Comparison of Fecal Calprotectin and Myeloperoxidase in Predicting Outcomes in Inflammatory Bowel Disease.

BACKGROUND: Biomarkers have been proposed as surrogate treatment targets for the management of inflammatory bowel disease (IBD); however, their relationship with IBD-related complications remains unclear. This study investigated the utility of neutrophil biomarkers fecal calprotectin (fCal) and fecal myeloperoxidase (fMPO) in predicting a complicated IBD course. METHODS: Participants with IBD were followed for 24 months to assess for a complicated IBD course (incident corticosteroid use, medication escalation for clinical disease relapse, IBD-related hospitalizations/surgeries). Clinically active IBD was defined as Harvey-Bradshaw index >4 for Crohn's disease (CD) and simple clinical colitis activity index >5 for ulcerative colitis (UC). Area under the receiver-operating-characteristics curves (AUROC) and multivariable logistic regression assessed the performance of baseline symptom indices, fCal, and fMPO in predicting a complicated disease IBD course at 24 months. RESULTS: One hundred and seventy-one participants were included (CD, n = 99; female, n = 90; median disease duration 13 years [interquartile range, 5-22]). Baseline fCal (250 µg/g; AUROC = 0.77; 95% confidence interval [CI], 0.69-0.84) and fMPO (12 µg/g; AUROC = 0.77; 95% CI, 0.70-0.84) predicted a complicated IBD course. Fecal calprotectin (adjusted OR = 7.85; 95% CI, 3.38-18.26) and fMPO (adjusted OR = 4.43; 95% CI, 2.03-9.64) were associated with this end point after adjustment for other baseline variables including clinical disease activity. C-reactive protein (CRP) was inferior to fecal biomarkers and clinical symptoms (pdifference < .05) at predicting a complicated IBD course. A combination of baseline CRP, fCal/fMPO, and clinical symptoms provided the greatest precision at identifying a complicated IBD course. CONCLUSIONS: Fecal biomarkers are independent predictors of IBD-related outcomes and are useful adjuncts to routine clinical care.

Elevated seminal plasma myeloperoxidase is associated with a decreased sperm concentration in young men.

Myeloperoxidase is a major neutrophil protein which generates oxidants that are highly reactive, and if present in seminal fluid, could be potentially damaging to spermatozoa. We recruited young males aged 18-35 years, unscreened for fertility status, for a pilot study measuring seminal plasma myeloperoxidase. On three occasions, over a 3-month period, we measured parameters of semen quality and correlated these with seminal myeloperoxidase protein and activity. After baseline measurement, participants were supplemented daily with 250 mg of vitamin C, a potent scavenger of reactive oxygen species with antiinflammatory activities. Seminal plasma from eight of the 12 participants had measurable concentrations of myeloperoxidase protein, across a broad range (15-250 ng/mL). Median myeloperoxidase protein concentrations were ~45-fold higher in semen samples with low vs. high sperm concentrations. Seminal plasma myeloperoxidase protein concentration was inversely correlated with the percentage of rapidly motile spermatozoa assessed by computer-assisted sperm analysis, and the total number of spermatozoa per ejaculate, but positively correlated with sperm maturity, measured by DNA staining ability. We measured an inverse correlation between semen vitamin C concentration and seminal plasma myeloperoxidase protein concentration, although vitamin C supplementation had no effect on semen quality. Our pilot data suggest that high concentrations of myeloperoxidase were present in the seminal plasma of many of our young participants, and that this may be associated with decreases in semen quality. A larger study is required to confirm these findings.

Superoxide enhances hypochlorous acid production by stimulated human neutrophils.

Stimulated neutrophils undergo a respiratory burst discharging large quantities of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and Cl- to hypochlorous acid. Human neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in Cl(-)-free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalase, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorbol myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.

The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon.

