Published online February 1, 2008
PEDIATRICS Vol. 121 No. 2 February 2008, pp. e253-e259 (doi:10.1542/peds.2007-0056)
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ARTICLE

Pepsin, a Marker of Gastric Contents, Is Increased in Tracheal Aspirates From Preterm Infants Who Develop Bronchopulmonary Dysplasia

Sabeena Farhath, MDa, Zhaoping He, PhDa, Tarek Nakhla, MDb, Judy Saslow, MDb, Sam Soundar, PhDa, Jeanette Camacho, MDc, Gary Stahl, MDb, Stephen Shaffer, MDa, Devendra I. Mehta, MDd and Zubair H. Aghai, MDb

a Division of Gastroenterology and Nutrition and Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, Delaware
b Departments of Pediatrics/Neonatology
c Pathology, Cooper University Hospital-UMDNJ-Robert Wood Johnson Medical School, Camden, New Jersey
d Division of Gastroenterology and Nutrition, Nemours Children's Clinic, Orlando, Florida


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE. The objective of this study was to study the association between pepsin in tracheal aspirate samples and the development of bronchopulmonary dysplasia in preterm infants.

METHODS. Serial tracheal aspirate samples were collected during the first 28 days from mechanically ventilated preterm neonates. Bronchopulmonary dysplasia was defined as the need for supplemental oxygen at 36 weeks’ postmenstrual age. An enzymatic assay with a fluorescent substrate was used to detect pepsin. Total protein was measured by the Bradford assay to correct for the dilution during lavage. Immunohistochemistry using antibody against human pepsinogen was performed in 10 lung tissue samples from preterm infants.

RESULTS. A total of 256 tracheal aspirate samples were collected from 59 preterm neonates. Pepsin was detected in 234 (91.4%) of 256 of the tracheal aspirate samples. Twelve infants had no bronchopulmonary dysplasia, 31 infants developed bronchopulmonary dysplasia, and 16 infants died before 36 weeks’ postmenstrual age. The mean pepsin concentration was significantly lower in infants with no bronchopulmonary dysplasia compared with those who developed bronchopulmonary dysplasia or developed bronchopulmonary dysplasia/died before 36 weeks’ postmenstrual age. Moreover, the mean pepsin level was significantly higher in infants with severe bronchopulmonary dysplasia compared with moderate bronchopulmonary dysplasia. The mean pepsin level in tracheal aspirate samples from the first 7 days was also lower in infants with no bronchopulmonary dysplasia compared with those who developed bronchopulmonary dysplasia or developed bronchopulmonary dysplasia/died before 36 weeks’ postmenstrual age. Pepsinogen was not localized in the lung tissues by immunohistochemistry.

CONCLUSION. The concentration of pepsin was increased in the tracheal aspirate of preterm infants who developed bronchopulmonary dysplasia or died before 36 weeks’ postmenstrual age. Recovery of pepsin in tracheal aspirate samples is secondary to gastric aspiration, not by hematogenous spread or local synthesis in the lungs. Chronic aspiration of gastric contents may contribute in the pathogenesis of bronchopulmonary dysplasia.

Key Words: gastric aspiration • gastroesophageal reflux • pepsin • preterm infants • bronchopulmonary dysplasia

Abbreviations: BPD—bronchopulmonary dysplasia • GER—gastroesophageal reflux • TA—tracheal aspirate • PMA—postmenstrual age • GA—gestational age • BSA—bovine serum albumin

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease during infancy and is associated with significant mortality and morbidity in preterm infants. Twenty to 40% of ventilated preterm neonates develop BPD.1 Multiple factors, including exposure to a high fraction of inspired oxygen, ventilator-induced lung injury, symptomatic patent ductus arteriosus, various lytic proteinases, infection, and nutritional deficiencies, have been implicated in the etio-pathogenesis of BPD, but the exact cause is still unknown.1 Despite the use of prenatal steroids and surfactant replacement therapy, the incidence of BPD has not changed significantly in the past decade.2 Moreover, a new chronic lung disease called the "new BPD" is emerging in preterm infants, not related to oxygen therapy or mechanical ventilation.3

