Introduction 1 2 4 5 6 7 8 9 10 13 14 16 The aim of the present study was to analyze data of pH and bile monitoring in a collective of healthy age- and gender-matched controls and patients with Barrett’s esophagus. Subjects and Methods Subjects Selection of controls was carried out according to a strict protocol. Healthy volunteers treated from 1999 to 2000 between the ages of 40 and 60 years were included in the study. None of the controls were on acid suppressing or gut motility medications, had a history of upper gastrointestinal disease, had undergone upper or major abdominal surgery, or had had therapeutic endoscopic procedures of the upper gastrointestinal tract. Diagnostic endoscopy and barium swallows were not performed, but gallstone disease was excluded by ultrasound scan. From 1999 to 2002, 24 patients with histologically confirmed Barrett’s mucosa were included in the study. For additional comparison, we include a group of patients with esophagitis (stage I to III according Savary and Miller) without Barrett’s esophagus, which had the same diagnostic procedures before planned laparoscopic fundoplication. During the aforementioned time span, 21 patients age older than 40 years were available for this study. Exclusion criteria were history of esophageal, gastric, or biliary surgery, history of abdominal or thoracic radiotherapy, or presence of peptic ulcer disease, active gastrointestinal bleeding, esophageal or fundic varices, esophageal or upper small intestine chronic disease, or neoplastic disease. All drugs potentially affecting gastrointestinal motility and secretion were discontinued at least 1 week before the study. Upper Gastrointestinal Endoscopy All patients underwent classical upper gastrointestinal endoscopy. If sedation was necessary, intravenous administration of propofol (up to 200 mg) was normally used, or occasionally, midazolam (up to 5 mg) was used. During endoscopy, the presence and extent of esophagitis, Barrett’s esophagus, and hiatal hernia was noted. Biopsies were taken from the Barrett’s mucosa. Ambulatory Esophageal/Gastric pH and Bile Monitoring All groups underwent esophageal manometry and 24-h pH and simultaneous bile monitoring using a standardized protocol. Ambulatory pH monitoring was performed using a transnasally inserted antimony pH electrode with a separate skin reference electrode (Synetics Medical, Stockholm, Sweden). The data were stored on a portable digital recorder (Digitrapper MkIII, Synectics Medical Stockholm, Sweden). Before each study, the pH probe was calibrated in buffer solutions of pH 7 and 1. An episode of acid reflux was defined as a decrease in esophageal pH to less than 4 for more than 10 s. 14 17 1 Figure 1 Position of the pH- and bilirubin-probes in the stomach and in the esophagus. (UES = upper esophageal sphincter; LES = lower esophageal sphincter).  The simultaneous biliary and pH monitoring was done with administration of a colorless “white diet” (WD) including liquid and solid foods with a maximum in vitro bile absorbance of 0.25 [absorbance scale ranging from 0 (plain water) to 1 (total screen)]. The meals included water, milk, toast, potatoes, chicken, dry biscuits, and fish. Data Acquisition and Interpretation After completion of the measurements, probes were withdrawn from the patients, and data were stored via interface on an IBM-compatible computer equipped with Polygram® software (Medtronic). The data of each second of the 24-h measurements were used for analysis. To assess the presence of gastric or esophageal biliary reflux, the percentage of time when absorbance was greater than 0.25 was calculated for the following periods: total supine, upright, and postprandial. The postprandial period was defined as 2 h after the end of meals. The percentage of time with esophageal pH lower than 4 and median gastric pH and the percentage of time with gastric pH measuring 1, 2, 3–7, and >7 was also calculated for the above periods. The mean duration of the ambulatory pH and Bilitec monitoring study was 22 h, 40 min in patients and 23 h, 44 min in the controls. Statistical Analysis The SPSS (version 11.0, Chicago, Illinois) program was used to analyze the results. For graphical presentation, we used the program MedCalc for Windows, (Version 9.0, MedCalc Software, Belgium). Median, interquartile range (IQR or 25th to 75th percentile) values were established. The nonparametric tests (Mann–Whitney and Kruskal–Wallis analysis) were used to assess the relationship between variables. Box and Whisker plots were used to present some of the data. In these plots, the box represents the IQR, and the Whiskers represent the highest and lowest values. Outliers are also plotted, defined as more than 1.5 times the IQR from the 75th centile. Extreme values were defined as more than three times the IQR from the 75th centile. 