INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Surfactant preparations containing the hydrophobic surfactant proteins B and C (SP-B and SP-C) absorb rapidly to an air-liquid interface. After intratracheal instillation in sufficient amounts, exogenous surfactant can effectively compensate for surfactant deficiency in premature infants and animals with respiratory distress syndrome (RDS). The efficacy of a surfactant preparation depends on the relative proportion of the hydrophobic surfactant proteins it contains (1). The presence of SP-B in particular is critical to surfactant function, and SP-B deficiency due to a mutation in the SP-B gene can lead to fatal respiratory disease in newborn infants (2). The amounts of SP-B and SP-C in animal-derived surfactant preparations vary considerably (3, 4). Survanta (Ross Laboratories, Columbus, OH), a modified bovine lung extract and an effective surfactant replacement in premature infants with RDS (5), contains < 0.1% SP-B, versus > 1% in surfactant preparations derived from lung lavage (4). Increasing the SP-B content of this clinical surfactant might therefore improve its in vivo function. Supplementation of Survanta with native SP-B increases dynamic compliance and deflation lung volume in 27-d gestation premature rabbits (10). In premature infants, instillation of Survanta leads to a rapid improvement in oxygenation, followed by a more gradual increase in lung compliance, measurable only after 2 to 24 h (11, 12). The surfactant-deficient rat model offers the opportunity to measure both oxygenation and lung function, as is the case in the premature infant.

Synthetic surfactant peptides based on the human SP-B and SP-C sequence mimic the structural and functional properties and the in vivo function of the native lung surfactant proteins (13), and offer the possibility of designing or modifying synthetic surfactant preparations (16). Using the synthetic surfactant peptides B and C, we tested the hypothesis that the in vivo function of Survanta can be improved by supplementation with synthetic surfactant peptides in a surfactant-deficient rat model.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials

FastMoc amino acids and peptide coupling reagents, and deprotection-cleavage reagents were obtained from Applied Biosystems (Foster City, CA). High-performance liquid chromatography (HPLC) solvents, including acetonitrile, water, and trifluoroacetic acid, were supplied by Fisher Chemical Co. (Pittsburgh, PA). Trifluoroethanol and hexafluoroisopropanol were obtained from Aldrich Chemical Co. (Milwaukee, WI), and sodium dodecyl sulfate (SDS) from Bio-Rad (Richmond, CA). Dipalmitoyl phosphatidylcholine (DPPC) and phosphatidyl glycerol were obtained from Avant Polar Lipids (Alabaster, AL). Survanta is a commercial surfactant containing SP-B and SP-C, obtained by organic solvent extraction of bovine lung mince, and supplemented with DPPC, palmitic acid, and tripalmitin. All surfactant preparations were used at a lipid concentration of 2.5 mg/ml in surface activity studies, and of 25 mg/ml in the in vivo studies. Adult male Sprague-Dawley rats weighing 200 to 250 g were obtained from B&K Universal Inc. (Fremont, CA).

Synthesis and Purification of Surfactant Peptides B, C and KL₄

Full-length SP-B_1-78 (B) and palmitoylated SP-C_1-35 (C), and synthetic KL₄ peptide were each synthesized on a 0.25-mmol scale with a peptide synthesizer (Perkin Elmer Model 431A; Applied Biosystems) using FastMoc chemistry (17). The monomeric B peptide based on the 78-residue human sequence (Figure 1) was synthesized, purified, and oxidized in a manner similar to that described by Lipp and colleagues (18). A prederivatized Fmoc-Wang resin (Applied Biosystems-Perkin Elmer), with the C-terminal serine, was used for synthesis. Amino-acid residues 26 to 59 of the B peptide were double coupled (chemical formation of peptide bond) to ensure a high degree of peptide bonding, whereas all other residues were single coupled (17). After cleavage from the resin and single-pass purification with reverse-phase HPLC, the molecular mass of the reduced monomer was confirmed by electrospray mass spectrometry (UCLA Center for Molecular and Medical Sciences), and the product was twice lyophilized from acetonitrile: 10 mM HCl (1:1, vol/vol) to remove acetate counter ions. The peptide was then oxidized with EKATHOX resin (Ekagen, Palo Alto, CA) through the addition of a 10-fold molar excess of resin-active groups to 1 mM B-peptide thiol in trifluoroethanol: water (8:2, vol/ vol) for 6 h, followed by removal of the resin from the peptide solution by centrifugation (1,000 × g for 10 min). The degree of disulfide formation by the oxidized peptide was estimated by 5,5'-dithionitrobenzoic acid titration of the peptide (19), and the mass of monomeric oxidized peptide was confirmed by Matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry (UCLA Center for Molecular and Medical Sciences).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Amino acid sequences of synthetic surfactant peptides derived from human SP-B1-78 and SP-C1-35 and the synthetic peptide KL4.

