Bile acid composition of gallbladder contents in dogs with gallbladder mucocele and biliary sludge

Toshiaki Kakimoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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Hideyuki Kanemoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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Kenjiro Fukushima Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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Koichi Ohno Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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Hajime Tsujimoto Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

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Abstract

OBJECTIVE To examine bile acid composition of gallbladder contents in dogs with gallbladder mucocele and biliary sludge.

ANIMALS 18 dogs with gallbladder mucocele (GBM group), 8 dogs with immobile biliary sludge (i-BS group), 17 dogs with mobile biliary sludge (m-BS group), and 14 healthy dogs (control group).

PROCEDURES Samples of gallbladder contents were obtained by use of percutaneous ultrasound-guided cholecystocentesis or during cholecystectomy or necropsy. Concentrations of 15 bile acids were determined by use of highperformance liquid chromatography, and a bile acid compositional ratio was calculated for each group.

RESULTS Concentrations of most bile acids in the GBM group were significantly lower than those in the control and m-BS groups. Compositional ratio of taurodeoxycholic acid, which is 1 of 3 major bile acids in dogs, was significantly lower in the GBM and i-BS groups, compared with ratios for the control and m-BS groups. The compositional ratio of taurocholic acid was significantly higher and that of taurochenodeoxycholic acid significantly lower in the i-BS group than in the control group.

CONCLUSIONS AND CLINICAL RELEVANCE In this study, concentrations and fractions of bile acids in gallbladder contents were significantly different in dogs with gallbladder mucocele or immobile biliary sludge, compared with results for healthy control dogs. Studies are needed to determine whether changes in bile acid composition are primary or secondary events of gallbladder abnormalities.

Abstract

OBJECTIVE To examine bile acid composition of gallbladder contents in dogs with gallbladder mucocele and biliary sludge.

ANIMALS 18 dogs with gallbladder mucocele (GBM group), 8 dogs with immobile biliary sludge (i-BS group), 17 dogs with mobile biliary sludge (m-BS group), and 14 healthy dogs (control group).

PROCEDURES Samples of gallbladder contents were obtained by use of percutaneous ultrasound-guided cholecystocentesis or during cholecystectomy or necropsy. Concentrations of 15 bile acids were determined by use of highperformance liquid chromatography, and a bile acid compositional ratio was calculated for each group.

RESULTS Concentrations of most bile acids in the GBM group were significantly lower than those in the control and m-BS groups. Compositional ratio of taurodeoxycholic acid, which is 1 of 3 major bile acids in dogs, was significantly lower in the GBM and i-BS groups, compared with ratios for the control and m-BS groups. The compositional ratio of taurocholic acid was significantly higher and that of taurochenodeoxycholic acid significantly lower in the i-BS group than in the control group.

CONCLUSIONS AND CLINICAL RELEVANCE In this study, concentrations and fractions of bile acids in gallbladder contents were significantly different in dogs with gallbladder mucocele or immobile biliary sludge, compared with results for healthy control dogs. Studies are needed to determine whether changes in bile acid composition are primary or secondary events of gallbladder abnormalities.

Gallbladder mucocele in dogs is generally defined as the accumulation of abnormally gelatinous bile within the gallbladder, and the frequency of diagnosis of GBM has increased during the past 2 decades.1–3 Some epidemiological studies have been conducted on risk factors for GBM. For example, Pomeranians, American Cocker Spaniels, Shetland Sheepdogs, Miniature Schnauzers, and Chihuahuas are predisposed to developing GBM.3,4 Hypothyroidism, hyperadrenocorticism, abnormal lipid metabolism, and a decrease in postprandial gallbladder emptying have also been reported as predisposing factors for development of GBM,4–6 although pathogenic mechanisms for the condition remain unclear.

