ANNALS OF B[OMEDICAL ENGINEER[NG 3, 119--159 (1975)

Pulmonary Surfactant: A Surface Chemistry Viewpoint R. H. NOTTER Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 AND

P. E. MORROW Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 Received April 16, 1975 In this paper, we attempt to review and evaluate p u l m o n a r y surfactant research from what is essentially a surface chemistry viewpoint. T h e physiological importance of the s y s t e m underlies the discussion, but this is coupled with the surface chemical consideration of p u l m o n a r y surfactant as a complex multicomponent s y s t e m of soluble and insoluble surfactants at a liquid-air interface. T h e s y s t e m in vivo is at a constant temperature (37~ and an essentially constant bulk pressure, but is subject to large variations in surface cycling frequency and in m a x i m u m and m i n i m u m surface area. In this context, then, the p r e s e n t level of knowledge of the s y s t e m is discussed, and areas of uncertainty or conflict which require further study are isolated. In addition, experimental or analytical approaches useful for future surface studies are given w h e r e v e r possible.

I. INTRODUCTION The human lungs are composed of about 300 million tiny air sacs (alveoli) which provide the huge surface area required for sufficient pulmonary gas exchange. The importance of the surface tension of the liquid-air interface in the alveoli to pulmonary mechanics has been known since the pioneering work of yon Neergaard in 1929. However, the existence of surface-active molecules in the alveolar lining layer, and the role that they play in controlling alveolar surface tension, was largely unsuspected for the next quarter-century. It is now well known that the lungs of mammals contain a complex system of surfactant molecules which act, at least in part, to stabilize the lung during respiration. The existence of such materials in the alveolar lining layer was first given quantitative verification in 1955 by the experiments of Pattle on air bubbles squeezed from lung slices. By relating the lifetime or "stability" of these bubbles to surface tension effects, Pattle was able to show that there was material in the lung which significantly lowered alveolar surface tension. Pattle's work was extended by Clements (1957), who studied the effect of minced lung extracts on surface tension in a modified Wilhelmy surface balance. Clements (1957) was the first pulmonary researcher to make use of this kind of surface balance, the principles of which had been elucidated by the premier surface chemist Irving Langmuir forty years before. Essentially, this apparatus consists of a hydrophobic trough, usually teflon or plexiglas, which is 119 Copyright 9 1975 by AcademicPress, Inc. All rights of reproduction in any form reserved.

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filled with a liquid subphase. A surfactant film is spread on the surface of this subphase, and is then compressed by means of hydrophobic barriers which lie in the surface. During this compression, the surface tension is monitored by means of a thin slide (e.g., platinum) that is dipped in the surface. By this method, curves of surface pressure versus surface area (~--A curves) can be obtained that characterize the effectiveness of the given surfactant. The surface pressure ~- is traditionally defined as 7r = o-0 -- or, where o-0 is the surface tension of the pure liquid subphase, and o- is the surface tension when a surfactant monolayer is spread on the surface. The surface pressure is, then, the surface tension lowering generated by the surfactant. For experiments with a water or saline subphase, o-0 is approximately 72 dyn/cm at room temperature, and the surface pressure can thus vary from 0 to 72 dyn/cm. The most meaningful way to express the surface area A is in terms of the area available, on the average, to a single surfactant molecule on the surface, and is typically expressed in units of square Angstroms per molecule. This kind of description of surface area is valid for pure component films of insoluble surfactants and also for certain types of well-defined mixed films. However, for complex mixed films where the exact surfactant concentrations or degree of mixing in the film is unknown, A is often expressed in the more qualitative units of the percentage of the original trough area at a given Point during film compression or expansion. Since the initial pioneering experiments of Pattie (1955) and Clements (1957), literally hundreds of researchers have undertaken studies to elucidate the source, composition, and mechanism of action of the pulmonary surfactant system. Several review articles describing various aspects of this work are available, including those of Pattle (1965), Clements and Tierney (1965), Clements (1970), Morgan (I971), and Tierney (1974). Moreover, also available are an excellent research text by Scarpelli (1968) and a conference proceedings edited by Villee et al. (1973). However, these reviews have all concentrated primarily on the more direct biochemical or physiological aspects of pulmonary surfactant research. The importance of such research is undeniable, and it has resulted in an increased understanding of the mechanics of breathing and has led to several significant clinical advances in the treatment of respiratory distress in infants. Nonetheless, if one considers the general field of pulmonary surfactant research, it becomes clear that there remain numerous areas where conflicting research results, or a lack of either relevant experimental data or theoretical understanding, severely hampers further progress. This situation has arisen at least in part from a lack of knowledge concerning the fundamental interfacial mechanics of lung surfactant, and is responsible for the emphasis of the present paper. Thus, although several physiological considerations will be reviewed and discussed, we concentrate here primarily on a critical review of previous work that relates to the surface chemistry of pulmonary surfactant, and this is coupled with suggested areas where further interfacial studies would seem to be of particular benefit. The majority of the experimental studies with pulmonary surfactant have by necessity been in vitro, and they can be conveniently divided into several classifications which will be discussed in turn below. However, as noted above, a

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certain amount of the previous work on the surface properties of pulmonary surfactant is conflicting, and there are several areas of disagreement in the literature. Moreover, there are still other areas that have not yet received more than cursory experimental investigation. Consequently, the following discussion first concentrates on the general areas of agreement concerning the major features of the pulmonary surfactant system, and this is followed by a delineation of several specific areas where significant questions remain.

II. CHARACTERISTICS OF SURFACE TENSION LOWERING IN VITRO

A. General Areas o f Agreement for the Complete Surfactant System Most in vitro studies of the surface properties of films of surfactant washed or extracted from mammalian lungs have been carried out by spreading the surfactant in a Wilhelmy trough and determining the surface tension while compressing or expanding the surface film. Numerous studies of this kind have been carried out since 1958, and typical examples are the investigations of Lempert and Macklem (1971), Scarpelli et al. (1965), and Mendenhall et al. (1967). As discussed later, the extraction and preparation procedures used for such in vitro studies vary greatly and it is difficult to draw quantitative conclusions from them. Nonetheless, all lung films of this type have been shown to exhibit two characteristics which differentiate them from most "simple" surfactants. First, these films exhibit a large, reproducible hysteresis (i.e., a difference in film behavior on expansion from that observed on compression) when they are cycled on an electrolytic subphase in a surface balance, and second, the surface pressure or surface tension lowering attained at maximum compression is extremely high, of the order of 60-70 dyn/cm based on the o-0 of water. This kind of behavior has prompted many researchers to conceive of the pulmonary surfactant system in vivo as a monomolecular film at the liquid-air alveolar interface which acts to cause large or inflated alveoli to have a high surface tension ( - 5 0 dyn/cm) 1 while causing small or deflated alveoli to have a very low surface tension ( - 1-10 dyn/cm). Although this description may be somewhat simplistic, it does point out that the pulmonary surfactant system acts to help stabilize the alveolar structure of the lung. If the surfactant system were not present and the surface tension of all the different sized alveoli were constant, the Law of Young and LaPlace (~p = 2o-/R) would predict a higher pressure to open and maintain the small alveoli. Thus, on inspiration, only the larger alveoli would expand, and the lung would essentially be composed of a number of larger air sacs with the smaller alveoli in a collapsed or deflated state. In addition to explaining the lack of alveolar collapse (atelectasis) in the normal lung, several diseases have been linked to an alteration or deficiency of the pulmonary surfactant system. Chief among these is hyaline membrane disease, which strikes thousands of newborn infants each year. This disease, also i T h e maximum surface tension of 50 dyn/cm is due to the fact that lung extract films contain soluble as well as insoluble surfactants. It is these soluble or partially soluble surfactants which give the system a maximum surface tension of about 50 dyn/cm rather than the 72 dyn/cm of a pure water system.

