Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine

Z. Wang,1 A. L. Schwan,2 L. L. Lairson,2 J. S. O'Donnell,2 G. F. Byrne,2 A. Foye,1 B. A. Holm,3,4 and R. H. Notter1,5,6

Departments of 1Pediatrics, 5Environmental Medicine, and 6Biomedical Engineering, University of Rochester, Rochester 14642; Departments of 3Pediatrics and 4Obstetrics and Gynecology, State University of New York at Buffalo, Buffalo, New York 14214; and 2Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Submitted 16 October 2002 ; accepted in final form 23 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Surface activity and sensitivity to inhibition from phospholipase A2 (PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions <1 mN/m under dynamic compression on the Wilhelmy balance and on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression). DEPN-8 + 1.5% SP-B/C exhibited even greater adsorption and had overall dynamic surface tension lowering equal to CLSE on the bubble. In addition, films of DEPN-8 + 1.5% SP-B/C on the Wilhelmy balance had better respreading than CLSE after seven (but not two) cycles of compression-expansion at 23°C. DEPN-8 is structurally resistant to degradation by PLA2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2 led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.

exogenous surfactants; phospholipid analogs; phospholipase A2; phospholipase resistance; inhibition resistance; diether phosphonolipid; surfactant proteins


ENDOGENOUS LUNG SURFACTANT contains functionally important lipids and apoproteins (37), and its activity is compromised when these essential components are chemically degraded or altered. One important cause of such effects is through the action of phospholipases or proteases during inflammatory lung injury. Lytic enzymes of this kind can degrade and inactivate not only endogenous surfactant, but also exogenous lung surfactants used in treating clinical acute lung injury and the acute respiratory distress syndrome (ARDS). All current exogenous surfactant drugs contain substantial contents of glycerophospholipids including 1,2-dipalmitoyl-sn-3-phosphatidylcholine (DPPC), the most prevalent component of endogenous lung surfactant. Phospholipase-induced degradation of glycerophospholipids not only reduces the concentration of active surfactant but also generates byproducts like lysophosphatidylcholine (LPC) and fluid free fatty acids that can further decrease surface activity through biophysical interactions (21, 37, 59). Synthetic exogenous surfactants containing novel lipid components resistant to degradation by phospholipases have the potential to maintain their activity in the presence of these enzymes.

The present study investigates a synthetic phospholipase-resistant C16:0 diether phosphonolipid analog of DPPC (designated DEPN-8) originally synthesized by Turcotte, Notter, and coworkers (51, 52). DEPN-8 is studied both as a pure compound and in combination with column-purified mixed hydrophobic surfactant proteins (SP)-B and SP-C from calf lung surfactant extract (CLSE). Complementary biophysical methods (adsorption with subphase stirring, pulsating bubble surfactometer, Wilhelmy balance) are used to assess a range of relevant surface properties. Experiments examine the adsorption, dynamic surface tension lowering, and film respreading of DEPN-8 + 1.5% (by weight) SP-B/C, along with its resistance to chemical degradation by phospholipase A2 (PLA2) and its sensitivity to biophysical inhibition by C16:0 and C18:1 LPC and serum albumin. CLSE (equivalent to the clinical exogenous surfactant Infasurf) is used as a comparative standard of known high surface activity and inhibition resistance. The surface behavior of pure DEPN-8 is also examined relative to DPPC and column-purified bovine lung surfactant phospholipids (PPL).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Glycerophospholipids, PLA2, and Inhibitors

DPPC (>99% pure) was obtained from Avanti Polar Lipids (Alabaster, AL). DPPC gave a single defined spot on thin-layer chromatography (TLC) with a solvent system of 30:9: 25:7:25 (by volume) chloroform-ethanol-2-propanol-watertriethylamine (49). PLA2 (Naja naja venom, 2,500 units/mg protein), bovine serum albumin (BSA, essentially fatty acid free prepared from fraction V albumin), and C16:0 and C18:1 LPC were obtained from Sigma Chemical (St. Louis, MO) and were used without further purification.

Synthesis and Purification of DEPN-8

The diether phosphonolipid DEPN-8 was synthesized by a modification of the methods of Turcotte et al. (51, 52). The following reaction intermediates and synthesis/purification protocols were used.

(±)-1-Hexadecyloxy-2,3-propanediol. To DMSO (75 ml) was added solketal (5.00 g, 37.8 mmol) and finely divided (mortar and pestle) KOH (7.40 g, 132 mmol). This mixture was stirred for 10 min at room temperature, and C16H33Br (23.9 g, 78.3 mmol) was introduced. The two-phase mixture was stirred for 18 h at room temperature and then heated at 60°C for 90 min. After cooling, water (135 ml) was added, and the solution was extracted with an EtOAc/hexanes mixture (1:1 vol/vol, 3x 75 ml). The combined extracts were washed with water, dried over MgSO4, filtered, and concentrated to afford 13.9 g of crude (±)-4-(hexadecyloxy)methyl-2,2-dimethyl-1,3-dioxolane. The major portion of this latter crude compound was dissolved in a solution of MeOH (71.5 ml) and concentrated HCl (7.15 ml), and the mixture was stirred at room temperature for 18 h. Ether (250 ml) was added, and the mixture was washed with aqueous NaHCO3 (5x 100 ml) and water (5x 100 ml). The remaining ether was dried over MgSO4, filtered, and concentrated. The crude residue was recrystallized with hexanes to afford pure (±)-1-hexadecyloxy-2,3-propanediol (7.23 g, 62% yield over two steps).