Gallbladder mucosal cystic hyperplasia as well as mucus hypersecretion and accumulation of mucus within the gallbladder lumen are characteristic histopathologic findings in dogs with GBM.2,7–9 Investigators of 1 study10 found that the bile acids taurochenodeoxycholic acid and deoxycholic acid stimulate gallbladder epithelial cells of dogs to accelerate mucin secretion. Investigators of another study11 reported that mucus hypersecretion from the gallbladder epithelium is associated with increased unconjugated gallbladder bile acid concentrations in dogs with experimentally induced gallstones. Furthermore, gallbladder concentrations of unconjugated chenodeoxycholic acid and deoxycholic acid are elevated in dogs with experimentally induced hyperadrenocorticism, which is regarded as one of the risk factors for development of GBM.12

Gallbladder hypomotility is another feature of GBM and is thought to play a role in the pathogenesis or progression of GBM in dogs.6 Taurochenodeoxycholic acid causes gallbladder muscle dysfunction in guinea pigs by stimulating H2O2 production,13 and bile acids regulate gallbladder motility in mice by inducing synthesis of fibroblast growth factor-15 in the ileum.14

Abnormal lipid metabolism is another predisposing factor for development of GBM.4 Bile acids can regulate triglyceride and cholesterol homeostasis. Bile acids are ligands for farnesoid × receptor, and activated farnesoid × receptor interferes with the sterol regulatory element-binding protein 1c, which is the master regulator of fatty acid and triglyceride biosynthesis.15 Because hydrophobic bile acids have more affinity for farnesoid × receptor than do hydrophilic bile acids, changes to bile acid composition might lead to abnormal lipid metabolism. Although the aforementioned results provide insight into the involvement of changes to bile acid composition in the development of GBM, these associations have not been fully evaluated.

The 2 primary bile acids of dogs, cholic acid and chenodeoxycholic acid, are synthesized from cholesterol and conjugated to taurine or glycine in the liver. After a dog consumes a meal, these bile acids are released from the gallbladder into the intestines, where these conjugated primary bile acids are deconjugated and converted to secondary bile acids (deoxycholic acid, ursodeoxycholic acid, and lithocholic acid) by the intestinal microbiota. It has been reported that the 3 major bile acids (taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid) account for 72.8%, 20.3%, and 6.2% of total bile acids, respectively, in the gallbladder of healthy dogs.16 However, the authors are not aware of any reports on changes to gallbladder bile acid concentrations or compositional ratios in dogs with GBM. Therefore, the purpose of the study reported here was to investigate changes in bile acid concentrations in dogs with GBM and biliary sludge. We hypothesized that there would be differences in bile acid composition in gallbladder contents between dogs with GBM and healthy dogs.

Materials and Methods

Animals

Dogs included in the study (n = 57) were owned by clients, the University of Tokyo, or university staff. Informed consent was obtained for participation of each client- or staff-owned dog. All experimental and animal care procedures complied with policies outlined in the Guide to Animal Use and Care of the University of Tokyo (P14–927).

The dogs represented 4 groups. Healthy dogs (control group; n = 14) had no abnormalities detected during physical examination and laboratory testing; ultrasonographic examination confirmed that the dogs had no gallbladder abnormalities. The control group consisted of 11 males (7 castrated and 4 sexually intact) and 3 spayed females. All 14 dogs were Beagles. Median age of control dogs was 4 years (range, 3 to 8 years).

Dogs with mobile biliary sludge (m-BS group; n = 17) were identified during ultrasonographic examination by the presence of mobile echogenic material without acoustic shadowing in the gallbladder or a large amount of sludge-like material in the gallbladder bile (or both). The m-BS group consisted of 8 castrated males and 9 females (6 spayed and 3 sexually intact). There were 13 Beagles, 1 Dachshund, 1 Italian Greyhound, 1 Miniature Schnauzer, and 1 Toy Poodle. Median age was 7 years (range, 4.00 to 12.75 years).

