In the present study, we explored the potential utility of measures of oxidative stress in monitoring disease activity, and in the diagnosis of complications, in BD patients. We applied a broad panel of widely adopted oxidative stress-associated measures to plasma, serum and erythrocyte samples. These measures were carried out in conjunction with both standard laboratory assays of the inflammatory response, such as CRP and ESR, and clinical assessments. We aimed to assess the additional clinical information that measures of oxidative stress might provide in terms of disease activity or the diagnosis of complications.
As expected, the group of BD patients showed evidence of a substantial inflammatory response, as indicated by high median levels of CRP, ESR, blood leukocyte count, MPO and IL-18, compared with healthy, age- and sex-matched control individuals. Moreover, when the BD group was subdivided into active disease and remission subgroups, some of these inflammatory measures were clearly elevated in the active disease subgroup compared with the remission subgroup. In contrast - and as expected - measures of inflammation provided no obvious indication of clinical complications or disease duration. Thus, our findings with standard inflammatory measures are consistent with their known nonspecific increase in BD. None of these markers can be defined to be a specific measure for disease activity or an indicator of any complication. 
TNF-α is an important proinflammatory cytokine and it has been implicated in the pathogenesis of many inflammatory and autoimmune diseases such as sepsis, inflammatory intestinal disease  and RA . Similar to these studies, Oztas et al  have shown the role of this cytokine in BD patients. Our study found no statistically significant evidence of elevated TNF-α concentrations in serum samples from patients versus healthy subjects, nor in BD patients with active disease versus patients in remission. This might be due to the presence of other proteins released in the active period which interfere with the TNF-α measures or lack of ability of the current assay to detect TNF-α that had been complexed by the soluble TNF receptor. Our results demonstrate that serum TNF-α is not a reliable biomarker of either the presence of BD or of disease activity.
Recently, Musabak and colleagues  and Lee and colleagues  independently showed the role of another proinflammatory cytokine, IL-18, in the pathogenesis of BD. It was proposed that IL-18 probably directs the immune response in BD toward the T helper 1 type pathway. This hypothesis was supported by high serum levels of IL-18 in inactive patients, indicating an ongoing T helper polarization and subclinical inflammation during the inactive period. Interestingly, we also observed higher IL-18 levels in the BD patient group when compared with controls. In regard to IL-18 concentrations in active and remission periods, the difference between the median values in the two groups was less marked, although still statistically significant. This may be due to the subclinical inflammation during the inactive period, as proposed previously .
Increased neutrophil activation is observed in BD patients and this can directly be determined with MPO activities. In accordance with previous studies [25, 26], leukocyte MPO activity was higher in the BD patient group than the control group in our study. In addition, the weak positive correlation found between leukocyte MPO activity and serum TNF-α in BD patients (r = 0.232, p = 0.041) demonstrates the concomitant increase in neutrophil activation and inflammation markers in BD.
The strong negative correlation between leukocyte MPO activity and FRAP (r = -0.343, p = 0.001) in this study shows the importance of the oxidant/antioxidant balance in BD patients. In inflammatory diseases such as BD, MPO production from activated neutrophils is increased, which could be consistent with oxidative stress. Also consistent with oxidative stress, plasma antioxidant activity in BD is reportedly decreased .
Although increased oxidative stress has been suggested to be another hallmark of BD [2, 28, 29], the present study provides little evidence to support this idea, despite the clearly inflamed status of the BD group. Previously, a number of studies have shown an increase in serum TBARS and eTBARS concentrations in BD patients [27, 30, 31]. In contrast, our study showed no evidence that eTBARS levels were higher in patients than controls.
In the present study, when patients were grouped for ocular, vascular, articular and mucocutaneous involvements, the measured parameters did not show statistically significant differences between groups. In contrast, Taysi et al.  have shown a high serum MDA level and SOD activity in patients with ocular involvement. We also found no correlations between measures of oxidative stress and disease duration. In this respect, our results are in concordance with other related literature .
It has been proposed that NO plays a role in the pathogenesis of BD. Laroux et al.  showed that NO modulates the adhesion molecules induced by cytokines in inflammatory processes and Gunduz et al.  futher suggested that increased NO release from the endothelium is expected in BD patients due to enhanced leukocyte-endothelium interactions. Previously increases in NO production due to various forms of the tissue injury in inflammation have been shown . In recent years studies showed that S-nitrosothiols, formed by the reaction of reactive nitrogen species (derived from NO) with free thiols, are increased in biologic systems as a response to inflammatory reactions [5, 37]. In the current study RSNOs in the plasma of BD and control subjects were not within the detection limit of the analytical technique (EPR spectrometry with spin trapping). The technique was further applied with various preanalytical and analytical modifications such as ultrafiltration, incubating at alternative temperatures and time, and enzymatic proteolysis. Despite all these modifications, RSNOs could not be detected and this is attributed to their very low levels in BD plasma. Previously, RSNOs were detected in plasma and synovial fluid from rheumatoid arthritis patients, but seldom in healthy control subjects .
Recently 3-NT, which is produced by nitration of tyrosine by reactive nitrogen species has been suggested to be a useful marker for inflammation and NO-mediated tissue injury [38, 39]. Both the free and protein-bound nitrotyrosine forms are increased in various tissues in inflammatory disorders such as atherosclerotic plaques, intestinal tissue of inflammatory bowel disease patients, and lung tissue of adults with respiratory distress syndrome [40–42]. In our study, we aimed to determine whether the local formation of 3-NT in the inflammatory processes in BD is also systemically detectable. In our previous work investigating the 3-NT levels in RA patients , a proportion of both plasma and SF samples from RA patients had detectable 3-NT levels with the ELISA sandwich method used. However, in the current study no detectable 3-NT was observed in BD patients or controls with the same method. This was also in contrast to the findings of epnr et al .
In conclusion, in agreement with previous studies [22, 23, 25, 27], significant increases in BD patients compared with healthy control subjects, were observed in ESR, CRP, leukocyte count, IL-18, and MPO enzyme activity, indicating the activation of leukocytes and macrophages in the inflammatory process. However, in contrast to previous studies, we found that the assays used appeared to offer no potential for providing additional insight into disease activity or the diagnosis of disease complications. RSNOs and 3-NT, which are linked to NO metabolism and are proposed to play a role in the pathogenesis of BD, were not proved to be markers for the active period of BD. We propose that the search for a biochemical marker that will indicate the active period in BD should be continued with new (more specific and sensitive) measures of oxidative stress, and in larger studies.