- Email: [email protected]
Surfactant function in respiratory distress syndrome
Surfactant function in respiratory distress syndrome Airway samples from infants with respiratory distress syndrome were recovered by suction immediately after tracheal intubation for respiratory failure. The minimum surface tension of these airway samples was 27.3 +_ 3.0 dynes/era. Surfactant with low surface tension (1.4 +_ 1.0 dynes/cm) was recovered from these samples by centrifugation; the supernatant fractions from the samples had high minimum surface tensions. The supernatant fractions contained soluble proteins that inhibited the surface tension-lowering properties of natural sheep surfactant. Similar supernatant fractions collected from infants intubated for reasons other than respiratory distress syndrome were much less inhibitory to sheep surfactant. The minimum surface tension of sequential daily airway samples from infants with respiratory distress syndrome fell progressively to 5.7 +_ 2.4 dynes/cm on the day of extubation. These results document the presence of proteins in the airways of infants with respiratory distress syndrome that inhibit the surface tension lowering properties of surfactant. (J PEDIArR 102:443, 1983)
Machiko Ikegami, M.D., Harris Jacobs, M.D., and Alan Jobe, M.D., Ph.D. Torrance, Calif.
SALINE EXTRACTS OF LUNG MINCES from infants who die of respiratory distress syndrome contain less phospholipid and have higher minimum surface tensions than similar extracts from the lungs of infants who die from other causes5 -3 Saturated phosphatidyleholine is the principal surface-active component of surfactant,4 and the amount of saturated phosphatidylcholine recovered by lavage from lungs of infants who died of respiratory distress syndrome was far below that necessary to stabilize the air spaces. 2 Thus respiratory distress syndrome has been considered to result from inadequate amounts of surfactant. The proteinaceous pulmonary edema and the hyaline membranes are considered to be secondary to atelectasis and lung damage resulting from respiratory failure and therapeutic interventions?
Pattle 6 and Clements7 have suggested that a second function of the surface tension-lowering properties of surfactant may be to prevent transudation of fluid into the alveoli. Several authors have commented that substances released into the alveoli may inactivate surfactant and contribute to functional surfactant deficiency.8-~zWe have demonstrated that after treatment of prematurely delivered lambs with sheep surfactant, respiratory failure occurs after three to four hours, concurrent with the appearance in the airways of inhibitors of surface tensionlowering properties of surfactant. ~3 We document the presence of functionally similar inhibitory substances in the airways of premature infants with respiratory distress syndrome. MATERIALS AND METHODS
From the Fetal-Maternal Research Laboratories, Department of Pediatrics, UCLA School of Medicine. Reprint requests: Machiko lkegami, M.D., Building A-17, Harbor-UCLA Medical Center, 1000 W. Carson St., Torrance, CA 90509. Supported by a grant from the March of Dimes--Birth Defects Foundation; by Grant HD-12714 from the Department of Health and Human Development; by the Perinatal Clinic Study Center at Harbor-UCLA Medical Center; and by Research Career Development Award HD-HL0252 to Dr. ,lobe.
Patients, We studied 10 infants with severe respiratory distress syndrome admitted from our delivery service or by transport to the neonatal intensive care unit at HarborUCLA Medical Center. The diagnosis of respiratory distress syndrome was based on chest radiographs, symptoms, and clinical course of the disease, The mean gestational age of the 10 infants was 31.7 _+ 0.6 weeks (range 28 to 34 weeks); the mean birth weight was 1.48 • 0.14 kg (range 0.93 to 2.24 kg). Intubation and ventilation for
TheJournalofPEDIATRICS
443
444
lkegami, Jacobs, and Jobe
dynes/cm
The Journal of Pediatrics March 1983
13 RDS
40
~
t-
(n= 8)
NO RDS (n=lO) "r
.o 30 u {3 '-'c
20 E 9-E-E
r ,m
Table. Percent composition of phospholipids in surfactant recovered from initial airway samples
10
0 Airway Sample
Isolated Surfactant
Supernatant
Fig. 1. Minimum surface tensions of initial airway samples from infants with and infants without respiratory distress syndrome (RDS). Minimum surface tensions of eight airway samples were measured with the dynamic alveolar model. After isolation of surfactant and supernatant fractions by centrifugation, the minimum surface tension of each was measured.
