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PubMed1990

Luminol chemiluminescence reaction catalyzed by a microbial peroxidase.

A peroxidase produced by microorganisms belonging to the genera Arthromyces and Coprinus was found to be a potent catalyst for the chemiluminescent oxidation of luminol, the luminescence produced per unit of microbial peroxidase protein being well over 100 times as strong as that produced by horseradish peroxidase. No large difference in Km value for H2O2 in the presence of luminol was found between Arthromyces ramosus peroxidase and horseradish peroxidase (7.0 and 15.5 microM, respectively), but Vmax of the Arthromyces peroxidase was 500 times greater than that of the horseradish peroxidase. It was also found that the Arthromyces peroxidase surpasses, beyond expectation, the horseradish peroxidase in the initial velocity of the chemiluminescence reaction with the stopped-flow method. The Arthromyces peroxidase was used for the glucose and cholesterol assays, which were notably more sensitive than the corresponding assays involving the horseradish peroxidase.

Calibration↗
PubMed1991

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.

2,2'-Dipyridyl↗
PubMed1991

Oxygen radical production during ischemia-reperfusion in the isolated perfused rat liver as monitored by luminol enhanced chemiluminescence.

We have applied the Luminol enhanced chemiluminescence technique to the isolated perfused rat liver during ischemia and reperfusion to monitor the production of oxygen radicals in tissue. Livers under perfusion with Luminol-containing buffer were subjected to 30 minutes of global ischemia followed by 60 minutes of reperfusion. Their chemiluminescence was continuously monitored to obtain the time course of oxygen radical production. Transient bursts of oxygen radical production were observed in the livers as indicated by chemiluminescence changes on reperfusion. Superoxide dismutase treatment abolished while catalase treatment enhanced the reperfusion-induced chemiluminescence transient.

Animals↗
PubMed1985

Direct measurement of phagosomal reactive oxygen by luminol-binding microspheres.

A new method, utilizing microsphere-bound luminol, which makes possible the direct measurement of highly reactive oxygen within phagosomes, was studied. When Freund's complete adjuvant-elicited mouse peritoneal macrophages and luminol-binding microspheres were mixed, the microspheres were engulfed in macrophages and enclosed in phagosomes, where chemiluminescence (CL) was generated, showing the generation of highly reactive oxygen. The reactive oxygen could be quantitatively assayed by measuring the intensity of CL. The addition of cytochalasin B inhibited the CL. CL production by the thioglycollate-elicited macrophages was found to be only a ninth of that by Freund's complete adjuvant-elicited macrophages, though the phagocytic activities were almost equivalent in both cases.

Animals↗
PubMed1988

Luminol-dependent chemiluminescence microassay for phagocytic function.

A luminol-dependent chemiluminescence (CL) microassay was developed to measure phagocytic function of peripheral blood leukocytes. Buffy coats, obtained by centrifugation of only 100 microliter of whole blood, provided an enriched population of polymorphonuclear leukocytes (PMNs). The total reaction mixture, consisting of leukocytes-luminol-inducer (opsonized zymosan), was 450 microliter. Peak CL activity was seen 5 min after addition of inducer at 37 degrees C with cells tested within 60 min after collection. Tests to determine precision and reproducibility of the microassay gave a coefficient of variation of 8.5% and 11%, respectively. There was no significant difference between the mean peak CL values for 20 healthy adult donors compared to 14 premature neonates, however, the newborns' CL activity declined more rapidly; CL activity was severely depressed in cells obtained from a patient with chronic granulomatous disease. Results suggest that this microassay provides a simple, rapid, and reliable test of phagocytic function in cases where the amount of blood available for testing is limited.

Humans↗
PubMed1980

Hydroxyl radical involvement in the luminol chemiluminescence from the reaction of arachidonic acid with sheep vesicular gland microsomes.

The luminol chemiluminescence associated with the reaction between vesicular gland microsomes and arachidonic acid was found to be inhibited by superoxide dismutase, catalase and hydroxyl radical scavengers. It was also found that diethylenetriamine pentaacetic acid caused only slight inhibition. This is interpreted as showing the formation of hydroxyl radicals in a Haber-Weiss reaction and the interaction of luminol with the hydroxyl radicals. The Haber-Weiss catalyst is taken to be heme iron as the luminescence was not inhibited by diethylenetriamine pentaacetic acid. A model system involving lipoxygenase, haematin and arachidonic acid was shown to be similar, the hydroxyl radicals coming from the interaction of haematin with the hydroperoxides, produced from the lipoxygenase-arachidonic acid reaction.

