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Surfactant and surfactant inhibitors in meconium aspiration syndrome
Surfactant and surfactant inhibitors in meconium aspiration syndrome Peter A. Dargaville, FRACP, Michael South, MD, FRACP, and Peter N. McDougall, FRACP
Surfactant indices and inhibitors were measured in lung lavage fluid from 8 infants with meconium aspiration syndrome (MAS) who were receiving mechanical ventilation and 11 healthy control subjects. Surfactant phospholipid and surfactant protein A content in MAS was not different from that of control subjects, but concentrations of total protein, albumin, and membrane-derived phospholipid were elevated. All infants with MAS had hemorrhagic pulmonary edema. These findings reinforce the notion of MAS as a toxic pneumonitis with epithelial disruption and proteinaceous exudation. (J Pediatr 2001;138:113-5)
The pathophysiology of meconium aspiration syndrome is diverse and includes airway obstruction, epithelial injury, and surfactant dysfunction.1 Perturbations of the surfactant system are prominent in MAS, although the available measurements in humans are from infants receiving extracorporeal membrane oxygenation at the time of lavage sampling.2-4 We therefore studied surfactant indices and potential surfactant inhibitors in MAS, using
From the Department of Neonatology and University Department of Paediatrics, Royal Children’s Hospital, Melbourne, Australia.
Supported by Murdoch Childrens Research Institute, Parkville, Victoria, Australia (Dr Dargaville’s research salary) and Teijin Limited, Iwakuni-city, Yamaguchi-ken, Japan (SP-A immunoassay kits). Submitted for publication Jan 14, 2000; revision received June 1, 2000; accepted June 19, 2000. Reprint requests: Peter Dargaville, FRACP, Department of Neonatology, Royal Children’s Hospital, Flemington Rd, Parkville, Victoria 3052, Australia. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/22/109602 doi:10.1067/mpd.2001.109602
lavage fluid from infants who were receiving mechanical ventilation, but not treated with ECMO.
METHODS Newborn infants receiving mechanical ventilation were enrolled in this study if they fulfilled all of the following diagnostic criteria for MAS: (1) history of meconium-stained amniotic fluid, (2) onset of respiratory distress soon after delivery, (3) either meconium suctioned from beyond the cords after delivery or chest x-ray findings typical of MAS. Infants with normal lungs, anesthetized and intubated for elective surgery, were the control group. Informed parental consent was obtained in all cases, and the protocol was approved by our institutional ethics in human research committee. Lung fluid samples were collected by non-bronchoscopic bronchoalveolar lavage with 3 aliquots of 1 mL/kg saline solution.5 In the MAS group, the BAL sample was taken soon after admission or intubation. Most recent plasma urea concentration and results of arterial blood
gas analysis were recorded, and oxygenation index was calculated, where Oxygenation index = (Mean airway pressure × Fraction of inspired oxygen × 100)/PaO2. BAL fluid was centrifuged at 150g and 4°C, and blood-staining of the centrifugation pellet was quantified by using a 0-3 scale, as previously described.5 In the BAL supernatant, concentrations of the following analytes were determined: disaturated phosphatidylcholine (osmium tetroxide method), surfactant protein A (immunoassay), total protein (bicinchoninic acid assay after delipidation with Triton X-100), albumin (immunoturbidimetry), and urea (urease method). Phospholipid composition was analyzed by means of high-performance liquid chromatography.6 Recovery of epithelial lining fluid was quantified5 according to the formula: ELF volume (per mL of return fluid) = ([Urea]LAVAGE/[Urea]PLASMA) The ELF concentration of all analytes was calculated, and geometric means were compared in the MAS and control groups. BAL DSPC ECMO ELF MAS SP-A
Bronchoalveolar lavage Disaturated phosphatidylcholine Extracorporeal membrane oxygenation Epithelial lining fluid Meconium aspiration syndrome Surfactant protein A
RESULTS Concentrations of surfactant indices and potential surfactant inhibitors are indicated in the Figure. These infants with MAS were comparable with others in terms of duration of mechanical 113
DARGAVILLE, SOUTH, AND MCDOUGALL
THE JOURNAL OF PEDIATRICS JANUARY 2001 serine, were considerably elevated in infants with MAS. Additionally, BAL fluid from each infant with MAS had a blood-staining score ≥2, whereas all control samples had a score of zero. Recovery of ELF tended to be higher in the MAS group compared with the control group (97 vs 51 µL of ELF/mL lavage fluid, P = .058).
