High-throughput evaluation of pulmonary surfactant adsorption and surface film formation

Abstract

The assessment of new therapeutic strategies to cure surfactant-associated lung disorders would greatly benefit from assay systems allowing routine evaluations of surfactant functions. We present a method to measure surfactant adsorption kinetics into interfacial air-liquid interfaces based on fluorescence microplate readers. The principle of measurement is simple, robust, and reproducible: Wells of a microtiter plate contain an aqueous solution of a light-absorbing agent. Fluorescence is excited and collected from the top of the wells so that fluorescently labeled surfactant injected into the bulk can be detected only once adsorbed into the air-liquid interface. Mass transfer from the bulk to the interface is achieved by orbital shaking implemented in the plate reader instrument. The method has been tested and validated by using phospholipids or surfactants of different origins, by using albumin as surfactant inhibitor, and by comparison of results with Wilhelmy balance measurements. The method is suited for implementation in high-throughput screening routines for conditions affecting, or improving, surfactant film formation. In contrast to surface tension measurements, our method gives a direct readout of the amount of surfactant adsorbing into the interface, including the functionally important amount of material firmly associating with the interfacial film.-Ravasio, A., A. Cruz, J. Pérez-Gil, and T. Haller. High-throughput evaluation of pulmonary surfactant adsorption and surface film formation. J. Lipid Res. 2008Res. . 49: 2479Res. -2488

Surface film formation in vitro by infant and therapeutic surfactants: role of surfactant protein B

“…These results indicate that budesonide supplementation does not interfere with film formation in vitro . By contrast, addition of albumin, a known inhibitor of surfactant function, to calfactant at 33% PL significantly increased the ½Max value (data not shown), confirming published results (10). …”

“…(10). We found that steady-state surface film PL content was equivalent with addition of labeled surfactant to the bottom of the well vs. application to the surface, that the presence of unlabeled surfactant at the surface markedly lowered signal from labeled, bottom-loaded surfactant, and that absence of rotational shaking did not alter results.…”

“…Recently, Ravasio et al . (10) described a technique for measuring PL surface film formation in multiwell plates containing fluorescence-labeled surfactant and a light quencher that allows high-throughput kinetic analyses at various concentrations of subphase surfactant; they established the principles of the assay, optimized the experimental conditions, and analyzed variables related to lipid composition in natural surfactant and synthetic lipid mixtures. In the current study, we tested the applicability of this new in vitro assay for both clinical samples and component-specific pharmacological testing, focusing on the role of the lipophilic surfactant proteins.…”

Lamellar Bodies Form Solid Three-dimensional Films at the Respiratory Air-Liquid Interface

“…3). This hypothesis may gain additional support by the facts that lipid sorting into lamellar bodies is an ATP-and thus energy-driven process (27), that lamellar bodies appear to be under tension, which is partially relieved when the hemifusion state is reached (28), and that released LBPs show a faster initial rate of adsorption than natural surfactant (29). Such potential energy-loaded state of LBPs could also enable them to push and pack material within the preformed structures, creating condensation of ordered phases, and the projection of multilayered structures beyond the interfacial plane.…”

Effect of whole body hypothermia on inflammation and surfactant function in asphyxiated neonates

“…The serum/BAL urea ratio was used to correct for dilution and obtain epithelial lining fluid (ELF) concentrations [12]. Surfactant function in BAL was studied via fluorescence analysis of adsorption kinetics into the air/liquid interface, as described previously [13]. Results are provided as background-corrected relative fluorescent units.…”