Detection of protein-bound 3-nitrotyrosine in the plasma of pediatric patients with severe ARDS and avian influenza virus infection

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Nitric oxide (NO) and reactive oxygen species (ROS) may be involved in the pathogenesis of various diseases, including microbial infections, inflammatory diseases, and cancer. 3-Nitrotyrosine (3-NT) produced by NO and ROS is considered a biomarker of oxidative stress. Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease and is associated with the excessive production of NO and ROS. Immunohistochemical analyses showed that 3-NT may be produced in the lungs of patients with ARDS. We have identified the extensive and NO-dependent formation of 3-NT in the lungs of mice with ARDS caused by the influenza virus (IFV). However, the biochemical and quantitative aspects of 3-NT formation in patients with ARDS remain poorly understood. Thus, we investigated the levels of plasma protein-bound 3-NT in pediatric patients with severe ARDS using a reverse-phase high performance liquid chromatography (HPLC) coupled with electrochemical detector (ECD). The plasma samples of 40 patients with influenza-negative ARDS (non-IFV-ARDS group) and of 7 patients with influenza-positive ARDS (IFV-ARDS group) were analyzed. IFV-ARDS group consisted of two patients with highly pathogenic avian influenza (A/H5N1) and 5 patients with seasonal influenza (A/H1N1 and A/H3N2). Twenty-five patients without ARDS were used as control (non-ARDS group). Patients in the IFV-ARDS group had significantly higher 3-NT levels (median: 0.350 µmol/mol) than those in the non-ARDS group (median: 0.210; p = 0.046). Moreover, the 3-NT levels were significantly higher in the non-IFV-ARDS group (median: 0.270; p = 0.039) than in the non-ARDS group. However, the difference was not significant, the survivors had higher 3-NT levels than non-survivors, and the 3-NT levels were higher in patients without multiple organ failure (MOF) than those with MOF. Moreover, the survival rate was more likely higher in the high 3-NT level group than in the low 3-NT level group, indicating the protective role of NO/ROS in the pathogenesis of ARDS. Using this method, we could successfully detect 3-NT from the plasma of patients with ARDS. This method is convenient, specific, and sensitive for 3-NT quantification that is applicable on clinical specimens; hence, it may help in the further understanding of the pathological roles of NO/ROS formation in ARDS.