Surfactant function and composition after free radical exposure generated by transition metals

Surfactant dysfunction in acute lung injury has been postulated as a result of free radical damage to lipid and protein components. This study examines whether transition metals with different redox potentials and different binding affinities for lipids and proteins affect interfacial properties differently. Purified whole calf lung surfactant (CLS) was incubated with 0.125 mM Fe2+, Fe3+, Fe3+-EDTA complex, or Cu2+ either alone or with 0.25 mM H2O2 or H2O2 plus 0.25 mM ascorbate for 4 and 24 h. Lipid peroxidation was assessed by measurement of thiobarbituric acid-reactive substances (TBARS), and free radical-mediated alterations in protein structure were assessed by fluorescamine assay and Western blot analysis. Function was assayed by pulsating bubble surfactometry. Lipid peroxidation was detected in samples incubated with Fe2+, Fe3+, and Fe3+-EDTA but not with Cu2+. All transition metal-based free radical systems affected surfactant protein composition by fluorescamine assay, indicating free radical-mediated modification of protein side chains. Western blot analysis demonstrated surfactant protein A modification, with the generation of higher- and lower-molecular-mass immunoreactive products. Despite biochemical evidence of lipid and protein modification, surfactant dysfunction was minimal and was manifest as an increase in the compression ratio required to achieve surface tension < 1 dyn/cm. This dysfunction was readily reversed by the addition of 5 mM Ca2+ either before or after oxidation. These data indicate that copper- and iron-based free radical-generating systems modify the lipid and protein components of surfactant differently but suggest that these changes have little effect on surfactant function.