Chlorination of monochlorodimedon is routinely used to measure the production of hypochlorous acid catalysed by myeloperoxidase from H2O2 and Cl-. We have found that the myeloperoxidase/H2O2/Cl- system, at pH 7.8, catalysed the loss of monochlorodimedon with a rapid burst phase followed by a much slower steady-state phase. The loss of monochlorodimedon in the absence of Cl- was only 10% of the steady-state rate in the presence of Cl-, which indicates that the major reaction of monochlorodimedon was with hypochlorous acid. During the steady-state reaction, myeloperoxidase was present as 100% compound II, which cannot participate directly in hypochlorous acid formation. Monochlorodimedon was necessary for formation of compound II, since it was not formed in the presence of methionine. Both the amount of hypochlorous acid formed during the burst phase, and the steady-state rate of hypochlorous acid production, increased with increasing concentrations of myeloperoxidase and with decreasing concentrations of monochlorodimedon. Inhibition by monochlorodimedon was competitive with Cl-. From these results, and the ability of myeloperoxidase to slowly peroxidase monochlorodimedon in the absence of Cl-, we propose that the reaction of monochlorodimedon with the myeloperoxidase/H2O2/Cl- system involves a major pathway due to hypochlorous acid-dependent chlorination and a minor peroxidative pathway. Only a small fraction of compound I needs to react with monochlorodimedon instead of Cl- at each enzyme cycle, for compound II to rapidly accumulate. Monochlorodimedon, therefore, cannot be regarded as an inert detector of hypochlorous acid production by myeloperoxidase, but acts to limit the chlorinating activity of the enzyme. In the presence of reducing species that act like monochlorodimedon, the activity of myeloperoxidase would depend on the rate of turnover of compound II. Components of human serum promoted the conversion of ferric-myeloperoxidase to compound II in the presence of H2O2. We suggest, therefore, that in vivo the rate of turnover of compound II may determine the rate of myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.

A pulse radiolysis investigation of the reactions of myeloperoxidase with superoxide and hydrogen peroxide.

Using pulse radiolysis, the rate constant for the reaction of ferric myeloperoxidase with O2- to give compound III was measured at pH 7.8, and values of 2.1.10(6) M-1.s-1 for equine ferric myeloperoxidase and 1.1.10(6) M-1.s-1 for human ferric myeloperoxidase were obtained. Under the same conditions, the rate constant for the reaction of human ferric myeloperoxidase with H2O2 to give compound I was 3.1.10(7) M-1.s-1. Our results indicate that although the reaction of ferric myeloperoxidase with O2- is an order of magnitude slower than with H2O2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.

Neutrophils convert tyrosyl residues in albumin to chlorotyrosine.

Hypochlorous acid chlorinates tyrosyl residues in small peptides to produce chlorotyrosine. Detection of chlorotyrosine has the potential to unequivocally identify the contribution hypochlorous acid makes to inflammation. I have developed a selective and sensitive HPLC assay for measuring chlorotyrosine. When albumin was exposed to reagent hypochlorous acid, or that produced by myeloperoxidase and stimulated neutrophils, tyrosyl residues in the protein were converted to chlorotyrosine. About 2% of the hypochlorous acid generated by neutrophils was accounted for by the formation of chlorotyrosine. These results demonstrate that chlorotyrosine will be a useful marker for establishing a role for hypochlorous acid in host defence and inflammation.

Biomarkers of myeloperoxidase-derived hypochlorous acid.

Hypochlorous acid is the major strong oxidant generated by neutrophils. The heme enzyme myeloperoxidase catalyzes the production of hypochlorous acid from hydrogen peroxide and chloride. Although myeloperoxidase has been implicated in the tissue damage that occurs in numerous diseases that involve inflammatory cells, it has proven difficult to categorically demonstrate that it plays a crucial role in any pathology. This situation should soon be rectified with the advent of sensitive biomarkers for hypochlorous acid. In this review, we outline the advantages and limitations of chlorinated tyrosines, chlorohydrins, 5-chlorocytosine, protein carbonyls, antibodies that recognize HOCl-treated proteins, and glutathione sulfonamide as potential biomarkers of hypochlorous acid. Levels of 3-chlorotyrosine and 3,5-dichlorotyrosine are increased in proteins after exposure to low concentrations of hypochlorous acid and we conclude that their analysis by gas chromatography and mass spectrometry is currently the best method available for probing the involvement of oxidation by myeloperoxidase in the pathology of particular diseases. The appropriate use of other biomarkers should provide complementary information.Keywords-Free radicals, Myeloperoxidase, Neutrophil oxidant, Hypochlorous acid, Chlorotyrosine, Chlorohydrin, Oxidant biomarker

Assays using horseradish peroxidase and phenolic substrates require superoxide dismutase for accurate determination of hydrogen peroxide production by neutrophils.

We used horseradish peroxidase and either scopoletin, homovanillic acid, or phenol red to measure hydrogen peroxide generated by human neutrophils. With these assays, superoxide dismutase significantly increased the amount of hydrogen peroxide detected. In contrast, it had no effect when the accumulation of hydrogen peroxide was measured with a hydrogen peroxide electrode. We propose that superoxide interferes with horseradish peroxidase-dependent assays so that hydrogen peroxide is underestimated. Thus, when using these assays, superoxide dismutase must be added to neutrophils to ensure that all the hydrogen peroxide they produce is detected.