respiratory failure were carried out at times varying from birth to 15 hours of age. In three cases the L/S ratios were less than 2.0 before delivery; amniotic fluid for the other infants was not tested. All infants were given respiratory assistance by a standard methodJ 4 After intubation and instillation of 0.5 ml isotonic saline, the airways were suctioned with either 6 or 8 French suction catheters attached to 10 ml De Lee suction traps to recover the airway samples. Initial samples were obtained at the time of intubation in eight of the 10 infants. Sequential daily airway samples subsequently were collected in four of the babies initially sampled and in the two other infants until extubation after resolution of the respiratory distress syndrome. All airway samples were frozen at - 2 0 ~ until assay. One airway sample was collected similarly from 10 other intubated infants without respiratory distress syndrome. These infants ranged in weight from 0.68 to 4.60 kg. Diagnoses in these infants included meningitis-sepsis (two infants), prematurity without respiratory distress syndrome (three), meeonium aspiration (two), asphyxia in a term infant (one), gastroschisis (one), and pyloric stenosis (one). Analysis of airway samples. The minimum surface tension of each airway sample was measured using the dynamic alveolar modelJ 3.~5This method measures surface tensions by recording the pressure-volume relationships of a microbubble oscillating 16 times per minute. Minimum surface tension was calculated from bubble diameter, and the pressure was measured at the fifth cycle, using the
Phospholipid
Infants with RDS (n = 7)
Infants without RDS (n = 6)
Phosphatidylcholine Phosphatidylglycerol Phosphatidylinositol Phosphatidylethanolamine Sphingomyelin Phosphatidylserine
84.0 _+ 0.8 0.6 _+ 0.4 8.4 + 1.4 2.8 _+ 0.5 2.8 _+ 0.5 0.5 _+ 0.4
86.0 _+ 2.6 6.5 _+ 1.7 5.0 _+ 2.2 1.0 + 0.5 0.7 _+ 0.2 0.7 _+ 0.1
Laplace formula. The minimum surface tension is independent of surfactant concentration, if the phosphatidylcholine content is >~ 0.5 #mole/ml. Minimal surface tensions are reported only for samples containing >_ 1 #mole/ml. In all instances the initial sample taken at intubation from infants with respiratory distress syndrome was sufficiently concentrated for the measurement of surface tension. Samples collected on subsequent days often were too dilute. To assess phosphatidylcholine concentrations, aliquots of all samples were extracted with chloroform-methanol (2: 1). Phosphatidylcholine was isolated from the lipid extracts by one-dimensional thin-layer chromatography ~6 and quantified by phosphate assayJ 7 Protein concentration was measured according to Lowry et al., ~8 using bovine serum albumin as a standard. "Surfactant" was isolated from initial airway samples collected from infants with respiratory distress syndrome by layering part of each sample over 0.7 M sucrose in saline. The gradient was centrifuged at 8000 X g for 30 minutes. The supernatant overlying the interface was aspirated and saved. The "surfactant" identified as a white band at the interface was recovered as a pellet after centrifugation at 27,000 • g for 20 minutesJ 3 This procedure for surfactant isolation is equivalent to that used to recover surfactant from lung lavage. 19After determination of phosphatidylcholine and protein content in the isolated surfactant and supernatant fractions, minimum surface tensions were measured. The composition of phospholipids in airway samples and isolated surfactant was measured by assay of phosphate for the phospholipids separated by two-dimensional thin-layer chromatographyJ 6 Testing for inhibitors. Surfactant was isolated from lavage fluid from adult sheep lungsJ 9 This surfactant gives a minimum surface tension of 0 dyne/cm at a concentration of 0.5 #moles phosphatidylcholine/ml with the dynamic alveolar modelJ 3 We held the final concentration of surfactant constant at 1 #mole/ml and added increasing amounts of the supernatant fractions isolated from airway samples to this surfactantJ 3 Minimum surface tensions
Volume 102 Number 3
were expressed versus the amount of supernatant protein added to the sheep surfactant. Partial eharaeterization of inhihitors. Aliquots of pooled supernatant fractions were dialyzed in tubing that retained molecules > 12,000 molecular weight, boiled for 10 minutes, treated with protease at 37~ for three hours, and successively extracted with butanol-isopropyl ether (2:3) to remove lipids. Polyethylene glycol (nominal molecular weight 3350) was used to successively fractionate pooled supernatant fractions initially at pH 7.4 in a manner analogous to that used to fractionate plasma. Polyethylene glycol was added to the solution to a concentration of 20%; the precipitate was recovered by centrifugation. Polyethylene glycol was then added to a concentration of 25% and the pH was adjusted to pH 5.4. Following recovery of the precipitate, a final precipitate was recovered from a 50% polyethylene glycol solution. All fractions were tested for inhibition activity. All measurements are expressed as mean _+ SEM. RESULTS Initial airway samples recovered from eight infants after intubation for respiratory failure resulting from respiratory distress syndrome had a mean minimal surface tension of 27.3 _+ 3.0 dynes/cm (Fig. 1). A highly surface-active surfactant fraction with a minimal surface tension of 1.4 _+ 1.0 dynes/cm was isolated from each sample by centrifugation. The supernatants from the airway samples had a mean minimal surface tension of 35.0 _+ 1.4 dynes/ cm. In contrast, the mean minimal surface tension of airway samples from 10 infants without respiratory distress syndrome was 6.3 + 1.1 dynes/cm. Surfactant isolated from these samples had a minimal surface tension of 0.3 _+ 0.3 dynes/cm, and the supernatant fractions had mean minimal surface tensions of 33.9 _+ 2.4 dynes/cm. The phospholipids in the surfactant from infants with respiratory distress syndrome contained no or only trace amounts of phosphatidylglycerol, whereas surfactant from all infants in the comparison group contained measurable amounts of phosphatidylglycerol (Table). The sphingomyelin and phosphatidylinositol contents of the surfactant from infants with the respiratory distress syndrome also were higher relative to those of the comparison group. The initial airway samples from infants with the respiratory distress syndrome contained 0.43 _~ 0.16 #moles phosphatidylcholine/mg protein. The surfactant and supernatant fractions recovered from these samples had 14.1 _+ 3.4 and 0.17 _+ 0.06 #moles phosphatidylcholine/mg protein, respectively. Thus surfactant fractions contained 33 times more phosphatidylcholine than protein relative to the airway samples. The airway samples from the infants without respiratory distress syndrome had 2.13 _+ 0.58
Surfactant function in respiratory distress syndrome
445
dynes/cm
g
2O
/.Psi1 o
10 .A (NO RDS) n~5
-
E "E ~E "I" .1
T .3
T .6
I ]
l 2
mg
Protein Fig. 2. Inhibition of minimal surface tensions by supernatant fractions. After measurement of protein content, increasing amounts of the supernatant fractions from the initial airway samples were added to natural sheep surfactant, so that the final solution contained a constant amount of sheep surfactant (1 #mole phosphatidylcholine/ml).
