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PubMedDate not supplied

Chemiluminescence assay of lipase activity using a synthetic substrate as proenhancer for luminol chemiluminescence reaction.

A novel chemiluminescence (CL) assay method for lipase (triacylglycerol lipase, E.C.3.1.1.3) activity was developed by using the lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI) as a substrate. The method is based on the enhanced CL reaction of luminol-hydrogen peroxide-horseradish peroxidase (HRP) with HDI that is liberated from the substrate by enzymatic hydrolysis. To simplify the assay procedure, both the hydrolysis of the substrate and the enhanced CL reaction were performed in the same reaction mixture. Lipases from Candida cylindracea and porcine pancreas were successfully determined with the detection limits (blank signal + 3 SD) of 0.05 and 50.0 mU/tube, respectively. The method is simple and rapid, permitting the completion of single assay within 5 min. The reproducibilities obtained with replicate assays were relative standard deviations (RSDs) of <=> 4.7% for within-day and <=> 6.0% for between-day assays.

Animals↗
PubMedDate not supplied

Interaction of metals with peroxidase--mediated luminol-enhanced, chemiluminescence (PLmCL).

The peroxidase-mediated luminol-enhanced chemiluminescence (PLmCL) method has been used to study the in vitro effect of contaminants such as heavy metals on the reactive oxygen species production by immunocytes. We were interested to know whether metals could directly affect peroxidase-mediated luminescence, taking horseradish peroxidase (HRP) as a model enzyme, since this could contribute to the inhibition of immunocyte LmCL. Copper inhibited PLmCL in a dose-dependent manner, while cadmium, iron, silver and lead only partly decreased the signal in the concentration range tested. In contrast, zinc enhanced the signal at high concentrations. Eventually, chromium, mercury and aluminium did not affect PLmCL. It is suggested that these effects reflect the ability of the metals to interact with the active site of the peroxidase. These results demonstrate that such interactions have to be considered when interpreting the effects of metals on immunocytes using the LmCL method.

Animals↗
PubMedDate not supplied

Post-chemiluminescence behaviour of Ni2+, Mg2+, Cd2+ and Zn2+ in the potassium ferricyanide-luminol reaction.

A new chemiluminescence (CL) reaction was observed when Ni2+, Mg2+, Cd2+ or Zn2+ was injected into the reaction mixture after the finish of the CL reaction of alkaline luminol and potassium ferricyanide. This reaction is described as a post-chemiluminescence (PCL) reaction. The possible mechanism for the PCL was proposed based on studies of the CL kinetic characteristic and the CL spectra. The experimental conditions of the CL reactions were optimized and the feasibility of using the reaction to analyse these metal ions was evaluated. The PCL reaction method operates in the ranges: 1 x 10(-7)-8 x 10(-6) g/L Ni2+; 3 x 10(-6)-2 x 10(-4) g/L Mg2+; 8 x 10(-7)-1 x 10(-4) g/L Cd2+; and 2 x 10(-4)-2 x 10(-3) g/L Zn2+, with detection limits of 4 x 10(-8) g/mL, 1 x 10(-6) g/mL, 3 x 10(-7) g/mL, 8 x 10(-5) g/mL, respectively.

Cadmium↗
PubMedDate not supplied

Flow-injection determination of streptomycin residues in milk using the luminol-periodate-Mn2+ chemiluminescence system.

A novel flow-injection method with chemiluminescence detection was developed for the determination of streptomycin residues in milk, based on the enhancement by streptomycin of the luminol-potassium periodate-Mn(2+) chemiluminescence system. The assay allowed analysis of streptomycin residues in whole milk samples ( approximately 3.5% fat) after sample work-up procedures. The limit of detection was 5.16 x 10(-9) mol/L for milk, and was far below the regulations of maximum residue limits (MRLs). The relative standard deviation (RSD) for 8.66 x 10(-8) mol/L streptomycin was 1.94% (n = 15) in milk. The results obtained for the assay of streptomycin residues in milk was comparable with those obtained by the official methods (ELISA) and demonstrated good accuracy and precision. The possible mechanism is briefly discussed.