Neutrophil-mediated activation of mitoxantrone to metabolites which form adducts with DNA.

In this study we have shown that phorbol ester-stimulated human neutrophils are able to oxidatively activate mitoxantrone and result in covalent incorporation of the drug into cellular DNA. The use of the myeloperoxidase inhibitor sodium azide confirmed that the activation and covalent binding of mitoxantrone to cellular DNA was due to its metabolism by the haem enzyme myeloperoxidase. Phorbol ester-stimulated neutrophils were also able to oxidatively metabolise mitoxantrone and facilitate extracellular covalent binding of the drug to calf thymus DNA. These results suggest that myeloperoxidase may contribute to the mode of action of mitoxantrone.

Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs.

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils, and should therefore be expected to contribute to the damage caused by these inflammatory cells. It is produced from H2O2 and Cl- by the heme enzyme myeloperoxidase (MPO). We used a H2O2-electrode to assess the ability of a variety of anti-inflammatory drugs to inhibit conversion of H2O2 to HOCl. Dapsone, mefenamic acid, sulfapyridine, quinacrine, primaquine and aminopyrine were potent inhibitors, giving 50% inhibition of the initial rate of H2O2 loss at concentrations of about 1 microM or less. Phenylbutazone, piroxicam, salicylate, olsalazine and sulfasalazine were also effective inhibitors. Spectral investigations showed that the inhibitors acted by promoting the formation of compound II, which is an inactive redox intermediate of MPO. Ascorbate reversed inhibition by reducing compound II back to the active enzyme. The characteristic properties that allowed the drugs to inhibit MPO reversibly were ascertained by determining the inhibitory capacity of related phenols and anilines. Inhibition increased as substituents on the aromatic ring became more electron withdrawing, until an optimum reduction potential was reached. Beyond this optimum, their inhibitory capacity declined. The best inhibitor was 4-bromoaniline which had an I50 of 45 nM. An optimum reduction potential enables inhibitors to reduce MPO to compound II, but prevents them from reducing compound II back to the active enzyme. Exploitation of this optimum reduction potential will help in targeting drugs against HOCl-dependent tissue damage.

The activation of gold complexes by cyanide produced by polymorphonuclear leukocytes. III. The formation of aurocyanide by myeloperoxidase.

There is considerable evidence that the anti-rheumatic gold complexes are activated by their conversion to aurocyanide. In order to understand the mechanism of production of aurocyanide, we investigated the involvement of myeloperoxidase in the reaction. This haem enzyme of neutrophils and monocytes uses hydrogen peroxide to oxidise chloride and thiocyanate to hypochlorous acid and hypothiocyanite, respectively. When aurothiomalate (10 microM) was incubated with thiocyanate (200 microM), hydrogen peroxide (100 microM) and myeloperoxidase (20 nM), it was transformed to a product that was spectrally identical to authentic aurocyanide. Aurothiomalate was quantitatively converted to aurocyanide in about 10 min at pH 6.0 and in 40 min at pH 7.4. Aurocyanide formation occurred after myeloperoxidase had used all the hydrogen peroxide available to produce hypothiocyanite. Thus, the cyanide must have formed from the slow decomposition of hypothiocyanite. The rate of aurocyanide production was increased in the presence of 100 mM chloride, which indicates that hypochlorous acid accelerates the formation of cyanide. Hypochlorous acid (100 to 400 microM) reacted non-enzymatically with thiocyanate (200 microM) and aurothiomalate (10 microM) to produce aurocyanide. Thus, aurocyanide is produced by two processes, involving both the formation of hypothiocyanite and hypochlorous acid. Aurocyanide is an effective inhibitor of the respiratory burst of neutrophils and monocytes and the proliferation of lymphocytes. Therefore, aurothiomalate may attenuate inflammation by acting as a pro-drug which is reliant on neutrophils and monocytes to produce hypothiocyanite. When the hypothiocyanite decays to hydrogen cyanide, the pro-drug is converted to aurocyanide which then suppresses further oxidant production by these inflammatory cells.

Oxidative metabolism of mitoxantrone by the human neutrophil enzyme myeloperoxidase.

