Assessment of a standardized ROS production profile in humans by Electron Paramagnetic Resonance (EPR)

Despite the growing interest in the role of reactive oxygen species (ROS) in health and disease, reliable quantitative methods for the assessment of oxidative stress in humans are still lacking. EPR, coupled to specific spin trapping molecular probes, could be the method of choice to gain a direct measurement of ROS in biological fluids and tissue. Differently from currently available ‘a posteriori’ assays of ROS-induced damage by means of bio-molecules such as proteins, lipids and DNA, spin-trapping EPR provides direct evidence of the ‘instantaneous’ presence of radical species in the sample. Moreover, in EPR spectra, signal areas are proportional to the number of excited electron spin, potentially leading to absolute concentration levels, when adopting a stable radical compound as reference. Methods: A recently developed bench top continuous wave system i.e. the E-Scan EPR scanner (Bruker) was used. It operates at the common X-Band microwave frequency (~ 9.8 GHz) dealing with very low concentration levels of paramagnetic species in small (50l) samples. Results The developed method allows among others reliable, quick and quantitative determinations of ROS in human venous or peripheral blood, provided all variables influencing the measurement (PO2, temperature, pH, probe concentration, acquisition and elaboration parameters) are standardized. The statistical correlation between EPR ROS results and data obtained by various conventional enzymatic assays such as Total antioxidant capacity (TAC) Protein Carbonyls (PC) and Thiobarbituric acid reactive substances (TBARS) in healthy subjects of different age, gender, training conditions has been determined in total blood, plasma and erythrocytes samples.