Diving mammals that descend to depths of 50-70 m or greater fully collapse the gas exchanging portions of their lungs and then reexpand these areas with ascent. To investigate whether these animals may have evolved a uniquely developed surfactant system to facilitate repetitive alveolar collapse and expansion, we have analyzed surfactant in bronchoalveolar lavage fluid (BAL) obtained from nine pinnipeds and from pigs and humans. In contrast to BAL from terrestrial mammals, BAL from pinnipeds has a higher concentration of phospholipid and relatively more fluidic phosphatidylcholine molecular species, perhaps to facilitate rapid spreading during alveolar reexpansion. Normalized concentrations of hydrophobic surfactant proteins B and C were not significantly different among pinnipeds and terrestrial mammals by immunologic assay, but separation of proteins by gel electrophoresis indicated a greater content of surfactant protein B in elephant seal surfactant than in human surfactant. Remarkably, surfactant from the deepest diving pinnipeds produced moderately elevated in vitro minimum surface tension measurements, a finding not explained by the presence of protein or neutral lipid inhibitors. Further study of the composition and function of pinniped surfactants may contribute to the design of optimized therapeutic surfactants.
- surfactant protein
- sea lion
the lung surfactant system is of critical importance in maintaining patency of distal air spaces during tidal breathing (8). Surfactant function may be inadequate in the immature lung and is effectively restored by administration of exogenous surfactant (17). Adults with acute respiratory distress syndrome have an acute inflammatory lung injury in which lung surfactant function is also impaired through a variety of mechanisms (38). Although extensive preclinical investigation suggests that administration of exogenous surfactant might also benefit these patients, such a benefit has not yet been conclusively demonstrated and may await the use of surfactants with optimal efficacy.
Surfactants that have been investigated in the treatment of acute respiratory distress syndrome include those derived from bovine or porcine lungs or synthesized by using component mixtures that may contain proteins or peptides (38). The composition of phosphatidylcholine (PC) molecular species in these substances differs significantly, and, partially as a consequence, their in vitro surface tension lowering functions also differ (5).
Recent data (2, 4) suggest that surfactant composition is adapted to the differing anatomic and physiological conditions of the respiratory system, both across vertebrate species and during postnatal development. Animals that repetitively collapse and then rapidly expand a fully alveolarized lung must repetitively overcome surface tension forces that impede lung expansion. Thus these animals should require a surfactant that is optimized to cope with such physiological conditions.
Compelling evidence, both physiological and anatomic, supports the hypothesis that diving mammals collapse the gas-exchanging portions of their lungs when diving to depths of ≥50-70 m. Scholander (36) proposed that alveolar collapse would occur in diving mammals at ∼100 m. Such collapse would prohibit gas exchange and possibly protect against the bends and nitrogen narcosis. This hypothesis is supported by radiographic evidence in Weddell seals (Leptonychotes weddellii) during simulated dives to a depth of 300 m (19) by minimal elevations in arterial pN2 during free or simulated dives of Weddell seals and elephant seals (11) and by development of pulmonary shunting in harbor seals and sea lions during simulated dives (20, 21). Lung collapse is estimated to occur by 50 m in these species. Studies in dolphins are also consistent with cessation of gas exchange by a depth of 70 m (32, 33).
Experiments reported here test the hypothesis that surfactant from several species of diving mammals has a composition that is distinct from that of terrestrial mammals and may be uniquely suited to repetitive collapse and expansion of the lung. Knowledge of such surfactants might contribute to optimal formulation of surfactant for therapeutic use.
Sample acquisition and preparation. We studied three California sea lions (Zalophus californianus, 24-27 kg), five northern elephant seals (Mirounga angustirostris, 55-80 kg), and one harbor seal (Phoca vitulina, 18 kg). These young (3-8 mo old) animals were obtained from a rehabilitation program at Sea World (San Diego, CA), were maintained in a ring tank facility, and were released at the completion of studies. Observations by others (1, 16) document body temperatures in the range of 36-38°C in these animals. All procedures involving animals and humans were approved through both local review and a federal marine mammal permit. Animals were anesthetized by mask induction with 5% isoflurane-95% oxygen, endotracheally intubated, and maintained on 1-2% isoflurane. Two elephant seals were presedated with 1 mg/kg ketamine intramuscularly. A 4.8-mm outer diameter fiberoptic bronchoscope (Olympus America, Melville, NY) was passed via the endotracheal tube and wedged in a right upper lobe segmental bronchus. Bronchoalveolar lavage (BAL), using five 30-ml aliquots of 37°C 150 mM NaCl, was performed, and the returned volumes were pooled, placed on ice, and subsequently strained through a 100-μm cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ). The total and differential BAL cell counts were determined (23), and the fluid was centrifuged (100 g for 20 min at 4°C) to remove formed elements. An aliquot of the resulting supernatant was centrifuged (40,000 g for 15 min at 4°C) to prepare a surfactant-rich pellet that was resuspended in 0.6 ml of 150 mM NaCl and subsequently used for analysis of surfactant lipid composition and function. Samples were stored at -70°C.
For comparison, samples from patients referred to the University of California, San Diego, for pulmonary artery thromboendarterectomy were obtained before surgery. Informed consent was obtained from all subjects. BAL was performed in a lingular or right middle lobe segment, and samples were then prepared as described. These patients have marked pulmonary vascular obstruction but no clinical evidence of parenchymal inflammation. In addition, analysis of phospholipid (PL) molecular species composition was performed on surfactant obtained from BAL from four freshly killed pigs, as described previously, because the composition of human and porcine surfactant phospholipids is quite similar (3, 29). In some cases, insufficient surfactant was available for all assays, and thus group numbers vary modestly in the results we report.
Surfactant composition. Aliquots of BAL fractions were extracted (6), PL phosphorus content was measured (34), and the fraction of surfactant PL presented as large aggregates (i.e., in the 40,000-g pellet) was calculated. The fractional content of the various PL classes was analyzed by thin-layer chromatography (9). BAL total protein concentration was measured by using a bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) with bovine serum albumin as a standard.
Neutral lipid content of surfactant from the 40,000-g pellet was determined by a modification of the method of White et al. (41). Surfactant was extracted, dried under nitrogen, and an amount containing 150 nmol of phosphorus was resuspended in 35 μl of chloroform and applied 1 cm from the bottom of 20 × 20 cm plates of high-performance thin-layer chromatography silica gel 60 (Analtech, Newark, DE). Dilutions of a mixture of neutral lipid standards, including diolein, monoleoyl-rac-glycerol, palmitic acid, triolein, cholesteryl palmitate, and cholesterol (all from Sigma Chemical, St. Louis, MO), containing 5, 2.5, and 1.25 μg/ml of each lipid, were applied similarly to the high-performance thin-layer chromatography plate. Plates were placed in a tank containing chloroform-methanolacetic acid (90:10:1, vol/vol/vol), and the solvent was allowed to ascend 4 cm. Plates were dried, then placed in a tank containing hexane-diethylether-acetone (60:40:5, vol/vol/vol), and the solvent was allowed to ascend 15 cm. Plates were again dried and then placed in a third tank containing hexane-diethylether (97:3, vol/vol), and the solvent was allowed to ascend to 18 cm. Finally, the plate was dried and placed in a fourth tank containing hexane, and solvent was allowed to ascend 20 cm. After drying, the plates were then dipped in 0.1% 8-anilino-1-naphthalensulfonic acid for 30 min, removed, dried, and analyzed on a ChemiImiger 4400 (Innotech, San Leandro, CA) equipped for fluorescent detection. As diglycerides and free fatty acids comigrate under these conditions, they were specifically separated by using the method of R. Schmidt (personal communication), which also allows resolution of 1,2- and 1,3-diglycerides. Samples were applied to plates prerun in chloroform and placed in a tank containing hexane-diethylether-formic acid (80:20:2, vol/vol/vol), dried, and then rerun. After thorough drying, they were stained and analyzed as described above. The ChemiImager signal was linear over the range of analysis. Sufficient surfactant was available to perform analyses on samples from all pinnipeds, except one elephant seal, and nine patients.
The molecular composition of surfactant phospholipids was determined by electrospray ionization mass spectrometric analysis as described previously (31). Samples were analyzed in the positive ionization mode for PC species as their sodium adducts (M+22) and in the negative ionization mode for phosphatidylglycerol (PG) and phosphatidylinositol (PI) species. Data were recorded at atomic resolution with a signal average of 20 scans/collection. Data were processed by using BioMultiview software (Perkin Elmer-Sciex, Toronto, Canada). After correction for 13C isotope effects, molecular species of PC, PG, or PI were expressed as mole percentages of total PC, PG, or PI, respectively, in the sample.
Concentrations of surfactant proteins (SP) B and C in BAL were measured by ELISA, as previously described (22, 35). For analysis of SP-B, the primary antibody was a murine monoclonal anti-porcine SP-B antibody, a generous gift of Dr. Y. Suzuki (14), and the SP-B standard (a generous gift of R. Schmidt) was purified human dimeric SP-B from BAL fluid. For analysis of SP-C, the primary antibody was a rabbit anti-recombinant human SP-C (a generous gift of W. Steinhilber, ALTANA Pharma AG, Konstanz, Germany), and the SP-C standard was purified recombinant human SP-C (dipalmitoylated) (a generous gift of W. Steinhilber). In addition to the above measurements, SPs in aliquots of the (unextracted) high-speed BAL supernatant containing 1 nmol PL phosphorus were separated by sodium dodecyl sulfate Novex 10-20% tricine gel (Invitrogen, Carlsbad, CA) electrophoresis under reducing conditions and detected by silver staining (SilverXpress kit, Invitrogen). Band intensity was quantified by a ChemiImager 4400 and averaged for the three elephant seal and three human samples analyzed, and the seal-to-human signal ratios were calculated for SP-B and SP-C. Immunoblots of elephant seal and human BAL and relevant standards were performed as previously described (25) and confirmed the localization of SPs detected on silver-stained tricine gels.
Surfactant function. Surfactant surface tension-lowering function of the large-aggregate containing fraction was determined in a pulsating bubble surfactometer (Electronetics, Buffalo, NY) (10) at a concentration of 1 mg PL/ml and a temperature of 37°C as previously described (39). A bubble of 0.55-mm radius was formed, and oscillation between 0.55- and 0.40-mm bubble radius (50% compression) at a rate of 20 bubbles/min was initiated. Maximum and minimum surface tensions were recorded after 10 min of oscillation.
Statistics. Results from groups containing three or more animals were analyzed statistically; larger group size was not possible due to the limited number of animals available for study. Differences among normally or nonnormally distributed groups were detected by using the ANOVA or Kruskal-Wallis statistic, respectively; differences between groups were detected by using Student's t-test with Bonferroni correction for multiple comparisons or the Mann-Whitney U test. A P value of <0.05 was taken as indicating significance. Values are presented as means ± SE unless otherwise noted.
Although we compare results of the analysis of pinniped BAL to results from clinical and porcine samples, there are differences in the lavage procedure that may affect results. First, only upper lobes of the pinniped lung were within reach of the bronchoscope, whereas either middle or lingular segments were lavaged to obtain clinical samples. Porcine BAL was from whole lung lavage. Second, although identical lavage procedures and instilled volumes (five 30-ml lavages) were used, the percent return was significantly greater for pinnipeds than for patients (76 ± 6 vs. 22 ± 2%, respectively; P < 0.0001). To deal with the differences in lavage techniques and subsequent BAL surfactant concentration, analyses including gel electrophoriesis and determination of surfactant protein content were normalized to surfactant PL content.
