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Mucin gene expression in bile of patients with and without gallstone disease, collected by endoscopic retrograde cholangiography

AIM: To investigate the pattern of mucin expression and concentration in bile obtained during endoscopic retrograde cholangiography (ERC) in relation to gallstone disease. METHODS: Bile samples obtained at ERC from 29 consecutive patients, 17 with and 12 without gallstone disease were evaluated for mucin content by gel filtration on a Sepharose CL-4B column. Dot blot analysis for bile mucin apoproteins was performed with antibodies to Mucin 1 (MUC1), MUC2, MUC3, MUC5AC, MUC5B and MUC6. Staining intensity score (0-3) was used as a measure of antigen expression. RESULTS: MUC1, MUC2, MUC3, MUC5AC, MUC5B and MUC6 were demonstrated in 34.4%, 34.4%, 51.7%, 51.7%, 55.1% and 27.5% of bile samples, respectively. The staining intensity scores were 0.62 ± 0.94, 0.58 ± 0.90, 0.79 ± 0.97, 1.06 ± 1.22, 1.20 ± 1.26 and 0.41 ± 0.73, respectively. Mean mucin concentration measured in bile by the Sepharose CL-4B method was 22.8 ± 24.0 mg/mL (range 3.4-89.0 mg/mL). Mean protein concentration was 8.1 ± 4.8 mg/mL (range 1.7-23.2 mg/mL). CONCLUSION: High levels of MUC3, MUC5AC and MUC5B are expressed in bile aspirated during ERC examination. A specific pattern of mucin gene expression or change in mucin concentration was not found in gallstone disease.

Mucins can be divided into two classes: gel-forming and membrane-associated. Bile mucin has two main domains: one rich in serine, threonine and proline, which contains the majority of the covalently-bound carbohydrates; and another, nonglycosylated domain, enriched in serine, glutamic acid, glutamine and glycine, which binds hydrophobic ligands such as bilirubin. An increased expression of gel-forming mucin, such as MUC5AC and MUC2, was found in patients with hepatolithiasis [15] . Although bile-duct mucin production has been extensively studied in malignant diseases [16][17][18][19][20][21][22] , little is known about mucin synthesis and expression in cholelithiasis, choledocholithiasis and cholangitis.
The aim of the present study was to examine mucin concentration and specific expression in bile samples of patients underg oing endoscopic retrograde cholangiography (ERC) for the evaluation of symptomatic bile duct disease, and to investigate the possible association between mucin expression and the clinical states of gallstone disease.

Sampling
Twenty-nine patients who underwent ERC due to symptomatic bile duct disease were included in the study. Background data and results for ultrasound examinations and liver function tests were obtained from the files. Bile was collected by aspiration, as completely as possible, after the papilla was cannulated and before proceeding to any other procedures, such as papillotomy or choledochal stone removal. The Institutional Review Board (Ethical Committee) of Rabin Medical Center approved the study.

Bile analysis
To determine mucin concentration in the bile, we used the gel-filtration technique, as previously described [23,24] . Briefly, after centrifugation to remove debris, samples of bile were subjected to gel filtration on Sepharose CL-4B columns (1 × 40 cm). We used the closed-column system of Pharmacia Biotech (Cambridge, MA, USA): peristaltic pump, P-1; columns and adapters, C10; fraction collector, Redifrac and Ultraspec 1000; VV/ visible spectrophotometer; and chart reader, 80-2109-03. Samples of 2 mL were applied to the columns and eluted with 10 mmol/L Tris-HCl buffer at pH 8.0. Fractions of 1 mL were collected, and optical density was determined at a wavelength of 280 nm. Findings were correlated with a standard curve of readings of mucin purified from porcine stomach (1% bound sialic acid), purchased from Sigma (St. Louis, MO, USA). The amount of protein was estimated by the Laury method.

Dot blot analysis
Samples were subjected to dot blot analysis on nitrocellulose membranes. Membranes were incubated with monoclonal antibodies to Mucin 1 (MUC1), MUC2, MUC3, MUC5AC, MUC5B and MUC6 (all mouse), followed by incubation with anti-mouse and IgG labeled with biotin. Antibody binding was detected with streptavidin-horseradish peroxide and chemiluminescent reagents (EZ-ECL, Beit-Haemek, Israel). Monoclonal antibodies were purchased from Neomarkers (Fremont, CA, USA). Staining intensity was scored (0-3) as a measure of antigen expression.

Statistical analysis
All results are expressed as mean ± SD. The analyses included descriptive statistics, χ 2 test, Student's t-test, and linear regression analysis. P < 0.05 was considered significant.

Study population
The study group consisted of 13 men and 16 women aged 64.5 ± 16.8 years (Table 1). A gallstone disease was diagnosed in 17 patients and excluded in 12 patients. The indications for ERC, abdominal ultrasound results, and liver function test results before ERC are presented in Table 1.

