FFA, the products of sPLA2 reaction, were measured using a colorimetric assay  having as detection range was 7-1,000 ��M. Surfactant Protein-A (SP-A) was measured using an ELISA kit having as detection range 0.16/500 ng/mL . These selleckchem assays (Biovendor Laboratorni, Brno, Czech Republic) had coefficients of variations ��10%.Proteins and urea were measured, as previously described [22,23]. Serum urea values obtained during the routine clinical tests in the same day of the lavage were used to calculate the serum-to-BALF urea ratio and obtain epithelial lining fluid (ELF) concentrations.Biophysical studySixteen BALF specimens consisted of a remaining volume ��500 ��L, beyond that used for the above-described assays. These specimens were immediately frozen and sent under dry ice to the biophysical laboratory.
Total native surfactant (large plus small aggregates) was purified by ultracentrifugation (100,000 g; 4��C; 1 h), used in aqueous suspension and diluted with Tris buffer (5 mM; pH 7) containing 150 mM NaCl to the final desired phospholipids concentration, without organic extraction. Phospholipids were measured as previously published ; detection limit was 11 ��g/mL.Surfactant activity was then evaluated using captive bubble surfactometry (CBS), as previously described [25,26]. In detail, CBS chamber was thermostated at 37��C and contained 5mM Tris-HCl (pH 7), 150 mM NaCl, and 10% sucrose. After a small air bubble (0.035-0.040 cm3) was formed, approximately 150 nL of surfactant (10mg/mL) was deposited below the bubble surface with a transparent capillary, as previously described .
Following the introduction of surfactant, changes in surface tension were monitored for 5 min (initial adsorption) from the change in the bubble shape . The chamber was then sealed and the bubble was quickly (1s) expanded to 0.15 cm3, to record post-expansion adsorption during 5 min. Then quasi-static cycles started, where the bubble size was first reduced and then enlarged in a stepwise fashion. There was 1′ delay between the four quasi-static cycles and the following dynamic cycles. During dynamic cycles the bubble was continuously compressed and expanded at a rate of 20 cycles/min.All laboratory assays were performed in triplicate by investigators blinded to the clinical data.Clinical dataClinical data, PRISM-III24 , and derived predicted mortality were recorded.
Single breath static respiratory system compliance (Crs) was measured, just before the lavage, under controlled ventilation, Entinostat using a passive exhalation following end-inspiratory occlusion . 15′ before lavage, routine gas analysis was also performed from an indwelling arterial line: PaO2/FiO2 and oxygenation index (OI= ((mean airway pressure) x FiO2/PaO2)) were recorded. Severity of ARDS was assessed using the Murray’s lung injury score modified for children .