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See detailPossible intracellular effect of the new water-soluble form of curcumin (NDS27) on the oxidant response of stimulated neutrophils
Derochette, Sandrine ULg; Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette et al

Poster (2012, April 18)

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative ... [more ▼]

Neutrophils (PMNs) are involved in host defense against infections through the production of reactive oxygen species (ROS) to kill pathologic agents. But, an excessive ROS production, called “oxidative stress” is associated with tissue damages and development of chronic or acute inflammatory diseases. PMNs are prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenolic molecules to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27 (1), a new highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs. NDS27 (10-6 to 10-4 M) was pre-incubated with cells and eliminated before their activation. The ability of NDS27 to enter into the cells was checked by HPLC from the cellular extracts. The intracellular ROS production by phorbol myristate acetate (PMA) stimulated PMNs was measured by fluorescence using 2’,7’-dichlorofluorescin diacetate. Lucigenin dependent chemiluminescence was used to measure extracellular ROS production. Additionally, the effect of NDS27 was tested on the activity of myeloperoxidase (MPO), a hemic enzyme contributing to the oxidant response of neutrophils. The activity of the released MPO by cytochalazine B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated PMNs was measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”) (2). The HPLC results showed that NDS27 enters into PMNs and interacts with their membrane. NDS27 significantly and dose-dependently inhibited the ROS production in neutrophils without affecting their viability. Likewise, the activity of MPO released by PMNs was lowered by NDS27. Overall, our findings demonstrate that the membrane of neutrophils is permeable to NDS27 or interacts with the drug, suggesting that its inhibitory effect on ROS production is mainly associated to an intracellular effect probably by acting on the enzymes implied in respiratory burst like NADPH oxidase and MPO. The modulatory effect of NDS27 towards the oxidant activity of cells involved in immune and inflammatory response open therapeutic perspectives to control equine or human pathologies with excessive inflammatory reactions. 1. Neven et al. 2011, Patent Application Publication: US2011/0257126 A1 2. Serteyn et al. 2005, European Patent Specification : EP1711817 B1 [less ▲]

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See detailAssessment of reactive oxygen species production in cultured equine skeletal myoblasts in response to conditions of anoxia followed by reoxygenation with or without exposure to peroxidases.
Ceusters, Justine ULg; Mouithys-Mickalad, Ange ULg; de la Rebière de Pouyade, Geoffroy ULg et al

in American Journal of Veterinary Research (2012), 73(3), 426-434

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the ... [more ▼]

Objective—To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. Animals—5 healthy horses (5 to 15 years old). Procedures—Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding. Results—Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. Conclusions and Clinical Relevance—Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation– treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses. [less ▲]

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See detailAntioxidant and anti-inflammatory like properties of benzoic acid analogs on the oxidant response of neutrophils: structure/redox potential relationship study.
Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg; Robert, Thierry ULg et al

in Free Radical Biology & Medicine (2012), 53(supplement 1),

We investigated the antioxidant capacity of phenolic acid derivatives by measuring their capacity to prevent ABTS oxidation and evaluating their anti-inflammatory like-properties on the oxidant response ... [more ▼]

We investigated the antioxidant capacity of phenolic acid derivatives by measuring their capacity to prevent ABTS oxidation and evaluating their anti-inflammatory like-properties on the oxidant response of neutrophils, especially on superoxide anion production and the activity of myeloperoxidase (MPO), an oxidant enzyme present and released by the primary granules of neutrophils. The superoxide anion production by PMA-stimulated neutrophils was measured by lucigenin-enhanced chimiluminescence (CL) and the activity of MPO by SIEFED to study the potential interaction of a molecule with the enzyme without interferences due to medium. The antioxidant and anti-inflammatory activities of the phenolic compounds were correlated to their redox potentials measured by voltammetry method, and discussed in relation to their molecular structure. The ability of the phenolic molecules to decrease ABTS oxidation and CL production was inversely correlated to their redox potential increasing as follows: propyl gallate > gallic acid > caffeic acid > 3,4-dihydroxybenzoic acid > ferulic acid > syringic acid > 2,6-dihydroxybenzoic acid > salicylic acid > benzoic acid. The number of hydroxyl groups (3) and their position (catechol) were essential for the efficacy of the molecules as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential and increased as follows: gallic acid > caffeic acid > 2,6-dihydroxybenzoic acid > propyl gallate > ferulic acid = syringic acid > 3,4-dihydroxybenzoic acid = salicylic acid > benzoic acid. The number of OH groups and the elongation of the carboxyl group were essential for the inhibition of MPO activity, probably by facilitating the interaction with the MPO active site or structure. The redox potential measurement seems to be a good technique to select stoichiometric antioxidants, but not anti-catalytic ones. [less ▲]