Dogs with immobile biliary sludge (i-BS group; n = 8) were identified during ultrasonographic examination by the presence of echogenic material without acoustic shadowing in the gallbladder or a large amount of sludge-like material in the gallbladder bile (or both) that did not move, regardless of the dog's position. The i-BS group consisted of 5 males (2 castrated and 3 sexually intact) and 3 spayed females. There was 1 dog each of Beagle, Bichon Frise, Chihuahua, Miniature Dachshund, Pomeranian, Shih Tzu, Toy Poodle, and Yorkshire Terrier. Median age was 10 years (range, 8.00 to 13.17 years).

Dogs with GBM (group GBM; n = 18) were identified on the basis of immobile and finely striated or stellate (or both) bile patterns within the gallbladder lumen during ultrasonographic examination1 and pathological findings of an accumulation of a green-black, bile-laden, semisolid-to-immobile mucoid mass within the gallbladder and cystic mucinous hyperplasia of the gallbladder epithelium.3,17 The GBM group consisted of 8 males (6 castrated and 2 sexually intact) and 10 females (7 spayed and 3 sexually intact). There were 6 Chihuahuas, 3 Pomeranians, 2 American Cocker Spaniels, 2 Shiba Inus, 2 Toy Poodles, 1 American Eskimo Dog, 1 Miniature Schnauzer, and 1 mixed-breed dog. Median age was 11 years (range, 4.00 to 13.08 years).

Sample collection and bile acid analysis

A blood sample (1 mL) was obtained from each dog at the same time as collection of samples of gallbladder contents and at least 12 hours after feeding. Samples of gallbladder contents for control, m-BS, and i-BS groups were collected by use of ultrasound-guided percutaneous fine-needle aspiration, as described in another report,18 or during necropsy. Gallbladder contents for GBM and some of the i-BS group were collected during cholecystectomy or necropsy. Gelatinous or highly viscous gallbladder contents of those GBM and i-BS dogs were aspirated by use of a 10-mL syringe directly from the gallbladder after it had been incised or after it had been removed. All collected samples of gallbladder contents were frozen immediately at −80°C.

Separation and determination of bile acid fractions were performed by use of an HPLC system with a 3α-hydroxysteroid dehydrogenase columna by personnel at a laboratory,b as described previously.19 Gelatinous or highly viscous gallbladder contents for the GBM group and some of the i-BS group had to be diluted to restore fluidity for HPLC analysis. Briefly, 100 mg of gelatinous or highly viscous gallbladder contents was diluted in 4 mL of distilled water and homogenized for 30 seconds on ice. To avoid quantification failure attributable to the dilution process, detection sensitivity of HPLC was enhanced to 1,000 times that for bile acid fractions of clinically normal dogs. Concentrations of 15 bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycoursodeoxycholic acid, lithocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid, tauroursodeoxycholic acid, and ursodeoxycholic acid) were determined, and the compositional ratio for each bile acid was calculated.

Dogs that were receiving ursodeoxycholic acid were included in the m-BS, i-BS, and GBM groups. These dogs were categorized into a ursodeoxycholic acid treatment subgroup, and bile acid fractions for this subgroup were compared with values for the subgroup of nontreated dogs in the m-BS, i-BS, and GBM groups.

Statistical analysis

Data were analyzed by use of statistical software.c The lower limit of detection of the bile acid concentration was estimated as 0 for statistical analysis. Hematologic examination results as well as bile acid concentrations and compositions were compared among the 4 groups by use of the Steel-Dwass test. Bile acid concentrations and compositions were compared between the ursodeoxycholic acid treatment and nontreated subgroups by use of the Wilcoxon rank sum test. Values of P < 0.05 were considered significant.

