Lucigenin- and luminol-enhanced chemiluminescence in turkey monocytes

Monocytes from 10 week-old specific pathogen-free turkeys were isolated from peripheral blood by density centrifugation and assayed for their oxidative activity by means of a luminometer. Chemiluminescence (CL) properties after stimulation with different soluble and particulate stimuli were compared in lucigenin- and luminol-enhanced assays. A distinct response could be measured with 12-phorbol 13-myristate acetate (PMA) and Zymosan A, but only a weak signal was obtained with calcium ionophore A23187. No oxidative activity could be induced with N-formyl-methionyl-phenylalanine. Peak maxima for both lucigenin- and luminol-enhanced CL were ranked: PMA>Zymosan A>calcium ionophore. The velocity of the lucigenin- and luminol-enhanced responses induced by calcium ionophore were of similar magnitude, but the lucigenin-enhanced responses of Zymosan A and PMA-stimulated monocytes were respectively about 5 and 10 times higher than those obtained in luminol-enhanced assays. No peroxidase activity could be detected in the purified turkey monocytes. As luminol-enhanced CL primarily results from the peroxidase activity, this lack of myeloperoxidase may explain the observed lower responses to the different stimuli, in the presence of a luminol. In contrast, lucigenin-enhanced CL is not related to peroxidase activity, but is a selective probe of oxidase activity. Irrespective of the myeloperoxidase deficiency, different soluble and particulate stimuli induced a significant and reproducible CL response in turkey monocytes, in the presence of both chemiluminigenic probes, lucigenin and luminol. The possibility of measuring the phagocyte oxygenation activity of turkey monocytes represents a useful tool for the study of monocyte mediated host defence in the turkey. © 1997 John Wiley & Sons, Ltd.     

Effect of a new fluorinated quinolone (ofloxacin) on the chemiluminescence of phagocytosing human neutrophils

We measured the chemiluminescence (CL) of human neutrophils (PMNLs) exposed to different concentrations of ofloxacin (2, 4, and 6 μg/ml) readily achievable in therapy. CL reaction during zymosan phagocytosis by PMNLs obtained from human healthy volunteers was registered in a computer-linked LKB 1251 luminometer. Ofloxacin did not induce significant variations on the respiratory burst of PMNLs.     

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

A two-intermediate model for imidazole-promoted peroxyoxalate chemiluminescence

A mechanism is proposed for imidazole-catalysed peroxyoxalate chemiluminescence. The reaction model includes a sequential formation of 1-aroxalylimidazole and 1,1′-oxalyldiimidazole as light-producing reaction intermediates. The suggestion is supported by the kinetic data obtained for the reaction of imidazole with bis(4-nitrophenyl) oxalate and on the recently reported ability of 1,1′-oxalyldiimidazole to function as an efficient chemiluminescence reagent. The relative contributions of different catalytic pathways and hydrolytic side-reactions are discussed © 1997 John Wiley & Sons, Ltd.     

Morpho‐functional modifications of human neutrophils induced by aqueous cigarette smoke extract: comparison with chemiluminescence activity

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke‐related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Application of optimized chemiluminescence assay for determination of the antioxidant capacity of herbal extracts

A chemiluminescence (CL) assay for the determination of antioxidant capacity (AOC) has been optimized and applied to analyses of herbal extracts in the present study. The optimal concentrations of reagents (luminol, H₂O₂, horseradish peroxidase) have been determined, as well as the optimal reaction conditions (wavelength, pH, temperature, sample volume). All of the measurements were performed at the emission maximum of the oxidized form of luminol (425 nm). The optimal concentrations of the reagents were determined as follows: 1.6 mmol/L luminol, 7.5 mmol/L H₂O₂ and 0.14 U/mL horseradish peroxidase activity in the reaction mixture. Analyses were carried out in phosphate buffer, pH 7.4, at room temperature. With the optimized CL assay, the AOCs of various water and methanol herbal extracts were determined (dog rose hips, plantain leaves and coltsfoot and thyme flowers) and the results were compared to those obtained by other classical methods for the evaluation of antioxidants. Strong correlations (r > 0.9) with the Folin–Ciocalteau assay and the 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH)^● assay are confirmed, although there is no correlation between AOC and the concentration of ascorbic acid in the samples analysed. This optimized CL assay is simple, rapid and reliable, and it represents a good alternative to classical methods (Folin–Ciocalteau, DPPH^●) for the determination of AOC of herbal extracts and other food samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Electrogenerated chemiluminescence from violanthrone

Violanthrone, an emitter of exceptionally bright chemiluminescence, was examined in dimethylformamide solution to determine whether it also emits particularly bright electrogenerated chemiluminescence (ECL). The ECL measurements were carried out using a cycled potential which was applied to platinum electrodes. At the maximum sweep rate of 80 Vs^?1 available, the intensity of the violanthrone ECL was still increasing with sweep rate and was c. 56% of that from rubrene, a bright, commonly used emitter of ECL. Furthermore, assuming that the emission arises from radical anioncation recombination, the sweep rate dependence showed that the least stable radical ion (probably the cation) decays with a half-life shorter than 0.2s.     

