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 oftuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k = (1.22 0.05) 106 M 1 s 1 ] but reacted less favorably with compound II [(9.8 0.6) 102 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

Myeloperoxidase and oxidation of uric acid in gout: implications for the clinical consequences of hyperuricaemia

Objectives. The aims of this study were to establish whether, in patients with gout, MPO is released from neutrophils and urate is oxidized to allantoin and if these effects are attenuated by allopurinol. Methods. MPO, urate, allantoin and oxypurinol were measured in plasma from 54 patients with gout and 27 healthy controls. Twenty-three patients had acute gout, 13 of whom were receiving allopurinol, and 31 had intercritical gout, 20 of whom were receiving allopurinol. Ten additional gout patients had samples collected before and after 4 weeks of allopurinol. Results. Plasma MPO and its specific activity were higher (P < 0.05) in patients with acute gout not receiving allopurinol compared with controls. MPO protein in patients’ plasma was related to urate concentration (r = 0.5, P < 0.001). Plasma allantoin was higher (P < 0.001) in all patient groups compared with controls. In controls and patients not receiving allopurinol, allantoin was associated with plasma urate (r = 0.62, P < 0.001) and MPO activity (r = 0.45, P < 0.002). When 10 patients were treated with allopurinol, it lowered their plasma urate and allantoin (P = 0.002). In all patients receiving allopurinol, plasma allantoin was related to oxypurinol (r = 0.65, P < 0.0001). Oxypurinol was a substrate for purified MPO that enhanced the oxidation of urate. Conclusion. Increased concentrations of urate in gout lead to the release of MPO from neutrophils and the oxidation of urate. Products of MPO and reactive metabolites of urate may contribute to the pathology of gout and hyperuricaemia. At low concentrations, oxypurinol should reduce inflammation, but high concentrations may contribute to oxidative stress

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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead