Structures of pulmonary surfactant films adsorbed to an air-liquid interface in vitro.

Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air-fluid interface.

Interfacial free energies of intact and reconstituted erythrocyte surfaces. Implications for biological adhesion.

Model cell surfaces consisting of phospholipids or phospholipids and the erythrocyte membrane glycoprotein glycophorin have been formed at an oil/water interface. Interfacial free energies have been estimated from surface wetting by both hydrophobic and hydrophilic test droplets on both the model surfaces and on intact erythrocytes. The use of a dense fluorocarbon oil to form the oil/water interface facilitates analysis by minimising surface deformation by the test drop. Hydrophobic test droplets (polar hydrocarbon oils) show increasing contact angles (decreasing wetting) with increasing hydrophilicity (decreasing interfacial free energy) of the model interface. Hydrophilic test droplets (phase separated aqueous polymer systems) show the opposite behaviour spreading more as the interfacial free energy is decreased. Both systems give similar estimates of the interfacial free energy. Glycophorin reproduces the wetting properties of intact cell surfaces by reducing the lipid-water interfacial free energy from 5.10(-3) J . m-2. From molecular considerations it is concluded that 'cell surface free energy' is an ambiguous term; its magnitude depends on the location of the interface in question. Thus, in a thermodynamic analysis of interactions at biosurfaces (such as cellular adhesion, chemotaxis or membrane fusion), the interfacial free energies may vary by more than three orders of magnitude depending on the location of the particular interface.

The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations.

For measuring the properties of lung surfactant, we provide formulas for calculating the surface tension, area, and volume of captive air bubbles in aqueous media. Only measurements of bubble height (h) and diameter (d) are required. Data processing has been automated using standard video capture hardware and software, and our own image-processing and -analyzing programs. Our polynomials in h/d describe the ratios of actual bubble surface area and volume to that of a spherical bubble having the same diameter. For surface tension, a polynomial in h/d describes the ratio of the surface tension of a flat semi-infinite bubble to the surface tension of the measured bubble of the same height. Coefficients for the area and volume polynomials were obtained from h and d, and measured areas and volumes of revolution of calibrating bubbles. Coefficients for the surface tension polynomial were obtained analytically from a published polynomial in h/d [3]. Results using these polynomials agree satisfactorily with those obtained independently [4] using bubble perimeter measurements.

Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hysteresis, stability.

Surface tension-area relations from pulmonary surfactant were obtained with a new apparatus that contains a leak free captive bubble of controllable size. Rat pulmonary surfactant was studied at phospholipid concentrations of 50, 200 and 400 micrograms/ml. At the highest concentration, adsorption was rapid, reaching surface tensions below 30 mN/m within 1 s, while at the lowest concentration, approximately 3 min were required. Upon a first quasi static or dynamic compression, stable surface tensions below 1 mN/m could be obtained by a film area reduction of approximately 50%. After three to four cycles the surface tension-area relations became stationary, and the tension fell from 25-30 to approximately 1 mN/m for a film area reduction of less than 20%. Hysteresis became negligible, provided the films were not collapsed by further area reduction. Under these conditions, the films could be cycled for more than 20 min without any noticeable loss in surface activity. After only three to four consecutive cycles, surfactant films exhibited the low surface tensions, collapse rates and compressibilities characteristic of alveolar surfaces in situ. Remarkably, surface tension and area are interrelated in the captive bubble which may promote low and stable surface tensions. If the surface tension of the captive bubble suddenly increases ('click') because of mechanical vibration or unstable surfactant, the bubble shape changes from flat to more spherical. The associated isovolumetric decrease in surface area prevents the surface tension from rising as much as it would have in a constant-area situation. This feedback mechanism may also have a favorable effect in stabilizing alveolar surface tension at low lung volumes.

A synthetic emulsion reproduces the functional properties of pulmonary surfactant.

An effective substitute for pulmonary surfactant must be able to reduce water-vapour surface tensions to under 1 mN/m and it must spread rapidly and spontaneously at the air interface from the aqueous phase. Pure dipalmitoylphosphatidylcholine meets the former requirement, but not the latter. A synthetic surfactant is described which meets both of these criteria. The surfactant is prepared as a DPPC monolayer stabilizing an aqueous emulsion of an inert fluorocarbon oil; it spreads rapidly at the air/liquid interface, lowers the surface tension to under 1 mN/m during compression at the air/liquid interface and restores normal pressure-volume characteristics to surfactant-depleted lungs.

Formation and structure of surface films: captive bubble surfactometry.

The adsorption model for soluble surfactants has been modified for suspensions of pulmonary surfactant. The dynamic adsorption behavior may be governed by a two-step process: (1) the transfer of molecules between the surface layer and the subsurface layer, which has a thickness of a few molecular diameters only; (2) the exchange of molecules between the subsurface and the bulk solution. The first step is an adsorption process and the second step is a mass transfer process. Between the subsurface and the bulk solution is an undisturbed boundary layer where mass transport occurs by diffusion only. The thickness of this boundary layer may be reduced by stirring. Rapid film formation by adsorption bursts from lipid extract surfactants, as observed in the captive bubble system, suggests that the adsorption process as defined above is accompanied by a relatively large negative change in the free energy. This reduction in the free energy is provided by a configurational change in the association of the specific surfactant proteins and the surfactant lipids during adsorption. The negative change in the free energy during film formation more than compensates for the energy barrier related to the film surface pressure. In the traditional view, the extracellular alveolar lining layer is composed of two parts, an aqueous subphase and a surfactant film, believed to be a monolayer, at the air-water interface. The existence and continuity of the aqueous subphase has recently been demonstrated by Bastacky and coworkers, and a continuous polymorphous film has recently been shown by Bachofen and his associates, using perfusion fixation of rabbit lungs with slight edema. In the present chapter, we have described a fixation technique using a non-aqueous fixation medium of perfluorocarbon and osmium tetroxide to fix the peripheral airspaces of guinea pig lungs. A continuous osmiophilic film which covers the entire alveolar surface, including the pores of Kohn, is demonstrated. By transmission electron microscopy, the surface film frequently appears multilaminated, not only in the alveolar corners or crevices, but also at the thin air-blood barrier above the capillaries. Disk-like structures or multilamellar vesicles appear partially integrated into the planar multilayered film. In corners and crevices, tubular myelin appears closely associated with the surface film. Tubular myelin, however, is not necessary for the generation of a multilaminated film. This is demonstrated in vitro by the fixation for electron microscopy of a film formed from lipid extract surfactant on a captive bubble. Films formed from relatively high surfactant concentration (1 mg/ml of phospholipid) are of variable thickness and frequent multilayers are seen. In contrast, at 0.3 mg/ml, only an amorphous film can be visualized. Although near zero minimum surface tensions can be obtained for both surfactant concentrations, film compressibility and mechanical stability are substantially better at the higher concentrations. This appears to be related to the multilaminated structure of the film formed at the higher concentration.

Determination of cell/medium interfacial tensions from contact angles in aqueous polymer systems.

The contact angles on cell layers of a series of polymeric droplets from aqueous two-phase systems of dextran and poly(ethylene glycol) have been used to determine the critical or limiting interfacial tension for spreading on the cell layers. Test droplets of the denser dextran-rich phase were formed in the lighter poly(ethylene glycol)-rich phase. The interfacial tensions, gamma, between the phases were determined with the pendant drop method, and a linear relationship was found between gamma-1/2 and the cosine of the angle the droplets made with the cell layers (Good-Girifalco plot). We were thus able to determine the limiting or critical interfacial tension, gamma c, for spreading on the cell layers. The value of gamma c is a measure of the interfacial energy of the cell/bathing medium interface. Values of gamma c obtained by this method include the following: 0.65 and 0.84 microN . m-1 for human erythrocytes and neutrophils, respectively; 0.93 microN . m-1 for porcine pulmonary macrophages; 0.75--3.60 microM . m-1 for various transformed murine lymphoid cell lines, and 2.53 microN . m-1 for Balb/c murine spleen lymphocytes. Exposure to various agents has differing effects on gamma c. Concanavalin A reduces gamma c, and bacterial lipopolysaccharide increases gamma c of murine spleen lymphocytes. The calcium ionophore, A23187, increases gamma c of both porcine pulmonary macrophages and murine spleen lymphocytes. This new method provides a quantitative approach to the cell surface energy and hydrophobicity which are thought to play an important role in membrane-mediated phenomena and in cell adhesion.

Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability.

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.

Surface activity and film formation from the surface associated material of artificial surfactant preparations.

Surfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL(4)) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02--0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5--10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5--2.0 mN/m was least for the KL(4) preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL(4) preparation incorporation of excess material took place at a surface tension of 25--35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30--40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40--50 mN/m. With 2% SP-C/BR incorporation took place at 40--45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated.

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos.

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatous and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 +/- 1.5 (S.D.) days compared to 27.3 +/- 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 +/- 0.7 (S.D.) days compared to 48.5 +/- 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.

A study of surface property changes in rat mesothelial cells induced by asbestos using aqueous two-phase polymer solutions.

Intraperitoneal (i.p.) injection of crocidolite asbestos was used to induce mesotheliomas in rats. The morphological changes of the mesothelial cells were studied by light and electron microscopy and by cytologic examination of peritoneal washings. After injection, the asbestos fibres stimulated an acute inflammatory response and were rapidly phagocytosed by the mesothelial cells and incorporated into the submesothelial tissues. At 7 days, the normal microvillous surface of the mesothelium was replaced with a syncytium of proliferating mesothelial cells showing extensive loss of microvilli. Nine months or so later, multifocal mesothelial tumours arose within the peritoneal cavity. The surface thermodynamic properties of normal, asbestos-stimulated mesothelial cells and of mesothelial tumour cells in culture were studied using an aqueous two-phase system containing 4% Dextran T-2000 and 4% poly (ethylene glycol) (PEG) w/w. After asbestos stimulation, there was a significant (P < 0.01) increase in contact angle between the dextran-rich phase and the mesothelial cell surface. These changes were even greater for the mesothelial tumours. The results indicate that the work of adhesion for asbestos-stimulated mesothelial cells and mesothelial tumours is lower than in normal tissue. These findings may be relevant to the process of tumour spread in the serosal cavities and to the development of distant metastases.

Membrane glycoprotein and surface free energy changes in hypoxic fibroblast cells.

Hypoxia affects the biochemistry of mammalian cells and thus alters their sensitivity to subsequent chemo- and radiotherapy. When V79 Chinese hamster lung fibroblasts were grown under conditions of extreme hypoxia (less than 10 ppm O2) there was a significant shift in the membrane glycoprotein composition. Scanning electron microscopy revealed altered cell surface morphology including loss of pseudopodial projections. Experiments to determine changes in interfacial free energy of these cells using equilibrium two phase systems of poly(ethylene glycol) (PEG) and dextran were carried out. Test fluid droplets of the denser dextran-rich phase were formed on layers of cells in the PEG-rich phase as the bathing medium, and the contact angles the droplets made with the cell layers were measured from photomicrographs. The contact angles on cells in the plateau phase increased significantly with time of exposure to hypoxia, from 25 degrees (zero time) to 35 degrees (6 h) to 60 degrees (9 h). Contact angles on cells in the exponential phase increased from 80 degrees (zero time) to 150 degrees after 20 h of hypoxia. It appears that the altered contact angles reflect changes in cell surface hydrophobicity that may, in part, reflect alterations in the membrane glycoprotein composition.

Interfacial tensions in healthy and atherosclerotic rabbit aortae. Higher values and lesion surfaces.

Interfacial tensions at the saline/arterial wall interface were determined by measuring contact angles between various test fluid droplets and the walls of rabbit aortae immersed in physiological saline. These contact angles and the interfacial tensions of the test fluid/bathing fluid interface (measured by the Du Noüy ring method) were converted to saline/arterial wall interfacial tensions by applying Neumann's equation of state. Four diseased animals, fed an atherogenic diet for 6-8 weeks and 6 controls formed the experimental group. A significantly higher interfacial tension (P < 0.001), was determined for lesion surfaces in atherosclerotic arteries (0.36 +/- 0.08 (SEM) mM . M-1, n = 13) compared to both the surrounding undisturbed regions (0.035 +/- 0.01 mN . m-1, n = 14) and the intact surface of control vessels (0.060 +/- mN . m-1, n = 48). This increase may reflect a change in the strength of hydrophilic interactions associated with the lesion surface in atherogenesis.

Surfactant and inhaled particles in the conducting airways: structural, stereological, and biophysical aspects.

