Krishnan, Usha*; Mitchell, John D.†; Messina, Isabella‡; Day, Andrew S.§; Bohane, Timothy D.∥
Pulmonary aspiration is a recognized and important complication of gastroesophageal reflux (GER) in children (1,2) and adults (3,4). Current techniques for assessing reflux, such as esophageal pH monitoring and esophagoscopy with esophageal biopsy, identify a group of patients considered at increased risk of pulmonary aspiration, but obtaining proof of such aspiration remains elusive. Clinical history, pattern of radiologic abnormalities on repeated chest radiographs, and observation of tracheobronchial aspiration during barium contrast studies may suggest reflux aspiration, but these investigations lack sensitivity and specificity (5). Gastric scintiscan has also proven insensitive (6,7), and the lack of specificity of fat-laden macrophages in tracheal aspirate limits the clinical value of this method of assessment (8,9).
Because gastric pepsin is absent from respiratory secretions (10), we surmised that the finding of this enzyme in tracheal aspirate would be a marker for pulmonary aspiration of gastric contents. This study assesses pepsin activity in tracheal aspirates to identify reflux aspiration in infants and children undergoing endotracheal intubation, comparing the assay result against the perceived likelihood of aspiration suggested by a history of clinically significant reflux and/or chronic respiratory symptoms.
MATERIALS AND METHODS
Ninety-eight infants and children undergoing general anesthesia requiring endotracheal intubation at Sydney Children's Hospital were recruited in this prospective case-controlled study. Of the 98 children, 84 were children undergoing elective or emergency gastrointestinal endoscopies over a 12-month period. Of these 84 children, 64 were being assessed or reassessed for clinically significant GER, and the remaining 20 for a variety of other reasons (inflammatory bowel disease, n = 12; bleeding per rectum, n = 4; foreign body removal, n = 2; and celiac disease, n = 2). The remaining 14 study subjects were undergoing anesthesia for elective cardiac surgery (n = 8), ear/nose/throat surgery (n = 4), orthopedic surgery (n = 1), or general surgical procedures (n = 1).
All children were allocated before anesthesia to one of four clinical groups depending on presence or absence of clinically significant reflux (GER+ or GER−, respectively) or chronic respiratory symptoms (RS+ or RS−, respectively), (Tables 1 and 2). A questionnaire modified from Orenstein's validated Infant Gastroesophageal Reflux Questionnaire (11) was used to determine the presence of clinically significant reflux. Children were categorized as having “chronic respiratory symptoms” based on the presence of persistent cough and/or asthma for more than 3 months or recurrent episodes of “bronchitis” or pneumonia. A history of chronic respiratory symptoms was obtained in 45 of the 98 children (Table 2). Details of previous investigations (chest radiograph, barium meal, pH probe, and/or endoscopy) were completed with the help of the parent or guardian and patient records (Table 3). The timing of investigations before the date of collection of tracheal aspirate samples varied from 1 week to, on one occasion, 2 years (this was a pH probe study in a neurologically delayed child with symptoms of reflux). However, the median time between the timing of investigations and the collection of tracheal aspirate was 3 months.
Reflux esophagitis was judged to be present if the basal layer of esophageal squamous cells was greater than 25% of the total epithelial thickness, if papillary height was greater than 50% of epithelial thickness, and if eosinophils or ulcerations were present. Children were categorized as having chronic respiratory symptoms based on the presence of persistent cough and/or asthma for more than 3 months or recurrent episodes of “bronchitis” or pneumonia. Two children with difficult-to-treat cystic fibrosis lung disease were included in this group as they had both reflux and respiratory symptoms.
The Research and Ethics Committee of the South Eastern Sydney Area Health Service approved the study, and informed consent was obtained from a child's parent or guardian before inclusion in the study.
Tracheal aspirates obtained from the same group of patients was also assayed for fat-laden macrophages to reevaluate their role in diagnosing reflux aspiration (data to be published as a separate study).
Collection of Tracheal Aspirates
After induction of anesthesia by mask, venous access was obtained. Patients were then paralyzed with nondepolarizing muscle relaxants and intubated. Time between commencement of induction and intubation varies between 3 and 15 minutes, usually dependent on venous access. After intubation and before the start of the procedure or operation, an appropriately sized “Y” suction catheter, with one limb connected to a small sterile mucous trap, was passed through the endotracheal tube into the trachea proximal to the carina. Two milliliters of normal saline was instilled through the catheter and then rapidly suctioned into the mucous trap. The aspirate sample was immediately frozen and stored at −80°C. The amount of tracheal aspirates collected ranged from 500 μL to 1 mL, with a median volume of 750 μL.