(±)-2,3-Bis(hexadecyloxy)-1-propanol. Tritylation of (±)-1-hexadecyloxy-2,3-propanediol at the 3-position was performed as previously outlined (47) to give (±)-3-hexadecyloxy-1-trityl-2-propanol, the alkylation of which proceeded as follows. To DMSO (50 ml) was added (±)-1-hexadecyloxy-3-trityl-2-propanol (9.90 g, 17.7 mmol) and finely divided KOH (4.97 g, 88.6 mmol). This mixture was stirred for 10 min at room temperature, and C16H33Br (10.8 g, 35.4 mmol) was introduced. The two-phase mixture was stirred for 18 h at room temperature and then heated to 60°C for 90 min. After cooling, an EtOAc/hexanes mixture (1:1 vol/vol, 400 ml) was added, and the solution was washed with water (2x 100 ml) and brine (100 ml). The aqueous layers were back extracted with an EtOAc/hexanes mixture (1:1 vol/vol, 100 ml), and the combined extracts were dried over MgSO4. The solution was dried, concentrated, and subjected to flash chromatography on silica gel (100% hexanes, then 5% EtOAc in hexanes, vol/vol) to give pure (±)-2,3-bis(hexadecyloxy)-1-trityloxypropane (12.1 g, 87%), which was detritylated (5) to yield (±)-2,3-bis(hexadecyloxy)-1-propanol.

(±)Trimethyl(3-phosphonopropyl)ammonium, mono[2,3-bis(hexadecyloxy)propyl]ester (DEPN-8). Following closely the protocol of Turcotte et al. (52), 3-(bromopropyl)phosphonochloridic acid was prepared from 3-bromopropylphosphonic acid (2.00 g, 9.86 mmol) and PCl5 (2.46 g, 11.8 mmol). The crude 3-(bromopropyl)phosphonochloridic acid was mixed in a three-neck flask with CHCl3 (25 ml) by stirring under nitrogen in an ice bath. Solutions of (±)-2,3-bis(hexadecyloxy)-1-propanol (2.66 g, 4.93 mmol) in CHCl3 (25 ml) and Et3N (1.38 ml, 9.91 mmol) in CHCl3 (20 ml) were added simultaneously via dropping funnels over the course of 1.5 h. The solution was stirred for 48 h, at which time water (2.5 ml) was added and stirring continued for an additional 1 h. The mixture was concentrated, and a solution of CHCl3/MeOH/H2O (19:19:2, 40 ml) was added. To this mixture was added Amberlite exchange resin (47 ml), and the resulting solution was stirred for 1 h. The mixture was then filtered, taken up in CH2Cl2 (60 ml), washed with water (30 ml) and brine (30 ml), and dried over MgSO4. The material was filtered and concentrated to give 3.2 g of crude 2,3-bis(hexadecyloxy)-1-propyl hydrogen (3-bromopropyl)phosphonate, which was used immediately without purification in the next step. All of the crude phosphonate and a solution of 40% aqueous Me3N (21.4 ml, 142 mmol) were dissolved in a solution of CHCl3/isopropanol/MeCN (1.7:1.7:1, 64 ml), and this solution was stirred at 60°C for 48 h. After cooling, the solution was concentrated and taken up in CHCl3/MeOH (9:1, 30 ml), washed with a minimum of water, and dried over MgSO4. After concentration, the crude solid was taken up in CHCl3/MeOH/H2O (10:10:1, 100 ml), and after the addition of Amberlite (50 ml), the mixture was stirred for 1 h. After filtration, the Amberlite was rinsed with the same solvent, and the combined solutions were concentrated. The material was then partially dried by the successive addition and removal of benzene (3x 60 ml) on the rotary evaporator. The residue was taken up in CHCl3 and applied to a flash chromatography column containing flash-grade silica gel and CHCl3. After initial elution of 100 ml of CHCl3, the eluent was changed to CHCl3/MeOH/H2O (60:35:5), and elution under flash chromatography conditions continued until all the lipid was recovered. Concentration of lipid-containing fractions was followed by partial drying by the addition and removal of benzene (6x 60 ml) on the rotary evaporator to give 2.11 g of damp lipid. This material was dried to constant weight under vacuum at 50°C in a rotating tube oven in the presence of P2O5 to afford 1.89 g (yield 55% over two steps) of pure DEPN-8, melting point 208°C decomposition; literature 195°C decomposition (51).

CLSE, Hydrophobic Surfactant Proteins (SP-B/C), and Column-Purified Surfactant Phospholipids (PPL)

Large-aggregate surfactant was pelleted from calf lung lavage by centrifugation at 12,000 g for 30 min, followed by extraction with chloroform-methanol to obtain CLSE (18,37). Isolates of hydrophobic SP-B/C and of PPL were separated from CLSE by gel permeation chromatography on a 1.5 x 50-cm column packed with Sephadex LH-20 (Pharmacia-LKB Biotechnology, Piscataway, NJ) (15). The elution solvent was 1:1 by volume chloroform and methanol plus 5% 0.1 N HCl. Pooled fractions from the first and second column passes were extracted with chloroform-methanol (4) to remove acid. Final protein isolates contained only SP-B/C by SDS-PAGE and NH2-terminal amino acid sequencing and had no detectable phospholipid by phosphorus assay (1). Final PPL isolates had a protein content below the limits of detection of the assay of Lowry et al. (34) modified by the addition of 15% SDS.

Analysis of PLA2-Mediated Degradation of Surfactant Mixtures

Surfactants of interest (CLSE or DEPN-8 + 1.5% SP-B/C) in chloroform were dried under nitrogen and resuspended by vortexing in 5 mM Tris (hydroxymethylaminomethane) buffer containing 5 mM CaCl2 (pH 7.4). Lyophilized PLA2 (0.1 units/ml) suspended in the same buffer was added, and the mixture was incubated for 30 min at 37°C. Phospholipid (phosphonolipid) classes including LPC were analyzed by TLC using a solvent system of chloroform-methanol-2-propanol-water-triethylamine (30:9:25:7:25 by volume) (49). The weight percentage of each class was determined relative to total phospholipid (phosphonolipid) based on the assay of Ames (1).

Surface Activity Measurements

Adsorption experiments were done at 37 ± 0.5°C in a Teflon dish with a 35-ml subphase of buffered saline (10 mM HEPES, 1.5 mM CaCl2, and 150 mM NaCl, pH 7.0) stirred to minimize diffusion resistance as described previously (36, 39). At time zero, a bolus of surfactant dispersion (2.5 mg phospholipid in 5 ml of buffer) was injected into the stirred subphase, and adsorption surface pressure (surface tension lowering below that of the pure subphase) was measured as a function of time by the force on a partially submerged, sandblasted platinum Wilhelmy slide (36, 39). The final surfactant concentration for adsorption studies was uniform at 0.0625 mg of phospholipid/ml (2.5 mg surfactant phospholipid/40 ml of final subphase).