Results

Animals

Results of hematologic examination for representative hepatobiliary and lipid metabolism markers in each group were summarized (Table 1). Of the 17 dogs in the m-BS group, 5 had a concurrent disease, which included mast cell tumor (n = 1), chronic enteritis (1), immune-mediated thrombocytopenia (1), hepatosplenic lymphoma (1), and splenic tumor (1). The other 12 dogs with m-BS did not have clinical abnormalities other than biliary sludge. The 8 dogs in the i-BS group also had extrahepatic biliary obstruction (n = 5), cholelithiasis (2), cholecystitis (2), hyperadrenocorticism (1), and bacterial peritonitis (1). The 18 dogs in the GBM group also had hypothyroidism (1), hyperadrenocorticism (1), allergic dermatitis (1), hypercholesterolemia (5), hypertriglyceridemia (5), epilepsy (2), cholangitis (3), chronic enteritis (1), and multicentric lymphoma (1). Two dogs in the m-BS group, 4 dogs in the i-BS group, and 9 dogs in the GBM group had been administered a corticosteroid. Two of 17 dogs in the m-BS group, 5 of 8 dogs in the i-BS group, and 12 of 18 dogs in the GBM group were receiving ursodeoxycholic acid tablets at the time that samples of gallbladder contents were collected.

Table 1—

Results for hematologic examination of 4 groups of dogs.*

VariableReference intervalControlm-BSi-BSGBM
ALP (U/L)47–254223 (96–294)244 (98–1,959)3,761 (203–12,350)2,423 (514–16,827)
ALT (U/L)17–7846 (30–95)54 (34–440)615 (49–1,173)421 (40–1,699)
GGT (U/L)5–148 (6–12)8 (6–13)125 (9–158)54 (13–584)
T-Bil (mg/dL)0.1–0.50.2 (0.I-0.4)0.2 (0.I-0.6)5.1 (0.2–20.I)0.5 (0.2–4.4)
T-Cho (mg/dL)113–312190 (101–294)180 (111–264)356 (l94–483)292 (l53–450)
TG (mg/dL)30–13353 (26–95)67 (39–212)73 (22–161)129 (75–1,699)

Values are expressed as median (range). Blood samples were obtained at the same time as collection of samples of gallbladder contents and at least 12 hours after feeding.

Control group consisted of 14 healthy dogs, m-BS group consisted of 17 dogs with mobile biliary sludge detected during ultrasonographic examination, i-BS group consisted of 8 dogs with immobile biliary sludge (regardless of a dog's position) during ultrasonographic examination, and GBM group consisted of 18 dogs with GBM on the basis of immobile and finely striated or stellate (or both) bile patterns within the gallbladder lumen during ultrasonographic examination and pathological findings of an accumulation of a green-black, bile-laden, semisolid-to-immobile mucoid mass within the gallbladder and cystic mucinous hyperplasia of the gallbladder epithelium.

ALP = Alkaline phosphatase. ALT = Alanine aminotransferase. GGT = γ-Glutamyltransferase. T-Bil = Total bilirubin. T-Cho = Total cholesterol. TG = Triglyceride.

Gallbladder contents collected from 5 dogs in the i-BS group were submitted for bacterial culture, and on the basis of culture results, it was determined that 2 of these dogs were infected with Pseudomonas aeruginosa and Enterococcus spp, respectively. Gallbladder contents collected from 11 dogs in the GBM group were submitted for bacterial culture, and results indicated that 2 of these dogs were infected with glucose nonfermentative rods and Klebsiella pneumoniae, respectively.

Bile acid concentrations

Bile acid concentrations in gallbladder contents were determined for each group (Table 2). Concentrations of glycodeoxycholic acid, glycochenodeoxycholic acid, glycolithocholic acid, and lithocholic acid were below the lower limit of detection in all dogs or detected in < 3 dogs in each group. Two samples for the i-BS group and 16 samples for the GBM group were diluted in distilled water to restore fluidity for HPLC analysis. Concentrations of most of the bile acids in the GBM group were significantly lower than concentrations in the control and m-BS groups. Concentrations of taurochenodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid, and ursodeoxycholic acid were significantly lower in the i-BS group than in the control group. Although concentrations of tauroursodeoxycholic acid and total bile acids were typically higher in ursodeoxycholic acid–treated dogs, compared with concentrations in nontreated dogs in the GBM and i-BS groups, respectively, there were no significant differences in bile acid concentrations between the ursodeoxycholic acid–treated and nontreated dogs in the i-BS and GBM groups.

