Abstract
We investigated the effect of ultrasonic nebulization versus instillation of exogenous surfactant on gas exchange abnormalities provoked by detergent inhalation in perfused rabbit lungs. Ventilation-perfusion (V˙ a/Q˙) distribution was assessed by the multiple inert gas elimination technique. For nebulization of natural bovine surfactant (AIveofact ®), an ultrasonic device was placed in line with the inspiratory gas flow tubing, manufacturing particles with a mass median aerodynamic diameter of ∼ 4.5 μ M and high aerosol concentration. In vitro studies demonstrated biochemical and biophysical integrity of postnebulization surfactant. Lung aerosol deposition was monitored by a laserphotometric technique. In lungs with sham inhalation of saline, tracheal instillation of surfactant ( ∼ 11 mg/kg body weight, infused over 50 min) provoked substantial V˙ a/Q˙ mismatch and limited shunt flow, whereas lung surfactant deposition by ultrasonic nebulization ( ∼ 7 to 9 mg/kg body weight; nebulization time, 50 min) did not interfere with physiologic gas exchange. Tween 20 inhalation provoked severe V˙ a/Q˙ mismatch with predominant shunt-flow ( ∼ 21%). This was not reversed by “rescue” application of instilled surfactant, but largely reversed by nebulized surfactant (shunt reduced to 5.5%; p < 0.01). Analysis of postaerosol lavage fluid demonstrated partial reconstitution of surface activity by nebulized surfactant. We conclude that ultrasonic nebulization may be employed for efficient delivery of functionally intact natural surfactant to the distal bronchoalveolar space. This approach effects rapid improvement of gas exchange in a model of acute homogeneous lung injury.
Gas exchange disturbances in the acute respiratory distress syndrome of the adult (ARDS) are characterized by ventilation-perfusion mismatch with predominance of shunt-flow (1, 2). Surfactant abnormalities have long been implicated in these gas exchange disturbances, evidence by the facts that (1) biochemical and biophysical surfactant alterations are encountered under clinical conditions of ARDS (3, 4), (2) induction of surfactant alterations reproduces gas exchange disturbances in different animal models (5), and (3) transbronchial administration of surfactant may improve gas exchange in models of ARDS. In the majority of these studies, natural surfactant was directly instilled into the airways, but this procedure is hampered by nonuniform distribution of the surfactant material, and a high dosage regimen is required, which surpasses manifoldly the quantity of the endogenous surfactant pool (6-9). An appealing alternative represents the surfactant delivery as aerosol, and by employing jet nebulization this approach was shown to improve gas exchange in experimental states of surfactant deficiency by Lewis and coworkers (7-10). Jet nebulization is, however, hampered by the limited efficacy in aerosol production and the constant and high gas flow necessary for its generation. Accordingly, in the cited studies, the lung surfactant deposition ranged only between ∼ 0.15 and 1.6 mg/kg body weight per hour. To put this value in perspective, 50 to 100 mg/kg surfactant is usually instilled in preterm infants with surfactant deficiency, and even 300 to 500 mg/kg (instilled in the trachea or applied via bronchsocope) has been reported to be necessary to achieve an acute improvement of arterial oxygenation in patients with ARDS, possessing high surfactant inhibitory capacity in the alveolar space (11, 12). In a recent study with continuous jet nebulization of a synthetic surfactant material for as long as 5 d in patients with ARDS, in which no benefit was found, the investigators estimated that they achieved a lung surfactant lipid deposition of < 5 mg per kg body weight per day despite the ongoing nebulization procedure (13). A nebulization technique effecting lung deposition of intact surfactant with higher efficacy would thus be highly desirable. In the current study we employed an ultrasonic nebulization device, which allowed us to increase the surfactant “load” of inhaled air approximately 8-fold as compared with standard jet nebulizers. Using this device in a model of surfactant inhibition by detergent inhalation we found that (1) postnebulization surfactant is functionally intact, (2) ∼ 10 mg surfactant/kg body weight per hour is deposited in the lungs by this technique, and (3) detergent-elicited severe gas exchange abnormalities are acutely reversed. Further evaluation of this approach may be worthwhile to improve the preconditions for surfactant administration via the inhalative route.
Alveofact®, a calf lung surfactant extract commercially available for treatment of the neonatal respiratory distress syndrome, was a gift from Dr. Karl Thomae GmbH (Biberach an der Riss, Germany). Polyoxyethylene sorbitan monolaurate (Tween 20) was obtained from Sigma (Deisenhofen, Germany). Halothane was supplied by Hoechst AG (Frankfurt am Main, Germany). Gas mixtures of sulfur hexafluoride (SF6), ethane, and cyclopropane (10, 20, and 70%; quantified by gas chromatography) were from Messer Griesheim (Frankfurt-Griesheim, Germany). All other analytic grade biochemicals, including diethyl ether and acetone, were purchased from Merck (Darmstadt, Germany). Columns for gas chromatography, already packed with Hayesep Q mesh 80/100 (FID) and a 5-Å molecular sieve, were obtained from Chrompack (Frankfurt am Main, Germany). The ultrasonic nebulizers Portasonic II (Model 500G) were graciously supplied by DeVilbiss Medizinische Produkte GmbH (Langen, Germany).
The perfused lung model has been previously described (14). Briefly, rabbits of either sex (weighing 2.5 to 3.1 kg) were anaesthetized intravenously with ketamine/xylazine and anticoagulated with heparin (1,000 U/kg). Tracheostomy was performed and the animals were ventilated with room air, using a Harvard respirator (cat ventilator; Hugo Sachs Elektronik, March Hugstetten, Germany). After midsternal thoracotomy, catheters were placed into the pulmonary artery and the left atrium, and perfusion with Krebs Henseleit buffer, containing 4% wt/vol hydroxyethylamylopectine (KHHB), was started. For washout of blood, perfusate was initially not recirculated, and the lungs were removed without interruption of ventilation and perfusion. The lungs were placed in a temperature-equilibrated chamber at 37.5° C, freely suspended from a force transducer for monitoring of organ weight. In a recirculating system the flow was slowly increased to 150 ml/min (total volume, 350 ml). Left atrial pressure was set at 2 mm Hg in all experiments. In parallel with onset of artificial perfusion, an air mixture of 80.5% N2, 15% O2, and 4.5% CO2 was used for ventilation. Tidal volume (11 ml/kg) and frequency (10 to 13 breaths/min) were adapted to maintain the pH of the recirculating buffer in the range between 7.35 and 7.37. A positive end-expiratory pressure of 1 mm Hg was used throughout. The Po 2 and Pco 2 values in the postlung buffer fluid ranged between 100 and 120 mm Hg and 38 and 43 mm Hg, respectively. Pressures in the pulmonary artery, the left atrium, and the trachea were registered by means of small-diameter tubing threaded into the perfusion catheters and the trachea and connected to pressure transducers (zero referenced at the hilus). Perfusate samples were taken by use of these tubes. Gas samples were taken from the outlet of a copper expiration gas mixing box. The whole system was heated to 37.5° C and monitored by temperature-sensoring in the pulmonary artery catheter and the expiration gas mixing box.