ERC results
The ERC findings are shown in Table 2. Linear regression analysis revealed a positive correlation between the mean common bile duct (CBD) width measured on abdominal ultrasound and ERC. There was also a positive correlation between ultrasound findings of cholelithiasis and ERC findings of dilated CBD; between the presence of a clinical syndrome of cholangitis and ultrasound findings of pancreatitis; and between increased concentrations of serum direct bilirubin and ERC findings of CBD stricture. A wider CBD was demonstrated in patients with evidence of choledocholithiasis on ERC (10.90 ± 4.97 mm) than in patients without CBD stones (7.44 ± 1.98 mm). Information from the ultrasound studies and ERC results was used to stratify the patients into a group with gallstone related disease, and a group without gallstone disease.

Mucin expression in bile
The expression of the mucin genes examined by dot blot analysis is shown in Table 3 Table 4.

Comparison of patients with and without gallstone disease
Summarizing the clinical and imaging data allowed the patients to be stratified into a group with diagnosed gallstone disease (n = 17), and a group with no evidence of gallstone disease (n = 12). There were no significant differences in gender, age, laboratory results, ultrasound finding, indication and results of ERC, except in the presence of gallstone disease (Table 5). Mucin concentration in bile was similar in both groups (21.68 ± 7.87 mg/mL vs 24.54 ± 24.10 mg/mL, P = 0.759), as was mucin gene expression (Table 5).

DISCUSSION
Different mucin genes are expressed in bile, and the role of each is unclear. Bile mucin is derived from pure hepatic bile, gallbladder-concentrated bile, and mucin secreted by the bile duct epithelium. Ko et al [25] found that in patients with biliary sludge, mucin concentration was higher in bile collected by ERC than in gallbladder bile. They concluded that the biochemical composition of hepatic bile is modified during residence in the gallbladder, contributing to sludge formation, and that hepatic bile samples are therefore inappropriate for microscopic detection of microlithiasis. However, although the mucin concentration in hepatic bile in the present study was similar to that reported by Ko et al [25] (22.8 ± 24.0 mg/mL vs 20 ± 30 mg/mL), the concentration of mucin in gallbladder bile in our previous study was 17.5 ± 16.4 mg/mL [26] , close to that of hepatic bile and much lower than the 450 ± 290 mg/mL found by Ko et al [25] . Thus, our studies do not support the assumption of Ko et al [25] , and this controversy requires further investigation. We demonstrated a higher expression of two secretory mucin proteins, MUC5AC and MUC5B, and the membrane-bound protein, MUC3. MUC5AC and MUC5B are both gel-forming mucins that may increase the viscosity of bile in cases of symptomatic bile duct disease. Since we could not find a change in mucin concentration or in these specific genes expressions in bile derived from patients with or without gallstone disease, our findings do not support a role for MUC5AC or MUC5B in the etiopathogenesis of gallstones.
Zen and coworkers described a lipopolysaccharideinduced increase in MUC2 and MUC5AC expression in cultured murine biliary epithelial cells, which was mediated by tumor necrosis factor alpha [27] . They concluded that since lipopolysaccharide is a bacterial component, bacterial infection may be involved in the altered mucin secretion in the intrahepatic biliary tree and, thereby, in the lithogenesis of hepatolithiasis. Wandenhaute and coworkers noted a strong mRNA expression of MUC5B, MUC3 and MUC6, and a weak expression of MUC1, MUC2 and MUC5AC, in biliary epithelial cells [28] . Lee and Liu found that MUC3 and MUC5B were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls [29] . Mucin gene expression  was altered in dysplastic preneoplastic cells.
The main weakness of our study is the absence of healthy controls. We could not compare mucin concentration and gene expression in the cholestatic situation with that of normal bile collected in ERC, since ERC is usually performed with therapeutic intent in symptomatic patients.
In the present study, we obser ved a positive correlation between MUC1 expression in bile and the expression of all the other mucin genes examined. Wang and coworkers reported a similar result in mice [30] . They described a positive correlation between MUC1 and MUC5AC expression, indicating a gene-gene interaction that might affect the accumulation of mucin gel and cholesterol gallstone formation.
In summary, we could not demonstrate a change in mucin secretion and expression between patients with and without gallstone disease, or support the role of mucin in the etiopathogenesis of biliary sludge or stone formation.

Background
Secretory mucins are gel-forming and may increase bile viscosity. The biochemical composition of hepatic bile is modified during residence in the gallbladder, contributing to sludge formation. An increased expression of gelforming mucin, such as MUC5AC and MUC2, was found in patients with hepatolithiasis. Little is known about mucin synthesis and expression in cholelithiasis, choledocholithiasis and cholangitis.

Innovations and breakthroughs
High levels of MUC3, MUC5AC and MUC5B are expressed in bile aspirated during endoscopic retrograde cholangiography examination. A specific pattern of mucin gene expression or change in mucin concentration was not found in gallstone disease.

Applications
Expression of other mucin genes or changes in concentration should be investigated in gallstone disease. The role of mucin synthesis and secretion in gallstone formation is still unknown.

Peer review
The manuscript by Vilkin