Effect of cholesterol on the biophysical and physiological properties of a clinical pulmonary surfactant

Abstract

Pulmonary surfactant is a complex mixture of lipids and proteins that forms a surface-active film at the air-water interface of alveoli capable of reducing surface tension to near 0 mN/m. The role of cholesterol, the major neutral lipid component of pulmonary surfactant, remains uncertain. We studied the physiological effect of cholesterol by monitoring blood oxygenation levels of surfactant-deficient rats treated or not treated with bovine lipid extract surfactant (BLES) containing zero or physiological amounts of cholesterol. Our results indicate no significant difference between BLES and BLES containing cholesterol immediately after treatment; however, during ventilation, BLES-treated animals maintained higher PaO2 values compared to BLES+cholesterol-treated animals. We used a captive bubble tensiometer to show that physiological amounts of cholesterol do not have a detrimental effect on the surface activity of BLES at 37 degrees C. The effect of cholesterol on topography and lateral organization of BLES Langmuir-Blodgett films was also investigated using atomic force microscopy. Our data indicate that cholesterol induces the formation of domains within liquid-ordered domains (Lo). We used time-of-flight-secondary ion mass spectrometry and principal component analysis to show that cholesterol is concentrated in the Lo phase, where it induces structural changes.

FIGURE 1

Mean arterial blood oxygenation (PaO₂) after air bolus (▴, n = 5), 20 mg phospholipids (PL)/kg body weight BLES (♦, n = 5) and 20 mgPL/kg BLES + 20 mol % cholesterol (▪, n = 5). Animals were ventilated with 100% oxygen. a = p < 0.05 BLES or BLES + cholesterol versus air bolus, b = p < 0.05 BLES versus BLES + cholesterol by two-way ANOVA.

FIGURE 2

Adsorption isotherms of BLES containing increasing amounts of cholesterol. Isotherms were constructed by monitoring adsorption of 500 μg/ml BLES in the absence or presence of cholesterol in the CBT at 37°C.

FIGURE 3

Quasistatic compression (•)-expansion (○) isotherms of BLES containing increasing amounts of cholesterol. Isotherms were constructed by averaging three independent experiments for the first and fourth compression-expansion cycles. An intercycle delay of 4 min was used to allow the system to reequilibrate.

FIGURE 4

Dynamic compression (•)-expansion (○) isotherms of BLES containing increasing amounts of cholesterol. Isotherms were constructed by averaging three independent experiments for the first and 21st compression-expansion cycles. Adsorbed films were cycled at 25 cycles/min.

FIGURE 5

AFM height images of BLES (A and B) and BLES + 20 mol % cholesterol (D and E). Samples were spread on a liquid-air interface and compressed to a surface pressure of 30 mN/m at 24°C. G and H are AFM height images of BLES + 20 mol % cholesterol exposed to 1 mM methyl β-cyclodextrin for 1 h at 24°C. Film transfer was performed using the LB method onto mica. The film was scanned in air in contact mode. Images A, D, and G are 60 × 60 μm², B and E are 4 × 4 μm², and H is 8 × 8 μm², and were collected from the boxed areas shown. C, F, and I are height analysis profiles for B, E, and H, respectively, along the lines shown.

FIGURE 6

ToF-SIMS negative ion images of two BLES LB films containing 0 mol % (A and C) and 30 mol % (B and D) cholesterol-d7. M255 corresponds to the palmitate group, M2 to deuterium present in the tail of cholesterol-d7. Images E–H were obtained by subjecting A–D, respectively, to a smoothing function available as part of the ION-TOF software and show more clearly the location of the palmitate group and deuterium. Images are 50 × 50 μm², 256 × 256 pixels with 1 shot/pixel.

FIGURE 7

(A) Image scores for PC2 obtained from PCA of ToF-SIMS negative ion mode images of a BLES + 30 mol % cholesterol-d7 LB film. Image is 50 × 50 μm². (B) Corresponding loadings for PC2 where negative loadings correspond to the liquid-ordered domains shown in blue, and positive loadings correspond to the liquid-disordered phase shown in yellow.

FIGURE 8

(A and B) AFM height images of BLES + 50 mol % cholesterol. Samples were spread on a liquid-air interface and compressed to a surface pressure of 30 mN/m at 24°C. Film transfer was performed using the LB method onto mica. The film was scanned in air in contact mode. Image A is 60 × 60 μm² and B is 4 × 4 μm² and was collected from the boxed area shown in A. C is a height analysis profile for B along the line shown. (D and E) ToF-SIMS negative ion images of BLES + 50 mol % cholesterol-d7. M255 corresponds to the palmitate group, M2 to deuterium present in the tail of cholesterol-d7. Images F and G were obtained by subjecting A and B, respectively, to a smoothing function available as part of the ION-TOF software and show more clearly the location of the palmitate group and deuterium. Images D–G are 50 × 50 μm², 256 × 256 pixels with 1 shot/pixel.