18 The assumptions for calculation of the required sample size were alpha = 0.05, beta = 0.80, and that a test is only valid for daily use if less than 20% of the healthy controls and at least 80% of the patients have positive test results. The calculated sample size for each group was 20. Ethics The study protocol was approved by the ethics committee of the University of Cologne. Each subject gave written informed consent. Results 1 Table 1 Demographic Data of Patients with Barrett-Mucosa or Esophagitis and Healthy Volunteers Parameters n n n Significance Pat. with Barrett vs Controls Age (median)  57 years 58 years 51 years – Min–max 29–75 years 42–77 years 39–62 years  Gender m:f 16:8 11:10 11:8 n.s. BMI (median) min–max 2 2 2 p Smokers (%) n n n n.s. No alcohol % n n n n.s. BMI Acidic Gastric Reflux (AGR) p p 2 Table 2 Median of Acidic Gastric Reflux into the Esophagus in Patients with BM or with Esophagitis and in Healthy Controls Parameters n n n Significance Pat. with Barrett vs Controls Percentage of total measuring time pH < 4 (%) 10.6 (6.2–38.3) 19.9 (1.6–71.7) 3.2 (0.9–5.5) p Percentage of upright measuring time pH < 4 (%) 11.7 (6.03–6.4) 18.9 (8.7–60.8) 2.4 (0.9–6.1) p Percentage of supine measuring time pH < 4 (%) 10.9 (0.4–27.1) 6.3 (0.0–13.3) 0.3 (0.0–4.2) p LQ UQ 24-h Intragastric pH and Bile Monitoring 3 3 2 Table 3 Results of 24-H Intragastric pH and Bile Monitoring in Patients with Barrett Esophagus and Healthy Controls Parameters n n Significance Median of intragastric pH during TMT 1.3 (1.0–1.4) 1.4 (1.1–1.7) n.s. Bilirubin exposure percentage (%) of TMT  7.8 (1.6–17.8) 0.0 (0.0–1.0) p Bilirubin exposure percentage (%) of upright time 6.9 (0.1–12.9) 0.0 (0.0–1.3) p Bilirubin exposure percentage (%) of supine time 2.0 (0.0–28.6) 0.0 (0.0–0.0) p LQ UQ TMT Figure 2 24h intragastric pH- and bile monitoring in a patient with Barrett’s esophagus demonstrating the duodenogastric reflux in the early morning. a. Bilitec®-monitoring, b. pH-monitoring.  Bilirubin Exposure of the Esophagus p 3 Figure 3 Results of the esophageal bile-monitoring in 24 patients with Barrett’s esophagus, 21 patients with esophagitis and 19 healthy controls a) total measuring period (Kruskal-Wallis Test = 0.01) b) supine period (Kruskal-Wallis Test p = 0.01).  4 Figure 4 ROC-curve with 95% confidence intervals for pathologic bile-monitoring in patients with Barrett esophagus compared to age and sex matched healthy controls.  p Discussion The results of our study confirm that patients with Barrett’s esophagus have significantly more frequent duodenogastric reflux into the esophagus than age- and sex-matched healthy controls. In addition, this reflux, measured by acid and bilirubin exposure, remains longer in the esophagus, especially during sleep. 13 19 20 21 In our study, the control group of patients with different grades of esophagitis showed no significantly different measurements of acidic or bile reflux into the stomach or the esophagus compared to Barrett’s patients. This may be caused by selection of patients with esophagitis, which were candidates for fundoplication, but both groups of patients differed significantly compared to healthy controls. Therefore, our results are of great clinical relevance especially for preoperative diagnostic. 22 14 23 6 8 10 13 24 17 25 27 16 28 29 Although about 30% of the healthy controls showed acid reflux in pH monitoring, patients with BM had significantly more acid reflux during all measured periods. Healthy controls did not have relevant duodeno-gastric-esophageal reflux measured by bilirubin absorbance. Especially during the supine period, there was no bile reflux. The optimal threshold for pathological bile reflux is 1.1 % (bile monitoring with an absorbance of 0.25).