Molecular and functional changes of pulmonary surfactant in response to hyperoxia.

Surfactant comprises phosphatidylcholine (PC) together with anionic phospholipids, neutral lipids, and surfactant proteins SP-A to-D. Its composition is highly specific, with dipalmitoyl-PC, palmitoyl-myristoyl-PC, and palmitoyl-palmitoleoyl-PC as its predominant PC species, but with low polyunsaturated phospholipids. Changes in pulmonary metabolism and function in response to injuries depend on their duration and whether adaptation can occur. We examined in rats prolonged (7 days) versus acute (2 days) exposure to non-lethal oxygen concentrations (85%) with respect to the composition and metabolism of individual lung phospholipid molecular species. Progressive inflammation, structural alteration, and involvement of type II pneumocytes were confirmed by augmented bromodeoxyuridine incorporation, broadening of alveolar septa, and increased granulocyte, macrophage, SP-A, and SP-D concentrations. Surfactant function was impaired after 2 days, but normalized with duration of hyperoxia, which was attributable to inhibition but not to alteration in SP-B/C concentrations. Phospholipid pool sizes and PC synthesis by lung tissue, as assessed by [methyl-(3)H]-choline incorporation, were unchanged after 2 days, although after 7 days they were elevated 1.7-fold. By contrast, incorporation of labeled PC into tissue pools of surfactant and lung lavage fluid decreased progressively. Moreover, concentrations of arachidonic acid containing phospholipids were augmented at the expense of saturated palmitoyl-myristoyl-PC and dipalmitoyl-PC. We conclude a persisting impairment in the intracellular trafficking and secretion of newly synthesized PC, accompanied by a progressive increase in alveolar arachidonic acid containing phospholipids in spite of recovery of acutely impaired surfactant function and adaptive increase of overall PC synthesis.

Mass spectrometric analysis of surfactant metabolism in human volunteers using deuteriated choline.

Surfactant reduces surface tension at pulmonary air-liquid interfaces. Although its major component is dipalmitoyl-phosphatidylcholine (PC16:0/16:0), other PC species, principally palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC, and palmitoyloleoyl-PC, are integral components of surfactant. The composition and metabolism of PC species depend on pulmonary development, respiratory rate, and pathologic alterations, which have largely been investigated in animals using radiolabeled precursors. Recent advances in mass spectrometry and availability of precursors carrying stable isotopes make metabolic experiments in human subjects ethically feasible. We introduce a technique to quantify surfactant PC synthesis in vivo using deuteriated choline coupled with electrospray ionization tandem mass spectrometry. Endogenous PC from induced sputa of healthy volunteers comprised 54.0 +/- 1.5% PC16:0/16:0, 9.7 +/- 0.7% palmitoylmyristoyl-PC, 10.0 +/- 1.0% palmitoylpalmitoleoyl-PC, and 13.1 +/- 0.3% palmitoyloleoyl-PC. Infusion of deuteriated choline chloride (3.6 mg/kg body weight) over 3 hours resulted in linear incorporation into PC over 30 hours. After a plateau of 0.61 +/- 0.04% labeled PC between 30 and 48 hours, incorporation decreased to 0.30 +/- 0.02% within 7 days. Compared with native PC, fractional label was initially lower for PC16:0/16:0 (31.9 +/- 8.3%) but was higher for palmitoyloleoyl-PC (21.0 +/- 1.2%), and equilibrium was achieved after only 48 hours. We conclude that infusion of deuteriated choline and electrospray ionization tandem mass spectrometry is useful to investigate surfactant metabolism in humans in vivo.

Surfactant from diving aquatic mammals.

Diving mammals that descend to depths of 50-70 m or greater fully collapse the gas exchanging portions of their lungs and then reexpand these areas with ascent. To investigate whether these animals may have evolved a uniquely developed surfactant system to facilitate repetitive alveolar collapse and expansion, we have analyzed surfactant in bronchoalveolar lavage fluid (BAL) obtained from nine pinnipeds and from pigs and humans. In contrast to BAL from terrestrial mammals, BAL from pinnipeds has a higher concentration of phospholipid and relatively more fluidic phosphatidylcholine molecular species, perhaps to facilitate rapid spreading during alveolar reexpansion. Normalized concentrations of hydrophobic surfactant proteins B and C were not significantly different among pinnipeds and terrestrial mammals by immunologic assay, but separation of proteins by gel electrophoresis indicated a greater content of surfactant protein B in elephant seal surfactant than in human surfactant. Remarkably, surfactant from the deepest diving pinnipeds produced moderately elevated in vitro minimum surface tension measurements, a finding not explained by the presence of protein or neutral lipid inhibitors. Further study of the composition and function of pinniped surfactants may contribute to the design of optimized therapeutic surfactants.