The overall surface tension-lowering ability of surfactant dispersions was measured during cycling at a physiological rate of 20 cycles/min at 37 ± 0.5°C on a pulsating bubble surfactometer (General Transco, Largo, FL) based on the design of Enhorning (9). Surface tension at minimum bubble radius (minimum surface tension, 50% area compression) was calculated as a function of time of pulsation from the measured pressure drop across the bubble interface using the Laplace equation for a spherical interface (9, 14). Surfactant concentration was 1.0 or 2.5 mg phospholipid/ml in 10 mM HEPES, 1.5 mM CaCl2, and 150 mM NaCl, pH 7.0. In experiments on inhibition, PLA2, albumin, or C16:0 or C18:1 LPC were initially added at the desired concentration to surfactant in buffer and incubated for 30 min at 37°C before bubble measurements.

Surface pressure-area isotherms for solvent-spread interfacial films were measured on a custom-designed Wilhelmy surface balance with a Teflon trough and ribbon barrier designed to minimize leakage at high surface pressure (48). Surfactants in 9:1 (vol/vol) hexane-ethanol were spread drop-wise from a syringe at the surface of a buffered saline subphase (10 mM HEPES, 1.5 mM CaCl2, 150 mM NaCl, pH 7.0) in the balance trough. A uniform "surface excess" initial film concentration of 15 Å2/molecule was used to emphasize molecular behavior during dynamic cycling in the collapse regime. After a 10-min pause for solvent evaporation, films were cycled with a compression ratio of 4.35:1 at a rate of 5 min/cycle at 23 ± 1°C. Surface pressure was measured from the force on a sandblasted platinum Wilhelmy slide dipped into the ribbon-confined interface, and a second slide outside the ribbon monitored film leakage (not present in any experiments reported). Film respreading was quantitated as ratios of compression collapse plateaus on the surface pressure-area isotherm for cycle 2 or cycle 7 relative to cycle 1 (37, 40, 52). A value of 1 for the ratio of compression collapse plateaus on cycle 2/cycle 1 or cycle 7/cycle 1 indicated complete respreading, whereas a ratio of 0 indicated no respreading of ejected film material (37, 40, 52).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The adsorption and overall dynamic surface activity of DEPN-8 and DEPN-8 + 1.5% SP-B/C relative to DPPC, PPL, DPPC + 1.5% SP-B/C, and CLSE are shown in Figs. 1 and 2. Pure DEPN-8 adsorbed better than DPPC, but not as well as PPL (Fig. 1). The adsorption of both DEPN-8 and DPPC was greatly improved by combination with 1.5% by weight of purified bovine hydophobic surfactant proteins. DEPN-8 + 1.5% SP-B/C rapidly adsorbed to final equilibrium surface pressures near 48 mN/m, behavior equivalent to solvent-extracted large-aggregate calf lung surfactant (CLSE, Fig. 1). In pulsating bubble assessments of overall dynamic surface tension lowering in surfactant dispersions, DEPN-8 reached much lower minimum surface tensions than DPPC or PPL (Fig. 2). Dispersions of DEPN-8 + 1.5% SP-B/C also had greater dynamic surface activity than DPPC + 1.5% SP-B/C. The overall dynamic surface tension-lowering ability of DEPN-8 + 1.5% SP-B/C on the pulsating bubble apparatus was equivalent to the highly active lung surfactant extract CLSE (Fig. 2).



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Fig. 1. Adsorption of DEPN-8 and glycerophospholipids with and without hydrophobic surfactant proteins. Data are adsorption surface pressure as a function of time for dispersions in a dish with a stirred subphase of buffered 0.15 M NaCl (37°C; pH, 7.0). Surfactants are DEPN-8 ± 1.5% (by wt) bovine SP-B/C, DPPC ± 1.5% SP-B/C, column-purified bovine surfactant phospholipids (PPL), and calf lung surfactant extract (CLSE). The latter extract contains all of the hydrophobic components of lavaged large-aggregate bovine lung surfactant. Final subphase surfactant concentration was always 0.0625 mg phospholipid/ml. Data are means ± SE for n = 3-4.

 


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Fig. 2. Dynamic surface activity of DEPN-8 and glycerophospholipids with and without added hydrophobic surfactant proteins. Data are minimum surface tensions as a function of time on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression) at a uniform phospholipid concentration of 1 mg/ml. Surfactant mixtures are the same as in Fig. 1. Data are means ± SE for n = 4-8.

 

Representative surface pressure-area isotherms for solvent-spread films of DEPN-8, DPPC, PPL, DEPN-8 + 1.5% SP-B/C, and CLSE during compression and expansion in a Wilhelmy surface balance are shown in Fig. 3. Maximum surface pressures and respreading ratios (means ± SE) for multiple independent experiments for each film type are given in Table 1. All the different spread films studied on the Wilhelmy balance had high maximum surface pressures near 72 mN/m (minimum surface tensions <1 mN/m) at the experimental temperature of 23°C. However, distinct differences in dynamic respreading during cycling were present (Table 1). Films of DEPN-8 had much better respreading than DPPC on the basis of collapse plateau ratios for compression curves 2/1 and 7/1. The relative respreading of these two compounds was assessed at room temperature but would not be expected to change appreciably at body temperature, since both DEPN-8 and DPPC would still be below their gel-to-liquid crystal transitions (45 and 41°C, respectively) (33, 52). The greatest overall dynamic respreading was found in films of DEPN-8 + 1.5% SP-B/C and CLSE (Table 1). Films of DEPN-8 + 1.5% SP-B/C had lower respreading than CLSE on cycle 2/cycle 1, but substantially better respreading on cycle 7/cycle 1 (Table 1).



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Fig. 3. Representative surface pressure-area isotherms for spread films of DEPN-8, DEPN-8 + 1.5% SP-B/C, DPPC, PPL, and CLSE in a Wilhelmy balance. Representative isotherms show the 1st, 2nd, and 7th compressions, and the 1st expansion, for films spread in hexane-ethanol (9:1 by volume) to a surface-excess concentration of 15 Å2/molecule in a modified Wilhelmy balance. Cycling rate was 5 min/cycle at 23°C. Table 1 gives maximum surface pressures and respreading ratios for the complete sets of isotherms studied for each film type (means ± SE, n = 3-5).