dynes~m 3O
(n=6)
g o
E
20
io
0
--5
~ -4 Days
-3 to
-2
, -1
extubate
Extubation
Fig. 3. Sequential minimal surface tensions of airway samples from infants with respiratory distress syndrome. Airway samples were collected daily until the day of extubation in six infants. Minimum surface tensions are shown for the five days before extubation and on the day of extubation. Several of the infants were intubated for longer than five days; the earlier samples had high surface tensions.
#moles phosphatidylcholine/mg protein, a significantly higher ratio than in the samples from infants with respiratory distress syndrome (P < 0.02). Supernatant fractions recovered from airway samples of infants with respiratory distress syndrome inhibited the minimal surface tension of sheep surfactant (Fig. 2). When increasing amounts of supernatant were added to a final
446
Ikegami, Jacobs, and Jobe
concentration of 1 #mole/ml sheep surfactant, the minimal surface tension increased in a relatively linear fashion at protein concentrations between 0.3 and 2 mg/ml. Five of the supernatant fractions from airway samples from infants without respiratory distress syndrome contained sufficient protein to test for inhibition of minimum surface tension of sheep surfactant. No inhibition was detected at 0.6 mg protein/ml, and much less inhibition occurred at 1 and 2 mg protein/ml. The inhibitory activity in pooled supernatant fractions was not inactivated by boiling, was completely inactivated by protease, and was retained by dialysis tubing, indicating a molecular weight > 12,000. Extraction with lipid solvents did not diminish the inhibitory activity of the resulting lipid-free water phase. The mean minimum surface tension of sheep surfactant after addition of aliquots from three different pools of supernatant fractions at 1 mg protein/ml was 10.2 _~ 0.5 dynes/cm. After successive precipitation steps with polyethylene glycol, no inhibitory activity was detected in precipitates from 0 to 20% or 20 to 25% polyethylene glycol; these two fractions together contained 87 + 3% of the protein initially present in the supernatant fraction. All of the inhibitory activity precipitated between 25 and 50% polyethylene glycol. The proteins from this precipitate at a concentration of 50 ~g/ml increased the minimum surface tension of natural sheep surfactant to 12.3 _+ 1.1 dynes/cm. Thus the polyethylene glycol precipitation procedure resulted in an approximate 20-fold increase in inhibitory activity per milligram protein. In six of the infants with respiratory distress syndrome, airway samples were collected daily until the day of extubation. The mean minimal surface tensions of these airway samples decreased over the six days preceding extubation (Fig. 3). The curve documents the change in surface tension during the course of the respiratory distress syndrome. At extubation the minimal surface tension was 5.7 _+ 2.3 dynes/cm, a value similar to that measured for infants without respiratory distress syndrome. DISCUSSION Surfactant from lung specimens of infants with respiratory distress syndrome had high surface tensions, 1 and lungs from these infants also had decreased absolute amounts of lung -3 and airway-associated phospholipid.2 More recently the composition of phospholipids in airway samples was shown to change as the infants recovered from the respiratory distress syndrome. 2~ At birth, neither the amniotic fluid nor the airway samples contain phosphatidylglycerol; this phospholipid appears as the disease resolves. The fatty acid composition of the phospholipids in airway samples also changes with time to contain more
The Journal of Pediatrics March 1983
saturated fatty acids. 2~ Concomitant with these biochemi. cal changes in surfactant phospholipids with resolution of respiratory distress syndrome, the pool size of the alveolar surfactant is assumed to increase? The infants in our study group had little or no phosphatidylglycerol in surfactant isolated from airway samples, supporting the diagnosis of respiratory distress syndrome. Moreover, L / S ratios for those infants tested before delivery were < 2. Although the dynamic alveolar model requires a very small sample size to measure surface tensions, sufficient material must be present to quantify the phosphatidylcholine content of each sample. Unless the phosphatidylcholine content is _> 0.5 ~mole/ml, the surface tension measurement may be concentration-dependent. The airway samples contain material recovered at some distance from the alveoli and might not be truly representative of the surface tensions in the alveoli. However, samples collected in a similar manner from premature lambs demonstrate close correspondence in minimal surface tensions between airway samples and samples recovered by alveolar lavage. 13 The possibility that the surface tension-lowering properties of surfactant can be inhibited in vivo has been suggested by a number of in vitro experiments. Taylor and Abrams ~~demonstrated that fibrinogen raised the surface tension of surfactant, whereas albumin, plasminogen, and globulin did not. They proposed that surfactant could activate plasma constituents in the alveoli and thus be inactivated concurrently with the formation of hyaline membranes. Tierney and Johnson 8 found that serum or blood usually raised the minimum surface tension of surfactant. Balis et al. ~2 thought that inactivation of surfactant by serum was reversible because a surfaceactive fraction could be recovered by centrifugation. Miyahara ~ further characterized the inhibitory effects of blood fractions on surfactant and proposed that this inactivation was directly responsible for the atelectasis in hyaline membrane disease. We found that very premature lambs with respiratory failure responded to treatment with natural surfactant with a prompt improvement in pulmonary function; however, the response lasted only about three hours? 