Lungs included in the study had (1) a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis; (2) pulmonary artery and ventilation pressures in the normal range; (3) were isogravimetric (lung weight gain < 0.3 g/h) during an initial steady-state period of at least 45 to 60 min. According to these criteria, 2 to 5% of all lung preparations were discarded before entering the study.
The continuous V˙a/Q˙ distributions were determined by the multiple inert gas elimination technique (MIGET) as described by Wagner and coworkers (15), which has been adapted to assess gas exchange of blood-free perfused rabbit lungs (16). Briefly, six inert gases (sulfur hexafluoride [SF6], ethane, cyclopropane, halothane, diethylether, and acetone) were dissolved in KHHB and continuously infused at a rate of 0.5 ml/min. After an equilibration period of at least 40 min, 10-ml perfusate samples were simultaneously collected from the pulmonary artery and the left atrium. A corresponding 30-ml gas sample was drawn from the heated expiration gas mixing chamber. Extraction of the gases dissolved in the buffer fluid was carried out by equilibration (40 min) with nitrogen in a shaking water bath (37.5° C). The gas phases after equilibration of the buffer samples, as well as the exhaled gases were analyzed by gas chromatography. Separation and quantification of ethane, cyclopropane, halothane, diethylether, and acetone were performed with a gas chromatograph equipped with a flame ionization detector (Model 3300; Varian Associates, Palo Alto, CA), using a commercially available Hayesep Q column. SF6 was measured by a gas chromatograph fitted with an electron capture detector (Fractovap; Carlo Erba, Milano, Italy), using a 5 Å molecular sieve. The ratios of left atrial to mixed venous partial pressures (retention) and of expired to mixed venous partial pressures (excretion) were calculated for each gas and were plotted against buffer-gas partition coefficients (retention-solubility and excretion-solubility curves). The duplicate samples of each set of the retentions and the excretions were treated separately, resulting in two V˙a/Q˙ distributions by least-squares analysis with enforced smoothing, using a computer programme graciously supplied by P. D. Wagner. The final data were the average of variables determined from both distributions. The position of the distribution was also described by the mean V˙a/Q˙ ratio for perfusion (Q˙ mean) and ventilation (V˙a mean) and their dispersion by the log standard deviation of both perfusion and ventilation (log SDQ˙, log SDV˙a). These parameters of dispersion do not take into account either shunt or dead space. The residual sum of squares (RSS) was the result of testing the compatibility of the inert gas data to the derived V˙a/Q˙ distribution by the least square method. An indication of acceptable quality of the V˙a/Q˙ distributions is a RSS of 5.348 or less in half of the experimental runs (50th percentile) or 10.645 or smaller in 90% of the experimental runs (90th percentile) (17). In the present study, 69.8% of RSS were less than 5.348, and 97.7% were less than 10.645.
For delivery of saline, Tween, and surfactant, Portasonic II ultrasonic nebulizers were employed. This nebulizer produces an aerosol with a mass median aerodynamic diameter (MMAD) of 4.5 μm and a geometric standard deviation (GSD) of 2.6, as measured with a laser-diffractometer (HELOS; Sympatec, Clausthal-Zellerfeld, Germany). In order to assess the influence of ultrasonic nebulization on surfactant composition and biophysical function in vitro, Alveofact (10 mg/ml) was nebulized for 2 h (temperature in the nebulizer 37° C) with room air serving as carrier gas. Original surfactant material, material remaining in the nebulizer chamber, and trapped aerosol were analyzed for phospholipid classes by high-performance thin-layer chromatography as described (4). Aerosol was trapped in an ice-water-cooled bubble trap, which was connected to the inspiratory limb of the ventilation system. For fatty acid analysis, lipids were treated with 2 N HCl/ methanol, and the resulting fatty acid methyl esters (FAME) were applied to gas chromatography (Carlo Erba FID gas chromatograph, Fractovap 2150). SP-B was measured by a solid-phase adsorption ELISA technique (18). Surfactant biophysical properties were assessed as described below. For use in the isolated lung experiments, one Portasonic nebulizer (nebulization of Tween and saline) or two nebulizers (placed in parallel for nebulization of surfactant) were located between the ventilator and the lung to be passed by the inspiration gas. The tubings between ventilator, nebulizer, and lung and the laserphotometer and pneumotachygraph were heated to 40° C to prevent condensation. Surfactant was nebulized at a phospholipid concentration of 10 mg/ml throughout, which resulted in an optimal performance of the nebulization device and a surfactant “load” of the postnebulizer gas of ∼ 1.3 mg/L.
The amount of substances (surfactant, Tween) deposited in the rabbit lungs was evaluated as follows (Table 1). The mass of aerosolized liquid was calculated by weighing the nebulizers before and after nebulization. Aerosol losses in the delivery system were measured in separate experiments using 99mTc-enriched saline or surfactant. These experiments revealed that a mean of 50.5% nebulized mass reached the trachea. The deposition fraction of this aerosol in the lung was determined on-line by use of a laserphotometric technique recently published (19). Briefly, aerosol concentration (laserphotometer) and flow rate (pneumotachograph) were continuously monitored in inspiration and expiration at the entry of the trachea. Computer-assisted processing of these signals, with a correction for hygroscopic particle growth in the tracheobronchial space, allowed the breath-by-breath calculation of deposition fraction. During aerosol delivery, the breathing pattern was altered to enhance deposition (frequency was increased from 11 ± 2 to 40 ± 2 breaths/min and tidal volume from 11 to 14 ml/kg): no V˙a/Q˙ measurements were undertaken during the nebulization periods. In previous studies, this technique was found to result in superimposable deposition curves, when compared with on-line measurement of γ-counts upon use of a 99mTc-labeled saline aerosol (19).