The anti-cancer drug mitoxantrone is readily oxidized by the human heme enzyme myeloperoxidase (MPO) and H2O2. Direct oxidation yielded up to three products, which depended on the ratio of H2O2 to mitoxantrone. At an H2O2: mitoxantrone ratio of 1.0, one major product was obtained, with a spectrum and HPLC retention time identical to that resulting from oxidation by horseradish peroxidase. This metabolite is a substituted hexahydronaphtho[2,3-f]quinoxaline-7,12-dione and has been discovered in the urine of patients treated with mitoxantrone, hence implicating MPO in the in vivo metabolism of mitoxantrone. At higher concentrations of H2O2, the oxidation of mitoxantrone was more complex, with two further metabolites being identified. When mitoxantrone was incubated with neutrophils that had been stimulated with phorbol myristate acetate, it was oxidized by an MPO-dependent mechanism. Therefore, it appears that MPO may play a significant role in the clinical activity displayed by mitoxantrone against acute myelogenous leukemias, as neutrophils, monocytes and their bone marrow precursors contain high levels of the enzyme.

Superoxide is an antagonist of antiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase.

Myeloperoxidase, the most abundant enzyme in neutrophils, catalyses the conversion of hydrogen peroxide and chloride to hypochlorous acid. This potent oxidant has the potential to cause considerable tissue damage in many inflammatory diseases. We have investigated the ability of dapsone, diclofenac, primaquine, sulfapyridine and benzocaine to inhibit hypochlorous acid production by stimulated human neutrophils. The drugs were also tested against purified myeloperoxidase using xanthine oxidase to generate hydrogen peroxide and superoxide. The inhibitory effects of the drugs on hypochlorous acid production, either by cells stimulated with phorbol myristate acetate or by myeloperoxidase and xanthine oxidase, were significantly less than those determined with myeloperoxidase and reagent hydrogen peroxide. Comparable potency was observed only when superoxide dismutase was present to remove superoxide. We also observed that with the xanthine oxidase system, inhibition of hypochlorous acid production by dapsone decreased markedly as the concentration of myeloperoxidase increased. Dapsone was a poor inhibitor of hypochlorous acid production by neutrophils stimulated with opsonized zymosan, regardless of the presence of superoxide dismutase. With this phagocytic stimulus, catalase inhibited hypochlorous acid formation by only 60%, which indicates that a substantial amount of the hypochlorous acid detected originated from within phagosomes. Thus, it is apparent that dapsone is unable to affect intraphagosomal conversion of hydrogen peroxide to hypochlorous acid. All the drugs inhibit myeloperoxidase reversibly by trapping it as its inactive redox intermediate, compound II. We propose that superoxide limits the potency of the drugs by reducing compound II back to the active enzyme. Furthermore, under conditions where the activity of myeloperoxidase exceeds that of the hydrogen peroxide-generating system, which is most likely to occur in phagosomes, partial inhibition of myeloperoxidase need not affect hypochlorous acid production. We conclude that drugs that inhibit myeloperoxidase by converting it to compound II are unlikely to be effective against hypochlorous acid-mediating tissue damage.

Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase.

Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.

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.

Myeloperoxidase: a key regulator of neutrophil oxidant production.

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. This heme enzyme uses hydrogen peroxide and chloride to catalyze the production of hypochlorous acid, which is the major strong oxidant generated by neutrophils in appreciable amounts. In addition to chlorination, myeloperoxidase displays several other activities. It readily oxidizes thiocyanate to hypothiocyanite, converts a myriad of organic substrates to reactive free radicals, and hydroxylates aromatic compounds. Depending on the concentration of its competing substrates and the conditions of the local environment, myeloperoxidase could substantially affect oxidant production by neutrophils. Superoxide is undoubtedly a physiological substrate for myeloperoxidase. Its interactions with the enzyme are key factors in determining how neutrophils use superoxide to kill pathogens and promote inflammatory tissue damage. Superoxide modulates the chlorination and peroxidation activities of myeloperoxidase. It also reacts with the enzyme to form oxymyeloperoxidase which is catalytically active and hydroxylates phenolic substrates. Myeloperoxidase reacts rapidly with nitric oxide and peroxynitrite so that at sites of inflammation there is a strong possibility that these reactions will impact on oxidative damage caused by neutrophils. Under certain conditions, many substrates of myeloperoxidase act as inhibitors and regulate oxidant production by the enzyme. Given the numerous reactions of myeloperoxidase, all its activities should be considered when assessing the injurious oxidants produced by neutrophils.