Phase Separation in Monolayers of Pulmonary Surfactant Phospholipids at the Air-Water Interface: Composition and Structure

The phase behavior of monolayers containing the complete set of purified phospholipids (PPL) obtained from calf surfactant was investigated as a model for understanding the phase transitions that precede compression of pulmonary surfactant to high surface pressure. During compression, both fluorescence microscopy and Brewster angle microscopy (BAM) distinguished domains that separated from the surrounding film. Quantitative analysis of BAM grayscales indicated optical thicknesses for the PPL domains that were similar to the liquid condensed phase for dipalmitoyl phosphatidylcholine (DPPC), the most abundant component of pulmonary surfactant, and higher and less variable with surface pressure than for the surrounding film. BAM also showed the optical anisotropy that indicates long-range orientational order of tilted lipid chains for the domains, but not for the surrounding film. Fluorescence microscopy shows that addition of DPPC to the PPL increased the area of the domains. At fixed surface pressures from 20 - 40 mN/m, the total area of each phase grew in proportion with the mol fraction of DPPC. This constant variation allowed analysis of the DPPC mol fraction in each phase, construction of a simple phase diagram, and calculation of the molecular area for each phase. Our results indicate that the phase surrounding the domains is more expanded and compressible, and contains reduced amounts of DPPC in addition to the other phospholipids. The domains contain a mol fraction for DPPC of at least 96%. The structure of the interfacial film of pulmonary surfactant is a critical determinant of its physiological function. Pul- monary surfactant is the mix of lipids and proteins that coats the alveolar air spaces. It adsorbs to the surface of the thin liquid layer that lines the alveoli, and the resulting film lowers interfacial tension and stabilizes small alveoli. Dur- ing the expiratory phase of the respiratory cycle, the de- creasing alveolar surface area compresses the film, and measurements in the lung indicate that its surface pressure achieves high values approaching 70 mN/m (Schurch, 1982). Lung surfactant's physiological function then de- pends on the formation of a film that remains stable at such high pressures without collapse from the interface. Most investigators have attributed this stability to the presence of a highly ordered liquid-condensed (LC) phase (Keough, 1992). The most prevalent component of surfactant, di- palmitoyl phosphatidylcholine (DPPC), occurs as the LC phase in single-component monolayers at physiologic tem- peratures and can achieve the pressures measured in the lung. This constituent, however, represents only a third of surfactant (Kahn et al., 1995), and both the structure and the composition of the functional film in situ remain uncertain. We have undertaken studies of the phase behavior of surfactant monolayers at pressures below the equilibrium spreading pressures of approximately 45 mN/m. Phase be- havior under these conditions provides important informa- tion for understanding the development of the structure and composition of the functional film at higher pressures. Our studies emphasize the full complexity of the surfactant