DISCUSSION
Figure. ELF concentration of surfactant indices and inhibitors in BAL fluid. Geometric mean, error bars indicate 1 SD. Solid bars, MAS; stippled bars, control. Upper panel, ELF concentrations of DSPC, phosphatidylinositol (PI), protein, and albumin. Lower panel, ELF concentrations of phosphatidylglycerol (PG), SP-A, sphingomyelin (SM), and phosphatidylserine (PS). *Differs from value in control group, P < .05; **P < .01. Results with IgA secretory component as a dilutional marker are virtually identical (data not shown).
Table. Demographic and disease data
Infants with MAS No. Gestation (wk) Age (d)* Weight (kg)* Type of ventilation* OI*† Duration of ventilation (d)†
8 40 (38-41) 2.7 (0.68-6.9)‡ 3.4 (2.3-4.0) 5 CV, 3 HFOV 12 (4.5) 5.2 (3.5)
Control subjects 10 39 (34-40) 23 (1.0-120) 3.7 (2.7-7.0)
Values are expressed as medians with ranges in parentheses, unless otherwise stated. CV, Conventional ventilation; HFOV, high-frequency oscillatory ventilation; OI oxygenation index. *At initial sampling. †Mean (SD). ‡Differs from control group, P < .05, Mann-Whitney U test.
ventilation and requirement for highfrequency ventilation,7,8 and all had moderately severe lung disease with fraction of inspired oxygen ≥0.6 (Table). Despite this, ELF concentrations of DSPC, as well as phos114
phatidylglycerol, phosphatidylinositol, and SP-A, were not different from those found in the normal lung. By contrast, concentrations of known inhibitors of surfactant, including total protein, albumin, and phosphatidyl-
We found that infants with MAS receiving mechanical ventilation have normal concentrations of surfactant phospholipid and SP-A in the alveolar space but high concentrations of potential surfactant inhibitors in association with hemorrhagic pulmonary edema. These findings reinforce the notion of MAS as a toxic pneumonitis with consequent disruption of the alveolar capillary barrier, which occurs in a mature lung in which the machinery for surfactant synthesis, secretion, and recycling is largely intact. The normal concentration of surfactant phospholipid and SP-A in this study is at odds with previous data in infants with MAS, all of whom were receiving ECMO at the time of lavage sampling.2-4 These investigations demonstrated lavage concentrations of DSPC3,4 and SP-A2,3 that were lower at the beginning of ECMO than immediately before decannulation, and it was inferred that surfactant was deficient at the height of disease, with recovery as the lung injury resolved. This conclusion may be correct; by virtue of the need for ECMO, those infants almost certainly had more severe lung disease than infants in this study. Those infants may have developed a deficiency of surfactant secondary to alveolar epithelial injury. Interpretation of those studies is complicated by the absence of pre- and post-ECMO lavage samples, and the contribution of the ECMO therapy to the findings cannot be determined. It is possible, for example, that the initial ECMO-induced inflammatory re-
DARGAVILLE, SOUTH, AND MCDOUGALL
THE JOURNAL OF PEDIATRICS VOLUME 138, NUMBER 1 sponse, or the application of a “lungrest” ventilatory strategy, may have decreased surfactant synthesis at the onset of ECMO, resulting in the low surfactant concentration in the lavage samples. Although surfactant concentration was unaffected in infants with MAS in this study, concentrations of protein and albumin were more than 3 times those found in the normal lung, and phosphatidylserine content was also elevated. The inhibitory effects of serum-derived proteins and membrane phospholipid on surfactant function are well documented,9,10 and our data suggest that both groups of inhibitors may contribute to surfactant dysfunction in infants with MAS. The direct inhibitory effect of meconium on surfactant must also be considered, and it may be that the combined inhibitory effects of serum protein, membrane phospholipid, and meconium are