SIGNIFICANCE: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. RECENT ADVANCES: an increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. CRITICAL ISSUES: the literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. FUTURE DIRECTIONS: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker.

Advanced oxidation protein products (AOPP) are reportedly elevated in the plasma of patients with a number of diseases, including diabetes mellitus, that involve oxidative stress. However, the accurate measurement of AOPP in human plasma is hampered by the formation of a precipitate following the addition of potassium iodide and glacial acetic acid according to the published assay procedure. Here we describe a modification of the AOPP assay which eliminates interference by precipitation and provides a robust, reliable, and reproducible protocol for the measurement of iodide oxidising capacity in plasma samples (intra-assay CV 1.7-5.3%, interassay CV 5.3-10.5%). The improved method revealed a significant association of AOPP levels with age (p

Background: Recent findings suggest a role of oxidative stress in the pathogenesis of Behcet's disease (BD), but the utility of oxidative stress-associated assays in offering diagnostic information or in the monitoring of disease activity is largely unassessed. Objective and methods. We aimed to measure oxidative and inflammatory markers, along with the markers of reactive nitrogen species, S-nitrosothiols and 3-nitrotyrosine, in BD patients (n = 100) and healthy volunteers (n = 50). These markers were evaluated in regard to their role in the pathogenesis of BD as well as their relation to clinical presentation, disease activity and duration. Results: Median values for erythrocyte sedimentation rate (ESR), C-reactive protein, leukocyte count, and IL-18 levels, as well as myeloperoxidase (MPO) activity, were statistically higher in the patient group compared to controls. Some inflammation markers (ESR, neutrophil and leukocyte counts) were statistically higher (p < 0.05) in the active period. In contrast, oxidative stress-associated measures (erythrocyte lipid peroxidation, antioxidant enzymes and measures of serum antioxidant capacity), revealed no statistically significant differences between the median values in BD patients versus healthy control subjects (p > 0.05 in all statistical comparisons), nor was there any difference in median levels of these oxidative stress markers in active disease versus disease remission. S-nitrosothiols and 3-nitrotyrosine were undetectable in BD plasma. Conclusions: the application of oxidative stress-associated measures to BD blood samples offered no supplemental diagnostic or disease activity information to that provided by standard laboratory measures of inflammation. S-nitrosothiols and 3-nitrotyrosine appeared not to be markers for active BD; thus the search for biochemical markers that will indicate the active period should be continued with larger studies. © 2012 Akcay et al; licensee BioMed Central Ltd.

Apoptosis of neutrophils and their subsequent phagocytosis is critical to the successful resolution of inflammation. During inflammation, activated inflammatory cells generate reactive oxygen and nitrogen species, including nitric oxide (NO) and superoxide anion (O(2)(•-)), which rapidly combine to generate peroxynitrite (ONOO(-)). NO and ONOO(-) are proapoptotic in human neutrophils. This study examines the effects of NO and ONOO(-) on caspase activation and mitochondrial permeability in human neutrophils and determines the ability of these species to evoke apoptosis in human monocyte-derived macrophages (MDMs). NO or ONOO(-) release from donor compounds was characterized by electrochemistry and electron paramagnetic resonance. Neutrophils and MDMs isolated from the peripheral blood of healthy volunteers were exposed to NO or ONOO(-) before analysis of apoptosis by caspase activation, mitochondrial permeability, and annexin V binding. Both NO and ONOO(-) induced apoptosis via rapid activation of caspases 2 and 3 in neutrophils. In contrast, only ONOO(-) promoted apoptosis in MDMs, whereas a variety of NO donors were ineffective at inducing apoptosis in this cell type. We propose that human macrophages are refractory to NO-stimulated apoptosis in order that they persist long enough within the inflammatory focus to phagocytose apoptotic neutrophils, thereby ensuring successful resolution of inflammation.

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO•, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.

Neutrophil-derived granule enzymes, oxidants, and mediators have been implicated in the pathogenesis of a variety of inflammatory diseases. Neutrophil apoptosis is associated with the loss of expression of adhesion molecules and greatly reduced responsiveness to external stimuli, so that these cells become functionally isolated from their environment. In contrast with necrosis, apoptosis is associated with preservation of plasma membrane integrity, so that release of harmful neutrophil contents is limited, and the inert neutrophils are phagocytosed by local macrophages. Furthermore, phagocytosis of apoptotic neutrophils by human macrophages in vitro suppresses release of macrophage-derived pro-inflammatory mediators. In this way, by downregulating neutrophil functions and triggering "silent" clearance by phagocytes, apoptosis provides a mechanism for the safe disposal of potentially destructive inflammatory cells. Many of the molecular events involved in the apoptosis pathway have been identified and several complementary methods may be employed to identify and quantitate neutrophil apoptosis. This chapter will discuss analysis of neutrophil morphology, DNA fragmentation, membrane changes, mitochondrial alterations, caspase activation, and phagocytosis of apoptotic neutrophils by macrophages.

Apoptosis may be regulated by oxidants such as peroxynitrite (ONOO(-)). The tumour suppressor, p53, has been reported to play a crucial role in apoptosis induced by oxidants, therefore we assessed the ability of a ONOO(-) donor, GEA 3162, to activate caspases and induce mitochondrial permeability in a p53-deficient murine bone marrow cell line, Jaws II. Furthermore, these cells were stably transfected with Bcl-2, in order to investigate the impact of this survival protein on ONOO(-)-induced apoptosis. GEA 3162 activated caspases and induced loss of mitochondrial membrane potential in Jaws II cells. In particular, caspases 3 and 2 were activated, alongside minor activation of caspases 8 and 9, and apoptosis was partially dependent upon p38 MAP kinase activation, with little or no role for JNK. Overexpression of Bcl-2 abolished activation of all caspases and reduced the change in mitochondrial membrane potential. Thus, we have demonstrated that the ONOO(-) donor, GEA 3162, induces apoptosis in Jaws II murine myeloid cells despite lacking functional p53, via a pathway that principally involves caspases 2 and 3 and mitochondrial changes. This is blocked by overexpression of Bcl-2 via a mechanism that does not appear to merely reflect stabilisation of the mitochondrial membrane.