The synthetic SP-C mimic peptide (15, 20) was based on the human SP-C sequence, with a periodic substitution of alanine for valine (residues 15, 17, 19, 21, 23, 25, and 27) (Figure 1) in the hydrophobic polyvaline segment in order to enhance the helical propensity of the molecule in a manner similar to that observed in signal sequences (21). This modification insured that the SP-C mimic had a helical structure and in vitro-in vivo function resembling that of acylated native SP-C (22), in contrast to the nonacylated dimeric beta-sheet native variant of the protein (23). A prederivatized Fmoc Wang resin (Applied Biosystems-Perkin Elmer) was used for synthesis of the peptide. Residues 11 to 28 of the hydrophobic polyvaline stretch were double coupled using the conventional FastMoc cycles with the ABI 431A synthesizer, whereas all other residues were single coupled. Alternatively, the hydrophobic sequence was synthesized manually, using an 80-ml reaction vessel for small-scale peptide synthesis (AnaSpec, San Jose, CA). The resin was washed four times with dimethylformamide (DMF), and the Fmoc protecting group was removed with 20% piperidine in DMF for 30 min with shaking. After removal of the protecting group, the reaction vessel was drained and the resin washed four times with DMF. The coupling step was performed with 1 mmol of Fmoc amino acid, 2 mmol of 0-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 348 µl of N,N-diisopropylethylamine (DIEA) (2 mmol) in 2 ml of 0.45 M 1-hydroxybenzotriazole (HOBT) (Applied Biosystems) with shaking for 30 min. The resin was then drained and washed four times with DMF, and recoupled as described for the first coupling step.

After cleavage of the peptide from the resin, the crude material was purified by reverse-phase HPLC with a Vydac C4 column (The Separations Group, Hesperia, CA), using a water-acetonitrile:isopropanol (1:1, vol/vol) gradient containing 0.1% trifluoroacetic acid. Palmitoylated SP-C was obtained through a modification of the protocol of Lapidot and colleagues (24). Synthetic SP-C peptide was reacted with two-fold amounts of N-(palmitoyl) succinimide in trifluoroethanol:10 mM phosphate buffer, pH 7 (9:1, vol/vol) for 24 h. The molecular weight of the palmitoylated product was determined by fast-atom-bombardment mass spectrometry.

The KL₄ sequence in Figure 1 has been described by Cochrane and associates (25) as a paradigm for SP-B protein because of the amphipathic repeat sequence of lysine and leucine residues. The mimetic peptide (UCLA. KL₄) was assembled with a prederivatized Fmoc-Wang resin (AnaSpec) on a 0.25 mmol scale with the ABI 431A synthesizer. The first amino-acid residue was double coupled, whereas all other residues were single coupled using a FastMoc strategy. Cleavage-deprotection, purification, and confirmation of the molecular mass of the peptide were the same as described for the B peptide.

Experimental Surfactant Preparations

Synthetic surfactant peptides were added to Survanta through two techniques. In the first, Survanta was extracted with 2:1 chloroform:methanol (vol/vol), the chloroform phase was isolated, and the organic solvent was removed by flash evaporation. This lipid fraction was thoroughly dried overnight under vacuum, left unmodified or enriched with 1% B, 2% B, or 2% B plus 1% C in lyophilized form, and rehydrated in 0.9% NaCl at 37° C for 48 h with gentle mixing. The four resulting preparations are hereafter referred to as extracted Survanta (ES). In the second technique, Survanta was not extracted but was enriched by direct addition of 2% B, 2% B plus 1% C, or 2% KL₄ (modified Survanta, S). In this case, the peptides were dried down as a film, Survanta was added on top, and the peptides were taken up by the surfactant liposomes through vortexing. All surfactant preparations were prepared at a concentration of 25 mg of phospholipids/ml and administered in a dose of 100 mg of phospholipids /kg of body weight.

