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

Abstract

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.