The activity of neutrophil myeloperoxidase in patients with lung carcinoma

MPO activity in the neutrophils has been evaluated in 25 patients with lung carcinoma. Surprisingly for us we observed no differences between patients and controls in the total MPO activity. However, we noted the increase in the percentage granulocytes with the first and fourth degree of scores in cancer patients in comparison to normal adults. The highest activity was characterized by the distribution of this enzyme not only in granules but also outside them. Any changes in the total activity of MPO may reflect the balance between the factors which may stimulate it as tumor promoters enhancing the production of superoxide anion or the inhibitory effect of plasma. Diffuse enzymatic reaction may be due to some functional abnormalities of phagocytosis or membrane damage by the MPO system. The role of MPO in anticancer mechanisms is briefly discussed.     

An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid,and its application in finding inhibitors which interact directly with the enzyme

Abstract

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme.

The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content.

To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation.

The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.     

The activity of neutrophil myeloperoxidase in patients with Hodgkin's disease.

Neutrophil myeloperoxidase activity has been studied in twenty one patients diagnosed with Hodgkin's disease. The presented data indicate no differences in total MPO activity, whereas we observed some differences in the percentage of granulocytes with different degree of scores. Changes in the intensity of reaction may indicate the possibility of exocytosis as a mechanism releasing MPO from the cell to the surrounding area. In peripheral, circulating neutrophils, such a phenomenon seems to be of no avail and may disorganise anti-cancer defence.     

Inhibition of myeloperoxidase-catalyzed tyrosylation by phenolic antioxidants in vitro

We have developed an in vitro assay system for the evaluation of the inhibitory effects of phenolic antioxidants on myeloperoxidase (MPO) activity. The formation of dityrosine from the MPO/H2O2/L-tyrosine system was used as an indicator of the MPO activity. Because the buffer system used does not include chloride ion, this assay has the advantage of exclusion of direct reaction between an antioxidant and HOCl. In this assay, ferulic acid, gallic acid, and quercetin strongly inhibited the dityrosine formation, and curcumin and caffeic acid were also effective.     

A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.

Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.     

Inhibition of peroxidase-catalyzed reactions by deferoxamine

Phagocytes generate superoxide (O2-.) and hydrogen peroxide (H2O2) and their interaction in an iron-catalyzed reaction to form hydroxyl radicals (OH.) (Haber-Weiss reaction) has been proposed. Deferoxamine chelates iron in a catalytically inactive form, and thus inhibition by deferoxamine has been employed as evidence for the involvement of OH. generated by the Haber-Weiss reaction. We report here that deferoxamine also inhibits reactions catalyzed by the peroxidases of phagocytes, i.e., myeloperoxidase (MPO) and eosinophil peroxidase (EPO). The reactions inhibited include iodination in the presence and absence of chloride and the oxidation of guaiacol. Iodination by MPO and H2O2 is stimulated by chloride due to the intermediate formation of hypochlorous acid (HOCl). Iodination by reagent HOCl also is inhibited by deferoxamine with the associated consumption of HOCl. Iron saturation of deferoxamine significantly decreased but did not abolish its inhibitory effect on iodination by MPO + H2O2 or HOCl. Deferoxamine did not affect the absorption spectrum of MPO, suggesting that it does not react with or remove the heme iron. The conversion of MPO to Compound II by H2O2 was not seen when H2O2 was added to MPO in the presence of deferoxamine, suggesting either that deferoxamine inhibited the formation of Compound II by acting as an electron donor for MPO Compound I or that deferoxamine immediately reduced the Compound II formed. Iodination by stimulated neutrophils also was inhibited by deferoxamine, suggesting an effect on peroxidase-catalyzed reactions in intact cells. Thus deferoxamine has multiple effects on the formation and activity of phagocyte-derived oxidants and therefore its inhibitory effect on oxidant-dependent damage needs to be interpreted with caution.     

Plasma myeloperoxidase activity as a criterion of therapeutic effectiveness for patients with cardiovascular diseases

A significant increase in the myeloperoxidase (MPO) activity has been found in plasma of patients with stable angina and with acute coronary syndrome (ACS) in comparison with the control group. MPO concentration was significantly increased in plasma of ACS patients. Reduced MPO activity in the treated ACS patients correlated with a favorable outcome of the disease. Generally, changes in plasma MPO concentration coincided with changes in lactoferrin concentration thus confirming the role of neutrophil degranulation in the increase of plasma concentrations of these proteins. The increase in MPO activity was obviously determined by modification of the MPO protein caused by reactive oxygen species and halogen in the molar ratio of 1: 25 and 1: 50. The decrease in plasma MPO activity may be associated with increased plasma concentrations of the physiological inhibitor of its activity, ceruloplasmin, and also with modification of the MPO protein with reactive oxygen species and halogen at their molar ratio of 1: 100 and higher. Thus, MPO activity may be used for evaluation of effectiveness of the treatment of cardiovascular diseases.     

