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A2332 - Pulmonary Alveolar Proteinosis: A Compositional and Biophysical Functional Analysis of Its Effects on Lung Surfactant
Author Block: J. Perez-Gil1, C. Ballester2, M. Echaide2; 1Biochemistry and Molecular Biology, Faculty of Biology - UCM, Madrid, Spain, 2Biochemistry and Molecular Biology, Faculty of Biology - UCM, MADRID, Spain.
Rationale. Alveolar proteinosis is a chronic rare disease, which has different origins but it is always characterized by an abnormal accumulation of pulmonary surfactant at the alveolar spaces. Several studies have reported important alterations in the composition of surfactant from these patients, but there is no evidence on how these changes affect the biophysical behavior of surfactant. Methods. Purified porcine native and human PAP surfactant were subjected to organic extract and molecular exclusion chromatography to separate hydrophobic components. Surfactant phospholipid concentration was estimated by phosphorous analysis. Total protein quantitation was performed by Lowry and cholesterol was quantitated with a commercial enzyme kit. Lipid composition was analyzed by thin layer chromatography and protein aggregates studied by western blot. Surfactant functional analysis was performed in a captive bubble surfactometer (CBS) operated to mimic breathing dynamics. Results. This study presents the compositional and functional analysis of surfactant from a patient suffering from alveolar proteinosis (P) in comparison with that of a typical native surfactant obtained from animal (porcine) lungs (NS). As previously shown, the protein-to-lipid ratio was 3.5 times higher in P, while western blot analysis showed an excess of all the surfactant proteins analyzed, SP-A, SP-B and SP-C, also indicating the presence of aggregate forms of the proteins. Thin layer chromatography revealed substantial qualitative and quantitative differences in phospholipid components, while cholesterol/phospholipid ratio was highly increased in P. Functional characterization in a CBS revealed that P surfactant exhibited a poor behavior both regarding its adsorption at the air-liquid interface and the dynamical properties of interfacial films once subjected to breathing-like compression-expansion cycling. Samples reconstituted from the organic extract of P surfactant, containing the lipids and the hydrophobic proteins, showed an improvement in adsorption but still a deficient dynamical function. To evaluate the particular implication in the proteinosis-originated surfactant dysfunction of different altered components such as the hydrophobic proteins, the lipid fraction, or the amount of cholesterol, we have compared the functional behavior of samples reconstituted from different fractions of P surfactant obtained upon LH20 cromatography. Conclusions. -PAP altered proteins contribute importantly to the dysfunctional of surfactant, probably due to the alteration of protein structure. - Despite that phospholipid composition was altered in P, it did not collaborate substantially to PAP-associated surfactant malfunction. - Excess of cholesterol contributed significantly to biophysical impairment, probably due to alteration of the mechanical properties of surfactant films. (Supported by Grant BIO2015-67930-R from Spanish Ministry of Economy).