The recovery of pepsin in tracheal aspirates has already been demonstrated in several studies in adults, children, and neonates, and its presence is thought to denote aspiration (1–4). These findings led to gastroesophageal reflux disease (GERD) being implicated in asthma, otitis media, sinusitis, morbidity in lung transplant recipients, and bronchopulmonary dysplasia (BPD) (5–10). Elabiad et al (11) detected pepsin in 77% of tracheal aspirate samples collected from premature infants in the first 56 days of life. Sixteen of the samples were collected in the first 3 days of life and all but 1 (94%) were positive for pepsin (unpublished data). In a similar study on premature ventilated infants, Farhath et al detected pepsin in 92% of samples collected (2). With pepsin used as proof for the presence of gastric fluid, both studies suggested that it is extremely common to have aspiration in intubated premature infants as early as the first day of life.
Another possible explanation for the common recovery of pepsin stems from the scientific method used in the detection assay. Both studies used an enzymatic method to detect pepsin. In this method, the pepsin concentration in the sample is based on its proteolytic activity to digest a known amount of protein, and the concentration is then compared with standards. This is a frequently used technique to measure pepsin (2,4–6,9–11). We speculated about whether this method of detection is measuring a molecule of airway origin that has known similar activity to gastric pepsin. Pepsinogen C is a molecule produced by the stomach and the lung (12,13) and has functions similar to pepsinogen A, the precursor molecule of gastric pepsin (14,15).
We hypothesized that, in infants, the high prevalence of pepsin activity in tracheal aspirates is the result of the presence of variable amounts of 2 proteolytic enzymes, the first being pepsin A after being converted from pepsinogen A by stomach acidity, and the second being pepsin C from pepsinogen C being produced and converted locally or even refluxed and aspirated with pepsin A. To evaluate our hypothesis, we performed a pilot study in which we stained for both pepsinogen types on lung samples collected at autopsy.
PATIENTS AND METHODS
This study was conducted on patients who had died and on whom an autopsy was performed between June and December 2009 at the Neonatal Intensive Care Unit at the Regional Medical Center, Memphis, Tennessee. The gestational age, sex, and survival time of each patient are listed in Table 1. The study protocol was approved by the institutional review board at the University of Tennessee Health Science Center.
Sixteen paraffin blocks containing bronchi and lung parenchyma and 13 blocks containing stomach tissue from 16 patients were processed. No stomach tissue was sampled at autopsy in 2 cases, and a third case showed severe mucosal autolysis. All of the slides from each block were 4-μm thick. After deparaffinization, rehydration, and blockage with conditioner, slides were then incubated with 1 of the primary antibodies: monoclonal antipepsinogen A and C with dilution 1:10 in pepsinogen A and 1:20 in pepsinogen C (cat no. P3272–04X and P3272–07X, USBiological, Swampscott, MA). The secondary antibody and detection were via the iVIEW DAB detection system with biotinylated general immunoglobulin (Ventana Medical System Inc, Tucson, AZ). The immunostaining was performed in the Ventana Benchmark XT system (Ventana Medical System). After immunostaining, the slides were counterstained with hematoxylin and covered with coverslip for evaluation. Human adult stomach antrum was used as positive control, and tested lung and stomach tissues without primary antibody served as negative control.
Sixteen patients with diverse causes of death were evaluated (Table 1). Three patients did not have simultaneous staining done on a stomach section. Pepsinogen A was detected in the chief cells in 12 of 13 stomach sections. No pepsinogen A was detected in any of the 16 lung sections. Pepsinogen C was detected in both chief cells and adjacent mucus cells in all 13 stomach sections. Pepsinogen C was detected mainly in type II pneumocytes in 9 of 16 lung sections (Fig. 1). Pepsinogen C was not detected in lung sections from cases with a gestational age <23 weeks. It was detected as early as 21 weeks’ gestational age on stomach sections.
