Primary Motility  Disorders of the  Esophagus
 The Esophageal
 Esophagogastric  Junction

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Volume: Barrett's Esophagus
Chapter: Etiology and origins of Barrett's epithelium

Composition of the refluxate

D. Nehra (Carshalton)

Bile, pancreatic and duodenal secretions all contain potentially injurious constituents. Pepsin is secreted in the gastric juice as an inactive precursor, pepsinogen. It is converted into a pepsin-inhibitor complex and various small peptides spontaneously at pH below 6.0 slowly but almost instantaneously at pH 2.0. At pH below 5.4 the inhibitor dissociates from pepsin. Pepsin is a very acidic protein with an isoelectric point less than pH 1 and an optimum pH of 1.5-2.5. It is stable in acid solution but is rapidly inactivated in neutral or alkaline solutions.

Trypsin is secreted by the pancreas as an inactive precursor trypsinogen which is activated by the enzyme enterokinase, secreted by the intestinal mucosa. The optimum pH for trypsin is in the range 7-9 and it has a low Michaelis constant indicating that the substrate is firmly bound to the enzyme.

Bile acids are a class of endogenous organic anions that are the principal physiologically active constituent of bile. The main functions of bile acids are to aid fat digestion by forming micelles with fatty acids and monoglycerides, and the formation of mixed micelles with phospholipids to permit the excretion of cholesterol in bile. The products of cholesterol metabolism are cholic acid and chenodeoxycholic acid, and these are referred to as the "primary" bile acids. Two "secondary" bile acids are produced in the colon by bacterial dehydroxylation of the primary bile acids to form deoxycholic acid and lithocholic acid. After re-absorption the secondary bile acids are conjugated with either glycine or taurine and join the primary bile acids as components of bile.

The agents involved in the etiology of reflux esophagitis are multifactorial but considerable controversy exists in regard to what ingredient in the refluxing fluid produces mucosal injury. Although evidence that acid plays a significant role remains undisputed, the attention has been focused on the effects of duodenal contents on the esophageal mucosa. Most of the evidence, mainly from esophageal perfusion studies in animal models, supports injury by bile acids [1, 2] though pepsin [3] and trypsin [4, 5] have also been implicated. Damage by bile acid reflux is unique in that the concentrations of bile acids and its toxic effects are known to vary with pH depending on the degree of ionisation. Unconjugated bile acids and glycine conjugates whose pKa values are more than 4 and 6 respectively precipitate in solutions with pH less than 4, whilst taurine conjugates are freely soluble even at pH 2 [6, 7]. Esophageal perfusion studies in animal models have shown that unconjugated bile acids cause mucosal damage selectively in alkaline solutions whereas taurine conjugates were toxic in acidic conditions [8, 9].

The difficulty lies in monitoring "non acid" reflux due to its intermittent nature which involves surges of small volumes of fluid into the esophagus. Direct aspiration of the esophageal contents [10-12] has been attempted to determine the composition of the refluxate however results are conflicting due to the variable duration of study and pooling of aspirates into single aliquot. Detailed bile acid fraction analysis have be done only in two studies show a predominance of the conjugated bile acids, taurocholic and glycocholic [11] or glycine conjugates [12]. These bile acids were found to be significantly higher in the patients with esophagitis and Barrett's but only in the postprandial period. Only in one study [10] was pepsin detected and found to be in higher concentrations in patients with strictures and Barrett's. Trypsin was seldom found.

Spectrum of bile acids in the esophageal refluxate using a real time automated esophageal aspiration technique

A new technique of combined esophageal sampling and pH monitoring with a purpose built automated suction device [13] was developed to obtain bile acid profiles comprising of fourteen individual fractions in patients with varying grades of esophagitis and the concentrations correlated with the esophageal pH. Ten asymptomatic subjects and thirty patients with symptoms of gastroesophageal reflux disease (minimal mucosal injury, erosive esophagitis - Grade 2 or 3 Savary-Miller-, Barrett's/ stricture; n = 10 in each group) underwent 15 hour continuous esophageal aspiration with simultaneous pH monitoring. A combined 14F double channel sump tube (Zinetics Medical, Salt Lake City, Utah) incorporating an antimony pH detector was passed nasally and the tip positioned to lie 5 cm above the manometrically located lower esophageal sphincter. This tube was attached to the suction channel of the purposely built automated suction device and a solid state pH logger (Oakfield Instruments, Oxon UK) was used to monitor esophageal pH simultaneously. Timed samples of esophageal aspirate were collected in vials and stored at -20 degrees C. These were later assayed for bile acids using a modified high performance liquid chromatography [14]. The methodology allowing a clear resolution of 14 bile acid fractions with high specificity and sensitivity. Bile acid concentrations of the esophageal samples were matched with the pH profile The results of the esophageal bile acid assay, expressed as the medians (range) of the peak concentrations of individual bile acid fractions and the median esophageal acid exposure time in each group are shown in Table I. The total bile acid concentration in the patient groups with erosive esophagitis (median 124 mmol/l) and Barrett's/stricture (median 181 mmol/l) were significantly greater than the group with minimal injury (median 14 mmol/l). Five patients in the Barrett's group had reflux episodes with bile acid concentrations in excess of 200 mmol/l. The control group (asymptomatic subjects) had negligible bile acid reflux. Esophageal acid exposure time was greater with increasing grade of mucosal injury with significantly higher DeMeester acid scores in the esophagitis and Barrett/stricture group.