Animals↗
PubMedDate not supplied

Inhibition and enhancement by organic compounds of luminol-KIO4-H2O2 chemiluminescence.

The effects of 36 organic compounds on luminol-KIO(4)-H(2)O(2) chemiluminescence (CL) were studied. It was found that most of the tested compounds could inhibit or enhance the CL intensity. The activities of such inhibitors or enhancers were related to the pH of the CL system and the number and position of functional groups such as -OH and -NH(2) on aromatic rings. The mechanism of the CL inhibition and enhancement was considered. Based on the CL inhibition or enhancement, the possibility of analytical applications was explored. The results demonstrated that numerous compounds were detectable at the ng/mL level using the CL system.

Hydrogen Peroxide↗
PubMedDate not supplied

Chemiluminescence flow-injection analysis of beta-lactam antibiotics using the luminol-permanganate reaction.

A new flow injection chemiluminescence (CL) method has been developed for the determination of six beta-lactam antibiotics, including amoxicillin, cefadroxil, cefoperazone sodium, cefazolin sodium, cefradine and ceftriaxone sodium. When the antibiotic was injected into a stream of KMnO4 with alkaline luminol, a strong CL signal was produced. The method allows the measurements of 0.1-50.0 mg/L amoxicillin, 0.1-80.0 mg/L cefadroxil, 1.0-30.0 mg/L cefoperazone sodium, 1.0-30.0 mg/L cefazolin sodium, 3.0-50.0 mg/L cefradine and 3.0-50.0 mg/L ceftriaxone sodium. The detection limits are 0.05 mg/L for amoxycillin, 0.05 mg/L for cefadroxil, 0.4 mg/L for cefoperazonum sodium, 0.4 mg/L for cefazolin sodium, 0.8 mg/L for cefradine and 0.8 mg/L for ceftriaxone sodium. The relative standard deviations in 11 repeated measurements are 0.6%, 0.8%, 1.5%, 1.2%, 0.4% and 0.3% for 3.0 mg/L amoxicillin, 1.0 mg/L cefadroxil, 10.0 mg/L cefoperazone sodium, 10.0 mg/L cefazolin sodium, 10.0 mg/L cefradine and 10.0 mg/L ceftriaxone sodium, respectively. The method was successfully applied to the determination of amoxicillin in pharmaceutical preparations. A possible CL reaction mechanism is also discussed.

Amoxicillin↗
PubMedDate not supplied

A sensitive inhibition chemiluminescence method for the determination of trace tannic acid using the reaction of luminol-hydrogen peroxide catalysed by tetrasulphonated manganese phthalocyanine.

A novel CL method for the determination of tannic acid (TA) was established based on TA inhibition of the chemiluminescence (CL) emission of the luminol-hydrogen peroxide CL system catalysed by tetrasulphonated manganese phthalocyanine (MnTSPc) under alkaline conditions. The peak height is proportional to the natural logarithm of concentration of TA in the range 1.0 x 10(-7)-3.0 x 10(-10) mol/L with a detection limit of 5.6 x 10(-11) mol/L and the relative standard deviation was 2.5% for the determination of 1.0 x 10(-8) mol/L TA (n = 11). The proposed method has been successfully applied to the determination of TA in Chinese gall and industrial wastewater.

Catalysis↗
PubMedDate not supplied

Studies on PVP hydrogel-supported luminol chemiluminescence: 1. Kinetic and mechanistic aspects using multivariate factorial analysis.

The chemiluminescent oxidation of luminol by hydrogen peroxide in the presence of hemin is revisited in an UV-C cross-linked PVP hydrogel. Chemiluminescence properties such as initial light intensity (I(0)), area of emission (S) and observed rate constants (k(obs)) are studied, varying the concentration of all reactants using a multivariate factorial approach.