 

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Table 1. Respreading ratios and maximum surface pressures for surfactant films studied on the Wilhelmy surface balance in Fig. 3

 

Additional experiments investigated the effects of PLA2 on the adsorption and dynamic surface activity of DEPN-8, DEPN-8 + 1.5% SP-B/C, and CLSE (Figs. 4 and 5). DEPN-8 and DEPN-8 + 1.5% SP-B/C were unchanged in adsorption following incubation with PLA2, whereas the adsorption of CLSE was slightly decreased (Fig. 4 vs. Fig. 1). The minimum surface tension reached by CLSE dispersions on the pulsating bubble apparatus was significantly increased by PLA2 (0.1 unit/ml) (Fig. 5 vs. Fig. 2). In contrast, DEPN-8 and DEPN-8 + 1.5% SP-B/C maintained their ability to reduce surface tension to <1 mN/m on the bubble apparatus in the presence of PLA2 (Fig. 5). To help assess mechanisms contributing to these surface activity findings, we examined the ability of PLA2 to chemically degrade DEPN-8 + 1.5% SP-B/C and CLSE. CLSE and DEPN-8 + 1.5% SP-B/C were incubated with PLA2 (0.1 unit/ml) for 30 min at 37°C, followed by compositional analysis of phospholipid (phosphonolipid) classes by TLC. A significant content of LPC was found following incubation of CLSE with PLA2, indicating substantial enzyme-induced degradation of glycerophospholipid (Table 2). In contrast, incubation of DEPN-8 + 1.5% SP-B/C with PLA2 did not result in any enzyme-induced degradation (100% of lipid material ran as phosphatidylcholine on TLC, consistent with intact DEPN-8) (Table 2).



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Fig. 4. Effects of phospholipase A2 (PLA2) on the adsorption of DEPN-8, DEPN-8 + 1.5% SP-B/C, and CLSE. Adsorption isotherms of surface pressure vs. time are shown for dispersions of DEPN-8, DEPN-8 + 1.5% SP-B/C, and CLSE in the presence of 0.1 units/ml of PLA2. Adsorption isotherms for these surfactants in the absence of PLA2 are in Fig. 1. Final subphase phospholipid concentration was uniform at 0.0625 mg/ml in buffered 0.15 M NaCl at 37°C and pH 7.0. Data are means ± SE for n = 3.

 


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Fig. 5. Effects of PLA2 on the dynamic surface tension lowering of DEPN-8, DEPN-8 + 1.5% SP-B/C, and CLSE. Minimum surface tensions as a function of time of pulsation on a bubble surfactometer (37°C, 20 cycles/min, 50% area compression) are shown for DEPN-8, DEPN-8 + 1.5% SP-B/C, and CLSE (1 mg phospholipid/ml) in the presence of PLA2 (0.1 unit/ml). Dynamic surface tension lowering data for these surfactants in the absence of PLA2 are in Fig. 2. Data are means ± SE for n = 4-8.

 

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Table 2. Effect of PLA2 in degrading extracted bovine lung surfactant (CLSE) and DEPN-8 + 1.5% bovine SP-B/C

 

LPC produced by the activity of PLA2 indicates not only that functional surfactant glycerophospholipids have been degraded but also can itself impair surface activity through biophysical interactions (21). The relative sensitivity of DEPN-8 + 1.5% SP-B/C and CLSE to biophysical inhibition by C16:0 and C18:1 LPC is shown in Figs. 6 and 7. In pulsating bubble studies, C18:1 LPC had a greater inhibitory effect than C16:0 LPC on the dynamic surface tension-lowering ability of dispersions of CLSE and DEPN-8 + 1.5% SP-B/C (Fig. 6, B vs. A). The two surfactants were qualitatively similar in being inhibited more severely in dynamic surface activity when the content of C18:1 or C16:0 LPC was increased from 15 to 30% by weight relative to phospholipid. However, DEPN-8 + 1.5% SP-B/C was quantitatively more resistant than CLSE to the inhibitory effects of a given amount of either C18:1 or C16:0 LPC on the pulsating bubble (Fig. 6, A and B). The inhibitory effects of C18:1 LPC on adsorption were less pronounced than on dynamic surface activity and did not differ significantly between DEPN-8 + 1.5% SPB/C and CLSE (Fig. 7).



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Fig. 6. Effects of lysophosphatidylcholine (LPC) on the dynamic surface activity of DEPN-8 + 1.5% SP-B/C relative to CLSE. A: C16:0 LPC; B: C18:1 LPC. Minimum surface tension is plotted as a function of time for dispersions of DEPN-8 + 1.5% SP-B/C and CLSE mixed with LPC on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression, 1 mg phospholipid/ml). Data are means ± SE (n = 5-9 for A and 4-8 for B).

 


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Fig. 7. Effects of LPC on the adsorption of DEPN-8 + SP-B/C relative to CLSE. Curves show the effects of C18:1 LPC (15 or 30% by weight) on the surface pressure-time adsorption of DEPN-8 + 1.5% SP-B/C and CLSE. Final surfactant phospholipid concentration was 0.0625 mg/ml in buffered 0.15 M NaCl at pH 7.0 and 37°C. Data are means ± SE for n = 3-4.

