Luminol Chemiluminescence Method Research Articles

Антиоксидантный профиль ретроплацентарной крови при физиологической беременности и преэклампсии

The pathogenesis of pre-eclampsia (PE) is based on the disorders of placental circulation, which develop due to the impaired migration of extravillous trophoblast. Local processes at the maternal-fetal interface can be assessed through the retroplacental blood (RB) which flows during placental separation. Objective. To study the parameters of antioxidant status of RB in uncomplicated pregnancy and PE. Patients and methods. The study included 50 pregnant women: 36 women with uncomplicated pregnancy and 14 women with PE. Total antioxidant capacity (TAC) of plasma was measured by the luminol chemiluminescence method. Results. In uncomplicated pregnancy, total antioxidant capacity of RB was increased compared to peripheral blood (PB) (1236 ± 344 sec vs. 852 ± 246 sec; p < 0.0001), which indicates that RB provides a balance of pro-oxidants and antioxidants at the maternal-placental-fetal interface. The increase in total antioxidant capacity of RB plasma in PE compared to uncomplicated pregnancy (1716 ± 922 sec vs. 1236 ± 344 sec; p = 0.007) may be both compensatory and pathogenetic mechanisms of the disease. Thus, the prevention and treatment of PE with antioxidants should be discussed. The increase in total antioxidant capacity of RB plasma was observed in severe PE (2304 ± 948 sec vs. 1236 ± 344 sec, p < 0.0001), which is associated with an unfavorable prognosis. Conclusion. Our pilot study of the antioxidant defense status of retroplacental blood shows that pre-eclampsia leads to a significant imbalance between pro-oxidants and antioxidants at the maternal-placental-fetal interface. Key words: antioxidants, antioxidant capacity, pre-eclampsia, retroplacental blood, chemiluminescence

Simultaneous and Sensitive Determination of Levodopa and Carbidopa in Pharmaceutical Formulation and Human Serum by High Performance Liquid Chromatography with On-Line Gold Nanoparticles-Catalyzed Luminol Chemiluminescence Detection

Abstract Levodopa, the metabolic precursor of dopamine, is usually administrated in combination with carbidopa to control dopamine levels in an appropriate manner and reduce side effects in the treatment of Parkinson's disease. In this study, a selective and sensitive high performance liquid chromatography coupled with on-line gold nanoparticles-catalyzed luminol chemiluminescence method for simultaneous determination of levodopa and carbidopa was developed. This method was based on the strongly enhanced chemiluminescence signal of on-line gold nanoparticles-catalyzed luminol-H2O2 system by levodopa and carbidopa. The possible enhancement mechanism was attributed to that levodopa and carbidopa could promote the on-line formation of a large number of gold nanoparticles, which catalyzed the luminol-H2O2 chemiluminescence reaction. The good separation of levodopa and carbidopa was achieved with isocratic elution using a mixture of methanol and 0.2% aqueous phosphoric acid (5:95, V/V) within 10.5 min. Under the optimal conditions, the linear ranges of levodopa and carbidopa were 2.24–448 ng mL−1 and 4.32–1080 ng mL− with the detection limits of 0.89 and 1.08 ng mL− (S/N = 3), corresponding to 17.92 and 21.60 pg for 20 μL sample injection, respectively. The validated method was successfully applied to simultaneous quantification of levodopa and carbidopa in controlled-release tablets (Sinemet®) and human plasma. The average recoveries of levodopa and carbidopa in the tablets were 100.5% and 103.1% with the precisions (RSDs) of 2.4% and 4.0%. The recoveries of levodopa and carbidopa in human plasma ranged from 97.0% to 103.5% with RSDs of no more than 3.3%.

GROUNDING OF POSSIBILITIES OF MORPHOLOGICAL INVESTIGATION METHODS APPLICATION IN TERMS OF PROCESS OF FREE RADICAL OXIDATION OF BIOPOLYMERS (LITERATURE REVIEW)

The conducted research revealed the fact that particular parts of free radical processes can currently be investigated and qualitatively calculated by means of morphological methods: relative concentration of nitro peroxides (luminol chemiluminescence method for frozen histological sections combined with computer assessment of the intensity of luminescence) oxidative modification of proteins (histochemical reaction “acidic” and “basic” proteins with blue Bromo by Mikel Calvo combined with microspectrophotometry at a rate of R/B), oxidation of sulfhydryl groups (histochemical reaction by Lillie combined with computer microdensitometry) antioxidant substances – total glutathione, glutathione-S-transferase, protein Bcl-2 (immunohistochemical method combined with computer microdensitometry).

