Main

Since the discovery of pulmonary surfactant in the 1950s(1, 2), its functional composition has been of concern and interest to researchers seeking to understand its biophysical activity. A particularly critical aspect of this functional composition is the content of DPPC. Although lung surfactant is now known to be a complex mixture of phospholipids and specific proteins, DPPC is its single most prevalent component as identified in early compositional studies(3, 4). This disaturated phospholipid has a liquid-crystal transition temperature of 41-42°C and in the rigid gel phase at body temperature is almost certainly responsible for the ability of interfacial films of lung surfactant to reduce surface tension to very low values during dynamic compression(5–9).

Although it is agreed that DPPC is the most abundant constituent of lung surfactant, widely varying DPPC levels of ≈40-70% of total PC or ≈30-60% of total surfactant lipid have been reported (see e.g.Refs. 10–13 for reviews). Moreover, even higher DPPC contents of 70-90% by weight have been used in the majority of surface activity studies with synthetic exogenous lung surfactants over the past several decades (seeRefs. 14–16 for reviews). The present study seeks to define more precisely the DPPC content of an extract of calf lung surfactant and to investigate the relationship of this variable to the surface active properties of model mixtures of phospholipids and hydrophobic SP-B and -C.

A number of analytical methods have been used to determine the phospholipid composition of lung surfactant, most commonly based on chromatography. One common method of approximating the DPPC content of lung surfactant mixtures is the DSPC isolation procedure defined by Mason et al.(17). This DSPC assay utilizes osmium tetroxide to oxidize unsaturated acyl groups, thereby allowing the unaffected disaturated molecules to be isolated by thin layer chromatography and quantified colorimetrically. Alternatively, the fatty acid chains of lung surfactant phospholipids in a given class (such as the PC class) can be defined by GC. Yuet al.(18) and Possmayer et al.(19), for example, have used GC to measure the DPPC content of extracted bovine lung surfactant. They reported probable values well below 50% for the DPPC content of lung surfactant PC(18, 19), near the lower end of the reported range and much lower than the DSPC content found using the osmium tetroxide method(20). In the present study, we applied both GC methodology and the osmium tetroxide assay to study the DPPC/DSPC content of CLSE. Differences between the GC and osmium tetroxide results were further assessed using synthetic unsaturated and mixed chain PC molecules to define their contributions, if any, to DSPC assay results.

To complement and correlate with compositional measurements, surface activity studies were done on a series of model mixtures containing synthetic phospholipids with a range of DPPC contents from 40-80%, combined with hydrophobic SP-B and -C. Model phospholipid/aproprotein mixtures were studied for the time dependence of adsorption to the air-water interface after dispersion in the aqueous phase and for dynamic behavior in spread surface films. Biophysical comparisons with CLSE emphasized interfacial behaviors important for lung surfactant function, including surface tension lowering and dynamic film respreading(6–8, 15). Results showed that the model mixture with 40% DPPC most closely approximated the surface activity of CLSE in both films and dispersions, consistent with GC measurements of DPPC content in the surfactant extract.

METHODS

CLSE and hydrophobic apoproteins. CLSE was prepared by organic solvent extraction(21) of bronchoalveolar lavage material obtained from lungs from freshly killed calves (Conti Packing Co., Henrietta, NY), as previously described by Notter and co-workers(22–24). Phospholipid content was determined from the phosphorus content(25) of extracted samples. The composition of phospholipid head groups was determined by thin layer chromatographic analysis of samples using solvent system C of Touchstoneet al.(26). Protein was assayed by the method of Lowry et al.(27) modified by the inclusion of 0.1% (wt/vol) SDS to eliminate turbidity. The measured protein content of CLSE was in agreement with the value of 1.3% by weight reported previously by Hall et al.(28), comprising a mixture of hydrophobic SP-B and -C by ELISA, SDS-PAGE, and N-terminal amino acid sequence analysis.