| Groups | Materials Applied during the First and Second Nebulization Period | Total Nebulized Volume (ml) | Deposition Fraction (%) | Deposited Material (mg) | Deposited Material/Body Weight (mg/kg BW) | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Saline-Aer. | 1. Saline | 2.07 ± 0.17 | 45.6 ± 0.4 | — | — | |||||
| 2. Saline | 6.97 ± 0.44 | 46.2 ± 0.3 | — | — | ||||||
| Surf.-Inst. | 1. Saline | 3.18 ± 0.46 | 46.3 ± 0.7 | — | — | |||||
| 2. Surf.-Inst. | — | — | 30.0 ± 0.0 | 12.5 ± 1.9 | ||||||
| Surf.-Aer. | 1. Saline | 3.32 ± 0.44 | 45.6 ± 0.7 | — | — | |||||
| 2. Surf.-Aer. | 8.83 ± 0.69 | 45.8 ± 0.4 | 20.4 ± 1.5 | 7.8 ± 0.6 | ||||||
| Tween | 1. Tween | 2.62 ± 0.35 | 49.6 ± 0.5 | 32.8 ± 3.7 | 13.0 ± 1.4 | |||||
| 2. Saline | 8.08 ± 0.63 | 45.4 ± 0.4 | — | — | ||||||
| Tween/Surf.-Inst. | 1. Tween | 3.18 ± 0.46 | 51.2 ± 0.8 | 41.1 ± 4.8 | 13.9 ± 1.8 | |||||
| 2. Surf.-Inst. | — | — | 30.0 ± 0.1 | 11.5 ± 1.2 | ||||||
| Tween/Surf.-Aer. | 1. Tween | 2.97 ± 0.86 | 51.0 ± 0.6 | 38.2 ± 4.8 | 13.6 ± 1.8 | |||||
| 2. Surf.-Aer. | 9.58 ± 1.23 | 47.0 ± 0.2 | 22.7 ± 2.6 | 8.7 ± 1.0 |
Instillation of surfactant was undertaken via a small-diameter tubing in the trachea, ending 1 cm above the tracheal bifurcation. 30 mg surfactant (10 mg/ml in saline) were administered by slow infusion over a 50-min period.
After termination of the experiments, the entire bronchoalveolar space was lavaged with 150 ml saline in three fractions, each fraction being injected and reaspirated three times. The total recovery of lavage fluid was ∼ 95%. The lavage fluid was immediately cooled and centrifuged at 300 × g for 10 min (5° C) to remove cells.
For determination of phospholipid contents, the lavage fluids were extracted according to Bligh and Dyer (20) and phosphorus was quantified employing a colorimetric assay according to Rouser and coworkers (21). Lavage fluids were then centrifuged at 48,000 × g at 4° C for 1 h to pellet surfactant for biophysical characterization. The pellets were again analyzed for the phospholipid content, adjusted to a phospholipid concentration of 2 mg/ml, preincubated for 30 min at 37° C, and then analyzed for surface tension-reducing properties employing the pulsating bubble surfactometer as described (22). The surface tension after 12 s of adsorption period (γads) and after 5 min of film oscillation and at minimal bubble radius (γmin) are given. In the experiments with Tween nebulization, the concentration of the detergent in the original lavage fluids as well as in the 48,000 × g pellets was measured by gas chromatography. For this purpose, Bligh and Dyer extracts were directly methylated (2N hydrochloric acid/methanol) and applied to a Carlo Erba FID gas chromatograph (Fractovap 2150) equipped with a capillary column (Chrompack CP-Sil 88). The 12:0 FAME was separated from other fatty acid methyl esters, and quantification was achieved by comparison with a standard FAME solution. In lungs with Tween administration, the 48,000 × g pellets displayed lower Tween-surfactant ratios than did the original lavage fluids. Therefore, before performing biophysical measurements in the surfactant pellets, these were enriched with Tween to restore the Tween-surfactant ratio of the individual original lavage fluid.
A total of 42 isolated lung experiments was performed. After an initial steady-state period of at least 40 min, samples for estimation of V˙a/Q˙ relationships were drawn. Further V˙a/Q˙ measurements were undertaken according to the different protocols:
Control (n = 6). No interventions were undertaken. V˙a/Q˙ measurements were carried out at 0, 10, 40, 100, and 130 min.
Nebulization of saline (Saline-Aer.) (n = 6). Saline was nebulized over a 10-min period, and time was set at zero. A second nebulization of saline was carried out over a 50-min period (40 to 100 min). Detailed data on nebulized volume are given in Table 1. V˙a/Q˙ measurements were performed at 10, 40, 100, and 130 min.
Nebulization of surfactant (Surf.-Aer.) (n = 6). After initial nebulization of saline, surfactant was aerosolized over a 50-min period (40 to 100 min). V˙a/Q˙ measurements were undertaken as in Saline-Aer.
Instillation of surfactant (Surf.-Inst.) (n = 6). After initial nebulization of saline, instillation of 30 mg surfactant in 3 ml volume was performed over a 50-min period (40 to 100 min). V˙a/Q˙ measurements were undertaken as in Saline-Aer.
Nebulization of Tween (Tween) (n = 6). Tween (5% in saline) was nebulized over a 10-min period, and time was set at zero. This was followed by a 50-min nebulization of saline (40 to 100 min). V˙a/Q˙ measurements were performed as in Saline-Aer.
Nebulization of Tween and instillation of surfactant (Tween/Surf.-Inst.) (n = 6). After initial 10-min nebulization of Tween, instillation of 30 mg surfactant in 3 ml volume was performed over a 50-min period (40 to 100 min). V˙a/Q˙ measurements were undertaken as in Saline-Aer.
Nebulization of Tween and nebulization of surfactant (Tween/ Surf.-Aer.) (n = 6). After initial 10-min nebulization of Tween, surfactant was aerosolized over a 5-min period (40 to 100 min). V˙a/Q˙ measurements were undertaken as in Saline-Aer.
Data are given as means ± SE. For analyzing statistical difference, Student's two-tailed t test for unpaired samples was performed. After Bonferroni's correction a p value < 0.05 was considered significant.