Gastroesophageal reflux (GER) is also common in preterm neonates.4–6 In a recent study,7 we showed that pepsin, a marker of gastric contents, is present in >92% of tracheal aspirate (TA) samples from preterm ventilated infants. Aspiration of gastric contents is implicated in the induction or exacerbation of asthma and is a recognized risk factor for ventilator-associated pneumonia in critically ill patients.8–10 The role of gastric aspiration as a cause of lung injury in preterm infants and its relationship to the development of BPD has not been previously investigated. Chronic aspiration of gastric contents may contribute to worsening lung disease in preterm infants.

The aim of this study was to evaluate whether there is a correlation between the concentration of pepsin in TA samples and the development of BPD in preterm infants. We hypothesized that pepsin, a marker of gastric contents, is increased in preterm infants who develop BPD.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Group
The study was conducted in a 39-bed level III NICU at Cooper University Hospital (Camden, NJ) between March 2003 and October 2006. The institutional review committee approved the study, and parents signed a written informed consent. Infants who were born before 32 weeks’ gestation and required mechanical ventilatory support were eligible for inclusion. Relevant clinical data, including the infant's demographics, clinical parameters, and data on respiratory support, were collected from the patient's chart. BPD was defined as the need for supplemental oxygen at 36 weeks’ postmenstrual age (PMA). We separated infants who developed BPD into moderate and severe categories on the basis of National Institute of Child Health and Human Development/National Heart, Lung, and Blood Institute Workshop's proposed severity-based definition of BPD for infants <32 weeks’ gestational age (GA; moderate: need for <30% O2 at 36 weeks’ PMA or discharge; severe: need for ≥30% O2 and/or positive pressure at 36 weeks’ PMA or discharge).11

Sample Collections
TA samples were collected on days 1, 3, 5, 7, 14, 21, and 28 while the infant was mechanically ventilated. TA samples were collected ~3 hours after feeding. Samples were obtained by instilling 0.5 mL of normal saline into the infant's endotracheal tube and suctioning the residue with a 5-F suction catheter after 2 or 3 ventilator breaths. The suction catheter was passed to a standardized length of 0.5 to 1 cm beyond the tip of the endotracheal tube. This method of collection is used widely in neonates to collect TA samples,12–14 and it is well tolerated by even the most critically ill neonates. The procedure was repeated 3 times, and replicates were pooled. The suction catheter was flushed with 0.5 mL of normal saline after each suctioning episode to collect the residual sample in the catheter. Samples were immediately transported to the laboratory on ice and processed within 30 min. The samples were centrifuged at 4°C for 10 minutes at 3000 x g. The supernatant was collected, divided into aliquots, and stored at –70°C for future use.

Stored blocks of lung tissues from 10 preterm infants, unrelated to the study population, were used for immunohistochemistry to localize pepsinogen. Three infants were born at borderline gestation and had no resuscitation done, 2 died of sepsis, 1 had anencephaly and received comfort care only, 2 had respiratory distress syndrome, and 1 each had birth asphyxia and BPD.