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See detailPolyphenol Content and Modulatory Activities of Some Tropical Dietary Plant Extracts on the Oxidant Activities of Neutrophils and Myeloperoxidase
Tsumbu, César Ndele ULg; Deby-Dupont, Ginette; Tits, Monique ULg et al

in International Journal of Molecular Sciences (2012), 13(1), 628-650

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See detailAntioxidant and Antiradical Activities of Manihot esculenta Crantz (Euphorbiaceae) Leaves and Other Selected Tropical Green Vegetables Investigated on Lipoperoxidation and Phorbol-12-myristate-13-acetate (PMA) Activated Monocytes
Tsumbu, César Ndele ULg; Deby-Dupont, Ginette; Tits, Monique ULg et al

in Nutrients (2011), 3(9), 818-838

Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by ... [more ▼]

Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration. [less ▲]

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See detailSpecific Immuno Extraction Followed by Enzymatic Detection (SIEFED), a new tool to measure active myeloperoxidase in complex media and to screen potential inhibitors of this enzyme.
Serteyn, Didier ULg; Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette et al

in The 7th International Human Peroxidase Meeting, 22-25 May 2011, Brussels (2011, May 25)

A method called SIEFED (“Specific Immuno Extraction Followed by Enzymatic Detection”) was developed for the specific measurement of myeloperoxidase (MPO) activity without interference of the sample medium ... [more ▼]

A method called SIEFED (“Specific Immuno Extraction Followed by Enzymatic Detection”) was developed for the specific measurement of myeloperoxidase (MPO) activity without interference of the sample medium. In this method, MPO is first extracted out of aqueous or biological samples by its capture by immobilized anti-MPO antibodies. A washing step then allows to eliminate the solution or biological fluid with interfering materials (proteins, lipids, reducing or oxidizing molecules, ...) and in a last step the activity of MPO bound to its antibodies is measured by using a sensitive detection system containing a fluorogenic substrate, hydrogen peroxide and nitrite as reaction enhancer. SIEFED is a method of choice to measure easily, quantitatively, and specifically the active part of MPO present in biological samples or complex media. Results obtained with this technique applied to biological samples emphasize the importance to distinguish the total MPO concentration of a sample obtained by ELISA from the active part of MPO, which is the real witness of the oxidant potential of the enzyme. SIEFED is also a powerful tool to study compounds or natural extracts that could have an inhibitory effect on the activity of MPO. Since the potential inhibitor is first incubated with the enzyme solution, and further eliminated by washing after the immunocapture of MPO, the tested compound or extract cannot interfere with the chromogenic substrate used to measure the activity of MPO or with products derived from the enzyme activity. Thus, an inhibitory effect could only be attributed to a direct interaction of the tested compound with the enzyme, either on the active site or another key position of the structure. In conclusion, the SIEFED technique opens new perspectives to study pathologies in which the release of active MPO is relevant and to select interesting compounds or extracts able to modulate the MPO activity. [less ▲]

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See detailEffect of benzoic acid analogs on myeloperoxidase activity measured by a new technique to study their direct interaction with the enzyme.
Franck, Thierry ULg; Mazloum, Ali; Mouithys-Mickalad, Ange ULg et al

Poster (2011, May)

Myeloperoxidase (MPO) plays a key role in inflammatory response and constitutes a target for new drug development. The effects of some benzoic acid analogs were studied on the specific activity of human ... [more ▼]

Myeloperoxidase (MPO) plays a key role in inflammatory response and constitutes a target for new drug development. The effects of some benzoic acid analogs were studied on the specific activity of human MPO measured by SIEFED (“Specific Immunologic Extraction Followed by Enzymatic Detection”), an original method that consists of incubation of the compound with MPO, followed by capture of the enzyme by specific antibodies, washing (elimination of the compounds) and enzymatic detection of the immunocaptured enzyme. The compounds tested at 10-4, 10-5 and 10-6 M were studied in terms of structure activity relationship. Gallic acid (3,4,5-trihydroxybenzoic acid) with 3 hydroxyl groups had an important dose dependent inhibitory effect on MPO activity. Other molecules with less or without hydroxyl groups [3,4-dihydroxybenzoic acid, 2-hydroxybenzoic acid (salicylic acid) and benzoic acid] had rather an activator effect at 10-5 and 10-6 M. 2,4,6-Trihydroxybenzoic acid, with two hydroxyl groups adjacent to the carboxyl group, had a less efficient inhibitory effect. Caffeic acid (3,4-dihydroxycinnamic acid) with a propenoic acid group presented a dose dependent inhibitory effect on MPO activity contrary to its analog 3,4-dihydroxybenzoic acid. The esterification of the carboxyl group of gallic acid to obtain propyl gallate induced an activation of MPO at 10-5 and 10-6 M. Finally, the substitution of one or two hydroxyl groups by methoxyl ones (ferulic and syringic acids) decreased the efficiency of the molecules on the enzyme inhibition. The SIEFED technique appears as an innovative pharmacological tool to study the direct interaction of compounds with MPO. Number and position of hydroxyl groups and the extension of the carboxyl group of benzoic acid play a crucial role in the inhibition of MPO activity probably by facilitating the interaction with the active site or another elements of the enzyme structure. [less ▲]

