Diagnostic Potential of Salivary Bile Acid for Laryngopharyngeal Reflux

Introduction: Reflux monitoring is the reference standard for diagnosing laryngopharyngeal reflux (LPR) and non-invasive diagnostic methods are needed. Gastric refluxate contains bile acids, which can reflux in the oropharynx in LPR and cause symptoms; therefore, measurement of salivary bile acids has potential for diagnosing LPR. The aim of this study was to measure and compare salivary bile acid concentrations across healthy controls and symptomatic patients with or without objective evidence of LPR/GERD. Methods: This prospective study enrolled adults over one year into 3 groups: 1) GERD symptoms: heartburn, regurgitation, and/or chest pain; 2) LPR symptoms: cough, throat clearing, sore throat, and/or dysphonia; 3) healthy volunteers. Participants provided 2 saliva samples for bile acid analysis using ultraperformance liquid chromatography tandem mass spectrometry methods (Charles River Laboratory) and pepsin analysis using Peptest lateral flow device (RD Biomed Ltd.). Symptomatic patients underwent reflux monitoring off acid suppression [acid exposure time (AET) < 4% normal and ≥ 4% elevated]. Primary analysis compared salivary bile acids between healthy volunteers, symptomatic with normal AET (no GERD/LPR), GERD symptoms with elevated AET (objective GERD), and LPR symptoms with elevated AET (objective LPR). Results: 35 participants were enrolled [mean age 47.4 years (SD 18.9), 19 (54%) female]: 10 healthy volunteers, 6 GERD symptoms, 19 LPR symptoms. Of the symptomatic groups: 9 were no GERD/LPR, 5 objective GERD, and 11 objective LPR. Total salivary bile acid concentration was highest in the objective LPR group (24.2nM (SD 24.7)) compared to other groups (no GERD/LPR [3.1 (4.4); P=0.04]; healthy controls [5.8 (6.0)]; objective GERD [7.1 (7.1)]) (Figure 1). Bile acids were elevated ( > 20nM) in 45% (5/11) of the objective LPR group compared to 0% of the other 3 groups (P < 0.01) (Table 1). Among those with LPR symptoms, AET was elevated in 5/5 (100%) with elevated total bile acid versus 6/14 (43%) with normal total bile acids (P=0.04). Total salivary bile acid was 45% sensitive and 100% specific for elevated AET. Conclusion: Salivary bile acid concentration was higher among patients with objective LPR versus other groups and was 100% specific for objective LPR (elevated acid exposure) among patients with laryngeal symptoms. Salivary bile acids are a quantifiable non-invasive biomarker with diagnostic potential for LPR.Figure 1.: Comparing salivary bile acids between symptomatic cohorts and by AET. Panel A compares salivary bile acids between groups. Panel B compares salivary bile acids to AET. Table 1. - Comparing controls, symptomatic patients without pathologic AET and GERD/LPR patients with pathologic AET Controlsn 10 Symptomatic (No GERD/LPR)n 9 Objective GERDn 5 Objective LPRn 11 P-value Demographics Age (years) 37.0 (16.6) 43.3 (14.1) 49.8 (23.6) 59.1 (17.5) 0.04* Male sex 5 (50%) 2 (22%) 4 (80%) 5 (45%) 0.23 Body mass index (kg/m2) -- 23.9 (4.1) 24.2 (2.0) 30.6 (8.1) 0.02‡ Physiologic Testing Acid exposure time (%) -- 1.2 (1.1) 8.2 (2.2) 7.0 (1.7) < 0.01†‡ Total salivary bile acids (nM) 5.8 (6.0) 3.1 (4.4) 7.1 (7.1) 24.2 (24.7) 0.02‡ Elevated total salivary bile acids ( > 20nM) 0 (0%) 0 (0%) 0 (0%) 5 (45%) < 0.01*‡ Total unconjugated salivary bile acids 0.2 (0.4) 0.3 (0.4) 0.6 (0.8) 1.0 (1.2) 0.20 Total conjugated salivary bile acids 2.5 (2.5) 1.2 (2.1) 2.7 (2.5) 10.1 (12.1) 0.03‡ Salivary pepsin (ng/mL) 91.3 (58.7) 97.8 (83.2) 63.2 (78.3) 87.9 (50.1) 0.82 Hiatal Hernia -- 2 (22%) 0 (0%) 3 (30%)n 10 0.68 Esophagitis -- 0 (0%) 0 (0%) 1 (10%)n 10 1.00 Symptoms GerdQ 6.3 (0.7) 8.1 (2.0) 8.4 (1.5) 8.0 (1.8) 0.04 Reflux symptom index score 0.5 (0.7) 17.4 (10.3) 7.0 (2.2) 18.5 (7.9) < 0.01*£¥§ ANOVA/Fisher’s. If P-values <0.05, post-hoc analyses performed and adjusted P-values significant at <0.05 denoted via symbols: *Comparing controls with objective LPR; ¥ Comparing controls with objective GERD; £ Comparing controls with no GERD/LPR; † Comparing no GERD/LPR and objective GERD; ‡ Comparing no GERD/LPR and objective LPR; §Comparing objective GERD and objective LPR.