As detailed in Table 2, a 2-h ultrasonic nebulization period did not affect the biochemical properties (phospholipid classes, SP-B content) of the surfactant material, whether remaining in the nebulization chamber or being trapped after nebulization. This was also true for the adsorption facilities and the surface tension-lowering properties of the surface-active material.
| Prenebulization | Postremnant | Postaerosol | ||||
|---|---|---|---|---|---|---|
| PC, % of total PL | 87.7 ± 0.6 | 87.0 ± 1.2 | 88.3 ± 2.0 | |||
| PC-16:0, % of total PC | 68.9 ± 4.2 | 69.6 ± 3.9 | 73.8 ± 4.4 | |||
| PG, % of total PL | 8.8 ± 1.2 | 9.2 ± 1.8 | 8.8 ± 1.5 | |||
| PG-18:1, % of total PG | 46.7 ± 3.5 | 45.5 ± 2.9 | 43.1 ± 4.1 | |||
| SP-B, % of PL | 3.5 ± 1.5 | 3.3 ± 0.3 | 3.2 ± 0.2 | |||
| γmin, mN/m | 0.0 | 1.1 ± 0.7 | 0.0 | |||
| γads, mN/m | 21.8 ± 0.35 | 22.4 ± 0.3 | 22.2 ± 0.3 |
After termination of the steady-state period, all lungs displayed mean pulmonary arterial pressures of 6 to 10 mm Hg. Baseline V˙a/Q˙ measurements revealed physiologic V˙a/Q˙ distribution in all experiments. Unimodal narrow distribution of perfusion to midrange V˙a/Q˙ areas (0.1 < V˙a/Q˙ < 10) was observed throughout. Shunt flow (V˙a/Q˙ < 0.005) and perfusion to poorly ventilated regions (0.005 < V˙a/Q˙ < 0.1) ranged below 2% and no perfusion to high V˙a/Q˙ regions (10 < V˙a/Q˙ < 100) was observed. Ventilation of both dead space (V˙a/Q˙ > 100) and midrange V˙a/Q˙ areas (0.1 < V˙a/Q˙ < 10) ranged at about 50%. The low log standard deviation of ventilation (0.3 to 0.4) and perfusion (0.3 to 0.5) indicated narrow dispersion of ventilation and perfusion in midrange V˙a/Q˙ areas. All lungs were isogravimetric (lung weight gain < 0.3 g/h). In control lungs not undergoing any intervention, all variables remained constant over the entire 135-min observation period.
Both nebulization periods did not affect the pulmonary artery pressure. A total weight gain of ∼ 7 g was monitored, which slightly surpassed the total deposited aerosol mass (Table 1 and Table 3). In response to the first nebulization period, only a slight leftward shift of mean perfusion (Q˙ mean) and a slight broadening of perfusion distribution in midrange V˙a/Q˙ areas (log SDQ˙) were noted (not depicted). These changes progressed during the second nebulization period, and some perfusion of areas with low V˙a/Q˙ ratios (maximum ∼ 3%) as well as some shunt flow (maximum ∼ 4%) became apparent (Figures 1 and 3). The mean V˙a/Q˙ ratio of ventilation distribution (V˙a mean) and the dispersion of the ventilation (log SDV˙a) remained unaltered.
| Time (min) | Saline-Aer. | Surf.-Aer. | Surf.-Inst. | Tween | Tween/Surf.-Inst. | Tween/Surf.-Aer. | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| −15 | Ppa, mm Hg | 7.8 ± 0.3 | 8.1 ± 0.3 | 8.1 ± 0.5 | 8.0 ± 0.4 | 8.4 ± 0.2 | 8.4 ± 0.4 | |||||||
| ΔW, g | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | ||||||||
| 45 | Ppa, mm Hg | 8.2 ± 0.4 | 8.4 ± 0.3 | 8.0 ± 0.4 | 8.1 ± 0.3 | 7.9 ± 0.2 | 8.3 ± 0.4 | |||||||
| ΔW, g | 2.7 ± 0.2 | 2.6 ± 0.3 | 1.7 ± 0.6 | 2.6 ± 0.4 | 2.4 ± 0.6 | 3.3 ± 0.2 | ||||||||
| 105 | Ppa, mm Hg | 8.2 ± 0.4 | 8.4 ± 0.3 | 8.1 ± 0.4 | 8.2 ± 0.3 | 8.0 ± 0.4 | 8.4 ± 0.5 | |||||||
| ΔW, g | 7.5 ± 0.6 | 6.7 ± 0.4 | 3.6 ± 0.3† | 7.7 ± 0.6 | 7.0 ± 0.8 | 7.6 ± 0.4 | ||||||||
| 135 | Ppa, mm Hg | 8.0 ± 0.3 | 8.3 ± 0.4 | 8.1 ± 0.5 | 8.3 ± 0.3 | 7.3 ± 0.4 | 8.5 ± 0.6 | |||||||
| ΔW, g | 7.9 ± 0.6 | 7.7 ± 0.4 | 4.2 ± 0.4† | 8.5 ± 0.7 | 8.6 ± 0.8 | 8.2 ± 0.4 |
Fig. 1. Intrapulmonary shunt flow in response to saline nebulization with subsequent application of a second saline aerosol dose (Saline-Aer.), nebulization of surfactant (Surf.-Aer.), or surfactant instillation (Surf.-Inst.). The intervention maneuvers are indicated; time was set at zero after the first nebulization period. Mean ± SEM of six independent experiments are given; error bars are missing when falling into symbol. Shunt, percentage of perfusion on nonventilated areas (V˙a/Q˙ < 0.005). †p < 0.05 as compared with Saline-Aer. lungs.
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Fig. 3. Percentage of perfusion of poorly ventilated areas (Low V˙a/Q˙ ) in response to saline nebulization, with subsequent application of a second saline aerosol dose (Saline-Aer.), nebulization of surfactant (Surf.-Aer.), surfactant instillation (Surf.-Inst.). The intervention maneuvers are indicated; time was set at zero after the first nebulization period. Mean ± SEM of six independent experiments are given; error bars are missing when falling into symbol. Low V˙a/Q˙, percentage of perfusion of poorly ventilated areas (0.005 < V˙a/Q˙ < 0.1); †††p < 0.001 as compared with Saline-Aer. lungs.