Cross-Linking Reagents↗
PubMedDate not supplied

High performance liquid chromatography of penicillins with penicillin-enhanced luminol chemiluminescence detection.

A high performance liquid chromatographic method with chemiluminescence detection for the determination of penicillin G and ampicillin is reported. The method is based on the enhancement of the luminol chemiluminescence with beta-lactam antibiotics. The linear relationship was obtained between the peak height and the concentration of penicillin G or ampicillin up to 15 nmol per 20 microL injection. Detection limits were 1 nmol for penicillin G and 0.5 nmol for ampicillin with a signal-to-noise ratio of 2. Relative standard deviations for five replicate measurements of 4 nmol/injection each of penicillin G and ampicillin were 2.1 and 2.3%, respectively.

Ampicillin↗
PubMed1995

The increasing effect of some synthetic peptides on luminol-dependent chemiluminescence of mouse blood.

Some synthetic peptides increased the luminol-dependent chemiluminescence of mouse blood during phagocytosis. It is suggested that the levels of antimicrobial activity of the neutrophil peroxidase system can be raised very quickly (within some dozen of seconds) by these peptides. This raises the possibility of finding a new approach to the therapy for infectious diseases.

Amino Acid Sequence↗
PubMed1989

Characterization of the luminol-amplified light-generating reaction induced in human monocytes.

Increased production of oxidative metabolites following interaction between mononuclear phagocytes and soluble stimuli can be measured as luminol-amplified chemiluminescence (CL). The effects of superoxide dismutase (SOD), catalase, and azide on the monocyte CL response were investigated. Azide, a myeloperoxidase (MPO) inhibitor, reduced the CL reaction by more than 80%, which indicates that the CL reaction is dependent on the granule enzyme MPO. Because SOD and catalase only partly inhibited the monocyte CL response, the authors propose that part of the monocyte CL response is of intracellular origin. This conclusion is further supported by the effects on the CL response obtained by adding extra peroxidase and the lack of correlation with techniques measuring only extracellular generated metabolites. However, it should be pointed out that the relation between extracellular and intracellular activity is stimulus dependent. Furthermore, even if quantitative differences exist between monocyte and granulocyte CL, the mechanism for the light-generating reaction seems to be the same in both cell types.

Azides↗
PubMed1994

Inhibition of luminol-enhanced chemiluminescence by reduced pterins.

Luminol-enhanced chemiluminescence, initially induced by exogenous H2O2 or enzymatic reactions which generate reactive oxygen species in situ, is inhibited by reduced pterins. Except dihydroneopterin which is as effective as tetrahydroneopterin, all tetrahydropterins are more effective than dihydropterins, which in turn is more effective than oxidized pterins in removing reactive oxygen species such as O2-. and H2O2. The antioxidant efficacy of some reduced pterins is better than ascorbic acid and L-glutathione. Reduced pterins may act as physiological scavengers for reactive oxygen species, and play an important role in cellular defense against oxidative damage.

Antioxidants↗
PubMed1995

Luminescent visualization of low amounts of cytochrome P450 and hemoproteins by luminol in acrylamide gels.

In this paper we apply luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), a light-emitting substrate, in conjunction with H2O2 to the luminescence labeling of hemoproteins. We describe in detail a photodetection device which permits an efficient recording of the light emitted by heme-containing proteins resolved in acrylamide gels. The sensitivity of this procedure, when compared to the classical 3,3'-5,5'-tetramethylbenzidine staining method, results in a 5- to 20-fold enhancement with standard hemoproteins (lactoperoxidase, catalase, cytochrome P450 2B4, horseradish peroxidase, hemoglobin, myoglobin, and cytochrome b5). The potential applications of this technique are illustrated by the detection of cytochrome P450 in microsomes from plant as well as from animal extrahepatic tissues which possess low amounts of this cytochrome.