Pepsin Enzymatic Method
The enzymatic method was modified according to the assay developed by Krishnan et al.15 Porcine pepsin (Sigma-Aldrich, St Louis, MO) standards (12.5–400 ng/mL) were prepared in 0.1 mg/mL bovine serum albumin (BSA) with saline. Gastric fluid from positive control patients was diluted in the same BSA/saline solution, and the enzymatic reactions were conducted in a 96-well microplate. Fifty microliters of standard or sample was pipetted into the microplate wells. Sample blanks were prepared by incubating the standard or sample on a 100°C dry block for 5 minutes to inactivate the enzymatic activity. To each well, 23 µL of 129 mM HCl was added to adjust the pH to 2.0 and left on ice for 15 minutes to inactivate the lysosomal acid hydrolase (cathepsin D) and to convert pepsinogen to active pepsin.15 Next, 20 µL of 0.5% fluorescein isothiocyanate casein (Sigma-Aldrich) was added to each well and incubated for 3 hours at 37°C. The plates were transferred back to the ice tray, and 90 µL of 1.2 mg/mL BSA and 30 µL of 20% trichloroacetic acid were added to each well for trichloroacetic acid precipitation. The plates were centrifuged for 90 minutes at 3500 rpm and 8°C in a Sorvall centrifuge. Thirty-eight microliters of the supernatant was transferred to a new, clean, flat-bottomed microplate, and 212 µL of 500 mM Tris was added to each well. The plate was read in a spectrofluorometer at excitation (485 nm) and emission (530 nm), and the net fluorescent intensity was subtracted from the blanks. The final pepsin concentration of the sample was determined on the basis of the net fluorescent intensity of the known concentrations of the standards. The subjective pepsin level (defining positive from negative) of an aspirate was set at the lower limit (12.5 ng/mL) of sensitivity of the assay.

Protein Assay
Total protein concentration was measured in each TA sample by the Bradford assay (Bio-Rad, Richmond, CA) to correct for dilution during the lavage procedure. The level of pepsin is expressed as nanograms per milligram of protein.

Immunohistochemistry
Immunohistochemical staining for localization of pepsinogen was performed on the lung tissue using monoclonal mouse anti-human pepsinogen I antibodies (US Biological, Swampscott, MA). Detection used a labeled streptavidin-biotin method using chemicals from Zymed Laboratories (San Francisco, CA), diaminobenzidine chromogen from BioFx (Owings Mills, MD), and a Dako Autostainer (DakoCytomation, Carpinteria, CA). Localization of pepsinogen was detected as brown-stained cells. Negative controls were performed using nonimmunized mouse immunoglobulin G1 in place of the pepsinogen antibody. Tissue from human stomach was used as a positive control.

Statistical Analysis
Statistics were performed using Sigma Stat 3.1 for Windows statistical package (Systat Software, Inc, Point Richmond, CA). The level of pepsin between the groups (no BPD versus BPD, no BPD versus BPD or died, and moderate versus severe BPD) was compared using Mann-Whitney U test. Comparisons between the group's clinical characteristics (Table 1) were done using Student's t test (birth weight, GA, and Survanta doses), Mann-Whitney U test (days on ventilator, days on oxygen, and length of hospitalization), {chi}2 (gender, race, and prenatal steroids), and Fisher's exact test (postnatal steroids and H2 blockers). The difference was considered significant at P < .05.


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  		TABLE 1 Demographic and Clinical Characteristics of the Study Group

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 256 TA samples were collected from 59 preterm neonates (birth weight: 777 ± 183 g; GA 25.6 ± 1.7 weeks). Clinical characteristics of the study group are summarized in Table 1.

Pepsin was detected in 234 (91.4%) of 256 of TA samples. None of the infants in this study had below detectable levels of pepsin in all of their TA samples. The median pepsin level of all TA samples was 283.21 ng/mg protein (range: 0–2441 ng/mg protein). Pepsinogen was localized in the chief cells of human stomach tissue by immunohistochemistry, but none of the 10 lung tissues from preterm infants had brown staining indicating the presence of pepsinogen (Fig 1).


Figure 1
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  		FIGURE 1 Localization of pepsinogen shown as a brown staining on immunohistochemistry using monoclonal mouse anti-human antibodies against pepsinogen I. A and B, Human adult stomach tissue. C and D, Lung tissue from a preterm infant. Pepsinogen is localized as a brown staining in chief cells of the stomach tissue, but no localization of pepsinogen is observed in lung tissue. Magnifications: x20 in A and C; x40 in B and D.

