Analysis of selected bile acids in saliva by high-performance liquid chromatography-mass spectrometry as a potential new non-invasive diagnostic method for Barrett ’s esophagus



Year of publication 2019
Type Conference abstract
MU Faculty or unit

Faculty of Medicine

Description Introduction: Barrett’s esophagus (BE) is defined as the replacement of squamous epithelium in the distal esophagus with metaplastic intestinal columnar epithelium and develops because of chronic inflammation resulting from gastroesophageal reflux disease. BE is associated with an increased risk of developing esophageal adenocarcinoma (EAC). Experimental, clinical and immunohistochemical studies show that one of the factors in the pathogenesis of esophageal injury, BE and EAC is duodenal reflux and the associated synergistic damage to the esophagus by bile acid (BA) and gastric acid reflux. Oxidative stress secondary to bile acids (BAs) exposure has been associated with metaplastic degeneration of normal esophageal mucosa into BE cells and eventually EAC. At the current time, a diagnosis of Barrett’s esophagus can only be made using endoscopy and detecting a change in the lining of the esophagus. The definitive diagnosis of Barrett’s esophagus requires biopsy confirmation of the change in the lining of the esophagus. The Prague classification system is universally accepted standardized endoscopic grading system for BE and uses the “C” value as the “circumferential extent” and the “M” value as “maximal extent” of BE above the gastroesophageal junction in centimeters for endoscopic standardization of BE lengths. Aims & Methods: We studied the content of selected BAs in saliva samples in a cohort of 15 patients with endoscopically and histologically proven BE (Prague Classification C: 1-10, M: 2-13) and a control group consisting of10 subjects without known medical history or treatment for GERD or BE at least in the last 5 years. Concentration of major salivary BAs (glycocholic acid, glycodeoxycholic acid, and glycochenodeoxycholic acid) in saliva were analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) detection. Saliva samples were collected from healthy individuals and patients with BE during the day, but all subjects were asked not to eat, drink nor brush their teeth at least 3 hours before sampling. Afterwards were samples stored at -80 °C. Before the measurement saliva samples were let to thaw at room temperature. After that 900 ul of MeOH was added to 300 ul of saliva sample for protein precipitation. This mixture was vortexed, sonicated and centrifugated and 1 mL of the supernatant was transferred to a new vial. Supernatant was dried under stream of nitrogen. Dried sample was re-dissolved in 300 ul of 80% MeOH, vortexed, sonicated and the supernatant was analyzed by HPLC-MS. The analysis was completed under 4 minutes. Results: We compared the concentrations of selected BAs in both groups of patients, and we found significantly increased concentrations of glycochenodeoxycholic acid (p<0.01) in the group of patients with BE. Significantly better statistical differentiation of samples was observed by using principal component analysis, especially in patients with high C and M values of BE length by Prague classification. Conclusion: Analysis of selected bile acids in saliva by HPLC-MS appears to be a potential new non-invasive diagnostic method for detection of Barrett’s esophagus and for estimation of its severity. HPLC-MS is a highly sensitive method to measure low amounts of bile acids in human saliva. The analysis is fast, but the sample preparation is more complicated. Further refinement of this new diagnostic method is needed.
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