We have investigated the displacement into the sol phase of inhaled particles deposited in the intrapulmonary conducting airways. Hamsters inhaled an aerosol of monodisperse polystyrene particles of 6 microns diameter. Their lungs were fixed by intravascular perfusion, and light and electron microscopy was used to study the epithelial coating. The surfactant film at the wall-air interface was investigated by measuring its surface tension. The number of particles retained was determined stereologically. In addition we investigated the displacement of spherical particles in vitro on a DPPC monolayer in a Langmuir-Wilhelmy surface balance and determined the surface tension in vivo in the horse trachea by video bronchoscopy, applying the droplet spreading method. We found that particles deposited onto a surfactant film were pulled into the aqueous subphase, and we concluded that surface forces due to the airway surfactant likely displace deposited particles into the periciliary fluid (sol phase). Comparing lungs fixed immediately after inhalation with lungs fixed 24 hr after inhalation revealed that 86% of the particles retained in the intrapulmonary conducting airways immediately after inhalation had been cleared within 24 hr. One-third of the particles of the lungs fixed immediately after inhalation was phagocytized. The combination of structural and stereological analyses with in vitro and in vivo measurements has led to new insights into the role of airway surfactant with respect to the fate of inhaled particles, which may have important consequences regarding the effects of hazardous particles. These studies may also help to evaluate the deposition pattern and clearance of therapeutic particles, with important implications for their therapeutic use.

Disturbance of alveolar lining layer: effects on alveolar microstructure.

To further study the influence of altered surface tensions on alveolar micromechanics, we analyzed the structure-function relationships in excised rabbit lungs filled with or rinsed by a fluorocarbon (approximately 15 mN/m) or by hexadecane (approximately 25 mN/m). The lungs were fixed and dehydrated by vascular perfusion, and the tissue samples were analyzed by light, transmission, and scanning electron microscopy. We made three observations. 1) Pressure-volume (P-V) loops hexadecane-filled lungs are shifted to the left and coincide with those of saline-filled lungs, indicating near-zero interfacial tension. In accordance, the alveolar microstructure and surface area of hexadecane-filled lungs resemble those of saline-filled lungs. 2) The P-V loops of fluorocarbon-filled lungs are not shifted to the left but coincide with those of fluorocarbon-rinsed lungs. Under both conditions, the alveolar microstructure is qualitatively identical and the alveolar surface areas are markedly reduced compared with normal air-filled lungs. These findings show that fluorocarbon-filled or fluorocarbon-rinsed lungs are subjected to similar interfacial tensions at the alveolar level. 3) Hexadecane-rinsed lungs show a pear-shaped P-V curve and a complex surface texture of peripheral air spaces. These results, together with in vitro observations, suggest a metamorphic interplay between lung surfactant and hexadecane in lining the surface and determining the surface tension. Evidently, the effects of foreign liquids introduced into the lung on the structure-function relationship cannot accurately be predicted from their in vitro surface tensions. This fact should be considered in the development of artificial surfactants.

Effect of budesonide and salbutamol on surfactant properties.

The objective of this study was to evaluate the in vitro effect of budesonide and salbutamol on the surfactant biophysical properties. The surface-tension properties of two bovine lipid extracts [bovine lipid extract surfactant (BLES) and Survanta] and a rat lung lavage natural surfactant were evaluated in vitro by the captive bubble surfactometer. Measurements were obtained before and after the addition of a low and high concentration of budesonide and salbutamol. Whereas salbutamol had no significant effect, budesonide markedly reduced the surface-tension-lowering properties of all surfactant preparations. Surfactant adsorption (decrease in surface tension vs. time) was significantly reduced (P < 0.01) at a high budesonide concentration with BLES, both concentrations with Survanta, and a low concentration with natural surfactant. At both concentrations, budesonide reduced (P < 0.01) Survanta film stability (minimal surface vs. time at minimum bubble volume), whereas no changes were seen with BLES. The minimal surface tension obtained for all surfactant preparations was significantly higher (P < 0.01), and the percentage of film area compression required to reach minimum surface tension was significantly lower after the addition of budesonide. In conclusion, budesonide, at concentrations used therapeutically, adversely affects the surface-tension-lowering properties of surfactant. We speculate that it may have the same adverse effect on the human surfactant.

Effects of fixatives on function of pulmonary surfactant.

