INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Because of its extremely high surface activity under normal conditions, adulteration of mammalian lung surfactant by other materials almost always impairs surface tension lowering ability. A variety of compounds, including plasma proteins, hemoglobin, cell membrane lipids, fatty acids, phospholipases, and proteases, have been shown to decrease the ability of pulmonary surfactant to reduce surface tension in vitro (1- 12). Lung surfactant inhibition of this kind, also called surfactant inactivation or surfactant dysfunction, contributes to the pathophysiology of several pulmonary diseases, most notably the acute respiratory distress syndrome (ARDS) (13). The clinical syndrome of ARDS is associated with acute lung injury from multiple etiologies, leading to severe respiratory failure and significant mortality in both adult and pediatric patients (17, 18). Improved knowledge about the mechanisms and interactions of lung surfactant inhibitors is important for understanding and treating surfactant dysfunction in conditions such as ARDS.

Several mechanisms of action have been defined by which protein and nonprotein inhibitors can reduce the surface activity of lung surfactant (6, 11, 19). Enzymatic proteins such as proteases (19) or phospholipases (20, 21) act by chemically degrading essential lung surfactant components. However, a variety of other substances impair lung surfactant activity through biophysical interactions. Many biologic molecules, including tissue and blood proteins, lipids, and fatty acids, have intrinsic surface activity. Like the lipids and proteins that constitute lung surfactant, these substances adsorb to the air-water interface and form surface films. If such compounds are present in a mixture with lung surfactant, their intrinsic surface active behavior can reduce the adsorption of lung surfactant constituents into the interface, or alter film properties during dynamic cycling, or both. The blood proteins albumin and hemoglobin have been shown to compete with and hinder the entry of lung surfactant components into the interface during adsorption (6). Albumin has smaller detrimental effects on surface tension lowering if added directly at the surface with phospholipids (22), indicating that its inhibitory actions in the interfacial film itself during dynamic compression are less pronounced. In contrast, a major part of the inhibitory action of free fatty acids appears to result from mixing with lung surfactant phospholipids in the interfacial film so that surface tension lowering during dynamic compression is impaired (11). Although many surface-active inhibitors of lung surfactant have been studied as single substances, their effects when combined together have not been well characterized. The differing mechanisms of action of such compounds may give additive or even synergistic detriments to lung surfactant activity if mixtures of inhibitors are present in acute lung injury.

To help assess the magnitude and additivity of interactions between protein and nonprotein inhibitors of lung surfactant, we studied the effects on surface activity of a series of physiologically relevant compounds alone and in mixtures. Inhibitors investigated were the blood proteins albumin and hemoglobin (Hb), as well as C16:0 and C18:1 lysophosphatidylcholine (LPC), extracted red blood cell membrane lipids (RBCML), and the fatty acids palmitoleic acid (PA), arachidonic acid (AA), and oleic acid (OA). Lung surfactants studied were endogenous lung surfactant (LS) lavaged from calves and an organic solvent extract of this material designated calf lung surfactant extract (CLSE). CLSE is equivalent to the clinical exogenous surfactant Infasurf, which has been widely used to prevent and treat respiratory distress syndrome (RDS) in premature infants and has also been studied in patients with ARDS-related acute lung injury (reviewed in Reference 16). The effects of inhibitors on the surface activity of LS and CLSE were defined by experiments on a pulsating bubble apparatus incorporating the combined influence of adsorption and dynamic film compression at physical conditions relevant for the lung (37° C, saturated humidity, 20 cycles/min, 50% area compression). Detriments to the time course of surface tension lowering and/or the minimum surface tension achieved during cycling were assessed at several surfactant and inhibitor concentrations to define additive interactions and the ease of reversal of surfactant dysfunction by selected mixtures of protein and nonprotein inhibitors.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Natural and Extracted Calf Surfactant

Endogenous LS was obtained by bronchoalveolar lavage (BAL) of freshly excised calf lungs with 0.15 M NaCl as described previously (2, 4). BAL was centrifuged at 250 × g for 10 min to remove cells, and the supernatant was centrifuged at 12,500 × g for 30 min to pellet whole LS. This material was resuspended, and extracted in chloroform:methanol by the method of Bligh and Dyer (23) to obtain CLSE. The percentage and distribution of phospholipids in CLSE by phosphate assay (24) and one-dimensional thin layer chromatography with solvent system C of Touchstone and colleagues (25) was equivalent to previously published reports (2, 4). LS and CLSE contained ~ 8% and 1.3% protein by weight, respectively, measured by the assay of Lowry and colleagues (26) modified by the addition of 1% sodium dodecyl sulfate (SDS) to eliminate turbidity.

