Assessment of Reproducibility of Exhaled Hydrogen Peroxide Concentration and the Effect of Breathing Pattern in Healthy Subjects

Background: Hydrogen peroxide (H2O2) is detectable in exhaled breath condensate (EBC) and has been proposed to be a surrogate marker of oxidative stress in the airways. In this study we tested whether the breathing pattern during EBC collection influences the concentration of exhaled H2O2. Methods: EBC was collected during (1) tidal breathing and (2) breathing with increased tidal volume for 10 min from 16 healthy volunteers. On-line H2O2 measurement was performed by the EcoCheck™ biosensor system. Repeated measurements were also conducted to assess intrasubject reproducibility. Results: Minute ventilation, tidal volume, expiratory flow rate were all increased significantly when subjects were asked to perform breathing with increased tidal volume. In parallel, EBC volume increased (1413 ± 59 vs. 1959 ± 71 μL, p < 0.001), whereas exhaled H2O2 levels decreased significantly (1400 ± 170 vs. 840 ± 130 nmol/L, p < 0.001). H2O2 levels did not correlate with any individual breathing parameters (p > 0.05). Assessment of intersubject variability of H2O2 measurements during the two types of breathing revealed a coefficient of variation of 49 and 54%, respectively. The EBC H2O2 measurement was highly reproducible (888 ± 176 vs. 874 ± 156 nmol/L) as tested during normal breathing. Conclusions: These data demonstrate that the concentration of H2O2 in EBC depends on the ventilatory pattern during sample collection that has to be taken into consideration in all EBC H2O2 assays.