Captive Bubble Surfactometer Research Articles

Interfacial Behavior of Murine Pulmonary Surfactant Expressing Different Human Surfactant Protein SP-A Variants

Pulmonary surfactant is a lipoprotein complex which reduces surface tension to prevent alveolar collapse. At the same time, surfactant also contributes to the protection of the large respiratory surface from the entry of pathogens. It is composed of around 92% lipids and 8% of specific surfactant proteins by mass. One of these proteins is SP-A, a hydrophilic glycoprotein of the collectin family. The main function of this protein is related with host defense. However, previous studies showed that SP-A also aids in the formation and surface behavior of surfactant films at the air-water interface. Humans possess two SP-A genes expressing protein variants SP-A1 and SP-A2. Although their specific or combined activity is not well understood, it has been shown that both gene products are necessary for tubular myelin formation, an extracellular structural form of surfactant. The goal of the present study was to investigate potential differences in the biophysical behavior of surfactants containing human SP-A1, SP-A2, or both. For this purpose we have studied pulmonary surfactant from humanized transgenic mice expressing hSP-A1, hSP-A2, or both human SP-As instead of the endogenous murine protein, in the Captive Bubble Surfactometer (CBS), which simulates alveolar interfacial compression-expansion dynamics. We observed that surfactant containing both hSP-A1 and hSP-A2 reaches lower surface tension after initial and post-expansion interfacial adsorption than surfactants containing only hSP-A1 or hSP-A2. Under interfacial cycling conditions (Quasi-static or Dynamic compression-expansion regimes) surfactant films containing both hSP-A1 and hSP-A2 also showed better performance than surfactant containing only SP-A1 or SP-A2. We conclude that the simultaneous presence of hSP-A1 and hSP-A2 proteins permits surfactant to adopt a particularly favorable structure with optimal functional properties.NIH HL34788

Membrane-Perturbing Activities of KL4-Related Surfactant Peptides

KL4 is a 21-residue peptide proposed as a potential substitute of pulmonary surfactant protein SP-B in synthetic surfactants, intended for the treatment of respiratory pathologies. The peptide, composed by leucines interrupted by lysine every four residues, was synthesized to simulate C-terminal amphipathic helical segments of SP-B. Once incorporated into lipid-protein complexes, KL4 promotes formation of interfacial films that produce and maintain surface tensions below 5 mN/m during compression-expansion cycling. Although KL4 was designed as an amphipathic helix at the membrane surface, the data on orientation and interactions of the peptide in membranes are contradictory. In the present work the surface activity and interaction with membranes of KL4 was compared with the behavior of two other peptides, KL2A2 and KL4PQ, to gain information about the properties defining the potential of KL4 as surfactant additive.The surface activity of the different peptides in lipid suspensions mimicking surfactant composition (DPPC/POPC/POPG 50:25:15 w/w/w), was analyzed in a captive bubble surfactometer (CBS). Only lipid suspensions containing KL4 adsorbed to the air-liquid interface with kinetics comparable to those containing SP-B, with KL4PQ and specially KL2A2 showing less activity. The interaction of the three peptides with membranes was analyzed by DSC, Laurdan fluorescence and ATR-FTIR. Perturbations induced by peptides on the structure and permeability of giant unilamellar vesicles (GUVs) were also analyzed and compared with the behavior of natural SP-B.The results indicate that the extent of penetration of the peptides in bilayers and their ability to perturb membrane structure correlate with their different interfacial activity.

Exposure to Polymers Reverses Inhibition of Pulmonary Surfactant by Serum, Meconium, or Cholesterol in the Captive Bubble Surfactometer

Dysfunction of pulmonary surfactant in the lungs is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome. Serum, cholesterol, and meconium have been described as inhibitory agents of surfactant’s interfacial activity once these substances appear in alveolar spaces during lung injury and inflammation. The deleterious action of these agents has been only partly evaluated under physiologically relevant conditions. We have optimized a protocol to assess surfactant inhibition by serum, cholesterol, or meconium in the captive bubble surfactometer. Specific measures of surface activity before and after native surfactant was exposed to inhibitors included i), film formation, ii), readsorption of material from surface-associated reservoirs, and iii), interfacial film dynamics during compression-expansion cycling. Results show that serum creates a steric barrier that impedes surfactant reaching the interface. A mechanical perturbation of this barrier allows native surfactant to compete efficiently with serum to form a highly surface-active film. Exposure of native surfactant to cholesterol or meconium, on the other hand, modifies the compressibility of surfactant films though optimal compressibility properties recover on repetitive compression-expansion cycling. Addition of polymers like dextran or hyaluronic acid to surfactant fully reverses inhibition by serum. These polymers also prevent surfactant inhibition by cholesterol or meconium, suggesting that the protective action of polymers goes beyond the mere enhancement of interfacial adsorption as described by depletion force theories.

