Stephan Kohnen

A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-enzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diodearray spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Abstract Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS•+ radical cation: 65% and 89% inhibition for compound [3] at 10–4 M and 5 × 10–5 M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.

Investigation of the reaction of peroxynitrite with propofol at acid pH: Predominant production of oxidized, nitrated, and halogenated derivatives

We reported here the reaction, in acidic conditions, of peroxynitrite (ONOO(-)) with the anaesthetic agent propofol (2,6-diisopropylphenol, PPF). The most interesting finding is that peroxynitrite is able to nitrate and to oxidize propofol leading to 4-nitropropofol, quinone, and diphenylquinone as the major products. More surprisingly, we also found that peroxynitrite is capable of halogenating propofol in the presence of halide ions, suggesting the formation of nitrosyl chloride (NOCl) or nitryl chloride (NO(2)Cl) from the reaction of peroxynitrite with chloride ions. A significant enhancement of the halogenation yield is observed with a simultaneous decrease of the yields of the other products in the presence of methanol or hydrogen peroxide. Increased halogenation of PPF probably results from the formation of peroxynitrate (O(2)NOO(-)), that further oxidizes chloride ions in hypochlorous acid (HOCl) or molecular chlorine (Cl(2)). Spontaneous decay of peroxynitrate is relatively slow in acidic medium, thus explaining the decrease of the yields of the other products. By direct EPR techniques, we also observed that this reaction occurs via a radical pathway.

Modulatory activities of Agelanthus dodoneifolius (Loranthaceae) extracts on stimulated equine neutrophils and myeloperoxidase activity

Agelanthus dodoneifolius DC Danser (Loranthaceae) is used for the treatment of various diseases including asthma. The aqueous and hydroalcoholic extracts have been reported to have anti-inflammatory, spasmolytic and bronchorelaxant activities. The present study investigates the effects of the aqueous decoction and the diethyl ether, ethyl acetate and butanolic fractions of Agelanthus dodoneifolius DC Danser (Loranthaceae) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by phorbol 12-myristate 13-acetate (PMA)-stimulated equine neutrophils and on purified equine MPO activity. ROS production and MPO release by the PMA-stimulated neutrophils were measured by the lucigenin-enhanced chemiluminescence and ELISA assays, respectively. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to specifically measure the equine MPO activity. Identification and quantification of the individual and total phenolic and flavonoid compounds were performed using UPLC-MS/MS equipment and colorimetric methods involving Folin-Ciocalteu and AlCl₃, respectively. All the tested extracts displayed dose-dependent inhibitory effects on the oxidant activities of neutrophils; a stronger effect was observed with the organic fractions than the aqueous decoction. These findings could be correlated with a high content of phenolic and flavonoid compounds. The results confirm the previously shown anti-inflammatory effect of Agelanthus dodoneifolius and its potential use for the treatment of neutrophil-dependent inflammatory diseases.

Evaluation of antiradical and anti-inflammatory activities of ethyl acetate and butanolic subfractions of agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) using equine myeloperoxidase and both PMA-activated neutrophils and HL-60 cells

The ethyl acetate and n-butanolic subfractions of Agelanthus dodoneifolius were investigated for their antioxidant and antimyeloperoxidase (MPO) activities. The reactive oxygen species (ROS) generation was assessed by lucigenin-enhanced chemiluminescence (CL) and dichlorofluorescein- (DCF-) induced fluorescence techniques from phorbol myristate acetate- (PMA-) stimulated equine neutrophils and human myeloid cell line HL-60, respectively. In parallel, the effects of the tested subfractions were evaluated on the total MPO release by stimulated neutrophils and on the specific MPO activity by means of immunological assays. The results showed the potent activity of the butanolic subfraction, at least in respect of the chemiluminescence test (IC50 = 0.3 ± 0.1 µg/mL) and the ELISA and SIEFED assays (IC50 = 2.8 ± 1.2 µg/mL and 1.3 ± 1.0 µg/mL), respectively. However, the ethyl acetate subfraction was found to be the most potent in the DCF assay as at the highest concentration, DCF fluorescence intensity decreases of about 50%. Moreover, we demonstrated that the ethyl acetate subfraction was rich in catechin (16.51%) while it was not easy to identify the main compounds in the butanolic subfraction using the UPLC-MS/MS technique. Nevertheless, taken together, our results provide evidence that Agelanthus dodoneifolius subfractions may represent potential sources of natural antioxidants and of antimyeloperoxidase compounds.

Effect of honey on oxidation, chlorination and nitration by purified equine myeloperoxidase

Saad Aissat, Hama Benbarek, Thierry Franck, Stephan Kohnen, Didier Serteyn, Moussa Ahmed, Ange Mouithys-Mickalad Institute of Veterinary Sciences, Ibn Khaldoun University, Tiaret 14000, Algeria Pharmacognosy and Api-Phytotherapy Research Laboratory, Mostaganem University, Mostaganem, Algeria Faculty of Sciences of Nature and Life, Mascara University, Mascara 29000, Algeria Centre for Oxygen, Research and Development (CORD), Institute of Chemistry B6a, University of Liège, Sart Tilman, 4000 Liège 1, Belgium Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine, B41, University of Liège, Sart Tilman, 4000 Liège 1, Belgium Celabor, Centre de Services Scientifique et Technique aux Entreprises, Zoning Industriel de Petit-Rechain, Avenue du Parc 38, 4650 Herve, Belgium Journal of Coastal Life Medicine 2017; 5(9): 398-402

Effect of honey on purified equine myeloperoxidase activity and superoxide radical production in activated Polymorphonuclear neutrophils

Three Algerian honey samples (nectar honey, mixed honey and honeydew honey) were analysed for their total phenolic content (TPC) using the Folin–Ciocalteu reagent and total flavonoid content (TFC) by the aluminium chloride method. The antioxidant activity was tested both against reactive oxygen species (ROS) produced by phorbol 12-myristate 13-acetate (PMA)-stimulated equine polymorphonuclear neutrophils (PMNs) and on purified equine myeloperoxidase (MPO) enzyme activity. The ROS production by stimulated PMNs was measured by lucigenin-enhanced chemiluminescence. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to measure specifically the activity of equine MPO. Nectar honey, mixed honey and honeydew honey had TPCs of 59.52 ± 1.31, 68.36 ± 1.20 and 122.76 ± 4.20 mg gallic acid equivalents (GAE)/100 g honey, respectively (mean ± SD). As for the TPC, the highest TFC was observed for honeydew honey [20.55 ± 0.72 mg catechine equivalents (CE)/100 mg honey], followed by mixed honey (7.47 ± 0.07 CE/100 mg honey) and nectar honey (4.80 ± 0.08 CE/100 mg honey). All the tested honeys displayed a dose-dependent inhibitory effect on the oxidant activities of PMNs. By the SIEFED technique, it was shown that most of the honeys interact directly with MPO: the light honeys (nectar honey and mixed honey) were inhibitors of MPO activity, while the dark honey had less inhibiting effect and even behaved as an enhancer of MPO activity at high concentrations.