Animals↗
PubMedDate not supplied

Effect of venom from Cerastes cerastes (Egyptian sand viper) on luminol-dependent chemiluminescence of human blood phagocyte cells.

Cerastes cerastes venom added in vitro to human whole blood caused a marked inhibitory effect on the luminol-dependent chemiluminescence induced by phorbol myristate acetate (PMA) or opsonized zymosan on phagocyte cells. The inhibitory effect produced by the venom was both dose- and time-dependent when PMA was used as the stimulant of the oxidative burst. Similarly, the venom produced a significant inhibitory effect when added to isolated polymorphonuclear leukocytes (PMNs). Incubation of the isolated PMNs with the highest concentration of the venom used (1000 micrograms/ml), however, did not result in any significant disruption of the membranes of these cells. The effect of the venom on the isolated PMNs was also reversible following washing of the venom-treated cells with phosphate-buffered saline. The scavenger of reactive oxygen species such as superoxide dismutase, catalase and dimethyl sulphoxide potentiated the effect of the venom on the luminol-dependent chemiluminescence of human phagocyte cells. Sodium azide, the myeloperoxidase inhibitor, and sodium benzoate produced a similar potentiating effect. The results suggest that some of the toxicity of the venom may be mediated by an action on the phagocytic immune system.

Animals↗
PubMed1991

A rapid microassay for measuring the luminol-dependent chemiluminescent response in canine whole blood.

A microassay for the luminol-dependent chemiluminescence (CL) response in canine whole blood was developed to measure indirectly the oxidative metabolism of peripheral blood leukocytes. Fifty microliters of blood were mixed with 705 microliters of Hank's balanced salt solution containing 25 mM Hepes and 1.3 x 10(-4) M luminol. This mixture was allowed to equilibrate for 5 min after which 60 microliters of latex beads (0.801 microns diameter) were added as a stimulant, and the CL response was monitored continuously for 5 min at 37 degrees C using a luminometer. The whole blood CL response was significantly correlated (r = 0.784, P less than 0.01, n = 14) with the number of neutrophils in the peripheral blood. Further, the whole blood CL response was abolished by the depletion of neutrophils after passing the blood through an adherence column and by the addition of sodium azide. The relative chemiluminescent light unit (RCLU) was a reliable marker for comparing each peak value in different samples. The coefficient of variation (CV) of repetitive samples was 9.87%, and the CV of 14 normal dogs was 15.7%. This method is useful and applicable for screening the CL response in canine whole blood.

Animals↗
PubMed1991

High-performance liquid chromatographic investigation of product formation in the horseradish peroxidase-enhanced chemiluminescence of luminol with different enhancers.

A reversed-phase high-performance liquid chromatographic method for the investigation of the chemiluminescence-producing oxidation of luminol by the enzyme horseradish peroxidase is presented. Both the kinetics and the mechanism of product formation can be monitored. Special attention is paid to the mechanism of enhancement of the chemiluminescence by phenolic compounds, such as p-iodophenol, p-hydroxycinnamic acid and 6-hydroxybenzothiazole. The function of the enhancers was elucidated partially by the observation of a higher degradation rate of luminol. In addition, it was concluded that the mechanism of enhancement is probably different for the various enhancers, based on the product formation.

Chromatography, High Pressure Liquid↗
PubMed1990

Luminol chemiluminescence using xanthine and hypoxanthine as xanthine oxidase substrates.

Luminol chemiluminescence induced by the xanthine or hypoxanthine-O2-xanthine oxidase system is analyzed and compared. Characteristics of the light emission curves were examined considering the conventional reaction scheme for the oxidation of both substrates in the presence of xanthine oxidase. The ratio of the areas of the rate of superoxide production during substrate oxidation to uric acid. The O2-. to uric acid ratio for each substrate can account for differences in xanthine and hypoxanthine-supported light emission, since uric acid is a strong inhibitor of O2-.-dependent luminol chemiluminescence. These results are consistent with a free radical scavenging role for uric acid. A similar but weaker scavenging effect of xanthine may also contribute to the observed differences in chemiluminescent yields between both substrates.