 
Twelve infants had no BPD, 31 infants developed BPD, and 16 infants died before 36 weeks’ PMA. Clinical characteristics of the study group are summarized in Table 1. Birth weight was higher in infants with no BPD compared with infants who developed BPD or died. The duration of mechanical ventilation, oxygen therapy, and hospitalization was also higher in infants with BPD. Although the duration of mechanical ventilation, oxygen therapy, and hospitalization was also lower in infants who died compared with infants in BPD group, this difference was attributable to early death and may not reflect the severity of illness; however, there was no significant difference in GA and other demographics in infants who developed BPD and those who did not. The mean pepsin level was significantly lower in infants with no BPD (425 ± 451 ng/mg protein) compared with those who developed BPD (606 ± 349 ng/mg; P = .036) or developed BPD/died before 36 weeks’ PMA (866 ± 998 ng/mg; P = .02; Fig 2). On the basis of the severity, a posthoc analysis was done to compare mean pepsin level in infants with moderate BPD (oxygen requirement <30% at 36 weeks’ PMA) and severe BPD (>30% oxygen requirement and/or ventilatory or pressure support at 36 weeks’ PMA). Of 31 infants who developed BPD, 20 infants developed moderate BPD and 11 had severe BPD. The mean pepsin level was significantly higher in infants with severe BPD (856 ± 431 ng/mg) compared with moderate BPD (469 ± 197 ng/mg; P = .002; Fig 3). The median pepsin concentration was lower in infants with no BPD (Fig 2) compared with infants with moderate BPD (Fig 3); however, this difference was not statistically significant, probably because of the small sample size. Because the infants who developed BPD were on mechanical ventilation for a longer period, the number of TA samples collected from these infants with BPD was higher (total of 167 samples from 31 infants; median: 6; range: 1–7) compared with infants with no BPD (total of 38 from 12 infants; median: 3; range: 1–7) and infants who died (total of 51 from 16 infants; median: 4; range: 1–6). For correction of this disparity, the mean pepsin levels were compared from the first-week samples in the groups. There was no significant difference in the number of samples collected during the first week of life in infants with no BPD (total of 28; median: 2; range: 1–4), BPD (total of 93; median: 3; range: 0–4) and infants who died before 36 weeks’ PMA (total of 44; median: 3; range: 1–4). The mean pepsin level in TA samples from the first 7 days was also lower in infants with no BPD (269 ± 197 ng/mg) compared with those who developed BPD (684 ± 440 ng/mg; P = .004) or developed BPD/died before 36 weeks’ PMA (865 ± 1108 ng/mg; P = .002; Fig 4).


Figure 2
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  		FIGURE 2 Mean pepsin concentration in TA from infants with no BPD (n = 12), from infants with BPD (n = 31), and from infants who had BPD or died before 36 weeks’ PMA (n = 47). The level was significantly higher in infants who developed BPD compared with those who did not develop BPD (P = .036, Mann-Whitney U test; A). Similarly, the mean pepsin concentration was significantly higher in infants who developed BPD or died before 36 weeks’ PMA compared with those who survived without BPD (P = .02, Mann-Whitney U test; B). Box plot: the boundary of the box indicates the 25th and 75th percentiles, the line within the box marks the median value, and the error bars indicate the 5th and 95th percentiles.

 

Figure 3
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  		FIGURE 3 Mean pepsin concentration in preterm infant with moderate BPD (n = 20) and severe BPD (n = 11). The concentration of pepsin was significantly higher in infants with severe compared with moderate BPD (P = .002, Mann-Whitney U test).

