INTRODUCTION

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Keywords: surfactant; lung injury; polyethylene glycol

Exogenous surfactant administration is currently being evaluated as a therapeutic intervention for patients with the acute respiratory distress syndrome (ARDS). Although results of clinical trials have shown promise, there is significant variability in patient responses to this therapy (1). As a result, several in vitro and in vivo animal studies have been performed to determine the mechanisms responsible for surfactant alterations, and consequently, the lung dysfunction associated with lung injury (5, 6).

Inactivation of surfactant by serum proteins leaking into the airspace of the injured lung is one important mechanism leading to surfactant dysfunction (7, 8). This inhibition can be overcome either by administering relatively large quantities of surfactant into the lung (9) or by adding surfactant-associated protein A (SP-A) to the administered material (10). Unfortunately, exogenous surfactant preparations containing SP-A do not currently exist for human use, and the requirement of large quantities of exogenous surfactant has several drawbacks. Specifically, with the potentially large patient population that would ultimately benefit from this therapy, resource limitations, particularly for the natural surfactant preparations, as well as expense, are two factors that may limit its potential.

These concerns have led to the evaluation of substances that may augment the functional properties of exogenous surfactant within the injured lung, such as nonionic polymers. Previous studies have shown that some of these polymers, such as polyethylene glycol (PEG) and dextran, reversed the inhibitory effects of serum proteins, meconium, and lysophosphatidylcholine on surfactant function when tested in vitro (11). In fact, these agents were shown to be more effective than adding SP-A to the surfactant preparation. Recently, PEG was also shown to augment the effects of exogenous surfactant in vivo when tested in a rat model of meconium aspiration (12). Based on these data, it was suggested that these compounds may be useful in the clinical setting for patients with ARDS by improving the efficacy of exogenous surfactant and potentially decreasing the amount of surfactant required to treat these patients. We hypothesized that the addition of PEG to an exogenous surfactant preparation lacking SP-A would augment the physiologic response observed with surfactant alone in an adult rabbit model of acute lung injury. Furthermore, we postulated that the mechanisms responsible for these superior responses would be related to the alveolar metabolism of the administered surfactant. To address the latter hypothesis, alveolar surfactant pool sizes, surfactant composition, and surfactant aggregate metabolism were analyzed in these animals.

	METHODS

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Induction of Lung Injury

Adult New Zealand White rabbits were anesthetized, catheterized, and mechanically ventilated. Lung injury was induced via whole lung saline lavage as previously described (15), and animals were mechanically ventilated for a period of 60 min after the final lavage. These interventions resulted in stable gas exchange parameters, thereby representing a consistent model of lung injury suitable for evaluating the efficacy of various exogenous surfactant treatment strategies (1, 15, 16). Animals meeting predefined oxygenation criteria were then randomized to one of eight treatment groups, which were instituted 60 min after the final lavage.

Treatment Groups and Administration

Group 1 was a nonsurfactant-treated, lung-injured control group (No Rx), Group 2 received PEG only (PEG), Group 3 received 100 mg lipid/kg of exogenous surfactant (100 mg/kg), Group 4 received 100 mg/kg surfactant plus PEG (100 + PEG), Group 5 received 25 mg lipid/kg surfactant (25 mg/kg), Group 6 received 25 mg/kg surfactant plus PEG (25 + PEG), and Groups 7 and 8 involved normal, noninjured rabbits ventilated in a fashion similar to other animals, but given 25 mg/kg surfactant ± PEG.

The surfactant suspension (Survanta, Ross Laboratories, Columbus, OH) was diluted appropriately so that all animals received the same volume (4.4 ml/kg body weight) to standardize the distribution of the administered material within the lung (16, 17).

PEG (mol wt 10K, 5% wt/vol) was prepared the same as in other studies (13, 14), and all solutions (surfactant ± PEG) were administered as previously described (15).

Lung Lavage Analyses

After sacrifice, lungs were lavaged and aliquots removed for isolation of both large (LA) surfactant and small (SA) surfactant aggregates via centrifugation procedures (15, 18). Phospholipid-phosphorus measurements were performed on lipid-extracted aliquots of total lung lavage, LA and SA fractions (19), and protein recovery was measured in whole lung lavage (20). The surface tension of LA samples was measured using the pulsating bubble surfactometer (21) and the in vitro conversion of isolated LA (including input samples and LA recovered from animals) was assessed using the surface area cycling technique as previously described (22, 23). Briefly, the exogenous surfactant (± PEG) solutions were resuspended in conversion buffer, placed in plastic tubes, and cycled at 40 rpm at 37° C for various periods up to 3 h. After cycling, samples were centrifuged for separation of LA and SA and then assessed for phospholipid content as described previously (19).

