Measurement of Biomarkers of Oxidative Stress in Plasma, Respiratory Fluids and Tissues Collected From Recurrent Airway Obstruction Affected Horses and Their Controls
Multiple biomarkers of oxidative stress have been measured and used in human medicine to diagnose and monitor airway disease. The purpose of the study was to determine if relationships existed between clinical status of RAO-affected horses or controls and; concentrations of isoprostanes and isofurans in plasma, EBC and BALF; mRNA expression of interleukin 4 (IL4) and gamma interferon (INFγ) in airway inflammatory cells of BALF; and mRNA expression of inducible nitric oxide synthase (iNOS), extracellular glutathione peroxidase (GPx-3), and cytosolic superoxide dismutase (SOD-1) from bronchial mucosal biopsies. In addition, EBC was evaluated as a non-invasive method to diagnose and monitor RAO-affected horses.
Eight pairs of non-RAO-affected (controls) and RAO-affected horses were used in this study. Horses were maintained on pasture for a minimum of 4 weeks to minimize exposure to respirable debris and sample 1 (remission) was taken after horses were determined to be in remission based on clinical parameters. Environmental challenge was performed one week later and continued until RAO-affected horses reached a clinical score of at least 5 (out of 8) or they stayed in the barn greater than 72 hours, at which time sample 2 (challenge) was collected. Sample 3 (recovery) was post-environmental challenge collected after paired horses were returned onto pasture with reduction in clinical score by 2 points, or after 1 week. Plasma, serum, EBC, and BALF were collected at each sample time. Bronchial mucosal biopsies were collected at challenge. A stable isotope dilution gas chromatography negative ion chemical ionization mass spectrometry (GC-MS) method was used to measure isoprostanes and isofurans. Measurement of IL4 and INFγ in BALF cells was by real time PCR, and iNOS, GPx-3, and SOD-1 from bronchial mucosal biopsies by quantitative PCR.
Isofurans could only be detected in EBC in three samples from RAO-affected and five samples from control horses. Isoprostanes were not detected in any EBC sample. In BALF and plasma no significant difference in isoprostanes or isofuran concentrations were found between either group or sample time. mRNA expression of IL4 and INFγ from cells harvested from BALF and iNOS, GPx-3, and SOD-1 from bronchial mucosal biopsies were also not significantly different.
None of the biomarkers measured could differentiate disease status of RAO-affected horses from their controls. This may indicate lack of test sensitivity, study power or a minimal role of the selected biomarkers in RAO. The use of EBC to measure isoprostanes or isofurans did not yield additional diagnostic information.