Table 2—

Concentrations of bile acids (mmol/L) in gallbladder contents of 4 groups of dogs.*

VariableControlm-BSi-BSGBM
GUDCA0 (0–0.20)0 (0–0.20)0 (0–0.2l)0 (0–0.30)
TUDCA0.35 (0.10–0.80)0.40 (0.l0–40.80)3.55 (0.05–2l.50)0.37 (0–57.00)
UDCA0.10 (0–0.20)0.10 (0–0.l0)0 (0–0)0 (0–0.20)
GCA1.55 (0–2.60)2.20 (0–4.l0)0.25 (0–3.40)0 (0–0.80)§
TCA74.05 (55.70–154.00)9l.60 (50.80-l72.50)99.40 (0.43–2l2.l0)5.02 (0.03-l07.20)§
CA0.25 (0–3.00)0.l0 (0–6.70)0 (0–0.40)0 (0–0.02)§
TCDCA16.80 (7.80–20.50)l3.00 (5.00–29.70)6.50 (0.03–28.70)1.20 (0–9.59)§
TDCA31.10 (l8.00–73.70)36.20 (0.20–99.80)0.30 (0–67.80)0.03 (0–3.50)§
CDCA0 (0–0)0 (0-l.60)0 (0–0)0 (0–0.02)
DCA0 (0–0)0 (0–0.40)0 (0–0)0 (0–0)
TLCA0.55 (0-l.50)0.20 (0–4.40)0 (0–1.00)§0 (0–0)§
Total bile acids125.75 (85.50–229.70)l72.60 (80.90–280.60)l25.l5 (0.96–3l4.00)11.95 (0.48-l47.90)§

Values are expressed as median (range). Concentrations of glycodeoxycholic acid, glycochenodeoxycholic acid, glycolithocholic acid, and lithocholic acid are not reported because they were below the lower limit of detection in all dogs or detected in < 3 dogs in each group.

Value differs significantly (P < 0.05), compared with the value for the control group.

Value differs significantly (P < 0.01), compared with the value for the control group.

Value differs significantly (P < 0.01), compared with the value for the m-BS group.

Value differs significantly (P < 0.05), compared with the value for the m-BS group.

CA = Cholic acid. CDCA = Chenodeoxycholic acid. DCA = Deoxycholic acid. GCA = Glycocholic acid. GUDCA = Glycoursodeoxycholic acid. TCA = Taurocholic acid. TCDCA = Taurochenodeoxycholic acid. TDCA = Taurodeoxycholic acid. TLCA = Taurolithocholic acid. TUDCA = Tauroursodeoxycholic acid. UDCA = Ursodeoxycholic acid.

See Table 1 for remainder of key.