9 The short response was not related primarily to removal of surfactant from the airways; rather, inhibitors of surfactant function entered the airways. ~3Lung lavages from the lambs contained inhibitory substances analogous to the inhibitors in the airway samples from the infants with respiratory distress syndrome. These inhibitory substances are also present in the fetal lung fluid of lambs; they will inactivate an artificial surfactant at much lower concentrations than natural surfactant. 22 This study demonstrates that highly surface-active sur-
Volume 102 Number 3
factant fractions can be recovered from airway samples of infants early in the course of respiratory distress syndrome if the surfactant lipids are separated from soluble proteins by centrifugation. Precipitation with polyethylene glycol was used to fractionate the soluble proteins in a m a n n e r analogous to the fractionation of plasma into its protein components. The precipitation of the inhibitory proteins only at high concentrations of polyethylene glycol indicates that the inhibitors are not albumin, globulin, or fibrinogen. Sequential airway samples d o c u m e n t a gradual decrease in surface activity until the respiratory distress syndrome has resolved sufficiently to permit extubation of the infant. W e assume t h a t either sufficient s u r f a c t a n t was present to overcome the effects of the inhibitors or t h a t the inhibitors had been cleared from the airways. The source (lung tissue or blood) of the inhibitory substances remains speculative. M a n y perinatal insults increase the severity of respiratory distress syndrome (hypothermia, shock, asphyxia, the presence Of a patent ductus arteriosus). W e suspect t h a t the c o m m o n result of these occurrences is an increase in alveolar epithelial permeability with the entrance of increased amounts of proteins into the airways. T h e relatively small pool size of surfactant present in the infant with respiratory distress syndrome may be uniquely susceptible to functional inhibition by protein inhibitors. Thus the a m o u n t of surfactant, the functional m a t u r i t y of t h a t surfactant, and the quantity of protein will interact to result in variable severity of respiratory distress syndrome. REFERENCES 1. Avery ME, Mead J: Surface properties in relation to atelectasis and hyaline membrane disease. Am J Dis Child 97:51, 1959. 2. Adams FH, Fujiwara T, Emmanouilides G, Raiha H: Lung phosphoholipid of human fetuses and infants with and without hyaline membrane disease. J PEDIATR 77:833, 1970. 3. Brumley G, Hodson AD, Avery ME: Lung phospholipid and surface tension correlations in infants with and without hyaline membrane disease and adults. Pediatrics 40:13, 1967. 4. Van Golde LMG: Metabolism of phospholipid in the lung. Am Rev Res Dis 114:977, 1976.
Surfactant function in respiratory distress syndrome
447
5. Farrell PM, Avery ME: Hyaline membrane disease. Am Rev Res Dis 111:657, 1975. 6. Pattie RE: Properties, function and origin of the alveolar lining layer. Proc R Soc London [Biol] 1438:217, 1958. 7. Clements JA: Puhnonary edema and permeability of alveolar membranes. Arch Environ Health 2:280, 1961. 8. Tierney DF, Johnson RP: Altered surface tension of Iung extracts and lung mechanics. J Appl Physiol 20:1253, 1965. 9. Avery ME, Said S: Surface phenomena in lungs in health and disease. Medicine 44:503, 1965. 10. Taylor F, Abrams M: Effect of surface active lipoprotein on clotting and fibrinolysis, and of fibrinogen on surface tension of surface active lipoprotein. Am J Med 40:346, 1966. I I. Miyahara T: A study of the pathogenesis of hyaline membrane disease in the newborn infant. Kyushu Univ J 60:95, 1969. 12. Balls J, Shelley S, McCue M, Rappaport E: Mechanisms of damage to the lung surfactant system. Exp Mol Path 14:243, 1971. 13. Ikegami M, Jobe A, Glatz T: Surface activity following natural surfactant treatment of premature lambs. J AppI Physiol $1:306, 1981. 14. Mannino FL, Gluck L: The management of respiratory distress syndrome. In Thibeault D, Gregory G, editors: Neonatal pulmonary care. Reading, Mass., 1979, AddisonWesley, p 261. 15. Nozaki M: Pressure-volume relationships of a model alveolus. Tohoku J Expl Med 101:271, 1970. 16. Jobe A, Kirkpartrick E, Gluck L: Labeling of phospholipid in the surfactant and subcellular fractions of rabbit lung. J Biol Chem 253:3810, 1978. 17. Bartlett GR: Phosphorus assay in column chromatrography. J Biol Chem 234:466, 1959. 18. Lowry OH, Rosebrough N J, Farr AL, Randall R J: Protein measurement with the folin phenol reagent. J Biol Chem 193:265, 1951. 19. Jobe A, lkegami M, Glatz T, Yoshida Y, Diakomanolis E, Padbury J: Duration and characteristics of treatment of premature Iambs with natural surfactant. J Clin Invest 67:370, 1981. 20. Hallman M, Feldman B, Kirkpatrick E, Gluck L: Absence of phosphatidylglycerol in respiratory distress syndrome in the newborn. Pediatr Res !!:714, 1977. 21. Shelly S, Kovacevic M, Paciga J, Balls J: Sequential changes of surfactant phosphatidylcholine in hyaline-membrane disease of the newborn. N Engl J Med 300:112, 1979. 22. lkegami M, Jobe A, Jacobs H, Jones S: Sequential treatments of premature lambs with an artificial surfactant and natural surfactant. J Clin Invest 68:491, 1981.