multiplicative rather than additive. Taken together, the high ELF concentration of serum-derived proteins and the uniform finding of bloodstained lavage return fluid suggest that hemorrhagic pulmonary edema is an important element of the pathophysiology of MAS. In addition to the inhibition of surfactant, edema fluid in the alveolar space may directly affect gas exchange in MAS. Given that low alveolar surface tension is important for the clearance of fluid from the alveolar space, surfactant dysfunction will perpetuate and potentially amplify the edema that occurs in MAS. The findings of this study highlight some of the important considerations that need to be taken into account in ap-
plying exogenous surfactant therapy to MAS. Unlike hyaline membrane disease, in which much of the physiologic benefit of exogenous surfactant can be attributed to reversal of surfactant deficiency, in MAS, surfactant dysfunction must be overcome if clinical improvement is to be seen. The persistence of surfactant inhibition may explain why many infants with MAS do not respond to normal doses of surfactant7 and why those nearing ECMO criteria appear not to benefit from the therapy in terms of reduction in duration of ventilation (in days) or pulmonary complications.8 By virtue of the capacity to remove surfactant inhibitors, therapeutic pulmonary lavage with dilute surfactant may be more effective in MAS than bolus surfactant therapy.11 Alternatively, therapy with exogenous surfactant containing polymers to counteract surfactant inhibition may prove useful.12 These therapeutic possibilities deserve further exploration in the treatment of this complex lung disease. We thank Mr Peter Vervaart for his technical assistance in performing the urea assay.
REFERENCES 1. Wiswell TE, Bent RC. Meconium staining and the meconium aspiration syndrome. Unresolved issues. Pediatr Clin North Am 1993;40:955-81. 2. Lotze A, Whitsett JA, Kammerman LA, Ritter M, Taylor GA, Short BL. Surfactant protein A concentrations in tracheal aspirate fluid from infants requiring extracorporeal membrane oxygenation. J Pediatr 1990;116:435-40. 3. Bui KC, Walther FJ, David-Cu R, Garg M, Warburton D. Phospholipid and surfactant protein A concentrations
4.
5.
6.
7.
8.
9.
10.
11.
12.
in tracheal aspirates from infants requiring extracorporeal membrane oxygenation. J Pediatr 1992;121:271-4. Lotze A, Stroud CY, Soldin SJ. Serial lecithin/sphingomyelin ratios and surfactant/albumin ratios in tracheal aspirates from term infants with respiratory failure receiving extracorporeal membrane oxygenation. Clin Chem 1995;41:1182-8. Dargaville PA, South M, McDougall PN. Comparison of two methods of diagnostic lung lavage in ventilated infants with lung disease. Am J Respir Crit Care Med 1999;160:771-7. Andrews AG. Estimation of amniotic fluid phospholipids by high-performance liquid chromatography. J Chromatogr B 1984;336:139-50. Halliday HL, Speer CP, Robertson B. Treatment of severe meconium aspiration syndrome with porcine surfactant. Collaborative Surfactant Study Group. Eur J Pediatr 1996;155:1047-51. Lotze A, Mitchell BR, Bulas DI, Zola EM, Shalwitz RA, Gunkel JH, et al. Multicenter study of surfactant (beractant) use in the treatment of term infants with severe respiratory failure. Survanta in Term Infants Study Group. J Pediatr 1998;132:40-7. Fuchimukai T, Fujiwara T, Takahashi A, Enhorning G. Artificial pulmonary surfactant inhibited by proteins. J Appl Physiol 1987;62:429-37. Holm BA, Notter RH. Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity. J Appl Physiol 1987;63:1434-42. Cochrane CG, Revak SD, Merritt TA, Schraufstätter IU, Hoch RC, Henderson C, et al. Bronchoalveolar lavage with KL4-surfactant in models of meconium aspiration syndrome. Pediatr Res 1998;44:705-15. Taeusch HW, Lu KW, Goerke J, Clements JA. Nonionic polymers reverse inactivation of surfactant by meconium and other substances. Am J Respir Crit Care Med 1999; 159:1391-5.