Interfacial behavior and structural properties of a clinical lung surfactant from porcine source

Surfacen® is a clinical surfactant preparation of porcine origin, partly depleted of cholesterol, which is widely used in Cuba to treat pre-term babies at risk or already suffering neonatal respiratory distress. In the present study we have characterized the interfacial behavior of Surfacen in several in vitro functional models, including spreading and compression–expansion cycling isotherms in surface balances and in a captive bubble surfactometer, in comparison with the functional properties of whole native surfactant purified from porcine lungs and its reconstituted organic extract, the material from which Surfacen is derived. Surfacen exhibited similar properties to native porcine surfactant or its organic extract to efficiently form stable surface active films at the air–liquid interface, able to consistently reach surface tensions below 5mN/m upon repetitive compression–expansion cycling. Surfacen films, however, showed a substantially larger and stable compression-driven segregation of condensed lipid phases than exhibited by films formed by native surfactant or its organic extract. In spite of structural differences observed at microscopic level, Surfacen membranes showed a similar thermotropic behavior to membranes from native surfactant or its organic extract, characterized by calorimetry or fluorescence spectroscopy of samples doped with the Laurdan probe. On the other hand, analysis by atomic force microscopy of films formed by Surfacen or by the organic extract of native porcine surfactant revealed a similar network of interconnected condensed nanostructures, suggesting that the organization of the films at the submicroscopic level is the essential feature to support the proper stability and mechanical properties permitting the interfacial surfactant films to facilitate the work of breathing.

Properties of modified natural surfactant after exposure to fibrinogen in vitro and in an animal model of respiratory distress syndrome

Plasma proteins are known to interfere with pulmonary surfactant. Studies have proven the hypothesis that fibrinogen preserves exogenous surfactant subjected to long-term surface area cycling. The exogenous surfactant Curosurf was subjected to long-term surface area cycling without or with fibrinogen (ratio 2:1 w/w) and was tested by captive bubble surfactometer and on newborn premature rabbits. Surface tension increased in Curosurf (80 mg/ml) samples without fibrinogen after 6-12 d of cycling. In samples with fibrinogen the cycling time had no effect on surface tension. Addition of fibrinogen to surfactant prevented lipid peroxidation. Lung gas volumes of animals with noncycled Curosurf or Curosurf cycled with fibrinogen for 6 d were comparable and higher than in rabbits with Curosurf cycled without fibrinogen. Alveolar volume density was higher in groups with noncycled Curosurf or Curosurf cycled with fibrinogen than in Curosurf cycled without fibrinogen (both P < 0.001). The effect of fibrinogen on pulmonary surfactant cycled at 37 °C depends both on surfactant concentration and cycling time. At high phospholipid concentration used in clinical practice fibrinogen has a protective effect on biophysical and physiological properties of natural modified surfactant subjected to surface area cycling. This effect is partially mediated by reduction in lipid peroxidation.

Effects of KL4-Type Peptides on the Surface Activity and Stability of Pulmonary Surfactant Films as Evaluated in the Captive Bubble Surfactometer

Although SP-B is the most critical protein in lung surfactant, recombinant or synthetic forms of SP-B as a basis for the development of therapeutic surfactants are still not available. An alternative is the design and production of peptides mimicking the structure and general properties of essential motifs in SP-B.In the present study the surface activity of different KL4-derived peptides, as sequence variations of the original peptide designed to replicate a general amphipathic motif of SP-B [1], has been assessed in the captive bubble surfactometer. The peptides were reconstituted in a surfactant lipid matrix: DPPC/POPC/POPG (50:25:15, w/w/w). This mixture was selected because it offers a fluid environment where the interfacial stability of surfactant films has to be provided primarily by the SP-B mimetic and not by the lipid moiety.Presence of just 1% (w/w) peptide KL4 (KLLLLKLLLLKLLLLKLLLLK) provided to the lipid mixture similar ability to adsorb at the interface than the presence of 1% native SP-B purified from porcine lungs. Films made of DPPC/POC/POPG/KL4 showed also similar ability to reach very low surface tension with limited compression than exhibited by films containing native SP-B, both under quasi-static and dynamic compression-expansion cycling. A KL4 peptide with amidated end showed similar surface activity, as well as a KL4 version including a PQ insertion in the middle of the sequence to break the alpha-helical conformation. In contrast, variants with reduced numbers of leucine residues showed significantly reduced ability to promote interfacial adsorption and much worse activity under compression-expansion cycling. These results support the concept that hydrophobicity and potential leucine-promoted peptide-peptide interactions are more important than helicity for SP-B-like surface activity.[1] Cochrane and Revak (1991), Science 254, 566-568