Inhibitor Compounds

Bovine serum albumin (BSA), bovine Hb, synthetic C16:0 and C18:1 LPC, and fatty acids (PA, AA, and OA) were purchased from the Sigma Chemical Co. (St. Louis, MO) and were used without further purification in inhibitor studies. BSA was essentially free of fatty acid, prepared from fraction V albumin. LPC, PA, AA, and OA were reagent grade ( 99% pure). RBCML were prepared by extraction from rabbit blood as follows. Blood was drawn from an adult New Zealand rabbit into heparinized saline, followed by probe sonication (Model W-220F; Heat Systems-Ultrasonic Inc., Farmington, NY) on ice to disrupt cells, and extraction into chloroform-methanol (23) to give RBCML. The molar concentration of phospholipid in RBCML was defined by phosphate assay (24), with an average molecular weight of 750 assumed for weight conversions. The final RBCML preparation contained 46.9% by weight phospholipid, 50.6% neutral lipid (cholesterol and cholesterol esters), and ~ 2.5% protein (24, 26). The phospholipid fraction of RBCML was found by thin layer chromatography to be ~ 8% LPC, 11% sphingomyelin, 51% phosphatidylcholine, 24% phosphatidylethanolamine, and 6% other (25).

Oscillating Bubble Surfactometer

The dynamic surface activity of lung surfactant dispersions in the presence and absence of inhibitors was defined by a pulsating bubble surfactometer (Electronetics Corp., Buffalo, NY) (27). Surface tension at minimum bubble radius (minimum surface tension) was calculated from the measured pressure drop at end-compression and the Laplace equation for a spherical interface. The accuracy of the Laplace equation for minimum surface tension calculations with this apparatus, despite significant nonspherical deformations of the tiny air bubble at low surface tensions, has been defined previously (28). In experiments where the air bubble deformed sufficiently to detach from the capillary tip, subsequent minimum surface tensions were recorded as < 1 mN/m. All bubble experiments utilized a subphase of 0.15 M NaCl, 5 mM CaCl₂, and 10 mM HEPES at pH 7.0, and were done at 37.0 ± 0.5° C with a pulsation rate of 20 cycles/min. In a typical experiment, an appropriate amount of an inhibitor or mixture of inhibitors dispersed in buffer (or in ethanol in the case of the fatty acids PA, OA, and AA) was added to LS or CLSE in buffer, followed by gentle vortexing. Aliquots of surfactant/inhibitor mixtures were always examined on the bubble apparatus within 2 h of preparation. Pulsation was begun immediately after formation of an air bubble in a given dispersion in the instrument sample chamber. Control experiments demonstrated that the small amount of ethanol present along with fatty acid inhibitors did not by itself affect the surface tension lowering of either LS or CLSE at the concentrations studied.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The inhibitory effects of Hb and AA on the surface tension lowering of CLSE and LS are shown in Figure 1. When present alone, Hb (2.5 mg/ml) or AA (0.15 mM) increased the time course over which CLSE (0.75 mg/ml) lowered surface tension to < 1 mN/m (Figure 1, top panel). When Hb and AA were mixed at the same concentrations (Hb, 2.5 mg/ml; AA, 0.15 mM), the minimum surface tension of CLSE after 20 min of cycling was raised to 7.2 ± 2.2 mN/m (Figure 1, top panel), indicating that some additive inhibition was present. When inhibitor concentration in the mixture was halved (Hb, 1.25 mg/ ml; AA, 0.075 mM), the time course of surface tension lowering was similar to that found when the most severe individual inhibitor was present alone at higher concentration (AA, 0.15 mM), and the minimum surface tension reached after 20 min was elevated slightly from < 1 to ~ 2 mN/m. However, although some additive inhibition was present in mixtures of Hb and AA at low CLSE concentrations, inhibition was largely overcome by a relatively small increase in CLSE concentration, from 0.75 to 1.375 mg/ml (Figure 1, top panel). Synergy, in the sense of a substantial change in the character and extent of the inhibitory process, was not observed.