Statistics

All data are presented as means ± standard error of the mean (SEM). Comparisons among the various experimental groups were made using an analysis of variance. Comparisons within a treatment group were made using an analysis of variance followed by a Tukey's post hoc test. Comparison between two groups was made using the Student's t test. All analyses were performed using SPSS software with a probability value below 0.05 considered significant.

	RESULTS

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Physiological Parameters

Thirty-nine of the 70 animals that underwent the multiple saline lavage procedure and subsequent ventilation met the predefined oxygenation inclusion criteria and were randomized to either one of the six experimental groups (n = 6-7 animals per group). There were no significant differences in mean body weights of these animals (2.3 ± 0.1 kg), and no significant differences in mean Pa_O2 values among groups over the 45-min period of ventilation up to and immediately before randomization at Time 0. Figure 1 shows the Pa_O2 values for the six groups of animals after randomization. Animals that did not receive exogenous surfactant (No Rx and PEG-only groups) had oxygenation values that remained below 100 mm Hg over the 180 min of ventilation after randomization. There were no significant differences in oxygenation values either within or between these groups. All four surfactant-treated groups had immediate and significant improvements in Pa_O2 values within 5 min after treatment (p < 0.05 versus respective pretreatment values) with no significant differences observed among these groups at this time point. Shortly thereafter, however, oxygenation values started to differ among groups. Animals given surfactant alone had slight but nonsignificant decreases in their Pa_O2 values over the first 60 min after treatment but then stabilized and tended to increase over the latter 60 min of ventilation.

View larger version (23K): [in this window] [in a new window]   Figure 1.   Mean PaO2 values before treatment (Time 0) and over the 3 h after treatment in the various treatment groups. Animals given surfactant alone at doses of 100 mg/kg and 25 mg/kg had significant improvements in oxygenation, which were sustained over the 3-h monitoring period. Animals given polyethylene glycol (PEG) with the exogenous surfactant showed deterioration in oxygen values for both the 100 mg/kg + PEG group (100 + PEG) and the 25 mg/kg + PEG group (25 + PEG). Animals given PEG alone (PEG) and animals not receiving treatment (No Rx) had no significant change in oxygenation values over the 3-h period. In general, animals given surfactant + PEG had inferior responses compared with the respective surfactant-only treatment groups.

Animals given exogenous surfactant mixed with PEG, however, showed a deterioration in oxygenation values over time such that by the end of the experiment, these two groups had significantly lower Pa_O2 values than both surfactant-treated groups without PEG (p < 0.05). The 25 mg/kg + PEG group had oxygenation values that were significantly lower than the three other treatment groups (p < 0.05) and not significantly different than the two nonsurfactant treatment groups at the 180-min time point.

Mean Pa_CO2 values for the various groups are shown in Figure 2. Before randomization, Pa_CO2 values were elevated in all groups due to the lung injury induced in these animals, with no significant differences observed among these groups. After randomization, Pa_CO2 values increased in the two nonsurfactant treatment groups (No Rx and PEG-only groups) to levels greater than their respective pretreatment values (p < 0.05), with no significant differences noted between these two groups. In all animals receiving surfactant, Pa_CO2 values decreased immediately after treatment. The surfactant-only treatment groups (25 and 100 mg/kg) had significantly lower Pa_CO2 values at 180 min compared to their respective pre-treatment values at Time 0 (p < 0.05). Animals that received PEG with surfactant had Pa_CO2 values that gradually increased over the latter part of the experiment, such that values for the 25 mg/kg + PEG group were similar to the nonsurfactant treatment groups at 180 min.

View larger version (27K): [in this window] [in a new window]   Figure 2.   Mean PaCO2 values for the various treatment groups before treatment (Time 0) and over the 3-h monitoring period after treatment. Animals given both doses of surfactant alone (25 mg/kg, 100 mg/kg) had lower PaCO2 values at 3 h compared with their respective pretreatment values. Animals given PEG with the surfactant (100 + PEG, 25 + PEG) had higher PaCO2 values than the surfactant-only groups, with values for the 25 + PEG group similar to the nonsurfactant treatment groups. The two latter groups had PaCO2 values that increased significantly by 3 h compared with their respective pretreatment values.