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known as the respiratory distress syndrome, attacks premature infants and is characterized by pronounced atelectasis, decreased lung compliance, and by lung extract films which exhibit a decreased surface activity (Avery and Mead, 1959) when studied in vitro. In fact, the relationship of this disease to the pulmonary surfactant system is the central theme of the book of Villee et al. (1973). Some progress has been made recently in the treatment of hyaline membrane disease, primarily by the postnatal application of "positive end expiratory pressure" respiration, and by the prenatal injection of glucocorticoids into the mother before a premature birth, as detailed by Liggins and Howie (1972) and Avery et al. (1973). These exciting advances have had a good deal of clinical impact. Nonetheless, a greater knowledge of the fundamental surface behavior of the pulmonary surfactant system should aid in the determination of the underlying cause of hyaline membrane disease and perhaps suggest even more effective measures for its treatment and prevention. This kind of information may also be needed for the effective treatment of more general pathologic conditions such as pulmonary edema, which has also been linked to a deficiency, alteration, or breakdown of the lung surfactant system (Pattie, 1958), as discussed later. The rather satisfying but simplistic picture of the pulmonary surfactant system as a monomolecular film being compressed and expanded in the alveoli during the breathing cycle has been modified in the light of recent work. Among others, the biochemical studies of Frosolono et al. (1970), King and Clements (1972b), and Galdston, Shah, and Shinowara (1969) have shown quantitatively that many chemical species are present in the alveolar lining layer. This material includes saturated and unsaturated phospholipids, cholesterol, triglycerides, and various proteins, although there is a question about the presence of proteins (e.g., Clements, 1970; Scarpelli and Colacicco, 1970), as will be discussed later. Some of these surfactant molecules are partially soluble in water and are undoubtedly present not only at the liquid-air alveolar interface, but also in the underlying subphase as well. Moreover, work by Clements et al. (1970) on a number of mammalian species, including man, indicates that the lungs contain more surfactant material than necessary to cover the alveolar surface area with a monomolecular film at end expiration. Finally, the system is further complicated by the fact that in the living organism, these surfactants are being depleted and replenished by metabolic processes over a period of days and perhaps hours. It is clear that a really thorough study of the pulmonary surfactant system must include consideration of the film subphase and the interaction of subphase molecules with the surface, as well as the interaction of different film molecules with each other. This kind of information cannot be obtained solely from inherently qualitative studies of lung extract films. B. General Areas of Agreement for Films of Pulmonary Surfactant Components Another class of in vitro surface balance studies that have been carried out in the last decade has centered on films of highly purified, synthetic, 1,2 diacyl phosphatidylcholines (lecithins). Specifically, the compound most extensively studied has been 1,2 dipalmitoyl phosphatidylcholine (often called dipalmitoyl

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lecithin and abbreviated DPL) which is the primary phospholipid constituent of pulmonary surfactant (e.g., Frosolono et al., 1970; King and Clements, 1972b). The advantage of this kind of pure component study is that the surface film is well defined, and quantitative conclusions can be drawn concerning the behavior of a given component of lung surfactant. Several careful studies of the surface pressure-area (~r-A) behavior of D P L films have been made, including those of Vilallonga (1968), Phillips and Chapman (1968), Watkins (1968), Shah and Schulman (1965, 1967a), Galdston and Shah (1967), Berg (1971), Hayashi et al. (1972), and Tabak and Notter (1975). In addition to studies with pure D P L films, a few investigations of the ~--A behavior of !,2 dipalmitoyl phosphatidylethanolamine (DPPE) films have been performed (Standish and Pethica, 1968; Hayashi et al., 1972; Tabak and Notter, 1975). This species, like DPL, is present in pulmonary surfactant in significant concentrations (Morgan et al., 1965; Triiuble et al., 1974). Also, Van Deenan et al. (1962), Ghosh et al. (1971), and Tinoco and Mclntosh (1970), among others, have investigated the rr-A behavior of films of several phosphatidylcholines and phosphatidylethanolamines with fatty acid chains other than palmitic acid. Most of these studies, however, concerned film compression only and concentrated on the characteristics of the compression curve prior to film collapse. In general, two major features are apparent from studies of pure component phospholipid films, especially those concerned with DPL. First, films of D P L are able to sustain extremely high surface pressures of the order of 70 dyn/cm at maximum film compression on a water or saline subphase. This corresponds to surface tensions of only about 2 dyn/cm or less and is really a dramatic surface tension lowering. By comparison, more "normal" surfactants such as long-chain fatty acids and alcohols are only able to lower the surface tension of water to about 30 dyn/cm at maximum compression. Not all of the saturated phospholipids are as effective as D P L in lowering surface tension. For example, D P P E films are found to reach a collapse surface pressure of about 55 dyn/cm (Tabak and Notter, 1975), which is some 15 dyn/cm lower than that found for DPL. However, other pulmonary phospholipids such as sphingomyelin have also been found to generate collapse pressures of the order of 70 dyn/cm (Colacicco et al., 1974; Tabak and Notter, 1975). Consequently, it is now widely accepted that it is the saturated phospholipids such as DPL, either by themselves or in combination with other components of the system, that give pulmonary surfactant its extraordinary surface tension lowering effect. A second feature of these pure component saturated phospholipid studies has equally important consequences. A few studies with D P L or D P P E (Galdston and Shah, 1967; Watkins, 1968; Berg, 1971; Tabak and Notter, 1975) have been carried out during both compression and expansion, and can thus be compared with similar studies of the complete pulmonary surfactant system (e.g., Scarpelli et al., 1965; Lempert and Macklem, 1971). As stated earlier, one of the primary characteristics of the ~--A curves of lung extracts or washings is the large r e p r o d u c i b l e hysteresis observed on compression-expansion. The most attractive explanation for this hysteresis is that surfactant molecules are somehow expelled from the surface monolayer during film compression. For example, sur-

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factant molecules may be squeezed out from the surface into regions of multilayered film, or they may be pushed into the underlying bulk solution as micelles. At any rate, the net effect of molecules being squeezed from the surface film on compression is to cause the film to exhibit different ~--A characteristics when it is allowed to expand. Moreover, the fact that this hysteresis is reproducible for lung extract films on successive cycles of the surface appears to indicate that the "squeezed out" surfactant molecules are able to re-enter the surface film during the immediately following expansion. This re-entry is an extremely important characteristic, because otherwise surfactant would be continuously depleted at the alveolar-air interface. Moreover, the surface entry property is also important in terms of fresh surfactant being able to penetrate and enter the interface after being produced by alveolar cells. It is now clear that single-component films of D P L and DPPE do not exhibit the kind of reproducible hysteresis that is observed for the complete pulmonary surfactant system. These pure component phospholipid films do exhibit an initially large hysteresis, but rather than being reproducible, it decreases continuously with successive cycles of the film (e.g., Galdston and Shah, 1967; Berg, 1971; Tabak and Notter, 1975). This indicates that molecules of D P L and DPPE will apparently not easily respread if squeezed out of the surface. This point is of basic importance, and it may be postulated that the role of some of the other molecular species in pulmonary surfactant may be to aid the saturated phospholipid molecules to re-enter the surface if they are squeezed out during film compression. This explanation finds support among researchers today, but the specific components of pulmonary surfactant that are responsible for the effect are not yet known. The preceding discussion has been intended to describe the major approaches and areas of agreement with regard to in vitro surface pressure research on pulmonary surfactant. Now attention is turned to areas where conflicting results or lack of knowledge point out the need for further research. C. General Areas o f Conflict or Lack o f Knowledge

The review of these areas will be broken down into four areas of research which will be discussed in turn. 1. Composition and surface behavior o f the complete system. One of the basic problems in studying films washed or extracted from mammalian lungs is the determination of the chemical species present in the film and their concentration. Several comprehensive studies have been carried out on the composition of lung surfactant extracted from different species by varying techniques (Frosolono et al., 1970; Pawlowski et al., 1971; King and Clements, 1972a, 1972b; King et al., 1973; Galdston et al., 1969; Hurst et al., 1973; Scarpelli et al., 1967; Abrams, 1966; Morgan et al., 1965; Tr~iuble et al., 1974). These studies generally agree on the high concentration of phospholipids in lung extracts obtained from mincing or homogenation of lung tissue and from lung alveolar washings obtained by perfusing the lung with saline. In particular, all these studies have found large amounts of D P L in lung surfactant. Also, the studies of King and Clements (1972b), Frosolono et al. (1970), Pawlowski et al. (1971),