Synthetic phospholipids and model surfactants. 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), egg PC, and egg PG were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). All phospholipids were purchased as >99% pure and were used without further purification in surface or chromatographic studies. Biophysical experiments(see below) used three SPL mixtures: 80:10:10 (molar ratio) DPPC/egg PC/egg PG; 60:30:10 DPPC/egg PC/egg PG; and 40:50:10 DPPC/egg PC/egg PG. SPL mixtures were studied alone and in combination with purified hydrophobic SP at the same level of 1.3% (by weight) present in CLSE. Hydrophobic SP for combination with SPL were purified from CLSE by column chromatography(28), and stored at -20°C in 1:1 chloroform:methanol before use. SPL in chloroform and SP in chloroform:methanol (1:1) were combined and dried under nitrogen before being dissolved in spreading solvent or dispersed in the aqueous phase for biophysical study.

DSPC assay. DSPC was assessed in phospholipid mixtures by the method of Mason et al.(17). Briefly, 1 mg of CLSE or a phospholipid standard was reacted with 3.2 mg of osmium tetroxide in 0.5 mL of carbon tetrachloride for 15 min. The carbon tetrachloride was then removed using a nitrogen stream, and the dried sample was resuspended in chloroform:methanol (1:2). The samples were then spotted on thin layer chromatography plates and run in a solvent system of chloroform/methanol/7 M ammonium hydroxide. The spots were scraped and analyzed for lipid phosphorus content.

GC. Gas chromatographic analyses of PC fatty acids in CLSE were performed using a wide-bore capillary gas chromatograph and comparison to known standards, after PC was isolated from other phospholipids by preparative thin layer chromatography. To determine the composition of fatty acids in thesn-2 position of PC, PLA2 was added (20 U/mL, 5 mM Tris, pH 7.4, containing 5 mM CaCl2), followed by incubation for 2 h to ensure complete reaction(29). After PLA2 treatment and incubation, FFA were extracted into methylene chloride and analyzed by GC as described by Yu et al.(18).

Oscillating bubble methods. Measurements of surface tension lowering during dynamic cycling were made on an oscillating bubble surfactometer (Electronetics Corp, Amherst, NY), modified from the design of Enhorning(30). A small air bubble, communicating with ambient air, was formed in a surfactant dispersion in a sample chamber mounted on a pulsating unit. Immediately after formation, bubble oscillation was begun at a rate of 20 cycles/min between minimum and maximum radii of 0.4 and 0.55 mm, respectively, at 37°C. The pressure drop across the air-liquid interface was measured by a pressure transducer, and surface tension was calculated at the end points of compression and expansion from the equation of Young and Laplace for a spherical interface. A surfactant concentration of 1.25 mg of phospholipid/mL was used in bubble studies. Dispersion was in buffer (10 mM HEPES, 1.5 mM CaCl2, 150 mM NaCl, pH 7.0) by probe sonication on ice with three 10-s bursts (Heat Systems probe sonicator model W-220).

Adsorption methods. The adsorption of dispersed surfactants was measured at 37°C in a Teflon dish, with the subphase stirred with a Teflon-coated magnetic stirring bar to minimize diffusion resistance(24, 31). Surfactant samples containing 2.5 mg of phospholipid were dispersed in 10 mL of buffer by sonication on ice, as in oscillating bubble methods, and were added to 70 mL of buffered subphase in the Teflon dish (final concentration 0.0312 mg of phospholipid/mL). Surface pressure, calculated as the surface tension of the pure subphase (70 mN/m at 37°C) minus the surface tension with surfactant present, was monitored continuously from the force on a sandblasted platinum slide dipped into the interface(31).

