Irada Khalilova

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Background: Myeloperoxidase promotes oxidative stress during inflammation by producing hypochlorous acid. Results: Ceruloplasmin was a potent inhibitor of myeloperoxidase and slowed its activity in plasma from wild type mice compared with ceruloplasmin knock-out animals. Conclusion: Ceruloplasmin is a physiologically relevant inhibitor of myeloperoxidase. Significance: Ceruloplasmin will provide a protective shield against oxidant production by myeloperoxidase during inflammation. 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 FeV redox intermediate of myeloperoxidase, to Compound II, which has FeIV 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.

Measuring chlorine bleach in biology and medicine

BACKGROUND Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid. SCOPE OF REVIEW We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated. MAJOR CONCLUSIONS Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide. GENERAL SIGNIFICANCE These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Oxidation contributes to low glutathione in the airways of children with cystic fibrosis

Glutathione is an important antioxidant in the lungs but its concentration is low in the airways of patients with cystic fibrosis. Whether this deficit occurs from an early age or how oxidative stress contributes to lowering glutathione is unknown. We measured glutathione, its oxidation products, myeloperoxidase, and biomarkers of hypochlorous acid in bronchoalveolar lavage from children with cystic fibrosis and disease controls using mass spectrometry and immunological techniques. The concentration of glutathione was lower in bronchoalveolar lavage from children with cystic fibrosis, whereas glutathione sulfonamide, a specific oxidation product of hypochlorous acid, was higher. Oxidised glutathione and glutathione sulfonamide correlated with myeloperoxidase and a biomarker of hypochlorous acid. The percentage of glutathione attached to proteins was higher in children with cystic fibrosis than controls. Pulmonary infections in cystic fibrosis resulted in lower levels of glutathione but higher levels of oxidised glutathione and glutathione sulfonamide in bronchoalveolar lavage. The concentration of glutathione is low in the airways of patients with cystic fibrosis from an early age. Increased oxidation of glutathione by hypochlorous acid and its attachment to proteins contribute to this deficiency. Therapies targeted against myeloperoxidase may boost antioxidant defence and slow the onset and progression of lung disease in cystic fibrosis. The antioxidant glutathione is low in the airways of children with CF due to oxidation by hypochlorous acid http://ow.ly/tKfyX

Isoniazid as a substrate and inhibitor of myeloperoxidase: identification of amine adducts and the influence of superoxide dismutase on their formation.

Neutrophils ingest Mycobacteria tuberculosis (Mtb) in the lungs of infected individuals. During phagocytosis they use myeloperoxidase (MPO) to catalyze production of hypochlorous acid (HOCl), their most potent antimicrobial agent. Isoniazid (INH), the foremost antibiotic in the treatment of tuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k = (1.22 ± 0.05) × 10(6) M(-1) s(-1)] but reacted less favorably with compound II [(9.8 ± 0.6) × 10(2) M(-1) s(-1)]. Oxidation of INH by MPO and hydrogen peroxide was unaffected by chloride, the physiological substrate for compound I, and the enzyme was partially converted to compound III. This indicates that INH is oxidized outside the classical peroxidation cycle. In combination with superoxide dismutase (SOD), MPO oxidized INH without exogenous hydrogen peroxide. SOD must favor reduction of oxygen by the INH radical to give superoxide and ultimately hydrogen peroxide. In both oxidation systems, an adduct with methionine was formed and it was a major product with MPO and SOD. We show that it is a conjugate of an acyldiimide with amines. INH substantially inhibited HOCl production by MPO and neutrophils below pharmacological concentrations. The reversible inhibition is explained by diversion of MPO to its ferrous and compound III forms during oxidation of INH. MPO, along with SOD released by Mtb, will oxidize INH at sites of infection and their interactions are likely to limit the efficacy of the drug, promote adverse drug reactions via formation of protein adducts, and impair a major bacterial killing mechanism of neutrophils.