 

Figure 4
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  		FIGURE 4 Mean pepsin concentration in TA samples from the first 7 days of life from infants with no BPD (n = 12), from infants with BPD (n = 30), and from infants who had BPD or died before 36 weeks’ PMA (n = 46). The level was significantly higher in infants who developed BPD compared with those who did not develop BPD (P = .004, Mann-Whitney U test; A). Similarly, the mean pepsin concentration was significantly higher in infants who developed BPD or died before 36 weeks’ PMA compared with those who survived without BPD (P = .002, Mann-Whitney U test; B).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Preterm infants are predisposed to GER and are at increased risk for pulmonary aspiration. Because of difficulties in diagnosis, the true incidence of GER and gastric aspiration in preterm infants is unknown. Recent studies have shown that modified pepsin is a sensitive and specific marker of gastric aspiration.15–17 By using a similar enzymatic assay, we have demonstrated that aspiration of gastric contents into the lung is a widespread phenomenon in mechanically ventilated preterm infants.7 The possible sources of pepsin in TA samples are (1) aspiration of gastric contents into the lungs, (2) hematogenous spread of pepsinogen/pepsin to the lungs, and (3) local synthesis of pepsinogen/pepsin. In our previous study,7 we measured pepsin in 10 serum samples from 8 preterm infants, collected at the same time of TA collection. Undetectable pepsin in serum samples excluded hematogenous spread as a source of pepsin in TA.7 Similarly, local synthesis of pepsinogen was excluded by the inability to localize pepsinogen in lung tissue; therefore, the most likely source of pepsin in TA samples in our study population is the aspiration of gastric contents into the lungs.

Preterm ventilated infants are more likely to develop pulmonary aspiration (as a result of prone position, presence of nasogastric tubes, and use of uncuffed endotracheal tubes), but the tools to recognize such aspiration are limited. Investigators have used dye studies, lipid-laden macrophages, and glucose and lactose assays for detection of gastric aspiration, but they lack the sensitivity and specificity.17–21 In clinical practice, technetium scintigraphy is used to detect pulmonary aspiration; however, this study is performed during and for 2 to 3 hours after a single feed and therefore has limited ability to detect aspiration of gastric contents.22 Recent studies7,15–17 suggested that the detection of pepsin in TA samples may be a more reliable and specific marker of gastric aspiration. An added advantage of measuring pepsin in TA samples for assessing aspiration is that TA samples can be collected easily at the bedside as a noninvasive procedure and is more feasible in ventilated preterm infants.

The aspiration of gastric contents can potentially cause mechanical obstruction and chemical injury to the airways and initiate an inflammatory response in the lungs. In animal models of gastric aspiration, gastric particulates altered the pulmonary mechanics, increased pulmonary inflammatory cells, released proinflammatory mediators, and inactivated surfactant.23,24 Gastric aspiration also decreased pulmonary bacterial clearance and contributed to the development of bacterial pneumonia.25

In this study, pepsin was used as a marker of the presence of gastric contents in the airways. We investigated the relationship between gastric aspiration and the development of BPD in preterm infants. Our results indicated that preterm infants, who developed BPD or died with respiratory insufficiency had elevated concentrations of pepsin in their TA samples. Increased concentrations of pepsin in TA samples were also associated with increased severity of BPD. Elevated level of pepsin in TA samples may indicate a higher degree of GER and gastric aspiration. Pepsin along with other gastric contents (low pH and bile salts) can potentially damage the airways and initiate an inflammatory process by increasing the number of inflammatory cells with the release of proinflammatory mediators.23,26 Recent evidence27,28 suggested that hyperoxia exacerbates lung injury after gastric aspiration. More recently, Hermon et al29 reported that mechanical ventilation also aggravated aspiration-induced lung injury in rabbits.

The literature is sparse correlating GER with BPD in preterm infants. Fuloria et al30 reported that preterm infants who developed BPD were more often treated for GER than control subjects. Akinola et al31 did not find any correlation between acid reflux and BPD in preterm infants; however, the majority of GER in preterm infants is nonacidic because 90% of the time in preterm neonates, gastric pH is >4.32,33 O'Hare et al34 showed that aspiration of human breast milk in a rabbit model caused severe lung injury with an increase in alveolar-arterial oxygen gradient, decrease in dynamic compliance, and increase in circulating neutrophil count. The severity of lung injury after instillation of human breast milk was similar at pH values between 1.8 and 7.0.