BAL cell and protein content. As shown in Table 1, BAL obtained from elephant seals was significantly more cellular and had a higher protein concentration than BAL obtained from patients. Macrophages were the predominate cell type in BAL obtained from all species.
Surfactant concentration and composition. The PL concentration of BAL was significantly greater in fluid recovered from pinnipeds than from patients (Table 2). Although not significant, the large aggregate surfactant fraction was greater in samples from elephant seals and sea lions than from patients. Surfactant from all pinnipeds contained a lesser fraction of PG than was found in surfactant from patients, and this difference was significant for elephant seals (Table 2).
The complete fatty acid composition of the PC, PG, and PI molecular species of pinniped surfactants compared with porcine surfactant is shown in Table 3, and dominant species are shown in Figs. 1 and 2. Observations in sea lions were limited by the amount of PL available for analysis. Pinniped PC was 20-25% dipalmitoylated, whereas that from pigs was ∼37%. PI in samples from elephant seals lacked a number of lower moleculear weight species found in porcine PI. In addition, distearyl PI was abundant in pinniped surfactant from all three species studied, whereas it was minimally present in porcine PI. In general, pinniped surfactant phospholipids contained significantly more docosahexaenoic acid (22:6) than was found in porcine surfactant.
Significant differences in the concentrations of the cholesterol, diglycerides, and triglycerides were detected (Figs. 3, 4, 5). The amount of cholesterol and triglycerides present in surfactant from pinnipeds was greater than in samples from patients, and this difference was significant for sea lions (Figs. 3 and 5). In addition, diglycerides were detected in all three sea lions and two humans but were not detected in any other patient samples or other samples from pinnipeds (Fig. 4). Diglycerides from sea lions were entirely of the 1,3 species, whereas diglycerides from the two patients were present ∼35% as the 1,3 and 65% as the 1,2 species. Monoglyceride content ranged from 20 to 40 ng/nmol PL for elephant seals, sea lions, and patients, and was not different among groups. Cholesterol ester content was <1.4 ng/nmol PL and also did not differ among groups.
The concentrations of the hydrophobic proteins SP-B and SP-C in BAL from elephant seals and sea lions were significantly greater than in samples from patients (Table 4). However, when the concentrations were expressed relative to either PL or protein, no significant differences were detected by immunologic assay. Separation of proteins by gel electrophoresis under reducing conditions showed roughly equivalent amounts in BAL of SP-C per nanomole of PL from humans and seals (seal-to-human ratio = 1:4) but substantially more SP-B monomer per nanomole of PL (seal-to-human ratio = 6:6) (Fig. 6). SP-A was present in approximately equal amounts in pinneped and human BAL, as estimated from bands on silver-stained tricine gels, which, by immunoblotting, were identified as SP-A.
Surfactant function. As shown in Table 5, the minimum surface tension measured in vitro was significantly greater in all samples from elephant seals than in samples from other pinnipeds or from patients. Maximum surface tension after 10 min of oscillation was not different among groups.
The pinniped species in this study exhibited a wide range of diving behaviors. The elephant seal, a member of the phocid family, is the premier pinniped diver with routine 20-min dives to depths of 400 m and maximum dive duration and depth of 2 h and 1,581 m, respectively (24, 40). Surface intervals averaged ∼2 min, and 80-90% of time at sea is spent beneath the surface. In contrast, the harbor seal, another phocid, routinely dives to depths of <50 m for 1- to 3-min periods (7), and the California sea lion, a member of the otariid family, predominantly dives to depths of <70 m, although dives as deep as 274 m have been reported (13). Thus, if alveolar collapse occurs in the range of 70-100 m, one might expect that adapted surfactant function, which allows for rapid and atraumatic expansion of collapsed alveoli, might be found in the elephant seal and sea lion.
