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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
exogenous surfactants; phospholipid analogs; phospholipase A2; phospholipase resistance; inhibition resistance; diether phosphonolipid; surfactant proteins
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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).
|
|
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).
|
|
|
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).
|
|
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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|