View larger version (29K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   Figure 1.   Effect of Hb and AA on the surface tension lowering ability of CLSE and LS. Top: CLSE; bottom: Whole LS. Data are minimum surface tension as a function of time for dispersions of surfactant plus inhibitors in an oscillating bubble surfactometer (20 cycles/min at 37° C) (see METHODS). Hb concentration: 2.5 mg/ml; AA concentration: 0.15 mM; Hb + AA (L): Hb, 1.25 mg/ml; AA, 0.075 mM. Surfactant phospholipid concentration: 0.75 or 1.375 mg/ml for CLSE (top panel ) and 0.5 or 1.0 mg/ml for LS (bottom panel ). Data are mean ± SEM, n = 4 to 6.

Hb and AA inhibited LS (Figure 1, bottom panel) with an identical pattern to that found for CLSE. The only difference was that the LS concentration of 0.5 mg/ml in Figure 1, bottom panel, is lower than the concentration used for CLSE in Figure 1, top panel, reflecting the slightly improved ability of whole surfactant to resist inactivation compared with that of surfactant extract (8). When present alone, Hb (2.5 mg/ml) or AA (0.1 mM) extended the time course by which LS (0.5 mg/ml) lowered surface tension to < 1 mN/m (Figure 1, bottom panel). Inhibition was increased when the Hb and AA were mixed together at the same concentrations, with the magnitude of increase again relatively small and minimum surface tension raised only slightly compared with AA alone (from < 1 to ~ 3 mN/m) (Figure 1, bottom panel). The inhibitory effects of the mixture of Hb and AA were then overcome by raising the concentration of LS from 0.5 to 1 mg/ml.

To assess inhibitor additivity further, the surface activity of CLSE was examined in the presence of higher Hb and AA concentrations of 5 mg/ml and 0.3 mM, respectively (Figure 2). At these higher concentrations, both Hb and AA significantly impaired the surface tension lowering of CLSE (0.75 mg/ml). After 20 min of cycling, minimum surface tension was raised to 15.4 ± 1.5 mN/m by Hb (5 mg/ml) and to 19.4 ± 1.4 mN/m by AA (0.3 mM). However, the additivity of Hb and AA was less apparent at these higher inhibitor concentrations. The minimum surface tension generated by the mixed inhibitors was 22.6 ± 1 mN/m (Figure 2), a relatively small increase compared with the value resulting from AA alone at the same concentration. The surface activity detriment from the mixed inhibitors was largely overcome by raising the concentration of CLSE to 1.375 mg/ml, although the time course of surface tension lowering was slightly prolonged compared with that in Figure 1, top panel, at lower inhibitor concentration. Inhibition by Hb (5 mg/ml) and AA (0.3 mM) was abolished at a CLSE concentration of 1.625 mg/ml (Figure 2).

View larger version (33K): [in this window] [in a new window]   Figure 2.   Effect of Hb and AA at higher concentration on CLSE activity. Hb concentration, 5 mg/ml; AA concentration, 0.3 mM; CLSE concentration, 0.75, 1.375, or 1.625 mg/ml as shown. Other details as in Figure 1 legend. Data are mean ± SEM, n = 4 to 6.

To investigate if the protein inhibitor influenced additivity, experiments similar to those in Figure 1 investigated the effects of albumin and AA on the activity of CLSE and LS (Figure 3). In this case, inhibitor interactions were found to be antagonistic rather than additive. BSA (2.5 mg/ml) or AA (0.15 mM) alone prolonged the time course over which CLSE lowered surface tension to < 1 mN/m, with the fatty acid giving the largest individual effect (Figure 3). When BSA and AA were combined at the same concentrations, however, inhibition was less than that generated by AA alone and was instead very similar to that found for albumin alone (Figure 3, top panel). This same behavior was also found when albumin and AA were studied with whole LS rather than CLSE (Figure 3, bottom panel), or when a fatty acid other than AA was used (Table 1). When mixed with Hb (2.5 mg/ml), PA or OA (0.25 mM) caused a prolonged time course of surface tension lowering to < 1 mN/m compared with the fatty acid or Hb alone (Table 1). However, when PA was combined with BSA (2.5 mg/ml), the detriments to surface tension lowering from the mixture were less than for PA alone and were instead essentially equivalent to results found for BSA alone (Table 1). As discussed later, this antagonistic interaction between albumin and free fatty acids is most likely due to the binding of the latter molecules by the plasma protein.