Ultrasensitive assay of clindamycin in medicine and bio-fluids with chemiluminescence detection.

A simple chemiluminescence (CL) method using flow injection has been developed for the determination of clindamycin, based on the inhibitory effect of clindamycin on the CL generated from the luminol-K(3)Fe(CN)(6) system in alkaline medium. It was found that the decrement of CL intensity was linear with the logarithm of clindamycin concentration over the range 0.7-1000 ng/mL. The detection limit was 0.2 ng/mL with a relative standard deviation (RSD) of <5.0% (n = 7). At a flow rate of 3.0 mL/min, a complete analytical process could be performed within 0.5 min, including sampling and washing. The proposed procedure was applied successfully to the determination of clindamycin in pharmaceutical preparations and human urine without pretreatment.     

Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils

A fast and sensitive chemiluminescence assay for the determination of H₂O₂ in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H₂O₂ concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H₂O₂ without influence of H₂O₂ decomposition by cellular enzymes or newly produced H₂O₂ due to dismutation of superoxide anion radicals. Concentrations of H₂O₂ were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H₂O₂ concentration upon stimulation could be observed at 3000 cell/mL.     

A chemiluminescence sensor array based on nanomaterials for discrimination of teas

In recent years, electronic tongue and nose devices have been developed that consist of an array of cross‐responsive sensors. In this study, we report a chemiluminescence (CL) sensor array based on oxidation at twelve different catalytic nanomaterial locations for the discrimination of eight teas. CL response patterns or “fingerprints” were obtained for a given compound on the sensor array and then discriminated through linear discriminant analysis. The experiments demonstrate that the sensor array had excellent differentiability and reversibility. Copyright © 2012 John Wiley & Sons, Ltd.     

Inhibition of monocyte luminol-dependent chemiluminescence by tetrahydrobiopterin, and the free radical oxidation of tetrahydrobiopterin, dihydrobiopterin and dihydroneopterin

Luminol-dependent chemiluminescence of normal human monocytes activated by zymosan is demonstrated to be inhibited by tetrahydrobiopterin in a concentration-dependent manner. The reduced pterins tetrahydrobiopterin, dihydrobiopterin, and dihydroneopterin are all shown to be readily oxidized by the hydroxyl radical. The susceptibility of reduced pterins to free radical attack may explain the inhibition of chemiluminescence observed and an additional role of reduced pterins as free radical scavengers in tissues is considered.     

Effects of granulocytes on human neuroblastoma cells measured by chemiluminescence and chromium-51 release assay

We investigated whether polymorphonuclear leukocytes (PMN) are able to kill human neuroblastoma cells either directly or if coated with antibody MAb 14.18 that recognizes ganglioside GD₂ present on the cell surface of most neuroblastoma cells. Neuroblastoma cells could not be destroyed directly, whereas in the antibody-dependent reaction (ADCC-reaction) they were easily eliminated. In order to answer the question whether reactive oxygen intermediates are involved in this process, chemiluminescence measurements were performed. Compared to the signals that could be measured using opsonized zymosan as stimulus, only weak CL-signals could be registered during the ADCC reaction. Pretreatment of PMN with granulocyte-macrophage colony stimulating factor (GM-CSF) enhanced the CL-signals, catalase and SOD reduced it; however, cell killing was only slightly influenced in the presence of catalase and superoxide dismutase. These data suggested that reactive oxygen compounds do not play a prominent role in the killing process. Definitive evidence for this suggestion could be obtained using PMN from a patient with chronic granulomatous disease (CGD): MAb 14.18 coated neuroblastoma cells could be killed effectively, but no CL-signal could be registered, either in the ADCC-reaction or using opsonized zymosan as stimulus.     

