Effect of cholesterol on the molecular structure and transitions in a clinical-grade lung surfactant extract

Abstract

The lipid-protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of 1 H-13 C solid-state nuclear magnetic spectroscopy, small-and wide-angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, L α(o) , at physiological temperatures. This L α(o) phase gradually changes into a liquid-disordered lamellar phase, L α(d) , when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one L α(d) phase. Remarkably, it was possible to measure a large set of order parameter magnitudes |S CH | from the liquiddisordered and -ordered lamellar phases and assign them to specific C-H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological selfassembly compositions for treatment of pathological states such as respiratory distress syndrome.T he air-alveoli interface of human lungs consists of ∼100 m 2 of surface area covered by a 1-to 2-µm-thick film rich in lipids, generally referred to as lung surfactant (LS) (1, 2). The alveolar interfacial film has several important functions, including stabilizing the interface, allowing for area expansions and controlling diffusional transport (3-7). These functions strongly rely on the self-assembly structure, as well as on the mechanical, barrier, and surface properties of the dynamic LS interfacial layer, which in turn depend on its chemical composition and external conditions in terms of compression, hydration, and so on. The main components of LS are phospholipids, cholesterol, and the so-called surfactant proteins representing ∼80, 10, and 10 wt %, respectively (2).Deficiency of LS in the alveoli, or an imbalance of its chemical composition, induces a number of pathological conditions (8). For instance, neonatal respiratory distress syndrome affects prematurely born infants when LS is not yet matured or is completely lacking; this deficiency may be lethal if left untreated. In these cases, the most common treatment is intratracheal administration of an exogenous LS into the lungs (9). In case of acute respiratory distress syndrome the mortality is 35 to 45% (10) and an effective treatment is clearly lacking (8). An accurate characterization of the molecular structure and dynamics of endogenous an...

Self-Assembly in Ganglioside‒Phospholipid Systems: The Co-Existence of Vesicles, Micelles, and Discs

“…The crowded spectral region around 30.5 ppm is indicative of the acyl chain trans/gauche (TG) conformations, which is expected for a fluid bilayer phase. This can be distinguished from the all-trans (AT) conformation that would be expected for a solid (gel phase) bilayer, which gives rise to a peak centered around 33.5 (AT) ppm [42]. For a system with co-existing solid and fluid domains, one should expect to distinguish both AT and TG crowded spectra regions [42,45].…”

“…These experiments provide information on the mobility and conformation in different segments of the lipid molecules with close-to-atomic resolution [37,38]. The same experimental approach has previously been used to yield atomically resolved information on molecular dynamics in lipids and surfactants [38,39] and in amyloid fibrils [40,41], as well as in samples with a complex composition from lung surfactant [42], cartilage [43], and skin stratum corneum [44]. Three different NMR experiments were performed for each sample: DP (direct polarization), CP (cross-polarization), and INEPT (insensitive nuclei enhanced by polarization transfer).…”

“…This can be distinguished from the all-trans (AT) conformation that would be expected for a solid (gel phase) bilayer, which gives rise to a peak centered around 33.5 (AT) ppm [42]. For a system with co-existing solid and fluid domains, one should expect to distinguish both AT and TG crowded spectra regions [42,45]. INEPT experiments the polarization transfer occurs via bond couplings.…”

The Impact of Nonequilibrium Conditions in Lung Surfactant: Structure and Composition Gradients in Multilamellar Films

“…We also evaluate the impact of cholesterol on the structure of lung surfactant multilamellar films reconstructed from Curosurf. Indeed, the cholesterol content in endogenous lung surfactant has previously been shown to have large consequences for self-assembly in bulk and monolayer conditions, 37 − 39 and it has been linked to the in vivo performance. 40 Finally, our setup makes it possible to make an evaluation of the deposition process of lung surfactant at the air–liquid interface, in a situation that is similar to the clinical one.…”

Subphase Material Stabilizes Films of Pulmonary Surfactant

“…Physiological levels of cholesterol convert the fixed, irregular domains in the phospholipid films to circular shapes that can reconfigure rapidly (26). The solid, TC domains change to the fluid characteristics of the L o phase (63). The prior and current studies, using different experimental approaches (64), both conclude that the cholesterol in CLSE converts TC domains to L o (65).…”