Peroxiredoxin I in sperm and reactive oxygen species in seminal plasma in patients with idiopathic asthenozoospermia

To explore the relationship between peroxiredoxin I expression and seminal reactive oxygen species (ROS) in patients with idiopathic asthenozoospermia. Twenty-six infertile male patients were selected from the Reproductive Medical Center, Xiangya Hospital, from September to December in 2012. Fresh semen was collected from an experimental group (26 idiopathic asthenozoospermia patients) and a control group (15 men with fertility history and normal semen). Luminol chemiluminescence method was applied to detect the seminal ROS level. Western blot was used to detect the peroxiredoxin I expression. 1)The seminal ROS level in the experimental group was significantly increased compared with that in the control group (P<0.05), and the seminal ROS level was negatively correlated with mobility of the sperm (r=-0.777, P<0.01). 2) Compared with the control group, the peroxiredoxin I expression was significantly downregulated in the experimental group (P<0.05). The content of sperm peroxiredoxin I in the 2 groups was negatively correlated with the seminal ROS level (r=-0.474, P<0.01). 3) The content of peroxiredoxin I had a positive correlation with human sperm motility(r=0.779, P<0.01). The decline of peroxiredoxin I expression may be one of the crucial factors that leads to idiopathic asthenozoospermia. High level of ROS may be one of the main reasons for sperm vitality decline in patients with idiopathic asthenozoospermia.

A luminol chemiluminescence method for sensing histidine and lysine using enzyme reactions

The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl–tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid–aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0–30μM histidine and 1.0–60μM lysine.

Effects of KW-2228 on the function of polymorphonuclear neutrophils in rats for the phagocytosis and killing activity and the production of active oxygen

KW-2228 is a novel derivative of a recombinant human granulocyte colony-stimulating factor (rhG-CSF) with a modification. KW-2228 has some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. In the present study, we investigated the effect of KW-2228 on phagocytic capacity and killing action of polymorphonuclear neutrophils (PMNs) obtained from normal rats which had been treated with KW-2228 using various microorganisms, such as Candida albicans, Escherichia coli and Pseudomonas aeruginosa. We also investigated the effect of KW-2228 on the production of superoxide anion using a luminol-chemiluminescence method. KW-2228 enhanced the phagocytosis and killing activity of PMNs of rats against C. albicans and E. coli. PMNs treated with KW-2228 showed some bactericidal activity against P. aeruginosa. These data obtained with PMNs treated with KW-2228 show a correlation between the bacterial susceptibility to phagocytosis and killing action and the lunimol-dependent chemiluminescence response. These results suggest the significance and the efficacy of KW-2228 used in an additional therapy to that of antibiotics in the treatment of infections diseases. KW-2228 is currently in Phase III clinical trials in Japan.

Development of Selective Determination Methods for Quinones with Fluorescence and Chemiluminescence Detection and Their Application to Environmental and Biological Samples

Quinones are compounds that have various characteristics such as a biological electron transporter, an industrial product and a harmful environmental pollutant. Therefore, an effective determination method for quinones is required in many fields. This review describes the development of sensitive and selective determination methods for quinones based on some detection principles and their application to analyses in environmental, pharmaceutical and biological samples. Firstly, a fluorescence method was developed based on fluorogenic derivatization of quinones and applied to environmental analysis. Secondly, a luminol chemiluminescence method was developed based on generation of reactive oxygen species through the redox cycle of quinone and applied to pharmaceutical analysis. Thirdly, a photo-induced chemiluminescence method was developed based on formation of reactive oxygen species and fluorophore or chemiluminescence enhancer by the photoreaction of quinones and applied to biological and environmental analyses.

