Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.     

Lysophosphatidic acid and sphingosine 1-phosphate: two lipid villains provoking cardiovascular diseases?

Several plant-derived polyphenolic compounds are considered to possess anticancer and apoptosis-inducing properties in cancer cells. Such compounds are recognized as naturally occurring antioxidants but also exhibit prooxidant properties under appropriate conditions. Evidence in the literature suggests that the antioxidant properties of polyphenolics such as gallotannins, curcumin, and resveratrol may not fully account for their chemopreventive effects. We propose a mechanism for the cytotoxic action of these compounds against cancer cells that involves mobilization of endogenous copper and the consequent prooxidant action.     

Radical chemistry of epigallocatechin gallate and its relevance to protein damage

The radical chemistry of the plant polyphenolics epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were investigated using electron paramagnetic resonance spectroscopy. Radical species formed spontaneously in aqueous solutions at low pH without external oxidant and were spin stabilized with Zn(II). The spectra were assigned to the gallyl radical and the anion gallyl radical, with only 10% of the signal assigned to a radical from the galloyl ester. Spectral simulations were used to establish a pK(a) of 4.8 for the EGCG radical and a pK(a) of 4.4 for the EGC radical. The electrochemical redox potentials of EGCG and EGC varied from 1000 mV at pH 3 to 400 mV at pH 8. The polyphenolics did not produce hydroxyl radicals unless reduced metal ions such as iron(II) were added to the system. Zinc(II)-stabilized EGCG radicals were more effective protein-precipitating agents than unoxidized EGCG and produced irreversibly complexed protein. EGCG and other naturally occurring polyphenolics are effective radical scavengers but their radical products have the potential to damage biological molecules such as proteins.     

Propofol inhibits the myeloperoxidase activity by acting as substrate through a redox process

BackgroundPropofol (2,6-diisopropylphenol) is frequently used as intravenous anesthetic agent, especially in its injectable form (Diprivan), to initiate and maintain sedative state during surgery or in intensive care units. Numerous studies have reported the antioxidant and anti-inflammatory effect of propofol. The oxidant enzyme myeloperoxidase (MPO), released from activated neutrophils, plays a key role in host defense. An increase of the circulating MPO concentration has been observed in patients admitted in intensive care unit and presenting a systemic inflammatory response related to septic shock or trauma.MethodsThis study investigates the immunomodulatory action of propofol and Diprivan as inhibitor of the oxidant activity of MPO. The understanding of the redox action mechanism of propofol and Diprivan on the myeloperoxidase chlorination and peroxidase activities has been refined using the combination of fluorescence and absorption spectroscopies with docking and cyclic voltammetry.ResultsPropofol acts as a reversible MPO inhibitor. The molecule interacts as a reducing substrate in the peroxidase cycle and promotes the accumulation of compound II. At acidic pH (5.5), propofol and Diprivan do not inhibit the chlorination activity, but their action increases at physiological pH (7.4). The main inhibitory action of Diprivan could be attributed to its HOCl scavenging property.General significancePropofol can act as a reversible MPO inhibitor at clinical concentrations. This property could, in addition to other previously proven anti-inflammatory actions, induce an immunomodulatory action, beneficial during clinical use, particularly in the treatment of systemic inflammation response syndrome.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1 x 10(6) M(-1)s(-1)), it reacts sluggishly with compound II (kII 7 M(-1)s(-1)). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 microM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 microM) unless superoxide dismutase was present (I50 5 microM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Phagocytes produce 5-chlorouracil and 5-bromouracil,two mutagenic products of myeloperoxidase,in human inflammatory tissue

Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism involves myeloperoxidase, a hemeprotein secreted by human phagocytes. In this study, we demonstrate that human neutrophils use myeloperoxidase to oxidize uracil to 5-chlorouracil in vitro. Uracil chlorination by myeloperoxidase or reagent HOCl exhibited an unusual pH dependence, being minimal at pH approximately 5, but increasing markedly under either acidic or mildly basic conditions. This bimodal curve suggests that myeloperoxidase initially produces HOCl, which subsequently chlorinates uracil by acid- or base-catalyzed reactions. Human neutrophils use myeloperoxidase and H2O2 to chlorinate uracil, suggesting that nucleobase halogenation reactions may be physiologically relevant. Using a sensitive and specific mass spectrometric method, we detected two products of myeloperoxidase, 5-chlorouracil and 5-bromouracil, in neutrophil-rich human inflammatory tissue. Myeloperoxidase is the most likely source of 5-chlorouracil in vivo because halogenated uracil is a specific product of the myeloperoxidase system in vitro. In contrast, previous studies have demonstrated that 5-bromouracil could be generated by either eosinophil peroxidase or myeloperoxidase, which preferentially brominates uracil at plasma concentrations of halide and under moderately acidic conditions. These observations indicate that the myeloperoxidase system promotes nucleobase halogenation in vivo. Because 5-chlorouracil and 5-bromouracil can be incorporated into nuclear DNA, and these thymine analogs are well known mutagens, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.     

Salivary myeloperoxidase of young adult humans

Leukocytes, principally polymorphonuclear leukocytes (PMNs), enter the oral cavity where they release a portion of their constituents, including myeloperoxidase, into oral fluids. A greater number of PMNs in the oral cavity are associated with oral inflammation. However, the quantitative contribution of the PMN to oral fluids, including saliva, during various conditions is poorly understood. An assay method based on the adsorbance loss at 278 nm from the reaction of the myeloperoxidase product hypochlorous acid with monochlorodimedon to yield dichlorodimedon was developed for the quantitation of salivary myeloperoxidase. Myeloperoxidase was determined in supernatants of whole saliva obtained at low and moderate flow rates and in parotid saliva collected during moderate and pronounced stimulation from young adults with minimal oral inflammation. The greatest myeloperoxidase activity was in whole saliva supernatants collected at low flow rates where PMN products have an opportunity to accumulate. Lesser quantities of myeloperoxidase were found in both the whole saliva supernatants and parotid saliva obtained at the faster flow rates. Low flow rate whole saliva supernatants contained about 25% of the myeloperoxidase in the PMNs which enter the oral cavity. Myeloperoxidase is responsible for a significant portion (15-20%) of the total peroxidase activity in supernatants of whole saliva obtained at low flow rates. Preliminary results indicate that young adults with phenytoin-associated gingival overgrowth or who smoke have more myeloperoxidase activity in low flow rate whole saliva.     

The effects of disodium pamidronate on human polymorphonuclear leukocytes and platelets: An <Emphasis Type="Italic">in vitro</Emphasis> study

Recent reports have indicated that, as well as having antiresorptive effects, bisphosphonates could have an application as anti-inflammatory drugs. Our aim was to investigate whether this anti-inflammatory action could be mediated by the nitric oxide (NO) released by the leukocytes migrating to the site of inflammation. In particular, we investigated in vitro the intracellular calcium concentration ([Ca²⁺]_i), the level of NO released by PMN and platelets, and the PMN myeloperoxidase activity after incubation with disodium pamidronate, since there was a postulated modulatory effect of this aminosubstituted bisphosphonate on leukocytes both in vitro and in vivo. Our data shows that the pamidronate treatment provoked a significant increase in the [Ca²⁺]_i parallel to the enhancement in NO release, suggesting a possible activation of constitutive nitric oxide synthase, while the myeloperoxidase activity was significantly reduced. In conclusion, we hypothesized that treatment with pamidronate could stimulate NO-production by cells present near the bone compartment, thus constituting a protective mechanism against bone resorption occurring during inflammation. In addition, PMN- and platelet-derived NO could act as a negative feed-back signal to restrict the inflammatory processes.     

N-(3-(aminomethyl)benzyl)acetamidine, an inducible nitric oxide synthase inhibitor, decreases colonic inflammation induced by trinitrobenzene sulphonic acid in rats.

