Ontogeny of the pulmonary surfactant and antioxidant enzyme systems in the viviparous lizard,Tiliqua rugosa

The antioxidant enzyme (AOE) system protects the lung from oxidative damage. The pulmonary surfactant (PS) system lowers the interfacial pressure within the lung, improving lung compliance and aiding lung clearance. In mammals, the AOE and PS systems develop in tandem during the final 10%-20% of gestation. Here, we investigated the development of these systems in the viviparous skink, Tiliqua rugosa. The content of total phospholipid (PL), disaturated phospholipid (DSP), and cholesterol (Chol) increased in lung washings from foetal lizards with advancing gestational age. Similarly, the relative saturation of the PLs increased throughout gestation, with mid-stage 40 foetuses having a DSP/PL equivalent to newborns and adults. Maternal lizards had significantly less total PL, DSP, and Chol than nongravid and newborn lizards; however, the relative composition did not differ from nongravid animals. This presumably results from compression of the lungs under the bulk of the developing foetus. The Chol/PL and Chol/DSP ratios declined early in development such that mid-stage 40 embryos had comparable ratios to both newborns and adults. Thus, it appears that the PS system matures in a similar manner in skinks and in mammals. However, the composition of surfactant is complete some weeks before parturition, probably to enable improved survivorship of the precocial young in the event of premature birth. Unlike the surfactant lipids, the AOEs, catalase, superoxide dismutase, and glutathione peroxidase did not differ appreciably throughout gestation. It appears therefore that like the surfactant lipids the AOE system is in readiness for air breathing throughout the latter stages of gestation, possibly in preparation for premature birth. Unlike mammals, the PS and AOE systems develop independently from one another.     

Development of the pulmonary surfactant system in two oviparous vertebrates

In birds and oviparous reptiles, hatching is often a lengthy and exhausting process, which commences with pipping followed by lung clearance and pulmonary ventilation. We examined the composition of pulmonary surfactant in the developing lungs of the chicken, Gallus gallus, and of the bearded dragon, Pogona vitticeps. Lung tissue was collected from chicken embryos at days 14, 16, 18 (prepipped), and 20 (postpipped) of incubation and from 1 day and 3 wk posthatch and adult animals. In chickens, surfactant protein A mRNA was detected using Northern blot analysis in lung tissue at all stages sampled, appearing relatively earlier in development compared with placental mammals. Chickens were lavaged at days 16, 18, and 20 of incubation and 1 day posthatch, whereas bearded dragons were lavaged at day 55, days 57-60 (postpipped), and days 58-61 (posthatched). In both species, total phospholipid (PL) from the lavage increased throughout incubation. Disaturated PL (DSP) was not measurable before 16 days of incubation in the chick embryo nor before 55 days in bearded dragons. However, the percentage of DSP/PL increased markedly throughout late development in both species. Because cholesterol (Chol) remained unchanged, the Chol/PL and Chol/DSP ratios decreased in both species. Thus the Chol and PL components are differentially regulated. The lizard surfactant system develops and matures over a relatively shorter time than that of birds and mammals. This probably reflects the highly precocial nature of hatchling reptiles.     

The roles of cholesterol in pulmonary surfactant: insights from comparative and evolutionary studies

In most eutherian mammals, cholesterol (Chol) comprises approximately 8-10 wt.% or 14-20 mol.% of both alveolar and lamellar body surfactant. It is regarded as an integral component of pulmonary surfactant, yet few studies have concentrated on its function or control. Throughout the evolution of the vertebrates, the contribution of cholesterol relative to surfactant phospholipids decreases, while that of the disaturated phospholipids (DSP) increases. Chol generally appears to dominate in animals with primitive bag-like lungs that lack septation, in the saccular lung of snakes or swimbladders which are not used predominantly for respiration, and also in immature lungs. It is possible that in these systems, cholesterol represents a protosurfactant. Cholesterol is controlled separately from the phospholipid (PL) component in surfactant. For example, in heterothermic mammals such as the fat-tailed dunnart, Sminthopsis crassicaudata, and the microchiropteran bat, Chalinolobus gouldii, and also in the lizard, Ctenophorus nuchalis, the relative amount of Chol increases in cold animals. During the late stages of embryonic development in chickens and lizards, the Chol to PL and Chol to DSP ratios decrease dramatically. While in isolated lizard lungs, adrenaline and acetylcholine stimulate the secretion of surfactant PL, Chol secretion remains unaffected. This is also supported in isolated cell studies of lizards and dunnarts. The rapid changes in the Chol to PL ratio in response to various physiological stimuli suggest that these two components have different turnover rates and may be packaged and processed differently. Infusion of [3H]cholesterol into the rat tail vein resulted in a large increase in Chol specific activity within 30 min in the lamellar body (LB) fraction, but over a 48-h period, failed to appear in the alveolar surfactant fraction. Analysis of the limiting membrane of the lamellar bodies revealed a high (76%) concentration of LB cholesterol. The majority of lamellar body Chol is, therefore, not released into the alveolar compartment, as the limiting membrane fuses with the cell membrane upon exocytosis. It appears unlikely, therefore, that lamellar bodies are the major source of alveolar Chol. It is possible that the majority of alveolar Chol is synthesised endogenously within the lung and stored independently from surfactant phospholipids. The role of cholesterol in the limiting membrane of the lamellar body may be to enable fast and easy processing by maintaining the membrane in a relatively fluid state.     