115
Surfactant indices and inhibitors were measured in lung lavage fluid from 8 infants with meconium aspiration syndrome (MAS) who were receiving mechanical ventilation and 11 healthy control subjects. Surfactant phospholipid and surfactant protein A content in MAS was not different from that of control subjects, but concentrations of total protein, albumin, and membrane-derived phospholipid were elevated. All infants with MAS had hemorrhagic pulmonary edema. These findings reinforce the notion of MAS as a toxic pneumonitis with epithelial disruption and proteinaceous exudation. (J Pediatr 2001;138:113-5)
The pathophysiology of meconium aspiration syndrome is diverse and includes airway obstruction, epithelial injury, and surfactant dysfunction.1 Perturbations of the surfactant system are prominent in MAS, although the available measurements in humans are from infants receiving extracorporeal membrane oxygenation at the time of lavage sampling.2-4 We therefore studied surfactant indices and potential surfactant inhibitors in MAS, using
From the Department of Neonatology and University Department of Paediatrics, Royal Children’s Hospital, Melbourne, Australia.
Supported by Murdoch Childrens Research Institute, Parkville, Victoria, Australia (Dr Dargaville’s research salary) and Teijin Limited, Iwakuni-city, Yamaguchi-ken, Japan (SP-A immunoassay kits). Submitted for publication Jan 14, 2000; revision received June 1, 2000; accepted June 19, 2000. Reprint requests: Peter Dargaville, FRACP, Department of Neonatology, Royal Children’s Hospital, Flemington Rd, Parkville, Victoria 3052, Australia. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/22/109602 doi:10.1067/mpd.2001.109602
lavage fluid from infants who were receiving mechanical ventilation, but not treated with ECMO.
METHODS Newborn infants receiving mechanical ventilation were enrolled in this study if they fulfilled all of the following diagnostic criteria for MAS: (1) history of meconium-stained amniotic fluid, (2) onset of respiratory distress soon after delivery, (3) either meconium suctioned from beyond the cords after delivery or chest x-ray findings typical of MAS. Infants with normal lungs, anesthetized and intubated for elective surgery, were the control group. Informed parental consent was obtained in all cases, and the protocol was approved by our institutional ethics in human research committee. Lung fluid samples were collected by non-bronchoscopic bronchoalveolar lavage with 3 aliquots of 1 mL/kg saline solution.5 In the MAS group, the BAL sample was taken soon after admission or intubation. Most recent plasma urea concentration and results of arterial blood
gas analysis were recorded, and oxygenation index was calculated, where Oxygenation index = (Mean airway pressure × Fraction of inspired oxygen × 100)/PaO2. BAL fluid was centrifuged at 150g and 4°C, and blood-staining of the centrifugation pellet was quantified by using a 0-3 scale, as previously described.5 In the BAL supernatant, concentrations of the following analytes were determined: disaturated phosphatidylcholine (osmium tetroxide method), surfactant protein A (immunoassay), total protein (bicinchoninic acid assay after delipidation with Triton X-100), albumin (immunoturbidimetry), and urea (urease method). Phospholipid composition was analyzed by means of high-performance liquid chromatography.6 Recovery of epithelial lining fluid was quantified5 according to the formula: ELF volume (per mL of return fluid) = ([Urea]LAVAGE/[Urea]PLASMA) The ELF concentration of all analytes was calculated, and geometric means were compared in the MAS and control groups. BAL DSPC ECMO ELF MAS SP-A
Bronchoalveolar lavage Disaturated phosphatidylcholine Extracorporeal membrane oxygenation Epithelial lining fluid Meconium aspiration syndrome Surfactant protein A
RESULTS Concentrations of surfactant indices and potential surfactant inhibitors are indicated in the Figure. These infants with MAS were comparable with others in terms of duration of mechanical 113
DARGAVILLE, SOUTH, AND MCDOUGALL
THE JOURNAL OF PEDIATRICS JANUARY 2001 serine, were considerably elevated in infants with MAS. Additionally, BAL fluid from each infant with MAS had a blood-staining score ≥2, whereas all control samples had a score of zero. Recovery of ELF tended to be higher in the MAS group compared with the control group (97 vs 51 µL of ELF/mL lavage fluid, P = .058).