Because the clinical and pinniped lavage techniques differed, significantly greater volumes were recovered from pinnipeds. The recovery of more dilute lavage fluid from patients may also be explained by these technical differences. Given this variability, we have found it useful to compare results from different species by expressing surfactant PL components as a percent of the total PL present, expressing fatty acid components as mole percent, and normalizing surfactant protein and neutral lipid content to PL content.
Our observations disclose several differences beween surfactant from the aquatic mammals we have studied and terrestrial mammals. The cell population in BAL from elephant seals and the harbor seal was composed of 95% or more macrophages, which is consistent with findings from normal terrestrial mammals. That a higher percentage of polymorphonuclear leukocytes was seen in patients suggests underlying exposure to irritants (e.g., urban air pollution and cigarette smoke) or pathology not evident clinically. Because the pinnipeds might have had a prior pneumonitis, it is reassuring that the cell population did not suggest residual infection, and thus the measurements of surfactant composition are likely to represent findings in healthy animals.
The BAL PL concentration in elephant seals and in sea lions was greater than in patients both when expressed on an absolute basis or when normalized to BAL protein concentration (Table 2), suggesting larger surfactant pool sizes in these animals compared with adult humans. In addition, the large aggregate fraction, in which surface tension-lowering activity is predominately found, tended to be greater in these animals (Table 2). Finally, the fractional content of PG was signifi-cantly lower in elephant seals compared with patients.
The molecular species composition of pinniped surfactant PLs differed in several ways from that of pigs. Dipalmitoyl PC (PC16:0/16:0), the dominant PL in pulmonary surfactants from adult mammals, was a significantly smaller fraction of total PC in pinniped surfactant than in surfactant from pigs (Table 3) and humans (30). In contrast, pinniped surfactant was signifi-cantly enriched in the fluidic PC species palmitoylmyristoyl-PC (PC16:0/14:0) when all nine pinnipeds were considered as a group. The minor constituant alkyl/acyl species PC 16:0/16:1 was significantly increased in pinniped surfactant, and the amount of the diacyl PC 16:0/16:1 species also tended to be greater in pinniped surfactant. These findings contrast sharply with those in avian species (duck and chicken) in which there is relative enrichment in dipalmitoyl PC (PC16:0/16:0) and a scarcity of PC16:0/14:0 and PC16:0/16:1 species relative to porcine surfactant (2). Interestingly, the fractional concentrations of PC16:0/16:0 correlate inversely with airliquid interface dynamics, whereas those of PC16:0/14:0 and PC16:0/16:1 correlate directly with this parameter(4). Considering the need for aquatic mammals to rapidly expand a collapsed lung, the requirement for rapid spreading may be critical, and the inclusion of fluidic species in their surfactant may provide another example of molecular adaptation of surfactant to specific physiological conditions.
Finally, all pinniped surfactant PC contained significantly more docosahexaenoic acid (22:6) than was found in porcine surfactant. Because fish contain relatively large amounts of 22:6 fatty acid, and seal blubber and milk fatty acid profiles have been shown to reflect dietary fatty acid intake (18, 37), it is tempting to hypothesize that 22:6 fatty acid is also present in the minor fractions of surfactant lipids as a result of its dietary abundance.