[More] [Minimize]Similar to that in the preceding group, a total weight gain of ∼ 7 g was noted, without any change in vascular perfusion pressure (Table 3). The initial saline nebulization period again provoked a decrease in Q˙ mean and a slight increase in log SDQ˙. This trend did not, however, continue during the second nebulization period, in which surfactant was administered, and there was no significant appearance of shunt flow or perfusion of low V˙a/Q˙ areas (Figures 1 and 3).
The instillation of 30 mg surfactant in 3 ml saline was reflected by a corresponding increase in lung weight, again without influencing the pulmonary artery pressure (Table 3). This maneuver, however, substantially affected the gas exchange conditions: both shunt flow (maximum ∼ 9%) and perfusion of areas with low V˙a/Q˙ ratios (maximum ∼ 19%) increased significantly (Figures 1 and 3). In parallel, a leftward shift of mean perfusion (Q˙ mean) and a marked broadening of perfusion dispersion (increase in log SDQ˙) were noted, in conjunction with a bimodal distribution of perfusion in five of the six lungs (not shown in detail). In addition, there was a rightward shift of mean ventilation (V˙a mean) and a broadening of ventilation distribution in midrange V˙a/Q˙ areas (log SDV˙a, not depicted). Dead space remained unchanged.
The sequential nebulization of first Tween 20 and then saline provoked a weight gain of ∼ 8 g without changes in perfusion pressure (Table 3). This was accompanied by dramatic disturbances in gas exchange: shunt flow increased to a maximum of ∼ 21%, and perfusion of low V˙a/Q˙ regions appeared with a maximum of ∼ 10% (Figures 2 and 4). These alterations were again accompanied by a decrease of Q˙ mean and a marked broadening of the perfusion distribution in midrange V˙a/Q˙ areas (increase in log SDQ˙). There was some minor increase in the mean V˙a/Q˙ ratio of ventilation distribution (Va mean), and the dispersion of ventilation (log SDV˙a) remained unchanged (data not given in detail). Ventilation of high V˙a/Q˙ areas was not detected.
Fig. 2. Intrapulmonary shunt flow in response to Tween nebulization with subsequent application of a saline aerosol dose (Tween), nebulization of surfactant (Tween/Surf.-Aer.), or surfactant instillation (Tween/Surf.-Inst.). The intervention maneuvers are indicated; time was set at zero after the first nebulization period. Mean ± SEM of six independent experiments are given; error bars are missing when falling into symbol. Shunt, percentage of perfusion on nonventilated areas (V˙a/Q˙ < 0.005). ‡‡p < 0.01 as compared with the Tween group.
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Fig. 4. Percentage of perfusion of poorly ventilated areas (Low V˙a/Q˙) in response to Tween nebulization with subsequent application of a saline aerosol dose ( Tween), nebulization of surfactant (Tween/Surf. -Aer.), or surfactant instillation (Tween/Surf.-Inst.). The intervention maneuvers are indicated; time was set at zero after the first nebulization period. Mean ± SEM of six independent experiments are given; error bars are missing when falling into symbol. Low V˙a/Q˙, percentage of perfusion of poorly ventilated areas (0.005 < V˙a/Q˙ < 0.1); ‡p < 0.05 as compared to Tween group.
[More] [Minimize]The “rescue” application of 30 mg surfactant, instilled after the preceding Tween nebulization, exerted only marginal influence on the detergent-elicited alterations in gas exchange: the shunt flow increased to a maximum of ∼ 17% (instead of 21% in the absence of surfactant), but there was a higher rate in perfusion of low V˙a/Q˙ areas (maximum 20% instead of 10%, Figures 2 and 4). The Tween-elicited leftward shift of perfusion (decrease in Q˙ mean) and broadening of perfusion distribution in midrange V˙a/Q˙ areas (increase in log SDQ˙) were even further increased by the subsequent surfactant instillation (not given in detail). This was also true for the rise in the mean V˙a/Q˙ ratio of ventilation distribution (V˙a mean), and some increased dispersion of ventilation distribution became evident in response to surfactant instillation in these lungs. The total weight gain in these lungs approximated 8.5 g (Table 3).
As in the preceding groups, Tween nebulization provoked a rapid increase in shunt flow and perfusion of low V˙a/Q˙ areas (Figures 2 and 4), paralleled by a broadening and leftward shift or perfusion distribution (increase in log SDQ˙ and decrease in Q˙ mean; not given in detail). These alterations were significantly reversed by the subsequent “rescue” nebulization of surfactant (total deposited amount, 22.7 ± 2.6 mg): the shunt flow was reduced to ∼ 5.5% and the perfusion of regions with low V˙a/Q˙ ratios to ∼ 3% at the end of experiments, as compared with 21% shunt and 7% low V˙a/Q˙ areas at this time point in the Tween group (Figures 2 and 4). Concomitantly, the leftward shift of perfusion distribution was partially reversed and a more narrow distribution of perfusion developed in response to surfactant nebulization in the detergent-pretreated lungs (depicted in Figure 5). Similarly, the Tween-elicited increase in the mean V˙a/Q˙ ratio of ventilation distribution was reversed by subsequent surfactant nebulization. The total weight gain in these lungs again approximated 8 g (Table 3).
Fig. 5. Ventilation perfusion matching in response to Tween nebulization and subsequent aerosol application of surfactant (one example of the Tween/Surf.-Aer. group). The V˙a/Q˙ distribution was assessed under baseline conditions, 45 min after termination of Tween nebulization, and at the end of the experiment.