DISCUSSION
Figure. ELF concentration of surfactant indices and inhibitors in BAL fluid. Geometric mean, error bars indicate 1 SD. Solid bars, MAS; stippled bars, control. Upper panel, ELF concentrations of DSPC, phosphatidylinositol (PI), protein, and albumin. Lower panel, ELF concentrations of phosphatidylglycerol (PG), SP-A, sphingomyelin (SM), and phosphatidylserine (PS). *Differs from value in control group, P < .05; **P < .01. Results with IgA secretory component as a dilutional marker are virtually identical (data not shown).
Table. Demographic and disease data
Infants with MAS No. Gestation (wk) Age (d)* Weight (kg)* Type of ventilation* OI*† Duration of ventilation (d)†
8 40 (38-41) 2.7 (0.68-6.9)‡ 3.4 (2.3-4.0) 5 CV, 3 HFOV 12 (4.5) 5.2 (3.5)
Control subjects 10 39 (34-40) 23 (1.0-120) 3.7 (2.7-7.0)
Values are expressed as medians with ranges in parentheses, unless otherwise stated. CV, Conventional ventilation; HFOV, high-frequency oscillatory ventilation; OI oxygenation index. *At initial sampling. †Mean (SD). ‡Differs from control group, P < .05, Mann-Whitney U test.
ventilation and requirement for highfrequency ventilation,7,8 and all had moderately severe lung disease with fraction of inspired oxygen ≥0.6 (Table). Despite this, ELF concentrations of DSPC, as well as phos114
phatidylglycerol, phosphatidylinositol, and SP-A, were not different from those found in the normal lung. By contrast, concentrations of known inhibitors of surfactant, including total protein, albumin, and phosphatidyl-
We found that infants with MAS receiving mechanical ventilation have normal concentrations of surfactant phospholipid and SP-A in the alveolar space but high concentrations of potential surfactant inhibitors in association with hemorrhagic pulmonary edema. These findings reinforce the notion of MAS as a toxic pneumonitis with consequent disruption of the alveolar capillary barrier, which occurs in a mature lung in which the machinery for surfactant synthesis, secretion, and recycling is largely intact. The normal concentration of surfactant phospholipid and SP-A in this study is at odds with previous data in infants with MAS, all of whom were receiving ECMO at the time of lavage sampling.2-4 These investigations demonstrated lavage concentrations of DSPC3,4 and SP-A2,3 that were lower at the beginning of ECMO than immediately before decannulation, and it was inferred that surfactant was deficient at the height of disease, with recovery as the lung injury resolved. This conclusion may be correct; by virtue of the need for ECMO, those infants almost certainly had more severe lung disease than infants in this study. Those infants may have developed a deficiency of surfactant secondary to alveolar epithelial injury. Interpretation of those studies is complicated by the absence of pre- and post-ECMO lavage samples, and the contribution of the ECMO therapy to the findings cannot be determined. It is possible, for example, that the initial ECMO-induced inflammatory re-
DARGAVILLE, SOUTH, AND MCDOUGALL
THE JOURNAL OF PEDIATRICS VOLUME 138, NUMBER 1 sponse, or the application of a “lungrest” ventilatory strategy, may have decreased surfactant synthesis at the onset of ECMO, resulting in the low surfactant concentration in the lavage samples. Although surfactant concentration was unaffected in infants with MAS in this study, concentrations of protein and albumin were more than 3 times those found in the normal lung, and phosphatidylserine content was also elevated. The inhibitory effects of serum-derived proteins and membrane phospholipid on surfactant function are well documented,9,10 and our data suggest that both groups of inhibitors may contribute to surfactant dysfunction in infants with MAS. The direct inhibitory effect of meconium on surfactant must also be considered, and it may be that the combined inhibitory effects of serum protein, membrane phospholipid, and meconium are multiplicative