Jörg Flemmig

Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase

In Mediterranean folk medicine Olea europaea L. leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine K(i) values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a K(i) value of 0.52 μM. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a K(i) of 7.3 μM. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher K(i) value of 53.0 μM, it still displayed a significant inhibition of XO. Furthermore, caffeic acid (K(i) of 11.5 μM; 1.89% of the extract), luteolin-7-O-β-D-glucoside (K(i) of 15.0 μM; 0.86%) and luteolin (K(i) of 2.9 μM; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed mode of inhibition. Tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-β-D-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-β-D-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a rational basis for the traditional use of olive leaves against gout in Mediterranean folk medicine.

Non-Vital Polymorphonuclear Leukocytes Express Myeloperoxidase on their Surface

The heme-containing enzyme myeloperoxidase (MPO) becomes expressed to the cell surface of non-vital polymorphonuclear leukocytes (PMNs) as evidenced by flow cytometry analysis and confocal fluorescence microscopy. While only a very small percentage of freshly isolated cells was able to bind the MPO antibody, PMN suspensions cultured for 36 h or longer time periods contained an increasing number of cells able to interact with these antibodies. Two distinct patterns of fluorescence for the MPO antibodies were observed. Antibodies were localised either in surface patches or distributed over the whole cell body. The latter type dominated in cell samples cultured for more than three days, while the first type was predominantly found in samples cultured for lower time periods. We observed also two peaks for fluorescence distribution by flow cytometry after addition of MPO antibodies to PMNs. Myeloperoxidase was localised at phosphatidylserine epitopes at the surface of non-vital PMNs as evidenced by coincubation with fluorescent MPO antibodies and FITC-labelled annexin V. Myeloperoxidase bound to the outer surface of PMNs uses hydrogen peroxide as a substrate as shown by appearance of an intense chemiluminescence using the impermeable luminescent protein Pholasin. Thus, myeloperoxidase becomes expressed to the surface of non-vital polymorphonuclear leukocytes colocalised with phosphatidylserine that may indicate a role of myeloperoxidase in apoptosis of PMNs.

The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

Background: Aminophenyl fluorescein was developed to detect the halogenating activity of myeloperoxidase. Results: Aminophenyl fluorescein can also be used to quantify the formation of HOBr by eosinophil peroxidase. Conclusion: Aminophenyl fluorescein is suitable to evaluate the halogenating activity of eosinophil peroxidase and peroxidase activity modulators. Significance: Eosinophil peroxidase, a mammal heme peroxidases family member, may contribute to the regulation of inflammation. The specific detection of peroxidase activity in human granulocytes is essential to elucidate their role in innate immune responses, immune regulation, and inflammatory diseases. The halogenating activity of myeloperoxidase in neutrophils can be determined by the novel fluorescent probe aminophenyl fluorescein (APF). Thereby non-fluorescent APF is oxidized by HOCl to form fluorescein. We successfully verified that APF equally detects the hypobromous acid (HOBr)-producing activity of eosinophil granulocytes. This was revealed by three different approaches. First, we investigated the conversion of non-fluorescent APF into fluorescein by HOCl and HOBr by means of fluorescence and mass spectrometry approaches. Thereby comparable chemical mechanisms were observed for both acids. Furthermore in vitro kinetic studies were used to detect the halogenating activity of myeloperoxidase and eosinophil peroxidase by using APF. Here the dye well reflected the different substrate specificities of myeloperoxidase and eosinophil peroxidase regarding chloride and bromide. Finally, peroxidase activities were successfully detected in phorbol ester-stimulated neutrophils and eosinophils using flow cytometry. Thereby inhibitory studies confirmed the peroxidase-dependent oxidation of APF. To sum up, APF is a promising tool for further evaluation of the halogenating activity of peroxidases in both neutrophils and eosinophils.

Epicatechin enhances the chlorinating activity of human myeloperoxidase

The heme-containing enzyme myeloperoxidase (MPO) accumulates at inflammatory sites and is able to catalyse one- and two-electron oxidation reactions. Here it is shown that (-)-epicatechin, which is known to have numerous beneficial health effects, in low micromolar concentration enhances the degradation of monochlorodimedon (MCD) or the chlorination of taurine in a concentration-dependent bell-shaped manner whereas at higher concentrations it sufficiently suppresses the release of hypochlorous acid. Presented reaction mechanisms demonstrate the efficiency of micromolar concentrations of the flavan-3-ol in overcoming the accumulation of compound II that does not participate in the chlorination cycle. In case of MCD the mechanism is more complicated since it also acts as peroxidase substrate with very different reactivity towards compound I (3 x 10(5) M(-1) s(-1)) and compound II (8.8M(-1)s(-1)) at pH 7. By affecting the chlorinating activity of myeloperoxidase (-)-epicatechin may participate in regulation of immune responses at inflammatory sites.

Epicatechin regenerates the chlorinating activity of myeloperoxidase in vitro and in neutrophil granulocytes

The heme-containing enzyme myeloperoxidase (MPO) is mainly expressed in polymorphonuclear leukocytes (PMNs), the most abundant immune cell type in the blood. Accordingly, MPO is classically attributed to the innate immune response against pathogens. Yet, new results also show immune-regulatory functions of the halogenating MPO activity including the formation of anti-inflammatory mediators. In this work we tested the ability of the flavonoid (-)-epicatechin to regenerate this enzymatic activity both in vitro at the isolated MPO-H2O2-Cl(-) system and ex vivo in human PMNs. For all experiments the non-fluorescent dye aminophenyl fluorescein (APF) was used. Upon oxidation by the MPO, the halogenation product hypochlorous acid (HOCl) fluorescein is formed which can be detected e.g. by flow cytometry. The in vitro- and ex vivo-results concordantly show that (-)-epicatechin is a suitable substrate to overcome a compound II accumulation of MPO which was experimentally forced by applying excess hydrogen peroxide. Thereby concentration-dependent effects of the flavan-3-ol were found in both cases and confirmed the proposed mode of action of (-)-epicatechin. The results are in accordance with previous stopped-flow kinetic studies which showed a high reactivity of the polyphenol with MPO compound II. The obtained data may contribute to the explanation of the well-known health promoting effects of (-)-epicatechin. Moreover, the presented study provides new insights into the role of MPO during inflammation.