Free radical injury from oxygen therapy and volutrauma/barotrauma from mechanical ventilation are major contributing factors in the cause of BPD. We now present evidence that gastric aspiration may also play a key role in the development of BPD. Gastric aspiration can worsen the lung disease in preterm infants by directly inducing lung injury and aggravating hyperoxia and mechanical ventilation-induced lung damage. Despite gentle ventilation and close monitoring of oxygen saturation, the incidence of BPD is not significantly changed, and a new chronic lung disease, "the new BPD," is emerging in preterm infants.4 Charafeddine et al35 reported that 31% of BPD in a group of preterm infants with birth weight of <1251 g was atypical BPD, occurring without preceding respiratory distress or after recovery from respiratory distress. The new BPD or atypical BPD develops in preterm neonates with no or minimal initial lung disease and presents with gradual worsening of respiratory failure. We speculate that chronic aspiration of gastric contents may be contributing to the worsening of lung disease in preterm infants and playing a major role in the development of new BPD.

We recognize some important limitations of this study:

  1. Pepsin was measured in TA samples and was potentially diluted. D'Angio et al36 showed that TA specimen may be a suitable substitute for bronchoalveolar lavage samples in preterm infants. It is controversial whether TA samples should be corrected for dilution in neonates.37 Investigators have used protein,36,38 secretory immunoglobulin A,39–42 and serum urea13,43 to correct for dilution during the lavage procedure. We opted to normalize our data using total protein in TA samples.
  2. Using pepsin as a marker of gastric aspiration may underestimate the true incidence of aspiration because reports have shown that pepsin secretion is inconsistent and decreased during infancy.44,45
  3. Although the data were analyzed using mean pepsin levels from each infant, more TA samples were collected from preterm infants who developed BPD because they were on the ventilator for a longer period. For correction of this disparity, the data were also analyzed using TA samples collected from the first 7 days of life.
  4. The approach of using immunohistochemistry to localize pepsinogen in the lung tissue and excluding it as a source of the TA pepsin is a negative-result study. More appropriate study is to probe for evidence of pepsinogen mRNA in the lung tissue by mRNA isolation and quantification techniques.

Despite these limitations, this is the first study to demonstrate the possible relationship between gastric aspiration and the development of BPD in preterm infants. Additional studies are required in preterm infants to investigate the role of gastric aspiration in acute lung injury and its relationship with proinflammatory mediators and the effect of preventing GER and aspiration on the development of BPD.


   ACKNOWLEDGMENTS
 
Part of this work was supported by a grant from American Lung Association of New Jersey.

We thank Charlene Martin, RN, Jane Hasson, RN, Valerie Gibson, RN, and Lois Meyer, RN, for help in screening infants for enrollment and collecting TA samples. We also thank Kee Pyon, PhD, for help in analyzing the data and reviewing the manuscript.


   FOOTNOTES
 
Accepted Jul 6, 2007.

Address correspondence to Zubair H. Aghai, MD, Division of Neonatology, 755 Dorrance, One Cooper Plaza, Camden, NJ 08103. E-mail: aghai-zubair{at}cooperhealth.edu

The authors have indicated they have no financial relationships relevant to this article to disclose.

This work was presented in part at the annual meeting of the North American Society for Pediatric Gastroenterology Hepatology and Nutrition; October 19–22, 2006; Orlando, FL; the annual meeting of the Eastern Society for Pediatric Research; March 9–11, 2007; Philadelphia, PA; and the annual meeting of Pediatric Academic Societies; May 5–8, 2007; Toronto, Ontario, Canada.


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 METHODS
 RESULTS
 DISCUSSION
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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics

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