View larger version (24K): [in this window] [in a new window]   View larger version (24K): [in this window] [in a new window]   Figure 3.   Effect of BSA and AA on the surface tension lowering ability of CLSE and LS. Top: CLSE; bottom: Whole LS. BSA concentration, 2.5 mg/ml; AA concentration, 0.15 mM. Surfactant phospholipid concentration, 0.75 mg/ml for CLSE (top panel ) and 0.5 mg/ml for LS (bottom panel ). Data are mean ± SEM for n = 4 to 6.

Although BSA and Hb differed in their pattern of inhibition in mixtures with fatty acids, the behavior of these proteins was the same when they were combined with either C16:0 or C18:1 LPC (Table 2). LPC shares with fatty acids the ability to penetrate CLSE surface films (29), but unlike free fatty acids it does not bind appreciably to albumin. As single inhibitors, C16:0 or C18:1 LPC increased the time course over which CLSE lowered surface tension to < 1 mN/m on the oscillating bubble (Table 2). When combined with either Hb or BSA, these LPC compounds exhibited additivity in giving more severe detriments in CLSE activity than when they were present alone (Table 2). Again, the observed reduction in surface activity from all protein/LPC mixtures could be overcome by raising CLSE concentration from 0.75 to 1.375 mg phospholipid/ml (Table 2).

A final inhibitory material studied was RBCML, itself a complex mixture of erythrocyte membrane constituents, including phospholipids, neutral lipids, and a small amount of protein or protein hydrophobic fragments (see METHODS) (Figures 4 and 5). RBCML of this kind have previously been shown to give a strong inhibition of lung surfactant activity (4), and their additivity with BSA and Hb was investigated further here. RBCML (0.1 mM) raised the minimum surface tension reached by CLSE (0.75 mg/ml), from < 1 to 5.7 ± 1 mN/m after 20 min of bubble pulsation (Figure 4). RBCML also prolonged the time course of surface tension lowering of LS (0.5 mg/ml), although minimum surface tension remained < 1 mN/m (Figure 5). When combined with either Hb or BSA, RBCML exhibited additivity in inhibiting the surface tension lowering ability of CLSE and LS. Mixtures of RBCML (0.1 mM) with either Hb or albumin (2.5 mg/ml) raised the minimum surface tension after 20 min of cycling to ~ 16 mN/m for CLSE (Figure 4, top and bottom panels) and to ~ 6 mN/m for LS (Figure 5, top and bottom panels). As with the other mixtures of inhibitors studied, the detrimental effects of RBCML and blood proteins could be overcome by raising the concentration of LS or CLSE.

View larger version (27K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   Figure 4.   Effect of BSA or Hb combined with RBCML on the surface tension lowering ability of CLSE. Top panel: BSA plus RBCML; bottom panel: Hb plus RBCML. BSA concentration, 2.5 mg/ml; Hb, 2.5 mg/ml; RBCML, 0.1 mM. CLSE phospholipid concentration, 0.75 or 1.75 mg/ml. Data are mean ± SEM for n = 4 to 8.

