Purification of surfactant from lung washings and washings contaminated with blood constituents

Abstract A rapid and simple method capable of purifying surfactant from rabbit alveolar washings and from washings contaminated with serum has been developed. The sample, containing 16% NaBr, is placed beneath a two‐layer discontinuous gradient of NaBr. After centrifugation, the surfactant is found near the top of the gradient tube at a density of 1.085 at 4 C while the contaminating material remains near the bottom. The lipid composition of surfactant from lung washings of normal animals isolated by this method compares quite favorably with surfactant isolated by much more elaborate and time consuming methods. Surfactant purified from mixtures of3H‐palmitate labeled rabbit serum and lung washings (1.6 mg serum phospholipid: 1 mg washing phospholipid) contained less than 3% of the phospholipid radioactivity. The phospholipid composition of this band was quite similar to that of surfactant from normal lung washings, but the protein content was much higher. A second density gradient centrifugation removed 90% of this protein resulting in a surfactant fraction with a phospholipid to protein ratio similar to that of surfactant from normal lung washings. These findings demonstrate that this purification method is capable of removing a large proportion of both serum phospholipids and proteins from lung washings contaminated with serum, making this method uniquely suitable for evaluation of surfactant in pathologic conditions of the lung.