Alterations in pulmonary surfactant after rapid arousal from torpor in the marsupial Sminthopsis crassicaudata.

Torpor in the dunnart, Sminthopsis crassicaudata, alters surfactant lipid composition and surface activity. Here we investigated changes in surfactant composition and surface activity over 1 h after rapid arousal from torpor (15-30 degrees C at 1 degrees C/min). We measured total phospholipid (PL), disaturated PL (DSP), and cholesterol (Chol) content of surfactant lavage and surface activity (measured at both 15 and 37 degrees C in the captive bubble surfactometer). Immediately after arousal, Chol decreased (from 4.1 +/- 0.05 to 2.8 +/- 0.3 mg/g dry lung) and reached warm-active levels by 60 min after arousal. The Chol/DSP and Chol/PL ratios both decreased to warm-active levels 5 min after arousal because PL, DSP, and the DSP/PL ratio remained elevated over the 60 min after arousal. Minimal surface tension and film compressibility at 17 mN/m at 37 degrees C both decreased 5 min after arousal, correlating with rapid changes in surfactant Chol. Therefore, changes in lipids matched changes in surface activity during the postarousal period.     

Surfactant in the gas mantle of the snail Helix aspersa

Surfactant occurs in cyclically inflating and deflating, gas-holding structures of vertebrates to reduce the surface tension of the inner fluid lining, thereby preventing collapse and decreasing the work of inflation. Here we determined the presence of surfactant in material lavaged from the airspace in the gas mantle of the pulmonate snail Helix aspersa. Surfactant is characterized by the presence of disaturated phospholipid (DSP), especially disaturated phosphatidylcholine (PC), lavaged from the airspace, by the presence of lamellated osmiophilic bodies (LBs) in the airspaces and epithelial tissue, and by the ability of the lavage to reduce surface tension of fluid in a surface balance. Lavage had a DSP/phospholipid (PL) ratio of 0.085, compared to 0.011 in membranes, with the major PL being PC (45.3%). Cholesterol, the primary fluidizer for pulmonary surfactant, was similar in lavage and in lipids extracted from cell homogenates (cholesterol/PL: 0.04 and 0. 03, respectively). LBs were found in the tissues and airspaces. The surface activity of the lavage material is defined as the ability to reduce surface tension under compression to values much lower than that of water. In addition, surface-active lipids will vary surface tension, increasing it upon inspiration as the surface area expands. By these criteria, the surface activity of lavaged material was poor and most similar to that shown by pulmonary lavage of fish and toads. Snail surfactant displays structures, a biochemical PL profile, and biophysical properties similar to surfactant obtained from primitive fish, teleost swim bladders, the lung of the Dipnoan Neoceratodus forsteri, and the amphibian Bufo marinus. However, the cholesterol/PL and cholesterol/DSP ratios are more similar to the amphibian B. marinus than to the fish, and this similarity may indicate a crucial physicochemical relationship for these lipids.     