 

A final set of studies examined the inhibitory effects of BSA (which does not act by chemical degradation or generate LPC) on the surface activity of DEPN-8 + 1.5% SP-B/C and CLSE (Figs. 8 and 9). At phospholipid (phosphonolipid) concentrations of 1 and 2.5 mg/ml on the pulsating bubble surfactometer, DEPN-8 + 1.5% SP-B/C and CLSE were inhibited in equivalent fashion by BSA (0.5 and 3 mg protein/ml at the low and high surfactant concentrations, respectively) (Fig. 8). Albumin-induced inhibition in bubble studies was seen as an extended time course of surface tension lowering, although minimum surface tensions <1 mN/m were still reached by DEPN-8 + 1.5% SP-B/C and CLSE. The two preparations were also similar in being unaffected in dynamic surface activity by BSA (0.5 mg/ml) at the higher surfactant concentration of 2.5 mg phospholipid/ml on the bubble (data not shown in Fig. 8). In addition, DEPN-8 + 1.5% SP-B/C and CLSE were equivalent in their sensitivity to inhibition by BSA (50% by weight relative to phospholipid) in adsorption studies at low surfactant concentration (Fig. 9). The albumin-induced decrease in adsorption for both surfactants was greater than for LPC in Fig. 7, consistent with the known ability of albumin to adsorb at the air-water interface and hinder the entry of lung surfactant components into the surface (19, 21).



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Fig. 8. Effects of bovine serum albumin (BSA) on the dynamic surface activity of DEPN-8 + SP-B/C relative to CLSE. Minimum surface tensions are shown as a function of time of pulsation on a bubble surfactometer (37°C, 20 cycles/min, 50% area compression, 1 or 2.5 mg phospholipid/ml) for DEPN-8 + 1.5% SP-B/C and CLSE in the presence and absence of BSA (0.5 or 3 mg protein/ml). Subphase was buffered 0.15 M NaCl (pH 7.0). Data are means ± SE for n = 4-10.

 


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Fig. 9. Effects of BSA on the adsorption of DEPN-8 + SP-B/C relative to CLSE. Adsorption isotherms (surface pressure vs. time) are shown for DEPN-8 + 1.5% SP-B/C and CLSE in the presence and absence of BSA (50% by wt). Final subphase surfactant concentration was always 0.0625 mg phospholipid/ml in buffered 0.15 M NaCl at pH 7.0 and 37°C. Data are means ± SE for n = 3-4.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study demonstrates that DEPN-8, a phospholipase-resistant diether phosphonolipid analog of DPPC, can be combined with bovine SP-B/C to form a highly active synthetic exogenous surfactant. DEPN-8 as a pure compound had greater adsorption and film respreading than DPPC, and its surface activity was increased further by combination with bovine hydrophobic surfactant proteins. DEPN-8 + 1.5% SP-B/C had adsorption and dynamic surface activity equal to CLSE, a chloroform-methanol extract of lavaged calf lung surfactant equivalent to the clinical exogenous surfactant Infasurf (Figs. 1, 2, 3, Table 1). CLSE and Infasurf have previously been shown to be highly active in reversing surfactant deficiency and dysfunction in animals and humans (see Refs. 37, 38, 41 for review). In addition, DEPN-8 + 1.5% SP-B/C maintained its chemical integrity and high surface activity in the presence of PLA2 (Figs. 4 and 5; Table 2). In contrast, glycerophospholipids in CLSE were degraded by PLA2, and its surface activity was impaired in the presence of this enzyme. DEPN-8 + 1.5% SP-B/C also resisted inhibition by LPC more effectively than CLSE (Figs. 6 and 7), and the two preparations had similar inhibition sensitivity to albumin (Figs. 8 and 9).

There is significant interest in new synthetic lung surfactants containing lipids plus recombinant surfactant proteins or related synthetic peptides (see Refs. 22, 24, 25, 37, 58 for review). The majority of prior work in this area has focused on the protein rather than the lipid constituents of such materials. The results here demonstrate that synthetic surfactants with high surface activity and inhibition resistance can be formed by combining novel lipids with bovine SP-B/C. Surfactants containing nonhuman SP-B/C can be administered clinically, but ideal synthetic lipid/protein surfactants would use human-sequence recombinant proteins (6, 13, 53, 55, 61) or synthetic peptides (12, 24, 25, 37, 54, 56-58, 60). The latter are particularly attractive because they can be synthesized and purified with relative ease by solid-state technology. However, to be useful clinically, synthetic peptides must approach or exceed native surfactant proteins in activity. The present study did not examine human-sequence proteins or peptides in combination with DEPN-8, and further studies including physiological activity assessments on such surfactants appear warranted.

The synthesis and characterization of DEPN-8 have been reported previously by Turcotte, Notter, and coworkers (29-33, 46, 51, 52). DEPN-8 contains saturated C16:0 alkyl moieties in analogy with the acyl chains of DPPC and has the same choline N-head group. The saturated C16:0 chains of DEPN-8 promote the formation of tightly packed surface films that generate very low surface tensions under dynamic compression (Figs. 2 and 3; Table 1). DEPN-8 also has ether, rather than ester, linkages between the fatty chains and the glycerol backbone, plus a methylene for oxygen substitution in the head group so that it is a phosphonolipid rather than a phospholipid (30, 51, 52). Ether linkages in DEPN-8 increase chain mobility and facilitate film respreading during cycling (31, 32, 52). Films of DEPN-8 + 1.5% SP-B/C in the present study had very impressive respreading, which exceeded that of CLSE, based on collapse plateau ratios for cycle 7/cycle 1 (but not cycle 2/cycle 1) (Table 1). DEPN-8 is also able to form interdigitated as well as normal opposed bilayers (46), which may help enhance its adsorption.

Several modifications of our prior synthesis and purification methods for DEPN-8 were employed here to improve efficiency and cost. One modification involved the synthesis of (±)-2,3-bis(hexadecyloxy)-1-propanol, a required intermediate that was previously prepared through alkylation of solketal [(±)-2,2-dimethyl-1,3-dioxolane-4-methanol] followed by a hydrolysis to (±)-1-hexadecyloxy-2,3-propanediol, tritylation of the 3-hydroxy group, alkylation of the 2-hydroxy, and detritylation (30, 51, 52). In the modified preparation scheme (MATERIALS AND METHODS), alkylation of the hydroxy group with hexadecyl bromide and KOH in DMSO (26) proved to be more convenient and economical. Two equivalents of hexadecyl bromide were necessary per mole of starting alcohol, but this alkylating agent is ~100 times cheaper than the corresponding mesylate (3, 30). Two additional adaptations of past procedures were used in generating final product DEPN-8. First, an intermediary purification originally used during installation of the polar head group was dropped for simplicity. Second, final purification of DEPN-8 was done by flash chromatography, avoiding the need for preparative HPLC and its attendant difficulties. These modifications improved the ease of final DEPN-8 production without a significant loss in yield or purity.