Surfactant in newborn compared with adolescent pigs: adaptation to neonatal respiration.

Surfactant composition and function differ between vertebrates, depending on pulmonary anatomy and respiratory physiology. Because pulmonary development in pigs is similar to that in humans, we investigated surface tension function, composition of phospholipid molecular species, and concentrations of surfactant protein (SP)-A to -D in term newborn pigs (NP) compared with adolescent pigs (AP), using the pulsating bubble surfactometer, mass spectrometry, high-performance liquid chromatography, and immunoblot techniques (IT). NP was more potent than AP surfactant in reaching minimal surface tension values near zero mN/m. Whereas SP-A and SP-D were comparable, SP-B and SP-C were increased 3- to 4-fold in NP surfactant. Moreover, fluidizing phospholipids such as palmitoylmyristoyl-PC (PC16:0/14:0) and palmitoylpalmitoleoyl-PC (PC16:0/16:1) were increased at the expense of PC16:0/16:0 (32.4 +/- 0.6 versus 44.5 +/- 3.2%, respectively). Whereas concentrations of total anionic phospholipids were similar in NP and AP surfactant (9.9 +/- 0.3 and 12.0 +/- 0.3%, respectively), phosphatidylinositol was the predominant anionic phospholipid in NP surfactant. We conclude that, compared with AP, NP surfactant displays better surface tension function under dynamic conditions, which is associated with increased concentrations of SP-B and SP-C, as well as fluidizing phospholipids at the expense of PC16:0/16:0.

Phosphatidylcholine metabolism of rat trachea in relation to lung parenchyma and surfactant.

Pulmonary surfactant prevents alveolar collapse and contributes to airway patency by reducing surface tension. Although alveolar surfactant, consisting mainly of phospholipids (PL) together with neutral lipids and surfactant-specific proteins, originates from type II pneumocytes, the contribution of airway epithelia to the PL fraction of conductive airway surfactant is still debated. We, therefore, analyzed the composition, synthesis, and release of phosphatidylcholine (PC) molecular species as the main surfactant PL of the rat trachea compared with the lung. Analyses of individual PC molecular species with HPLC and electrospray ionization mass spectrometry revealed that the rat trachea contained and synthesized much more palmitoyloleoyl-PC, palmitoyllinoleoyl-PC, and palmitoylarachidonoyl-PC, together with increased amounts of alkylacyl-PC, and less surfactant-specific species such as dipalmitoyl-PC than the lung. Organ cultures with [methyl-3H]choline as precursor of PC revealed that, in the trachea, synthesized PC was retained in the tissue, rather than secreted. [Methyl-3H]choline-labeled dipalmitoyl-PC was a negligible component in the trachea, and, in contrast to the lungs, palmitoyloleoyl-PC was enriched in tracheal secretions. We conclude that the surfactant fraction in the airways does not originate from the airways but is produced in the alveolar space and transported upward.

Molecular species compositions of lung and pancreas phospholipids in the cftr(tm1HGU/tm1HGU) cystic fibrosis mouse.

Fatty acid analysis of phospholipid compositions of lung and pancreas cells from a cystic fibrosis transmembrane regulator (CFTR) negative mouse (cftr(-/-))suggested that a decreased concentration of docosahexaenoate (22:6(n-3)) and increased arachidonate (20:4(n-6)) may be related to the disease process in cystic fibrosis (CF). Consequently, we have determined compositions of the major phospholipids of lung, pancreas, liver, and plasma from a different mouse model of CF, the cftr(tm1HGU/tm1HGU) mouse, compared with ZTM:MF-1 control mice. Electrospray ionization mass spectrometry permitted the quantification of all of the individual molecular species of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGly), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). There was no deficiency of 22:6(n-3) in any phospholipid class from lung, pancreas, or liver from mice with the cftr(tm1HGU/tm1HGU). Instead, the concentration of 20:4(n-6) was significantly decreased in plasma PtdCho species and in pancreas and lung species of PtdEtn, PtdSer, and PtdIns. These results demonstrate the variability of membrane phospholipid compositions in different mouse models of CF and suggest that in cftr(tm1HGU/tm1HGU) mice, the apparent deficiency was of 20:4n-6- rather than of 22:6n-3-containing phospholipid species. They highlight a need for detailed phospholipid molecular species analysis of cells expressing mutant CFTR from children with CF before the therapeutic effects of administering high doses of 22:6(n-3)-containing oils to children with CF can be fully evaluated.