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and Morgan et al. (1965) have found varying but significant percentages of 14:0 (14 carbon atoms, no double bonds), 16: 1, 18:0, and 18:1 phosphatidylcholines in lung surfactant. With regard to other phospholipid components, there are again variations in the relative amounts found in different studies. However, it does appear that sphingomyelins, phosphatidylethanolamines, and lysolecithins are present, as well as carbohydrates, triglycerides, and cholesterol. For more information regarding the composition of pulmonary surfactant, the reader is referred to the books by Scarpelli (1968) and Villee et al. (1973), as well as to a recent review of lung biochemistry by Tierney (1974). The largest area of disagreement concerning the composition of lung surfactant is the presence of proteins. For example, Galdston, Shah, and Shinowara (1969), Frosolono et al. (1970), King and Clements (1972a, 1972b), Tfiiuble et al. (1974), King et al. (1973), and Abrams (1966) have found proteins or lipoprotein complexes in lung surfactant, while Scarpelli et al. (1967, 1970), Scarpelli and Colacicco (1970), Steim et al. (1969), and Hurst et al. (1973) feel that proteins are either not present or not associated as lipoprotein complexes in pulmonary surfactant in vivo. One important reason for these conflicting opinions is that protein contamination from ceils or from blood is always possible in any extraction procedure for lung surfactant. The question of the in vivo presence of proteins in lung surfactant is a difficult one, and further sophisticated biochemical studies may be required to resolve this dispute completely, as addressed by Clements (1970). However, the recent studies of King et al. (1973), Colacicco et al. (1973), and Scarpelli et al. (1973) have indicated the presence of a nonserum protein in lung extracts. The latter two studies are particularly significant because of the previous views of these investigators (e.g., Scarpelli and Colacicco, 1970). Thus, biochemical evidence for the importance of protein in lung surfactant in vivo seems to be mounting. In addition to biochemical studies, surface chemical investigations of welldefined interactions of specific proteins with lung surfactant and its components should prove helpful in determining the necessity (or lack of it) for proteins in the mechanics of the pulmonary surfactant system. Some studies involving the interactions of various proteins with phospholipid monolayers have been done. For example, the penetration of various soluble proteins such as albumin into phosphatidyl serine monolayers has been studied (Kimelberg and Papahadjopoulos, 1971). In addition, protein penetration and interaction with egg lecithin was studied by Colacicco (1969), who also discussed the general use of monolayer techniques to study lipid-protein interactions. Bovine albumin-cholesterol interactions have also been studied (Vilallonga et al., 1967), as well as the effects of cytochrome C and albumin on surface balance measurements with D P L (Turner et al., 1974). However, because of the complexities inherent in working with proteins and protein films, it is difficult to interpret the results of such studies in an unambiguous manner. Moreover, as discussed by Pearson and Alexander (1968) and by Pearson (1968), a description of protein-surfactant interactions may in general require the simultaneous measurement of surface pressure, potential, and viscosity. Studies of this type involving the interactions of lung surfactant and its components with the apparent nonserum protein recently isolated by King et al. (1973) would seem to be particularly relevant.

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In addition to clarifying the importance of protein in lung surfactant as mentioned above, another reason to investigate specific protein-lung surfactant interactions concerns the effect of the plasma protein fibrinogen on pulmonary surfactant films. Taylor and Abrams (1966) showed that fibrinogen causes a significant decrease in both the surface pressure and hysteresis of films composed of lipoprotein extracted from mammalian lungs. Unfortunately, the study of Taylor and Abrams (1966) is limited in terms of experimental data, and contains no body temperature measurements or evaluations of the effect of fibrinogen on any of the components of pulmonary surfactant. Such studies of the effects of serum components on pulmonary surfactant appear to have relevance, not only for respiratory distress in infants, but also in the general problems of pulmonary alveolar proteinosis and edema. Moreover, the investigation of protein-phospholipid interactions has an overall relevance to the problem of the structure and mechanics of cell membranes. Aside from questions about the presence and effects of proteins, another area of conflict concerning lung surfactant is with regard to its temperature dependence. In particular, Lempert and Macklem (1971) found a significant decrease in the maximum surface pressure (increase in the minimum surface tension) achieved by lung alveolar wash films at body temperature as compared to room temperature results. However, other investigators, most recently King and Clements (1972c), have not found a significant change in the properties of surface tension lowering of lung surfactant at body temperature. This point has obvious importance in terms of extrapolation to the living system. One approach to determining whether or not lung surfactant films are indeed dependent on temperature is to examine the temperature dependence of the major components of lung surfactant in both pure and mixed films. This kind of study would then allow a firm basis for any temperature effects observed for the overall pulmonary surfactant system, and some work of this kind is discussed later. Not only are temperature effects critical in terms of trying to relate in vitro results to the living system; nonequilibrium effects are also of importance. The lung is a dynamic system, and it is indeed a valid question what relevance surface studies carried out at 1/2 to 1/4 cycles per minute have to the lung, where surfactant is being compressed and expanded at the rate of 15 cycles per minute. This point is discussed by Bienkowski and Skolnick (1972), who show that the hysteresis observed in pulmonary surfactant films is a function of cycling speed. This important effect should be the object of further studies that involve the cycling rate dependence, not only of pulmonary surfactant, but also of its components during both compression and expansion. Moreover, such dynamic cycling rate dependence studies should be done in conjunction with intermittent compression experiments which define the equilibrium state of the system. In this way, the dynamic behavior of the system can be characterized in terms of its departure from equilibrium. Although dynamic compression rate effects are undeniably important, it should also be noted that the compression ratio may be another significant parameter for the actual living system. This parameter has received little attention from surface chemistry researchers. Specifically, most in vitro surface bal-

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ance experiments have been carried out with compression ratios of 5 : 1 or higher (for example, a 5:1 ratio corresponds to compression to 20% of the initial trough area). However, the in vivo surface area change in the lung alveoli during the breathing cycle is almost certainly not as large as a 5:1 change. For example, the morphological study of Storey and Staub (1962) on frozen cat lungs estimates an increase of only about 70% in alveolar surface area between functional residual capacity and large lung volumes. Moreover, during normal breathing this change is even smaller, perhaps of the order of 25% (Weibel, 1964). Thus, although surface area changes are apparently present in vivo, the relevant magnitudes of these changes have not typically been reflected in surface balance studies in vitro. Consequently, in vitro surface balance experiments investigating film behavior for compression ratios of 2 : 1 or less, but at physiological cycling speeds of the order of 10 cycle/min, would seem to have definite relevance. 2. Single-component films of different molecular species in pulmonary surfactant. The foregoing discussion points out major areas of conflict or lack of knowledge about the in vitro surface behavior of the complete pulmonary surfactant system, and attention is now turned to pure films of its various components. As noted in Section liB above, there have been a number of careful, surface chemical investigations of the 7r-A behavior of some of the phospholipid components of lung surfactant, particularly DPL. Nonetheless, there are still significant areas where physiologically relevant data are not available. In particular, it would seem that not enough attention has been paid in these pure-component studies to temperature and cycling speed effects, or to the behavior of the films during both compression and expansion. Instead, many studies (Vilallonga, 1968; Phillips and Chapman, 1968; Shah and Schulman, 1965, 1967a; Standish and Pethica, 1968; Hayashi et al., 1972; Van Deenen et al., 1962) have been concerned only with film behavior during compression. Moreover, because of experimental problems associated with very high surface pressures (low surface tensions), most of these studies have not considered film behavior near collapse. Also, more fundamentally, another problem is that the very high surface pressure regime must be considered a thermodynamically unstable one if the surfactant film is above its equilibrium collapse pressure. Such high pressure regimes, however, may appear stable if the experimental time scale is not too long. This is apparently the case for D P L films which collapse at about 7r = 50 dyn/cm in static measurements but which can reach 7r > 70 dyn/cm during dynamic compression. The difference in the rr-A behavior of D P L during equilibrium compression and dynamic compression (e.g., Watkins, 1968; Berg, 1971; Tabak and Notter, 1975) is an effect that may be particularly relevant for the in vivo system, and it warrants further study. Only for pure D P L films at room temperature (-23~ is there anything approaching a well-defined body of knowledge concerning compression-expansion behavior and film collapse (Berg, 1971; Galdston and Shah, 1967; Tabak and Notter, 1975; Watkins, 1968). Even at room temperature, however, little attention has been given to such considerations as D P L hysteresis and rr-A behavior when a surface excess of phospholipid is present. Specifically, there is

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evidence that DPL gives a more reproducible hysteresis when cycled under surface excess conditions (Berg, 1971; Watkins, 1968). This effect may be particularly relevant for the behavior of pulmonary surfactant in vivo, and should be investigated in detail, particularly for D P L films at body temperature. For films of the other phospholipid components of pulmonary surfactant even less is known. Phosphatidylethanolamines (including DPPE) and phosphatidylcholines other than D P L have been studied during compression (Phillips and Chapman, 1968; Standish and Pethica, 1968; Ghosh et al., 1971; Tinoco and McIntosh, 1970; Hayashi et al., 1972; Van Deenen et al., 1962; Tabak and Notter, 1975), but relatively little is known about temperature dependence, behavior during expansion, film collapse regimes, and cycling rate dependence. 3. Multicomponent films o f molecular species in pulmonary surfactant. Mixed surfactant systems are very difficult to characterize, and the degree of difficulty is directly related to the number of components present in the mixed film. Studies of the complete pulmonary surfactant system involve films that are too complex to be described in a fundamental way, and so such studies do little to identify the interactions and importance of individual components. This kind of information must instead be gained from studies involving well-defined pure and simple mixed films of pulmonary surfactant components. Some binary mixed film studies related to this kind of work have been done, particularly because of the relevance to cell membrane structure and mechanics. Specifically, the effect on surface pressure of cholesterol in binary mixed films with phosphatidylcholines or phosphatidylethanolamines has been investigated by a number of workers including Van Deenen et al. (1962), Shah and Schulman (1967a), Demel et al. (1967, 1972a, 1972b), Chapman et al. (1966, 1969), Cadenhead and Phillips (1968), Tinoco and McIntosh (1970), Ghosh et al. (1971), Ghosh and Tinoco (1972), and Lusted (1973). A good review of work in this area prior to 1970 is given by Cadenhead (1970). One result indicated by such studies on the effect of cholesterol on phospholipid films is that cholesterol can act to fluidize saturated phospholipid films at temperatures below their bulk phase liquid-crystalline transition temperatures. Also, the addition of cholesterol leads to various area effects in binary mixed films with phospholipids, and these effects are most often an apparent condensation whose magnitude decreases as surface pressure increases (e.g., Chapman et at., 1969). For the discussion here, the property of cholesterol for fluidizing certain kinds of phospholipid monolayers is perhaps the most relevant. Interestingly, this surface or two-dimensional fluidization is apparently mirrored by the bulk phase effect of cholesterol on phospholipids. Specifically, differential scanning calorimetry, NMR, and X-ray studies of cholesterol-phospholipid-water systems (Ladbrooke et al., 1968; Darke et al., 1972) have shown that cholesterol fluidizes phospholipids at temperatures below their liquid crystalline transition temperature. This is interpreted by Ladbrooke, Williams, and Chapman (1968) as being due to cholesterol causing a reduction in the chain-chain interactions between the phospholipids. Thus, the bulk phase behavior of cholesterol correlates well with the observations of Shah and Schulman (1967a) that mixed monolayers of DPL with cholesterol are in a liquid film state instead of a solid