Hypoxic control of the development of the surfactant system in the chicken: evidence for physiological heterokairy

The surfactant system, a complex mixture of lipids and proteins, controls surface tension in the lung and is crucial for the first breath at birth, and thereafter. Heterokairy is defined as plasticity of a developmental process within an individual. Here, we provide experimental evidence for the concept of heterokairy, as hypoxia induces a change in the onset and rate of development of surfactant, probably via endogenous glucocorticoids, to produce individuals capable of surviving early hatching. Chicken eggs were incubated under normoxic (21% O(2)) conditions throughout or under hypoxic (17% O(2)) conditions from day 10 of incubation. Embryos were sampled at days 16, 18, and 20 and also 24 h after hatching. In a second experiment, dexamethasone (Dex), tri-iodothyronine (T(3)), or a combination (Dex + T(3)) was administered 24 and 48 h before each time point. Both hypoxia and Dex accelerated maturation of the surfactant lipids by increasing total phospholipid (PL), disaturated phospholipid (DSP), and cholesterol (Chol) in lavage at days 16 and 18. Maturation of surfactant lipid composition was accelerated, with day 16 %DSP/PL, Chol/DSP, and Chol/PL resembling the ratios of day 20 control animals. The effect of Dex + T(3) was similar to that of Dex alone. Hypoxia increased plasma corticosterone levels at day 16, while plasma T(3) levels were not affected. Hence, exposure to hypoxia during critical developmental windows accelerates surfactant maturation, probably by increasing corticosterone production. This internal modulation of the developmental response to an external stimulus is a demonstration of physiological heterokairy.     

Comparison of the respiratory transition at birth or hatching in viviparous and oviparous amniote vertebrates.

Regardless of the mode of reproduction, three things must occur at birth or hatching in amniote vertebrates: initiation of breathing, pulmonary fluid elimination and reabsorption, and adequate perfusion of pulmonary circulation. Although data on these events are few, there appears to be no fundamental difference in them that can be associated with the oviparity to viviparity transition. There are, however, differences in the timing of these events in oviparous and viviparous amniotes. The transition to neonatal respiration tends to be very quick in viviparous species because the vascular support for oxygen uptake provided by the mother is rapidly disassociated from the mechanism for uptake by the embryo. By contrast, hatching often is a slow process, taking 24 h or more in some species, as chorioallantoic blood flow slowly gives way to clearing of the lungs and pulmonary gas exchange. Little is known of the mechanisms of pulmonary fluid elimination and reabsorption or lung inflation in reptiles, but the cellular structures and surfactant systems are similar in all amniote vertebrates. Nevertheless, there are differences, particularly of timing and maturation of various systems, but there has been no exploration of the functional (or phylogenetic) bases of these differences. Perfusion of the neonatal pulmonary system to support respiration in reptiles remains to be investigated. In mammals and birds, closure of the ductus arteriosus is important, but the role played by the ductus arterioisus in reptilian hatching or birth is not known.     

Dipalmitoylphosphatidylcholine is not the major surfactant phospholipid species in all mammals

Pulmonary surfactant, a complex mixture of lipids and proteins, lowers the surface tension in terminal air spaces and is crucial for lung function. Within an animal species, surfactant composition can be influenced by development, disease, respiratory rate, and/or body temperature. Here, we analyzed the composition of surfactant in three heterothermic mammals (dunnart, bat, squirrel), displaying different torpor patterns, to determine: 1) whether increases in surfactant cholesterol (Chol) and phospholipid (PL) saturation occur during long-term torpor in squirrels, as in bats and dunnarts; 2) whether surfactant proteins change during torpor; and 3) whether PL molecular species (molsp) composition is altered. In addition, we analyzed the molsp composition of a further nine mammals (including placental/marsupial and hetero-/homeothermic contrasts) to determine whether phylogeny or thermal behavior determines molsp composition in mammals. We discovered that like bats and dunnarts, surfactant Chol increases during torpor in squirrels. However, changes in PL saturation during torpor may not be universal. Torpor was accompanied by a decrease in surfactant protein A in dunnarts and squirrels, but not in bats, whereas surfactant protein B did not change in any species. Phosphatidylcholine (PC)16:0/16:0 is highly variable between mammals and is not the major PL in the wombat, dunnart, shrew, or Tasmanian devil. An inverse relationship exists between PC16:0/16:0 and two of the major fluidizing components, PC16:0/16:1 and PC16:0/14:0. The PL molsp profile of an animal species is not determined by phylogeny or thermal behavior. We conclude that there is no single PL molsp composition that functions optimally in all mammals; rather, surfactant from each animal is unique and tailored to the biology of that animal.     