Bile acid compositional ratios

Bile acid compositional ratios for each group were summarized (Table 3). Compositional ratios for the 3 major bile acids in dogs (ie, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid) were plotted (Figure 1). Compositional ratios of glycodeoxycholic acid, glycochenodeoxycholic acid, glycolithocholic acid, and lithocholic acid were not estimated. The compositional ratio of tauroursodeoxycholic acid in the GBM group was significantly higher than in the control (P = 0.007) and m-BS (P = 0.039) groups and in the i-BS group than in the control group (P = 0.010). The ursodeoxycholic acid fraction was significantly lower in the m-BS (P = 0.003), i-BS (P = 0.001), and GBM (P = 0.001) groups, compared with the fraction in the control group. Compositional ratios of glycocholic acid, cholic acid, and taurolithocholic acid in the GBM group were significantly lower than the ratios in the control (P < 0.001, P = 0.003, and P < 0.001, respectively) and m-BS (P < 0.001, P = 0.008, and P < 0.001, respectively) groups. The glycocholic acid fraction in the i-BS group was significantly (P = 0.022) lower, compared with the fraction in the control group, and the taurolithocholic acid fraction was significantly lower than that in the control (P = 0.003) and m-BS (P = 0.045) groups. Compositional ratios of taurocholic acid were significantly (P = 0.035) higher in the i-BS group than in the control group, whereas the taurochenodeoxycholic acid ratio in the i-BS group was significantly (P = 0.029) lower than the ratio in the control group. Compositional ratios for taurodeoxycholic acid in the i-BS and GBM groups were significantly lower than those in the control (P = 0.001 and P < 0.001, respectively) and m-BS (P = 0.002 and P = 0.001, respectively) groups. In the i-BS group, there were no differences in bile acid fractions between ursodeoxycholic acid–treated and non-treated subgroups. On the other hand, in the GBM group, the taurochenodeoxycholic acid fraction was significantly (P = 0.013) lower in the ursodeoxycholic acid–treated subgroup (median, 11.83%; range, 0% to 41.32%) than in the nontreated subgroup (median, 40.87%; range, 14.56% to 56.72%). Although tauroursodeoxycholic acid fractions typically were higher in ursodeoxycholic acid–treated dogs than in nontreated dogs in the GBM and i-BS groups, there were no significant differences.

Table 3—

Compositional ratios of bile acids (%) in gallbladder contents of 4 groups of dogs.*

VariableControlm-BSi-BSGBM
GUDCA0 (0–0.23)0 (0–0.l0)0 (0–0.l2)0 (0–0.20)
TUDCA0.27 (0.05–0.65)0.2l (0.ll-26.09)7.10 (0.13–14.47)18.78 (0–93.55)
UDCA0.08 (0–0.22)0.04 (0–0.09)0 (0–0)§0 (0–0.l4)§
GCAl.l3 (0–l.75)l.32 (0–2.76)0.l5 (0–l.08)0 (0–0.54)§
CA0.23 (0–2.43)0.08 (0–3.49)0 (0–35.40)0 (0–0.03)§
CDCA0 (0–0)0 (0–0.8l)0 (0–0)0 (0–0.02)
DCA0 (0–0)0 (0–0.23)0 (0–0)0 (0–0)
TLCA0.49 (0-l.24)0.24 (0-l.8l)0 (0–0.32)§0 (0–0)§

Values are expressed as median (range).

Value differs significantly (P < 0.05), compared with the value for the control group.

Value differs significantly (P < 0.05), compared with the value for the m-BS group.

Value differs significantly (P < 0.01), compared with the value for the control group.

Value differs significantly (P < 0.01), compared with the value for the m-BS group.

See Tables l and 2 for remainder of key.

Figure 1—
Figure 1—

Comparison of the compositional ratios of the 3 major bile acids of dogs (taurocholic acid [TCA; A], taurodeoxycholic acid [TDCA; B], and taurochenodeoxycholic acid [TCDCA; C]) in samples of gallbladder contents obtained from 4 groups of dogs. The groups consisted of healthy dogs (control group; n = 14), dogs with mobile biliary sludge detected during ultrasonographic examination (m-BS group; 17), dogs with immobile biliary sludge (regardless of a dog's position) during ultrasonographic examination (i-BS group; 8), and dogs with GBM on the basis of immobile and finely striated or stellate (or both) bile patterns within the gallbladder lumen during ultrasonographic examination and pathological findings of an accumulation of a green-black, bile-laden, semisolid-to-immobile mucoid mass within the gallbladder and cystic mucinous hyperplasia of the gallbladder epithelium (GBM group; 18). Each symbol represents results for 1 dog. Notice that the scale on the y-axis differs among panels. *†Values differ significantly (*P < 0.05; †P < 0.01), compared with values for the control group. ‡§Values differ significantly (‡P < 0.05; §P < 0.01), compared with values for the m-BS group.

