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At least 19 recordsLinked to original sources

PubMed1991

On radical production by PMA-stimulated neutrophils as monitored by luminol-amplified chemiluminescence.

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2-. and H2O2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2-. production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2-. and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2-. for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2-. and H2O2. The failure of the CL assay to report O2-. and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2-. fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2-. and H2O2 generated by either stimulated neutrophils or in cell-free systems.

Cell-Free System↗

PubMed1990

Bimodal induction of sister-chromatid exchanges by luminol, an inhibitor of poly(ADP-ribose) synthetase, during the S-phase of the cell cycle.

The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2'-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dose-dependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such as cis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB)', an inhibitor of the NAD+ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the S-phase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.

Animals↗

PubMed1986

A demonstration that O2- is a crucial intermediate in the high quantum yield luminescence of luminol.

The chemiluminescence of luminol, due to its reaction with alkaline H2O2, is inhibited by superoxide dismutase or by hydroxyl radical scavengers. Hematin markedly enhances this H2O2-induced luminescence of luminol and lessens, but does not eliminate, the sensitivity towards these inhibitors. Reaction mechanisms are proposed to account for these results. Since luminol luminescence depends upon a reaction between the luminol radical and O2-, and since the luminol radical can reduce dioxygen to O2-, superoxide dismutase-inhibitable luminol luminescence cannot be reliably used as a detector of O2- production.

Hemin↗

PubMed1986

Variables in xanthine oxidase-initiated luminol chemiluminescence: implications for chemiluminescence measurements in biological systems.

We tested the effects of generally used chemiluminescence inhibitors on an example of luminol chemiluminescence elicited by xanthine oxidase/hypoxanthine system, and attempted to assess their capabilities in discovering the reaction pathways leading to chemiluminescence. Luminol itself is a xanthine oxidase inhibitor and its concentration affects the reaction mechanism. Maximal chemiluminescence response was observed at luminol concentration inhibiting urate production. Chemiluminescence was totally inhibited by superoxide dismutase, the inhibition by catalase depended on luminol concentration. Ferricytochrome c, a detector of superoxide, either stimulated or inhibited chemiluminescence in a concentration-dependent manner. Chemiluminescence was highly stimulated by peroxidases. A pronounced inhibition of chemiluminescence was caused by chelators; 1 mM desferal and 0.01 mM diethyldithiocarbamate. It is suggested that measurement of luminol chemiluminescence is not a suitable method for discrimination among individual reactive oxygen species and their quantitative determination in biological systems.

Catalase↗

PubMed1986

Aqualuminescence of alkaline luminol in the presence of fluorescein.

The light yield from both luminol and fluorescein is studied at a fixed concentration of luminol and by varying the concentration of fluorescein in 3 X 10(-2) M basic solution by direct chemical reaction as well as by aqualuminescence technique. The emission spectra of chemiluminescence and aqualuminescence of the alkaline luminol-fluorescein mixture are recorded on a Fuoss spectrograph. A 4-fold increase in the aqualuminescence intensity of luminol has been observed in the presence of fluorescein as compared to that of pure luminol. The results are explained on the basis of reactions of colour centres with the activators.

Fluorescein↗

PubMed1989

What do we measure by a luminol-dependent chemiluminescence of phagocytes?

The review presents a survey of published findings concerning the mechanism of luminol-dependent chemiluminescence in biological systems. The potential of various oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) to react with luminol is discussed. The ability of commonly used enzymes (superoxide dismutase, catalase), inhibitors, and oxygen radical scavengers to discriminate between individual oxygen species is assessed together with the potential of a variety of substances encountered in biological systems to interfere in luminol-dependent chemiluminescence reactions. It is concluded that luminol-dependent chemiluminescence gives at present very little ability to discriminate between individual oxygen or radical species. Furthermore, luminol-dependent chemiluminescence used in biological systems is extremely prone to many interferences, which are very difficult to control.

Animals↗

PubMed1989

Spontaneous and luminol-dependent chemiluminescences from tissue preparations of benzo[a]pyrene-injected mice.