Machiko Ikegami, M.D., Harris Jacobs, M.D., and Alan Jobe, M.D., Ph.D. Torrance, Calif.
SALINE EXTRACTS OF LUNG MINCES from infants who die of respiratory distress syndrome contain less phospholipid and have higher minimum surface tensions than similar extracts from the lungs of infants who die from other causes5 -3 Saturated phosphatidyleholine is the principal surface-active component of surfactant,4 and the amount of saturated phosphatidylcholine recovered by lavage from lungs of infants who died of respiratory distress syndrome was far below that necessary to stabilize the air spaces. 2 Thus respiratory distress syndrome has been considered to result from inadequate amounts of surfactant. The proteinaceous pulmonary edema and the hyaline membranes are considered to be secondary to atelectasis and lung damage resulting from respiratory failure and therapeutic interventions?
Pattle 6 and Clements7 have suggested that a second function of the surface tension-lowering properties of surfactant may be to prevent transudation of fluid into the alveoli. Several authors have commented that substances released into the alveoli may inactivate surfactant and contribute to functional surfactant deficiency.8-~zWe have demonstrated that after treatment of prematurely delivered lambs with sheep surfactant, respiratory failure occurs after three to four hours, concurrent with the appearance in the airways of inhibitors of surface tensionlowering properties of surfactant. ~3 We document the presence of functionally similar inhibitory substances in the airways of premature infants with respiratory distress syndrome. MATERIALS AND METHODS
From the Fetal-Maternal Research Laboratories, Department of Pediatrics, UCLA School of Medicine. Reprint requests: Machiko lkegami, M.D., Building A-17, Harbor-UCLA Medical Center, 1000 W. Carson St., Torrance, CA 90509. Supported by a grant from the March of Dimes--Birth Defects Foundation; by Grant HD-12714 from the Department of Health and Human Development; by the Perinatal Clinic Study Center at Harbor-UCLA Medical Center; and by Research Career Development Award HD-HL0252 to Dr. ,lobe.
Patients, We studied 10 infants with severe respiratory distress syndrome admitted from our delivery service or by transport to the neonatal intensive care unit at HarborUCLA Medical Center. The diagnosis of respiratory distress syndrome was based on chest radiographs, symptoms, and clinical course of the disease, The mean gestational age of the 10 infants was 31.7 _+ 0.6 weeks (range 28 to 34 weeks); the mean birth weight was 1.48 • 0.14 kg (range 0.93 to 2.24 kg). Intubation and ventilation for
TheJournalofPEDIATRICS
443
444
lkegami, Jacobs, and Jobe
dynes/cm
The Journal of Pediatrics March 1983
13 RDS
40
~
t-
(n= 8)
NO RDS (n=lO) "r
.o 30 u {3 '-'c
20 E 9-E-E
r ,m
Table. Percent composition of phospholipids in surfactant recovered from initial airway samples
10
0 Airway Sample
Isolated Surfactant
Supernatant
Fig. 1. Minimum surface tensions of initial airway samples from infants with and infants without respiratory distress syndrome (RDS). Minimum surface tensions of eight airway samples were measured with the dynamic alveolar model. After isolation of surfactant and supernatant fractions by centrifugation, the minimum surface tension of each was measured.
