Sequential isolation of lamellar bodies and surfactant fractions from rat lungs

Abstract

In the present study, we developed a simple procedure, shorter than most, which allows the sequential isolation of lamellar bodies and surfactant fractions from rat lungs. The surfactant was isolated from serial bronchoalveolar lavages obtained with a balanced salt solution (Ca2+ and Mg2+ free). The cell-free supernatants from the lavages were layered on 20% sucrose (0.58 M) and centrifuged at 4.9 × 106 g∙min. The interface was collected and washed by centrifugation. The enriched surfactant fraction was recovered as a tight pellet. From the same animals, the lavaged lungs were homogenized in 0.25 M sucrose and centrifuged at 2.6 × 105 g∙min. The upper loose pellet was layered on 0.58 M sucrose and centrifuged at 1.1 × 107 g∙min. As for the surfactant fraction, the lamellar bodies were also collected at a 0.25 M – 0.58 M sucrose interface, washed, and recovered as a tight pellet. Both electron microscopy and marker enzyme studies confirmed the structural integrity of the two fractions, as well as the relative absence of major contaminants such as lysosomes, mitochondria, and microsomes. Biochemical analysis such as phospholipid to protein ratios (surfactant, 8.0; lamellar bodies, 6.7), phospholipid compositions (phosphatidylcholines and phosphatidylglycerols: respectively, ~80 and ~10% for both fractions), fatty acid analysis (palmitic acid in phosphatidylcholines and phosphatidylglycerols: respectively, 69 and 62% for the lamellar bodies and 76 and 60% for the surfactant), and minimum surface tension measurements (10 mN∙m−1 for the surfactant and 12 mN∙m−1 for the lamellar bodies) confirmed the close relationship between the two lung fractions. From three major hydrolases detected in the lamellar bodies and surfactant fractions, only the alkaline phosphatase was enriched in the surface active material isolated from the alveolar lung lining. Our results indicate that this alkaline phosphatase may represent a potential marker by which the synthesis and secretion processes of lung surfactant could be monitored.