View larger version (28K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   Figure 5.   Effect of mixed inhibitors of BSA or Hb with RBCML on surface tension lowering ability of whole calf lung surfactant. Top panel: BSA plus RBCML; bottom panel: Hb plus RBCML. BSA concentration, 2.5 mg/ml; Hb, 2.5 mg/ml; RBCML, 0.1 mM. LS phospholipid concentration was 0.5 or 1 mg/ml as shown. Data are mean ± SEM for n = 4 to 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study has examined the additivity of several physiologically relevant proteins and lipids/fatty acids in inhibiting the surface activity of whole and extracted calf lung surfactant (LS and CLSE). Although many different endogenous compounds are known to be detrimental to the adsorption and/or dynamic surface tension lowering of pulmonary surfactant in biophysical studies in vitro (1), the majority of prior research has addressed the effects of specific inhibitors in isolation. Protein and nonprotein inhibitors can be present together in many clinically relevant acute lung injuries (13, 14), and it is important to understand the extent to which these compounds might interact to worsen lung surfactant dysfunction. Several combinations of protein and nonprotein inhibitors were shown here to reduce lung surfactant activity to a greater extent in mixtures than when present alone. This was the case when albumin or Hb was combined with C16:0 or C18:1 LPC or with RBCML (Figures 4 and 5 and Table 2), and additivity was also found when Hb was combined with fatty acids (Figure 1 and Table 1). However, the additive inhibition observed was, in general, small in magnitude when assessed relative to the effects of the most severe individual inhibitor present in a given mixture. Moreover, additivity was found to decrease as the concentration of inhibitor compounds was increased and their effects as individuals became more pronounced (Figure 2). Substantial synergy was not observed in the actions of any of the mixtures of protein and lipid/fatty acid inhibitors studied.

The degree of additivity displayed by different lung surfactant inhibitors is in principle dependent on their underlying mechanisms of action. The blood protein and lipid/fatty acid inhibitors in our study had antagonistic as well as complementary aspects in their biophysical interactions with lung surfactant. By adsorbing and blocking the interface (6), albumin or hemoglobin effectively limit the formation of the film structure on which lipid inhibitors act (11, 29), reducing the potential for synergy. Additivity may be more pronounced in mixtures of inhibitors that incorporate mechanisms such as chemical degradation and biophysical inactivation, which may prove more complementary. However, some inhibitors may prove to be less rather than more detrimental to surface activity in mixtures, as found here when albumin was mixed with arachidonic or palmitoleic acid (Figure 3 and Table 1). Our experiments did not address the specific mechanism responsible for this antagonistic interaction, but a presumptive explanation is that albumin acted to bind free fatty acid so that the effective concentration of this latter inhibitor was reduced. This would be consistent with the additivity observed when albumin was mixed with LPC or RBCML, which act similarly to fatty acids but are not bound significantly by albumin (Figures 4 and 5 and Table 2). Additivity was also observed when Hb, which inhibits lung surfactant by the same mechanism as albumin (6) but has much less binding affinity for free fatty acids, was mixed with PA, OA, or AA (Figures 1, 4, and 5 and Table 1). Additional studies comparing inhibition for mixtures of fatty acids with fatty-acid-saturated versus fatty-acid-free albumin would help to clarify the mechanistic importance of binding interactions.

Studies on lung surfactant inactivation are complicated by the often strong dependence of this phenomenon on inhibitor and surfactant concentrations. Depending on the specific inhibitors and surfactants used, relatively small changes in absolute or relative concentrations can significantly alter the consequences for surface activity. The present study is not immune from this complication. All of the inhibitors investigated could potentially be present in the alveoli during acute lung injuries of the ARDS type in vivo, but their concentrations will vary as a function of the amount of serum transudation, the severity of the inflammatory response, and a variety of other factors. The high plasma content and relatively low molecular weight of albumin make it a prevalent constituent in any permeability edema. Normal serum values for albumin (4 g/dl or 40 mg/ml) are substantially higher than the BSA concentration of 2.5 mg/ml investigated here (Tables 1 and 2 and Figure 3). Hb is also expected in significant levels in hemorrhagic edema where cell lysis occurs, and was studied at concentrations similar to albumin. LPC and fatty acid concentrations of 0.1 to 0.3 mg/ml are sufficiently low to be potentially generated in vivo by phospholipase action on lung surfactant and/or cell membrane phospholipids. Surfactant concentrations in the alveolar hypophase in vivo are not known precisely, but values of 0.5 to 1.75 mg phospholipid/ml as in our experiments are not unreasonable physiologically. Activity studies on the oscillating bubble indicate that lung surfactant concentrations of ~ 0.5 mg phospholipid/ml are near the lower limit where minimum surface tensions < 1 mN/m can be generated during prolonged dynamic cycling at physiologically relevant rates (20 cycles/min) and area compression (50% reduction) (4, 8, 16). In contrast, if LS concentration is raised to 5 to 10 mg/ml, surfactant inactivation is often not demonstrable even in the presence of very high inhibitor concentrations (4, 5, 7). Surfactant concentrations of 10 mg/ml or higher probably exceed those in vivo since the fact of surfactant inactivation and related lung functional and mechanical deficits is well documented in many animal models of lung injury (13).

