Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 +/- 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 +/- 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.     

Characterization of pulmonary surfactant protein D: its copurification with lipids

Surfactant protein D (SP-D) is a collagenous surfactant associated protein synthesized by alveolar type II cells. SP-D was purified from the supernatant of rat bronchoalveolar lavage fluids obtained by centrifugation at 33,000 x gav for 16 h. The contents of SP-D and SP-A in fractions obtained by the centrifugation of rat bronchoalveolar lavage were determined by enzyme-linked immunoassay. The total content of SP-D was approximately 12% of that of SP-A in these lavage fluids. 99.1% of SP-A was present in the 33,000g pellet, whereas 71.1% of SP-D was in the 33,000g supernatant. Analysis by high performance liquid chromatography reveals that lipids are copurified with isolated SP-D. Phosphatidylcholine accounted for 84.8% of the phospholipids copurified with SP-D. Unlike SP-A, SP-D in the purified and delipidated form failed to compete with 125I-labeled SP-A for phosphatidylcholine binding, and to aggregate phospholipid liposomes. The present study demonstrates that lipids are copurified with SP-D, that SP-D and SP-A distribute differently in rat bronchoalveolar lavage fluids, and that SP-D in the purified and delipidated form does not exhibit interaction with lipids in the same fashion as SP-A.     

Altered function of pulmonary surfactant in fatty acid lung injury

To determine whether acute fatty acid lung injury impairs pulmonary surfactant function, we studied anesthetized ventilated rabbits given oleic acid (55 mg/kg iv, n = 11) or an equivalent volume of saline (n = 8). Measurements of pulmonary mechanics indicated a decrease in dynamic compliance within 5 min of injury and a decrease in lung volume that was disproportionately large at low pressures, consistent with diminished surfactant activity in vivo. Bronchoalveolar lavage fluid obtained 1 h after injury had significantly increased erythrocytes and total leukocytes, largely polymorphonuclear cells. The phospholipid content and composition of the cell-free fraction had only minor changes from those of controls, but the protein content was increased 35-fold. Measurements of lavage surface activity in vitro showed an increase in average minimum surface tension from 1.3 +/- 0.4 (SE) dyn/cm in controls to 20.2 +/- 3.9 dyn/cm in injured animals. The alterations in static pressure-volume curves and decrease in lavage surface activity suggest a severe alteration of surfactant function in this form of lung injury that occurs despite the presence of normal amounts of surfactant phospholipids.     

Inhibition by colchicine of phospholipid secretion induced by lung distension

The effect of colchicine, a microtubule disruptor, on phospholipid secretion stimulated by distension of fetal rabbit lungs was investigated. After colchicine injection and breathing for 45 min, pups were killed and their lungs were lavaged with colchicine. Controls were injected and lavaged with saline. All lungs were given static air inflation and a final lavage, and the returns were analyzed for phospholipid DNA, and lactate dehydrogenase. The first lavage after breathing yielded 33% less phospholipid with colchicine, 3.83 compared with 5.72 mg/g dry lung wt (P less than 0.05). The postinflation phospholipid yield was also significantly reduced with colchicine from 1.04 to 0.70 mg/g dry lung wt (P less than 0.05). The postinflation DNA was significantly reduced with colchicine, from 1.26 to 0.44 micrograms (P less than 0.01), suggesting reduced alveolar macrophages. Colchicine did not change the recovery by lavage of exogenous radioactive phospholipid. As reflected by ATP and lactate levels, tissue metabolism was well maintained. The results are interpreted to mean that colchicine reduced simultaneously lavage-associated phospholipid secretion, inflation-produced phospholipid secretion, and macrophage migration.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.     

Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice

Targeted disruption of the surfactant protein (SP) D (SP-D) gene caused a marked pulmonary lipoidosis characterized by increased alveolar lung phospholipids, demonstrating a previously unexpected role for SP-D in surfactant homeostasis. In the present study, we tested whether the local production of SP-D in the lung influenced surfactant content in SP-D-deficient [SP-D(-/-)] and SP-D wild-type [SP-D(+/+)] mice. Rat SP-D (rSP-D) was expressed under control of the human SP-C promoter, producing rSP-D, SP-D(+/+) transgenic mice. SP-D content in bronchoalveolar lavage fluid was increased 30- to 50-fold in the rSP-D, SP-D(+/+) mice compared with the SP-D(+/+) parental strain. Lung morphology, phospholipid content, and surfactant protein mRNAs were unaltered by the increased concentration of SP-D. Likewise, the production of endogenous mouse SP-D mRNA was not perturbed by the SP-D transgene. rSP-D, SP-D(+/+) mice were bred to SP-D(-/-) mice to assess whether lung-selective expression of SP-D might correct lipid homeostasis abnormalities in the SP-D(-/-) mice. Selective expression of SP-D in the respiratory epithelium had no adverse effects on lung function, correcting surfactant phospholipid content and decreasing phosphatidylcholine incorporation significantly. SP-D regulates surfactant lipid homeostasis, functioning locally to inhibit surfactant phospholipid incorporation in the lung parenchyma and maintaining alveolar phospholipid content in the alveolus. Marked increases in biologically active tissue and alveolar SP-D do not alter lung morphology, macrophage abundance or structure, or surfactant accumulation.     

