In vitro inactivation of surface active properties of pulmonary surfactant by hydrogen peroxide and ferrous sulfate and possibility of prevention by antioxidant enzymes 1962

Cells exposed to hyperoxia or inflammatory cells can generate reactive oxygen species (ROS). These directly cause lipid peroxidation and inactivation of protein enzymes. Hydrogen peroxide at a high concentration was shown to inactivate pulmonary surfactant (PS). However, the most powerful agent known would be hydroxyl radicals, which can be produced through chemical reaction between hydrogen peroxide and ferrous ion. We hypothesized that in the presence of ferrous ions, which may be quite abundant in the lung, especially when there is pulmonary hemorrhage, hydroxyl radicals will be formed and will inactivate PS. We also evaluated the effect of pre- and post-treatment of antioxidant catalase. We used Surfacten^R (Tokyo-Tanabe, Japan) to prepare PS suspension of phospholipid (PL) concentration 0.5 mg/ml. H₂O₂ (final conc. 1 mM) was added to H group; FeCl₂(final conc. 1 mM) to F group; both H₂O₂ and FeCl₂ (final conc. 1 mM each) were added to HF group; and neither was added to the control(C) group. For each group catalase 10 U was added either before or after the addition of ferrous chloride and hydrogen perxide to PS. We measured in vitro minimum and maximum surface tensions(ST) and area-surface tension relationship using a Pulsating Bubble Surfactometer (Electronetics, NY). Results are 1) 5-minute minimum and maximum ST of H were higher than C (C: 4.7± 0.3 mN/m, 38.1 ± 0.1 mN/m; H: 15.1 ± 6.7 mN/m, 43.9± 5.9 mN/m); those of F were not much different from C; and those of HF were markedly increased to 15.4 ± 2.1 mN/m and 45.9 ± 6.1 mN/m similar to H, but the disruption of hysteresis curve was more prominent in HF group. The corresponding groups for which catalase was pretreated were safe from ROS inactivation of PS. But, when catalase was added after PS was incubated with hydrogen peroxide and/or ferrous chloride, the surface active properties were not recovered to the control levels. In conclusion, H₂O₂, which may be produced by SOD after exposure to hyperoxia, may further react with ferrous ion to form hydroxyl radicals, which aggravate the damage on the PS. This reaction may be prevented by pretreating an antioxidant such as catalase, which disintegrates hydrogen peroxide in advance. However, once damage has occurred to PS, the recovery of the original surface active properties of PS is not feasible.