Gastrointestinal inflammation has been associated with an increased generation of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS). Using an experimental model of colitis induced by trinitrobenzene sulphonic acid (TNBS), we sought to determine whether the administration of N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a specific inhibitor of iNOS, has a beneficial action on the colonic injury. 1400W (0.4 and 2 mg/kg/day) was administered intraperitoneally from day 5 to 10 after intrarectal instillation of TNBS. TNBS led to colonic ulceration and inflammation, an increase of colonic myeloperoxidase activity and the expression of the calcium-independent NOS from days 1 to 15. 1400W reduced the macroscopic damage and the histological changes induced by TNBS as well as the calcium-independent NOS activity and myeloperoxidase activity determined over 30 min after sacrifice. These findings indicate that the expression of iNOS accounts for most of the damage caused by TNBS and that the administration of 1400W after the onset of colitis has a beneficial action on the colonic injury.     

Macrophage activation includes high intracellular myeloperoxidase activity

Macrophages recovered from the peritoneum of mice, 48 h after concanavalin A administration, are primed and have a higher content of myeloperoxidase (MPO) than resident cells. The increase in MPO content is accompanied by an increased capability of macrophages generate hypochlorous acid and increased peroxidase activity. Contrary to the common sense, neutrophils is not the source of the MPO activity found in primed macrophages since macrophages recovered from mice treated with antigranulocyte antibody preserve the peroxidase activity. Given the broad spectrum of action of MPO, the preservation of MPO in primed macrophages might play a special role in the killing of pathogens and inflammation.     

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe^V redox intermediate of myeloperoxidase, to Compound II, which has Fe^IV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.     

三七总RNA提取方法的对比研究

比较利用改进的异硫氰酸胍一步法、异硫氰酸胍高盐法、CTAB法和Thomas’RNA提取法等4种方法提取三七根茎2个部位总RNA的可行性。结果表明,改进的异硫氰酸胍一步法和异硫氰酸胍高盐法能有效地抑制酚类物质、多糖及皂苷等次级代谢产物对总RNA的影响,可从三七根茎中获得质量高、完整性好的总RNA。RT—PCR分析显示提取的总RNA具有反转录活性。这2种方法具有快速、简单、有效的特点。     

Neuronal expression of myeloperoxidase is increased in Alzheimer's disease

Myeloperoxidase, a heme protein expressed by professional phagocytic cells, generates an array of oxidants which are proposed to contribute to tissue damage during inflammation. We now report that enzymatically active myeloperoxidase and its characteristic amino acid oxidation products are present in human brain. Further, expression of myeloperoxidase is increased in brain tissue showing Alzheimer's neuropathology. Consistent with expression in phagocytic cells, myeloperoxidase immunoreactivity was present in some activated microglia in Alzheimer brains. However, the majority of immunoreactive material in brain localized with amyloid plaques and, surprisingly, neurons including granule and pyramidal neurons of the hippocampus. Confirming neuronal localization of the enzyme, several neuronal cell lines as well as primary neuronal cultures expressed myeloperoxidase protein. Myeloperoxidase mRNA was also detected in neuronal cell lines. These results reveal the unexpected presence of myeloperoxidase in neurons. The increase in neuronal myeloperoxidase expression we observed in Alzheimer disease brains raises the possibility that the enzyme contributes to the oxidative stress implicated in the pathogenesis of the neurodegenerative disorder.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Superoxide radicals can act synergistically with hypochlorite to induce damage to proteins.

Activated phagocytes generate both superoxide radicals via a respiratory burst, and HOCl via the concurrent release of the haem enzyme myeloperoxidase. Amine and amide functions on proteins and carbohydrates are major targets for HOCl, generating chloramines (RNHCl) and chloramides (RC(O)NClR'), which can accumulate to high concentrations (>100 microM). Here we show that superoxide radicals catalyse the decomposition of chloramines and chloramides to reactive nitrogen-centred radicals, and increase the extent of protein fragmentation compared to that observed with either superoxide radicals or HOCl, alone. This synergistic action may be of significance at sites of inflammation, where both superoxide radicals and chloramines/chloramides are formed simultaneously.     