Surfactant subtypes in mice: characterization and quantitation

Surfactant obtained by bronchoalveolar lavage of normal adult mice was separated into subtypes by a one-step centrifugation to equilibrium on continuous sucrose gradients. Mouse surfactant resolved in this way exists in three subtypes with similar phospholipid compositions. A "light" subtype of buoyant density 1.027 +/- 0.012 (SD) g/ml comprises 43 +/- 18% of the total alveolar lavage phospholipid, has little surface activity, and consists exclusively of small unilamellar vesicles. A "heavy" subtype of buoyant density 1.055 +/- 0.016 g/ml comprises 48 +/- 11% of the total, is surface active, and consists of small amounts of tubular myelin among large empty vesicles. A third component, called "ultraheavy," comprises 9 +/- 4% of the total alveolar lavage phospholipid, has a density of 1.072 +/- 0.020 g/ml, is surface active, and consists of large aggregates of tubular myelin associated with lamellar bodylike structures. Labeling studies suggested that the ultraheavy material was labeled first and was of the same density as purified lamellar bodies. These results are consistent with the view that, in mice, surfactant is secreted into the alveolar compartment in an ultraheavy form, which evolves into the heavy and light forms.     

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.     

Metabolism and function of platelet-activating factor in fetal rabbit lung development

Previously, platelet-activating factor (PAF, PAF-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) had been identified in association with a lamellar-body-enriched fraction of human amniotic fluid obtained from women in labor. In consideration of the fact that fetal lung is the source of lamellar bodies, we have investigated the capacity of the developing lung to synthesize PAF. The specific activity of the PAF biosynthetic enzyme, 1-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase, increased from 116 pmol/min per mg protein in day 21 fetal rabbit lung to 332 pmol/min per mg protein by day 31. Although this enzymatic activity in fetal kidney also increased, it never reached the level found in lung. In contrast, the actyltransferase activity decreased by 80% in fetal liver between days 21 and 31. The acetyltransferase activity in lung was primarily localized in the microsomal fraction (105 000 X g pellet); however a significant proportion of the activity was found in the 18 000 X g pellet. The specific activity of acetyltransferase in adult alveolar type II rat pneumonocytes was significantly higher than that of adult rat lung or rat alveolar macrophages, suggesting that type II cells make a significant contribution to the actyltransferase activity of lung tissue. PAF acetylhydrolase remained relatively constant throughout the gestation in all tissues. The concentration of PAF in the fetal lung increased by 3-fold from 12 to 35 fmol/mg protein, between day 21 and day 31 of development. The concentrations of the PAF precursors, 2-lyso-PAF (1-alkyl-2-lyso-sn-glycero-3-phosphocholine) and the 2-acyl derivative, were several orders of magnitude higher than the PAF concentration. The pulmonary glycogen content decreased from 163 at day 21 to 35 micrograms/mg protein at day 31 of gestation. We suggest that the increase in PAF concentration may participate in the regulation of glycogen breakdown in fetal lung as it does in perfused rat liver (Shukla, S.D., Buxton, D.B., Olson, M.S. and Hanahan, D.J. (1983) J. Biol. Chem. 258, 10212-10214). The formation of PAF in the developing lung and its secretion, in association with lamellar bodies, into amniotic fluid is discussed in relation to parturition.     

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size,pool morphology and isoforms of the 32–38 kDa apolipoprotein

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0–24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 ± 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 ± 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32–38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.     

Induction of intra- and extra-cellular phospholipids in the lungs of rats exposed to silica.

Intracellular and extracellular compartments of phospholipids in the lungs of rats were examined 28 days after intratracheal injection of silica (200 mg/kg). All compartments containing phospholipids were elevated, but the largest increases were seen in the intracellular and extracellular pulmonary surfactant. Intracellular pulmonary surfactant increased 123-fold from 1.18 +/- 0.65 to 144.9 +/- 53.8 and the extracellular surfactant increased 22-fold from 1.17 +/- 0.04 to 25.1 +/- 7.1 mg per pair of rat lungs respectively. The phospholipid composition of intracellular and extracellular surfactant did not change in response to silica, except for an almost 2-fold increase in the percentage of total phosphatidylinositol in both compartments. The phospholipid content of the lungs increased from 24.9 +/- 4.6 to 268.6 +/- 20.8 mg, with the intracellular and extracellular surfactant accounting for 59.1 and 24.6% of this total increase respectively. These data demonstrate that the major increases in the phospholipid content of the lungs induced by silica is associated with the pulmonary-surfactant system.     