Oxidized glutathione and uric acid as biomarkers of early cystic fibrosis lung disease

BACKGROUND In cystic fibrosis (CF) there is an urgent need for earlier diagnosis of pulmonary infections and inflammation using blood- and urine-based biomarkers. METHODS Using mass spectrometry, oxidation products of glutathione and uric acid were measured in matched samples of bronchoalveolar lavage (BAL), serum and urine from 36 infants and children with CF, and related to markers of neutrophilic inflammation and infection in BAL. RESULTS Oxidation products of glutathione (glutathione sulfonamide, GSA) and uric acid (allantoin), were elevated in BAL of children with pulmonary infections with Pseudomonas aeruginosa (PsA) compared to those without (p<0.05) and correlated with other markers of neutrophilic inflammation. Serum GSA was significantly elevated in children with PsA infections (p<0.01). Urinary GSA correlated with pulmonary GSA (r=0.42, p<0.05) and markers of neutrophilic inflammation. CONCLUSIONS This proof-of-concept study demonstrates that urinary GSA but not allantoin shows promise as a non-invasive marker of neutrophilic inflammation in early CF lung disease.

Uric Acid and Thiocyanate as Competing Substrates of Lactoperoxidase

Background: Lactoperoxidase plays a key role in host defense by oxidizing thiocyanate to the bactericidal agent hypothiocyanite. Results: Urate is a good substrate for lactoperoxidase and competes with thiocyanate for oxidation in vitro. Conclusion: Urate is a likely physiological substrate for lactoperoxidase. Significance: Urate may influence the bactericidal activity of lactoperoxidase. The physiological function of urate is poorly understood. It may act as a danger signal, an antioxidant, or a substrate for heme peroxidases. Whether it reacts sufficiently rapidly with lactoperoxidase (LPO) to act as a physiological substrate remains unknown. LPO is a mammalian peroxidase that plays a key role in the innate immune defense by oxidizing thiocyanate to the bactericidal and fungicidal agent hypothiocyanite. We now demonstrate that urate is a good substrate for bovine LPO. Urate was oxidized by LPO to produce the electrophilic intermediates dehydrourate and 5-hydroxyisourate, which decayed to allantoin. In the presence of superoxide, high yields of hydroperoxides were formed by LPO and urate. Using stopped-flow spectroscopy, we determined rate constants for the reaction of urate with compound I (k1 = 1.1 × 107 m−1 s−1) and compound II (k2 = 8.5 × 103 m−1 s−1). During urate oxidation, LPO was diverted from its peroxidase cycle because hydrogen peroxide reacted with compound II to give compound III. At physiologically relevant concentrations, urate competed effectively with thiocyanate, the main substrate of LPO for oxidation, and inhibited production of hypothiocyanite. Similarly, hypothiocyanite-dependent killing of Pseudomonas aeruginosa was inhibited by urate. Allantoin was present in human saliva and associated with the concentration of LPO. When hydrogen peroxide was added to saliva, oxidation of urate was dependent on its concentration and peroxidase activity. Our findings establish urate as a likely physiological substrate for LPO that will influence host defense and give rise to reactive electrophilic metabolites.

A myeloperoxidase precursor, pro-myeloperoxidase, is present in human plasma and elevated in cardiovascular disease patients

Myeloperoxidase (MPO)-derived oxidants have emerged as a key contributor to tissue damage in inflammatory conditions such as cardiovascular disease. Pro-myeloperoxidase (pro-MPO), an enzymatically active precursor of myeloperoxidase (MPO), is known to be secreted from cultured bone marrow and promyelocytic leukemia cells, but evidence for the presence of pro-MPO in circulation is lacking. In the present study, we used a LC-MS/MS in addition to immunoblot analyses to show that pro-MPO is present in human blood plasma. Furthermore, we found that pro-MPO was more frequently detected in plasma from patients with myocardial infarction compared to plasma from control donors. Our study suggests that in addition to mature MPO, circulating pro-MPO may cause oxidative modifications of proteins thereby contributing to cardiovascular disease.