Applicability of FTIR-ATR Method to Measure Carbonyls in Blood Plasma after Physical and Mental Stress

INTRODUCTION: Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. METHODS: We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. RESULTS: The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. CONCLUSION: The FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.