In vitro Surface Activity

Surface-tension measurements were done on unbuffered 0.9% NaCl at room temperature. Dynamic respreading (i.e., the characteristic behavior of each preparation under conditions of repeated film compression) was assessed on a modified Langmuir-Wilhelmy surface balance, using 0.5 µg of phospholipids /cm², by measuring four parameters relative to the isotherms. The parameters considered were: (1) minimum surface tension; (2) maximum surface tension; (3) liftoff (i.e., the point during the first compression at which the isotherm deflects from the baseline); and (4) the percentage of the area below 10 mN /m (i.e., the portion of the isotherm encompassing surface tensions of 10 mN/m or less). The two latter measurements are expressed as percent of the total area. At identical lipid loads, a liftoff of 100% indicates that the dispersion spreads instantly as an interfacial film sufficient to decrease surface tension at the initial surface area, whereas liftoff values of less than 100% show the amount of compression necessary before surface activity is apparent (29). A liposomal dispersion of phospholipids and fatty acids typically shows 30 to 40% liftoff; maximum surface tension of 72 mN/m; and minimum surface tension varying from less than 5 mN/m to more than 65 mN/m, depending on the size of the liposomes and the age and hydration state of the dispersion. Minimum surface tensions of 10 mN/m and below are usually achieved only after extreme compression of the surface to less than 30% of the surface area. Whole natural bovine or porcine surfactant shows a unique isotherm, characterized by 100% liftoff, maximum surface tension below 50 mN/m, minimum surface tension below 5 mN/m, and an intermediate "squeezeout" plateau, typical of a two-phase coexistence region, probably representing the surface refinement occurring under compression, when lipid components characterized by lower collapse pressures are ejected from the monolayer into the subphase (30).

Animal Protocol

Rats were anesthetized with pentobarbital sodium, 35 mg/kg, and ketamine, 80 mg/kg, by intraperitoneal injection. After placement of a tracheal cannula, the rats were supported on a rodent ventilator (Harvard Apparatus, South Natick, MA) with 100% oxygen, a VT of 7.5 ml/kg, and a respiration rate of 60 breaths/min. An arterial line was placed in the abdominal aorta for serial measurements of arterial blood gases, and the rats were paralyzed with pancuronium bromide, 2 mg/kg intravascularly. Only animals with Pa_O2 values > 400 mm Hg while being ventilated with 100% oxygen were included in the experiments. The lungs were gently lavaged eight to 12 times with 8 ml of 0.9% NaCl warmed to body temperature. Five minutes after the Pa_O2 in 100% oxygen fell below 80 mm Hg, one of the experimental surfactant preparations was instilled intratracheally at a dose of 100 mg phospholipids/kg body weight. Rats treated with extracted Survanta preparations were ventilated for 15 min and had arterial samples for blood gas measurements drawn at 5-min intervals, whereas rats treated with plain or modified Survanta were ventilated for 60 min and had arterial blood gases determined at 15-min intervals. After 15 or 60 min of ventilation, the rats were killed with pentobarbital sodium, 100 mg/kg intravascularly, and exsanguination, and the lungs were degassed in situ. A pressure-volume (P-V) curve was constructed in situ for each pair of rat lungs in order to define lung mechanics. Lungs were inflated and deflated with a bidirectional Harvard pump coupled to a 50-ml glass syringe and pressure was continuously recorded on a Gould multichannel recorder (Cleveland, OH). Each P-V curve was corrected for the compliance of the system by subtracting the P-V curve of the pump/syringe unit, which was constructed before each lung P-V curve. Absence of air leaks was assessed by verifying that lung volumes changed by less than 0.1 ml/min over 3 min at 30 cm H₂O pressure. The lung volume measured at a pressure of 5 cm H₂O was used as an index of stability at low lung volumes, whereas lung volumes measured at a pressure of 30 cm H₂O were assumed as TLC. Each group treated with an experimental surfactant preparation consisted of seven to 10 animals, except for the air-placebo groups, which consisted of five animals. All experiments were performed humanely and with the approval of the Animal Care and Use Committee of the Harbor-UCLA Research and Education Insititute.

