Total Trypsin and Active Trypsin: could they be used as a Biomarker for Duodenal Reflux

Introduction: Extra-gastric duodenal reflux carries proteolytic enzyme trypsin to upper aero-digestive and lower respiratory tracts and so trypsin could be a reflux biomarker. The activity of trypsin, however, can be altered by gastric acidity and pepsin. This study aimed to measure trypsin in gastric juice of reflux patients and to analyse the eligibility of trypsin as a biomarker for duodenal reflux. Methods: Active trypsin was measured in 80 gastric juice samples, collected during gastroscopy, using a modified N-terminal assay. Total trypsin was also measured by a sandwich ELISA developed in our laboratory and compared with trypsin activity. The effect of gastric acidity and pepsin as well as bile salts on trypsin activity was studied in vitro. Results: Active and total trypsins were detected in 80% of gastric juice samples. Active trypsin progressively and irreversibly lost its activity when incubated with pepsin at pH 2 for 6 hour whereas total trypsin reactivity was reduced by only 25%. The content of active and total trypsin was not altered by incubation with bile salts at pH 7.4. Discussion: Refluxed trypsin is likely to be partially or completely inactivated by the low stomach pH and pepsin and this could invalidate the use of active trypsin as a reflux biomarker. Total trypsin is not significantly altered by gastric conditions and could then represent a more valid marker for duodenal reflux. The sensitivity of sandwich ELISA, however, needs to be improved to detect extra gastric trypsin. Conclusion: Total trypsin is a more stable parameter that, with refined techniques, could be used as a biomarker for duodenal reflux. Abbreviations: DR: Duodenal Reflux; TNBS: Trinitrobenzenesulfonic acid; SA: Succinyl Albumin; SDS: Sodium Dodecyl Sulphate; RC: Routine clinical; LTx: Lung Transplant; GJ: Gastric Juice; BALF: Broncho-Alveolar Lavage Fluid Glob J Otolaryngol 3(3): GJO.MS.ID.555611 (2017) 001 Global Journal of Otolaryngology How to cite this article: M S Ali, Shruti P, Jeffrey P P. Total Trypsin and Active Trypsin: could they be used as a Biomarker for Duodenal Reflux. Glob J Oto 2017; 3(3): 555611. DOI: 10.19080/GJO.2017. 03.555611. 002 The overall aim of this study was to measure and analyse trypsin contents in gastric juice of a cohort of patients with a variety of upper gastrointestinal diseases and lung transplant patients assessed for anti-reflux surgery. We measured trypsin activity by using a proteolytic activity assay and developed a sandwich ELISA for the measurement of total trypsin protein including its active and inactive forms in gastric juice. The effect of gastric environment and bile salts on trypsin activity was also studied. Materials and Methods Ethical approval was obtained from County Durham & Tees Valley 2 Research Ethics Committee. Fasting gastric juice samples were obtained during gastroscopy from 70 patients with a variety of upper gastrointestinal diseases and 10 lung transplant patients assessed for anti-reflux surgery. Unless stated otherwise, reagents used in this study came from SigmaAldrich UK Ltd., Fisher Scientific UK Ltd. or BDH Merck Ltd. Measurement of active trypsin in gastric juice This was performed at pH 7.4 using a modified colourimetric assay [9] based on the production of new N-terminal amino groups which react with trinitrobenzenesulfonic acid (TNBS) generating trinitrophenyl derivatives that can be measured spectrophotometrically. 500 μl of 8mg/ml succinyl albumin (SA) was added to test tubes each containing 200 μl of sample or standard pig trypsin (0-2.5μg/ml) and tubes were incubated at 37oC for 30 minutes. Proteolysis was stopped by the addition of 500 μl of 4% (w/v) NaHCO3followed by 500μl of 0.05% (w/v) TNBS and 10 minutes incubation at 50oC. 500μl of 10% (w/v) sodium dodecyl sulphate (SDS) was then added followed by 500μl of 1M HCl acid. Absorbance was read at 340nm. Negative controls were made by adding the substrate and immediate addition of NaHCO3. The effect of gastric pH (2.2 and pH 4.0) and pepsin (0.5 mg/ ml) at 37°C on trypsin (2.5 μg/ml) activity was monitored at several time points over 6 hours (extended to 7 days with pH 4.0). The effect of bile salts was also assessed, incubating 2.5μg/ ml trypsin at pH 7.4 overnight with 10 mM of a physiological bile salt mixture containing 50% cholic acid, 30 % chenodeoxycholic acid, 15 % deoxycholic acid and 5 % lithocholic acid [10] dissolved in methanol using trypsin activity at pH 7.4 as a standard in both experiments. Development of ELISA to measure total trypsin in gastric juice In initial experiments we used rabbit polyclonal antibody raised to bovine trypsin (Abcam ab1879) as the primary antibody. The large range of dilutions (1:8000 to 1:30000, in 0.1% BSA in PBS) suggested by the manufacturers was tested and we found that the optimal dilution 1:5000 was the best. Native trypsin has a significantly higher reactivity than heattreated trypsin (at 100oC for 5 minutes). However, trypsin levels in gastric juice obtained with this ELISA were too high (~4mg/ ml) to be physiologically possible suggesting interference in the assay. We then tried a mouse anti-trypsin monoclonal primary antibody (Abcam ab17263). However no colour development was obtained. Development of a sandwich ELISA to measure total trypsin in gastric juice We used the same mouse monoclonal antibody as the capture antibody bound to the ELISA plate and the rabbit polyclonal antibody (Abcam ab1879) as the detection antibody. For colour development a secondary (horseradish peroxidase conjugated polyclonal anti-goat, Sigma A6777, UK) antibody was used. The assay sensitivity was improved by increasing the concentration of detection antibody from a 1:5000 to a 1:2000 and this was the final version of sandwich ELISA used to measure total trypsin in gastric juice. 100 μl capture antibody (1:2000 dilution in 0.1% BSA in PBS) was coated onto a 96-well plate (Maxisorp, NUNC), covered and incubated at room temperature for 5 hours. The plate was then aspirated, blotted dry and blocked with 1% BSA in PBS overnight. After aspirating the plate and blotting dry, 100 μl of sample (1:25 dilution) or standards (rang 0-100 ng) made up in PBS were added to the wells and the plate was covered and incubated for 1.5 hours. The plate was then aspirated, washed three times with 0.05% Tween 20 in PBS followed by two changes of PBS and blotted dry. Detection antibody (100 μl, 1:2000 dilutionin 0.1% BSA in PBS) was added to each well and the plate was covered and incubated for 1.5 hours. The wash step was repeated and 100 μl of secondary antibody (1:5000 dilution) was added to each well. The plate was then covered and incubated for 1.5 hours. After washing again 100 μl of substrate solution 2, 2’-Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid was added to each well and the plate was covered and developed for 20 minutes. The reaction was stopped with 100 μl of 1% SDS and the absorbance measured at 405 nm with a plate reader (Bio-Tek EL808). Negative controls were made for standards and sample dilutions by omitting the detection antibody and incubating in antibody diluent instead. As previously done for the activity assay, we assessed if the reactivity of total trypsin to the antibody could be altered by gastric acidity, pepsin or bile salts. To test if long incubation times with pepsin at pH 4 could result in further loss of activity the inactivation experiment was extended up to 7 days comparing incubation at 4°C and -20°C, the temperature conditions gastric juice samples would be stored at after collection.