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[Chemiluminescence in the peroxidase oxidation of luminol with hydrogen peroxide in various media].

The effects of culture media of various compositions on chemiluminescence developing in peroxidase oxidation of luminol with hydrogen peroxide were under study. The findings evidence that the presence of carbonate and bicarbonate ions in the medium results in a two-staged chemiluminescence kinetics and in more intensive chemiluminescence in the peroxidase-luminol-hydrogen peroxide system. This fact has brought the authors to a conclusion that carbonate and bicarbonate-containing media are more effective for the detection of low peroxidase concentrations by the chemiluminescence technique.

Hydrogen Peroxide↗

[The efficiency of the action of alpha-tocopherol and its homologs on luminol-dependent chemiluminescence induced by (Fe2+ + NADP.H) and (Fe2+ + ascorbate) systems in rat liver microsomes].

The effects of alpha-tocopherol (C16) and its homologues with different chain length (6-hydroxychromanes-C1, C6, C11) on lipid peroxidation induced luminol-dependent chemiluminescence in rat liver microsomal suspensions were studied. It was shown that C1, C6 and C11 inhibited the (Fe(2+) + ascorbate)-and (Fe(2+) + NADP.H)-induced chemiluminescence. The inhibitory effect was decreased in the order: C1 C6 C11, C16 was not influenced chemiluminescence. The possible reason underlying these differences was discussed: different efficiency of interaction of C16 and its homologues with hydroxyl and superoxide radicals, which initiate the luminol-dependent chemiluminescence. It was concluded that C16 (in concentration below 0.5 mM) was not interacted with hydroxyl and superoxide free radicals, generated in microsomal suspensions under (Fe(2+) + ascorbate)- and (Fe(2+) + NADP.H)-dependent lipid peroxidation.

Animals↗

Effects of luminol on the subsequent analysis of bloodstains.

The effects of luminol upon additional presumptive chemical tests, subsequent confirmatory blood tests, species determination by immunoelectrophoresis, ABO typing by absorption elution, and genetic marker analysis by multienzyme system electrophoresis were examined. Results indicate that luminol does not affect additional presumptive chemical tests, confirmatory tests, species determination, or ABO typing, but does affect certain genetic marker systems.

ABO Blood-Group System↗

[Luminol and lucigenin-dependent chemiluminescence in 6-hydroxydopamine auto-oxidation].

It was shown by the methods of luminol- and lucigenin-dependent chemiluminescence that 6-hydroxydopamine 6-ONDA autooxidation is accompanied by generation of superoxide anion-radical which can further react with next 6-ONDA molecule. This fact is based on the observations that intensities of luminol- and lucigenin-dependent chemiluminescence during 6-ONDA autooxidation was increased in aprotonic aqueous solutions (with the decreasing of pH) and was extremely depended on 6-ONDA concentration. The obtained data allows to suggest that one of the possible mechanisms of 6-ONDA neurotoxic action includes the generation of superoxide, whose dismutation to hydrogen peroxide in the presence of transient valency ions gives rise to HO.

Acridines↗

[The effect of exogenous peroxidase on the intensity of luminol-dependent macrophage chemiluminescence].

The influence of exogenous horse-radish peroxidase on the capacity of mouse peritoneal macrophages to luminol-dependent chemiluminescence induced by zymosan was investigated. It was revealed that peroxidase (1-50 mg/ml) increased the chemiluminescence in a dose-dependent manner. The maximum increase of the response (4-6 times) was obtained with the enzyme concentration being 10 mg/ml. It is found that peroxidase acts as a co-oxidant of the peroxide-dependent extracellular luminol oxidation. The including of the enzyme into macrophages makes it possible to register the intracellular chemiluminescence.

Animals↗

Effects of extracellular pH on neutrophil superoxide anion production, and chemiluminescence augmented with luminol, lucigenin or DMNH.

When neutrophils were stimulated with f-met-leu-phe (fMLP) or phorbol myristate acetate (PMA) chemiluminescence (CL) augmented with luminol, lucigenin or 7-dimethylamino-naphthalene-1,2-dicarbonic acid hydrazide (DMNH) showed a strong dependence on the pH of the medium. CL increased in an exponential fashion when pH was increased from 6.5-9.2. All three luminescent intermediaries showed this pH-dependency but luminol was affected most. Unstimulated CL showed a similar increase with pH. Also neutrophil superoxide anion production (in the cytochrome C reduction assay) increased with pH, but not to the same extent as CL. CL induced by xanthine-oxidase plus hypoxanthine increased likewise with pH, and that increase was more pronounced than what was observed from the same reaction in the cytochrome C assay. The lucigenin-amplified neutrophil CL showed the best correlation to the superoxide production from neutrophils. Thus, several effects underlie the variation of CL with pH but the pH dependency of the luminescent intermediaries contribute substantially, which must be considered.