Wilhelmy balance methods. The interfacial characteristics of spread films during dynamic compression were measured on a Wilhelmy balance incorporating a ribbon barrier design to maximize confinement of films at high surface pressure(32). CLSE or model surfactants (SPL, SPL:SP) were dissolved in 9:1 hexane:ethanol, vol/vol, and were added in droplets to the surface of a buffered subphase confined within the Teflon ribbon in the balance. After allowing 10 min for evaporation of the spreading solvent, the surface area was compressed and expanded from 448.6 cm2(100% area) to 103.2 cm2 (23%) at a constant rate of 0.75 min per compression (1.5 min/complete cycle). Ambient and hypophase temperatures were maintained at 37.0 ± 0.2°C, and ambient humidity was kept fully saturated by dampened blotting paper and open dishes of water in the balance chamber. Surface pressure in the confined film was measured continuously during cycling from the force on a sandblasted platinum Wilhelmy plate, and a second plate outside the barrier ribbon monitored for leakage. Experiments in which leakage was detected at any surface pressure were discarded. Dynamic respreading during multiple film cycles was quantitated by calculations of surface pressure-area isotherm areas between compressions 2 and 1 and compressions 7 and 1, modified from the previous collapse plateau ratio criterion of Notter and co-workers(33–35). An isotherm area of zero between compressions 2 and 1 or between compressions 7 and 1 indicated complete respreading between the designated cycles, whereas the poor respreading of DPPC gave an expected upper limit on calculated isotherm areas(33).

RESULTS

Gas chromatographic analysis indicated that palmitic acid made up 65% of the total fatty acids in surfactant PC, consistent with a range of DPPC contents depending on the fatty chain distribution in the sn-1 andsn-2 positions (Table 1). A maximum DPPC content of 65% would exist if each of the PC molecules containing any palmitic chains was disaturated with C16:0 chains in both the sn-1 andsn-2 positions. Any distribution of C16:0 fatty chains into mixed chain PC molecules would reduce DPPC content. If maximum mismatching of C16:0 chains occurred, the DPPC content of PC from CLSE could be as low as 30% (100% of PC molecules having C16:0 chains in the sn-1 position, but only 30% also having C16:0 chains in the sn-2 position). Further GC analysis on fatty acids released from the sn-2 position of PC by PLA2 hydrolysis indicated that palmitic acid accounted for only 41%(n = 2, range 40-42), giving an upper limit on the possible content of DPPC.

Table 1 DPPC and DSPC content of the PC fraction of CLSE
Full size table

To complement GC measurements, the DSPC content of CLSE was defined by the spectrophotometric osmium tetroxide assay. This assay indicated that 69± 1% of the PC in CLSE was disaturated (n = 5). Lung surfactant PC is known to contain saturated fatty acids such as C14:0 and C18:0(18) that will elevate DSPC content relative to DPPC content. However, the relative amounts of nonpalmitic saturated chains in lung surfactant PC are insufficient to explain fully the difference between a value of nearly 70% for DSPC content and a value of ≤41% for DPPC content indicated by GC analysis of fatty acids released from the sn-2 position by PLA2 treatment.

To investigate other contributors to the potentially elevated DSPC assay results, PC standards having different fatty acid compositions (DPPC, POPC, and DOPC) were separately reacted with osmium tetroxide, and their subsequent recovery on thin layer chromatography was assessed (Table 2). The data showed that the DPPC molecule, containing two saturated fatty acids, was completely recovered after reaction with osmium tetroxide. As expected, the DOPC molecule, containing no saturated fatty acids, was completely oxidized by osmium tetroxide and therefore not recovered on subsequent thin layer chromatography analysis (Table 2). However, assay of POPC, which contains one saturated and one unsaturated fatty acid, yielded a 51% recovery (Table 2), even though the theory behind the assay would predict no PC recovery. All standards were completely recovered when not reacted with osmium tetroxide(Table 2).

Table 2 The recovery of synthetic phospholipids on thin layer chromatography after reaction with osmium tetroxide
Full size table

The data in Table 3 show that mixtures of the three standards DPPC, DOPC, and POPC gave analytic results similar to those found for each standard measured separately. In mixtures, DPPC was completely recovered, whereas recovery of DOPC was negligible (Table 3). However, the PC recovery in DPPC/POPC mixtures was consistently higher than the predicted values by a factor equivalent to 50% of the initial POPC level (Table 3). Because lung surfactant PC is known to contain many mono-unsaturated species including POPC(10–13), the osmium tetroxide assay is likely to give a significant overestimation of DSPC content. If DPPC content is estimated or inferred from the DSPC assay prediction, it too will be falsely elevated.