Deterioration of Pulmonary Surfactant by Volatile Anesthetics

Pulmonary surfactant (PS) is a lipid-protein mixture lining the alveolar air-water interface. By lowering surface tension, PS stabilizes the respiratory surface, thereby impeding alveolar collapse. Lack or alterations of PS properties are associated with acute lung injury (ALI). Sevoflurane is widely used in medical practice; however, some adverse properties were described in the lung function and PS [1,2]. The hypothesis of this study is that Sevoflurane affects native PS (NPS) system, which may contribute to ALI during anesthesia. In the present work, we have analyzed the functional, structural, and thermodynamic properties of NPS and its membranes reconstituted from the organic lipid extract (EPS) upon exposure to Sevoflurane. The compression-expansion properties of NPS films exposed to Sevoflurane were analyzed in a captive bubble surfactometer (CBS) as well as the effects of the anesthetic on the thermotropic properties of NPS and EPS membranes.Q-Static and dynamic cycles of NPS films were strongly affected upon exposure to Sevoflurane, which diminished the ability of the films to reach very low surface tension during compression. However, the equilibrium surface tension after adsorption and the maximum surface tension at the end of the expansion moieties of cycling isotherms were not affected by the presence of Sevoflurane. Spectroscopic analysis of surfactant complexes doped with LAURDAN or diphenylhexatriene indicate fluidification of EPS membranes upon exposure to Sevoflurane at room temperature. DSC experiments indicated that Sevoflurane induces a decrease of Tm and ΔH associated with the main thermotropic transition in PS membranes.In conclusion, the present results indicate that Sevoflurane impairs the biophysical properties of PS through perturbation of the lateral order of segregated phases, compromising correct compression along the respiratory cycles.[1] L. Malacrida et al. Am. J. Respir. Crit. Care Med.181;2010:A3631.[2] L. Malacrida et al. Biophysical J. 100(3);2011:505a-506a.

Structural and Functional Characterization of Native Complexes of Pulmonary Surfactant Proteins Purified with Detergents

Pulmonary surfactant is a specialized lipid-protein complex, which essential function is to stabilize the gas exchange surface of the respiratory epithelium against physical forces tending to its collapse. Hydrophobic surfactant proteins SP-B and SP-C are critical to promote very rapid adsorption of phospholipids into the interface and to stabilize the surface active film along the compression-expansion respiratory cycles. Mature SP-B is a homodimeric protein of approximately 18kDa (two 79 amino-acid polypeptides), while SP-C is an apparently monomeric lipopeptide of 35 residues (3.7kDa).To date, the classical method to extract and purify these proteins from surfactant membranes involves the use of organic solvents. This method can disrupt native supramolecular protein complexes. We have optimized the purification of surfactant membrane proteins upon solubilization of surfactant membranes by the zwitterionic detergent CHAPS. Different fractions have been purified from the solubilised material by gradient centrifugation and exclusion-size chromatography, showing the presence of two types of protein complexes in terms of size and density, heterogeneously composed of different oligomeric states of surfactant proteins.An ionic-exchange chromatography of solubilized surfactant yielded a fraction of cationic protein complexes consisting mainly of surfactant protein SP-B. Their structural characterization by circular dichroism and fluorescence emission revealed a structure entirely analogous to that of SP-B purified in organic solvents. However, detergent-purified SP-B showed a much more oligomeric structure, as analyzed by blue-native electrophoresis, analytical centrifugation or electron microscopy. This SP-B complex has been reconstituted into surfactant phospholipids and the resulting lipid/protein complexes showed in the captive bubble surfactometer (CBS) a similar surface active behaviour than provided by SP-B purified using the classic chloroform/methanol-based method.

Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology

Medical literature regularly reports on accidental poisoning in children after aspiration of combustibles such as lamp oils which usually contain hydrocarbons or rape methyl esters (RMEs). We aimed to analyze the toxic potential of alkanes and different combustible classes in vitro with regard to biologic responses and mechanisms mediating toxicity. Two different in vitro models were used, i.e. (i) a captive bubble surfactometer (CBS) to assess direct influence of combustibles on biophysical properties of surfactant film and (ii) cell cultures (BEAS-2B and R3/1 cells, primary macrophages, re-differentiated epithelia) closely mimicking the inner lung surface. Biological endpoints included cell viability, cytotoxicity and inflammatory mediator release. CBS measurements demonstrate that combustibles affect film dynamics, i.e. the surface tension/area characteristics during compression and expansion, in a dose and molecular chain length dependent manner. Cell culture results confirm the dose dependent toxicity. Generally, cytotoxicity and cytokine release are higher in short-chained alkanes and hydrocarbon-based combustibles than in long-chained substances, e.g. highest inducible cytotoxicity in BEAS-2B was for hexane 84.6%, decane 74% and hexadecane 30.8%. Effects of RME-based combustibles differed between the cell models. Our results confirm data from animal experiments and give new insights into the mechanisms underlying the adverse health effects observed.

Evaluation of Surfactant Function: In Vitro and In Vivo Techniques in Experimental Respirology

Evaluation of Surfactant Function: In Vitro and In Vivo Techniques in Experimental Respirology Pulmonary surfactant is present as the thin film of surface active material in the terminal airspaces and conducting airways. The major function of the surfactant film is to reduce the surface tension at the alveolar surface. Deficiency or dysfunction of pulmonary surfactant cause severe respiratory diseases that make the study of pulmonary surfactant not only of physiological but also of clinical importance. There are three main categories of methods for assessing the properties of pulmonary surfactant: in vitro, in situ and in vivo techniques. Pulsating bubble surfactometer (PBS) and captive bubble surfactometer (CBS) enables to study surfactant properties at spherical air-liquid interphase. Capillary surfactometer in contrast to alveolar models mimics the human terminal airways and evaluates surface properties required for airway patency. Each of above-mentioned methods enables to study exogenous surfactants, as well as surface activity of lavage fluids or tracheal aspirates. Biophysical characteristics should be reflected by lung compliance and, as a consequence, by improved blood oxygenation. Various animal models have been developed to evaluate the efficacy of surfactant replacement therapy on preterm and term animals. In vivo models can be divided in those, primarily involving surfactant deficiency, such as premature animal model, and those with secondary surfactant dysfunction or inactivation, such as meconium or acid aspiration models. This review is restricted to the in vivo and in vitro techniques handled by the authors in their research performed within the last years at both domestic and external laboratories.

Evaluation of Surfactant Inhibition and Reactivation by Captive Bubble Surfactometry

Pulmonary surfactant is a complex mixture of lipids and proteins, which forms a film at the air-water interface in the alveoli. Its main function is to lower the surface tension at this interface thus decreasing the work of breathing and preventing alveolar collapse. Dysfunction of surfactant in the lungs is associated with respiratory distress syndromes such as acute respiratory distress syndrome (ARDS) or meconium aspiration syndrome (MAS). Many substances have been described as inhibitory agents for pulmonary surfactant interfacial activity. Three of the most common in vivo are serum, cholesterol and meconium. The behaviour of surfactant films at the interface can be followed under physiologically relevant conditions in the Captive Bubble Surfactometer (CBS). This includes i) examination of rapid film formation by adsorption of surfactant into the interface of an air bubble, ii) monitoring re-adsorption of material from surface-associated reservoirs into the interface of the bubble upon expansion or iii) analysis of film dynamic properties during compression-expansion cycling of the interface. Using the CBS with a modified protocol that allows exposure of surfactant to high concentrations of inhibitory compounds, we have evaluated the effect of several agents on the biophysical properties of lung surfactant films under physiologically meaningful constraints. In this model assay, we observe significant differences in the interfacial performance of surfactant preparations in the presence or absence of inhibitory agents such as serum or meconium, which determine the importance of surfactant composition and structure on its susceptibility to inactivation. Moreover we have confirmed the higher resistance to inhibition of complexes of pulmonary surfactant with ionic and non-ionic polymers and the potential of these additives to design new therapeutic surfactant preparations.