The molecular structure of DEPN-8 makes it resistant to degradation by PLA1, -A2, and -D (51, 52), and partially resistant to PLC (29). The phospholipase resistance of DEPN-8 has direct implications for its use in synthetic surfactants for treating lung injury. PLA2 is thought to play important roles in the pathogenesis of meconium aspiration syndrome (27, 45) and ARDS (50). PLA2 is present in meconium (45) and is known to be inhibitory to surfactant function (2, 8, 10, 20, 45). PLA2 not only can degrade and deplete active surfactant glycerophospholipids but also produces reaction byproducts such as LPC and free fatty acids that interact biophysically with intact surfactant to further impair surface activity (16, 21, 59). LPC and free fatty acids can also injure the alveolo-capillary membrane and increase its permeability to exacerbate pulmonary edema (17, 35). Our experiments examined the direct effects of PLA2 on the composition and surface activity of surfactant mixtures, as well as biophysical inhibition by LPC. DEPN-8 + 1.5% SP-B/C was chemically resistant to PLA2 as expected (Table 2) and was not inhibited in surface activity by this enzyme (Figs. 4 and 5). Moreover, DEPN-8 + 1.5% SP-B/C was more resistant than CLSE to biophysical inhibition from LPC (Figs. 6 and 7). DEPN-8 + 1.5% SP-B/C and CLSE were equivalent in their sensitivity to inhibition by serum albumin (Figs. 8 and 9).

Although incubation with PLA2 significantly reduced the dynamic surface activity of CLSE, this enzyme had less effect on adsorption (Fig. 4 vs. 5). LPC at high concentrations similarly had a greater inhibitory effect on dynamic surface activity than on adsorption (Fig. 6 vs. 7). This likely reflects the fact that LPC and degradation-produced fluid free fatty acids are themselves able to adsorb rapidly to high surface pressures at the air-water interface (16, 21, 59). As a result, the presence of these substances in mixtures with CLSE or DEPN-8 + 1.5% SP-B/C tends to cause less apparent change in adsorption surface pressure than inhibitors like albumin. The effects of LPC or PLA2-induced fluid free fatty acids in raising minimum surface tension during cycling are more prominent, because this property depends strongly on molecular interactions within the dynamically compressed surface film. LPC and unsaturated fatty acids have previously been shown to mix into and fluidize lung surfactant films at the air-water interface, impairing their ability to reduce surface tension during dynamic compression (16, 21, 37, 59).

The molecular structural differences in DEPN-8 relative to native glycerophospholipids in principle affect its metabolic processing in vivo, but this was not investigated in the present study. PLA2 and other phospholipases may play a role in surfactant metabolism by degrading phospholipids (e.g., 11, 28), and the phospholipase resistance of DEPN-8 may affect its processing and recycling by alveolar type II pneumocytes. However, synthetic diether analogs similar to DEPN-8 have been shown to enter lung surfactant metabolic pathways (23, 42-44). Moreover, no evidence of short-term toxicity in animals has been found from tracheally instilled surfactants containing DEPN-8 (7). More detailed investigations on the pulmonary metabolism and long-term biological effects of DEPN-8 are needed before synthetic surfactants containing this compound can be used clinically. The activities of DEPN-8 + 1.5% SP-B/C and related peptide-containing surfactants also need to be studied in animal models of surfactant deficiency and surfactant dysfunction to extend the physicochemical assessments of the present work.