P. carinii induces selective alterations in component expression and biophysical activity of lung surfactant

Studies of Pneumocystis carinii pneumonia (PCP) suggest an important role for the surfactant system in the pathogenesis of the hypoxemic respiratory insufficiency associated with this infection. We hypothesized that PCP induces selective alterations in alveolar surfactant component expression and resultant biophysical properties. PCP was induced by intratracheal inoculation of 2 x 10(5) P. carinii organisms into C.B-17 scid/scid mice. Six weeks after inoculation, large (LA)- and small (SA)-aggregate surfactant fractions were prepared from bronchoalveolar lavage fluids and analyzed for expression of surfactant components and for biophysical activity. Total phospholipid content was significantly reduced in LA surfactant fractions from mice infected with PCP (53 +/- 15% of uninfected mice; P < 0.05). Quantitation of hydrophobic surfactant protein (SP) content demonstrated significant reductions of alveolar SP-B and SP-C protein levels in mice with PCP compared with those in uninfected mice (46 +/- 7 and 19 +/- 6%, respectively; P < 0.05 for both). The reductions in phospholipid, SP-B, and SP-C in LA fractions measured during PCP were associated with an increase in the minimum surface tension of LAs as measured by pulsating bubble surfactometer (13.1 +/- 1.1 vs. 5.4 +/- 1.8 mN/m; P < 0.05). In contrast to decreases in the hydrophobic SPs, SP-D content in the SA fraction was markedly increased (343 +/- 30% of control value; P < 0. 05) and SP-A levels in LA surfactant were maintained (93 +/- 26% of control value) during P. carinii infection. In all cases, the changes in SP content were reflected by commensurate changes in the levels of mRNA. We conclude that PCP induces selective alterations in surfactant component expression, including profound decreases in hydrophobic protein contents and resultant increases in surface tension. These changes, demonstrated in an immunologically relevant animal model, suggest that alterations in surfactant could contribute to the hypoxemic respiratory insufficiency observed in PCP.     

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.     

Secretory IgA is a component of rabbit lung surfactant

Secretory IgA is a major protein component of rabbit lung surfactant purified by NaBr density gradient centrifugation from endobronchial lavage and minced lung tissue. Secretory IgA was found in both surfactant and non-surfactant fractions obtained from endobronchial lung washings. By contrast in minced-lung washings, which are not contaminated with proteins from the upper respiratory tree, secretory IgA is prominent only in the surfactant fraction. These findings indicate that in rabbit lung secretory IgA is present in the alveoli and is intimately associated with the surfactant system.     

Neutrophils in reexpansion pulmonary edema

This study investigated the possible contribution of neutrophils to development of reexpansion pulmonary edema (RPE) in rabbits. Rabbits' right lungs were collapsed for 7 days and then reexpanded with negative intrathoracic pressure for 2 h before study, a model that creates unilateral edema in the reexpanded lungs but not in contralateral left lungs. Two hours after lung reexpansion, significant increases in lavage albumin concentration (17-fold), percent neutrophils (14-fold), and total number of neutrophils (7-fold) recovered occurred in the reexpanded lung but not in the left. After 2 h of reexpansion increased leukotriene B4 was detected in lavage supernatant from right lungs (335 +/- 33 pg/ml) compared with the left (110 +/- 12 pg/mg, P less than 0.01), and right lung lavage acid phosphatase activity similarly increased (6.67 +/- 0.35 U/l) compared with left (4.73 +/- 0.60 U/l, P less than 0.05). Neutropenia induced by nitrogen mustard (17 +/- 14 greater than neutrophils/microliters) did not prevent RPE, because reexpanded lungs from six neutropenic rabbits were edematous (wet-to-dry lung weight ratio 6.34 +/- 0.43) compared with their contralateral lungs (4.97 +/- 0.04, P less than 0.01). An elevated albumin concentration in reexpanded lung lavage from neutropenic rabbits (8-fold) confirmed an increase in permeability. Neutrophil depletion before reexpansion did not prevent unilateral edema, although neutrophils were absent from lung sections and alveolar lavage fluid from neutropenic rabbits.     

Lyophilization of rumen fluid for use in culture media

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Lyophilization of rumen fluid for use in culture media.

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ～80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ～50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.     

Changes in quantity, composition, and surface activity of alveolar surfactant at birth

We hypothesized that when the lung makes the transition from the fluid- to the air-filled state at birth, there are changes in physical and functional properties of the alveolar surfactant. To test this hypothesis, newborn rabbits were killed at different times in the first 24 h of life, their lungs lavaged with ice-cold saline, and the lavage fluid subfractionated by differential centrifugation. The phospholipid and protein content and composition and the kinetics of surface tension lowering of the subfractions were examined. We found that with the onset of breathing, shifts occur in the distribution of surfactant subfractions as a surfactant apoprotein-free phospholipid fraction is generated. The ratio of rapidly sedimentable apoprotein-rich to slowly sedimentable, apoprotein-free fractions decreases from 31 at birth to 4 at 24 h of life. Concurrently, rates of surface tension lowering by the subfractions increase with time. The results suggest that the adult pattern of pool sizes and surface activity of alveolar surfactant is not present at birth but evolves slowly over the 1st day of life.