Comparison of the respiratory transition at birth or hatching in viviparous and oviparous amniote vertebrates

Regardless of the mode of reproduction, three things must occur at birth or hatching in amniote vertebrates: initiation of breathing, pulmonary fluid elimination and reabsorption, and adequate perfusion of pulmonary circulation. Although data on these events are few, there appears to be no fundamental difference in them that can be associated with the oviparity to viviparity transition. There are, however, differences in the timing of these events in oviparous and viviparous amniotes. The transition to neonatal respiration tends to be very quick in viviparous species because the vascular support for oxygen uptake provided by the mother is rapidly disassociated from the mechanism for uptake by the embryo. By contrast, hatching often is a slow process, taking 24 h or more in some species, as chorioallantoic blood flow slowly gives way to clearing of the lungs and pulmonary gas exchange. Little is known of the mechanisms of pulmonary fluid elimination and reabsorption or lung inflation in reptiles, but the cellular structures and surfactant systems are similar in all amniote vertebrates. Nevertheless, there are differences, particularly of timing and maturation of various systems, but there has been no exploration of the functional (or phylogenetic) bases of these differences. Perfusion of the neonatal pulmonary system to support respiration in reptiles remains to be investigated. In mammals and birds, closure of the ductus arteriosus is important, but the role played by the ductus arterioisus in reptilian hatching or birth is not known.     

The composition and function of the pulmonary surfactant system during metamorphosis in the tiger salamander Ambystoma tigrinum

Mammalian lungs secrete a mixture of surface-active lipids (surfactant), which greatly reduces the surface tension of the fluid coating the inner lung surface, thereby reducing the risk of collapse upon deflation and increasing compliance upon inflation. During foetal lung maturation, these lipids become enriched in the primary and active ingredient, a disaturated phopholipid. However, disaturated phospholipids exist in their inactive gellike form at temperatures below 37°C and thus are inappropriate for controlling surface tension in the lungs of many ectotherms. We examined the development of the composition and function of the surfactant system of the tiger salamander (Ambystoma tigrinum) during metamorphosis from the fully aquatic larva (termed stage I) through an intermediate air-breathing larval form (stage IV) to the terrestrial adult (stage VII). Biochemical analysis of lung washings from these three life stages revealed a decrease in the percentage of disaturated phospholipid per total phospholipid (23.03 versus 15.92%) with lung maturity. The relative cholesterol content remained constant. The increased level of phospholipid saturation in the fully aquatic larvae may reflect their generally higher body temperature and the higher external hydrostatic compression forces exerted on the lungs, compared to the terrestrial adults. Opening pressure (pressure required for initial lung opening) prior to lavage decreased from larval to adult salamanders (7.96 versus 4.69 cm H₂O), indicating a decrease in resistance to opening with lung development. Opening pressure increased after lavage in older aquatic (stage IV) larvae (5.36 versus 9.80 cm H₂O) and in the adults (4.69 versus 7.65 cm H₂O), indicating that the surfactant system in salamanders may have an antiglue function which prevents apposing epithelial surfaces from adhering together.Abbreviations bm body mass - Chol cholesterol - DSP disaturated phospholipid - PC phosphatidylcholine - PL phospholipid - postlav postlavage - prelav prelavage - P-V pressure-volume - RH relative humidity - T_b body temperature - USP unsaturated phospholipid - WL wet lung mass     

Quantification of surfactant phospholipids in the dog lung

We quantified total phospholipid (PL), total and disaturated phosphatidylcholine (PC and DSPC), phosphatidylglycerol (PG), and total protein in alveolar washings and lung tissue in 22 dog lungs. Quantitative recovery of alveolar material and assessment of its possible contamination by blood lipids were important determinants of methodology. To remove blood, the vessels of half the lungs were perfused with a fluorocarbon emulsion before lavage. The volume of blood removed by perfusion and the quantity and fatty acid patterns of its whole blood and plasma PL and PC were determined. Washings of unperfused lungs contained means of 21% more PL and 24% more PC than those of perfused lungs. Although this excess could be accounted for by the PL and PC in pulmonary blood, the hemoglobin and total protein content of washings and their PC fatty acid patterns indicated that blood lipids were not a major source of the excess lipid in washings of unperfused lungs. Using more recent morphometric estimates rather than the indirect ones previously used by others, the quantity of alveolar DSPC (1 mg/g lung) is calculated to be 1.8 times the amount necessary to form a packed monolayer on the internal surface of the lung at functional residual capacity.     