respiratory failure were carried out at times varying from birth to 15 hours of age. In three cases the L/S ratios were less than 2.0 before delivery; amniotic fluid for the other infants was not tested. All infants were given respiratory assistance by a standard methodJ 4 After intubation and instillation of 0.5 ml isotonic saline, the airways were suctioned with either 6 or 8 French suction catheters attached to 10 ml De Lee suction traps to recover the airway samples. Initial samples were obtained at the time of intubation in eight of the 10 infants. Sequential daily airway samples subsequently were collected in four of the babies initially sampled and in the two other infants until extubation after resolution of the respiratory distress syndrome. All airway samples were frozen at - 2 0 ~ until assay. One airway sample was collected similarly from 10 other intubated infants without respiratory distress syndrome. These infants ranged in weight from 0.68 to 4.60 kg. Diagnoses in these infants included meningitis-sepsis (two infants), prematurity without respiratory distress syndrome (three), meeonium aspiration (two), asphyxia in a term infant (one), gastroschisis (one), and pyloric stenosis (one). Analysis of airway samples. The minimum surface tension of each airway sample was measured using the dynamic alveolar modelJ 3.~5This method measures surface tensions by recording the pressure-volume relationships of a microbubble oscillating 16 times per minute. Minimum surface tension was calculated from bubble diameter, and the pressure was measured at the fifth cycle, using the
Phospholipid
Infants with RDS (n = 7)
Infants without RDS (n = 6)
Phosphatidylcholine Phosphatidylglycerol Phosphatidylinositol Phosphatidylethanolamine Sphingomyelin Phosphatidylserine
84.0 _+ 0.8 0.6 _+ 0.4 8.4 + 1.4 2.8 _+ 0.5 2.8 _+ 0.5 0.5 _+ 0.4
86.0 _+ 2.6 6.5 _+ 1.7 5.0 _+ 2.2 1.0 + 0.5 0.7 _+ 0.2 0.7 _+ 0.1
Laplace formula. The minimum surface tension is independent of surfactant concentration, if the phosphatidylcholine content is >~ 0.5 #mole/ml. Minimal surface tensions are reported only for samples containing >_ 1 #mole/ml. In all instances the initial sample taken at intubation from infants with respiratory distress syndrome was sufficiently concentrated for the measurement of surface tension. Samples collected on subsequent days often were too dilute. To assess phosphatidylcholine concentrations, aliquots of all samples were extracted with chloroform-methanol (2: 1). Phosphatidylcholine was isolated from the lipid extracts by one-dimensional thin-layer chromatography ~6 and quantified by phosphate assayJ 7 Protein concentration was measured according to Lowry et al., ~8 using bovine serum albumin as a standard. "Surfactant" was isolated from initial airway samples collected from infants with respiratory distress syndrome by layering part of each sample over 0.7 M sucrose in saline. The gradient was centrifuged at 8000 X g for 30 minutes. The supernatant overlying the interface was aspirated and saved. The "surfactant" identified as a white band at the interface was recovered as a pellet after centrifugation at 27,000 • g for 20 minutesJ 3 This procedure for surfactant isolation is equivalent to that used to recover surfactant from lung lavage. 19After determination of phosphatidylcholine and protein content in the isolated surfactant and supernatant fractions, minimum surface tensions were measured. The composition of phospholipids in airway samples and isolated surfactant was measured by assay of phosphate for the phospholipids separated by two-dimensional thin-layer chromatographyJ 6 Testing for inhibitors. Surfactant was isolated from lavage fluid from adult sheep lungsJ 9 This surfactant gives a minimum surface tension of 0 dyne/cm at a concentration of 0.5 #moles phosphatidylcholine/ml with the dynamic alveolar modelJ 3 We held the final concentration of surfactant constant at 1 #mole/ml and added increasing amounts of the supernatant fractions isolated from airway samples to this surfactantJ 3 Minimum surface tensions
Volume 102 Number 3
were expressed versus the amount of supernatant protein added to the sheep surfactant. Partial eharaeterization of inhihitors. Aliquots of pooled supernatant fractions were dialyzed in tubing that retained molecules > 12,000 molecular weight, boiled for 10 minutes, treated with protease at 37~ for three hours, and successively extracted with butanol-isopropyl ether (2:3) to remove lipids. Polyethylene glycol (nominal molecular weight 3350) was used to successively fractionate pooled supernatant fractions initially at pH 7.4 in a manner analogous to that used to fractionate plasma. Polyethylene glycol was added to the solution to a concentration of 20%; the precipitate was recovered by centrifugation. Polyethylene glycol was then added to a concentration of 25% and the pH was adjusted to pH 5.4. Following recovery of the precipitate, a final precipitate was recovered from a 50% polyethylene glycol solution. All fractions were tested for inhibition activity. All measurements are expressed as mean _+ SEM. RESULTS Initial airway samples recovered from eight infants after intubation for respiratory failure resulting from respiratory distress syndrome had a mean minimal surface tension of 27.3 _+ 3.0 dynes/cm (Fig. 1). A highly surface-active surfactant fraction with a minimal surface tension of 1.4 _+ 1.0 dynes/cm was isolated from each sample by centrifugation. The supernatants from the airway samples had a mean minimal surface tension of 35.0 _+ 1.4 dynes/ cm. In contrast, the mean minimal surface tension of airway samples from 10 infants without respiratory distress syndrome was 6.3 + 1.1 dynes/cm. Surfactant isolated from these samples had a minimal surface tension of 0.3 _+ 0.3 dynes/cm, and the supernatant fractions had mean minimal surface tensions of 33.9 _+ 2.4 dynes/cm. The phospholipids in the surfactant from infants with respiratory distress syndrome contained no or only trace amounts of phosphatidylglycerol, whereas surfactant from all infants in the comparison group contained measurable amounts of phosphatidylglycerol (Table). The sphingomyelin and phosphatidylinositol contents of the surfactant from infants with the respiratory distress syndrome also were higher relative to those of the comparison group. The initial airway samples from infants with the respiratory distress syndrome contained 0.43 _~ 0.16 #moles phosphatidylcholine/mg protein. The surfactant and supernatant fractions recovered from these samples had 14.1 _+ 3.4 and 0.17 _+ 0.06 #moles phosphatidylcholine/mg protein, respectively. Thus surfactant fractions contained 33 times more phosphatidylcholine than protein relative to the airway samples. The airway samples from the infants without respiratory distress syndrome had 2.13 _+ 0.58
Surfactant function in respiratory distress syndrome
445
dynes/cm
g
2O
/.Psi1 o
10 .A (NO RDS) n~5
-
E "E ~E "I" .1
T .3
T .6
I ]
l 2
mg
Protein Fig. 2. Inhibition of minimal surface tensions by supernatant fractions. After measurement of protein content, increasing amounts of the supernatant fractions from the initial airway samples were added to natural sheep surfactant, so that the final solution contained a constant amount of sheep surfactant (1 #mole phosphatidylcholine/ml).
dynes~m 3O
(n=6)
g o
E
20
io
0
--5
~ -4 Days
-3 to
-2
, -1
extubate
Extubation
Fig. 3. Sequential minimal surface tensions of airway samples from infants with respiratory distress syndrome. Airway samples were collected daily until the day of extubation in six infants. Minimum surface tensions are shown for the five days before extubation and on the day of extubation. Several of the infants were intubated for longer than five days; the earlier samples had high surface tensions.