A comparative study on the peroxidase activity within the aqueous humor of the rabbit, guinea-pig and rat

Aqueous humor of the rabbit, guinea-pig and rat has been examined to determine if non-specific peroxidase activity is present within this ocular fluid. Substantial peroxidase activity was found within the aqueous humor of the rat. In contrast, no peroxidase activity could be detected in the aqueous humor of the rabbit or guinea-pig. Partial purification by gel chromatography revealed that the peroxidase within the aqueous humor of the rat has a mol. wt of approx. 45,000.     

Ceruloplasmin has two nearly identical sites that bind myeloperoxidase

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp–MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp–MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp’s crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp–MPO binding and rescues MPO activity. Collectively, our results confirm Cp–MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.     

The development of myeloperoxidase inhibitors

Myeloperoxidase (MPO), an abundant hemoprotein present in neutrophils and monocytes, plays a significant role in immune surveillance and host defense mechanisms. However, increased MPO activity has been linked to a number of pathologies with compelling evidence in initiation and progression of inflammatory events. As a result, search for active compounds that can efficiently inhibit MPO activity and subsequently decrease inflammatory events has been focus of the current research.This perspective provides an overview of the development of MPO inhibitors, their mechanism of action and the review of molecules that were in clinical trials as promising MPO inhibitors.     

PCR cloning and baculovirus expression of human lactoperoxidase and myeloperoxidase

Lactoperoxidase (LPO) and myeloperoxidase (MPO) have been identified previously in human milk. These peroxidases have antimicrobial activity and presumably contribute to the protective functions of milk. In this study, we amplified genes encoding LPO and MPO from human mammary gland cDNA by the polymerase chain reaction (PCR). These genes were expressed in a baculovirus-insect cell system. Peroxidase activity was observed in the culture supernatant of Tricoplusia ni cells infected with the recombinant viruses and the levels increased upon addition of delta-aminolevulinic acid. Purified recombinant human LPO and MPO, both with a molecular mass of about 80 kDa, showed properties similar to bovine LPO and human MPO, respectively, in terms of absorption spectrum, sensitivity to dapsone, specificity for chloride ions, and reactivity with anti-bovine LPO or anti-MPO antibodies. Our data suggest that this expression system is useful for studying the catalytic mechanism and biological significance of these human peroxidases.     

Protective Effect of Edible Marine Algae, Laminaria japonica and Porphyra haitanensis, on Subchronic Toxicity in Rats Induced by Inorganic Arsenic

The myeloperoxidase (MPO) activity and its corresponding mRNA expression as well as gene polymorphism were investigated in the population who live in the endemic fluorosis area. In the study, 150 people were selected from the coal-burning endemic fluorosis area and 150 normal persons from the non-fluorosis area in Guizhou province of China. The blood samples were collected from these people. The activity of MPO in the plasma was determined by spectrophotometer; the expression of MPO mRNA was measured by employing real-time polymerase chain reaction; DNAs were extracted from the leucocytes in blood and five SNP genotypes of MPO promoter gene detected by a multiplex genotyping method, adapter-ligation-mediated allele-specific amplification. The results showed that the MPO activity and its corresponding mRNA in blood were significantly increased in the population living in the area of fluorosis. The different genotype frequencies of MPO, including –1228G/A, –585T/C, –463G/A, and –163C/T, and the three haplotypes with higher frequencies, including –163C–463G–585T–1228G–1276T, –163C–463G–585T–1228G–1276C, and –163C–463G–585T–1228A–1276T, were significantly associated with fluorosis. The results indicated that the elevated activity of MPO induced by endemic fluorosis may be connected in mechanism to the stimulated expression of MPO mRNA and the changed gene polymorphism.     

Melatonin is a potent inhibitor for myeloperoxidase

Myeloperoxidase (MPO) catalyzes the formation of potent oxidants that have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Melatonin plays an important part in the regulation of various body functions including circadian sleep rhythms, blood pressure, oncogenesis, retinal function, seasonal reproduction, and immunity. Here, we demonstrate that melatonin serves as a potent inhibitor of MPO under physiological-like conditions. In the presence of chloride (Cl-), melatonin inactivated MPO at two points in the classic peroxidase cycle through binding to MPO to form an inactive complex, melatonin-MPO-Cl, and accelerating MPO compound II formation, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO compound II without any sign of compound I accumulation. This behavior indicates that melatonin binding modulates the formation of MPO intermediates and their decay rates. The Cl- presence enhanced the affinity of MPO toward melatonin, which switches the enzyme activity from peroxidation to catalase-like activity. In the absence of Cl-, melatonin served as a 1e- substrate for MPO compound I, but at higher concentration it limited the reaction by its dissociation from the corresponding complex. Importantly, melatonin-dependent inhibition of MPO occurred with a wide range of concentrations that span various physiological and supplemental ranges. Thus, the interplay between MPO and melatonin may have a broader implication in the function of several biological systems. This dual regulation by melatonin is unique and represents a new means through which melatonin can control MPO and its downstream inflammatory pathways.     

Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride.