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film state at high surface pressure, and with the finding of Joos (1970) that the surface viscosity of monolayers of distearoyl and oleoylstearoyl phosphatidylcholine is decreased by the addition of cholesterol. Since the transition temperature of D P L is 41~ (e.g., Phillips and Chapman, 1968), it may be expected that cholesterol will act to increase the fluidity of D P L films during dynamic compression-expansion at temperatures at or below body temperature, and hence that cholesterol will increase the respreadability of such films. Only a few studies of this type have yet been carried out, but the results are suggestive. In particular, Lusted (1973) has cycled multicomponent films of mixed lung phospholipids and cholesterol at 25~ and found a more reproducible hysteresis than that shown by the mixed phospholipids alone. Also, Tabak and Notter (1975) have found a similar effect of cholesterol on D P L films at body temperature (37~ Consequently, it appears likely that the presence of cholesterol in pulmonary surfactant in vivo would act to increase the respreadability of the high surface pressure generating saturated phospholipids such as DPL. However, cholesterol apparently accomplishes this increased respreadability with a concomitant decrease in the maximum surface pressure achieved by mixed DPL-cholesterol films in vitro (Tabak and Notter, 1975). The problem is not a simple one, and it is possible that the effect of some of the unsaturated phospholipids in increasing spreadability in pulmonary surfactant also may be important (Tr~iuble et al., 1974). However, more extensive dynamic cycling experiments are required before any of these effects can be characterized with certainty. In addition to cholesterol-phospholipid studies, there have been investigations of a number of other binary mixed film systems. For example, studies have been made on the surface tension characteristics of mixed films of phosphatidylcholine-di- and tri-glyceride (Finer and Phillips, 1973), phosphatidylcholinesterol (Ghosh and Tinoco, 1972; Demel et al., 1972b), and several phosphatidylcholines (Philips et al., 1970). Comprehensive reviews of this kind of work are given by Cadenhead (1970), Shah and Schulman (1968), and Shah (1970). Also included in these reviews is a description of work on the thermodynamics of surfactant mixtures, and other more recent studies involving mixed film thermodynamics are also available (e.g., see Gershfeld, 1970; Gershfeld and Pagano, 1972a, 1972b; Pagano and Gershfeld, 1972b; Eriksson, 1971; LucassenReynders, 1972, 1973a-1973c; Motomura, 1974; Motomura et al., 1974). Unfortunately, much of this previous experimental and theoretical mixed film work is not directly relevant to the pulmonary surfactant system, for several reasons. With regard to the theoretical work, one major problem is that equilibrium thermodynamic analyses are difficult to apply directly to a system undergoing dynamic compression-expansion. Moreover, even for the case of equilibrium measurements, many of the available thermodynamic treatments require experimental data that have not yet been determined for lung surfactant and its components in a meaningful, reproducible way. For example, Pagano and Gershfeld (1972a) and Gershfeld (1972) point out that the evaluation of the Gibbs excess free energy of mixing can require rather detailed knowledge of monolayer behavior in the region of large film area where the surface pressure ~-

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is very low. Such data are largely unavailable even for pure component films of the primary components of lung surfactant. In addition to the excess free energy, another parameter that may prove helpful for phospholipid-sterol films is the "percent condensation," defined by Cadenhead and Demchak (1969). In general, however, the most successful analytical procedure applied even to simple binary mixed films of phospholipids and sterols has been in terms of the deviations of the mixed films from additivity. Work of this kind is discussed and reviewed in some detail by Shah (1970) and by Cadenhead (1970). Also, Pagano and Gershfeld (1972b) and Gershfeld and Pagano (1972b) have recently pointed out some possible limitations of this kind of analysis, specifically with regard to cholesterol condensation effects. The available experimental mixed film studies also suffer from a lack of relevance to the behavior of pulmonary surfactant in the living system. In particular, almost all previous mixed film experimental studies have concentrated on film compression only, and in fact have usually not even covered the full range of the compression curve all the way to film collapse. Also, temperature effects, especially at body temperature, have received little attention. Moreover, to facilitate analysis, most of these mixed film experiments have been conducted under static or intermittent compression conditions to define the equilibrium state. However, this has not been supplemented with dynamic cycling studies. Thus, no relevant mixed film compression-expansion data are available, with the exception of the studies of Lusted (1973) on mixed phospholipid-cholesterol films, Colacicco et al. (1974) and Tabak and Notter (1975) on DPL-cholesterol films, and Watkins (1968) on mixed films with a surface excess of phosphatidylcholine or phosphatidylethanolamine. 4. In situ studies o f excised lungs. The discussion so far has been concerned with in vitro surface balance investigations of the surface tension behavior of films of pulmonary surfactant and its components, and this approach is of primary interest for the research reviewed here. However, another general approach for characterizing respiratory surface tension behavior is to consider the inflation of excised lungs in situ. Such studies are typified by the careful experiments of Bachofen et al. (1970), Horie and Hildebrandt (1971), and Fisher et al. (1970), which involve the inflation of excised lungs with liquid and with air. The difference in the pressure-volume characteristics found for the liquid and gas inflations is then used to obtain a measure of the contributions of tissue forces and surface tension forces to the mechanics of respiration. Moreover, if certain assumptions concerning lung geometry are made, it is possible to analyze the results of these in situ lung studies to obtain a measure of alveolar surface area and surface tension. This kind of procedure was first used by Radford (1954) and was modified by Brown (1957) and by Clements et al. (1958). Related studies prior to 1970 are noted by Bachofen et al. (1970). The surface tension-area characteristics found from in situ excised lung experiments show the large hysteresis and low minimum surface tension found in lung extract surface balance studies in vitro. However, the agreement between these two kinds of study is not exact. One reason for this is that the basis for the geometrical assumptions needed to analyze the in situ results is open to ques-

PULMONARY SURFACTANT

13 1

tion, particularly in terms of the assumed constancy of the relationship between lung volume and lung surface area throughout the breathing cycle (Bachofen et al., 1970). Moreover, the effect of neglecting the interfacial tension between the alveolar tissue and the liquid lining layer has been questioned recently by Bienkowski and Skolnick (1974). Nonetheless, in situ lung studies are attractive because they are one step closer than in vitro studies to the actual lung. They are mentioned here because such studies represent an alternative approach to the characterization of lung surfactant effects, and any discrepancies in alveolar surface tension behavior predicted from the two kinds of study need to be resolved. Moreover, the simultaneous consideration of pulmonary surfactant ~r-A behavior and bulk p - V behavior may yield interesting dividends. For example, Tr~iuble et al. (1974) have recently used such an approach to formulate the imaginative possibility of spontaneous opening and closing of some of the lung alveoli during respiration. D. Experimental Considerations for Surface Pressure Measurement We close this section on surface pressure with a brief discussion of experimental techniques. For further details, the reader is referred to any of several excellent texts on surface chemistry such as Gaines (1966) or Davies and Rideal (1961), or to review articles such as those of Shah (1970), Gershfeld (1972), and Cadenhead (1970). The most common problems in any surface chemistry study are the cleanliness of the equipment and the chemical purity of all the materials used, including the film subphase and spreading solvents as well as the surfactants being investigated. For the pulmonary surfactant system, such cleanliness and purity problems are increased greatly because of the complex multicomponent nature of the system. For example, proteins are present in many studies of lung extract films and such molecules require more abrasive cleaning procedures than normally used (Turner et al., 1974). Also, other sources of error present with lung extract studies, especially with regard to extract composition, are discussed by Pattle (1965) and Scarpelli (1968). Moreover, even comparatively well-defined studies with synthetic phospholipids have been questioned because of surfactant purity considerations (Watkins, 1968), and the same is true for surface potential studies, as will be discussed later (Colacicco, 1971, 1973). Finally, the kind of spreading solvent used to spread the phospholipid films may in some cases lead to apparent differences in surface behavior. For example, Cadenhead and Kellner (1974) have shown that hexane-ethanol (9: 1, v/v) leads to fewer effects in this regard than does chloroform as a spreading solvent for DPL. In addition to cleanliness and purity problems, the study of the pulmonary surfactant system poses several unique challenges to the surface chemist in terms of experimental equipment. In particular, two major problem areas involve barrier leakage or overflow in the surface balance, and contact angle hysteresis if a Wilhelmy slide is used to measure surface tensions. Measurements of the surface pressure-area characteristics of surfactant films are generally carried out in a surface balance with either a horizontal float system, as suggested by Langmuir (1917), or with a so-called Wilhelmy plate system (Harkins and Anderson,