Table I. Results: bile acid assay (median peak bile acid concentration and range; Ámol/l) and esophageal pH profile (median and interquartile range).

The predominant bile acid fractions detected in the patient groups were the primary bile acids, cholic acid, taurocholic and glycocholic acid. Although these primary bile acids were found in increasingly higher concentrations in patient groups with progressive esophageal mucosal injury only the taurocholic acid was significantly increased in the Barrett/stricture group compared to the minimal injury group. The dihydroxy- and the monohydroxysecondary bile acids appeared more frequently in the bile acid profiles of patients with severe esophagitis. Taurodeoxycholic were found in significantly higher concentrations in the refluxates of patients with erosive esophagitis and stricture/Barrett's while lower but significant levels of the taurolithocholic acid were detected in latter group. There was a significant preponderance of the unconjugated bile acids in the erosive and Barrett's groups. Other bile acids detected in low concentrations were the tauro- and glycochenodeoxycholic acid and glycodeoxycholic acid. The prevalence of mixed reflux (acid and bile acid) was the highest in the Barrett group (80%) while it occurred in four patients (40%) with erosive esophagitis and only in one patient (10%) in the minimal injury group. There was specifically a positive correlation between the concentration of taurine conjugated bile acid in the refluxate and percentage acid exposure (r = 0.58, p = 0.009).

In a separate study to investigate the effect of gastric acid suppression on the composition of bile acids in humans we found that bacterial overgrowth occurred when the pH of the gastric aspirate was > 3.8 and on analysing the aspirate for bile acids there was a significant shift in the spectrum of bile acids from the conjugated towards the unconjugated form of bile acids [15].


Using a new method of direct aspiration, reflux of bile acids in concentrations greater than 200 mmol/l has been shown to occur in 50% of the patients with severe esophagitis and Barrett's metaplasia. The most common bile acids present in the refluxate were taurocholic, glycocholic and cholic acid but a significant proportion of the bile acids in patients with extensive mucosal injury were the dehydroxylated; taurodeoxycholic acid and the unconjugated; cholic and deoxycholic acid. Bile acids refluxed over a wide pH range although the predominant pattern was that of mixed bile acid and acid reflux as observed in majority of the patients with Barrett's/stricture. Correlation study indicated a temporal relationship between taurine conjugated bile acids and the presence of acid. It can be speculated that by maintaining a high pH milieu it is possible that the toxicity of some of these bile acids, particularly the unconjugated fraction, could be potentiated. The pH range between 4 and 7 may represent the "danger zone" when most bile acids exist in a two phase state; the ionized and unionized phase. The unionized form of bile acids tend to diffuse through the mucosa more efficiently than the ionized form and are regarded as being more injurious. The results support the theory that pH of the refluxate modulates the toxic effects of bile acids by influencing their composition and concentrations and that it is possible for esophageal mucosal damage to occur at pH greater than 4. This may in part explain why overall 15-20% of the patients fail to respond to acid suppression therapy alone. Consistent finding of secondary bile acids in patients with Barrett's suggest that these bile acids may contribute to the metaplastic change. The significance of pepsin and trypsin in the refluxate needs to be further investigated.


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13. Nehra D, Watt P, Pye JK, Beynon J. Automated esophageal reflux sampler - A new device used to monitor bile acid reflux in patients with gastro-esophageal reflux disease. J Med Eng Technol 1997;21:1-9.

14. Nehra D, Howell P, Williams CP, Pye JK, Beynon J. Toxic bile acids in gastro-oesophageal reflux disease. Influence of gastric acidity. Gut 1999;44:598-602.

15. Theisen J, Nehra D, Citron D, Johannson J, Hagen J, Crookes PF, DeMeester SR, Bremner CG, DeMeester TR, Peters JH. Suppresssion of gastric acid secretion in patients with gastroesophageal reflux disease results in gastric bacterial overgrowth and deconjugation of bile acids. J Gastrointestinal Surg 2000;4:50-54

Publication date: August 2003 OESO©2015