic leukocytes (PMNLs) phagocytosed fluorescein-isothiocyanate (FITC)-labelled Staphylococcus aureus. Free bacteria, phagocytes, and nonphagocytes were discriminated and quantified by flow cytometry (FCM). The relative fluorescence of phagocyte-associated and free bacteria (Nf:N) was calculated by dividing the mean phagocyte fluorescence by that of the free bacteria and the number of phagocytosed bacteria. Bactericidal capacity and chemiluminescence were measured by standard methods. The red-to-green fluorescence ratio of acridine orange-stained PMNLs (R/G) was measured by FCM. Degradation of bacteria was monitored by the reduction in FITC and ethidiumbromide fluorescence of bacteria liberated from the phagocytes. Bacterial FITC fluorescence was pH dependent. Nf:N was 0.5 to 0.7. Using a standard curve for the interrelationship between bacterial fluorescence and pH, phagosomal pH was 5.0-5.5. Phagocytes, kept at 4 degrees C for 24 h had Nf:N approximately 1, did not degrade bacteria, but killed them and emitted chemiluminescence. NH4Cl increased phagocyte fluorescence by 27% and decreased R/G by 50%. Cyanide and azide did not affect Nf:N. Nf:N of phagocytes from a patient with chronic granulomatous disease was 32% below, and R/G was 32% higher than the controls. Acidification of the phagosomes seems to be related to discharge of PMNL granule contents and independent of the respiratory burst.