Our experiments did not attempt to compare in detail the relative severity and concentration-dependence of different protein and nonprotein inhibitors. The compounds studied, however, did exhibit some variation in their effects on lung surfactant activity. Albumin and hemoglobin gave a measureable reduction in the surface activity of LS (0.5 mg/ml) and CLSE (0.75 mg/ml) at protein concentrations of 2.5 mg/ml, equivalent to very low molar concentrations of ~ 0.04 mM (Figures 1 and 3). At the same surfactant concentrations, LPC impaired activity at 0.1 to 0.125 mg/ml or ~ 0.2 mM (Table 2), and fatty acids and RBCML were inhibitory at concentrations varying from 0.1 to 0.3 mM (Figure 1 and Table 1). However, other studies have indicated that inhibition by albumin and Hb is more easily overcome by raising surfactant concentration than is the case for RBCML (4) or fatty acids (11). Among the fatty acids investigated, arachidonic acid inhibited lung surfactant activity at a lower concentration than did palmitoleic acid or oleic acid, although the latter is known to have a severe inhibitory effect on endogenous LS during acute lung injury (11, 30). In all cases, the detrimental effects of inhibitors alone and in mixtures were overcome when the concentration of CLSE or LS was raised, indicating that supplementation with active exogenous surfactant would still be effective in reversing surfactant dysfunction from these compounds in acute lung injury.

The ability of different exogenous surfactants to reverse inhibition is particularly relevant for the effective application of surfactant replacement therapy to ARDS and related clinical acute lung injury syndromes. Surfactant inactivation has been well documented in vitro (1), in animal models of acute lung injury (31), and in human patients with ARDS (39- 41). It is crucial that exogenous surfactants used in ARDS not only have intrinsically high surface activity, but also the ability to overcome inhibition in the continued presence of inhibitor compounds. Surfactant mixtures that have apparently similar surface properties in the absence of inhibitors can exhibit large differences in inhibition resistance (12, 42). Our results show that CLSE, a preparation equivalent to the clinical exogenous surfactant Infasurf, was able to overcome inhibition by all of the inhibitors and mixtures studied (Figures 1-4). The ability of CLSE to resist inhibition is not quite as high as that of endogenous LS (Figure 1, top panel versus bottom panel, and Figure 4 versus Figure 5). This behavior is consistent with previous reports that the presence of surfactant protein A in whole LS acts to improve inhibition resistance relative to CLSE (8). Nonetheless, the inhibition resistance exhibited by CLSE is substantial (Figures 1-4), in agreement with several prior studies that have also found that this preparation has improved inhibition resistance over many other exogenous surfactants (12, 43). The present experiments suggest that exogenous CLSE can effectively reverse lung surfactant inactivation by mixtures of plasma proteins with fatty acids, LPC, or membrane lipids in ARDS-related acute lung injury in vivo.

In summary, the results of this study indicate that several mixtures of inhibitors containing a blood protein (albumin or hemoglobin) and a lipid or fatty acid (RBCML, LPC, AA, PA, or OA) exhibited additivity in being able to impair the surface activity of whole or extracted calf lung surfactant more severely than when the individual inhibitors were present alone. However, the magnitude of additivity was in general small compared with the action of the most severe individual inhibitor, and substantial synergy was not observed for any of the inhibitor compounds studied. In the special case of mixtures of albumin and fatty acids, inhibition was actually decreased relative to the effects of fatty acid alone, presumably because of albumin binding of fatty acid. In all cases, reductions in surface activity caused by the mixtures of protein and lipid/fatty acids were overcome by increasing lung surfactant concentration. These results suggest that lung surfactant inactivation in vivo may be influenced by additive interactions between inhibitors. However, the magnitude of such effects is likely to be limited for many mixtures of blood proteins with lipids/fatty acids and still responsive to exogenous surfactant supplementation with active materials such as CLSE.