Effect of pH and Bile Acids on Protease Activity: Implications for Mitigating Peristomal Skin Complications in Ostomy Patients

Each year, over 100,000 ostomy surgeries are performed in the U.S. to enhance the quality of life for those with severe digestive disorders. Peristomal Skin Complications (PSC) are a significant concern in >40% of such patients, often resulting from prolonged exposure to enzymatically active effluent containing pancreatic proteases (trypsin (TRP), chymotrypsin (CTRP), and elastase (ELA)) and unconjugated primary bile acid (BA), like chenodeoxycholic acid (CDCA). Our previous work demonstrated that CDCA induces oxidative stress and cytokine production in human colonic epithelial cells. Further, altered luminal pH and BA profiles, common in inflammatory bowel disease, may worsen skin damage. Based on these findings, we hypothesize that CDCA modulates pancreatic protease activity in a pH- and dose-dependent manner, and thereby may contribute to stoma-associated epithelial damage. Protease activity was measured across a pH range (4.0–8.3) using an Invitrogen EnzCheckTM fluorescence-based assays for TRP, CTRP, ELA individually and in combination. The effect of CDCA (50–250 µM) on protease activity at different pH values were measured in triplicates and then quantified. Enzyme kinetics parameters (Km and Vmax) were analyzed using Michaelis-Menten models and reported as percentages relative to their controls. Human colonic T84 cells were treated with CDCA (25–250 µM) and protease cocktail (10% - 25%; TRP+CTRP+ELA) at varying pH to evaluate cell death and apoptosis via Annexin-V (AV) and propidium iodide (PI) staining. Examining the effect of pH on protease activity revealed that it was pH-dependent, with maximum activity at pH 8.3. Reducing pH resulted in significant activity decreases (pH 8.3: 100%, 7.4: 85%, 6.0: 65%, 5.0: 58%, 4.0: 49%). Protease cocktail consistently exhibited the highest activity at pH 8.3. Next, we identified that CDCA dose-dependently inhibited protease activity, with a pronounced ~70% reduction at pH 4.0 and 250 µM CDCA. At all pH values, the Lineweaver-Burk plot shows that CDCA reduced both Vmax and Km values (>50%) values, indicative of mixed, non-competitive inhibition. CDCA induced dose-dependent apoptotic cell death in T84 cells, which was exacerbated by proteases (e.g., pH 7.4, CDCA 100 µM: 12 ± 4%; CDCA + 10% protease cocktail: 18 ± 4%). Interestingly, this apoptosis was slightly mitigated at lower pH (e.g., pH 5.0, CDCA + 10% protease cocktail: 15 ± 4%). Thus, at acidic pH and physiological dose, CDCA plays a role in reducing enzymatic activity and mitigating cell damage. Our findings suggest that mildly acidic conditions could protect peristomal skin by decreasing protease activity, potentially reducing the incidence and severity of PSC. Incorporating pH-lowering strategies for the peristomal area may serve as a novel approach to mitigate skin irritation and improve quality of life for ostomy patients. Ongoing studies in skin cell models will further elucidate these interactions and inform targeted interventions. Supported by Hollister Inc., Research and Development, University Collaborations Funds to AL, MP, NSSRP Funds, BenU to IH, AF, JS. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.