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See detailModulating effects of acepromazine on the reactive oxygen species production by stimulated equine neutrophils
Sandersen, Charlotte ULg; Mouithys-Mickalad, Ange ULg; de la Rebière de Pouyade, Geoffroy ULg et al

in Veterinary Anaesthesia & Analgesia (2011), 38

To investigate the effect of acepromazine (ACP) on reactive oxygen species (ROS) production by stimulated equine neutrophils.

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See detailProduction of free radicals and oxygen consumption by primary equine endothelial cells during anoxia-reoxygenation.
de la Rebière de Pouyade, Geoffroy ULg; Salciccia, Alexandra ULg; Ceusters, Justine ULg et al

in Open Biochemistry Journal (The) (2011), 5

The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function ... [more ▼]

The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced alpha-keto-gamma-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significantly decreased with the number of A/R cycles, and POBN-ESR spectra were specific of adducts formed in the cells from superoxide anion. After a one-hour A/R cycle, high intensity DMPO-ESR spectra were observed and assigned to superoxide anion trapping; the GC results confirmed an important production of ROS compared to normoxic cells. These results show that A/R induces mitochondrial alterations in endothelial cells, and strongly stimulates their oxidative activity as demonstrated by ESR and GC methods. [less ▲]

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See detailModulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity
Boly, Rainatou; Dessy, Stephanie; Kohnen, Stephan et al

in International Journal of Molecular Medicine (2011), 28

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See detailESR technique for non invasive way to quantify cyclodextrins effect on cell membranes
Grammenos, Angeliki ULg; Mouithys-Mickalad, Ange ULg; Guelluy, Pierre-Henri ULg et al

in Biochemical and Biophysical Research Communications (2010)

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of ... [more ▼]

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe,which can be further correlated with themicroviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes. [less ▲]

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See detailSynthesis of poly(vinyl alcohol)/C60 and poly(N-vinylpyrrolidone)/C60 nanohybrids as potential photodynamic cancer therapy agents
Hurtgen, Marie ULg; Debuigne, Antoine ULg; Mouithys-Mickalad, Ange ULg et al

in Chemistry : An Asian Journal (2010), 5(4), 859-868

Well-defined poly(vinyl acetate) (PVAc) and poly(N-vinylpyrrolidone)-co-poly(vinyl acetate) (PNVPco-PVAc) chains end-capped by Co-(acac)2 (acac=acetylacetonate) and prepared by cobalt-mediated radical ... [more ▼]

Well-defined poly(vinyl acetate) (PVAc) and poly(N-vinylpyrrolidone)-co-poly(vinyl acetate) (PNVPco-PVAc) chains end-capped by Co-(acac)2 (acac=acetylacetonate) and prepared by cobalt-mediated radical polymerization (CMRP) are grafted onto a fullerene. Homolytic Co-C bond cleavage of the polymer chain ends at 30°C releases the polymeric radicals that add onto C60, thereby leading to the corresponding PVAc/C60 and PNVP-co-PVAc/C60 nanohybrids. The [polymer–Co(acac)2]/[C60] molar ratio was varied to adjust the structure of the nanohybrids, and more particularly the number of grafted arms. Finally, the potential of the hydrosoluble PVOH/C60 nanohybrids, which result from the methanolysis of the ester groups of PVAc/C60, and of the PNVP-co-PVAc/C60 nanohybrids as photosensitizers for photodynamic therapy (PDT), was approached. First, photobleaching tests demonstrated the ability of these nanohybrids to produce singlet oxygen upon irradiation, which can play a role in cell damage. Second, cell viability assays demonstrated that both types of nanohybrids are deprived of intrinsic cytotoxicity in the dark, whereas they promoted significant cell mortality when subjected to light treatment. The selective response of these materials to irradiation makes them promising compounds for PDT. [less ▲]

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See detailLa microbiopsie musculaire : un nouvel outil pour le suivi sportif et la détection précoce des dysfonctions musculaires
Votion, Dominique ULg; Fraipont, Audrey ULg; Robert, Céline et al

in 36ème Journée de la Recherche Equine (2010)