In summary, this paper examines the surface activity and inhibition resistance of DEPN-8, a C16:0 diether phosphonolipid analog of DPPC, combined with 1.5% by weight of column-purified bovine SP-B/C. DEPN-8 + 1.5% SP-B/C had adsorption and overall dynamic surface tension-lowering ability equal to CLSE, the substance of the highly active clinical exogenous surfactant Infasurf. Films of DEPN-8 + 1.5% SP-B/C also had better respreading than CLSE after seven cycles (but not two cycles) of compression-expansion on the Wilhelmy balance. DEPN-8 + 1.5% SP-B/C was not degraded chemically by PLA2 and maintained high surface activity in the presence of this enzyme. CLSE was degraded by PLA2 and had significantly reduced surface activity in its presence. DEPN-8 + 1.5% SP-B/C was less sensitive than CLSE to biophysical inhibition by C16:0 and C18:1 LPC, and both surfactants were similar in their sensitivity to inhibition by serum albumin. The high activity and inhibition resistance of DEPN-8 + 1.5% SP-B/C make this or related surfactants of potential utility for treating surfactant dysfunction in inflammatory lung injury.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The financial support of National Heart, Lung, and Blood Institute Grant HL-56176 is gratefully acknowledged.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. H. Notter, Dept. of Pediatrics, Box 850, Univ. of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: robert_notter{at}urmc.rochester.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8: 115-118, 1966.
  2. Arbibe L, Koumanov K, Vail D, Rougeot C, Faure G, Havet N, Longacre S, Vargaftig BB, Voelker D, Wolf C, and Torqui L. Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 interaction. J Clin Invest 102: 1152-1160, 1998.[Abstract/Free Full Text]
  3. Baumann WJ and Mangold HK. Reactions of aliphatic methanesulfonates. II. Syntheses of long-chain di- and trialkyl glyceryl ethers. J Org Chem 31: 498-500, 1966.[ISI]
  4. Bligh EG and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917, 1959.[ISI]
  5. Browne JE, Freeman RT, Russell JC, and Sammes PG. On the preparation of some phospholipid analogues. J Chem Soc, Perkin 1: 645-652, 2000.
  6. Crouch E, Chang D, Rust K, Persson A, and Heuser J. Recombinant pulmonary surfactant protein D. Post-translational modification and molecular assembly. J Biol Chem 269: 15808-15813, 1994.[Abstract/Free Full Text]
  7. Dizon-Co L, Ikegami M, Ueda T, Jobe AH, Lin WH, Turcotte JG, Notter RH, and Rider ED. In vivo function of surfactants containing PC analogs. Am J Respir Crit Care Med 150: 918-923, 1994.[Abstract]
  8. Duncan JE, Hatch GM, and Belik J. Susceptibility of exogenous surfactant to phospholipase A2 degradation. Can J Physiol Pharmacol 74: 957-963, 1996.[ISI][Medline]
  9. Enhorning G. Pulsating bubble technique for evaluation of pulmonary surfactant. J Appl Physiol 43: 198-203, 1977.[Abstract/Free Full Text]
  10. Enhorning G, Shumel B, Keicher L, Sokolowski J, and Holm BA. Phospholipases introduced into the hypophase affect the surfactant film outlining a bubble. J Appl Physiol 73: 941-945, 1992.[Abstract/Free Full Text]
  11. Fisher AB, Dodia C, Chander A, and Jain M. A competitive inhibitor of phospholipase A2 decreases surfactant degradation by the rat lung. Biochem J 288: 407-411, 1992.[ISI][Medline]
  12. Gordon LM, Horvath S, Longo ML, Zasadzinski JAN, Taeusch HW, Faull K, Leung C, and Waring AJ. Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments. Protein Sci 5: 1662-1675, 1996.[Abstract/Free Full Text]
  13. Haas C, Voss T, and Engel J. Assembly and disulfide rearrangement of recombinant surfactant protein A in vitro. Eur J Biochem 197: 799-803, 1991.[Abstract]
  14. Hall SB, Bermel MS, Ko YT, Palmer HJ, Enhorning GA, and Notter RH. Approximations in the measurement of surface tension with the oscillating bubble surfactometer. J Appl Physiol 75: 468-477, 1993.[Abstract]
  15. Hall SB, Hyde RW, and Notter RH. Changes in subphase surfactant aggregates in rabbits injured by free fatty acid. Am J Respir Crit Care Med 149: 1099-1106, 1994.[Abstract]
  16. Hall SB, Lu ZR, Venkitaraman AR, Hyde RW, and Notter RH. Inhibition of pulmonary surfactant by oleic acid: mechanisms and characteristics. J Appl Physiol 72: 1708-1716, 1992.[Abstract/Free Full Text]
  17. Hall SB, Notter RH, Smith RJ, and Hyde RW. Altered function of pulmonary surfactant in fatty acid lung injury. J Appl Physiol 69: 1143-1149, 1990.[Abstract/Free Full Text]
  18. Hall SB, Wang Z, and Notter RH. Separation of subfractions of the hydrophobic components of calf lung surfactant. J Lipid Res 35: 1386-1394, 1994.[Abstract]
  19. Holm BA, Enhorning G, and Notter RH. A biophysical mechanism by which plasma proteins inhibit lung surfactant activity. Chem Phys Lipids 49: 49-55, 1988.[ISI][Medline]
  20. Holm BA, Kelcher L, Liu M, Sokolowski J, and Enhorning G. Inhibition of pulmonary surfactant by phospholipases. J Appl Physiol 71: 317-321, 1991.[Abstract/Free Full Text]
  21. Holm BA, Wang Z, and Notter RH. Multiple mechanisms of lung surfactant inhibition. Pediatr Res 46: 85-93, 1999.[Abstract]
  22. Holm BA and Waring AJ. Designer surfactants: the next generation in surfactant replacement. Clin Perinatol 20: 813-829, 1993.[ISI][Medline]
  23. Jacobs H, Jobe AH, Ikegami M, Miller D, and Jones S. Reutilization of phosphatidylcholine analogues by the pulmonary surfactant system. The lack of specificity. Biochim Biophys Acta 793: 300-309, 1984.[ISI][Medline]
  24. Johansson J, Curstedt T, and Robertson B. Synthetic protein analogues in artificial surfactants. Acta Paediatr 85: 642-646, 1996.[ISI][Medline]
  25. Johansson J, Gustafsson M, Zaltash S, Robertson B, and Curstedt T. Synthetic surfactant protein analogs. Biol Neonate 74, Suppl: 9-14, 1998.[ISI][Medline]
  26. Johnstone RAW and Rose ME. A rapid, simple, and mild procedure for alkylation of phenols, alcohols, amides, and acids. Tetrahedron 35: 2169-2173, 1979.[ISI]
  27. Kaapa P. Meconium aspiration syndrome: a role for phospholipase A2 in the pathogenesis? Acta Paediatr 90: 365-367, 2001.[ISI][Medline]
  28. Kishikawa T. Phospholipase activities of surfactant fractions and their role in the morphological change in surfactants in vivo. J Submicrosc Cytol Pathol 22: 507-513, 1990.[ISI][Medline]
  29. Lin WH, Cramer SG, Turcotte JG, and Thrall RS. A diether phosphonolipid surfactant analog, DEPN-8, is resistant to phospholipase-C cleavage. Respiration 64: 96-101, 1997.[ISI][Medline]