Table 3 The recovery of synthetic phospholipid mixtures on thin layer chromatography after reaction with osmium tetroxide
Full size table

To complement and extend the biochemical results on lung surfactant DPPC content, additional surface activity experiments investigated the adsorption and dynamic surface behavior of a set of synthetic surfactants containing 1.3% by weight hydrophobic SP combined with synthetic phospholipids with different percentages of DPPC. The adsorption of CLSE and SPL:SP mixtures to the air-water interface is shown in Figure 1. The model surfactant with a 40% DPPC content adsorbed faster than mixtures with higher percentages of DPPC, and had overall adsorption behavior very similar to CLSE (Fig. 1). Both CLSE and the SPL:SP mixture with 40% DPPC reached an equilibrium adsorption surface pressure of 46 mN/m (equilibrium surface tension of 24 mN/m at 37°C) after 3 min, whereas the SPL:SP mixture with 60% DPPC adsorbed to this equilibrium surface pressure at ≈10 min. The SPL:SP mixture with 80% DPPC reached a slightly lower equilibrium pressure after ≈15 min (Fig. 1).

Figure 1
figure 1

Adsorption isotherms for dispersions of CLSE and SPL combined with 1.3% hydrophobic surfactant proteins (SP-B/C). Adsorption experiments at 37°C were initiated at time 0 by addition of a surfactant dispersion (2.5 mg of phospholipid/10 mL of buffer) to a stirred, buffered 70 mL subphase (see “Methods”). Data are means of at least three independent experiments with a variation <2 mN/m.

Full size image

The maximum surface pressure and respreading behaviors of interfacial films of DPPC, CLSE, SPL, and SPL:SP (1.3% by weight) on the Wilhelmy balance are shown in Table 4. For both SPL and SPL:SP mixtures, there was a progressive improvement in film dynamic respreading as DPPC content was lowered, reflected by a decrease in calculated isotherm areas(Table 4). In films of SPL alone, respreading differences were most apparent between compressions 2 and 1. Films of SPL with 40% DPPC had 2/1 isotherm areas that were approximately 3-, 5-, and 10-fold lower than for 60% SPL, 80% SPL, and pure DPPC, respectively (Table 4). Films of SPL:SP had the same pattern of improved respreading with lowered DPPC content, but respreading differences were present throughout the full seven cycles studied. Comparisons of isotherm areas between compressions 1 and 7 for SPL:SP films showed that 40% SPL:SP approached CLSE in respreading, and was markedly improved in this film property compared with mixtures with higher DPPC contents (Table 4).

Table 4 Respreading and maximum surface pressure behavior of spread films of SPL, SPL:SP, CLSE, and DPPC on the Wilhelmy balance at 37°C
Full size table

The maximum surface pressures reached by films of CLSE, SPL, and SPL:SP(1.3 wt%) during compressions 1, 2, and 7 are also shown in Table 4. For both SPL and SPL:SP films, maximum surface pressure decreased as DPPC content decreased. However, although films containing SPL with 40% DPPC had lower maximum surface pressures, this behavior was also found with CLSE itself. At the relatively slow film compression rates in the Wilhelmy balance (1.5 min/cycle), CLSE films reached maximum pressures of 59.6-63.8 mN/m depending on the cycle(Table 4). Films of 40% SPL combined with the same 1.3% level of hydrophobic SP found in CLSE(28) had very similar maximum surface pressures on cycling, in contrast to the behavior of SPL:SP films with 60 and 80% DPPC (Table 4).