Conservation of Surfactant Protein A: Evidence for a Single Origin for Vertebrate Pulmonary Surfactant

Surface tension is reduced at the air–liquid interface in the lung by a mixture of lipids and proteins termed pulmonary surfactant. This study is the first to provide evidence for the presence of a surfactant-specific protein (Surfactant Protein A—SP-A) in the gas-holding structures of representatives of all the major vertebrate groups. Western blot analysis demonstrated cross-reactivity between an antihuman SP-A antibody and material lavaged from lungs or swimbladders of members from all vertebrate groups. Immunocytochemistry localized this SP-A–like protein to the air spaces of lungs from the actinopterygiian fish and lungfish. Northern blot analysis indicated that regions of the mouse SP-A cDNA sequence are complementary to lung mRNA from all species examined. The presence of an SP-A–like protein and SP-A mRNA in members of all the major vertebrate groups implies that the surfactant system had a single evolutionary origin in the vertebrates. Moreover, the evolution of the surfactant system must have been a prerequisite for the evolution of airbreathing. The presence of SP-A in the goldfish swimbladder demonstrates a role for the surfactant system in an organ that is no longer used for airbreathing. Received: 5 March 1997 / Accepted: 14 June 1997     

The role of extrinsic and intrinsic factors in the evolution of the control of pulmonary surfactant maturation during development in the amniotes

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar Type II cells. It reduces alveolar surface tension and hence the work of breathing. Despite the tremendous diversity of lung structures amongst the vertebrates, the composition of surfactant is highly conserved. Conserved elements of the surfactant system amongst distantly related species are likely to be crucial factors for successful lung development. Understanding the mechanisms by which the surfactant system becomes operational in animals with dramatically different birthing strategies and in distantly related species will provide important information about the role of the surfactant system in the commencement of air breathing and the processes regulating surfactant maturation and secretion. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones, and autonomic neurotransmitters. Here we review the mechanisms controlling the maturation of surfactant production, including birthing strategy, phylogeny, lung structure, and posthatching environment. Using four species of egg-laying amniote (chicken, dragon lizard, sea turtle, and crocodile) previously described in detail and the large amount of information available for mammals, we examine the hypothesis that the control of surfactant production is dependent on glucocorticoids (dexamethasone [Dex]), thyroid hormones (T3), and autonomic neurotransmitters (epinephrine and carbachol). We also examine whether the overall intrinsic pattern of the control of surfactant maturation is conserved throughout the vertebrate radiation and then how the environment (extrinsic factors) may account for the observed differences in the patterns of development. We also discuss the utility of a coculture system of embryonic Type II cells and fibroblasts to determine the evolutionary pattern behind the control of surfactant and to demonstrate that the surfactant system matures under multihormonal control. We demonstrate that Dex and T3 are stimulators of surfactant production during embryonic development, but they lose their efficacy closer to hatching or birth. Epinephrine stimulates surfactant secretion beyond 75% of development and also after hatching or birth. Carbachol stimulates surfactant secretion in the bearded dragon and saltwater crocodile but not in the sea turtle, chicken, or mammals. It is likely that the differences in control of surfactant development are likely to be primarily related to metabolic activity and the duration of incubation (i.e., the "speed" of development). Moreover, the hormones examined appear important in promoting development and therefore appear conserved within the amniotes. However, the autonomic neurotransmitters induced different responses in different species. Hence, some factors are crucial for the proper maturation of the surfactant system, whereas others vary throughout evolution without being detrimental to the overall function of the system.     

Control of pulmonary surfactant secretion: an evolutionary perspective

Pulmonary surfactant, a mixture consisting of phospholipids (PL) and proteins, is secreted by type II cells in the lungs of all air-breathing vertebrates. Virtually nothing is known about the factors that control the secretion of pulmonary surfactant in nonmammalian vertebrates. With the use of type II cell cultures from Australian lungfish, North American bullfrogs, and fat-tailed dunnarts, we describe the autonomic regulation of surfactant secretion among the vertebrates. ACh, but not epinephrine (Epi), stimulated total PL and disaturated PL (DSP) secretion from type II cells isolated from Australian lungfish. Both Epi and ACh stimulated PL and DSP secretion from type II cells of bullfrogs and fat-tailed dunnarts. Neither Epi nor ACh affected the secretion of cholesterol from type II cell cultures of bullfrogs or dunnarts. Pulmonary surfactant secretion may be predominantly controlled by the autonomic nervous system in nonmammalian vertebrates. The parasympathetic nervous system may predominate at lower body temperatures, stimulating surfactant secretion without elevating metabolic rate. Adrenergic influences on the surfactant system may have developed subsequent to the radiation of the tetrapods. Furthermore, ventilatory influences on the surfactant system may have arisen at the time of the evolution of the mammalian bronchoalveolar lung. Further studies using other carefully chosen species from each of the vertebrate groups are required to confirm this hypothesis.     

