Method for the isolation of surfactant from homogenates and lavages of lung of adult, newborn, and fetal rats.

A method is presented for the isolation of pulmonary surfactant from rat lung homogenates and lavages using differential discontinuous density gradient centrifugation. The isolated surfactant was characterized by electron microscopy, assay of enzyme markers for different subcellular organelles, and its chemical composition. Surfactant that was isolated from lung homogenates appeared to be free of other contaminating cellular organelles. Electron microscopic examination of surfactant isolated from whole homogenates showed the presence of lamellar bodies and tubular myelin figures and, hence, represented total lung surfactant. On the other hand, surfactant prepared from lung lavages had mostly tubular myelin figures and, thus, represented extracellular surfactant. Both preparations were rich in phospholipids, especially lecithins and phosphatidylglycerol, and showed high phospholipid to protein ratios. The method was used to study the quantity and composition of surfactant during lung development in rats. Surfactant was isolated from lung homogenates of fetuses at days 19 to 21 of gestation and from newborn and adult rats. During the period of 19 to 21 days of gestation, there is a 10- and 20-fold increase in the amount of protein- and phospholipids (milligrams per gram of wet lung), respectively. A further 2-fold increase occurs after birth. Phosphatidylcholines account for 65% of the total phospholipids at day 20, 71% on day 21, and 81% at term. A progressive increase in the phosphatidylcholine to sphingomyelin ratios and in the amount of phosphatidylglycerol occurs during this period. The amount of disaturated lecithins (expressed as percentage of total phosphatidylcholines) in the lung surfactant increases from 49% at day 20 to 53% at birth. There is no change in the amount of disaturated lecithins after the newborn stage. The present method for the isolation of lung surfactant is reproducible, is less time consuming, and can be used to isolate quantitatively surfactant from small lung aliquots.