The maximum surface pressures generated by surfactant films are a function of cycling rate. To supplement Wilhelmy balance experiments on spread films, surface tension lowering was also assessed for aqueous dispersions on the oscillating bubble surfactometer at 37°C (Table 5). Bubble experiments were done at a rate of 20 cycles/min, much faster than feasible with the Wilhelmy balance, and in the range of rates in the respiratory system in vivo. At this high cycling rate, dispersions of CLSE and all mixtures of SPL (40, 60, and 80% DPPC) with 1.3% SP rapidly lowered surface tension to <1 mN/m (1.25 mg phospholipid/mL,Table 5). However, although all mixtures had similar the surface tension lowering ability, the surface tension-time pattern of the model surfactant with 40% DPPC most closely resembled that of CLSE(Table 5).

Table 5 Time dependence of dynamic surface tension lowering for surfactant dispersions on an oscillating bubble surfactometer at 37°C
Full size table

DISCUSSION

The results of our study suggest that PC isolated from CLSE has a probable DPPC content near 40%, at the low end of the range of values reported for the content of this disaturated phospholipid in pulmonary surfactant PC(e.g. seeRefs. 10–13). GC analysis of PC from CLSE indicated a range of DPPC content from 30 to 65%, but additional GC studies of fatty acids released from PC by PLA2 treatment showed that only 41% of sn-2 fatty chains were palmitic acid(Table 1). If the upper limit on the DPPC content of surfactant PC is 41%, the DPPC content of whole surfactant will be even less than 40%, because PC accounts for only about 80% of total lung surfactant lipid(14–16). This emphasizes that, although DPPC is the single most abundant lung surfactant consitituent, its content is outweighed by additional phospholipids (secondary surfactant phospholipids) that along with surfactant proteins and possibly neutral lipids play important roles in overall system behavior.

Our biochemical studies also demonstrated that a spectrophotometric osmium tetroxide assay for DSPC(17), widely used in lung surfactant studies because of its relative rapidity and convenience, can significantly overestimate the content of disaturated PC if mono-unsaturated PC species are present (Tables 2 and 3). This assay indicated that the DSPC content of PC from CLSE was nearly 70%. Part of this difference was presumably due to other disaturated PC such as myristoyl/palmitoyl (C14:0/C16:0) PC(36, 37) known to exist in lung surfactant. However, our results indicate that a major factor influencing the DSPC assay results was falsely high readings generated by mono-unsaturated PC species in lung surfactant(10–13). One possible explanation for the observed contributions of monoenoic PC to the DSPC assay is that the presence of a single osmium double-bond complex is not sufficient to retard the flow of reacted material through the alumina columns, so that some monoenoic phospholipid remains associated with the disaturated fraction.

There have been a number of GC or HPLC studies showing a DPPC content of lung surfactant PC at or below the 50% level(18, 36, 38). Yu et al.(18) reported a value of 64% for the total content of palmitic acid chains in bovine lung surfactant PC, with palmitic chains at thesn-2 position present on 49.9% of PC molecules. Schlame et al.(38) found that DPPC constituted about 39% of total PC in rat lung surfactant using both GC and HPLC methodology. Huntet al.(36) also found by HPLC that the DPPC content of lung surfactant PC from neonatal guinea pigs and rats was about 43 and 45%, respectively, with slightly higher values for adult animals.

Surface activity studies on a series of phospholipid/apoprotein mixtures agreed with the GC findings, showing optimal activity at a DPPC content of 40%, compared with higher values of 60 or 80%. A mixture of synthetic phospholipids containing 40% DPPC combined with 1.3% hydrophobic SP consistently had surface behavior closer to CLSE than similar mixtures with higher DPPC contents. The SPL:SP mixture with 40% DPPC retained an ability to lower surface tension to <1 mN/m on rapid dynamic compression in an oscillating bubble apparatus (Table 5), while demonstrating improved adsorption and better film respreading compared with mixtures with a higher DPPC content (Table 4, Fig. 1). Moreover, in Wilhelmy balance studies at slower (nonphysiologic) compression rates, films of the 40% DPPC mixture exhibited a reduction in surface tension-lowering ability that paralleled the behavior of CLSE films.