[More] [Minimize]In lungs with saline nebulization, the mean lavage phospholipid concentration ranged between 110 and 150 μg/ml, corresponding to a total alveolar space phospholipid content of ∼ 7 mg/kg (Table 4). Excellent adsorption facilities and surface tension-lowering properties were noted when investigating this surfactant material in vitro. Tween nebulization did not significantly affect the lavage phospholipid content, but severely inhibited the surfactant function, with an increase in the minimal surface tension to nearly 20 mN/m and in the adsorption surface tension to ∼ 35 mN/m. As anticipated, surfactant nebulization augmented the total alveolar phospholipid pool, both in the lungs with preceding saline nebulization and with Tween application. Postsurfactant surface tension properties were still normal in the saline lungs. The deterioration of surfactant properties caused by Tween nebulization was significantly reduced, but not fully reversed, by the subsequent surfactant nebulization maneuver.
| Saline-Aer. | Surf.-Aer. | Tween | Tween/Surf.-Aer. | |||||
|---|---|---|---|---|---|---|---|---|
| PL in lavage, μg/ml | 128 ± 13 | 205 ± 14† | 156 ± 18 | 219 ± 15† | ||||
| Total recovered PL, mg | 18.2 ± 1.8 | 29.2 ± 1.9† | 22.2 ± 2.6 | 31.2 ± 2.1‡ | ||||
| γmin, mN/m | 1.1 ± 0.3 | 2.3 ± 0.5 | 18.6 ± 2.2‡ | 13.0 ± 3.5‡ | ||||
| γads, mN/m | 23.2 ± 1.5 | 21.7 ± 0.3 | 35.2 ± 2.9† | 27.7 ± 2.3 |
In a model of isolated perfused rabbit lungs, in which a detailed analysis of the gas exchange conditions was undertaken by use of the multiple inert gas elimination technique, inhalation of the detergent Tween 20 was employed for inhibition of surfactant function. This resulted in severe gas exchange disturbances, characterized by ventilation-perfusion mismatch and predominant shunt flow. Tracheal instillation of low quantities of natural surfactant did not improve gas exchange conditions in this model. In contrast, less than 1 h of ultrasonic nebulization of surfactant largely reversed the ventilation-perfusion mismatch and the shunt flow.
Detergents such as Tween 20 are known to interfere with natural surfactant function in vitro, increasing the minimal surface tension and reducing adsorption facilities. In pilot experiments, performed with different mixtures of Alveofact and Tween 20 in vitro, we found the dose-inhibition curves for both minimal and adsorption surface tension to range between a Tween/Alveofact ratio of 0.05 and 2 (data not given in detail). We ascertained that the surfactant inhibitory effect of Tween could be overcome by a mixture of additional functionally integer natural surfactant. On the basis of these pilot studies we aimed at a pulmonary deposition of Tween of ∼ 13 to 14 mg/kg body weight, thus surpassing the endogenous rabbit alveolar surfactant pool (∼ 7 mg/kg) (23, 24, and present study) nearly twofold. Accordingly, analysis of the post-Tween lavage fluid demonstrated a severe loss of minimal surface tension-lowering and adsorption facilities of the rabbit lung surfactant. Commencing immediately after the 10-min Tween nebulization period, progressive ventilation-perfusion mismatch was noted: there was marked broadening of the perfusion distribution in the midrange V˙a/Q˙ areas (> 3-fold increase in log SDQ˙), and perfusion of regions with low V˙a/Q˙ ratios appeared (maximum ∼ 10% after 90 min). In the further course, these low-ventilated (presumably partially atelectatic or dystelectatic) areas evidently fully collapsed, the percentage of low V˙a/Q˙ regions decreased, and shunt flow progressed to > 20% to be the predominant abnormality at the end of the experiments (130 min after Tween inhalation). The detergent-elicited profile of severe ventilation-perfusion mismatch and shunt-flow is well in line with the gas exchange disturbances encountered under clinical conditions of ARDS (1, 2, 25). There is good evidence that the abnormalities in lung function in this model were directly affected by the Tween-related changes in the alveolar surfactant system, and not by secondary lung injury events provoked by this agent. First, the dosage of Tween currently used was still low as compared with that in other studies (26). Second, the typical gas exchange disturbances commenced immediately after the 10-min Tween nebulization period. And third, the time course and total amount of weight gain in the Tween-nebulized lungs were undistinguishable from that in lungs undergoing sham nebulization with saline. This finding renders a less probable major contribution of detergent-elicited increase in microvascular and alveoloepithelial permeability (26-28) with secondary fluid leakage into the interstitial and alveolar spaces within the time frame of this short-term study. Overall, the appearance of severe gas exchange abnormalities in response to presumably selective surfactant inhibition by detergent inhalation, without any changes in pulmonary hemodynamics and without additional edema formation, underscores the essential physiologic role of the alveolar surfactant system in maintaining alveolar stability and enabling perfect ventilation-perfusion matching.
Tracheal instillation of surfactant in control lungs, in the absence of Tween nebulization, clearly interfered with the physiologic ventilation-perfusion matching. Marked appearance of low V˙a/Q˙ areas in the presence of only limited shunt flow was noted, along with broadened dispersion of both perfusion and ventilation in midrange V˙a/Q˙ areas and bimodality of V˙a/Q˙ distribution. This pattern of V˙a/Q˙ mismatch corresponds to that described in a model of multiple airway occlusion in the dog (29), thus favoring the assumption that occlusion of bronchial pathways may similarly occur in response to the tracheal instillation of surfactant material. The appearance of a bimodal V˙a/Q˙ distribution may be explained by the fact that after occlusion of small airways collateral ventilation prevents shunt perfusion at the expense of the appearance of low V˙a/Q˙ regions; this mechanism has been characterized in detail (30, 31). It is known that after instillation into the central airways, surfactant is distributed in an uneven fashion (9, 32). For technical reasons, since we could not rotate the isolated lungs during the surfactant instillation procedure, we chose the technique of continuous infusion of the material into the distal trachea, and this technique may give rise to a particularly nonuniform distribution (32), with airway occlusion in regions with higher fluid load. The nonuniform distribution, although not directly measured but deducted from the V˙a/Q˙ distribution and results from previous studies, also offers as the main reason for the lack of beneficial effect when the surfactant material was instilled after the preceding Tween inhalation. Overall, there was somewhat less shunt flow, but a higher pecentage of perfusion of low V˙a/Q˙ areas in these lungs, as compared with Tween administration alone. It is open for speculation whether the assumed beneficial effect of surfactant (restoration of surface activity in detergent-loaded areas) might have overcome the disadvantageous effect (airway occlusion caused by fluid instillation) if a higher dose of surfactant had been instilled in this model.