The roles of myeloperoxidase in coronary artery disease and its potential implication in plaque rupture

Atherosclerosis is the main pathophysiological process underlying coronary artery disease (CAD). Acute complications of atherosclerosis, such as myocardial infarction, are caused by the rupture of vulnerable atherosclerotic plaques, which are characterized by thin, highly inflamed, and collagen-poor fibrous caps. Several lines of evidence mechanistically link the heme peroxidase myeloperoxidase (MPO), inflammation as well as acute and chronic manifestations of atherosclerosis. MPO and MPO-derived oxidants have been shown to contribute to the formation of foam cells, endothelial dysfunction and apoptosis, the activation of latent matrix metalloproteinases, and the expression of tissue factor that can promote the development of vulnerable plaque. As such, detection, quantification and imaging of MPO mass and activity have become useful in cardiac risk stratification, both for disease assessment and in the identification of patients at risk of plaque rupture. This review summarizes the current knowledge about the role of MPO in CAD with a focus on its possible roles in plaque rupture and recent advances to quantify and image MPO in plasma and atherosclerotic plaques.     

Interaction of tgf-beta with immune cells in airway disease

Asthma, chronic obstructive pulmonary disorder (COPD), and cystic fibrosis (CF), chronic diseases of the airways, are characterized by symptoms such as inflammation of the lung tissue, mucus hypersecretion, constriction of the airways, and excessive fibrosis of airway tissue. Transforming growth factor (TGF)-beta, a cytokine that affects many different cell processes, has an important role in the lungs of patients with some of these chronic airway diseases, especially with respect to airway remodeling. Eosinophils can be activated by and are a major source of TGF-beta in asthma. The action of TGF-beta also shows associations with other cell types, such as T cells and neutrophils, which are involved in the pathogenesis of asthma. TGF-beta can perpetuate the pathogenesis of COPD and CF, as well, through its induction of inflammation via release from and action on different cells. The intracellular signaling induced by TGF-beta in various cell types has been elucidated and may point to mechanisms of action by TGF-beta on different structural or immune cells in these airway diseases. Some possible treatments, especially that prevent the deleterious airway changes induced by the action of either eosinophils or TGF-beta in asthma, have been investigated. TGF-beta-induced signaling pathways, especially those in different cell types in asthma, COPD, or CF, may provide potential therapeutic targets for the treatment of some of the most devastating airway diseases.     

Myricitrin as a substrate and inhibitor of myeloperoxidase: implications for the pharmacological effects of flavonoids

Flavonoids are increasingly being ingested by the general population as chemotherapeutic and anti-inflammatory agents. They are potentially toxic because of their conversion to free radicals and reactive quinones by peroxidases. Little detailed information is available on how flavonoids interact with myeloperoxidase, which is the predominant peroxidase present at sites of inflammation. This enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid, as well as to produce an array of reactive free radicals from organic substrates. We investigated how the flavonoid myricitrin is oxidized by myeloperoxidase and how it affects the activities of this enzyme. Myricitrin was readily oxidized by myeloperoxidase in the presence of hydrogen peroxide. Its main oxidation product was a dimer that underwent further oxidation. In the presence of glutathione, myricitrin was oxidized to a hydroquinone that was conjugated to glutathione. When myeloperoxidase oxidized myricitrin and related flavonoids it became irreversibly inactivated. The number of hydroxyl groups in the B ring of the flavonoids and the presence of a free hydroxyl m-phenol group in the A ring were important for the inhibitory effects. Less enzyme inactivation occurred in the presence of chloride. Neutrophils also oxidized myricitrin to dimers in a reaction that was partially dependent on myeloperoxidase. Myricitrin did not affect the production of hypochlorous acid by neutrophils. We conclude that myricitrin will be oxidized by neutrophils at sites of inflammation to produce reactive free radicals and quinones. It is unlikely to affect hypochlorous acid production by neutrophils.     

Effect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts

Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function.