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1937). These methods are discussed in detail, for example, by Gaines (1966). Effectively, the only major difference between the two techniques is that in the former the surface pressure is measured from the force on a horizontal float lying across the surface, while in the latter it is calculated from the force on a thin slide dipped into the surface. In both cases, the surfactant film is compressed in the surface balance by a barrier lying in the surface. The important point here is that phospholipid and pulmonary surfactant films can reach extremely low surface tensions (< 2 dyn/cm), and this causes the liquid subphase to be able to "wet" even the very hydrophobic materials, such as teflon, out of which most surface balances are constructed. Consequently, trough overflow or barrier leakage problems can arise. In fact, such overflow problems are largely responsible for the lack of high surface pressure data available in the binary mixed film studies discussed previously. Many of these experiments were carried out in surface balances designed to accommodate a horizontal float for surface tension measurement, and overflow problems are especially prevalent in such systems. Thus, to counteract overflow, many pulmonary surfactant researchers use a Wilhelmy plate method of surface tension measurement, coupled with a compression-expansion barrier that is recessed in the trough rather than one which lies across the trough surface. Such a design minimizes overflow and leakage problems but necessitates the consideration of contact angle effects because of the use of the Wilhelmy slide. One problem with the Wilhelmy slide method of surface tension measurement concerns the contact angle between the slide and the liquid surface. This angle appears in the force balance used to relate the force on the slide to surface tension (e.g., Gaines, 1966). In most applications of the Wilhelmy plate method, the contact angle is assumed to be zero, and this is equivalent to assuming that the liquid completely "wets" the slide. This is a relatively good assumption for many kinds of surfactants. Even more important, however, it can happen that the value of the contact angle may change during the course of a dynamic cycling experiment. This phenomenon is called "contact angle hysteresis," and it can result from several causes. For example, as the film is compressed and surface tension goes down, there may be better slide wetting. Thus, the contact angle can become zero even if it was not zero at the start of the compression. Also, the surfactant can actually become adsorbed on the slide surface during a compression, and this can change the slide contact angle during reexpansion of the film. One way in which contact angle effects (contact angle not equal to zero or not equal to a constant) can be minimized is to treat the surface of the Wilhelmy slide. For example, hand roughening of platinum Wilhelmy slides with fine sand paper has traditionally been used in the past to increase the wetting characteristics. However, hand roughening may not be sufficient, and it is recommended that platinum slides be roughened by sandblasting with a very small-grain sand. This has been shown to give a finely roughened, uniform surface that has good wetting characteristics (Berg, 1971). More important, the uniformity of the slide wetting is increased, and contact angle hysteresis effects are minimized. Probably the major effect of contact angle hysteresis, if it occurs in a dynamic surface tension study, is that a ~r-A curve found during film expansion returns to a higher baseline (i.e., to an apparent higher initial surface pressure) than the

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compression curve. In other words, the expansion curve will "cross-over" the compression curve and indicate an apparent new baseline for the next compression. It is now known that phospholipid films can be extremely sensitive to this kind of effect during dynamic cycling. Fortunately, treatment of the Wilhelmy slide with sandblasting (especially if applied prior to each run) seems to be sufficient to give data free from contact angle hysteresis effects for most phospholipid films. A final consideration with Wilhelmy slide measurements of surface pressure concerns the possible presence of surface tension gradients within the film during dynamic cycling. In most studies it is assumed that the surface pressure measured by the Wilhelmy slide is independent of the position or orientation of the slide in the film. However, Blank and Lee (1971) have used the simultaneous output of two Wilhelmy slides to demonstrate the existence of surface tension gradients of about 1 dyn/cm/cm in lung extract films. Such effects are obviously a function of cycling speed, and should be less prevalent in pure component or simple mixed films. However, more extensive data characterizing this phenomenon would appear pertinent. III. MEASUREMENT OF OTHER SURFACE PROPERTIES A. Surface Potential Measurements

To this point we have considered work involving the surface tension lowering characteristics of pulmonary surfactant, and this property has certainly been the object of most of the previous work with this system. However, some studies of other pulmonary surfactant surface properties have been carried out, and more work of this kind needs to be done in the future. In general, the surface potential is of particular use in studying ionic interactions in monolayers. A good discussion of the theoretical basis of surface potential measurements, and of the various kinds of experimental techniques, is given by Gaines (1966), Shah (1970), and Gershfeld (1972), among others. The conceptual basis for the existence of a surface potential in monolayer systems is the effect of the monolayer on the molecular alignment (and hence molecular dipoles) near the interface. However, the exact description of such effects is complex, and has not yet been treated in a completely rigorous way (Gershfeld, 1972). In general, the typical definition of surface potential is given as (e.g., Shah, 1970; Gaines, 1966) A V = Vy - Vo = (4~rntx]D) + qJo,

where Vo = Vf = n= D= tx = q~o=

(1)

interfacial potential without the film, interfacial potential with the film, number of molecules per cmz of film, dielectric constant, effective molecular dipole moment in vertical direction, potential due to the ionic double layer present if the monolayer is charged.

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In principle, surface potential measurements are especially useful in determining interactions in monolayers, particularly in terms of (AV/n), the average potential per molecule. For example, changes in the nature of the polar groups in a monolayer can influence the behavior of the surface potential, and the magnitude of the surface potential change can be used to indicate things such as the interaction of ions in the subphase with polar groups in the monolayer. For instance, Shah and Schulman (1965) found that calcium ions in the subsolution increased the surface potential of dicetyl phosphate monolayers by about 100 mV, and also increased the surface potential of DPL monolayers. These changes in the surface potential were then interpreted as an indication of the interaction of the calcium ions with ionic polar groups in the monolayer. However, as discussed by Colacicco (1971, 1973), the results of such surface potential measurements could be misinterpreted. For example, Colacicco discusses the case where acidic impurities present in otherwise neutral phospholipid films give rise to apparent surface potential changes with Ca ++ ions. According to Colacicco (1971, 1973), such changes denote effects due to the ionic impurities, and not the interaction of Ca ++ with the phospholipids, as proposed by Shah and Schulman (1965, 1967a, 1967b, 1968). This point is still open to question, but it does indicate that phospholipid purity considerations should be of special concern in future surface potential studies. In terms of pulmonary surfactant components, it is the phosphatidylcholines (PC) and phosphatidylethanolamines (PE) that have been most frequently studied in terms of their surface potential behavior. 2 Such studies have been carried out on PC or PE films by Shah and Schulman (1965, 1967a-1967c), Galdston and Shah (1967), Watkins (1968), Vilallonga (1968), Papahadjopoulos (1968), Standish and Pethica (1968), and Hayashi et al. (1972). The results of such studies on synthetic PC and PE film are reviewed by Cadenhead (1970), especially in terms of the variation of surface potential with film area, subphase composition, and pH. One important use of these results is trying to determine the molecular orientations present in pure component PC or PE films, in particular whether the zwitterionic headgroup of PC or PE is oriented in a parallel or vertical fashion to the surface. The question is a complex one because the orientation may be expected to depend on factors such as temperature, substrate pH, and composition, as well as on the degree of film compression. Although the answer is still not certain with regard to the most important lung phospholipids, it appears that at high surface pressure the D P L headgroup is vertically oriented to the surface (Shah and Schulman, 1967b, 1967c, 1968; Hayashi et al., 1972) while DPPE may have a more horizontal alignment (Standish and Pethica, 1968; Hayashi et al., 1972). These conclusions are also supported by bulk phase studies on phospholipid bilayers (Phillips et al., 1972). Another use of surface potential measurements is the characterizing of dipole and ionic interactions in mixed monolayers. For mixed monolayers, surface potential measurements can be an indispensable supplement to surface pressure 2 In general, measurements such as the surface potential are of primary use for well-defined systems. Thus, although some measurements of AV have been made for the complete pulmonary surfactant system (e.g., Watkins, 1%8; Blank et al., 1969), they are mainly of qualitative interest.