The importance of this study lies in the simultaneous staining for pepsinogen A and pepsinogen C in both lung and stomach at different gestational ages. We demonstrated that the same pepsinogen C molecule found in the lung is also found in the stomach. Our findings suggest that tracheal aspirates with positive enzymatic detection of pepsin may not be exclusively caused by gastroesophageal reflux, but rather to lung production of pepsinogen C. We also confirmed previous findings about the absence of pulmonary pepsinogen C in patients <23 weeks’ gestational age.
The evaluation of GERD has evolved from using 24-hour pH monitoring alone to using combined multichannel impedance and pH monitoring, which led to evidence that not all reflux is acidic and that nonacidic reflux in infants, for example, may be associated with apnea (16). With pepsin produced primarily by gastric-chief cells, it becomes an ideal marker of extraesophageal reflux. This marker is a major advantage during 24-hour pH monitoring alone and should offer comparable results to nonacidic reflux evaluation by using combined multichannel impedance and pH monitoring.
Pepsin detection has been evaluated using either the enzymatic or the immunologic method. The enzymatic method quantifies the amount of pepsin present in a sample based on the amount of protein digestion that sample can produce when combined with a known amount of protein. Other proteases with digestive characteristics similar to pepsin can cause interference and falsely increase the level of measured pepsin. This was negated previously by assuming that all of the other proteases would be inactivated by the acidity of the assay (10). On the contrary, pepsinogen C would be activated and may falsely contribute to reported pepsin levels. With more research, pepsinogen C levels at baseline and in disease may be described. A cutoff level may then be used to sort out its likely gastric or tracheal origin.
Pepsinogen C is involved in the proteolytic processing of surfactant protein B (13). Its expression occurs concomitantly with or in advance of surfactant protein B synthesis. The similarities in function between pepsinogen C and pepsinogen A may explain the high-pepsin prevalence in studies by Farhath et al (2) and Elabiad et al (11). Because both used the enzymatic method to indirectly measure pepsin, both could have measured lung pepsinogen C activity in addition to an unknown volume of pepsin A. This also may explain the findings of other studies in which a causal association was suggested between GERD and lung disease. The enzymatic pepsin detection assay was used to show an association between reflux and BPD (9) along with reflux and acute lung rejection (7). In infants with evolving BPD, surfactant turnover has been shown to increase (17). In these patients, pepsinogen C production and activity, as the molecule involved in surfactant protein B processing, would be expected to increase with surfactant turnover. There could well be an association between aspiration and development or worsening of BPD or aspiration asthma, but measuring pepsin by the enzymatic method actually could be measuring byproducts of higher surfactant turnover in an otherwise ailing lung.
Starosta et al (18) reached comparable conclusions when they found significant amounts of bronchoalveolar lavage fluid pepsin in some of the children without detectable proximal reflux. They also measured bronchoalveolar lavage fluid pepsin using the enzymatic method. They theorized that pepsinogen C produced by type II pneumocytes could have reduced the specificity of their assay.
The immunologic method most commonly uses an enzyme-linked immunosorbent assay to detect pepsin; however, the sensitivity and specificity of the method depend largely on the affinity and the specificity of the antibodies used. In our study, we used monoclonal antibodies to detect pepsinogen A and pepsinogen C. Using pepsinogen A antibody, the absence of pepsinogen A in the lung and its presence in the stomach means only that this particular pepsinogen A is not produced in the lung. Others have stained for pepsin A and showed that it is present in the upper airway in laryngeal and subglottic tissues (19), probably secondary to postaspiration endocytosis.
Pepsinogen C was not detected in lung tissue samples from infants with a gestational age <23 weeks. Our findings are similar to those published by Foster et al (12), in which pepsinogen C was not detected in any of 10 samples with a gestational age <23 weeks. This information delineates more the timing of pepsinogen C production and its role in the final stages of surfactant processing. We had 4 cases in which pepsinogen C was not detected, which is most likely associated with postmortem changes. Cases 11 and 13 were both born at 24 weeks’ gestation and with birth weight of 580 and 786 g, respectively. In cases 1 and 7, the majority of pneumocytes was detached and had accumulated in the air spaces. In the other cases, the architecture and cytology was well preserved. In the stomach, pepsinogen C was detected as early as 21 weeks’ gestational age.