Components of a standardised olive leaf dry extract (Ph. Eur.) promote hypothiocyanite production by lactoperoxidase.

We investigated in vitro the ability of a standardised olive leaf dry extract (Ph. Eur.) (OLE) as well as of its single components to circumvent the hydrogen peroxide-induced inhibition of the hypothiocyanite-producing activity of lactoperoxidase (LPO). The rate of hypothiocyanite (⁻OSCN) formation by LPO was quantified by spectrophotometric detection of the oxidation of 5-thio-2-nitrobenzoic acid (TNB). By using excess hydrogen peroxide, we forced the accumulation of inactive enzymatic intermediates which are unable to promote the two-electronic oxidation of thiocyanate. Both OLE and certain extract components showed a strong LPO-reactivating effect. Thereby an o-hydroxyphenolic moiety emerged to be essential for a good reactivity with the inactive LPO redox states. This basic moiety is found in the main OLE components oleuropein, oleacein, hydroxytyrosol, caffeic acid as well as in different other constituents including the OLE flavone luteolin. As LPO is a key player in the humoral immune response, these results propose a new mode of action regarding the well-known bacteriostatic and anti-inflammatory properties of the leaf extract of Olea europaea L.

Enhancing hypothiocyanite production by lactoperoxidase – mechanism and chemical properties of promotors

Background The heme enzyme lactoperoxidase is found in body secretions where it significantly contributes to the humoral immune response against pathogens. After activation the peroxidase oxidizes thiocyanate to hypothiocyanite which is known for its microbicidal properties. Yet several pathologies are accompanied by a disturbed hypothiocyanite production which results in a reduced immune defense. Methods The results were obtained by measuring enzyme-kinetic parameters using UV–vis spectroscopy and a standardized enzyme-kinetic test system as well as by the determination of second order rate constants using stopped-flow spectroscopy. Results In this study we systematically tested thirty aromatic substrates for their efficiency to promote the lactoperoxidase-mediated hypothiocyanite production by restoring the native ferric enzyme state. Thereby hydrophobic compounds with a 3,4-dihydroxyphenyl partial structure such as hydroxytyrosol and selected flavonoids emerged as highly efficient promotors of the (pseudo-)halogenating lactoperoxidase activity. Conclusions This study discusses important structure-function relationships of efficient aromatic LPO substrates and may contribute to the development of new agents to promote lactoperoxidase activity in secretory fluids of patients. Significance This study may contribute to a better understanding of the (patho-)physiological importance of the (pseudo-)halogenating lactoperoxidase activity. The presented results may in future lead to the development of new therapeutic strategies which, by reactivating lactoperoxidase-derived hypothiocyanite production, promote the immunological activity of this enzyme.

Lactoperoxidase as a potential drug target

Introduction: Lactoperoxidase (LPO) belongs to the immunologically relevant mammalian heme peroxidases. The enzyme contributes in external secretions to the humoral immune defense against pathogens by oxidation of thiocyanate (SCN–) and iodide (I–). The generation of oxidized thiocyanate and/or iodine species is also important in numerous biotechnological applications of LPO. Areas covered: In this review, we give an overview about the present knowledge of LPO concerning enzymatic structure, catalytic cycles and (pseudo-)halogenated species generated by the enzyme. Redox properties of LPO as well as kinetic aspects regarding the different enzymatic cycles are discussed in order to gain insights into the disturbance of the (pseudo-)halogenating enzyme activity under pathological conditions. Important structural features of LPO and crystallographic studies on the interaction and reaction of organic substrates with the enzyme are also summarized. A broad discussion is devoted to the binding and oxidation of substrates that either inhibit or promote LPO activity. Expert opinion: On the basis of these data, different strategies to further optimize LPO functions in humoral defense of mucous surfaces and biotechnological applications are discussed. In particular, hydrophobic organic substrates with a 3,4-dihydroxyphenyl partial structure considerably enhance the (pseudo-)halogenating activity of LPO. Their application provides, thus, a new strategy to enhance the anti-microbial activity of this enzyme.

Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein

Abstract The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.

SHORT COMMUNICATION: A phantom extinction? New insights into extinction dynamics of the Don-hare Lepus tanaiticus

The Pleistocene to Holocene transition was accompanied by a worldwide extinction event affecting numerous mammalian species. Several species such as the woolly mammoth and the giant deer survived this extinction wave, only to go extinct a few thousand years later during the Holocene. Another example for such a Holocene extinction is the Don‐hare, Lepus tanaiticus, which inhabited the Russian plains during the late glacial. After being slowly replaced by the extant mountain hare (Lepus timidus), it eventually went extinct during the middle Holocene. Here, we report the phylogenetic relationship of L. tanaiticus and L. timidus based on a 339‐basepair (bp) fragment of the mitochondrial D‐loop. Phylogenetic tree‐ and network reconstructions do not support L. tanaiticus and L. timidus being different species. Rather, we suggest that the two taxa represent different morphotypes of a single species and the extinction of ‘L. tanaiticus’ represents the disappearance of a local morphotype rather than the extinction of a species.