Mean peak inspiratory pressure (PIP) values for these groups are shown in Figure 3. There were no significant differences among groups up to and immediately before randomization. Thereafter, there was a general trend for PIP values to increase in all groups over the latter part of the experiment. In the two nonsurfactant treatment groups (No Rx and PEG alone), PIP values at 180 min were significantly higher than their respective pretreatment values at Time 0 (p < 0.05) and significantly higher compared with the four surfactant treatment groups (p < 0.05). There were no significant differences observed between these latter four groups at the 180-min time point, nor between any of these values and their respective pretreatment values.

View larger version (26K): [in this window] [in a new window]   Figure 3.   Mean peak inspiratory pressure (PIP) values for the various treatment groups before treatment (Time 0) and over the 3 h after treatment. The nonsurfactant-treated group (No Rx) and the PEG-only group (PEG) had higher PIP values than their respective pretreatment values, and higher values than the other four groups receiving surfactant ± PEG. There were no significant differences in PIP values between groups of animals given surfactant alone (100 mg/kg, 25 mg/kg) and those given surfactant + PEG (100 + PEG, 25 + PEG).

Mean arterial blood pressure was also monitored in these animals. There were no significant differences in blood pressure among these groups, either before, during, or up to 60 min after randomization. Thereafter, mean arterial pressure values in the two nonsurfactant-treated control groups decreased over the last 90 min of ventilation with mean pressures of 40 ± 7 mm Hg for the No Rx group and 38 ± 3 mm Hg for the PEG-only group. These values were significantly lower than their respective Time 0 values, and lower than the other four surfactant treatment groups at the 180-min time point (p < 0.05). Mean arterial pressure remained stable and above 55 mm Hg for all four lung-injured, surfactant treatment ± PEG groups, with no significant differences observed among these groups at any time point over the entire experimental period (data not shown).

In the series of experiments in which normal, noninjured rabbits were ventilated and given exogenous surfactant (25 mg/kg), there were no significant differences between the surfactant treatment group and the surfactant + PEG group for any of the measured physiological outcomes over 3 h. Oxygenation values remained above 400 mm Hg throughout the ventilation period, Pa_CO2 values remained within the 40-45 mm Hg range, and PIP values remained low and unchanged over the entire experimental period for these two groups (data not shown). In addition, there were no significant differences at the 180-min time point for each of these two groups compared with their respective pretreatment values.

Lavage Analyses

The total volumes of saline used for the final lavage procedures, as well as the percent recovery of the lavage volumes were similar in all groups. The total amount of surfactant in the lavage recovered from the various groups of lung-injured animals after sacrifice, and the pool sizes of both large (LA) and small (SA) surfactant aggregates are shown in Figure 4. As anticipated, the amount of surfactant recovered from all surfactant treatment groups (with or without PEG) was significantly greater than the nonsurfactant treatment groups (p < 0.01). The latter groups had very low pool sizes, with no significant differences observed between these two groups (Figure 4A). In addition, animals given the higher doses of surfactant (100 mg/kg ± PEG) had greater phospholipid pool sizes than both groups given the lower dose (25 mg/kg ± PEG, p < 0.05). Comparisons within these surfactant treatment groups revealed that animals given a particular dose of surfactant + PEG had lower total pool sizes than their respective surfactant-only treatment group, and this difference was significant for the 25 mg/kg + PEG group compared with the 25 mg/kg group (p < 0.05). Of note, preliminary experiments combining surfactant with PEG confirmed that the PEG did not interfere with the phosphorus assay used in this study (data not shown).