The ubiquitous free radical, nitric oxide (NO), plays an important role in many biological processes including the regulation of the inflammatory response. Alterations in NO synthesis by endogenous systems likely influence inflammatory processes occurring in a wide range of diseases including many in the cardiovascular system (e.g. atherosclerosis). Progression of inflammatory conditions depends not only upon the recruitment and activation of inflammatory cells but also upon their subsequent removal from the inflammatory milieu. Apoptosis, or programmed cell death, is a fundamental process regulating inflammatory cell survival and is critically involved in ensuring the successful resolution of an inflammatory response. Apoptosis results in shutdown of secretory pathways and renders effete, but potentially highly histotoxic, cells instantly recognisable for non-inflammatory clearance by phagocytes (e.g. macrophages). However, dysregulation of apoptosis and phagocytic clearance mechanisms can have drastic consequences for development and resolution of inflammatory processes. In this review we highlight the complexities of NO-mediated regulation of inflammatory cell apoptosis and clearance by phagocytes and discuss the molecular mechanisms controlling these NO mediated effects. We believe that manipulation of pathways involving NO may have previously unrecognised therapeutic potential for limiting or resolving inflammatory and cardiovascular disease.

The accumulation of neutrophils during inflammation is essential for the destruction and removal of invading microorganisms. However, for resolution of inflammation to occur, neutrophils must also be removed from the inflammatory site since these cells are capable of releasing tissue toxic molecules. Neutrophil removal has been shown to occur via apoptosis and phagocyte clearance of apoptotic cells. Therefore, manipulation of these processes is likely to be a key therapeutic strategy in the management of inflammatory disease. In this review, we examine mediators of neutrophil survival and apoptosis and the signalling pathways that regulate the balance between life and death in these cells.

1. GEA 3162 (1,2,3,4,-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-chloride), has powerful effects on neutrophil function and apoptosis, but the underlying mechanisms are unclear, particularly with respect to the possible roles of nitric oxide (NO) and/or peroxynitrite (ONOO(-)). 2. Our hypothesis was that GEA 3162 is a generator of ONOO(-) and that its biological effects on neutrophil apoptosis differ from those of a conventional NO donor. The effects of GEA 3162 were compared to those of the established ONOO(-) donor, 3-morpholinosydnonimine (SIN-1), and the NO donor, diethylamine diazeniumdiolate (DEA/NO) in neutrophils from healthy volunteers. Electrochemical detection and electron paramagnetic resonance were used to define the NO-related species generated from these agents. 3. GEA 3162 and SIN-1 influence neutrophil apoptosis differently from DEA/NO. All three compounds induced morphological neutrophil apoptosis. However, both GEA 3162 and SIN-1 paradoxically inhibited internucleosomal DNA fragmentation, whereas DEA/NO induced fragmentation compared to control. 4. In contrast to DEA/NO, generation of free NO was not detectable in solutions of GEA 3162 or SIN-1 (100 microm). However, Cu/Zn superoxide dismutase (SOD; 50-750 U ml(-1)) unmasked NO generated from these compounds in a concentration-dependent manner. GEA 3162 and SIN-1 oxidised the O(2)(-)- and ONOO(-)-sensitive dye, dihydrorhodamine 123 (DHR 123; 1 microm), suggesting that ONOO(-) released from these compounds is responsible for oxidation of DHR 123. 5. We conclude that GEA 3162 is an ONOO(-) donor with pro-apoptotic properties that more closely resemble SIN-1 than the NO donor, DEA/NO. Moreover, unlike NO, ONOO(-) induces apoptosis in neutrophils via a mechanism that does not require DNA fragmentation.

Apoptosis of inflammatory cells is a critical event in the resolution of inflammation, as failure to undergo this form of cell death leads to increased tissue damage and exacerbation of the inflammatory response. Many factors are able to influence the rate of apoptosis in neutrophils, eosinophils, monocytes and macrophages. Among these is the signalling molecule nitric oxide (NO), which possesses both anti- and proapoptotic properties, depending on the concentration and flux of NO, and also the source from which NO is derived. This review summarises the differential effects of NO on inflammatory cell apoptosis and outlines potential mechanisms that have been proposed to explain such actions.

The events of apoptotic cell death can be experimentally dissociated from each other in certain cell types. Here we demonstrate the ability of structurally diverse nitric oxide (NO) donating compounds to delay or enhance neutrophil apoptosis and to differentially influence distinct parameters of programmed cell death. We provide evidence that high concentrations of the NO donors GEA 3162, SPER/NO, and DEA/NO induce morphological and biochemical markers of neutrophil apoptosis, but that only DEA/NO causes a concomitant increase in DNA fragmentation as evidenced by nuclear propidium iodide intercalation and the classical laddering pattern of electrophoresed DNA. In contrast, both GEA 3162 and SPER/NO inhibit DNA cleavage in a time- and concentration-dependent manner. We are the first to show that DNA fragmentation can be dissociated from other changes of apoptosis in NO-treated neutrophils and that it may therefore be inappropriate to assess NO-induced apoptosis solely by measuring DNA fragmentation in this cell type.