A Direct Test of the “Squeeze-Out” Hypothesis of Lung Surfactant Function. External Reflection FT-IR at the Air/Water Interface

The current theory of pulmonary surfactant function requires that very low surface tension be achieved and maintained in the alveolar surface film during compression (expiration). To effect this condition, it has been hypothesized that the unsaturated and/or fluid components of surfactant are selectively excluded or “squeezed out” from mixed monolayers containing both saturated and unsaturated phospholipids, leaving a surface film of essentially pure 1,2-dipalmitoylphosphatidylcholine (DPPC). External reflection Fourier transform infrared (FT-IR) spectroscopy has been employed to quantitatively test this hypothesis. Mixed monolayer films of acyl chain-perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d₆₂) with 1,2-dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl, 2-oleoylPG (POPG), 1,2-dipalmitoylPG (DPPG) were examined in situ at the air/water interface as a function of surface pressure. The relative intensities of CD₂ (CM₂) stretching vibrations of the deuterated (proteated) components permitted quantitative determination of the relative concentrations of each in the film. For 7:1 (mokmol) mixtures of DPPC- d₆₂/DOPG, progressive, selective squeeze out of up to about 90% of the PG component is observed over a range of surface pressures from about 51 to 68 mN/m. The extent of maximal PG squeeze out was reduced to 61% for a 7:1 (mokmol) mixture of DPPCd₆₂/POPG. This phenomenon, which is at least partially reversible, appears to require relatively high rates of film compression. Squeeze out was reduced (<20%) for 7:1 (mokmol) mixtures of DPPC-d₆₂/DPPG or for 7:3 mixtures of DPPC-d₆₂/POPG. Squeeze out requires that the lipid mixture achieve surface pressures greater than about 50–60 mN/m along with unsaturation (or at least conformational disorder) in the acyl chains of the non-DPPC component. Examination of the CH₂ stretching frequencies of the unsaturated phospholipids showed them to be conformationally disordered at the highest surface pressures achievable in single-component films but conformationally ordered in 7:1 binary mixtures with DPPC-d₆₂ at high pressures. The DPPC-d₆₂ component is conformationally ordered throughout. The relevance of these observations to the mechanism of pulmonary surfactant action is discussed.