Circulating myeloperoxidase is elevated in septic shock and is associated with systemic organ failure and mortality in critically ill patients.

BACKGROUND: Neutrophils are elevated in critically ill patients during the systemic inflammatory response to trauma and sepsis. The neutrophil-derived enzyme myeloperoxidase generates reactive oxygen species which can react with host tissue resulting in cell damage and dysfunction. Thus, elevated myeloperoxidase in the circulation may be associated with adverse patient outcomes. METHODS: Circulating myeloperoxidase concentrations were measured in a cohort of 44 critically ill patients, 55% of whom were diagnosed with septic shock, and 44 healthy controls. Intensive care mortality prediction scores (SOFA, SAPS, APACHE) and ICU and hospital mortality were obtained from the patients' clinical notes. Hematological and biochemical assessments included blood cell counts, lactate, alanine transaminase, creatinine, bilirubin, C-reactive protein, and PaO2. Myeloperoxidase was measured using a commercial ELISA kit and cell free DNA was detected using SytoxGreen™ fluorescence staining. RESULTS: Myeloperoxidase concentrations were significantly higher in critically ill patients than control samples (234 ±â€¯30 ng/ml versus 15 ±â€¯4 ng/ml, p < 0.001), and were elevated in septic shock relative to non-septic patients (302 ±â€¯42 ng/ml versus 156 ±â€¯38 ng/ml, p = 0.02), despite neutrophil counts being comparable between the two subgroups (p = 0.6). Myeloperoxidase correlated with SOFA scores in the critically ill patients (r = 0.395, p = 0.02), and with markers of tissue dysfunction and injury such as lactate (r = 0.572, p < 0.001), log10 alanine transferase (r = 0.392, p = 0.016) and log10 cell free DNA (r = 0.371, p = 0.03). The subgroup of patients with higher than mean APACHE III scores (i.e. >78, n = 16) exhibited significantly elevated myeloperoxidase concentrations in the non-survivors compared with survivors (416 ±â€¯59 ng/ml versus 140 ±â€¯33 ng/mL, p = 0.001). Hospital mortality for the whole cohort was 27%; mortality in the high APACHE III subgroup was 38%, and when combined with higher than mean myeloperoxidase (i.e. >234 ng/mL), mortality increased to 71%. CONCLUSIONS: Myeloperoxidase is associated with markers of tissue injury and systemic organ failure, particularly in septic patients. The enzyme is also associated with mortality in patients with higher APACHE III scores, and thus has potential as an additional diagnostic marker to improve mortality prediction.

Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis.

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 µM, cross-linked calprotectin (5 µM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 µM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

Oxidative stress in early cystic fibrosis lung disease is exacerbated by airway glutathione deficiency.

Neutrophil-derived myeloperoxidase (MPO) is recognized as a major source of oxidative stress at the airway surface of a cystic fibrosis (CF) lung where, despite limited evidence, the antioxidant glutathione is widely considered to be low. The aims of this study were to establish whether oxidative stress or glutathione status are associated with bronchiectasis and whether glutathione deficiency is inherently linked to CF or a consequence of oxidative stress. MPO was measured by ELISA in 577 bronchoalveolar lavage samples from 205 clinically-phenotyped infants and children with CF and 58 children without CF (ages 0.2-6.92 years). Reduced glutathione (GSH), oxidized glutathione species (GSSG; glutathione attached to proteins, GSSP; glutathione sulfonamide, GSA) and allantoin, an oxidation product of uric acid, were measured by mass spectrometry. The odds of having bronchiectasis were associated with MPO and GSSP. GSH was low in children with CF irrespective of oxidation. Oxidized glutathione species were significantly elevated in CF children with pulmonary infections compared to uninfected CF children. In non-CF children, infections had no effect on glutathione levels. An inadequate antioxidant response to neutrophil-mediated oxidative stress during infections exists in CF due to an inherent glutathione deficiency. Effective delivery of glutathione and inhibition of MPO may slow the development of bronchiectasis.