Animals↗
PubMed1996

Synthesis and characterization of 4-iodophenylboronic acid: a new enhancer for the horseradish peroxidase-catalyzed chemiluminescent oxidation of luminol.

4-Iodophenylboronic acid has been synthesized and shown to be a potent enhancer of the chemiluminescent horseradish peroxidase (Type VI-A)-catalyzed oxidation of luminol and isoluminol. The enhancer was effective (>100-fold enhancement) in the concentration range 10-1500 microM. Light emission in the presence of the enhancer peaked at 5-10 min after initiation of the reaction and then decayed very slowly. 4-Iodophenylboronic acid also enhanced reactions catalyzed by horseradish peroxidase Type IX. The detection limit for Type VI-A horseradish peroxidase was 509 amol, and optimum signal enhancement was obtained at 769 microM compared to 154 microM for 4-iodophenol under the same conditions.

Blotting, Western↗
PubMed1995

Spontaneous luminol-dependent chemiluminescence of thrombocytes increases in hemorrhagic fever with renal syndrome.

Spontaneous luminol-dependent chemiluminescence (CL) of thrombocytes was studied in 12 patients with hemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus. CL in the acute phase of HFRS (median, 61 mV/10 sec; range, 22-91) was higher than that of healthy controls (median, 22 mV/10 sec; range, 18-29; P < 0.01) and patients with acute hepatitis B infection accompanied by thrombocytopenia (median, 33 mV/10 sec; range, 13-45; P < 0.01). CL of thrombocytes in the convalescence phase of HFRS (median, 31 mV/10 sec; range, 12-80) was significantly lower than that during the acute phase (P < 0.01). Neither was there a difference in CL response between HFRS patients with or without thrombocytopenia, but both groups produced a higher CL response than that of the control groups. Both the complement-containing and complement-free sera from acute HFRS patients (final dilutions: range 1:10-1:2560) exhibited no effect on CL response of normal thrombocytes. A possible role of increased oxygen radicals production by thrombocytes in pathogenesis of HFRS is discussed.

Animals↗
PubMed2002

N-acetylcysteine effect on the luminol-dependent chemiluminescence pattern of reactive oxygen species generation by human polymorphonuclear leukocytes.

The evidence of the effect of N-acetylcysteine on reactive oxygen species produced by human polymorphonuclear leukocytes (PMNs) is often contradictory, as thiol compounds may react with not only reactive oxygen and nitrogen species but also they may influence intracellular glutathione levels. The effect of 20, 100 and 200 microM N-aceylcysteine (NAC) on luminol dependent chemiluminescence (LDCL) of human PMNs (10(6) cells/ml phospate buffered saline (PBS)) and whole blood to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol-12-myristate-13-acetate (PMA) was studied. Further, the ability of NAC to increase PMNs intracellular thiols and affect subsequent PMNs, stimulation was assessed. NAC 100 and 200 microM, but not 20 microM, was found to attenuate the kinetic parameters of initial phase of fMLP-stimulated PMNs oxidative burst. NAC at the concentration of 100 and 200 microM decreased the rate, maximum and integrated value of PMNs response to fMLP. The integrated value of PMA-induced PMNs and fMLP-induced whole blood LDCL response was also decreased by 100 and 200 microM NAC. Furthermore, all tested NAC concentrations decreased LDCL of resting PMNs suspension. The chemiluminescence pattern of reactive oxygen species (ROS) generation by PMNs stimulated with fMLP or PMA did not differ significantly from those preincubated with either 20, 100, or 200 microM NAC. Similarly, NAC did not increase the concentration of intracellular thiols in resting PMNs. However, addition of 200 microM NAC to PMA-stimulated PMNs prevented the decline in intracellular thiols pool. Both PMA- and fMLP-activated PMNs oxidized extracellular NAC. These results indicate that NAC decreases the intensity of PMNs oxidative burst by direct scavenger activity.