Pepsin assay on tracheal aspirates was performed by one of the authors who was blinded to the clinical grouping of the patients. A modification of the proteolytic enzyme assay method of Twining (12) was used. A 0.5% wt/vol solution of fluorescein isothiocyanate–labeled casein (FITC casein) was prepared in 50 mmol/L sodium acetate (pH 5.3). Aliquots of 100 μL of tracheal aspirate were acidified to pH 1–2 by addition of 5 μL 1 mol/L HCl for 15 minutes at 4°C to inactivate lysosomal acid hydrolase (cathepsin D) and convert pepsinogen to active pepsin (13). The pH was then readjusted to 5.3 with 95 μL 1 mol/L sodium acetate (pH 5.8). Fifty microliters of the aspirate solution was added to polypropylene microcentrifuge tubes containing 20 μL of 50 mmol/L sodium acetate buffer (pH 5.3) and 20 μL FITC casein solution, thoroughly mixed and incubated for 3 hours at 37°C in a shaking water bath. The reaction was then stopped by addition of 5 μL of 20% wt/vol pepstatin A in dimethylsulphoxide, then 30 μL 20% trichloroacetic acid and 90 μL distilled water. Protein precipitates at 4°C during overnight standing, and the trichloroacetic acid insoluble fraction was sedimented by 5-minute centrifugation at 7,000 rpm in microfuge 12 (obtained from Beckman Instruments Pty Ltd, Bladesville, New South Wales, Australia). After mixing 680 μL of 500 mmol/L Tris buffer (pH 8.5) with 120 μL of supernatant, the relative fluorescence was measured in a spectrofluorometer (LS50B; Perkin Elmer, Gladesville, Sydney, Australia) at excitation 490 nm and emission 525-nm wavelength. Each assay was performed in duplicate with appropriate substrate blanks and enzyme controls. The interassay coefficient of variation was 5% for porcine pepsin and 16% for tracheal aspirate and human gastric juice. The intraassay coefficient of variation was 2.5% for porcine pepsin A and 7% for tracheal aspirate samples. In vitro studies on tracheal aspirates showed no significant decrease in peptic activity when held at 37°C for 3 hours or when stored frozen at −80°C for 6 months.
Assay of serial dilutions of porcine pepsin A (Lot 84M100, Sigma-Aldrich Pty, Castel hills, New South Wales, Australia) was performed to complete a standard curve. This was then used to convert fluorometric units to nanograms of pepsin. The arbitrary point delineating positive from negative assays of pepsin in tracheal aspirates was chosen to be at the lower limit of sensitivity of the assay, 12.5 ng/50 μL of sample assayed. A fluorometric assay (14) of pooled pepsin-positive tracheal aspirate samples did not detect any duodenal trypsin.
FITC casein (type III) and porcine pepsin A (Lot 84M100) were obtained from Sigma-Aldrich Pty.
The results were analyzed using the χ2 test, Student t test, and Fisher exact test. Probability statistics were calculated using SPSS statistical software (SPSS Inc., Chicago, IL).
The clinical details of the children involved in the study are shown in Tables 1and 2, and the findings of previous investigations relating to GER and/or aspiration, where performed, are shown in Table 3. The Tracheal pepsin assay results are given in Figure 1. There was a significant difference when we looked at the prevalence of tracheal pepsin in the clinical groups: GER+ versus GER− (P = 0.00025); GER+RS+ versus GER+RS−; GER−RS+ versus GER−RS−; GER+RS+ versus GER−RS− (in all 3 cases, P < 0.0001).
Tracheal pepsin was conspicuously absent in children without a history of significant GER or respiratory symptoms and therefore considered least likely to aspirate. The number of children who were positive for tracheal pepsin was low (7 of 27) in the group with GER symptoms alone (26%) and much higher (31 of 37) in both those with GER and respiratory symptoms (84%) and in those (7 of 8) with respiratory symptoms alone (87.5%). Seventy-six percent of 25 asthmatic patients in this study had positive results for tracheal pepsin.
Figure 2 shows the quantity of pepsin detected in individual aspirates. The pepsin level was higher (mean pepsin level in relative fluorescent units ± SEM) in children with GER and respiratory symptoms (354 ± 72) and the group with respiratory symptoms alone (282 ± 105), than the group with GER alone (20 ± 10, P < 0.0001).