View larger version (26K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Figure 4.   (A) Average total phospholipid pool sizes of animals in the various treatment groups. In general, animals receiving surfactant had significantly higher total pool sizes than those not receiving surfactant (No Rx, PEG). Groups of animals receiving the higher doses of surfactant (100 mg/kg, 100 + PEG) had pool sizes higher than those receiving the lower dose of surfactant (25 mg/kg, 25 + PEG). Pool sizes were also lower in animals given surfactant + PEG compared with their respective surfactant-only treatment group, which was significant for the 25 mg/kg dose of surfactant + PEG (25 + PEG) compared with the 25 mg/kg surfactant dose (25 mg/kg). (B) Average large aggregate (LA) and small aggregate (SA) surfactant pool sizes for the various treatment groups. Surfactant aggregate pool sizes were low in the nonsurfactant-treated groups (No Rx, PEG) compared with the four surfactant treatment groups. In addition, these former groups have relatively more of the surfactant recovered in SA forms compared with LA forms. In the surfactant treatment groups, large aggregate pool sizes were significantly higher in the surfactant-only groups (100 mg/kg, 25 mg/kg) compared with the similar dosage given with PEG (100 + PEG, 25 + PEG). Small aggregate pool sizes were not significantly different between the surfactant-only and surfactant + PEG groups for each of the doses tested.  LA;  SA.

Analyses of surfactant aggregate pool sizes revealed that relatively small amounts of both LA and SA were recovered in the nonsurfactant treatment groups (No Rx and PEG only), and that most of this surfactant existed in SA forms (approximately 75%) in these injured lungs (Figure 4B). Comparisons among the surfactant treatment groups revealed that both LA and SA pool sizes were higher in the 100 mg/kg ± PEG groups compared with the 25 mg/kg ± PEG groups, and that LA pool sizes were greater in those animals that received surfactant alone compared with groups given the same dose of surfactant mixed with PEG. This was significant for both the 25 and 100 mg/kg treatment groups (p < 0.05 versus their respective surfactant + PEG groups). There were no significant differences in SA pool sizes among the specific treatment groups, irrespective of PEG administration. Expressing these pool sizes as percent LA revealed that significantly more of the alveolar surfactant was recovered in LA forms for animals given surfactant alone (71% for 100 mg/kg and 51% for 25 mg/kg) compared with those given PEG with the surfactant (38% for 100 mg/kg + PEG and 30% for 25 mg/kg + PEG).

Analyses of the surfactant recovered from the two groups of normal, noninjured animals given the 25 mg/kg dose of surfactant ± PEG revealed that animals given surfactant alone had greater total (17.6 ± 1.3 mg/kg) and LA (8.6 ± 1.5 mg/kg) pool sizes than the normal group given surfactant + PEG (12.6 ± 0.5 and 4.8 ± 0.3 mg/kg, respectively). These differences were similar to those observed in the injured animals when comparing the surfactant-alone groups with the surfactant + PEG groups.

The total protein recovered from the six groups of lung-injured animals was high, and ranged from 180 to 282 mg/kg body weight, with no significant differences observed among these six groups. The total protein recovered from the two normal groups of animals in this study was only 18 ± 3 mg/kg.

Surface Activity and Cycling Analysis

Minimum surface tension values after 100 pulsations in the bubble surfactometer for the various samples of LA tested are shown in Figure 5. The first three bars show the surface activity of aliquots taken from the input samples used to treat the various groups of animals in this study. These samples involved material from the surfactant-alone (S) groups and the surfactant + PEG groups (S + P), including both the 25 mg/kg dose (25) and the 100 mg/kg dose (100). All samples were tested at a concentration of 2 mg lipid/ml. These values were all relatively low (< 5 mN/m) and there were no significant differences between samples. Aliquots of LA samples recovered after sacrifice from the four surfactant treatment groups are shown in the remaining bars. The minimum surface tension values of these samples, also analyzed at 2 mg/ml, were all significantly higher than the input samples (p < 0.05). In addition, LA from the 25 mg/kg ± PEG group tended to have higher surface tension values than the 100 mg/kg ± PEG group, but these differences were not significant. There were insufficient amounts of surfactant recovered after sacrifice from the saline lavaged, nonsurfactant treatment groups to assess surface activity.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Mean minimum surface tension values of the various samples tested for surface activity using the pulsating bubble surfactometer. Input samples consisted of aliquots of surfactant alone (Input S) and the input samples taken from the two doses of surfactant + PEG (Input S + P) administered to the lung-injured animals. Surface tension values were similar for the surfactant sample, the 100 mg/kg dose of surfactant + PEG (S+P[100]), and the 25 mg/kg dose of surfactant + PEG (S+P[25]). Surface tension for aliquots of large aggregate samples recovered from the various groups of animals after sacrifice are shown in the last four bars. Surface tension values were higher in these groups compared with the input samples, although there were no significant differences between these various treatment groups with respect to the function of their large aggregates after sacrifice.