Statistical Analysis

Surface-activity data are presented as mean ± SEM, with a minimum of four measures for each data point. The (a-A)PO₂ ratio was calculated with the values of Pa_O2, Pa_CO2, and FI_O2 and was used to express oxygenation. Oxygenation and data from the P-V curves are given as mean ± SEM, with a minimum of seven rats in each experimental group except for the air-placebo (n = 5) and nonlavaged (n = 3) groups. A two-tailed t test was used to compare two groups. The significance of differences between multiple experimental groups was compared through one-way analysis of variance (ANOVA). In those cases in which the F test showed a significant difference (p < 0.05) among groups, comparisons of different groups were made with the Student-Newman-Keuls test. A value of p < 0.05 was considered to indicate a significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The average lavage volume necessary to obtain a stable postlavage Pa_O2 < 80 mm Hg was 86 ± 2 ml. Two groups of five rats each were lavaged and treated with a bolus of air with a volume similar to the surfactant dose, and were used as controls to assure stability of the model. None of the rats in these air-placebo control groups showed a significant change in their Pa_O2 or Pa_CO2 values, or in the calculated (a-A)PO₂ compared with postlavage values during the ensuing 15- or 60-min period of ventilation. Two groups of three rats each were not lavaged or treated with surfactant or placebo, but were also ventilated for 15 or 60 min. These rats functioned as positive controls and had continuously high Pa_O2 values.

The four surfactant preparations based on extracted Survanta (i.e., ES, ES plus 1% B, ES plus 2% B, and ES plus 2% B and 1% C) had hystereses of 11 to 18 cm², a minimum surface tension of 1 to 7 mN/m on the first isotherm, and a reproducibility of 42 to 104% on the fifth isotherm (Figure 2). Thirty-six rats were treated with one of the four ES preparations. Their mean prelavage (a-A)PO₂ was 0.666 ± 0.022 in 100% oxygen, and decreased to 0.085 ± 0.005 following lavage (Figure 3). Surfactant instillation rapidly improved oxygenation, and 15 min after surfactant administration, the mean (a-A)PO₂ had increased over postlavage values by 88% with ES (n = 8), 116% with ES plus 1% B (n = 9), 185% with ES plus 2% B (n = 9), and 200% with ES plus 2% B and 1% C (n = 10) (Figure 3). The improvements in oxygenation with ES plus 2% B and ES plus 2% B and 1% C exceeded those with ES and ES plus 1% B at 5 min (p < 0.016), 10 min (p < 0.015), and 15 min (p < 0.007) after surfactant instillation. ES plus 2% B and 1% C not only improved Pa_O2, but also reduced Pa_CO2 values (29 ± 3 mm Hg versus 55 ± 11 mm Hg at 15 min after ES, p = 0.026). The mean deflation limbs of the P-V curves for the four ES preparations showed comparable TLC values measured at 30 cm H₂O, and stability at low lung volumes measured at a pressure of 5 cm H₂O (Figure 4). TLC in the ES groups exceeded that in the air-placebo group (p < 0.001) (Figure 4).

View larger version (28K): [in this window] [in a new window]   Figure 2.   In vitro surface activity of extracted Survanta (ES), ES plus 1% B, ES plus 2% B, and ES plus 2% B and 1% C, tested on a modified Langmuir-Wilhelmy balance.

View larger version (23K): [in this window] [in a new window]   Figure 3.   (a-A)PO2 ratios in nonlavaged rats (n = 3) and in lavaged rats treated with extracted Survanta (ES) (n = 8), ES plus 1% B (n = 9), ES plus 2% B (n = 9), ES plus 2% B and 1% C (n = 10), or air-placebo (n = 5) and ventilated with 100% oxygen for 15 min (*p < 0.016 versus ES and ES plus 1% B).

View larger version (22K): [in this window] [in a new window]   Figure 4.   Deflation limbs of the pressure-volume curves of nonlavaged rats and of lavaged rats treated with one of four surfactant preparations based on extracted Survanta (ES) or air-placebo and ventilated with 100% oxygen for 15 min.