A DPPC content near or below 40% in pulmonary surfactant emphasizes that, although this disaturated phospholipid is important in functional surface activity, significant concentrations of other system components are also necessary. It has been recognized for some time that DPPC does not exhibit all of the surface active behaviors of complex lung surfactant mixtures(5–7). Although DPPC is the primary surface tension-lowering component of functional surfactant(7, 8), it does not adsorb rapidly to the air-water interface at physiologic temperature (37°C)(31, 39), nor does it respread well in cycled interfacial films(34, 35). The DPPC content of the actual working film of alveolar surfactant in vivo almost certainly changes throughout each compression/expansion cycle. The surface film itself will become enriched with DPPC during each compression stroke, with more fluid components preferentially “squeezed-out” of the film to permit surface tension to be reduced to very low values at end-compression(5–8). However, if the DPPC content of the surfactant mixture as a whole were too high, crucial system behaviors such as rapid adsorption and good film respreading would be compromised.

A number of studies have shown that improved respreading in cycled interfacial films containing DPPC is associated with more fluid phospholipids(33, 35, 40–42), as well as the hydrophobic surfactant proteins(33, 43). The secondary lung surfactant phospholipids include many that are in the fluid liquid crystal state at body temperature, such as those with one or more unsaturated chains or saturated chains shorter than C16:0 (e.g. such as dimyristoyl PC or palmitoyl-myristoyl PC). A striking enhancement of film respreading by secondary surfactant phospholipids has been demonstrated in recent studies with purified lung surfactant subfraction films(33). Mixed chain and unsaturated phospholipids such as those in lung surfactant are also known to improve the adsorption of DPPC(44), as do the surfactant apoproteins.

One approach to the development of new exogenous surfactants for therapeutic use in RDS and ARDS involves the combination of synthetic phospholipids with cloned hydrophobic SP or model peptides. In developing such exogenous surfactants, synthetic phospholipids with a high DPPC content of 70-80% are by far the most widely studied to date. A substantially lower DPPC content of endogenous surfactant on the order of 40% suggests that future work might usefully investigate the in vitro and in vivo activity of synthetic mixtures with a similarly low content of DPPC. A reduction in DPPC content, for example, might allow for increased adsorption and respreading without a substantial detriment to the surface tension lowering ability of dynamically-compressed films. The useful percentage of DPPC in lung surfactant and exogenous replacement mixtures clearly has an upper and lower limit, and neither is known with precision. Current clinical exogenous surfactants for the therapy of RDS involve a range of DPPC contents, from a value over 80% in Exosurf (Burroughs Wellcome, Research Triangle Park, NC) to about 40% in Curosurf (Chiesi Pharmaceuticals, Parma, Italy)(37, 45), with Survanta (Ross Laboratories, Columbus, OH) having an intermediate level of DPPC. These clinical surfactants also contain additional constituents that differ among themselves and in comparison to CLSE. Berggren et al.(37) found that supplementing Curosurf with DPPC and PG gave a slight improvement in surface activity, but did not give improved efficacy in vivo when given to surfactant-deficient premature rabbit fetuses. Additional research on the DPPC content of exogenous surfactants, and how it affects biophysical and physiologic activity, needs to be carried out.

In summary, GC analysis of calf surfactant PC before and after phospholipase treatment was consistent with a DPPC content ≤41%. The widely used osmium tetroxide assay indicated a much higher DSPC content of 69%, but was shown to be inappropriately affected by monounsaturated phospholipids, giving a falsely elevated DSPC value for lung surfactant. Adsorption and dynamic surface studies with model surfactants containing synthetic phospholipids with a range of DPPC contents combined with hydrophobic surfactant proteins showed that the mixture with a 40% DPPC content consistently approached CLSE in behavior better than did preparations with higher DPPC contents of 60 and 80%. These results suggest that the study of mixtures with relatively low DPPC content might be useful in the future development of exogenous lung surfactants.