Aliquots of the input samples used to treat the surfactant ± PEG groups were also subjected to surface area cycling for 3 h to assess the in vitro conversion kinetics of these LA into SA. These data revealed no significant differences between the surfactant-alone samples and the surfactant + PEG samples. After 3 h of cycling, 51 ± 3% of the surfactant sample remained in LA forms compared with 54 ± 2% of the surfactant + PEG samples.

	DISCUSSION

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Contrary to our hypothesis, PEG added to exogenous surfactant attenuated the physiological improvements in lung function observed when the surfactant was administered alone in this model of acute lung injury. These effects of PEG were demonstrated for both doses of surfactant tested in this study. We chose to use PEG for these studies as it was the most effective polymer tested in previous studies, and this compound has been shown to be safe in humans when used in the concentrations reflected in these animal studies (11). Given the evidence that PEG actually augmented surfactant responses in the previous studies, the results of the present study were surprising. Both in vitro and in vivo studies had evaluated the potential of adding various nonionic polymers, including PEG and dextran, to exogenous surfactant. Taeusch and colleagues used a bubble surfactometer to show that these polymers added to surfactant prevented the inactivation of this surfactant when inhibitors such as protein, meconium, or lysophosphatidylcholine were added (14). Kobayashi and coworkers also showed in vitro that the addition of dextran to a mixture of an exogenous surfactant preparation plus albumin improved the surface tension properties of this mixture compared with a surfactant-albumin combination without dextran (11). The promising results of these in vitro experiments led to in vivo studies evaluating the combination of these nonionic polymers with surfactant preparations. Initially, dextran was added to a surfactant-albumin mixture and instilled into preterm rabbits with superior responses demonstrated in these animals compared with a group given the surfactant-albumin mixture alone (11). Lu and colleagues subsequently instilled a combination of either PEG or dextran plus an exogenous surfactant preparation into the lungs of adult rats 60 min after lung injury was induced in these animals via intratracheal administration of meconium (12). They found that PEG, but not dextran, improved the surfactant response over the subsequent 3 h of ventilation compared with surfactant alone. These authors suggested that although this therapeutic approach may have potential benefit in humans with acute lung injury, further studies were warranted not only to verify their results but also to evaluate potential mechanisms responsible for these observations.

One of our objectives was to evaluate the efficacy of PEG plus surfactant in an alternative model of acute lung injury involving a larger species. We also wanted to document what effects these polymers had on the administered surfactant in order to gain insight into potential mechanisms responsible for the physiologic responses observed. As noted, it is unknown how these polymers enhanced the function of surfactant in previous studies, and whether the metabolism of the surfactant was altered once deposited within the airspace. Based on information obtained in the present study, these interactions are obviously complex and likely depend on the nature of the injury itself, the specific polymer used, and the particular surfactant preparation administered.

Interestingly, the addition of PEG to surfactant in the present study did not affect the acute responses these animals exhibited to the surfactant immediately after instillation. Both doses of surfactant resulted in significant improvements in oxygenation shortly after the surfactant was instilled, with or without the addition of PEG. Previous studies showed that the beneficial effects of adding PEG to surfactant mainly involved peak oxygenation responses shortly after administration. Although peak responses were similar among groups in the present study, they were not sustained in the PEG-treated groups. The mechanisms responsible for these deleterious effects of adding PEG to exogenous surfactant, relative to the effects of the exogenous surfactant alone, are unknown. It is unlikely that the PEG itself was toxic or directly inhibited surfactant function. The group of normal animals tolerated the instillation of PEG plus surfactant well, and the PEG-alone treatment group of injured animals had responses similar to the No Rx group. Furthermore, in vitro surface tension values of the input samples of surfactant + PEG were similar to the surfactant-alone samples. It is also unlikely that the specific surfactant preparation utilized accounted for the inferior responses in the surfactant plus PEG groups. This surfactant and the techniques used to prepare the input samples were the same as in previous studies, which demonstrated beneficial effects of this polymer when added to this surfactant (12, 14).