  30. Lin WHC. Synthesis and purification of novel diether and ether-amide analogs of dipalmitoyl phosphatidylcholine (PhD thesis). Kingston, RI: Univ. of Rhode Island, 1989.
  31. Liu H, Lu RZ, Turcotte JG, and Notter RH. Dynamic interfacial properties of surface-excess films of phospholipids and phosphonolipid analogs. I. Effects of pH. J Colloid Interface Sci 167: 378-390, 1994.[ISI]
  32. Liu H, Turcotte JG, and Notter RH. Dynamic interfacial properties of surface-excess films of phospholipid and phosphonolipid analogs: II. Effects of chain linkage and headgroup structure. J Colloid Interface Sci 167: 391-400, 1994.[ISI]
  33. Liu H, Turcotte JG, and Notter RH. Thermotropic behavior of structurally-related phospholipids and phosphonolipid analogs of lung surfactant glycerophospholipids. Langmuir 11: 101-107, 1995.[ISI]
  34. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 132: 265-275, 1951.
  35. Niewoehner D, Rice K, Sinha A, and Wangensteen D. Injurious effects of lysophosphatidylcholine on barrier properties of alveolar epithelium. J Appl Physiol 63: 1979-1986, 1987.[Abstract/Free Full Text]
  36. Notter R, Taubold R, and Finkelstein J. Comparative adsorption of natural lung surfactant, extracted phospholipids, and synthetic phospholipid mixtures. Chem Phys Lipids 33: 67-80, 1983.[ISI][Medline]
  37. Notter RH. Lung Surfactants: Basic Science and Clinical Applications. New York: Dekker, 2000.
  38. Notter RH, Apostolakos M, Holm BA, Willson D, Wang Z, Finkelstein JN, and Hyde RW. Surfactant therapy and its potential use with other agents in term infants, children and adults with acute lung injury. Perspectives in Neonatol 1: 4-20, 2000.
  39. Notter RH, Smith S, Taubold RD, and Finkelstein JN. Path dependence of adsorption behavior of mixtures containing dipalmitoyl phosphatidylcholine. Pediatr Res 16: 515-519, 1982.[Abstract]
  40. Notter RH, Tabak SA, and Mavis RD. Surface properties of binary mixtures of some pulmonary surfactant components. J Lipid Res 21: 10-22, 1980.[Abstract]
  41. Notter RH and Wang Z. Pulmonary surfactant: physical chemistry, physiology and replacement. Rev Chem Eng 13: 1-118, 1997.[ISI]
  42. Rider ED, Ikegami M, and Jobe AH. Intrapulmonary catabolism of surfactant saturated phosphatidylcholine in rabbits. J Appl Physiol 69: 1856-1862, 1990.[Abstract/Free Full Text]
  43. Rider ED, Ikegami M, and Jobe AH. Localization of alveolar surfactant clearance in rabbit lung cells. Am J Physiol Lung Cell Mol Physiol 263: L201-L209, 1992.[Abstract/Free Full Text]
  44. Rider ED, Pinkerton KE, and Jobe AH. Characterization of rabbit lung lysosomes and their role in surfactant dipalmitoylphosphatidylcholine catabolism. J Biol Chem 266: 22522-22528, 1991.[Abstract/Free Full Text]
  45. Schrama AJJ, de Beufort AJ, Sukul YRM, Jansen SM, Poorthuis BJHM, and Berger HM. Phospholipase A2 is present in meconium and inhibits the activity of pulmonary surfactant: an in vitro study. Acta Paediatr 90: 412-416, 2001.[ISI][Medline]
  46. Skita V, Chester DW, Oliver CJ, Turcotte JG, and Notter RH. Bilayer characteristics of a diether phosphonolipid analog of the major lung surfactant glycerophospholipid dipalmitoyl phosphatidylcholine. J Lipid Res 36: 1116-1127, 1995.[Abstract]
  47. Stewart LC and Kates M. Synthesis and characterization of deuterium-labeled dihexadecylglycerol and diphytanylglycerol phospholipids. Chem Phys Lipids 50: 23-42, 1989.[ISI]
  48. Tabak SA and Notter RH. A modified technique for dynamic surface pressure and relaxation measurements at the air-water interface. Rev Sci Instrum 48: 1196-1201, 1977.[ISI]
  49. Touchstone JC, Chen JC, and Beaver KM. Improved separation of phospholipids in thin-layer chromatography. Lipids 15: 61-62, 1980.[ISI]
  50. Touqui L and Arbibe L. A role for phospholipase A2 in ARDS pathogenesis. Mol Med Today 5: 244-249, 1999.[ISI][Medline]
  51. Turcotte JG, Lin WH, Pivarnik PE, Sacco AM, Bermel MS, Lu Z, and Notter RH. Chemical synthesis and surface activity of lung surfactant phospholipid analogs. II. Racemic N-substituted diether phosphonolipids. Biochim Biophys Acta 1084: 1-12, 1991.[ISI][Medline]
  52. Turcotte JG, Sacco AM, Steim JM, Tabak SA, and Notter RH. Chemical synthesis and surface properties of an analog of the pulmonary surfactant dipalmitoyl phosphatidylcholine analog. Biochim Biophys Acta 488: 235-248, 1977.[ISI][Medline]
  53. Veldhuizen EJA, Batenburg JJ, Vandenbussche G, Putz G, van Golde LMG, and Haagsman HP. Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence. Biochim Biophys Acta 1416: 295-308, 1999.[ISI][Medline]
  54. Veldhuizen EJA, Waring AJ, Walther FJ, Batenburg JJ, van Golde LMG, and Haagsman HP. Dimeric N-terminal segment of human surfactant protein B (dSP-B1-25) has enhanced surface properties compared to monomeric SP-B1-25. Biophys J 79: 377-384, 2000.[Abstract/Free Full Text]
  55. Voss T, Melchers K, Scheirle G, and Schafer KP. Structural comparison of recombinant pulmonary surfactant protein SP-A derived from two human coding sequences: implications for the chain composition of natural human SP-A. Am J Respir Cell Mol Biol 4: 88-94, 1991.[ISI][Medline]
  56. Walther F, Hernandez-Juviel J, Bruni R, and Waring AJ. Protein composition of synthetic surfactant affects gas exchange in surfactant-deficient rats. Pediatr Res 43: 666-673, 1998.[Abstract]
  57. Walther FJ, David-Cu R, Leung C, Bruni R, Hernandez-Juviel J, Gordon LM, and Waring AJ. A synthetic segment of surfactant protein A: structure, in vitro surface activity, and in vivo efficacy. Pediatr Res 39: 938-946, 1996.[Abstract]
  58. Walther FJ, Gordon LM, Zasadzinski JM, Sherman MA, and Waring AJ. Surfactant protein B and C analogues. Mol Genet Metab 71: 342-351, 2000.[ISI][Medline]
  59. Wang Z and Notter RH. Additivity of protein and non-protein inhibitors of lung surfactant activity. Am J Respir Crit Care Med 158: 28-35, 1998.[ISI][Medline]
  60. Waring A, Faull L, Leung C, Chang-Chien A, Mercado P, Taeusch HW, and Gordon L. Synthesis, secondary structure and folding of the bend region of lung surfactant protein B. Pept Res 9: 28-31, 1996.[ISI][Medline]
  61. Yao L-J, Richardson C, Ford C, Mathialagan N, Mackie G, Hammond GL, Harding PG, and Possmayer F. Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs. Biochem Cell Biol 68: 559-566, 1990.[ISI][Medline]