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measurements in terms of interpreting mixed film interactions with respect to the various pure components. As discussed by Shah and Schulman (1968) and Cadenhead and Phillips (1968), among others, there are several ways in which equilibrium mixed film (~--A) and (AV]n-A) data can be analyzed in terms of the relevant pure components. Specifically, for an ideal mixed film, or for a film of immiscible components dispersed in each other, any total property is the sum of the mole fractions of the various components in the film times the pure component properties. Thus for such a binary mixed film of components 1 and 2 at fixed surface pressure, the average area per molecule in the mixed film, for example, is given by (Gaines, 1966)

A

= xlA

1

q- x2A2.

Here xl and x2 are mole fractions, and A1 and A2 are the areas per molecule found for pure component films of 1 and 2 at the given fixed value of surface pressure or potential. Experimentally determined values of A are usually plotted, at a fixed value of ~-, as a function of composition in the mixed film. If the film is ideal, or composed of immiscible components, the additivity rule given above says that the mixed film area A will fall on a straight line between the pure component areas A~ and A2. This line is called the additivity line. Deviations from this additivity line then indicate the various types of interactions which occur in the binary mixed films. Typically, in order to interpret equilibrium mixed film data in an unambiguous way, more than one mixed film property must be characterized (Shah and Schulman, 1968). For example, a mixed film may show negative deviations from ideality in terms of the area A (i.e., an apparent condensation), which would seem to indicate specific interactions between the mixed film components. However, an alternate explanation might simply be better molecular packing in the mixed film. Consequently, deviations from ideality of other film properties, such as the surface potential or viscosity, are necessary to substantiate that the film components are undergoing ion-ion or ion-dipole interactions.

B. Surface Viscosity Measurements The often striking analogies between two-dimensional monolayer behavior and three-dimensional bulk phase behavior are pointed out by the rheology of surface films. Specifically, it is possible to define both a dilatational surface viscosity as well as a surface shear viscosity "0s, which is the parameter most generally measured experimentally. The surface shear viscosity ~0s is defined as the ratio of the surface shear stress (tangential force per unit length) to the rate of flow of the monolayer (Gaines, 1966). Several types of surface viscometers have been developed, many of which are reviewed by Davies and Rideal (1963), Gaines (1966), and Joly (1964). Of the available methods, the canal viscometers are perhaps the most extensively used. One of the best of this kind of viscometer is the "viscous-traction" canal viscometer developed by Burton and Mannheimer (1967) and improved by Mannhelmet and Schechter (1970a, 1970b). Essentially, this apparatus consists of a circular canal, formed by two concentric stainless steel cylinders, which is posi-

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tioned inside a rotating dish so that the bottom edges of the cylinders almost touch the bottom of the dish. The speed of a teflon particle on the surface of the liquid that rises between the concentric cylinders is then determined. The difference between this surface speed on a clean and a film-covered surface can then be analyzed to give the surface viscosity. Measurements are usually taken at a fixed surface pressure as a function of shear rate. Then from a series of experiments of varying shear rate, at different fixed surface pressures, a relatively complete description of the surface viscosity characteristics can be obtained. An oscillating shear rate can also be applied to characterize further the rheological behavior (Mannheimer and Schechter, 1970b; Kott et al., 1974). Although the experimental techniques of surface viscosity measurement are available, very little work of this kind has been carried out for films of the pulmonary surfactant system or its components. Some workers have carried out a very crude and qualitative determination by looking at the surface mobility of sprinkled talc particles propelled by a gentle stream of air. As noted by Cadenhead (1970), such tests (Shah and Schulman, 1967a; Galdston and Shah, 1967; and Watkins, 1968) indicate that D P L films are in a solid state at high surface pressure, and in a more liquidlike state at low surface pressure. Also, Vilallonga (1968), Lim (1971), and Kott et al. (1974) have obtained quantitative measurements of DPL surface viscosity, with the latter two studies using the techniques of Mannheimer and Schechter (1970a, 1970b). In addition, Joos (1970) has used a modified Couette viscometer to demonstrate that cholesterol decreases the surface viscosity of phospholipid films, and hence acts as a film "liquifier." Aside from these few studies, there is a dearth of surface viscosity measurements for pure and mixed films of pulmonary surfactant components. This is unfortunate, because such studies can provide needed insight into the role of various components of lung surfactant. By contrast, surface viscosity studies of the complete lung surfactant system (Blank et al., 1969) are best looked on as of only qualitative interest. C. E l e c t r o n M i c r o s c o p e Studies

The surface pressure, potential, and viscosity are the major surface properties of importance for most studies. However, other techniques for monolayer study do exist and these include various optical techniques including light absorption, ellipsometry, light interference, and electron microscopy. Most of these techniques are described in detail elsewhere (Gaines, 1966, Chap. 3; Shah, 1970; Adamson, 1967). Of special interest here, however, are electron microscope techniques, because of their possible use for studying the collapse regime of surfactant films, where compression past the point of monolayer surface coverage has occurred. This regime is of importance for pulmonary surfactant because it is the collapse process that is thought to generate the observed hysteresis characteristics. Also as mentioned earlier, Clements et al. (1970) have found that more surfactant exists in the lungs than is needed to cover the surface with a monolayer at end expiration. Although all of this surfactant may not be available to the alveolar liquid-air interface at the same time, it is nonetheless possible that a surface excess of surfactant may exist during a significant part of the

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breathing cycle. Thus, the behavior of lung surfactant component films in the collapse regime is physiologically relevant, especially in terms of 7r-A behavior during dynamic cycling. However, it would also be meaningful if the collapse process could be studied directly. One way in which the collapse process can be studied directly is by electron microscopy. The most prevalent technique is that used by Ries and Kimball (1955, 1957) to study film collapse in fatty acid monolayers. The technique involves lifting the monolayer from the surface on a support (a collodion-covered screen), evaporation of the water substrate, and shadow casting with an appropriate alloy. The surface is then studied by electron microscopy. The homogeneity of the micrographs can be interpreted as indicating whether the collapse mechanism is that of squeeze out into multilayered lamina which lie on top of the monolayer, as opposed to bulk solution of film molecules as micelles. Because of the uncertainties associated with removing the surfactant film from the surface, and with the subsequent evaporation of any trapped subphase water, such electron microscopy studies are certainly not exact. In particular, because of problems associated with film cohesiveness at low pressures, it is difficult to use this technique to study low-pressure monolayer regimes (Pankhurst, 1955; Sheppard et al., 1965). However, the high surface pressure collapse region appears to be better suited for electron micrograph studies, and surprisingly goodquality micrographs can apparently be obtained (Ries and Kimball, 1955, 1957; Ries and Walker, 1961). Specifically, any collapse lamina are readily apparent, and height calculations based on shadowing angle show good agreement with a theoretical collapse model. This high surface pressure region is precisely the area of interest for pulmonary surfactant films in the collapse regime. To date, however, such studies have not been carried out. IV. EFFECTS OF GASES AND PARTICULATES ON PULMONARY SURFACTANT

The work above has largely been concerned with determining the behavior of pulmonary surfactant under normal conditions. Equally important, however, are the effects of various environmentally prevalent substances on the normal function of lung surfactant. In particular, a good deal of experimental work has involved a determination of the effects of various gases and particulates, and such research is reviewed in this section. A. General Effects

Since the epithelial surface of the alveolar membrane is an environmental interface, materials which are deposited upon or dissolved in the outermost layer of this surface would appear to have the maximum probability of biological interactions. It does not follow, however, that the most important biochemical and functional alterations will occur or involve the interfacial surface since the sensitivity and interdependence of all reaction sites must be considered. Modification of biochemical processes within the lungs by inhaled substances is well documented for hundreds of compounds, but the extent to which specific cause-effect relationships have been established is highly variable, and generally