Meconium Impairs Pulmonary Surfactant by a Combined Action of Cholesterol and Bile Acids

Mechanisms for meconium-induced inactivation of pulmonary surfactant as part of the meconium aspiration syndrome in newborn infants, to our knowledge, are not clearly understood. Here we have studied the biophysical mechanisms of how meconium affects surface activity of pulmonary surfactant and whether the membrane-perturbing effects of meconium can be mimicked by exposure of surfactant to a mixture of bile acids and cholesterol. Surface activity of pulmonary surfactant complexes purified from animal lungs was analyzed in the absence and in the presence of meconium in standard surface balances and in a captive bubble surfactometer. We have also evaluated accumulation of surfactant at the air-liquid interface by what we believe to be a novel microtiter plate fluorescent assay, and the effect of meconium components on surfactant membrane fluidity using Laurdan fluorescence thermotropic profiles and differential scanning calorimetry thermograms. Rapid interfacial adsorption, low surface tension upon film compression, efficient film replenishment upon expansion, and thermotropic properties of surfactant complexes are all adversely affected by meconium, and, in a similar manner, they are affected by cholesterol/taurocholate mixtures but not by taurocholate alone. We conclude that inhibition of surfactant by meconium can be mimicked by a bile salt-promoted incorporation of excess cholesterol into surfactant complexes. These results highlight the potential pathogenic role of cholesterol-mobilizing agents as a crucial factor resulting in cholesterol induced alterations of structure and dynamics of surfactant membranes and films.

Alterations of the C-terminal end do not affect in vitro or in vivo activity of surfactant protein C analogs

The secondary structure, orientation and hydrogen/deuterium exchange of SP-C33, a surfactant protein C analog, in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/egg phosphatidylglycerol (8:2, wt./wt.) bilayers, was studied by attenuated total reflection Fourier transform infrared spectroscopy. This showed a transmembrane α-helix, in which about 55% of the amide hydrogens do not exchange for up to 20h. Moreover, C-terminally modified SP-C33, either truncated after position 30, or having the methionine at position 31 exchanged for either lysine or isoleucine, showed the same secondary structure and orientation. The different peptides, suspended in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 68:31 (wt./wt.), were tested for surfactant activity in vitro in a captive bubble surfactometer and in vivo in an animal model of respiratory distress syndrome using premature rabbit fetuses. All preparations showed similar surface activity in the captive bubble surfactometer. Also, in the rabbit model, all preparations performed equally well and significantly better than non-treated controls, both regarding tidal volumes and lung gas volumes. Thus, truncation or residue replacements in the C-terminal part of SP-C33 do not seem to affect membrane association or surfactant activity.

335 Surface Activity of Small Volume Airway Aspirates Suctioned During Neonatal Resuscitation

Background: Only small amounts of surfactant are be needed for measuring biophysical activity in the captive bubble surfactometer (CBS). Using large sample volumes, amniotic fluid or gastric aspirates have been used before for prediction of lung maturity in neonates. With the CBS the analysis of surfactant samples from neonatal aspirates with small volumes and low amounts of phospholipids can be performed. Aims: To prepare surfactant samples from small volumes of oro-nasal aspirates suctioned during neonatal resuscitation, to determine the surface activity and to compare the results with clinical data. Methods: Suction aspirates and clinical data of 74 individuals were collected with parental consent, weighed and mucus, cell detritus plus large particles were removed. Subsequently the samples were ultracentrifugated. The pellet was resuspended at a ratio of 60µL/1g of original sample size. Then surface tension of 1-5µL of the samples was determined after 5 min adsorption to a ∽20µL bubble in the CBS using sucrose as a surfactant hypophase. Results: Surface tension of aspirates of neonates with a bw 2000g (35.8±9.9; p< 0.01). Absorption surface tension in non-ventilated neonates was significantly lower (34.1±9.8mN/m) compared to CPAP treated neonates (47.8±7.4 mN/m; p< 0.05) or mechanically ventilated individuals (48.4±5.6 mN/m; p< 0.01). Conclusions: Small volume airway samples from neonatal aspirates may be used to determine biophysical activity of the surfactant system quantitatively using the CBS. The obtained data correlate with the clinical course, birth weight and the mode of ventilatory support.