The four surfactant preparations based on modified Survanta (S, S plus 2% B, S plus 2% B and 1% C, and S plus 2% KL₄) had hystereses of 18 to 23 cm², a minimum surface tension of 4 to 9 mN/m on the first isotherm, and a reproducibility of 61 to 89% on the fifth isotherm (Figure 5). Thirty-one rats were treated with one of the four S preparations. Their mean (a-A)PO₂ decreased from 0.660 ± 0.021 to 0.102 ± 0.004 following lavage (Figure 6). Sixty minutes after surfactant instillation, the mean (a-A)PO₂ had increased by 64% with S (n = 9), 112% with S plus 2% KL₄ (n = 7), 166% with S plus 2% B (n = 7), and 222% with S plus 2% B and 1% C (n = 8) over postlavage values (Figure 6). The differences in oxygenation between rats treated with S plus 2% B and S plus 2% B and 1% C were not significant, but at 45 and 60 min after surfactant instillation, S plus 2% B outperformed S (p < 0.005), and S plus 2% B and 1% C outperformed both S (p < 0.001) and S plus 2% KL₄ (p < 0.05). Mean deflation limbs of the P-V curves for the four surfactant preparations reflected comparable TLC and stability, and TLC exceeded that of the air-placebo controls (p < 0.001) (Figure 7).

View larger version (26K): [in this window] [in a new window]   Figure 5.   In vitro surface activity of Survanta (S), S plus 2% B, S plus 2% KL4, and S plus 2% B and 1% C, tested on a modified Langmuir-Wilhelmy balance.

View larger version (26K): [in this window] [in a new window]   Figure 6.   (a-A)PO2 ratios in nonlavaged rats (n = 3) and in lavaged rats treated with Survanta (S) (n = 9), S plus 2% B (n = 7), S plus 2% KL4 (n = 8), S plus 2% B and 1% C (n = 7), or air-placebo (n = 5) and ventilated with 100% oxygen for 60 min (*p < 0.016 versus S; #p < 0.02 versus S plus 2% B and 1% C).

View larger version (23K): [in this window] [in a new window]   Figure 7.   Deflation limbs of the pressure-volume curves of nonlavaged rats and of lavaged rats treated with one of four surfactant preparations based on modified Survanta (S) or air-placebo and ventilated with 100% oxygen for 60 min.

On the Langmuir-Wilhelmy balance, hystereses on the first isotherm of the surfactant preparations based on modified Survanta were greater than those of the preparations based on extracted Survanta (18 to 23 cm² versus 11 to 18 cm²). These differences in hysteresis were not reflected in the mean (a-A)PO₂ values at 15 min after treatment with ES or S, ES plus 2% B and 1% C, or S plus 2% B and 1% C (Figures 3 and 6). TLC at 15 min after treatment with ES and ES plus 2% B and 1% C was greater than at 60 min after instillation of their equivalent S preparations (Figures 4 and 7). However, only the difference between ES plus 2% B and 1% C and S plus 2% B and 1% C was significant (p = 0.0145).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this surfactant-deficient rat model, tracheal instillation of ES or S enriched with a combination of 2% synthetic surfactant peptide B and 1% synthetic surfactant peptide C produced the greatest increase in oxygenation, closely followed by ES and S enriched with 2% B. The addition of 2% KL₄ to S had an effect intermediate between that of S and S plus 2% B, whereas the addition of 1% B to ES had only a minor effect on oxygenation. These data indicate that spiking of Survanta with synthetic surfactant peptides has an acute positive effect on oxygenation that depends on the type and quantity of synthetic surfactant peptides added. Supplementation with a combination of 2% B and 1% C had an effect on oxygenation that was clearly superior to that with the other Survanta modifications.

All modifications of Survanta used in this study were about equally surface active when tested on the Langmuir-Wilhelmy balance, and were not really different from the unmodified preparations. This was reflected in the P-V curves, which were similar among the ES and S groups, though clearly different from those of air-placebo controls. The similarity of the P-V curves in the treated groups indicates that the various surfactant preparations had a similar effect on pulmonary function. The clear differences in oxygenation among the various groups were not reflected in the in vitro surface activity of the various preparations, or in the P-V curves, reemphasizing that pulmonary function measurements alone are not the best measure of improvement immediately following surfactant treatment (31). Ventilator settings and oxygenation may actually be better clinical indicators of improved gas exchange and overall pulmonary function. All surfactant preparations examined in the present study had a rapid effect on TLC and compliance in the treated groups as compared with air-placebo controls, which is in sharp contrast with the observation that lung compliance in premature infants with RDS does not increase in the first 2 h following surfactant replacement therapy despite an early improvement in gas exchange (11, 12, 31, 32).