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.223

Discussion

Gallbladder mucocele is characterized by excess mucin secretion and abnormal accumulation in the gallbladder lumen in combination with hypomotility of the gallbladder. Cytotoxic effects of the hydrophobic bile acids, which cause gallbladder hypomotility and mucin hypersecretion from the gallbladder epithelial cells, have been described elsewhere.20–22 We hypothesized that changes in gallbladder bile acid composition might be responsible for the development of GBM. In the study reported here, we detected some compositional changes in the gallbladder bile acid content of dogs with biliary sludge and GBM.

Compositional ratios of the secondary hydrophobic bile acids, such as taurodeoxycholic acid and taurolithocholic acid, were significantly lower in the GBM and i-BS groups than in the control and m-BS groups. The lower compositional ratio of taurodeoxycholic acid, which is one of the major bile acids in dogs, was especially remarkable. Taurodeoxycholic acid is a taurine-conjugated form of deoxycholic acid, which is produced by the intestinal microbiota from cholic acid, and accounts for 20.3% of total bile acids in gallbladder bile of healthy dogs.16,23 The effect of a low concentration of taurodeoxycholic acid on the pathogenesis of GBM is unclear. Bile acids work as the signaling molecules that control fluid secretion into the gallbladder lumen.24 In the epithelium of the gallbladder of humans, activated TGR5 (a G-protein-coupled receptor) leads to an increase in intracellular cAMP concentrations, which activates the cystic fibrosis transmembrane conductance regulator that contributes to fluid and chloride secretion into the gallbladder lumen. The TGR5 is a bile acid receptor, and its major agonists are hydrophobic bile acids such as taurolithocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid.25–28 Because viscosity of gallbladder bile is increased in human patients with cystic fibrosis who have cystic fibrosis transmembrane conductance regulator gene mutations,29 the fluid secretion insufficiency caused by low taurodeoxycholic acid fractions, which are evident in dogs with GBM and immobile biliary sludge, might be associated with GBM formation in dogs.

The reason for the lower compositional ratio of secondary hydrophobic bile acids in the GBM and i-BS groups is not clear. Secondary bile acids, which are produced by intestinal microbiota, are returned to the liver through the enterohepatic circulation. In the present study, there was an elevation of plasma alkaline phosphatase and γ-glutamyltransferase activities and total bilirubin concentration, which indicated cholestasis in the GBM and i-BS groups. Defects of gallbladder emptying have been described in dogs with GBM and immobile biliary sludge.6 These results suggested that the decrease of the fractions of secondary bile acids, such as taurodeoxycholic acid, taurolithocholic acid, and ursodeoxycholic acid, in the gallbladder of dogs with GBM and immobile biliary sludge might be associated with cholestasis.

The fraction of taurodeoxycholic acid is low in dogs with germfree bile.23 In the present study, 6 of 8 dogs in the i-BS group and 13 of 18 dogs in the GBM group were given an antimicrobial agent such as metronidazole, ampicillin, or orbifloxacin. Although these antimicrobial agents might affect the intestinal microbiota and induce a decrease in the taurodeoxycholic acid fraction, there were no differences in taurodeoxycholic acid concentrations or compositional ratios between dogs in the GBM group that received an antimicrobial agent and those that did not receive one.

In the study reported here, 2 of 17 dogs in the m-BS group, 4 of 8 dogs in the i-BS group, and 9 of 18 dogs in the GBM group had received corticosteroids. In a previous report12 for which investigators administrated a corticosteroid (hydrocortisone; 8.5 mg/kg) twice daily for 84 days to healthy Beagles, increases in unconjugated bile acids concentrations were observed. However, we did not detect a similar pattern in the dogs of the present study that received corticosteroids. We assumed that one of the reasons for these differences in results might have been the dose of corticosteroids administered.