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very limited. Specifically, descriptions of the actions of inhaled gases and particles on lung surfactant are not exceptional despite a number of studies in both intact and isolated systems, and many results are largely of qualitative interest. For example, Scarpelli (1968) has summarized studies on cigarette smokers which indicated a fall in dynamic compliance (Miller and Sproule, 1966), a reduction of surface activity in endobronchial lavage fluid (Cook and Webb, 1966), and in vitro studies which showed that cigarette smoke modified the O'ma x and O'min of lung washings or extracts (Bondurant, 1960; Miller and Bondurant, 1962; Webb et al., 1967). Studies of other materials apparently affecting surfactant, cited by Scarpelli, include those on furniture polish (Giammona, 1967), oxygen (Giammona et al., 1965; Caldwell et al., 1963), sulfur dioxide (Kahana and Aronovitch, 1966), aluminum oxide dust (Rosenberg et al., 1962), and ozone (Mendenhall and Stokinger, 1962). Also, halothane and chloroform have been found to affect surfactant behavior (Woo et al., 1969). By contrast, Thomas (1966), Miller and Thomas (1967), and Evans et al. (1966) found no effect of halothane on surfactant. These three studies also drew similar conclusions about diethyl ether. Other materials, cited by Scarpelli as not having adverse effects on surfactant activity, included saline, distilled water, and tyloxipal (Modell et al., 1966). Uncertain or inconsistent effects were noted with aerosolized ethanol (Obenour et al., 1963); carbon dioxide (Schaefer et al., 1964); and "toxic smokes" (Pattie and Burgess, 1961). Studies by Alarie et al. (1972, 1975) are of special interest. In evaluating the toxicity of halogenated ethanes, e.g., 1,1,2,2-tetrachloro-l,2-difluorethane (TCDF) and 1,1,2-trichloro- 1,2,2-trifluorethane (TCTF), the authors established that the pressure-volume curves in lungs from treated and control animals were remarkably different when inflated and deflated with air, but virtually identical when measured with saline. The lungs from exposed animals were found to become atelectatic following inflation and deflation with air, and on the basis of functional and histological evaluations, they concluded that the halogenated ethanes cause atelectasis by virtue of a direct action on pulmonary surfactant. They found no evidence that these organic compounds modify the intrinsic elasticity of pulmonary tissue although actions involving the small airways could not be completely discounted. Treatment with T C D F utilized direct installation of the material, whereas T C T F was administered as a vapor; in neither case was the amount of material used indicated, so one cannot assume that occupational exposures of individuals to the same materials could evoke similar responses. Studies of oxygen toxicity have produced a wealth of information on in vitro and in vivo effects (Clark and Lambersten, 1971; Klaus et al., 1961; Brashear and Christian, 1973; and Adamson and Bowden, 1971). Ultrastructural examinations of the alveolar membrane indicate that the earliest sign of oxygen toxicity occurs in the interstitium and the capillary endothelium; somewhat later, changes in the membranous (type I) pneumocyte are seen (Kistler et al., 1967). These histological changes are associated with hyaline membrane formation and with both interstitial and alveolar edema, which implies a diminution or adversative alteration of the lung surfactant. In the recovery phase of oxygen toxicity,

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type 1I cell hyperplasia occurs, and this is viewed as part of a compensatory reaction to restore surfactant activity (Kanpanci et al., 1969; Adamson et al., 1970; Cross, 1974). The commonly described sequence of histological events infers that capillary leakage is the initiating action in oxygen toxicity, and that the inactivation of surfactant probably occurs secondarily. Beckman and Houlihan (1973) have presented experimental data from hyperbaric oxygen studies in cats which apparently contradict this viewpoint. They reported that surfactant changes occurred before pulmonary edema developed; their criteria being increased O'min and trmax of lung washes and increased lung weight, respectively. For rats, this temporal relationship was not proposed. However, the experimental design of their study is such that the conclusions must be challenged, since capillary epitheilial damage may have occurred well before there was an overt pulmonary edema. Moreover, lung weight as a criterion for pulmonary edema is insensitive, and there was an inconsistent temporal relationship found among species. Other reports on the actions (or lack of actions) of oxygen on surfactant demonstrate, at least with isolated preparations, that the method of extraction or preparation is probably responsible for the variabilities in experimental results (Scarpelli, 1968). The same general sequence and type of effects reported for oxygen toxicity have been noted with oxidant gases, e.g., NOn and ozone (Balchum et al., 1971; Brown, 1974; Bils, 1974; Stephens et al., 1974)and with irradiation of the lungs (Leroy et al., 1966). In each case, there is no convincing evidence that surfactant was the primary target of the injurious agent. Nevertheless, there have been numerous demonstrations that these various "oxidizing" agents lead to an alteration in surfactant activity, lung lipid metabolism, and in the cytodynamics of type II cells (Kilburn, 1974; Cross, 1974). Indeed, several studies infer that the production of tolerance to oxidant-induced pulmonary injury (acute edema) may well accompany type II cell hyperactivity and the associated increase in what Cross (1974) has termed the "antioxidant defense" of the lungs, e.g., glutathione, glutathione reductase, glucose-6-phosphate dehydrogenase, and other "reducing" substances. In addition to the above physiologically oriented experiments, some workers have looked directly at the effects of pollutants on monomolecular films of various lipids. Several of these studies with monomolecular films have demonstrated interactions with air pollutants (Mendenhall and Stockinger, 1962; Felmeister et al., 1968; Bondurant, 1960; and Weiner et al., 1969), and most are considered models for explaining the interaction between the lungs (lung surfactant) and air pollutants. That these are not entirely successful is exemplified by the study of Weiner et aL (1969), wherein D P L films were found resistant to NO2 action while unsaturated phospholipids were found to be affected by NO2 and cited as the more probable targets within the lungs. The authors also noted that protein, e.g., albumin, was reacted upon by NO2, but that DPL-albumin films were again resistant to NO2. Similarly, most of the studies of isolated and simple surfactant materials to date have failed to clarify the relationships between the oxidant pollutants and lung surfactant. Nevertheless they represent an important testing system.

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Silica (SiO2), or quartz dust, is one of the more intensively studied substances in the realm of pulmonary toxicology. Although the pathogenisis of silicosis has not centered on alveolar surfactant, there is striking interplay between SiO2 and the lipid metabolism of the lungs. The studies of Griinspan et al. (1973), KilroeSmith and Oxenham (1973), Lad~inyi et al. (1974), and Heppleston et al. (1972) all show important actions of SiO2 on lung phospholipids. Among this group, the approach of Ladfmyi et al. (1974) is unique in that they used a polarographic method for assessing surface activity. Perhaps the most provocative report is that of Heppleston et al. (1974) with specific pathogen free rats, which showed enhanced lipid and D P L synthesis, type II cell hyperplasia and hyperreactivity, alveolar lipoproteinosis, and an associated abnormality of surfactant activity in animals failing to develop a typical silicosis. The rate of D P L synthesis was found tripled, and the rate of its elimination doubled in SiO2-exposed animals; thus while both processes were augmented, the net accumulation of D P L was due to an imbalance of the two processes. In other silica studies, Heppleston and Young (1972) showed by electron microscopy that much of the intraalveolar phospholipid was in a liquid-crystalline phase and that a remarkable correspondence existed between the experimental disorder induced by SiO2 and human alveolar lipoproteinosis. Biochemical analyses of this alveolar material from the advanced experimental disease revealed that D P L was the principal constituent (Heppleston et al., 1970). As in the case of the oxidant gases, the primary actions of crystalline free silica and their temporal sequence remains uncertain and controversial. However, it is clear that the properties and biochemistry of surfactant are somehow modified by silica. Apart from silica, several other kinds of dusts have been investigated in relation to surfactant. Studies by Bondurant and co-workers (1960 and 1962), Robillard and Alarie (1963), Robillard et al. (1964a), and Miller and Sproule (1966) are typical of these investigations. In all instances, the inhalation of dusts was found to reduce the dynamic compliance of the lungs, and to increase the work of breathing or to deteriorate the surface activity of the pulmonary surfactant obtained by endobronchial lavage. Studies on isolated lungs imply that these materials effect this change in compliance by a nonspecific reaction, presumably with the surfactant layer. Among the materials investigated have been aluminum oxide, titanium dioxide, quartz, and tobacco smoke. A recent investigation of inhaled carbon on the surface properties of the lungs indicated no immediate action on the surfactant system, despite sufficient carbon dust deposition to produce varying degrees of alveolar membrane thickening and atelectasis (Rhoades, 1972). Rhoades concluded that a deficiency in surfactant does not appear to be an etiological factor as previously reported for the alveolar damage produced from certain types of inhaled dusts. However, this may not be the case with a material that is intrinsically toxic, such as silica. Despite the conflicting evidence, interest in the possibility of significant interaction between airborne dust and surfactant remains a field of investigative research with high priority. Relatively recent reports concerned with surfactant and bacterial strains (Jalowayski and Giammona, 1972), anesthetic gases (Cosmi