Animals↗
PubMed1994

On the use of the quenching of luminol luminescence to evaluate SOD activity.

Addition of horseradish peroxidase to a luminol solution (pH = 9.4) produces a burst of light followed by a steady luminescence that lasts for several minutes. This steady-state luminescence is readily quenched by SOD, with a Q1/2 concentration (the additive concentration needed to decrease by one-half the emitted luminescence intensity) of c.a. 4 ng/ml (14 mU/ml). The luminescence intensity decrease can then be employed to evaluate SOD activity in SOD-containing samples. However, the light intensity can also be quenched by additives, such as Trolox, that are able to trap luminol-derived intermediates. It is proposed that double quenching experiments must be performed in order to be able to relate the observed effect of an additive to its SOD-like activity.

Antioxidants↗
PubMed1991

Role of functional groups of human plasma and luminol in scavenging of NaOCl and neutrophil-derived hypochlorous acid.

Hypochlorous acid HOCl/OCl- and other oxidants derived from stimulated polymorphonuclear leukocytes are involved in tissue damage during a number of pathological processes. In order to obtain more detailed information on possible reactions of HOCl/OCl- the effects of both NaOCl and PMN-derived hypochlorous acid on functional groups of amino acid solutions and human plasma are studied. In valine and lysine solutions NaOCl diminishes the number of amino groups in a molar ratio of 1:1 between NaOCl and amino groups. In cysteine and methionine samples the decrease of amino groups starts only after all sulfhydryl or thioether groups are oxidized by NaOCl. If freshly prepared human plasma is treated with increasing amounts of NaOCl all plasma SH groups are oxidized first, then probably the thioether groups and only after this the amino groups are affected. Furthermore, it was found, that the reactivity of luminol against NaOCl is similar to that of amino groups. Increasing amounts of SH groups of components of human plasma are oxidized by incubation with PMA-stimulated polymorphonuclear leukocytes dependent on the incubation time. Plasma amino groups are not affected under the same experimental conditions. The addition of plasma to FMLP-stimulated PMN in the presence of luminol decreases that part of chemiluminescence caused by extracellularly generated hypochlorous acid. Plasma samples pretreated with NaOCl cause a lower inhibition of light generation in FMLP-stimulated PMN only when more than 4.10(-8) mol NaOCl per mg protein are used to pretreat plasma. It is assumed that in the development of tissue injuries caused by infiltrated PMN the following sequence of damage occurs in accessible tissue regions. First, the sulfhydryl groups are oxidized, then the thioether groups, and only after this amino and other target groups are affected.

Amino Acids↗
PubMed2004

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.

Animals↗
PubMed2005

Flow-injection chemiluminescence determination of phentolamine based on its enhancing effect on the luminol-potassium ferricyanide system.

It was found that the light emission produced by the oxidation of luminol by potassium ferricyanide in the basic medium was enhanced by phentolamine, a drug recently used to treatment of male and female sexual dysfunction. The optimum conditions for this chemiluminescent reaction were studied in detail by a flow-injection system. A new, simple and rapid method has been developed under the optimum conditions for determination of phentolamine. This method has the advantages of high sensitivity, good reproducibility and low detection limit. On the basis of investigation of chemiluminescent, fluorescent and UV spectra of phentolamine in basic solution containing potassium ferricyanide and luminol, a possible mechanism of this reaction was proposed. In the optimum conditions, CL intensities are proportional to concentrations of the phentolamine in the 0.01-1 microg/mL range. The limit of detection is 3.0 ng/mL for phentolamine. The method has been applied to the determination of phentolamine in the commercial preparations, synthetic samples and biological fluids with satisfactory results.

Calibration↗
PubMed2002

Luminol-type chemiluminescence derivatization reagents for liquid chromatography and capillary electrophoresis.

The present paper provides the principles for chemiluminescence of luminol-type compounds and their wide and powerful application to the detection system in liquid chromatography and capillary electrophoresis as derivatization reagents. The reagents can be classified into two types, chemiluminescence labeling and chemiluminogenic reagents. The former reagents are highly chemiluminescent themselves and used for tagging their intense chemiluminophores to analytes, whereas the latter are weakly chemiluminescent but generate intense chemiluminescence by reaction with analytes. The liquid chromatographic methods utilizing chemiluminescence derivatizing reactions with luminol-type reagents allow the analytes to be detected at pmol-sub-fmol levels. Furthermore, the chemiluminogenic reactions show high selectivity owing to their selective reaction against the analytes permitting facile and reproducible detection.