In the present study, concentrations of some bile acids, including glycocholic acid, cholic acid, taurocholic acid, and taurochenodeoxycholic acid, were significantly lower in the GBM group, compared with concentrations in the control and m-BS groups. As mentioned previously, excess mucin secretion and accumulation is one of the most notable features of GBM, and mucin plays a role in protecting the gallbladder epithelium against exposure to lumen bile acid.9 The low bile acid concentrations in the GBM group might have been attributable to a dilution effect of the mucin. These results suggested that it was unlikely that the gallbladder wall was exposed to concentrated bile acids in cases of GBM, and the changes in bile acid concentrations in the GBM group were believed to be secondary events.

High compositional ratios of taurocholic acid and low compositional ratios of taurochenodeoxycholic acid were detected in the i-BS group, compared with results in the control group. Although similar patterns have been reported in bile duct–ligated dogs,16 the precise mechanism for these changes in bile acid fractions could not be elucidated. The compositional ratios of tauroursodeoxycholic acid were higher in the GBM and i-BS groups. There also was a pattern of an increase in the concentrations and fractions of tauroursodeoxycholic acid in the ursodeoxycholic acid–treated dogs, compared with results for nontreated dogs in the GBM and i-BS groups, respectively. Although the reason for these results remains unclear, ursodeoxycholic acid treatment, which was given to some of the dogs in the GBM and i-BS groups, might have influenced the ratios. In the GBM group, taurochenodeoxycholic acid fractions in the ursodeoxycholic acid–treated subgroup were significantly lower than those in the nontreated subgroup. The reason for this difference is unknown, and further studies are necessary to evaluate the effect of ursodeoxycholic acid treatment on bile acid composition in dogs.

One of the limitations of the present study was the use of gelatinous or highly viscous gallbladder contents for bile acid analysis in dogs in the GBM group and some of the dogs in the i-BS group. It was possible that the bile acid concentrations were distributed unevenly within the gallbladder contents of those dogs in the GBM and i-BS groups because gallbladder contents of dogs with GBM consist of gelatinous content and bile. However, we assumed that results of this study reflected patterns for the entire gallbladder contents of dogs with GBM and immobile biliary sludge. In an unpublished preliminary study, we examined changes in serum bile acid compositional ratios of dogs with GBM. Several reports have indicated that determining the serum bile acid compositional ratio can aid in estimating the compositional gallbladder bile acid ratio.30,31 In that unpublished preliminary study, we also found significantly lower taurodeoxycholic acid fractions in the serum of dogs with GBM than in dogs of a control group, which indicated the validity of the results for the study reported here. Additional studies focusing on bile acids in various portions of gallbladder contents are needed.

A potential breed bias of each group was also a study limitation. Beagles owned by university staff were possibly overrepresented in the control (14/14 dogs) and m-BS (13/17 dogs) groups because it was difficult to obtain permission to collect bile samples from client-owned healthy dogs. Other limitations included the relatively small sample size of each group and the recruitment of dogs for the m-BS, i-BS, and GBM groups without taking into account concurrent diseases and drug administration. Dogs with extrahepatic biliary obstruction and cholecystitis were included, especially in the i-BS group. This might have contributed to values for hematologic examination results of hepatobiliary markers in the i-BS group.

In the present study, we did not examine gallbladder bile acid fractions during GBM development; therefore, we could not prove causality. The precise relationships between gallbladder bile acids and GBM or immobile biliary sludge remain unclear. Additional studies are necessary to clarify effects of the compositional changes of gallbladder bile acids on GBM in dogs.

Acknowledgments

Supported by a Grant-in-Aid for Young Scientists (No. 25850205) from the Japan Society for the Promotion of Science.

The authors declare that there were no financial conflicts or personal relationships with other people or organizations that would have inappropriately influenced or biased content of the manuscript.

ABBREVIATIONS

GBM

Gallbladder mucocele

HPLC

High-performance liquid chromatography

Footnotes

a.

Bilepak-II, JASCO Co, Tokyo, Japan.

b.

SRL Inc, Tokyo, Japan.

c.

JMP, SAS software, version 11.2, SAS Institute Inc, Cary, NC.

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