Although it is possible that differences in either the injury models utilized and/or the species involved may have accounted for the different results in these two studies, this too would seem unlikely. Because the major mechanism felt to be responsible for the beneficial effects of PEG was to reverse the inhibition of proteins on the function of the administered surfactant, improvements in the lung-injured rabbits utilized in the present study would be expected. These animals had marked elevations of serum protein recovered in the alveolar lavage after sacrifice compared with normal animals, but the addition of PEG made the situation worse. The saline lavage model is distinct from most models of acute lung injury due to the removal of the endogenous surfactant system via the lavage procedure. This model does result in a significant inflammatory response induced by the subsequent period of ventilation, however, and has been utilized extensively in previous studies evaluating physiologic responses to various surfactants, and has shown consistent results (15, 16). Nevertheless, it is possible that the presence of an endogenous surfactant system in injured lungs, albeit altered, may be important for the beneficial effects of PEG when added to exogenous surfactant. It should be noted, however, that previous in vitro studies that did not involve endogenous surfactant showed that PEG improved exogenous surfactant function, and that surfactant-deficient preterm rabbits also benefited from this treatment approach, casting doubt on this latter hypothesis (11, 14). To determine if the nature of the injury is indeed an important factor in a host's response to surfactant plus nonionic polymers, future studies should involve various models of lung injury involving different types of pulmonary insults.

Unlike previous studies, we evaluated the surfactant system of the various groups after sacrifice to gain insight into the mechanisms responsible for the effects of PEG on the administered surfactant. We found decreased total alveolar surfactant pool sizes as well as lower amounts of the large surfactant aggregates in animals that were given PEG with the surfactant compared with groups given surfactant alone. Moreover, the function of the isolated LA remaining in the lung was impaired in all animals, no doubt due to the large quantities of protein present within the airspaces of these lungs (7, 24). Although the function of whole lung lavage was not assessed in this study, we would anticipate similar, if not worse surface tension values compared with the LA fractions given the amount of protein recovered in these animals' lungs. We suggest that the observed differences in the quantity and function of the recovered surfactant likely contributed to the inferior physiologic responses observed in animals given PEG compared with the surfactant-only groups. Although the exact mechanism(s) leading to the differences in surfactant pool sizes are unknown, the observation that animals given PEG plus surfactant had immediate improvements in oxygenation but subsequently deteriorated suggests that PEG may have interfered with the alveolar metabolism of the administered surfactant over time rather than the ability of the surfactant to form an effective lining layer within the lung immediately after instillation. Because our initial results did not provide direct evidence that altered surfactant metabolism was specifically responsible for the lung deterioration or vice versa, we performed additional experiments in which exogenous surfactant with and without PEG was instilled into the lungs of normal, healthy rabbits. The results of these experiments showed no deleterious effects of adding PEG to surfactant but significant differences in surfactant pool sizes. Specifically, the changes in pool sizes in the normal animals were similar to those observed in the injured animals, indicating that the changes in pool size noted in the injured animals were independent of the presence of lung injury, rather than a result of the lung injury. As noted previously, however, surfactant metabolism within the mature lung is complex and may even vary with the amount of exogenous surfactant administered. This latter factor may explain why the recovered pool sizes in these animals were somewhat disproportionate for the two different doses given to these animals (Figure 4).

To further probe the possibility that PEG may have influenced alveolar surfactant metabolism, surface area cycling experiments were also performed. Previous studies evaluating surfactant metabolism in vivo using various models of acute lung injury have shown an increased conversion of functionally superior LA into functionally inferior SA over time (5, 15, 25, 26). This process results in marked changes in aggregate pool sizes, which, in turn, contributes to the lung dysfunction associated with the injury. The in vitro surface area cycling experiments performed with PEG added to the surfactant and cycled for various time periods revealed no effect of PEG on LA conversion in vitro, even after 3 h of cycling. These results, and the fact that SA pool sizes were similar between the surfactant plus PEG and surfactant-alone groups for the various doses, suggested that increased alveolar conversion of LA into SA was not a major factor contributing to the differences in LA pool sizes observed between the groups. We speculate that PEG may have affected LA forms within the airspace by enhancing tissue uptake and/or association, thereby resulting in a decreased recovery of LA at sacrifice and lower total pools. More elaborate in vivo metabolic studies are required to investigate these mechanisms further.

In summary, we have shown that the nonionic polymer PEG added to an exogenous surfactant preparation attenuated the efficacy of the surfactant in this model of acute lung injury. A possible mechanism responsible for these deleterious effects of PEG could be related to the effects of the polymer on the metabolism of the administered LA forms within the airspace. This may be model-specific, however, and these studies emphasize the need for further preclinical studies involving various models and species prior to embarking on human clinical trials.