The structure of pulmonary surfactant films remains ill defined. Although plausible film fragments have been imaged by electron microscopy, questions about the significance of the findings and even about the true fixability of surfactant films by the usual fixatives glutaraldehyde (GA), osmium tetroxide (OsO(4)), and uranyl acetate (UA) have not been settled. We exposed functioning natural surfactant films to fixatives within a captive bubble surfactometer and analyzed the effect of fixatives on surfactant function. The capacity of surfactant to reach near-zero minimum surface tension on film compression was barely impaired after exposure to GA or OsO(4). Although neither GA nor OsO(4) prevented the surfactant from forming a surface active film, GA increased the equilibrium surface tension to above 30 mN/m, and both GA and OsO(4) decreased film stability as seen in the slowly rising minimum surface tension from 1 to ~5 mN/m in 10 min. In contrast, the effect of UA seriously impaired surface activity in that both adsorption and minimum surface tension were substantially increased. In conclusion, the fixatives tested in this study are not suitable to fix, i.e., to solidify, surfactant films. Evidently, however, OsO(4) and UA may serve as staining agents.

Surface activity of lipid extract surfactant in relation to film area compression and collapse.

The physical properties of modified porcine surfactant (Curosurf), isolated from minced lungs by extraction with chloroform-methanol and further purified by liquid-gel chromatography, were investigated with the captive bubble technique. Bubble size, and thus the surface tension of an insoluble film at the bubble surface, is altered by changing the pressure within the closed bubble chamber. The film surface tension and area are determined from the shape (height and diameter) of the bubble. Adsorption of fresh Curosurf is characterized by stepwise decreases in surface tension, which can easily be observed by sudden quick movements of the bubble apex. These "adsorption clicks" imply a cooperative movement of large collective units of molecules, approximately 10(14) (corresponding to approximately 120 ng of phospholipid) or approximately 10(18) molecules/m2, into the interface during adsorption. Films formed in this manner are already highly enriched in dipalmitoyl phosphatidylcholine, as seen by the extremely low compressibility, close to that of dipalmitoyl phosphatidylcholine. Near-zero minimum tensions are obtained, even at phospholipid concentrations as low as 50 micrograms/ml. During dynamic cycling (20-50 cycles/min), low minimum surface tensions, good film stability, low compressibility, and maximum surface tensions between 30 and 40 mN/m are possible only if the films are not overcompressed near zero surface tension; i.e., the overall film area compression should not substantially exceed 30%.

A captive bubble method reproduces the in situ behavior of lung surfactant monolayers.

We tested a new captive bubble surface tensiometer with films adsorbed from aqueous suspensions of rabbit lung surfactant and a bovine lung surfactant lipid extract and with films of dipalmitoyl-sn-3-glycerophosphorylcholine (DPPC) spread from solvents. The lack of tubes penetrating the bubble surface eliminated potential leakage pathways for the surface film, which was compressed by increasing external pressure. Surface tensions and areas were calculated directly from bubble shapes without the need of pressure measurements. After only one to two compressions, the rabbit surfactant films exhibited the low surface tension, collapse rates, and compressibilities characteristic of the alveolar surface in situ and approached the behavior of spread DPPC films. The bubble "clicking" phenomenon described earlier by Pattle (Proc. R. Soc. Lond. B Biol. Sci. 148: 217-240, 1958) was also reproduced, but only with the bovine extract, which did not perform as well as the rabbit surfactant in surface tests. These findings suggest that surfactant apoprotein SP-A, which was probably present in the rabbit but not the bovine preparations, enhances both adsorption and stability of pulmonary surfactant monolayers.

Evaluation of pressure-driven captive bubble surfactometer.

We modified the captive bubble surfactometer [S. Schürch et al. J. Appl. Physiol. 67: 2389-2396, 1989] to facilitate the measurement of surface adsorption rates and to simplify its construction. We used a range of standards and monolayers of dipalmitoylphosphatidylcholine to check the calibration of the device against measurements made in a Wilhelmy surface balance and in the captive bubble by using a cathetometer, and we found good agreement. As a further test we measured the surface properties of rabbit lavage lung surfactant (60,000 x average g for 60 min) at 1.0 mg phospholipid/ml. This material adsorbed within 1 s to near-equilibrium surface tension, reached surface tensions of < 5 mN/m on the second compression, and formed very stable films. We conclude that a captive bubble surfactometer can provide accurate information about important surface properties of lung surfactant films.