Effects of cholesterol on the structure and collapse of DPPC monolayers

Abstract

Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, X_chol, exceeded 0.20. Structural measurements of grazing incidence X-ray diffraction at ambient laboratory temperatures and a surface pressure of 44 mN/m, just below the onset of collapse, showed that the major structural change in an ordered phase occurred at lower X_chol. A centered rectangular unit cell with tilted chains converted to an untilted hexagonal structure over the range of X_chol = 0.0-0.1. For X_chol = 0.1-0.4, the ordered structure was nearly invariant; the hexagonal unit cell persisted, and the spacing of the chains was essentially unchanged. That invariance strongly suggests that above X_chol = 0.1, cholesterol partitions into a disordered phase, which coexists with the ordered domains. The phase rule requires that for a binary film with coexisting phases, the stoichiometries of the ordered and disordered regions must remain constant. Added cholesterol must increase the area of the disordered phase at the expense of the ordered regions. X-ray scattering from dipalmitoyl phosphatidylcholine/cholesterol fit with that prediction. The data also show a progressive decrease in the size of crystalline domains. Our results suggest that cholesterol promotes adsorption not by altering the unit cell of the ordered phase but by decreasing both its total area and the size of individual crystallites.

Figure 1

Equilibrium spreading pressure (π_e) for films of extracted calf surfactant (calf lung surfactant extract) formed on the surface of captive bubbles. Films achieved the same final π during the following processes: adsorption to a constant surface area; slow compression of a monolayer, spread from organic solvent, across a plateau of relatively constant π, corresponding to collapse; and relaxation of a spread monolayer following a pulsed compression to a π slightly above π_e. Collapse at π_e limits access to the range of alveolar π, indicated by the vertical dashed line. Experimental conditions: temperature, 37°C; subphase, HSC; phospholipid concentration for adsorbed films, 0.5 mM phospholipid; A₀ is the area of the film when π = 46 mN/m; rates of compression: slow, 2.43 min⁻¹; pulsed, 14.4 min⁻¹. To see this figure in color, go online.

Figure 2

Measurements of isobaric compression for cholesterol-DPPC at X_chol = 0.10 and 23°C. (A) Temporal variation of surface area during the entire experiment. The film was subjected to the following: annealing by two cycles of slow expansion and compression at π < π_e; incubation at 45 mN/m to confirm the absence of collapse at that π (green); rapid compression to 52 mN/m (red); and then manipulation at that π (blue) to measure the rate of collapse. Area is expressed relative to the initial value (A_i) at which the film was spread. (B) π from the experiment in (A), expressed as a function of surface area. (C) Variation of area (left axis) and π (right axis) during the isobaric compression. Area and time are expressed relative to A_o and t_o when π first reached the isobaric value. To see this figure in color, go online.

Figure 3

Dose-response of collapse for DPPC with different levels of cholesterol. (A) Semilogarithmic plots of area versus time during isobaric collapse at 23°C and π = 52 mN/m. The legend indicates the content of cholesterol both as mol fraction and as percentage of cholesterol (w:w). (B) Rate constants, obtained from the slopes of plots in (A), as a function of the content of cholesterol. Temperature = 23°C. Mean ± SD. Some error-bars are smaller than the symbols. To see this figure in color, go online.

Figure 4

GIXD from monolayers of DPPC without and with (X_chol = 0.10) cholesterol. Measurements were made at π ≈ 44 mN/m, just below π_e ≈ 46.5 mN/m, and an ambient temperature of 23°C. Top panels: imaged intensities. Bottom panels: intensities integrated over q_z at each q_xy. Symbols indicate measured values. Continuous curves give the best fit to one or two Gaussian distributions. To see this figure in color, go online.

Figure 5

GIXD from films of DPPC with variable cholesterol. (A) X_chol = 0.00–0.10. Intensities are expressed relative to the maximum value of the fitted curve for each sample. (B) X_chol = 0.10–0.40. Intensities are normalized relative to the maximum value of the fitted curve for X_chol = 0.10. For each panel, symbols give measured values. Continuous curves provide the best fit to one or two Gaussian distributions. Temperature = 23°C; π = 45 mN/m. To see this figure in color, go online.

Figure 6

Dependence of the integrated, total scattered intensity (left axis) and correlation length (right axis) on content of cholesterol. Integration of the data in Fig. 5B for 1.3 ≤ q_xy ≤ 1.6 yielded the intensities. Calculation of L_xy from the Scherrer equation used the full width of the peak at the half-maximum from those fits. To see this figure in color, go online.