Acridines↗

Effect of Leishmania major on luminol-dependent chemiluminescence of human whole blood phagocytosis.

The aim of this study is to examine the effect of L. major on human whole blood phagocytosis. The phagocytosis was activated by phorbol myristate acetate (PMA) or opsonized zymosan. Various types of Leishmania antigens were tested on luminol-dependent chemiluminescence. In PMA-activated whole blood phagocytosis group, promastigotes and infected medium significantly (P less than 0.001) depressed the maximum peak of chemiluminescence to 5.77 +/- 1.6 and 5.65 +/- 0.45 mV respectively, compared to 9.85 +/- 22 in control group. In a similar fashion a significant reduction of the area under the curve was noted. On the other hand, opsonized zymosan-activated phagocytosis was significantly (P less than 0.025) inhibited in the promastigote treated group only. The maximum peak response was 4.68 +/- 1.5 and 10.8 +/- 0.46 mV in promastigote and control groups, respectively. The above data indicated; a) an inhibition of whole blood phagocytosis induced by Leishmania and b) a marked reduction of the oxygen-reactive metabolites radicals generated by whole blood phagocyte cells and measured by luminol-dependent chemiluminescence suggesting the important role of oxygen free radicals in the pathogenesis of leishmaniasis.

Animals↗

Chemiluminescence of luminol caused by interaction of cytochrome P-450 and cytochrome C with cumene hydroperoxide: comparative studies.

A comparative study of the interaction of cumene hydroperoxide with cytochrome P-450LM2 and with cytochrome C has been undertaken using the chemiluminescence method in the presence of luminol. Considerable differences in the mechanisms of action of both hemoproteins have been revealed with various inhibitors of radical stages, i.e. superoxide dismutase, mannitol, sodium azide, and alpha-tocopherol. It is shown that molecular oxygen participates in the process of hemoprotein-catalyzed hydroperoxide oxidation of luminol.

Animals↗

Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored enzyme immunoassays.

Certain phenol derivatives, including p-iodophenol and p-phenylphenol, enhance light emission from the horseradish peroxidase-catalyzed oxidation of cyclic diacyl hydrazides such as luminol. The light emission decays slowly (glowing for several minutes) and its intensity may be greater than 1000-fold that of the unenhanced reaction. The enhanced system enables rapid, sensitive assay of peroxidase conjugates. We describe its application in immunoassays for human choriogonadotropin, digoxin, and factor VIII-related antigen. Luminescent quantification of peroxidase labels has been directly incorporated into immunoassays based on beads, tubes, or microtiter plates, used in conjunction with photodetectors such as photomultiplier tubes or instant photographic film. Enhancement with phenol derivatives exceeds that achieved with 6-hydroxybenzothiazole derivatives and depends on pH and enhancer concentration. Emission spectra of phenol-enhanced and unenhanced reactions are remarkably similar, suggesting that the enhancers do not act as more efficient emitters but exert their action earlier in the complex reaction between peroxidase, oxidant, and luminol.

Antigens↗

Further evaluation of luminol-enhanced luminescence in the diagnosis of disorders of leukocyte oxidative metabolism: role of myeloperoxidase.

Chemiluminescence can be used to identify defects in the oxidative metabolism of granulocytes. This procedure has recently been adopted for use with microliter quantities of whole blood, appropriate for prenatal or neonatal study. Although the contribution of myeloperoxidase to the chemiluminescence assay has been noted, the possible diagnostic confusion between chronic granulomatous disease of childhood (which is rare and severe) and myeloperoxidase deficiency (which is common and of little clinical consequence) has not been stressed. We report a father and his infant daughter whose cells emitted no light in the luminol-enhanced luminescence assay; both patients are totally peroxidase deficient. These results emphasize the hereditary nature of myeloperoxidase deficiency, and the possibility for erroneous diagnosis of chronic granulomatous disease of childhood based on the luminol-enhanced luminescence test.