Palmitoylation of Pulmonary Surfactant Protein SP-C Is Critical for Its Functional Cooperation with SP-B to Sustain Compression/Expansion Dynamics in Cholesterol-Containing Surfactant Films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Combined and Independent Action of Proteins SP-B and SP-C in the Surface Behavior and Mechanical Stability of Pulmonary Surfactant Films

The hydrophobic proteins SP-B and SP-C are essential for pulmonary surfactant function, even though they are a relatively minor component (<2% of surfactant dry mass). Despite countless studies, their specific differential action and their possible concerted role to optimize the surface properties of surfactant films have not been completely elucidated. Under conditions kept as physiologically relevant as possible, we tested the surface activity and mechanical stability of several surfactant films of varying protein composition in vitro using a captive bubble surfactometer and a novel (to our knowledge) stability test. We found that in the naturally derived surfactant lipid mixtures, surfactant protein SP-B promoted film formation and reextension to lower surface tensions than SP-C, and in particular played a vital role in sustaining film stability at the most compressed states, whereas SP-C produced no stabilization. Preparations containing both proteins together revealed a slight combined effect in enhancing film formation. These results provide a qualitative and quantitative framework for the development of future synthetic therapeutic surfactants, and illustrate the crucial need to include SP-B or an efficient SP-B analog for optimal function.

Myristate is selectively incorporated into surfactant and decreases dipalmitoylphosphatidylcholine without functional impairment

Lung surfactant mainly comprises phosphatidylcholines (PC), together with phosphatidylglycerols and surfactant proteins SP-A to SP-D. Dipalmitoyl-PC (PC16:0/16:0), palmitoylmyristoyl-PC (PC16:0/14:0), and palmitoylpalmitoleoyl-PC (PC16:0/16:1) together comprise 75-80% of surfactant PC. During alveolarization, which occurs postnatally in the rat, PC16:0/14:0 reversibly increases at the expense of PC16:0/16:0. As lipoproteins modify surfactant metabolism, we postulated an extrapulmonary origin of PC16:0/14:0 enrichment in surfactant. We, therefore, fed rats (d19-26) with trilaurin (C12:0(3)), trimyristin (C14:0(3)), tripalmitin (C16:0(3)), triolein (C18:1(3)) or trilinolein (C18:2(3)) vs. carbohydrate diet to assess their effects on surfactant PC composition and surface tension function using a captive bubble surfactometer. Metabolism was assessed with deuterated C12:0 (ω-d(3)-C12:0) and ω-d(3)-C14:0. C14:0(3) increased PC16:0/14:0 in surfactant from 12 ± 1 to 45 ± 3% and decreased PC16:0/16:0 from 47 ± 1 to 29 ± 2%, with no impairment of surface tension function. Combined phospholipase A(2) assay and mass spectrometry revealed that 50% of the PC16:0/14:0 peak comprised its isomer 1-myristoyl-2-palmitoyl-PC (PC14:0/16:0). While C12:0(3) was excluded from incorporation into PC, it increased PC16:0/14:0 as well. C16:0(3), C18:1(3), and C18:2(3) had no significant effect on PC16:0/16:0 or PC16:0/14:0. d(3)-C14:0 was enriched in lung PC, either via direct supply or via d(3)-C12:0 elongation. Enrichment of d(3)-C14:0 in surfactant PC contrasted its rapid turnover in plasma and liver PC, where its elongation product d(3)-C16:0 surmounted d(3)-C14:0. In summary, high surfactant PC16:0/14:0 during lung development correlates with C14:0 and C12:0 supply via specific C14:0 enrichment into lung PC. Surfactant that is high in PC16:0/14:0 but low in PC16:0/16:0 is compatible with normal respiration and surfactant function in vitro.

SP-C Palmitoylation is Crucial for Stabilizing Cholesterol-Containing Surfactant Films during Continuous Compression/Expansion Cycling

