Günther Putz

Persistence of Phase Coexistence in Disaturated Phosphatidylcholine Monolayers at High Surface Pressures

Prior reports that the coexistence of the liquid-expanded (LE) and liquid-condensed (LC) phases in phospholipid monolayers terminates in a critical point have been compromised by experimental difficulties with Langmuir troughs at high surface pressures and temperatures. The studies reported here used the continuous interface of a captive bubble to minimize these problems during measurements of the phase behavior for monolayers containing the phosphatidylcholines with the four different possible combinations of palmitoyl and/or myristoyl acyl residues. Isothermal compression produced surface pressure-area curves for dipalmitoyl phosphatidylcholine (DPPC) that were indistinguishable from previously published data obtained with Langmuir troughs. During isobaric heating, a steep increase in molecular area corresponding to the main LC-LE phase transition persisted for all four compounds to 45 mN/m, at which collapse of the LE phase first occurred. No other discontinuities to suggest other phase transitions were apparent. Isobars for DPPC at higher pressures were complicated by collapse of the monolayer, but continued to show evidence up to 65 mN/m for at least the onset of the LC-LE transition. The persistence of the main phase transition to high surface pressures suggests that a critical point for these monolayers of disaturated phospholipids is either nonexistent or inaccessible at an air-water interface.

A Spreading Technique for Forming Film in a Captive Bubble

Mechanisms underlying the surface properties of lung surfactant are extensively studied in in vitro systems such as the captive-bubble surfactometer (CBS), the pulsating-bubble surfactometer, and the Wilhelmy balance. Among these systems, the CBS is advantageous when a leakproof system and high cycling rates are required. However, widespread application of the CBS to mechanistic studies of dynamic surfactant protein-phospholipid interactions of spread film and to comparative studies between spread and adsorbed film is hampered because spreading of film is difficult. In addition, when film is formed by adsorption, the amount of material required is fairly large. We have developed an easy spreading technique that allows routine formation of film by spreading of small amounts of surfactant components at the air-water interface of an air bubble in a CBS. The technique is reliable, precise, and accurate, and the biophysical activity of film formed by spreading is similar to that of film formed by adsorption. This method will be useful for mechanistic studies of surfactant components under dynamic conditions and for comparative studies of spread films and adsorbed films.

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.

In vitro and in vivo intrapulmonary distribution of fluorescently labeled surfactant.

ObjectiveTo determine the distribution of endotracheally administered surfactant at the alveolar level in an animal model of acute respiratory distress syndrome. DesignProspective, randomized animal study. SettingResearch laboratory of a university hospital. SubjectsSeventy-one male Sprague-Dawley rats, weighing 330–370 g. InterventionsTo measure surfactant distribution in vitro, a glass trough mimicking dichotomic lung anatomy was used to determine the spreading properties of bovine lung surfactant extract supplemented with fluorescent Bodipy-labeled surfactant protein B. To measure surfactant distribution in vivo, rats were anesthetized, and lipopolysaccharide was aerosolized (12 mg/kg body weight) to induce lung injury resembling acute respiratory distress syndrome; in control rats, buffered saline was aerosolized. Twenty-four hours later rats were anesthetized, tracheotomized, and mechanically ventilated (peak airway pressure = 20 mbar; positive end-expiratory pressure = 6 mbar; inspiration time = expiration time = 0.6 sec; Fio2 = 50%). Surfactant (bovine lung surfactant extract, supplemented with fluorescent Bodipy-labeled surfactant protein B; 50 mg/kg body weight) was applied as a bolus; in control rats, saline was administered as a bolus. Rats were ventilated for 5, 15, 30, or 60 mins (n = 8 or 9 for each group). Then, lungs were excised and sliced. Lung slices, divided into aerated (open), underinflated (dystelectatic), or collapsed (atelectatic) alveolar areas, were examined by both light and fluorescence microscopy. ResultsIn vitro experiments revealed that surfactant spread independent of glass trough geometry and lowered the surface tension to equilibrium values (25 mN/m) within a few seconds. In vivo experiments showed that administered surfactant distributed preferentially into underinflated and aerated alveolar areas. Furthermore, surfactant distribution was not affected by length of mechanical ventilation. ConclusionsWhen conventional mechanical ventilation was used in lipopolysaccharide-induced lung injury, surfactant preferentially distributed into underinflated and aerated alveolar areas. Because surfactant rarely reached collapsed alveolar areas, methods aiding in alveolar recruitment (e.g., open lung concept or body positioning) should precede surfactant administration.