In Table 4, the findings of previous investigations for assessing GER and/or aspiration of children in the study are correlated with their tracheal pepsin results. There were no differences (P > 0.05) in either the prevalence or the quantity of pepsin detected between the children either with or without esophagitis or GER seen during a barium study. However, the frequency and amount of pepsin was higher when children in either of the preceding groups also had respiratory symptoms. All 27 of the 64 children in the GER+ group who underwent a 24-hour pH probe were considered to have parameters outside the reference range, but those who also had respiratory symptoms were more likely to have pepsin-positive results than those without (P = 0.04).
Nine of 35 children in whom reports of a chest radiograph were available had changes consistent with pulmonary aspiration. All nine had symptomatic reflux and recurrent lower respiratory infections (Table 2), and all except one were positive for tracheal pepsin. The child with the negative result was neurologically developmentally delayed and had been noted to directly aspirate barium from the pharynx during swallowing, suggesting direct aspiration as a cause of this child's respiratory problems.
Pulmonary aspiration is a common cause of acute and chronic pulmonary disease and is a known cause of bronchospasm (15,16). There is evidence that gastric reflux into the esophagus may produce reflex bronchoconstriction both by a direct effect of acid and perhaps also by distension (17). However, the clinical significance of reflex bronchoconstriction caused by acid is in doubt, as there are conflicting reports on the effects of gastric acid suppression by omeprazole on lung function in adult asthmatic patients (18,19). A metaanalysis of all studies on the effects of medical antireflux therapy on asthma control suggest that, although asthma symptoms may be improved and asthma medication use decreased, there is little or no effect on lung function (20). Alternatively, intratracheal acidification during simultaneous esophageal and tracheal pH monitoring has been shown to produce a significant decrease in peak expiratory flow rate in asthmatic patients with reflux (21,22). This suggests that microaspiration of acid gastric contents is of potential importance as a cause of both chronic and recurrent pulmonary disease, including asthma.
The need for a simple and reliable test in clinical practice for accurately identifying aspiration of refluxed gastric material is clear. The detection of fat-laden macrophages in aspirated material from the trachea, although used for decades as a marker of reflux aspiration, has unacceptable specificity and sensitivity (8,9), except for reflux aspiration associated with severe lung disease (9). Simultaneous esophageal and tracheal pH monitoring is a recently reported technique for detecting acid aspiration (20,21). The technical difficulty of this procedure is likely to limit its routine clinical use and its utility in serial monitoring of reflux aspiration. The frequent use of medications inducing gastric acid suppression, and the buffering of gastric pH associated with frequent or continuous feeding techniques, are also factors that limit the usefulness of acid detection within the airways for diagnosing reflux aspiration. Detection of lactose in tracheal aspirates has been used to diagnose GER aspiration in ventilated and nasogastrically fed neonates (23). However, this and other methods (24) that rely on identifying the aspiration of specific food substances are likely to give false-negative results, especially after fasting, during sleep, and in the period leading up to the next meal, when the stomach is likely to contain gastric juice alone.
Although esophageal pH probe monitoring, barium contrast studies, and endoscopic grading of esophagitis are used to gauge the risk of pulmonary aspiration, these tests primarily assess esophageal events only. Conclusions made regarding an association between GER and aspiration using these methods usually are assumptions, since an alternative mechanism of direct aspiration from the oropharynx because of neurologic or anatomic abnormalities may be the sole or major cause of the problem. Intratracheal pepsin would be a more reliable and direct marker of aspiration of gastric material than methods relying on quantitation of GER. The reliability of pepsin as a marker for detecting reflux aspiration has been suggested in an animal study (25). These investigators were only able to detect peptic activity in bronchoalveolar lavage fluid after induced experimental gastric juice aspiration, but not after induced aspiration of normal saline in rabbits.
An association between GER and asthma (26), postulated for some years, has been confirmed by pH probe studies, especially those using simultaneous proximal and distal esophageal pH monitors (27,28). A direct link between microaspiration and asthma has also been shown by the intratracheal pH probe technique (20,21). The present study supports this association, as 76% of 25 asthmatic patients had positive results for tracheal pepsin.
The present study aimed to correlate the presence of pepsin in the tracheal aspirate with a history of GER and/or respiratory problems. The assay of fat-laden macrophages in tracheal aspirates, currently used for diagnosing GER aspiration, has an unacceptable false-positive rate (8), and it was therefore reassuring in the present study to find no tracheal pepsin in any of the 26 children with negative results for both GER and respiratory problems, supporting the validity of the procedure. Children with GER symptoms alone had a lower frequency of pepsin detection and a lower concentration of the enzyme in tracheal aspirates than that found in either the children with a combination of GER and respiratory symptoms or those with chronic respiratory symptoms alone. Thus, a respiratory history, alone or in combination with symptomatic GER, appears more likely to be associated with reflux aspiration in childhood than when there is a history of symptomatic GER only.