Chromatography, Liquid↗
PubMed2003

Real-time measurement of nitric oxide by luminol-hydrogen peroxide reaction in crystalloid perfused rat heart.

The objective of this study was to develop an assay system that allows continuous monitoring of nitric oxide (NO) released from crystalloid perfused hearts. We utilized chemiluminescence reaction between NO and luminol-H(2)O(2) to quantify the NO level in coronary effluent. Isolated rat hearts were subjected to ordinary Langendorff's perfusion, and the right ventricle was cannulated to sample coronary effluent. After equilibration, the coronary flow rate was set constant and the hearts were paced at 300 bpm. Coronary effluent was continuously sampled and mixed with the chemiluminescent probe containing 0.018 mmol/l luminol plus 10 mmol/l H(2)O(2). Chemiluminescence from the mixture of coronary effluent and the probe was continuously measured. NO concentration was calibrated by various concentrations (0.5-400 pmol/l) of standard NO solution. The lower detection limit of NO was 1 pmol/l. Basal NO release from isolated perfused rat heart was 0.41 +/- 0.17 pmol/min/g of heart weight, and that was significantly suppressed by 0.1 mmol/l of L-NAME to 0.18 +/- 0.10 pmol/min/g of heart weight (n = 7). Application of 0.1 and 0.3 micromol/l acetylcholine increased NO level in the coronary effluent, in a concentration-dependent manner, from 6.6 +/- 1.7 in a baseline condition to 16.3 +/- 7.4 and 30.3 +/- 16.1 pmol/l at each peak, respectively. Thrombin at 1 and 10 U/ml also increased NO level from 17.6 +/- 4.3 in control to 35.5 +/- 10.4 and 48.7 +/- 8.7 pmol/l at each peak, respectively (n = 7). Thus, this assay system is applicable to the continuous real-time measurement of NO released from crystalloid perfused hearts, and it may be useful for the study of physiological or pathophysiological role of NO in coronary circulation.

Acetylcholine↗
PubMed1985

Dissociation of natural killing and luminol-dependent chemiluminescence.

A long lasting luminol-dependent chemiluminescence was seen when mouse NK cell preparations or human NK cell preparations from Percoll gradients were mixed with NK susceptible targets. This CL could be abolished by extensive removal of adherent cells though normal NK cell activity remained. In addition, the NK cell line HY 3-Ag3 did not show any CL; although it expressed a very strong cytolytic activity. Thus, we conclude that there are two cells reacting with NK susceptible targets. First, the NK cell whose cytolytic activity does not necessarily depend on the formation of oxygen metabolites detectable by CL, and second, a more adherent cell population, found in NK cell preparations obtained after Percoll gradients, that reacts with NK-sensitive targets and leads to luminol-dependent CL. The observed CL paralleled the cytotoxicity measured by 51Cr release. The time course of the CL signal was similar in human and mouse. The maximal CL-signal obtained was about 10(5) cpm at 37 degrees C when 10(6) human effector cells were used at a ratio of 5:1 effector to target cells. The CL was shown to be highly temperature dependent.

Animals↗
PubMed2002

Determination of picomole amounts of thiamine through flow-injection analysis based on the suppression of luminol-KIO(4) chemiluminescence system.

A continuous flow sensor for the determination of thiamine was constructed by using controlled-reagent-release technology in a FIA-CL system. The analytical reagents, luminol and KIO(4), were both immobilized on an anion-exchange column. The CL signal produced by the reaction between luminol and KIO(4), which were eluted from the column through H(2)O injection, was decreased in the presence of thiamine. The decreased CL intensity was linear with thiamine concentration in the range 3.3 pmol ml(-1)-6.7 nmol ml(-1); and the limit of detection was 1.0 pmol ml(-1) (3.). The whole process, including sampling and washing, could be completed in 0.5 min with a relative standard deviation of less than 3.0%. The flow sensor showed remarkable stability and could be easily reused over 80 h. The sensor proposed was tested in determination of thiamine in pharmaceutical preparation and human urine samples.

Calibration↗