For aerosol application of surfactant we employed an ultrasonic nebulization device, placed in line with the inspiration air flow. By this technique we achieved a mean surfactant “load” of the gas passing the nebulization chamber of ∼ 1.3 mg/L, which is approximately 8-fold higher than the load we were able to establish with use of different jet nebulization devices. The increase in aerosol concentration is partly due to the fact that we aimed at a mass median aerodynamic diameter of the aerosol particles of ∼ 4.5 μM, thus surpassing the mean particle size of jet nebulizers (mostly ranging between 1 and 3.5 μM) and achieving a substantially higher mass per particle. The surfactant aerosol manufactured by ultrasonic nebulization turned out to be functionally fully active. In vitro, postnebulization material displayed surface tension lowering and adsorption facilities not different from the original material, and key biochemical variables were found to be unaltered. Efficacy of surfactant undergoing ultrasonic nebulization has also been shown in two recent reports (33, 34) In vivo, administered in control lungs, no disturbance of the physiologic ventilation-perfusion matching was noted as indicated by the sensitive MIGET technique. Thus, although a substantial percentage of the particles must be considered to be of a size beyond diameters that allow easy alveolar access (35-37), there was no evidence for occlusion of smaller airways. It may be assumed that the excellent adsorption characteristics and lateral spreading facilities of the exogenous surfactant might be sufficient to allow translocation of the material to the large alveolar surface even if the site of primary deposition is located in the distal airways. The total amount of surfactant definitely deposited in the bronchoalveolar space within the 50-min nebulization procedure ranged between 7 and 9 mg/kg body weight, which corresponds to the natural alveolar surfactant pool size of rabbits (23, 24, and present study). Performing a lavage of the entire bronchoalveolar space 30 min after termination of the nebulization procedure, approximately two thirds of the deposited surfactant was recovered. This datum is well in line with previous studies addressing lung uptake of exogenously administered surfactant, and reflects the rapid turnover of the surfactant system in the alveolar space (10, 38).
The most interesting finding of the present study was the fact that ultrasonic nebulization of Alveofact rapidly reversed the gas exchange abnormalities elicited by the preceding Tween inhalation. Perfusion of low V˙a/Q˙ areas, which had already amounted to ∼ 10% prior to surfactant nebulization, was reduced to ∼ 3%, and the shunt-flow at the end of experiments ranged at 5.5% in comparison with ∼ 21% in lungs with sole Tween nebulization, with a tendency to further decrease. Concomitantly, “rescue” nebulization of surfactant effected a better ventilation-perfusion matching in midrange V˙a/Q˙ areas, as evident from the narrowing of the perfusion distribution ( see Figure 5). Interestingly, these beneficial effects occurred even though the biophysical surfactant function was still not fully normalized. As anticipated from the in vitro studies with different Tween-surfactant mixtures, a shift in the mean Tween-surfactant ratio, from ∼ 2.0 to ∼ 1.0, because of the approximate doubling of the alveolar surfactant pool, clearly improved the surface tension-lowering and adsorption facilities assessed in the lavage fluid, but control surface activity was not yet achieved. Thus, marked improvement of gas exchange conditions may be achieved by nebulization of surfactant even if surfactant-inhibitory capacities in the alveolar space are not fully overcome by the deposited amount of exogenous surfactant. Surfactant nebulization was not followed by a decrease in lung weight, suggesting that there was no substantial reduction of pulmonary edema formation in response to surfactant nebulization within the time frame of the present experimental protocol.
Overall, the present findings support the observation of Lewis and coworkers (7-10) that even quite low amounts of inhaled surfactant might be more effective in improving gas exchange than large amounts of instilled substance. It is, nevertheless, obvious that the total amount of deposited material must be considered to be critical. Jet-nebulized surfactant has never been shown to worsen lung function, and we have demonstrated that this is also true for ultrasonic nebulization with use of larger particle size. The present study is limited in that a nonphysiologic mechanism of surfactant inhibition was employed. However, aspiration of detergents may occur accidentally in humans and result in severe lung injury, and this approach allowed calculated “titration” of surfactant inhibition and “back-titration” of the inhibitory mechanisms with exogenous surfactant in an intact lung. Although not verified by microscopic examination, the detergent-elicited changes must be assumed to be distributed in a homogeneous manner, and a nonhomogeneous model of lung injury may impose somewhat different challenges to a rescue application of surfactant. Notwithstanding these limitations, we consider the fact that ultrasonic nebulization may be employed for delivery of substantial quantities of surfactant to the peripheral lung within a reasonably short time period while maintaining in vitro and in vivo surfactant function alongside with rapid improvement of gas exchange to be encouraging and to warrant further development of this approach.
| 1. | Walmrath D., Schneider T., Pilch J., Grimminger F., Seeger W.Aerosolized prostacyclin reduces pulmonary artery pressure and improves gas exchange in the adult respiratory distress syndrome (ARDS). Lancet3421993961962 |
| 2. | Rossaint R., Falke K. J., López F., Slama K., Pison U., Zapol W. M.Inhaled nitric oxide for the adult respiratory distress syndrome. N. Engl. J. Med.3281993399405 |
| 3. | Gregory T. L., Longmore W. J., Moxley M. A., Whitsett J. A., Reed C. R., Fowler A. A., Hudson L. D., Maunder R. J., Crim C., Hyers T. M.Surfactant chemical composition and biophysical activity in acute respiratory disress syndrome. J. Clin. Invest.88199119761981 |