It is well known that positive end-expiratory pressure (PEEP) by itself has some beneficial effects for oxygenation, and in combination with surfactant treatment can reestablish gas exchange in the lung and increase lung volume (33). Omitting PEEP generally leads to a dampened response to surfactant treatment and an underestimation of in vivo surfactant function. Although we did not use PEEP in this surfactant-deficient rat model, we found distinct differences in the effects of various surfactant preparations on oxygenation. Work currently in progress in our laboratory, using surfactant preparations consisting of synthetic phospholipids and synthetic surfactant peptides in the same lavaged rat model used in the present study, suggests that the differences in oxygenation might have been even greater with the use of PEEP. This adult RDS model of surfactant deficiency differs from a premature newborn model with respect to the presence of excess lung fluid and its effects on lung function and possible differences in surfactant distribution. We therefore plan to test these designer surfactant preparations in premature rabbits with idiopathic RDS.

The effect of S plus 2% KL₄ was disappointing when compared with that reported in studies in which a surfactant preparation consisting of phospholipids with 3% of the synthetic KL₄ peptide was used to treat premature rabbits (26), rhesus monkeys (28), and premature infants (27). PEEP during instillation enhances the intrapulmonary distribution of phospholipids plus KL₄ peptide (35), but was not applied in the experiments described here. KL₄ is supposed to function in the same way as SP-B (26), but recent work shows that this 21-residue surfactant peptide is a transmembrane alpha-helix with a mixed nonpolar/polar surface (36). This observation suggests that KL₄ acts more like SP-C than SP-B, and may explain the difference in oxygenation response between the Survanta preparations enriched with the synthetic surfactant peptide B and those enriched with KL₄.

We used two techniques to add synthetic peptides to Survanta: the addition of lyophilized peptides to extracted and lyophilized Survanta, or vortexing of lyophilized peptides into original Survanta, in order to gain insight into possible methodologic differences in in vitro and in vivo surface activity. On the Langmuir-Wilhelmy balance, hysteresis of surfactant preparations based on S was greater than that of preparations based on ES on the first isotherm. This reflects a difference in spreading, which may be secondary to a loss of palmitic acid after extraction. These differences decreased during cycling, and had disappeared on the fifth isotherm. During dynamic recycling, progressive reduction of the area under the isotherm occurs to a variable degree with almost any surfactant tested, natural or synthetic. This phenomenon can be explained by the observation that in native surfactant, short-chain lipids leave the surface monolayer under compression and remain solubilized in the hypophase, thus refining the surface until a DPPC-enriched monolayer is produced. Since the number of surface-active molecules present at the air-water interface decreases progressively, subsequent cycles will require more and longer compression to achieve minimum surface tension and establish a collapse plateau. This explanation is supported, among other data, by the results of studies of pure monolayers of individual lipids used in the preparation of synthetic surfactants (30).

The oxygenation data suggest that tracheal instillation of extracted and modified Survanta preparations with a similar synthetic peptide composition led to a comparable response in the surfactant-deficient rats in the present study. In contrast, treatment with ES plus B and C resulted in a larger TLC than did treatment with S plus with B and C. However, these data were gathered 15 and 60 min after instillation, respectively, and are difficult to compare. It is conceivable that the extracted form of Survanta could outperform modified Survanta, but our data do not allow a firm conclusion on this issue, since we studied these two modalities over different time courses.

Synthetic surfactant peptides based on the human sequence of surfactant proteins confer surface activity on phospholipid mixtures. These synthetic "designer" preparations are effective surfactants in surfactant-deficient animals (13, 15), and will play an important role in the next generation of surfactant replacement therapy (16) because they offer the possibility of "tailoring" surfactant preparations to various states of surfactant deficiency, insufficiency, and inactivation. The present study delineates a role for these designer peptides in the optimization of commercial surfactants, derived from animal lungs, that are currently approved for use in a clinical setting.