An ultrasensitive and highly selective determination method for quinones by high-performance liquid chromatography with photochemically initiated luminol chemiluminescence

Quinones are a class of compounds of substantial toxicological and pharmacological interest. An ultrasensitive and highly selective chemiluminescence (CL) method was newly developed for the determination of quinones based on the utility of photochemically initiated luminol CL. The method involved ultraviolet (UV) irradiation of quinones to generate reactive oxygen species (ROS) through the unique photosensitization reaction accompanied with the photolytical generation of 3,6-dihydroxyphthalic acid (DHPA) from quinones. The photoproducts were detected by luminol CL reaction. Interestingly, it was noticed that DHPA had enhancement effect for the luminol CL. The generation of the enhancer (DHPA) in association with the oxidant (ROS) in the photochemical reaction greatly increases the sensitivity and selectivity of the proposed luminol CL method. In order to elucidate the type of ROS produced by the photosensitizaion reaction in relation to the proposed CL reaction, we investigated the quenching effect of selective ROS scavengers in the luminol CL. Although several ROS were generated, superoxide anion was the most effective ROS for the generated CL. Moreover, the enhancement mechanism of DHPA for luminol CL was confirmed. The enhancement was found to be through the formation of stabilized semiquinone anion radical that provided long-lived CL. The generation of the semiquinone radical was confirmed by electron spin resonance technique. Furthermore, we developed an HPLC method with on-line photochemical reaction followed by the proposed CL detection for the determination of four quinones. A luminol analogue, L-012, was used for its high sensitivity. The detection limits for quinones obtained with the proposed method (S/N = 3) were in the range 1.5–24 fmol that were 10–1000 times more sensitive compared with the previous methods. Finally, the developed HPLC-CL system was successfully applied for the determination of quinones in airborne particulate samples collected at Nagasaki city.

Impact of natural organic matter on H 2O 2-mediated oxidation of Fe(II) in a simulated freshwater system

The oxidation of Fe(II) by H 2O 2 has been studied in the presence of Suwannee River fulvic acid, a standard form of natural organic matter, by adding inorganic Fe(II) to solutions containing both H 2O 2 and fulvic acid and monitoring the total Fe(II) concentration using a luminol chemiluminescence method. At pH 8.4 and in the absence of competing metals, Suwannee River fulvic acid significantly retards the rate of Fe(II) oxidation due to gradual formation of a species that is oxidized more slowly than inorganic Fe(II) by both O 2 and H 2O 2. It is suggested that rapid formation of a weak Fe(II)–fulvic acid complex that is not readily oxidized by H 2O 2 is the cause of the reduction in the initial oxidation rate, and that the subsequent further reduction in oxidation rate is a result of the formation of a second type of Fe(II)–fulvic acid complex that is resistant to both O 2 and H 2O 2 oxidation. A kinetic model has been developed that supports this conceptual model. The results demonstrate that, under certain conditions, natural organic matter may stabilize Fe(II) in the presence of elevated H 2O 2 concentrations, significantly increasing the lifetime of ferrous iron and reducing the flux of hydroxyl radicals produced through this oxidation pathway.

UTILIZATION OF JAPANESE AROMATIC SUBSTANCES FOR MILK PRESERVATION AS ESTIMATED BY VAPOR CONTACT METHOD

ABSTRACT In order to study the possibility of utilizing aromatic materials for the preservation of milk, the antibacterial effects of various aromatic materials against Pseudomonas fluorescens inoculated in milk were investigated. The effects of aromatic materials were generated using the “vapor contact method” and bacterial growth was measured by the menadione‐catalyzed luminol chemiluminescence method. Wasabi (Wasabi japonica) and mustard (Brassica nigra L.) were found to be more effective than garlic (Allium sativum L.), ginger (Zingiber officinale Roscoe), sansho (Zanthoxylum piperitum) and peppermint (Mentha piperita). In addition to these materials, four types of commercially available food preservation sheets were tested for antibacterial activity. Sheets containing mustard extracts showed stronger antibacterial activities. Eucalyptus extract‐treated sheets showed a moderate effect.PRACTICAL APPLICATIONSThe vapor contact method was advantageous when compared with the direct contact method, as the flavor of the milk was affected little by the aromatic substances. This study suggests that aromatic materials can be used to preserve milk in areas where milk quality is low or sanitary conditions are insufficient. Experimental results of food preservation sheets suggest that such technique can be applied for package of dairy foods, too.