Currently in clinical practice, an association between reflux aspiration and asthma is suggested by the presence of reflux symptoms or abnormal reflux parameters (29) on esophageal pH monitoring. However, the occurrence of reflux microaspiration is not excluded by the observation of normal frequency of reflux on pH monitoring, since even infrequent episodes, within the normal range, may reach the laryngopharynx, especially at night (30). The assay of tracheal pepsin in such situations may have particular diagnostic and therapeutic value. Conversely, the absence of tracheal pepsin in the presence of respiratory symptoms and proven excessive GER may discourage surgical intervention, particularly in neurologically developmentally delayed individuals who may also directly aspirate during swallowing. One neurologically developmentally delayed child in this study with a history of recurrent aspiration pneumonia and who was orally fed had negative results for tracheal pepsin and improved (did not have any further episodes of pneumonia) subsequent to the placement of a feeding gastrostomy for bolus feeds without fundoplication, suggesting that the pepsin assay helped in distinguishing between reflux aspiration and direct aspiration caused by uncoordinated swallowing.
The known association between “silent reflux” (pH probe proven excessive GER with respiratory symptoms only) and bronchopulmonary aspiration (31) is confirmed in the current study, where a high rate of detection of tracheal pepsin was found in the small group of eight children with a respiratory history alone. Because seven of the children in this group were older than 4 years, we surmise that achievement of the upright posture progressively modifies the presentation of reflux aspiration from the frequent regurgitation and vomiting seen in infancy to one of “silent reflux” in older children.
Casein, the substrate used in this study to assay pepsin, although not a pepsin-specific substrate, has commonly been used for assay of the enzyme (13). Other nonpepsin proteases likely to be found in the normal lung, which during appropriate conditions might be detected using FITC casein as substrate, include matrix-degrading metalloproteases (MMPs), serine and cysteine proteases, and lysosomal acidic protease cathepsin D (10). In our assay, cathepsin D (13), cysteine protease cathepsin B, and serine protease trypsin are inactivated by acidification. The pH of the reaction mixture (pH 5.3) is well below the reported optimum for the activity of MMPs (32,33), and our assay does not include the activators (calcium and detergent); consequently, the likelihood of their inadvertent detection in tracheal aspirates is remote. The MMP inhibitor EDTA failed to inhibit the peptic activity of acidified tracheal aspirates using our assay. This suggests that there is very little or no MMP activity detected by our assay method. Similarly, serine and cysteine protease inhibitors antipain and leupeptin had no effect on enzyme activity measured in acidified tracheal aspirates by our assay, whereas commercial trypsin and cathepsin B were both inactivated by these inhibitors, suggesting the presence of little or none of these proteases in acidified tracheal aspirates. However, the aspartic protease inhibitor pepstatin inhibited the peptic activity of acidified tracheal aspirates to a similar degree to that found for human gastric juice.
The sensitivity of the assay is of importance because the volume of tracheal aspirate can vary considerably with very small quantities being obtained, especially in small infants. Twining (12) found the sensitivity of FITC casein for the assay of various proteases to equal that of radioimmunoassay methods, and this substrate in the present study proved capable of assaying pepsinlike activity in microliter samples of tracheal aspirates.
Pepsin in human gastric juice has been shown to be stable at pH 5.3 (34), and when stored frozen at −20°C for up to 6 weeks (35) and for 6 months when stored at −80°C. The substrate used for pepsin assay is commercially available, inexpensive, and stable to storage, and the assay itself can be performed on multiple stored specimens and requires little more than a microfuge and spectrofluorometer as equipment.
This assay currently requires endotracheal intubation of the patient for the collection of tracheal aspirate. Although tracheal intubation for a general anesthetic is standard procedure in our unit for endoscopy procedures, this would not occur if endoscopy was performed during sedation of the patient, as is the case in many pediatric gastroenterology units. Significant modification of the methods will be required if the procedure is to be performed other than by sample collection via endotracheal tube passed after induction of general anesthesia.
In conclusion, this study suggests that the assay of pepsin in tracheal aspirate may be a reliable marker of reflux aspiration. Tracheal samples are easily collected and stable on storage. The enzyme assay is inexpensive and easily performed on multiple specimens. Intratracheal pepsin, in combination with 24 hour pH probe and endoscopy, may allow a more reliable assessment of the relation between GER and chronic respiratory symptoms, and a more objective approach to the pharmacologic and surgical management of reflux disease.
The authors thank Dr. Keith Kelly (Department of Anesthesia, Sydney Children's Hospital) and Associate Professor Alan Stark (Department of Statistics, University of New South Wales).
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