As in most other animals, PG and PI molecular species compositions were quite distinct from those of PC, suggesting differential metabolism of diacylglycerols within the type II cells of these animals. Dipalmitoyl PG (PG16:0/16:0) was completely absent from pinniped surfactant but is a significant component of surfactant from pigs and humans (Table 3; Ref. 15), animals with similar respiratory physiology. The absence in pinniped surfactant of dipalmitoyl PG, together with a preponderance of highly unsaturated PG species, is consistent with the concept of a highly fluid surfactant. However, this observation contrasts with the finding of a high percentage of distearoylphosphoinositol (PI18:0/18:0). One might speculate that these findings result, on the one hand, from a rich concentration of highly unsaturated fatty acids in PG that are derived from the animals' food and, on the other hand, the necessity of compensating for the high fluidity resulting from that concentration. This speculation is supported by the observation that the fractional content of PI18:0/18:0 in pinniped surfactant is inversely related to the total amount of PI present (Tables 2 and 3), such that the total amount of PI18:0/18:0 in pininped surfactant is relatively constant. This amount is also quite low compared with the fluidic PGs, which comprise 0.8-1.9% of the total surfactant PL.
The content of hydrophobic surfactant proteins appeared increased in pinniped surfactant (Table 4). However, when the concentrations were normalized to PL or protein, there were no significant differences by ELISA. Results of gel electrophoresis gave conflicting information, showing distinctly greater amounts of SP-B in elephant seal compared with human BAL (Fig. 6). Differences in immunologic specificity may contribute to this discrepency. Standards used in the immunologic assays were human SP-B and SP-C. To the extent that pinniped SPs may have a different affinity for the antibodies employed, the concentrations may differ from those we measured. Although immunologic assays indicated that BAL SP-B concentrations, normalized to PL content, are three to nine times those of SP-C (Table 4), SP-C was more evident than SP-B on silver-stained gels of human BAL. Differences in avidity for silver stain may explain this observation (12).
Surfactant from elephant seals had a significantly greater minimum surface tension than was observed with samples from other pinnipeds or from patients (Table 5). This unexpected finding was present in all animals tested and was highly reproducible. Because an increased concentration of neutral lipids might explain this finding (27), we determined the content of those species in pinniped surfactant. Sea lion surfactant did contain significantly more cholesterol and triglyceride than patient surfactant (Fig. 3) and more diglyceride (Fig. 4) than either patient or elephant seal surfactant. However, sea lion surfactant exhibited surface tension-lowering properties similar to that of patients (Table 5) and pigs (2, 5), and thus the concentrations of neutral lipids that we detected do not appear to explain the modestly greater minimum surface tension seen in elephant seal surfactant. Although elephant seal surfactant contained less PG than that of other species, this deficit is also unlikely to explain the increased minimum surface tension that was observed (15, 26). Similarly, although protein may impair surfactant surface tension-lowering function, it is unlikely that the modest difference between patient and elephant seal BAL protein provides an explanation for altered function of elephant seal surfactant, especially since the protein content of harbor seal surfactant was equivalent to that of elephant seal surfactant, yet harbor seal surfactant had excellent surface tension-lowering properties.
The finding of increased cholesterol in pinneped surfactant (Fig. 3) recalls the observations of Orgeig et al. (28), who reported increased cholesterol content in vertebrates with simple saccular lungs, in aquatic species, and in species with low body temperature, and postulated that this cholesterol may make an important contribution to surfactant function.
In summary, we find that surfactant from these aquatic mammals has PL class and protein composition that is quite similar to that of pigs and patients. On the other hand, the molecular species composition of the constituent PLs is significantly different from that of pigs, perhaps to afford a more fluidic nature that would allow rapid respreading. The finding, in samples from the deepest diving group of pinnipeds, of modestly diminished surface tension-lowering function (as measured in vitro) was unexpected, was not explained by the presence of neutral lipids or protein, and remains unexplained. The unique composition of pinniped surfactant is consistent with previous studies that demonstrated molecular adaptation of the surfactant system to requirements imposed by respiratory physiology. Knowledge of such adaptations might provide a benefit to the design of therapeutic surfactants for clinical use.
This study was funded by National Heart, Lung, and Blood Institute Grant HL-23584 and National Science Foundation Grant IBN-0078540.
The authors are grateful to K. Harris and C. Acevedo for outstanding technical assistance and to T. Knower and D. H. Levenson for assistance with the care and anesthesia of the seals.
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.
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