Examination of the spectra of phagocytosing neutrophils and of myeloperoxidase present in the medium of neutrophils stimulated with phorbol myristate acetate has shown that superoxide generated by the cells converts both intravacuolar and exogenous myeloperoxidase into the superoxo-ferric or oxyferrous form (compound III or MPO2). A similar product was observed with myeloperoxidase in the presence of hypoxanthine, xanthine oxidase and Cl-. Both transformations were inhibited by superoxide dismutase. Thus it appears that myeloperoxidase in the neutrophil must function predominantly as this superoxide derivative. MPO2 autoxidized slowly (t 1/2 = 12 min at 25 degrees C) to the ferric enzyme. It did not react directly with H2O2 or Cl-, but did react with compound II (MP2+ X H2O2). MPO2 catalysed hypochlorite formation from H2O2 and Cl- at approximately the same rate as the ferric enzyme, and both reactions showed the same H2O2-dependence. This suggests that MPO2 can enter the main peroxidation pathway, possibly via its reaction with compound II. Both ferric myeloperoxidase and MPO2 showed catalase activity, in the presence or absence of Cl-, which predominated over chlorination at H2O2 concentrations above 200 microM. Thus, although the reaction of neutrophil myeloperoxidase with superoxide does not appear to impair its chlorinating ability, the H2O2 concentration in its environment will determine whether the enzyme acts primarily as a catalase or peroxidase.     

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.     

Prevalence of inherited myeloperoxidase deficiency in Japan

The microbicidal activity of the myeloperoxidase (MPO)-hydrogen peroxide-halide system has been implicated as the most efficient, oxygen-dependent antimicrobial component of neutrophil host defense. Unexpectedly, individuals with MPO deficiency suffer few clinical consequences. In order to understand better the clinical impact of MPO deficiency, we surveyed several clinical hematology laboratories in Japan to assess the prevalence of MPO deficiency in the general population. MPO activity was determined by flow cytometry using the Technicon H series of automated systems. We identified 26 cases of complete MPO deficiency, prevalence 1 in 57,135, and 129 cases of partial deficiency, prevalence 1 in 17,501. The distribution of complete and partial deficiencies differed among the laboratories studied.     

Discovery of a novel indole pharmacophore for the irreversible inhibition of myeloperoxidase (MPO)

Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.     

Assay method for myeloperoxidase in human polymorphonuclear leukocytes

A simple assay method for measuring myeloperoxidase (MPO) has been developed. MPO is found in polymorphonuclear leukocytes and is important as a bactericidal agent in the presence of H2O2 and halide ions. This improved assay method is based on work of Andrews and Krinsky using tetramethylbenzidine (TMB) a noncarcinogenic substrate. By assaying MPO under optimal conditions of TMB at 1.6 mM, H2O2 concentration of 0.3 mM, pH 5.4, and incubation temperature of 37 degrees C, sensitivity of MPO measurements increased eightfold in comparison with the original TMB method. A method has been established to determine absorbance at 655 nm of the reaction mixture by incubation for 3 min and then stopping the reaction by the addition of pH 3.0 buffer. An attempt was also made to raise the sensitivity by using 3,3'-dimethyoxybenzidine (DMB), a carcinogenic substrate. The improved TMB method was 34 times more sensitive than the DMB method.     

The determination of myeloperoxidase activity in liver

Myeloperoxidase (MPO) is an enzyme found in granulocytes of neutrophils, but not in mammalian tissues. Previous studies have directly correlated MPO activity with neutrophil accumulation in tissues. This study presents a method for determining MPO activity in liver. Neutrophil accumulation in rat liver was provoked by creating partial ischemia followed by reperfusion. Liver homogenates prepared by a standard procedure showed no MPO activity. The homogenate was applied to Sephadex G100 and DEAE Sepharose CL6B columns which separated MPO activity from inhibitory activity. The inhibitor was identified as catalase based upon its elution from the columns and removal with 3-amino- 1,2,4-triazole (AT), a catalase inhibitor. Based upon these findings, it was determined that full MPO activity can be assayed in unfractionated liver homogenates by first inactivating catalase with AT.     

Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence

We demonstrate that addition of H₂O₂ to a mixture of myeloperoxidase (MPO), chloride and luminol immediately evokes a short intense flash of chemiluminescence (CL). This flash is diminished in the absence of MPO or chloride, and in the complete system it is suppressed by an MPO inhibitor azide, hypochlorite scavengers taurine or methionine, or an MPO peroxidase-cycle substrate guaiacol. Hence, this CL is mostly due to the MPO halogenation function; a measure of this activity is provided by the integral CL. With three independent methods (CL, taurine chlorination, and peroxidase assay) it is shown that MPO activity is suppressed by ceruloplasmin (Cp). Lactoferrin has no effect either on MPO or on the MPO-Cp complex. It is also shown that peroxidase inhibition by Cp is the stronger the larger is the MPO substrate, which suggests steric hindrances to substrate binding in the MPO-Cp complex. Importantly, the conventional chlorination and peroxidase assays detect MPO inhibition by Cp only at a large excess of the latter, whereas the CL assay reveals it at stoichiometric ratios characteristic of the naturally occurring protein complexes.