Acetylcysteine↗
PubMed1998

Ortho-substituted polychlorinated biphenyls activate respiratory burst measured as luminol-amplified chemoluminescence in human granulocytes.

The effect of polychlorinated biphenyls (PCBs) on the activation of respiratory burst measured as luminol-amplified chemoluminescence in human granulocytes is elucidated here. Chemoluminescence was stimulated in a concentration-dependent manner (ED50 approximately 10 microM) by ortho-substituted PCB congeners, while meta- and para-substituted congeners had no significant effect. Two ortho-substituted PCB congeners were chosen for the mechanistic studies, namely 2,2',4,4'-TeCB and 2,2'-DCB, since they have been used in previous studies by others. In the absence of extracellular calcium, the respiratory burst in response to 2,2'-DCB and 2,2',4,4'-TeCB was reduced by 63% and 82%, respectively. Bisindolylmaleimide, which inhibits protein kinase C, reduced activated chemoluminescence by 2,2'-DCB, 2,2',4,4'-TeCB, N-formyl-methionyl-leucyl-phenylalanine, and phorbol 12-myristate 13-acetate. Neomycin, which inhibits phospholipase C, had a slight, but significant, effect on the 2,2',4,4'-TeCB-activated chemoluminescence but had a more pronounced effect on the 2,2'-DCB-activated chemoluminescence. 2,2'-DCB and 2,2',4,4'-TeCB significantly increased phospholipase D (PLD) activity measured as the amount of 14C-phosphatidylbutanol formed. Ethanol (1%), a phospholipase D modulator, reduced the response to 2,2'-DCB and 2,2',4,4'-TeCB by 72% and 75%, respectively. Furthermore, wortmannin (25 nM), a phosphatidylinositol 3-kinase, and genistein, a more unspecific tyrosine kinase inhibitor, reduced chemoluminescence in response to PCB. In conclusion, our results indicate that PCB-activated chemoluminescence is dependent on the Ca(2+)-dependent phospholipase D or phospholipase C, phosphatidylinositol 3-kinase, and protein kinase C activation prior to activation of the NADPH oxidase. Defects in neutrophhil functions upon exposure to PCB may render a greater susceptibility in the host to invading microorganisms or evoke inappropriate inflammatory responses leading to tissue injury.

Androstadienes↗
PubMed2005

Echinoderm reactive oxygen species (ROS) production measured by peroxidase, luminol-enhanced chemiluminescence (PLCL) as an immunotoxicological tool.

The importance of reactive oxygen species (ROS) production in invertebrate immunity prompted the use of this response in immunotoxicological studies in several taxa including marine organisms. In this chapter, we review the effects of environmental factors and contaminants such as heavy metals and polychlorinated biphenyls (PCBs) on the production of ROS by the main immune effector cells of echinoderms, the so-called amoebocytes. ROS production was measured by the peroxidase, luminol-enhanced chemiluminescence (PLCL) method. This method was found to predominantly reflect the production of superoxide anions and peroxides, among which hydrogen peroxide and peroxynitrite are the main species detected. Exogenous factors such as water temperature and salinity can influence this immune response in echinoderms. However, gender, handling stress and parasitism by a castrating ciliate apparently did not affect it. The impact of metals on ROS production differed greatly according to the duration and routes of exposure; in vitro and short-term in vivo exposures to metals caused an inhibition of this immune response, while the opposite effect was observed in a long-term in vivo exposure study. On the other hand, PCBs systematically had a stimulatory effect on ROS production independent of the echinoderm species or exposure routes. From the study of complex field contaminations, it appeared that contaminants released in the environment, such as metals, modulate starfish amoebocyte ROS production. This impact potentially represents a threat to the sustainability of natural populations of echinoderms and thereby to the stability of benthic ecosystems.

Allergy and Immunology↗