Lateral phase separation in interfacial films of pulmonary surfactant

Abstract

To determine if lateral phase separation occurs in films of pulmonary surfactant, we used epifluorescence microscopy and Brewster angle microscopy (BAM) to study spread films of calf lung surfactant extract (CLSE). Both microscopic methods demonstrated that compression produced domains of liquid-condensed lipids surrounded by a liquid-expanded film. The temperature dependence of the pressure at which domains first emerged for CLSE paralleled the behavior of its most prevalent component, dipalmitoyl phosphatidylcholine (DPPC), although the domains appeared at pressures 8-10 mN/m higher than for DPPC over the range of 20-37 degrees C. The total area occupied by the domains at room temperature increased to a maximum value at 35 mN/m during compression. The area of domains reached 25 +/- 5% of the interface, which corresponds to the predicted area of DPPC in the monolayer. At pressures above 35 mN/m, however, both epifluorescence and BAM showed that the area of the domains decreased dramatically. These studies therefore demonstrate a pressure-dependent gap in the miscibility of surfactant constituents. The monolayers separate into two phases during compression but remain largely miscible at higher and lower surface pressures.

Neutral Lipids Induce Critical Behavior in Interfacial Monolayers of Pulmonary Surfactant

“…2 Similar results have been reported previously for binary mixtures of DPPC and dioleoyl PC (22). This behavior, however, differs greatly from the much larger decrease observed for CLSE over a narrow range of surface pressure (1). Only N&PL produces a similarly abrupt drop in the nonfluorescent area.…”

“…N&PL produced the largest nonfluorescent phase, reaching a maximum of 26 ± 3% of the interface at 25 mN/m for which the domains in PPL occupied only 14 ± 1%. The domains in CLSE occupied 25 ± 5% of the interface at 35 mN/m, with areas at lower pressures generally lying intermediate to SP&PL and N&PL and comparable to values for PPL (1). The neutral lipids therefore increased the total area in N&PL relative to PPL and in CLSE relative to SP&PL.…”

“…The total area of the nonfluorescent phase in each case remains below or comparable to the values expected if all DPPC resided in LC domains. The fraction f c of constituents located within condensed domains with molecular area à c that occupy the interfacial fraction ϕ c of a film with average molecular areaà is given by (21) (1) Using values from DPPC isotherms for à c , this equation predicts that, at their maximum areas, the domains contain 27, 18, and 36% of the constituents in PPL, SP&PL, and N&PL, respectively. Prior quantitative analysis indicates that DPPC constitutes 33% of the phospholipids and 30% of the total lipids in CLSE (4).…”

“…Only films with the physical characteristics of a highly ordered phase seem likely to have sufficient rigidity to withstand compression to such high densities without collapse from the interface. The most prevalent component of surfactant, dipalmitoyl phosphatidylcholine (DPPC), 1 is capable of achieving the stable liquid condensed (LC) phase. When compressed in vitro, single component films of DPPC reach the high surface pressures implied by the measurements in situ.…”

Distribution of Coexisting Solid and Fluid Phases Alters the Kinetics of Collapse from Phospholipid Monolayers

“…49 Consequently, at 37 °C, where the main transition for DPPC begins at ~35 mN/m, the l o phase in CLSE appears just before the onset of collapse. 49 The dispersion of coexisting phases should, therefore, have little effect on the rates of collapse when that process first begins, although that distribution might become more important if exclusion of the l d phase would substantially increase the fractional area of the l o phase. Although the importance of phase separation for native pulmonary surfactant is therefore uncertain, we agree that the dispersion of coexisting phases might affect the behavior of therapeutic surfactants containing simpler mixtures.…”