#moles phosphatidylcholine/mg protein, a significantly higher ratio than in the samples from infants with respiratory distress syndrome (P < 0.02). Supernatant fractions recovered from airway samples of infants with respiratory distress syndrome inhibited the minimal surface tension of sheep surfactant (Fig. 2). When increasing amounts of supernatant were added to a final
446
Ikegami, Jacobs, and Jobe
concentration of 1 #mole/ml sheep surfactant, the minimal surface tension increased in a relatively linear fashion at protein concentrations between 0.3 and 2 mg/ml. Five of the supernatant fractions from airway samples from infants without respiratory distress syndrome contained sufficient protein to test for inhibition of minimum surface tension of sheep surfactant. No inhibition was detected at 0.6 mg protein/ml, and much less inhibition occurred at 1 and 2 mg protein/ml. The inhibitory activity in pooled supernatant fractions was not inactivated by boiling, was completely inactivated by protease, and was retained by dialysis tubing, indicating a molecular weight > 12,000. Extraction with lipid solvents did not diminish the inhibitory activity of the resulting lipid-free water phase. The mean minimum surface tension of sheep surfactant after addition of aliquots from three different pools of supernatant fractions at 1 mg protein/ml was 10.2 _~ 0.5 dynes/cm. After successive precipitation steps with polyethylene glycol, no inhibitory activity was detected in precipitates from 0 to 20% or 20 to 25% polyethylene glycol; these two fractions together contained 87 + 3% of the protein initially present in the supernatant fraction. All of the inhibitory activity precipitated between 25 and 50% polyethylene glycol. The proteins from this precipitate at a concentration of 50 ~g/ml increased the minimum surface tension of natural sheep surfactant to 12.3 _+ 1.1 dynes/cm. Thus the polyethylene glycol precipitation procedure resulted in an approximate 20-fold increase in inhibitory activity per milligram protein. In six of the infants with respiratory distress syndrome, airway samples were collected daily until the day of extubation. The mean minimal surface tensions of these airway samples decreased over the six days preceding extubation (Fig. 3). The curve documents the change in surface tension during the course of the respiratory distress syndrome. At extubation the minimal surface tension was 5.7 _+ 2.3 dynes/cm, a value similar to that measured for infants without respiratory distress syndrome. DISCUSSION Surfactant from lung specimens of infants with respiratory distress syndrome had high surface tensions, 1 and lungs from these infants also had decreased absolute amounts of lung -3 and airway-associated phospholipid.2 More recently the composition of phospholipids in airway samples was shown to change as the infants recovered from the respiratory distress syndrome. 2~ At birth, neither the amniotic fluid nor the airway samples contain phosphatidylglycerol; this phospholipid appears as the disease resolves. The fatty acid composition of the phospholipids in airway samples also changes with time to contain more
The Journal of Pediatrics March 1983
saturated fatty acids. 2~ Concomitant with these biochemi. cal changes in surfactant phospholipids with resolution of respiratory distress syndrome, the pool size of the alveolar surfactant is assumed to increase? The infants in our study group had little or no phosphatidylglycerol in surfactant isolated from airway samples, supporting the diagnosis of respiratory distress syndrome. Moreover, L / S ratios for those infants tested before delivery were < 2. Although the dynamic alveolar model requires a very small sample size to measure surface tensions, sufficient material must be present to quantify the phosphatidylcholine content of each sample. Unless the phosphatidylcholine content is _> 0.5 ~mole/ml, the surface tension measurement may be concentration-dependent. The airway samples contain material recovered at some distance from the alveoli and might not be truly representative of the surface tensions in the alveoli. However, samples collected in a similar manner from premature lambs demonstrate close correspondence in minimal surface tensions between airway samples and samples recovered by alveolar lavage. 13 The possibility that the surface tension-lowering properties of surfactant can be inhibited in vivo has been suggested by a number of in vitro experiments. Taylor and Abrams ~~demonstrated that fibrinogen raised the surface tension of surfactant, whereas albumin, plasminogen, and globulin did not. They proposed that surfactant could activate plasma constituents in the alveoli and thus be inactivated concurrently with the formation of hyaline membranes. Tierney and Johnson 8 found that serum or blood usually raised the minimum surface tension of surfactant. Balis et al. ~2 thought that inactivation of surfactant by serum was reversible because a surfaceactive fraction could be recovered by centrifugation. Miyahara ~ further characterized the inhibitory effects of blood fractions on surfactant and proposed that this inactivation was directly responsible for the atelectasis in hyaline membrane disease. We found that very premature lambs with respiratory failure responded to treatment with natural surfactant with a prompt improvement in pulmonary function; however, the response lasted only about three hours? 9 The short response was not related primarily to removal of surfactant from the airways; rather, inhibitors of surfactant function entered the airways. ~3Lung lavages from the lambs contained inhibitory substances analogous to the inhibitors in the airway samples from the infants with respiratory distress syndrome. These inhibitory substances are also present in the fetal lung fluid of lambs; they will inactivate an artificial surfactant at much lower concentrations than natural surfactant. 22 This study demonstrates that highly surface-active sur-