The objective of this study was to confirm the practical value of high-resolution respirometry (HRR) applied to biopsies to determine, in horses, the level of training, their athletic ability and for the ... [more ▼]

The objective of this study was to confirm the practical value of high-resolution respirometry (HRR) applied to biopsies to determine, in horses, the level of training, their athletic ability and for the early detection of muscular dysfunction. Materials and methods – The muscle mitochondrial respiration was determined by HRR in 20 endurance horses and 10 trotters sampled at the triceps brachii at different stages of their training. Results – Training increases mitochondrial respiration, in addition, the best performers had the highest rate of respiration. A trotter had abnormally low levels of muscle mitochondrial respiration for its level of training. This horse has presented several episodes of rhabdomyolysis during its racing season. Discussion – The biopsy is easily achievable by the attending veterinarian. This study shows that the athletic ability of horses is closely linked to respiratory muscle function, and suggests the value of HRR for performance prediction. In addition, the RHR has the ability to demonstrate mitochondrial dysfunction potentially responsible for exercise-induced rhabdomyolysis. [less ▲]

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See detailEffect of intensive exercise on plasmatic neutrophil elastase level in eventing and endurance horses
Lejeune, Jean-Philippe ULg; Sandersen, Charlotte ULg; Votion, Dominique ULg et al

in Equine Veterinary Journal. Supplement (2010), 48

Reasons for performing the study – Intensive exercise induces a systemic inflammatory response characterized by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase ... [more ▼]

Reasons for performing the study – Intensive exercise induces a systemic inflammatory response characterized by an increase of blood neutrophil count and myeloperoxidase (MPO) release. Neutrophil elastase (NE) could also contribute to tissues lesions by their proteinase activities. Objective – To compare plasmatic NE concentrations before and after different forms of intensive exercise. Materials and Methods – EDTA blood samples were taken from 51 eventing horses (EvH) and 32 endurance horses (EndH) were sampled before the race (T0). Blood sampling was performed 2 h (T1) after completing either phase D of a one or two star eventing competition (n=51) or a 120 or 160 km endurance race (n=32). Plasmatic NE and MPO were measured by a specific equine ELISA. Neutrophil counts and creatine kinase (CK) levels were also measured. A Wilcoxon test for paired samples was used to compare mean values of neutrophils, CK, MPO and NE at T0 and T1 in EvH and in EndH. Correlations were calculated between all the 4 parameters in EvH and EndH. Results – At T0, mean NE levels were 14.43 ± 3.63 ng/ml for EvH and 11.7 ± 2.11 ng/ml for EndH. The competition induced a significant increase of NE levels in (58.57 ± 24.06 ng/mL) EvH and (95.74 ± 22.70 ng/mL) EndH (p < 0,05). NE was significantly (p < 0,0001) correlated to MPO in EvH (r = 0.293) and EndH (0.594) and to CK (r = 0.297) in EndH (p<0.0001). Neutrophils, CK and MPO were significantly increased between T0 and T1 in both types of horses. Conclusions – Significant increase of NE was observed after intense exercise with a significant correlation between NE and MPO. The huge variability in MPO and ELT, indicates, that not all horses show the same intensity of systemic inflammatory response. [less ▲]

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See detailAlterations in mitochondrial respiratory function in response to endurance training and endurance racing
Votion, Dominique ULg; Fraipont, Audrey ULg; Goachet, Anne-Gaëlle et al

in Equine Veterinary Journal. Supplement (2010), 42(38), 268-274

Objectives: To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in horses with high-resolution respirometry (HRR).

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See detailAMADEOX method : combination of various techniques to distinguish stoichiometric and anticatalytic activities of antioxidant molecules or natural extracts
Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg; Kohnen, Stephan et al

in International Society of Antioxidants in Nutrition & Health, 5th International Conference on Polyphenols Applications, Bridging Bioefficacy to Innovations & Applications, October 29th-30th 2009, Malta (2009, October 29)

We developed an original method called AMADEOX (for « Action Modulatrice de l'Activité Des Enzymes Oxydantes ») able to distinguish stoichiometric, metabolic and anticatalytic activities of antioxidant ... [more ▼]

We developed an original method called AMADEOX (for « Action Modulatrice de l'Activité Des Enzymes Oxydantes ») able to distinguish stoichiometric, metabolic and anticatalytic activities of antioxidant molecules or natural extracts on stimulated neutrophils and purified myeloperoxidase (MPO), a key enzyme released by neutrophil and acting as one of the major ROS generating systems. [less ▲]

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