A Discrepancy in Superoxide Scavenging Activity between the ESR-Spin Trapping Method and the Luminol Chemiluminescence Method

In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric-protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric-protein complex, the effects of other ferric-protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicum and Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric-protein complex, and that an extract of C. ovata did not. These results suggest that the presence of such a ferric-protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.

Porphyrin-Induced Photogeneration of Hydrogen Peroxide Determined Using the Luminol Chemiluminescence Method in Aqueous Solution: A Structure-Activity Relationship Study Related to the Aggregation of Porphyrin

A luminol chemiluminescence method was used to evaluate the porphyrin-induced photogeneration of hydrogen peroxide (H2O2). This method enabled us to detect H202 in the presence of a high concentration of porphyrin, which was not possible using conventional colorimetry. The limit of detection was about 1 microM. We compared the ability to generate H2O2, using uroporphyrin (UP), hexacarboxylporphyrin (HCP), coproporphyrin (CP), hematoporphyrin (HP), mesoporphyrin (MP), and protoporphyrin (PP). The amount of H2O2 photoproduced was strongly related to the state of the porphyrin in the aqueous solution. UP and HCP, which existed predominantly in a monomeric form, had a good ability to produce H2O2. HP and MP, existing as dimers, showed weak activity. CP, forming a mixture of monomer and dimer, had a moderate ability to produce H2O2. PP, which was highly aggregated, had a good ability. These results demonstrated that the efficiency of porphyrins to produce H2O2 was strongly dependent on their aggregated form, and the dimer suppressed the production of H2O2.

Relationship between peroxyl radical scavenging capability measured by the chemiluminescence method and an aminocarbonyl reaction product in soy sauce

Oxygen-related free radicals have been suggested as a cause of aging and various diseases, for example, various cancers and rheumatoid arthritis. Because of this a radical scavenger as an antioxidant has been sought in food materials. Soy sauce is a traditional fermented seasoning of East Asian countries and is available throughout the world. The relationship between the peroxyl radical scavenging capability using the luminol chemiluminescence method and melanoidin, the main product from aminocarbonylation, i.e., Maillard reaction, from soy sauce was examined. In this study, we report that soy sauce has a very high antioxidative capacity and from the comparisons of the IC50 values of 26 soy sauces in the case of the optical density of the soy sauce's color being standardized, it was found that not only melanoidin is an antioxidative product, but also other products have strong antioxidative properties.

Peroxyl radical scavenging capability of fish sauces measured by the chemiluminescence method

Oxygen-related free radicals have been suggested as a cause of aging and various diseases, for example, various cancers and rheumatoid arthritis. A radical scavenger as an antioxidant has been sought in foods. Fish sauces are traditional Asian fermented seasonings. Using the luminol chemiluminescence method, the peroxyl radical scavenging capability of fish sauces was examined. From the IC50 values, many fish sauces have been shown to have a strong scavenging capability as well as soy sauces. A scavenging mechanism is also proposed.

Portable flow-injection analyzer with liquid-core waveguide based fluorescence, luminescence, and long path length absorbance detector

We describe a multifunctional flow analysis instrument that is portable ( 25 cm×20 cm×11 cm, 2.3 kg) for facile field deployment. Using a 50 cm long Teflon ® AF tubing as final reaction and optical measurement conduit, we combine a liquid-core waveguide (LCW) based fluorescence detector that is transversely illuminated by an addressable light emitting diode array, a chemiluminescence (CL) detector and an absorbance detector with a solid-state broadband (400–700 nm) source. Several illustrative experiments have been carried out to test the performance of the instrument in different detection modes. A S/N=3 limit of detection (LOD) of 0.25 μg l −1 for chromium(VI) was established using the diphenylcarbazide chemistry, and an LOD of 5 μg l −1 was similarly established for Al(III), using Pyrocatechol Violet (PCV) as the chelating chromogenic dye, in both cases using long path absorption detection. The LOD for aqueous hydrogen peroxide was 16 nM using a fluorescence method based on the formation of thiochrome from thiamine and 4 nM by a luminol chemiluminescence method. With a Nafion membrane diffusion scrubber (DS), the LOD was 8.0 pptv for gaseous hydrogen peroxide by the fluorescence method.