Adult↗

[Active oxygen and anti-inflammatory drug: measurement by luminol dependent chemiluminescence].

The active oxygen produced from stimulated phagocytic cells emits luminol-dependent chemiluminescence (CL) upon reaction with luminol. So the active oxygen was measured by using the CL and the results of this were compared with those by the LDH-NADH method. Moreover, effects of nonsteroidal anti-inflammatory drugs (NSAID) on the generation of active oxygen were studied by both methods. Rat peritoneal and pleural exudated cells (PEEC and PLEC) emitted strong CL on incubation with zymosan, but that from rat whole blood cells was very weak. The effects of superoxide dismutase, catalase, NaN3 and L-ascorbic acid on the generation of active oxygen from rat phagocytic cells were different between CL and LDH-NADH methods. These discrepancies seem to be due to the different kinds of active oxygen that can be measured by both methods. Except for BW-755C, most of the NSAID had only a slight inhibitory effect on the generation of active oxygen measured by both methods, and the ex vivo effect was the same as that observed in vitro. It may be considered that NSAID decrease the phagocytic function of cells by non-specifically stabilizing the biological membrane and inhibit slightly the generation of active oxygen from phagocytes. On the other hand, the CL method could be performed not only in PEEC and PLEC, but also in whole blood cells. From these results, it was suggested that CL measurement can be used as a simple and valuable method for the detection of all types of active oxygen including superoxide anion radical and its metabolites and for testing cellular functions and drug actions on them.

Animals↗

The effect of culture media on the production and measurement of luminol-dependent chemiluminescence.

The effect of various media constituents on the production and measurement of luminol-dependent chemiluminescence by neutrophils stimulated by opsonized zymosan was studied. Glucose, calcium ions and magnesium ions were found to be necessary for optimal chemiluminescence although high concentrations of glucose had a detrimental effect. For our luminol-dependent system, a pH of 8.5 give maximal readings. The use of simple balanced salt solutions gave higher chemiluminescence responses than did more complex media with added protein, amino acids, and vitamins. However, the latter were important in the maintenance of optimal cell function.

Amino Acids↗

The measurement of opsonic and phagocytic function by Luminol-dependent chemiluminescence.

Luminol-dependent chemiluminescence produced by peripheral blood phagocytic cells was measured using a light detecting instrument, the Luminometer 1250 (LKB Wallac). Although less sensitive than liquid scintillation counters, the Luminometer can measure chemiluminescence effectively and detect experimentally induced and clinical variations using small numbers of cells. It is small, inexpensive, simple to use and allows temperature within the reaction vial to be controlled accurately. Light emission can be recorded either graphically or by an intergrated digital printout. Luminol-dependent chemiluminescence is influenced by the presence of contaminating red blood cells and by composition of the medium, particularly presence of phenol red. HEPES buffer and foetal calf serum. Whereas clinical comparisons of opsonic activity are easily performed, comparisons of cellular activity require a high and uniform standard of cell preparation, characterization and maintenance.

Erythrocytes↗

[The effect of sodium butyrate and luminol on reciprocal exchanges and gene conversion during extrachromosomal DNA recombination in cultured animal cells].

For determination of the extrachromosomal homologous DNA recombination efficiency, somatic cells of various lines have been transformed with plasmid DNAs which contain copies of neo-gene with non-overlapping deletions. Reconstruction of the neo-gene functional activity, which imparts a geneticin-resistant phenotype to cells, indicates that recombination has occurred. If dP1 and dR copies of the neo-gene are used, a single (reciprocal) exchange is necessary for reconstruction of the neo-gene by homologous DNA recombination, but a double exchange (gene conversion) is needed in the case of dP1 and dS copies. It is shown that in human cells of line HeLa and in mouse cells of line LMtk-, in contrast to the Chinese hamster cells of line A238, the frequency of double exchanges is comparable to that of the single DNA exchanges which is an evidence of participation in DNA recombination of gene conversion in addition to a single exchange mechanism. The treatment of cells with sodium butyrate and luminol exerts different influences on the rate of the single DNA exchanges and on that of gene conversion (double exchanges) in cells of lines LMtk- and HeLa, respectively. Essential distinctions in correlation of the single DNA exchange frequency and the gene conversion frequency in cells of the studied lines, and the possibility to distinguish between these mechanisms of recombination, under the treatment by sodium butyrate and luminol, may suggest the existence of two mechanisms of homologous DNA recombination in cultured animal cells, which function independently of one another, to a considerable extent.