Effect of the main components of blood on pulmonary surfactant

The effect of the main blood constituents on surface activity of substrates containing pulmonary surfactant has been investigated. Mixing of hemolysate, serum, albumin, and fibrinogen with lung extracts and washings, their application in the form of a monolayer or administration into the hypophase of the washing monolayer raised the surface tension (ST) of these substrates. Hemoglobin, serum lipids and cholesterol exerted an opposite action. The contact of all above blood constituents, that elicited varied effects on the ST of the medium containing the surfactant with pulmonary vesicles led to an increase in their stability coefficient determined according to the method of Pattle.     

Evolution of the turtle bauplan: the topological relationship of the scapula relative to the ribcage

The turtle shell and the relationship of the shoulder girdle inside or ‘deep’ to the ribcage have puzzled neontologists and developmental biologists for more than a century. Recent developmental and fossil data indicate that the shoulder girdle indeed lies inside the shell, but anterior to the ribcage. Developmental biologists compare this orientation to that found in the model organisms mice and chickens, whose scapula lies laterally on top of the ribcage. We analyse the topological relationship of the shoulder girdle relative to the ribcage within a broader phylogenetic context and determine that the condition found in turtles is also found in amphibians, monotreme mammals and lepidosaurs. A vertical scapula anterior to the thoracic ribcage is therefore inferred to be the basal amniote condition and indicates that the condition found in therian mammals and archosaurs (which includes both developmental model organisms: chickens and mice) is derived and not appropriate for studying the developmental origin of the turtle shell. Instead, among amniotes, either monotreme mammals or lepidosaurs should be used.     

Dexamethasone and epinephrine stimulate surfactant secretion in type II cells of embryonic chickens

Pulmonary surfactant (PS), a mixture of phospholipids and proteins secreted by alveolar type II cells, functions to reduce the surface tension in the lungs of all air-breathing vertebrates. Here we examine the control of PS during lung development in a homeothermic egg-laying vertebrate. In mammals, glucocorticoids and autonomic neurotransmitters contribute to the maturation of the surfactant system. We examined whether dexamethasone, epinephrine, and carbamylcholine hydrochloride (agonist for acetylcholine) increased the amount of PS secreted from cultured type II cells of the developing chicken lung. In particular, we wanted to establish whether dexamethasone would increase PS secretion through a process involving lung fibroblasts. We isolated and cocultured type II cells and lung fibroblasts from chickens after 16, 18, and 20 days of incubation and from hatchlings (day 21). Epinephrine stimulated phosphatidylcholine (PC) secretion at all stages, whereas dexamethasone stimulated secretion of PC at days 16 and 18. Carbamylcholine hydrochloride had no effect at any stage. This is the first study to establish the existence of similar cellular pathways regulating the development of surfactant in chickens and eutherian mammals, despite the vastly different birthing strategies and lung structure and function.     

Isolation and Characterization of Proteins Associated with the Lung Surfactant System

Rabbit lung washings and purified lung surfactant were delipidated without precipitation or loss of protein. This enabled effective study of the proteins by electrophoretic and immunoelectrophoretlc techniques. The lung washings contained secretory immunoglobulin A and several serum proteins. The protein composition of purified lung surfactant was the same as the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with surfactant phospholipids obtained by alveolar lavage with isotonic saline.     

Isolation and characterization of proteins associated with the lung surfactant system

Rabbit lung washings and purified lung surfactant were delipidated without precipitation or loss of protein. This enabled effective study of the proteins by electrophoretic and immunoelectrophoretic techniques. The lung washings contained secretory immunoglobulin A and several serum proteins. The protein composition of purified lung surfactant was the same as the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with surfactant phospholipids obtained by alveolar lavage with isotonic saline.