Chemiluminescence (CL) from tissue preparations of mice i.p. injected with and without the chemical carcinogen benzo[a]pyrene (BP) was detected by a single photon counting apparatus. The spontaneously emitted CL (spontaneous CL) and the CL after luminol addition (luminol-CL) were measured for mice with and without previous induction of their liver mixed-function oxidases (MFO) by phenobarbital. In MFO-non-induced mice, although the spontaneous CL was not notably modified by BP injection, the kidneys presented three times greater luminol-CL after BP injection. On the other hand, MFO-induced mice had higher spontaneous CL of plasma, liver, kidneys, and lungs, as well as higher luminol-CL of liver after BP injection, when compared with the respective MFO-induced but BP-non-injected mice. The luminol-CL of liver was suppressed by scavengers of active oxygen and free-radicals such as TIRON, butylhydroxytoluene and in a less extent by superoxide dismutase. The CL detected from tissue preparations of mice after BP treatment is thought to reflect the formation of oxygen radicals and electronically excited-species during BP metabolism.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed1992

[The effect of monoclonal and polyclonal antibodies to peroxidase on combined peroxidase oxidation of 4-iodophenol with luminol and 4-aminoantipyrine].

The kinetics of peroxidase-dependent cooxidation for two substrate pairs [p-iodophenol + 4-aminoantipyrine (AAP) and p-iodophenol + luminol was studied both in the absence and presence of polyclonal antibodies (polyAB), three types of peroxidase-specific monoclonal antibodies (monoAB) and their double or triple mixtures in a wide range of H2O2 concentrations (0.01-10.0 mM). MonoAB 2C, 3E and 9D at concentrations of 0.05-500 nM inhibited the cooxidation of p-iodophenol + AAP at H2O2 concentration above 1.0 mM but activated the cooxidation of p-iodophenol + luminol. The double and triple mixtures of monoAB activated the cooxidation of p-iodophenol + AAP at the same H2O2 concentrations without any effect on the p-iodophenol + luminol cooxidation. PolyAB activated the cooxidation of p-iodophenol + AAP more effectively and only slightly activated (or inhibited) that of p-iodophenol + luminol. PolyAB diminished the values of rate constants for the interaction of the peroxidase active intermediates, E1 and E2, with p-iodophenol, AAP or luminol. Possible modes of monoAB and polyAB effects on the two substrate pair cooxidation are discussed.

Ampyrone↗

PubMed1987

Luminol dependent chemiluminescence and thiol group oxidation provoked by neutrophils is attributable to different oxidizing species.

Luminol-dependent chemiluminescence and thiol group oxidation of glutathione and human serum albumin were measured in order to demonstrate whether the inhibition of polymorphonuclear leukocyte chemiluminescence by albumin was attributable to thiol group oxidation. We have shown that: 1. thiol groups on glutathione and albumin are oxidized by PMNL stimulated by soluble and phagocytic stimuli; 2. thiol group oxidation in albumin and glutathione did not correlate with the inhibitory effects of these substances on luminol-dependent chemiluminescence with respect to time course, magnitude, effects of known scavengers or extracellular activity. It was therefore concluded that thiol group oxidation was not the cause of albumin inhibition of luminol-dependent chemiluminescence; 3. a metastable oxidant was identified after PMNL activation which was capable of oxidizing thiol groups but unable to elicit chemiluminescence from luminol.

Azides↗

PubMed1992

Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.

We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3',5,5' tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.

Acridines↗

PubMed1991

Penicillin-enhanced chemiluminescence of the luminol-H2O2-Co2+ system.

The luminol-H2O2-Co2+ system has been widely used in chemical and biological analysis. We report here an investigation of the observation that penicillins have the ability to prolong and enhance the intensity of chemiluminescence from luminol. The basis of this phenomenon appears, as revealed by difference spectroscopy, to be the formation of a complex between the beta-lactam and the superoxide ion. The latter is the oxidizing species responsible for the oxidation of luminol in alkaline solution and has a mean lifetime, in solution, of milliseconds. The stabilization of the superoxide ion by penicillin complexation extends the effective lifetime of the superoxide ion by a few orders of magnitude and thereby allows for more efficient oxidation of the beta-lactam. Several penicillins were determined by their enhancement of luminol chemiluminescence. A detection limit of 100 ng mL was obtained for penicillin G with a less-than-ideal detection system.

Catalysis↗

PubMed1987

Luminol-independent chemiluminescence by phagocytes is markedly enhanced by dexamethasone, not by other glucocorticosteroids.