Impaired reactive oxygen metabolism of phagocytic leukocytes in NIDDM patients. A role for non-enzymatic glycosylation of collagen

Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p < 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p < 0.05 and p < 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing. © 1998 John Wiley & Sons, Ltd.     

NMDA excitotoxicity and free radical generation in rat brain homogenates: Application of a chemiluminescence assay

NMDA, the specific agonist of glutamate gated ion channels permeable to calcium, is implicated as a causal factor in the pathogenesis of several neurobiological disorders such as stroke, seizures, ischemia, and chronic neurodegenerative disease. On the other hand, evidence on the roles of oxidative mechanisms involved in NMDA-induced neurotoxicity is accumulating. In this study, we have used chemiluminescence measurements as an easy, rapid and sensitive assay to investigate the effects of NMDA and oxidative stress on brain cell vulnerability. Rat brain homogenates were incubated with increasing concentrations of glutamate and NMDA. Production of reactive oxygen species was followed by single photon emission measurements using the specific enhancers luminol and lucigenin. Increases in emission were observed at excitotoxic concentrations of glutamate and NMDA. Other parameters of oxidative stress such as diene conjugates, TBARS and carbonyl groups were also investigated. Our results indicated that chemiluminescence measurements may be used to study involvement of oxidative stress in neurotoxicity.     

New chemiluminescence assay for linamarin

A new chemiluminescence assay was developed for the quantitative determination of linamarin, a cyanogenic glucoside present in cassava. The assay involved hydrolysis of linamarin by a specific enzyme, linamarase, to release glucose, which was then quantitated by a chemiluminescence system consisting of glucose oxidase-peroxidase-luminol. The new assay was more sensitive than the conventional spectrophotometric method for quantitating linamarin in cassava extracts. However, the following agents were found to interfere with the new assay: Vanadate, Mg2+, and Cu2+, were inhibitory to the luminescence of the H2O2-peroxidase-luminol system used in the coupling reaction, whereas EDTA and EGTA activated the system. In addition, Hg2+, which inhibits glucose oxidase, and Tris ion, which inhibits linamarase, both interfered with the new assay.     

Ultrasensitive chemiluminescence assay for cimetidine detection based on the synergistic improving effect of Au nanoclusters and graphene quantum dots

A novel and sensitive chemiluminescence (CL) procedure based on the synergetic catalytic effects of gold nanoclusters (Au NCs) and graphene quantum dots (GQDs) was developed for the reliable measurement of cimetidine (CM). The initial experiments showed that the KMnO₄‐based oxidation of alkaline rhodamine B (RhoB) generated a very weak CL emission, which was intensively enhanced in the simultaneous presence of Au NCs and GQDs. CL intermediates can be adsorbed and gathered on the surface of Au NCs, becoming more stable. GQDs participate in the energy transferring processes and facilitate them. These improving effects were simultaneously obtained by adding both Au NCs and GQDs into the RhoB‐KMnO₄ reaction. Consequently, the increasing effect of the Au NCs/GQDs mixture was more than that of pure Au NCs or GQDs, and a new nano‐assisted powerful CL system was achieved. Furthermore, a marked quenching in the emission of the introduced CL system was observed in the presence of CM, so the system was examined to design a sensitive sensor for CM. After optimization of influencing parameters, the linear lessening in CL emission intensity of KMnO₄‐RhoB‐Au NCs/GQDs was verified for CM concentrations in the range 0.8–200 ng ml^?1. The limit of detection (3S_b/m) was 0.3 ng ml^?1. Despite being a simple CL method, good sensitivity was obtained for CM detection with reliable results for CM determination in human urine samples.     

Detection of lipid hydroperoxides and hydrogen peroxide at picamole levels by an HPLC and isoluminol chemiluminescence assay

An isoluminol assay is utilized for the detection. of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described.     

A facile turn-on chemiluminescence probe for sensitive imaging on aminopeptidase N activity

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes ( APN-CL and BAPN-CL ) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN - CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN - CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN - CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN - CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.     

A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols

The lack of a simple assay for the quantification of S-nitrosothiols in complex biological matrices has hampered our understanding of their contribution to normal physiology and pathophysiological states. In this paper we describe an assay based upon the release of nitric oxide by reaction with a mixture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detect levels of S-nitrosothiols down to 5 nM in plasma. Following alkylation of free thiols with N-ethylmaleimide, and removal of nitrite with acidified sulfanilamide, we were able to measure known amounts of S-nitrosoalbumin added to plasma or whole blood, with an inter-assay variation for plasma S-nitrosothiols of ～4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28 ± 7 nM.