Animals↗

Effect of phenothiazines on activated macrophage-induced luminol-dependent chemiluminescence.

The inhibitory effect of some phenothiazine neuroleptics (chlorpromazine, levomepromazine, thioridazine, promethazine and trifluoperazine) on the ability of rat peritoneal macrophages to produce O2- during phagocytosis was investigated. The superoxide radical release was estimated by measuring the luminol-dependent chemiluminescence (CL). The effect of drugs was studied in the concentration range of 0.1-100 mumol/l. Additional experiments to determine the ability of the drugs to scavenge O2- were carried out. They included measuring the effect of phenothiazines on the luminol-dependent CL in systems with enzymatically (xanthine-xanthine oxidase) and non-enzymatically (KO2) generated O2-. The ability of phenothiazines to scavenge O2- was additionally tested by a "non-luminescence" method in which the superoxide concentration was determined spectrophotometrically by the reduction of nitro blue tetrazolium to formazan. All drugs tested decreased significantly CL of stimulated macrophages at concentrations greater than 1 mumol/l. The C50 values were between 0.45 and 1.74 mumol/l. Also phenothiazines were found to act as scavengers of O2-. However, this effect occurred at significantly higher drug concentrations. The C50 values for 50% scavenging of O2- in systems with different sources of O2- were in the concentration range of 5-160 mumol/l. These results suggested that phenothiazines predominantly affected the ability of macrophages to produce O2- during phagocytosis. The findings may provide some insight into the untoward effects of the drugs tested.

Animals↗

Increased release of free oxygen radicals by phagocytosing and nonphagocytosing cells from patients with active pulmonary sarcoidosis as revealed by luminol-dependent chemiluminescence.

Oxidative metabolism in phagocytes such as granulocytes, monocytes, and alveolar macrophages is becoming of increasing interest in efforts to determine the pathogenetic mechanisms in diseases related to tissue damage, e.g., sarcoidosis. The release of free oxygen radicals is dependent on the activation of the oxidative metabolism and can be measured by means of chemiluminescence. Basic luminol-dependent chemiluminescence released by monocytes and alveolar macrophages from 12 patients with untreated pulmonary sarcoidosis stage II was increased (p less than 0.01) compared with 12 control subjects. A less distinct difference could be observed in the chemiluminescence response of granulocytes (P less than 0.05). After stimulation with zymosan, alveolar macrophages and monocytes (P less than 0.01) as well as granulocytes (P less than 0.05) had an enhanced luminol-dependent chemiluminescence compared with the control group. Emission of chemiluminescence by alveolar macrophages was considerably lower than that of granulocytes and monocytes. No significant correlation could be demonstrated between chemiluminescence response of granulocytes and monocytes and cellular markers of sarcoidotic activity such as lymphocytosis in bronchoalveolar lavage and T-helper/T-suppressor ratio in the lavage fluid. In contrast to that, a significant correlation (P less than 0.01) could be observed both between nonstimulated chemiluminescence and stimulated chemiluminescence and lymphocytosis and T-helper/T-suppressor ratio in bronchoalveolar lavage. Enhanced chemiluminescence may indicate inflammatory activation in pulmonary sarcoidosis.

Adult↗

EMD 52692 (bimakalim), a new potassium channel opener, attenuates luminol-enhanced chemiluminescence and superoxide anion radical formation by zymosan-activated polymorphonuclear leukocytes.

We investigated the relationship of potassium channel activation on modulation of oxidative respiratory bursts in canine neutrophils. Generation of superoxide anion radicals in opsonized zymosan-activated cells was determined using the technique of ferricytochrome c reduction. Preincubation of cells with the selective potassium channel opener, EMD 52692 (1-100 microM), attenuated superoxide anion radical production. Furthermore, EMD 52692 also produced a concentration-dependent inhibition of luminol-enhanced chemiluminescence by activated neutrophils. Glyburide, a selective antagonist of ATP-sensitive potassium channels, prevented the modulatory effect of EMD 52692 on both superoxide anion generation and luminol-enhanced chemiluminescence. The results suggest that ATP-sensitive potassium channels may play a significant role in regulating oxygen-derived free radical production in neutrophil-induced tissue injury.

Animals↗