Cholesterol is critical to maintain a dynamic lateral structure in pulmonary surfactant membranes, including a defined fluid-ordered/fluid-disordered phase equilibrium and proper lateral sorting of surfactant proteins and lipids. However, an excess of cholesterol has been linked to impaired surface activity both in surfactant models and in surfactant from injured lungs. Surfactant protein C (SP-C), the smallest and most hydrophobic of all surfactant proteins, has been shown to interact with cholesterol and dual palmitoylation of its N-terminal segment has been shown to drive association with ordered phases in model membranes. Furthermore, it has been proposed that native palmitoylated SP-C can act in concert with surfactant protein B (SP-B) to permit cholesterol-containing surfactant films to reach very low surface tensions upon compression. In the present work, we report that palmitoylation of SP-C is important for its ability to counteract deleterious effects of cholesterol on surfactant film stability under continuous expansion/compression cycling, as evaluated in a captive bubble surfactometer (CBS) setup. Presence of 5% cholesterol impairs significantly the stability under quasi static and dynamic compression of films composed of DPPC/POPC/POPG/SP-B (50:25:15:1, w/w/w/w), which are able to reach tensions below 3 mN/m with only 20% compression and almost no hysteresis in the absence of cholesterol. Incorporation in the films of 2% native palmitoylated SP-C could alleviate these deleterious effects. However, recombinant non-palmitoylated SP-C was not able to reproduce the stabilizing effect of native SP-C, confirming that palmitoylation of SP-C at its N-terminal end is crucial for its potential function of stabilizing surfactant films during the respiratory cycles in the lung.

Critical Dependence of the Biophysical Activity of Pulmonary Surfactant Films on Temperature

Pulmonary surfactant is a lipid-protein complex that stabilizes the respiratory surface of lungs. Once secreted into the alveolar spaces, surfactant adsorbs rapidly at the air-liquid interface reducing surface tension upon compression to near 0 mN/m. Within a given animal species, surfactant composition is influenced by development, disease, respiratory rate, and/or body temperature. In principle, surfactant collected from animals functions optimally at the body temperature 3of the animal at the time of sample collection.Changes in temperature can alter the physical state and the molecular packing of surfactant membranes and films, potentially altering their biophysical performance. We have analyzed the effect of temperature on the structure of native surfactant, by differential scanning calorimetry (DSC) and fluorescence spectroscopy with the fluorescent probes DPH (Diphenylhexatriene) and Laurdan (6-Lauroyl-2-(N,N-dimethylamino)naphthalene). The spectral properties of these probes have been used to assess lipid packing and fluidity in surfactant as a function of temperature and compression state. The effect of temperature on the interfacial performance of surfactant has been evaluated by analyzing spreading capabilities in a surface balance and compression-expansion dynamics in a Captive Bubble Surfactometer. Native surfactant from porcine lungs showed optimal adsorption at temperatures around 37 oC, reaching minimal surface tensions below 2 mN/m upon quasi-static or dynamic compression-expansion cycling. Critical structural transitions at temperatures above 39oC led to reduced interfacial adsorption and impossibility of compressed films to reduce surface tensions below 20 mN/m, suggesting that surfactant composition has been optimized to work at a narrow interval of temperatures and that regulatory factors may be involved in adaptation of surfactant structures to changes in body temperature.

Anionic Polymers Reverse Serum Inhibition of Pulmonary Surfactant by Promoting Accumulation of Surfactant Near the Air-Liquid Interface

Acute respiratory distress syndrome (ARDS) is a common pathology, including a spectrum of respiratory diseases associated with lung injury, and exhibiting a high overall mortality and morbidity rate. Inactivation of surfactant by serum and inflammatory components leaked into the alveolar spaces is considered as an important pathogenic factor within ARDS.The mechanism by which inhibition is taking place depends on the nature of the inhibitory substance and could affect either the ability of surfactant to adsorb into the air-water interface or the ability of surfactant films themselves to reach the lowest surface tensions along the compression-expansion breathing cycles. Up to now, different polymers have proven to be useful to reverse or prevent inactivation of surfactant. We have explored the performance of inhibited surfactant and potential reactivating conditions using a fluorescent high-throughput method that detects and quantitates accumulation of surfactant near the air-liquid interface. This accumulation can be correlated in a first step with the concomitant decrease in surface tension that occurs when surface active lipids are transferred into the air-exposed side. Using this method we have evaluated inhibition of native porcine surfactant and of several clinical surfactants by serum, and the ability of hyaluronic acid (HA) to reverse or prevent this inhibition. A comparison was also made with the effect of other polymers. In general terms, presence of polymers in the subphase increases significantly the amount of surfactant associated with interfacial regions and seems to overcome, at least partially, the barrier to adsorption imposed by serum. Results obtained from a massive number of samples showed a very high reproducibility and a high correlation with data obtained using traditional methods to assess surfactant activity, such as surface balances or the Captive Bubble Surfactometer.