Surfactant protein B labelled with [99mTc(CO)3(H2O)3]+ retains biological activity in vitro

UNLABELLED Labelling of the hydrophobic surfactant protein B (SP-B) under non-reducing conditions was achieved with [(99m)Tc(CO)(3)(H2O)(3)](+) prepared according to Alberto et al. (JACS, 1998). The binding of radioactivity was protein-specific, with an overall radiochemical yield of 50%. Gel electrophoresis and Westernblot analyses showed no structural changes of SP-B. Spreading properties and surface activity of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. (99m)Tc-SP-B seems to be a promising agent to observe surfactant spreading under clinical conditions. BACKGROUND Therapeutic results for surfactant instillation in clinical trials are conflicting. The (99m)Tc-labelling of surfactant would allow to observe its spreading in the lung under clinical conditions. METHODS [(99m)Tc(CO)(3)(H2O)(3)](+) was prepared as described by Alberto et al. (JACS, 1998). This carbonyl complex was used for the direct labelling of surfactant protein B (SP-B) under non-reductive conditions by direct incubation with SP-B at elevated temperature followed by extraction into CHCl(3)/MeOH. RESULTS The hydrophobic protein SP-B was labelled with [(99m)Tc(CO)(3)(H2O)(3)](+). An overall radiochemical yield of about 50% was achieved. HPLC-analysis revealed a single radiolabelled species according to UV elution profile of SP-B, supported by paper and size exclusion chromatography. Gel electrophoresis confirmed that the dimer structure of SP-B was preserved. Spreading properties of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. Spreading of radioactivity observed in a glass trough of 26 cm x 27 cm with a gamma camera was completed during the first 7-9 sec after application of (99m)Tc-labelled SP-B. The corresponding decrease of surface tension to 45 mN/m at the peripheral surface tension sensors took 7 sec +/- 2 sec (MEAN +/- STD; n = 3). CONCLUSIONS Direct and specific (99m)Tc-labelling of the hydrophobic surfactant protein B was achieved using the [(99m)Tc(CO)(3)(H2O)(3)](+) precursor. This procedure can easily be used to prepare specifically labelled surfactant mixtures with spreading properties that coincide with those of unlabelled surfactant.

Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence

Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high alpha-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.

Hydrophobic lung surfactant proteins B and C remain associated with surface film during dynamic cyclic area changes.

The biophysical activity of lung surfactant depends, to a large extent, on the presence of the hydrophobic surfactant proteins B (SP-B) and C (SP-C). The role of these proteins in lipid adsorption and lipid squeeze-out under dynamic conditions simulating breathing is not yet clear. Therefore, the aim of this study was to investigate the interaction of spread hydrophobic surfactant proteins with phospholipids in a captive-bubble surfactometer during rapid cyclic area changes (6 cycles/min). We found that SP-B and SP-C facilitated the rapid transport of lipids into the air-water interface in a concentration-dependent manner (threshold concentration > or = 0.05:0.5 mol% SP-B/SP-C). Successive rapid cyclic area changes did not affect the concentration-dependent lipid adsorption process, suggesting that SP-B and SP-C remained associated with the surface film.

Gamma scintigraphic imaging of lung microvascular permeability in adult respiratory distress syndrome

The sequence of lung microvascular permeability (LMVP) changes in early direct posttraumatic and late indirect pancreatitis-induced adult respiratory distress syndrome (ARDS) was studied and compared with that of a control group, as well as non-ARDS ICU patients. A computerized large field of view gamma camera was used to measure LMVP simultaneously over both lungs by In 113m-labeled transferrin and Tc 99m-labeled erythrocytes. The LMVP index (LMVPI) (%/h) was used to quantify LMVP in the dynamic scintigraphic measurement. In the control group the LMVPI was 2.6 +/- 2.8%/h for the right and 2.0 +/- 2.8%/h for the left lung. Similar values were found in mechanically ventilated ICU patients without ARDS (group A) on admission (right LMVPI 3.2 +/- 2.6, left LMVPI 2.6 +/- 2.7%/h) and 4 days later (right LMVPI 3.9 +/- 2.6, left LMVPI 2.3 +/- 1.8%/h). Interestingly, the initial evaluation of patients with direct early posttraumatic ARDS (lung contusion) (group B) showed significantly (p less than .01) elevated LMVP for the contused side (LMVPI 10.8 +/- 5.1%/h), but normal values for the nontraumatized lung (LMVPI 3.9 +/- 3.4%/h), whereas 4 days later the LMVP increased significantly (p less than .05) on the primarily healthy side (LMVPI 8.0 +/- 5.0%/h) while remaining elevated for the traumatized lung (LMVPI 10.9 +/- 6.0%/h).(ABSTRACT TRUNCATED AT 250 WORDS)