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et al., 1970; Curti and Bracco, 1969), cigarette smoke (Giammona et al., 1971; Clements, 1972; Finley and L a dm an, 1972; B r a c c o e t al., 1969), silica (Gabor et al., 1971), aerosols (Curti and Camerota, 1972), and volatile oil aerosols (Stolz, 1972) all attest to the continuing interest. However, it is both surprising and unfortunate that studies of particulate and gas effects on lung surfactant have remained on such an empirical level. Presumably, greater attention will be given to the mechanistic bases when evidence is developed that a given in vivo interaction between an airborne substance and lung surfactant is of a primary nature and has a significant biomedical consequence. B. The Use o f Surfactant Aerosols as Therapeutic Agents It has been a challenge to some of those concerned with the respiratory distress syndrome to undertake methods of restoring or replacing the surfactant material which is believed to be deficient in the newborn infants who suffer this condition. Also, there are certain other circumstances in which the modification of surfactant is known to occur as part of the sequence of pulmonary injury, and the restoration of surfactant in these circumstances would likewise seem to be indicated. One of the more appropriate techniques for restoring surfactant to the alveolar lining would be the administration of a surfactant aerosol, with the proper particle size dimensions and concentration, so as to bring about a satisfactory deposition of the material within the alveolar spaces (Morrow, 1974). Robillard et al. (1964b) described the administration of synthetic L-a-lecithin (DPL) as a 0.25% mixture in aqueous propylene glycol by microaerosolization. This study of infants with respiratory distress was preceded by an earlier demonstration by Alarie and Robillard (1963) that D P L could decrease the alveolar surface tension when dispersed as an aerosol to rats. In the study with infants, the investigators believed that the clinical condition of most subjects appeared to be improved by the administration of the D P L aerosol. About 20 min after the initiation of therapy, the retraction score began to return to a lower number but the breathing difficulties did not entirely disappear. Improvement was generally at a maximum after 30 min of aerosolization, but the overall benefit and prognosis for these infants was difficult to assess. Besides the obvious difficulties of clinically evaluating infants who are in a respiratory distress syndrome before and after any therapy, there is the question about the efficacy of the aerosol administered both in terms of its value as an in vivo surfactant and in relation to the amount of material administered, i.e., whether or not it could have been an effective dose. In the 1964 investigation of Robillard et al., it is unlikely that the D-30 generator dispersed more than 7 mg of D P L over a 30-min period and only a small fraction of that reached the alveolar region. Consequently, as Bunnell and Shannon have indicated (1974), it is doubtful that an effective level of D P L was administered. In experimental animals, Shannon et al. (1969) attempted a similar type of experiment in which/~-y-dipalmitoyl lecithin (DPL) was prepared as a 2% solution in 0.15 M saline and aerosolized by an ultrasonic nebulizer. In this instance, isolated dog lungs were ventilated with 100% oxygen in an artificial thorax. When a consistent decrease in the slope of the deflation limb of the pressure-

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volume (P-V) curve was noted, then the D P L aerosol was delivered while the ventilation with oxygen was continued. The ultrasonic generator was estimated to have a volumetric output of 400 mg of DPL]hr. Continued ventilation with this D P L aerosol restored the deflation limbs to their initial slopes and increased the inflation-deflation pressure difference to initial values within 90 rain. A control aerosol consisting of 0.15 M saline did not produce these results, and therefore the authors concluded that nebulized D P L was capable of restoring the surfactant in isolated lungs. It should be noted that the output of the ultrasonic nebulizer in this instance was greater than that used by Robillard et al. (1964b); moreover, delivering an aerosol to an isolated lung is far more efficient in effecting alveolar deposition than in the actual living system. Although alveolar deposition was not determined in either this or the Robillard study, it is evident that a minimum of 30 min of inhalation with the D P L fog was required to produce even a small improvement in the P - V loop. In a recent study by Bunnell and Shannon (1974), an aerosol of 1% D P L in sterile water was administered using a positive-end-expiratory-pressure system. The experimental subjects were premature fetuses (55 days gestation) from beagle dogs and these were compared to premature puppies given only sterile water under identical conditions and to a second premature group which was only resusitated periodically and given no aerosol. On the basis of the dimensions of the D P L molecule and the surface area of the alveolar region, the investigators estimated that 3 mg DPL/kg body weight was necessary to provide a monolayer over the entire alveolar region. These calculations were based on the fact that alveolar surface area is roughly 1 m2/kg body weight for most mammals, and that D P L is in the most condensed monolayer state at 41 ,~2/molecule. Using this as an estimated effective dose (EED) of DP L, they determined that in puppies which received 84% of the EED, the mean lung compliance was about 28% better in the treated group than in either of the premature control groups and that the average opening pressure of the lungs in these animals was about 30% less than in premature control puppies. The differences in the characteristics of the DPL-treated premature puppies and normal puppies from a separate litter were not significantly different. From these results, the authors conclude that an aerosol prepared from 1% D P L in water can be effective in increasing lung compliance and reducing the work of breathing when the lung is deficient in its surface activity, provided a substantial part of the E E D is administered. Another D P L aerosol study in infants was reported by Chu et al. (1967). This was an extremely complex study involving a variety of treatment schedules and clinical evaluations. Insofar as the aerosol therapy was concerned, the D P L was prepared in a freon mixture and dispersed into a hood from which the infant breathed. The particular surfactant material used was evaluated on a LangmuirWilhelmy apparatus, bioassayed as an aerosol in rats, and its acute and subacute toxicity determined in several dog and rodent studies. An estimate of alveolar retention of the aerosol was obtained by adding a tracer of C14-1abeled palmitic acid to the DPL , and the C a4 recovered in isolated lungs of experimental animals was then quantified. On the basis of this test, between 2% and 6% of the aerosol

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was found to be Subject to alveolar deposition. Considering these factors and the requirement for a monolayer in an infant lung of - 0 . 3 /~g of D P L per cm 2 alveolar Surface, the investigators felt that the E E D could be delivered by a series of six 30-sec exposures given at 5-min intervals and the entire schedule repeated every 4 hr. As i n t h e case of the study by Robillard et al. (1964b) with infants, there was no attempt to perform control experiments and it was difficult to evaluate the overall impact of the D P L on the prognosis of the infants. In any case, on the basis of the infants studied, the authors conclude that an administration o f a D P L aerosol was followed by an increase in lung compliance even though in most instances it did not appear to be lifesaving to the infants. Infants showing an increase in lung compliance, but who had gas transfer difficulties, were not improved by the D P L administration. T h e authors appear to conclude that the treatment of idiopathic respiratory distress syndrome is so complex that it requires the simultaneous support of many systems. Especially evident to these investigators was the need for a pulmonary vasodilator since they felt ischemia presented a greater functional difficulty than did pulmonary atelectasis. T h e r e are many reasons to believe that surfactant aerosols may have an important role in the therapy of surfactant-deficient states, but clearly many additional factors should be assessed before a final evaluation is attempted. F o r example, attention must be given to the physical state of the surfactant aerosol and the limitation of the particulate form to revert effectively to a monolayer. As noted earlier in this paper, the ability of surfactant to respread or repenetrate to the interface is perhaps as critical as its ability to effect a significant surface tension lowering if initially spread on the surface. In this light it seems likely that one drawback to the administration of pure D P L aerosols is the lack of respreading ability apparent for D P L from monolayer studies. Thus, aerosol mixtures of D P L with other compounds known to enhance its respreadability, such as cholesterol, might prove to be more effective. Of course, any components added to increase spreadability must do so without a significant effect on surface tension lowering capability, and in this regard materials other than cholesterol could be preferable. This is an area that certainly seems worthy of further attention, in terms o f both surface balance studies and actual aerosol administration. V. RELATED AREAS We conclude this article with a look at several areas of research which have direct physiological relevance for pulmonary surfactant, and which involve concepts that may be significant in future work. A. Film Transport Resistance Effects It has been known since the studies o f A r c h e r and L a M e r (1955) that surfactant films o f fatty acids or alcohols can present a measurable resistance to mass transport. Specifically, these workers showed that monolayers of fatty acids and alcohols in the condensed surface state presented a specific resistanc& of from 1 to 20 sec/cm to the transport of water, which corresponds to a significant re3 The specific resistance is defined as the difference between the mass transfer driving force divided by the flux for a film-covered surface and for a clean surface (Archer and LaMer, 1955).

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duction in the water evaporation rate. Also, the addition of divalent metal ions such as Ca 2+ to the substrate beneath the film was found to increase monolayer transport resistances. Subsequent to the pioneering work of Archer and LaMer (1955), experiments by Blank and Roughton (1960) and Blank (1962) showed that monolayers which presented a transport resistance to water also presented a larger resistance, of the order of 100 sec/cm, to the transport of gases such as CO2. The primary reason that monolayers, in spite of their extremely small thickness, can present an actual diffusional barrier is the ordered molecular state present at the interface. From the work of Langmuir, Adam, and other researchers it is known that the surfactant molecules in a condensed monolayer at the air-water interface are highly ordered, with the polar head groups tightly packed in the water surface and the hydrocarbon tails sticking up into the gas phase. The large intermolecular forces that are present in this highly ordered and compressed state are responsible for the high diffusional resistance often exhibited by condensed monolayers. That the condensed film state is essential for a monolayer to present a large resistance is shown by the fact that, at the same surface pressure, condensed stearic acid monolayers greatly retard water transport while oleic acid monolayers, which are expanded, do not. No direct measurements of the transport resistance of film of pulmonary surfactant or its components to gases such as CO2 or oxygen have yet been carried out. However, Notter and Berg (1973) have determined the specific resistance of D P L films to water transport and found the resistance to be quite small (

Pulmonary surfactant: a surface chemistry viewpoint.

ANNALS OF B[OMEDICAL ENGINEER[NG 3, 119--159 (1975) Pulmonary Surfactant: A Surface Chemistry Viewpoint R. H. NOTTER Department of Chemical Engineeri...
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