rather than additive. Taken together, the high ELF concentration of serum-derived proteins and the uniform finding of bloodstained lavage return fluid suggest that hemorrhagic pulmonary edema is an important element of the pathophysiology of MAS. In addition to the inhibition of surfactant, edema fluid in the alveolar space may directly affect gas exchange in MAS. Given that low alveolar surface tension is important for the clearance of fluid from the alveolar space, surfactant dysfunction will perpetuate and potentially amplify the edema that occurs in MAS. The findings of this study highlight some of the important considerations that need to be taken into account in ap-
plying exogenous surfactant therapy to MAS. Unlike hyaline membrane disease, in which much of the physiologic benefit of exogenous surfactant can be attributed to reversal of surfactant deficiency, in MAS, surfactant dysfunction must be overcome if clinical improvement is to be seen. The persistence of surfactant inhibition may explain why many infants with MAS do not respond to normal doses of surfactant7 and why those nearing ECMO criteria appear not to benefit from the therapy in terms of reduction in duration of ventilation (in days) or pulmonary complications.8 By virtue of the capacity to remove surfactant inhibitors, therapeutic pulmonary lavage with dilute surfactant may be more effective in MAS than bolus surfactant therapy.11 Alternatively, therapy with exogenous surfactant containing polymers to counteract surfactant inhibition may prove useful.12 These therapeutic possibilities deserve further exploration in the treatment of this complex lung disease. We thank Mr Peter Vervaart for his technical assistance in performing the urea assay.
REFERENCES 1. Wiswell TE, Bent RC. Meconium staining and the meconium aspiration syndrome. Unresolved issues. Pediatr Clin North Am 1993;40:955-81. 2. Lotze A, Whitsett JA, Kammerman LA, Ritter M, Taylor GA, Short BL. Surfactant protein A concentrations in tracheal aspirate fluid from infants requiring extracorporeal membrane oxygenation. J Pediatr 1990;116:435-40. 3. Bui KC, Walther FJ, David-Cu R, Garg M, Warburton D. Phospholipid and surfactant protein A concentrations
4.
5.
6.
7.
8.
9.
10.
11.
12.
in tracheal aspirates from infants requiring extracorporeal membrane oxygenation. J Pediatr 1992;121:271-4. Lotze A, Stroud CY, Soldin SJ. Serial lecithin/sphingomyelin ratios and surfactant/albumin ratios in tracheal aspirates from term infants with respiratory failure receiving extracorporeal membrane oxygenation. Clin Chem 1995;41:1182-8. Dargaville PA, South M, McDougall PN. Comparison of two methods of diagnostic lung lavage in ventilated infants with lung disease. Am J Respir Crit Care Med 1999;160:771-7. Andrews AG. Estimation of amniotic fluid phospholipids by high-performance liquid chromatography. J Chromatogr B 1984;336:139-50. Halliday HL, Speer CP, Robertson B. Treatment of severe meconium aspiration syndrome with porcine surfactant. Collaborative Surfactant Study Group. Eur J Pediatr 1996;155:1047-51. Lotze A, Mitchell BR, Bulas DI, Zola EM, Shalwitz RA, Gunkel JH, et al. Multicenter study of surfactant (beractant) use in the treatment of term infants with severe respiratory failure. Survanta in Term Infants Study Group. J Pediatr 1998;132:40-7. Fuchimukai T, Fujiwara T, Takahashi A, Enhorning G. Artificial pulmonary surfactant inhibited by proteins. J Appl Physiol 1987;62:429-37. Holm BA, Notter RH. Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity. J Appl Physiol 1987;63:1434-42. Cochrane CG, Revak SD, Merritt TA, Schraufstätter IU, Hoch RC, Henderson C, et al. Bronchoalveolar lavage with KL4-surfactant in models of meconium aspiration syndrome. Pediatr Res 1998;44:705-15. Taeusch HW, Lu KW, Goerke J, Clements JA. Nonionic polymers reverse inactivation of surfactant by meconium and other substances. Am J Respir Crit Care Med 1999; 159:1391-5.
115