| 4. | Günther A., Siebert C., Schmidt R., Ziegler S., Grimminger F., Yabut M., Temmesfeld B., Walmrath D., Morr H., Seeger W.Surfactant alterations in severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema. Am. J. Respir. Crit. Care Med.1531996176184 |
| 5. | Lachmann B.Animal models and clinical pilot studies of sufactant replacement in adult respiratory distress syndrome. Eur. Respir. J. Suppl.3198998s103s |
| 6. | Loewen G. M., Holm B. A., Milanowski L., Wild L., Notter R. H., Matalon S.Alveolar hyperoxic injury in rabbits receiving exogenous surfactant. J. Appl. Physiol.66198910871092 |
| 7. | Lewis J. F., Ikegami M., Higuchi R., Jobe A. H., Absolom D.Nebulized vs. instilled exogenous surfactant in an adult injury model. J. Appl. Physiol.71199112701276 |
| 8. | Lewis J. F., McCaig L. A.Aerosolized versus instilled exogenous surfactant in a nonuniform pattern of lung injury. Am. Rev. Respir. Dis.148199311871193 |
| 9. | Lewis J. F., Tabor B., Ikegami M., Jobe A. H., Joseph M., Absolom D.Lung function and surfactant distribution in saline-lavaged sheep given instilled vs. nebulized surfactant. J. Appl. Physiol.74199312561264 |
| 10. | Lewis J., Goffin J., Yue P., McCaig L. A., Bjarneson D., Veldhuizen R. A. W.Evaluation of exogenous surfactant treatment strategies in an adult model of acute lung injury. J. Appl. Physiol.80199611561164 |
| 11. | Walmrath D., Günther A., Ghofrani H. A., Schermuly R., Schneider T., Grimminger F., Seeger W.Bronchoscopic surfactant administration in severe adult respiratory distress syndrome (ARDS). Am. J. Respir. Crit. Care Med.15419965762 |
| 12. | Gregory T. J., Gadek J. E., Weiland J. E., Hyers T. M., Crim C., Hudson L. D., Steinberg K. P., Maunder R. A., Spragg R. G., Smith R. M., Tierney D. F., Gipe B., Longmore W. J., Moxley M. A.Survanta® supplementation in patients with acute respiratory distress syndrome (ARDS) (abstract). Am. J. Respir. Crit. Care Med.1491994A567 |
| 13. | Anzueto A., Baughman R. P., Guntupalli K. K., Weg J. G., Wiedemann H. P., Raventos A. A., Lemaire F., Long W., Zaccardelli D. S., Pattishall E. N.Aerosolized surfactant in adults with sepsis- induced acute respiratory distress syndrome. N. Engl. J. Med.334199614171421 |
| 14. | Seeger W., Walmrath D., Grimminger F., Rosseau S., Schütte H., Krämer H. J., Ermert L., Kiss L.Adult respiratory distress syndrome: model systems using isolated perfused rabbit lungs. Methods Enzymol.2331994549584 |
| 15. | Wagner P. D., Saltzman H. A., West J. B.Measurement of continuous distributions of ventilation-perfusion ratios: theory. J. Appl. Physiol.361974588599 |
| 16. | Walmrath D., König R., Ernst C., Brückner H., Grimminger F., Seeger W.Ventilation-perfusion relationships in isolated blood-free perfused rabbit lungs. J. Appl. Physiol.721992374382 |
| 17. | Wagner, P. D., and J. B. West. 1980. Ventilation-perfusion relationships. In J. B. West, editor. Pulmonary Gas Exchange. Academic Press, New York. 233–235. |
| 18. | Krämer H.-J., Schmidt R., Günther A., Suzuki Y., Seeger W.Quantification of surfactant-protein B by enzyme-linked immunosorbent assay (ELISA). Am. J. Respir. Crit. Care Med.152199515401544 |
| 19. | Schmehl T., Gebhardt J., Schütte H., Scharmann A., Seeger W.On-line laser-photometric monitoring of aerosol deposition in ventilated rabbit lungs. J. Appl. Physiol.801996351356 |
| 20. | Bligh E. G., Dyer W. J.A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol.371959911917 |
| 21. | Rouser G., Fleischer S., Yamamoto A.Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids51969494496 |
| 22. | Seeger W., Grube C., Günther A., Schmidt R.Surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations. Eur. Respir. J.61993971977 |
| 23. | Berry D., Ikegami M., Jobe A.Respiratory distress and surfactant inhibition following vagatomy in rabbits. J. Appl. Physiol.61198617411748 |
| 24. | Lewis J. F., Ikegami M., Jobe A. H.Altered surfactant function and metabolism in rabbits with acute lung injury. J. Appl. Physiol.69199023032310 |
| 25. | Walmrath D., Schneider T., Schermuly R., Olschewski H., Grimminger F., Seeger W.Direct comparison of inhaled nitric oxide and aerosolized prostacyclin in acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med.1531996991996 |
| 26. | Bredenberg C. E., Paskanik A. M., Niemann G. F.High surface tension pulmonary edema. J. Surg. Res.341983515523 |
| 27. | Wang C. Z., Barrow R. E., Cox C. S., Yang S. F., Herndon D. N.Influence of detergent aerosol on lung microvascular permeability. J. Appl. Physiol.74199310161023 |
| 28. | Nilsson K., Wollmer P.Pulmonary clearence of Tc-99m-DTPA-albumin in rabbits with surfactant dysfunction and lung injury. Clin. Physiol.121992587 |
| 29. | Lee O., Ueno O., Wagner P. D., West J. B.Pulmonary gas exchange after multiple airway occclusion by beads in the dog. Am. Rev. Respir. Dis.140198912161221 |
| 30. | Wagner P. D., Dantzker D. R., Iacovoni V. E., Tomlin W. C., West J. B.Ventilation-perfusion inequality in asymptomatic asthma. Am. Rev. Respir. Dis.1181978511517 |
| 31. | Lagerstrand L., Dahlbäck M., Hedenstierna G.Gas exchange during simulated airway secretion in the anesthetized rabbit. Eur. Respir. J.5199212151222 |
| 32. | Ueda T., Ikegami M., Rider E. D., Jobe A. H.Distribution of surfactant and ventilation in surfactant-treated preterm lambs. J. Appl. Physiol.7619944555 |
| 33. | Henry M. D., Rebello C. M., Ikegami M., Jobe A. H., Langenback E. G., Davis J. M.Ultrasonic nebulized in comparison with instilled surfactant treatment of preterm lambs. Am. J. Respir. Crit. Care Med.1541996366375 |
| 34. | Ellyett K. M., Broadbent R. S., Fawcett E. R., Cambell A. J.Surfactant aerosol treatment of respiratory distress syndrome in the spontaneously breathing premature rabbit. Pediatr. Res.391996953957 |
| 35. | Huang Y. T., Caminiti S. P., Fawcett T. A., Moon R. E., Fracia P. J., Miller F. J., Young S. L., Piantadosi C. A.Natural surfactant and hyperoxic lung injury in primates. I. Physiology and biochemistry. J. Appl. Physiol.7619949911001 |
| 36. | Schlesinger R. B.Comparative deposition of inhaled aerosols in experimental animals and humans: a review. J. Toxicol. Environ. Health151985197214 |
| 37. | Morrow P. E.Aerosol characterization and deposition. Am. Rev. Respir. Dis.11019748897 |
| 38. | Ashton M. R., Postle A. D., Hall M. A., Austin N. C., Smith D. E., Normand I. C.Turnover of exogenous artificial surfactant. Arch. Dis. Child671992383387 |
This report includes portions of the doctoral thesis of Ralph Schermuly.
