Increased salivary hemoglobin in reflux protects against dental erosion

Objectives: Recently, we reported that gastroesophageal reflux disease (GERD) patients without erosive tooth wear (ETW) have increased concentrations of hemoglobin (Hb) in the acquired enamel pellicle. The present study compared the proteomic profile of saliva in volunteers 1) with GERD) and (ETW) (BEWE≥9; GE); 2) with GERD without ETW (BEWE=0; GNE) and 3) control (without GERD and BEWE=0; C). Additionally, a proof-of-concept in vitro study was conducted to evaluate the impact of hemoglobin (Hb) against in vitro initial erosion.
Methods: Unstimulated saliva was collected from patients from GE, GNE and C groups (8/group). Proteomic analysis was performed using label-free quantitative LC-ESI-MS/MS. In the in vitro study, bovine enamel specimens (n=15/group) were immersed for 2h at 37°C in different solutions: deionized water (negative control), 4mg/mL Hb, 2mg/mL Hb, 1mg/mL Hb, 0.1mg/mL Hb or 0.01mg/mL CaneCPI-5 (positive control). Specimens were then exposed to stimulated human saliva for 2h at 37°C to form the AEP and then incubated in 0.01M HCl solution (pH 2.3) for 10s. Specimens were treated once/day over 3 days and analyzed by surface hardness change. Data were analyzed by ANOVA and Tukey′s test (p〈0.05)
Results: GNE compared with GE volunteers had remarkable increase in distinct subunits of Hb (up to 22-fold). Other increased proteins were Serum albumin (4.5 fold) and isoforms of Cytoskeletal keratin type II (more than 3-fold). Proteinase inhibitors, such as Alpha-1-antitrypsin and Alpha-2-macroglobulin, were exclusively identified in the GNE group. In the in vitro study, Hb at 1.0 and 4.0 mg/mL significantly reduced initial enamel erosion after 3 days.
Conclusions: Our results indicate many proteins involved in the resistance of GERD volunteers to the occurrence of ETW, with especial emphasis on Hb, as confirmed in the in vitro study. Furthermore, these proteins are particular candidates to be included in dental products to protect against ETW.