Volume 102 Number 3
factant fractions can be recovered from airway samples of infants early in the course of respiratory distress syndrome if the surfactant lipids are separated from soluble proteins by centrifugation. Precipitation with polyethylene glycol was used to fractionate the soluble proteins in a m a n n e r analogous to the fractionation of plasma into its protein components. The precipitation of the inhibitory proteins only at high concentrations of polyethylene glycol indicates that the inhibitors are not albumin, globulin, or fibrinogen. Sequential airway samples d o c u m e n t a gradual decrease in surface activity until the respiratory distress syndrome has resolved sufficiently to permit extubation of the infant. W e assume t h a t either sufficient s u r f a c t a n t was present to overcome the effects of the inhibitors or t h a t the inhibitors had been cleared from the airways. The source (lung tissue or blood) of the inhibitory substances remains speculative. M a n y perinatal insults increase the severity of respiratory distress syndrome (hypothermia, shock, asphyxia, the presence Of a patent ductus arteriosus). W e suspect t h a t the c o m m o n result of these occurrences is an increase in alveolar epithelial permeability with the entrance of increased amounts of proteins into the airways. T h e relatively small pool size of surfactant present in the infant with respiratory distress syndrome may be uniquely susceptible to functional inhibition by protein inhibitors. Thus the a m o u n t of surfactant, the functional m a t u r i t y of t h a t surfactant, and the quantity of protein will interact to result in variable severity of respiratory distress syndrome. REFERENCES 1. Avery ME, Mead J: Surface properties in relation to atelectasis and hyaline membrane disease. Am J Dis Child 97:51, 1959. 2. Adams FH, Fujiwara T, Emmanouilides G, Raiha H: Lung phosphoholipid of human fetuses and infants with and without hyaline membrane disease. J PEDIATR 77:833, 1970. 3. Brumley G, Hodson AD, Avery ME: Lung phospholipid and surface tension correlations in infants with and without hyaline membrane disease and adults. Pediatrics 40:13, 1967. 4. Van Golde LMG: Metabolism of phospholipid in the lung. Am Rev Res Dis 114:977, 1976.
Surfactant function in respiratory distress syndrome
447
5. Farrell PM, Avery ME: Hyaline membrane disease. Am Rev Res Dis 111:657, 1975. 6. Pattie RE: Properties, function and origin of the alveolar lining layer. Proc R Soc London [Biol] 1438:217, 1958. 7. Clements JA: Puhnonary edema and permeability of alveolar membranes. Arch Environ Health 2:280, 1961. 8. Tierney DF, Johnson RP: Altered surface tension of Iung extracts and lung mechanics. J Appl Physiol 20:1253, 1965. 9. Avery ME, Said S: Surface phenomena in lungs in health and disease. Medicine 44:503, 1965. 10. Taylor F, Abrams M: Effect of surface active lipoprotein on clotting and fibrinolysis, and of fibrinogen on surface tension of surface active lipoprotein. Am J Med 40:346, 1966. I I. Miyahara T: A study of the pathogenesis of hyaline membrane disease in the newborn infant. Kyushu Univ J 60:95, 1969. 12. Balls J, Shelley S, McCue M, Rappaport E: Mechanisms of damage to the lung surfactant system. Exp Mol Path 14:243, 1971. 13. Ikegami M, Jobe A, Glatz T: Surface activity following natural surfactant treatment of premature lambs. J AppI Physiol $1:306, 1981. 14. Mannino FL, Gluck L: The management of respiratory distress syndrome. In Thibeault D, Gregory G, editors: Neonatal pulmonary care. Reading, Mass., 1979, AddisonWesley, p 261. 15. Nozaki M: Pressure-volume relationships of a model alveolus. Tohoku J Expl Med 101:271, 1970. 16. Jobe A, Kirkpartrick E, Gluck L: Labeling of phospholipid in the surfactant and subcellular fractions of rabbit lung. J Biol Chem 253:3810, 1978. 17. Bartlett GR: Phosphorus assay in column chromatrography. J Biol Chem 234:466, 1959. 18. Lowry OH, Rosebrough N J, Farr AL, Randall R J: Protein measurement with the folin phenol reagent. J Biol Chem 193:265, 1951. 19. Jobe A, lkegami M, Glatz T, Yoshida Y, Diakomanolis E, Padbury J: Duration and characteristics of treatment of premature Iambs with natural surfactant. J Clin Invest 67:370, 1981. 20. Hallman M, Feldman B, Kirkpatrick E, Gluck L: Absence of phosphatidylglycerol in respiratory distress syndrome in the newborn. Pediatr Res !!:714, 1977. 21. Shelly S, Kovacevic M, Paciga J, Balls J: Sequential changes of surfactant phosphatidylcholine in hyaline-membrane disease of the newborn. N Engl J Med 300:112, 1979. 22. lkegami M, Jobe A, Jacobs H, Jones S: Sequential treatments of premature lambs with an artificial surfactant and natural surfactant. J Clin Invest 68:491, 1981.