The effect of several glucocorticosteroids on the generation of reactive oxygen species (ROS) was examined. The ROS assessed were O-2, H2O2, OH., and chemiluminescence (CL) (determined in the presence or absence of luminol), generated by both opsonized zymosan-stimulated neutrophils or monocytes and by the xanthine-xanthine oxidase system. Except for luminol-independent CL, only high concentrations (10(-4) M) of steroids could decrease each ROS. In contrast, luminol-independent CL generation in the phagocyte system was increased in a dose-dependent manner by the addition of dexamethasone, but not by any other steroid. Further, in lymphocyte cultures stimulated with Con A for four days, luminol-independent CL generation was demonstrated and enhanced by the addition of dexamethasone, although CL generation was not detected in the absence of dexamethasone. These findings provide evidence that CL does not always represent light specific to ROS, and they suggest the possibility that dexamethasone induces emission of light at sites of inflammation.

Adrenal Cortex Hormones↗

PubMed1991

A comparative study of peroxidases from horse radish and Arthromyces ramosus as labels in luminol-mediated chemiluminescent assays.

The properties of a peroxidase from Arthromyces ramosus (ARP) in the chemiluminescent reaction of luminol oxidation have been studied. These were compared with the properties of horse radish peroxidase (HRP) in the cooxidation of luminol and p-iodophenol, the enhanced chemiluminescence (ECL) reaction. By means of the stop-flow technique, ARP was shown to have an enzymatic activity toward luminol higher than that toward HRP. ARP can efficiently catalyze luminol oxidation in the absence of substrate enhancer. pH and substrate concentrations were optimized to determine ARP with the highest sensitivity. The detection limit of ARP was 5 x 10(-13) M, the same as that for HRP in the ECL reaction. The data on the use of ARP as a label in enzyme immunoassay of human IgG are presented. ARP was shown to have all the advantages of HRP as a label in chemiluminescent enzyme immunoassays: (i) high signal intensity, (ii) slow decay of luminescence, (iii) high signal/noise ratio, and (iv) as a consequence of (i)-(iii), high detection sensitivity. However, the low thermostability of ARP can limit the potential fields of its application.

Enzyme Stability↗

PubMed1984

In vitro determination of phagocyte activity by luminol- and lucigenin-amplified chemiluminescence.

Amplified chemiluminescence (CL) detects most sensitively biologically important reactive oxygen species (ROS) which are generated by phagocytes by the respiratory burst permitting the determination of cell activity in vitro. Different murine phagocyte populations were used in combination with various ROS-catabolizing enzymes and some of their inhibitors to determine the possible advantages of one of the two main presently used amplifiers, i.e. luminol and lucigenin. Lucigenin appeared to react mainly with the first of the generated ROS the superoxide anion radical (O-.2) and thus records cell activity via the respiratory burst much more reliable than luminol. The more commonly employed luminol reacts mainly with hydrogen peroxide (H2O2) and probably the singlet oxygen (1O2) which result in photon emission. However, it seems not to react with the hydroxyl radical (OH.). The dependence of luminol-amplified CL upon the generation of the chain reaction intermediate H2O2 and its three main catalysts catalase, myeloperoxidase and glutathione makes this reaction prone to different artifacts if cell activity is to be determined. Lucigenin-amplified CL offers great advantages to study cell activating or inhibiting properties of drugs and kinetics in vitro because of the biological relevance of O-.2 determination, its sensitivity, reproducibility and ease in handling.

Acridines↗

PubMed1991

Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2.

Acridines↗

PubMed1989

Mechanisms for luminol-augmented chemiluminescence from neutrophils induced by leukotriene B4 and N-formyl-methionyl-leucyl-phenylalanine.

Neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4) generated kinetically distinctive luminol augmented chemiluminescence (LCL). Inhibitors of .O2- [superoxide-dismutase (SOD) or tiron], H2O2 (catalase), myeloperoxidase, MPO, (NaN3), HOCl (taurine) and .OH (mannitol) hampered LCL dose-dependently with similar characteristics for both stimuli. In cell free systems it was found that .O2- (generated in the xanthine/xanthine-oxidase reaction) or H2O2 produced LCL. Superoxide dismutase inhibited .O2- -induced LCL dose dependently. The MPO + H2O2 system, which generated more pronounced LCL than either component alone, was inhibited by catalase and taurine but not by SOD. When neutrophils, treated with luminol, but where extracellular luminol had been removed, were stimulated with fMLP or LTB4, they produced less than 2% of the LCL where luminol was present in the medium. When neutrophil LCL and superoxide formation by the cytochrome C method were assessed in parallel experiments, in all instances the peak LCL response coincided with the linear phase in that response. Thus, LCL, induced by LTB4 and the corresponding fMLP peak, are extracellular events with similar chemical backgrounds, closely related to generation of reactive oxygen species. Consequently, the kinetical differences in LCL between fMLP and LTB4 suggest that LTB4, by yet unknown mechanisms, activates the NADPH oxidase more rapidly than fMLP.

Drug Synergism↗

PubMed1978

In vitro evaluation of opsonic and cellular granulocyte function by luminol-dependent chemiluminescence: utility in patients with severe neutropenia and cellular deficiency states.

Actively phagocytizing polymorphonuclear leukocytes (PMN) emit light or chemiluminescence (CL) which has been shown to be linked to the oxidative activity of the PMN. The measurement of CL has been demonstrated to be a useful tool for the in vitro assessment of intracellular and opsonophagocytic function of PMN. We have increased the sensitivity of the CL measurement by the addition of luminol to the in vitro reaction of PMN, bacteria, and serum. The presence of luminol, which can be oxidized to emit light, amplifies the detection of CL and PMN cellular activity. This amplification effectively reduces the number of PMN that are necessary for assessment of PMN function from 1 x 10(7) to as low as 2 x 10(4) PMN/assay and permits the evaluation of PMN function in severely neutropenic patients (100 PMN/mm3) in whom cellular PMN function has been heretofore extremely difficult to assess by other methodology. When this luminol-dependent CL method was used, three of eight neutropenic leukemic patients with gram-negative septicemia were found to have deficient opsonic activity and/or increased or depressed cellular oxidative activity. Because the initial slope of CL is dependent on the amount of serum and heat-labile factors, this method can also be used effectively as a simple technique for the analysis of specific rates of opsonophagocytosis of various microorganisms. Additionally, this method can detect the cellular PMN abnormalities of chronic granulomatous disease and myeloperoxidase deficiency. The luminol-dependent CL method is a simple, sensitive, reproducible technique that provides useful information about PMN metabolic activity, particularly in studies in which the number of PMN is limited.

Agranulocytosis↗

PubMed1990

The relative contribution of bronchoalveolar macrophages and neutrophils to lucigenin- and luminol-amplified chemiluminescence.

The relationship between differential cell counts and latex-stimulated luminol and lucigenin-amplified chemiluminescence (CL) was investigated by mixing alveolar macrophages (AM) obtained at bronchoalveolar lavage (BAL) with allogeneic peripheral blood neutrophils (PMN) in varying proportions. In 5 non-asthmatic subjects, the mean luminol-amplified CL increased linearly from 2.1 (0.9 SEM) x 10(5) counts per second (cps) with less than 2% PMN, greater than 96% AM to 47.3 (11.1 SEM) x 10(5) cps with greater than 94% PMN, 0% AM (r = 0.996, p less than 0.001). The regression had a y-intercept indistinguishable from 0 cps, suggesting that luminol-amplified CL exclusively reflected PMN activity. Using the same technique, the mean lucigenin-amplified CL showed a fall from 35 (2.3 SEM) x 10(5) cps with a cell population of greater than 96% AM, less than 2% PMN to 20 (2.3 SEM) x 10(5) cps with 0% AM, greater than 94% PMN. Both PMN and AM appeared to contribute to lucigenin-amplified CL, with AM contributing approximately 1.7 times as much activity per cell as PMN. Lucigenin-amplified CL appeared to be an appropriate technique for measuring AM activity when the proportion of PMN in mixed cell populations was small. A linear relationship was found between percent PMN count and luminol-amplified CL measured in a mixed BAL cell population from asthmatic subjects (p less than 0.01) and non-asthmatic controls (p less than 0.01). The slope of this regression line was significantly greater for subjects with asthma than for control subjects (p less than 0.01), suggesting a uniform increase in PMN activity in cells obtained from asthmatic airways.

Acridines↗