Development of a deep-sea in situ Mn analyzer and its application for hydrothermal plume observation

Abstract This paper presents the first in situ flow-through chemical analyzer using a chemiluminescence (CL) method in the deep sea to a depth of 5200 m. The analyzer, called GAMOS (Geochemical Anomalies MOnitoring System), determines the concentration of dissolved manganese continuously using a H 2 O 2 –luminol CL method. A detection limit of 0.23 nM was obtained. The GAMOS was used successfully for hydrothermal plume observation.

Preparation of Antibody-Coupled Liposomes Containing Horseradish Peroxidase as a Marker Molecule

Horseradish peroxidase (HRP) was encapsulated in liposomes prepared by an extrusion technique. The liposomes were coupled covalently to anti-rabbit IgG using N-hydroxysuccinimide ester palmitic acid as a component of liposomes. The number of encapsulated HRP molecules per liposome was about 800. A large portion of HRP was encapsulated inside the liposomes for about one week at 4°C. The catalytic activity of HRP was measured by a luminol chemiluminescence (CL) method and was found to be almost constant during storage. The CL intensity per antibody in the detection of HRP encapsulated in the antibody-coupled liposomes was 125-times greater than that of HRP conjugated directly to the antibody.

Resolution of Amino Thiols in Time-Resolved Luminol Chemiluminescence Catalyzed by Peroxidases

Hydrogen peroxide (H2O2) formed during the course of the Cu(II)-catalyzed oxidation of amino thiols with oxygen was detected by the Arthromyces ramosus peroxidase-catalyzed luminol chemiluminescence (CL) method. Two peaks appeared in the CL response curve in the catalytic oxidation of both cysteine (CySH) and gluthathione (GSH) and in that of both cysteamine and GSH. The resolution of two peaks corresponding to CySH and GSH was better than that corresponding to cysteamine and GSH. Only a strong CL flash appeared in the catalytic oxidation of both CySH and cysteamine. The differences in the resolution of two peaks of amino thiols could be explained on the basis of the differences in the stabiliy constants between Cu(II) and amino thiols.

Phorbol 12-myristate 13-acetate (PMA)-induced oxyradical production in rheumatoid synovial cells.

We successfully detected the oxyradical production in human synovial A (macrophage-like) and B (fibroblast-like) cells by phorbol 12-myristate 13-acetate (PMA) using the luminol-chemiluminescence method. The PMA (0.1 microgram/ml)-induced photon generation was abolished by an O2- scavenger, superoxide dismutase, and an H2O2 scavenger, catalase, suggesting that the stimulus produced oxyradicals in synovial cells. Both of these responses were abolished by a protein kinase C (PKC) inhibitor, calphostine C, but unaffected by an intracellular Ca2+ chelator, BAPTA-AM, and Ca2+ removal from the extracellular medium. These findings suggest that synovial A and B cells produce oxyradicals through PKC-mediated and [Ca2+]i-independent mechanisms, probably through the activation of NADPH oxidase.

Phorbol 12-Myristate 13-Acetate (PMA)-Induced Oxyradical Production in Rheumatoid Synovial Cells

We successfully detected the oxyradical production in human synovial A (macrophage-like) and B (fibroblast-like) cells by phorbol 12-myristate 13-acetate (PMA) using the luminol-chemiluminescence method. The PMA (0.1 μg/ml)-induced photon generation was abolished by an O2− scavenger, superoxide dismutase, and an H202 scavenger, catalase, suggesting that the stimulus produced oxyradicals in synovial cells. Both of these responses were abolished by a protein kinase C (PKC) inhibitor, calphostine C, but unaffected by an intracellular Ca2+ chelator, BAPTA-AM, and Ca2+ removal from the extracellular medium. These findings suggest that synovial A and B cells produce oxyradicals through PKC-mediated and [Ca2+]i-independent mechanisms, probably through the activation of NADPH oxidase.