To summarize, because of the production of pepsinogen C in the lung, the enzymatic method for pepsin detection in the lung may give clinically inaccurate conclusions about the evaluation of GERD. With the availability of antibodies with higher affinity and specificity to the different pepsinogen and pepsin molecules, the immunologic method should be the method of choice or even combined with the enzymatic method to investigate the pathophysiology and relation between gastroesophageal reflux and lung disease. We suggest that future studies report levels of both pepsinogen A and pepsinogen C in their target sample.
1. Metheny NA, Clouse RE, Chang YH, et al. Tracheobronchial aspiration of gastric contents in critically ill tube-fed patients: frequency, outcomes, and risk factors. Crit Care Med
2. Farhath S, Aghai ZH, Nakhla T, et al. Pepsin, a reliable marker of gastric aspiration, is frequently detected in tracheal aspirates from premature ventilated neonates: relationship with feeding and methylxanthine therapy. J Pediatr Gastroenterol Nutr
3. Metheny NA, Chang Y-H, Ye JS, et al. Pepsin as a marker for pulmonary aspiration. Am J Crit Care
4. Gopalareddy V, He Z, Soundar S, et al. Assessment of the prevalence of microaspiration by gastric pepsin in the airway of ventilated children. Acta Pædiatr
5. He Z, O’Reilly RC, Bolling L, et al. Detection of gastric pepsin in middle ear fluid of children with otitis media. Otolaryngol Head Neck Surg
6. Ozmen S, Yucel OT, Sinici I, et al. Nasal pepsin assay and pH monitoring in chronic rhinosinusitis. Laryngoscope
7. Stovold R, Forrest IA, Corris PA, et al. Pepsin, a biomarker of gastric aspiration in lung allografts: a putative association with rejection. Am J Respir Crit Care Med
8. Ward C, Forrest IA, Brownlee IA, et al. Pepsin like activity in bronchoalveolar lavage fluid is suggestive of gastric aspiration in lung allografts. Thorax
9. Farhath S, He Z, Nakhla T, et al. Pepsin, a marker of gastric contents, is increased in tracheal aspirates from preterm infants who develop bronchopulmonary dysplasia. Pediatrics
10. Krishnan U, Mitchell JD, Messina I, et al. Assay of tracheal pepsin as a marker of reflux aspiration. J Pediatr Gastroenterol Nutr
11. Elabiad MT, Reynolds AM, Ryan RM, et al. Tracheal pepsin activity in intubated very low birth weight infants (abstract) Pediatric Academic Societies Meeting; 2006:5561.362.
12. Foster C, Aktar A, Kopf D, et al. Pepsinogen C: a type 2 cell-specific protease. Am J Physiol Lung Cell Mol Physiol
13. Gerson KD, Foster CD, Zhang P, et al. Pepsinogen C proteolytic processing of surfactant protein B. J Biol Chem
14. Gritti I, Banfi G, Roi GS. Pepsinogens: physiology, pharmacology pathophysiology and exercise. Pharmacol Res
15. Kageyama T. Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development. Cell Mol Life Sci
16. Wenzl TG, Schenke S, Peschgens T, et al. Association of apnea and nonacid gastroesophageal reflux in infants: investigations with the intraluminal impedance technique. Pediatr Pulmonol
17. Kimberly LS, James CZ, Bruce WP, et al. Substrate utilization and kinetics of surfactant metabolism in evolving bronchopulmonary dysplasia. J Pediatr
18. Starosta V, Kitz R, Hartl D, et al. Bronchoalveolar pepsin, bile acids, oxidation, and inflammation in children with gastroesophageal reflux disease. Chest
19. Johnston N, Wells CW, Samuels TL, et al. Rationale for targeting pepsin in the treatment of reflux disease. Ann Otol Rhinol Laryngol