Gallbladder mucoceles are characterized by an abnormal accumulation of intraluminal inspissated bile.1,2 Histologically, there is hyperplasia of the mucus-secreting glands of the gallbladder mucosa. Gallbladder mucoceles in dogs were rarely reported before 2000 but have since become known as one of the most common gallbladder diseases of that species. The cause of mucus hypersecretion has not yet been determined, although a striking association between hyperadrenocorticism and GBMs has been noted in recent years.1,3–6 A case-control study6 revealed that dogs with hyperadrenocorticism were 29 times as likely to have a GBM as dogs without hyperadrenocorticism. Some authors even reported rapid formation of a mature GBM after the initiation of glucocorticoid treatment.7 However, the role of steroid hormones in the etiopathogenesis of GBM remains unclear. Progestational hormones, which bind to cortisol receptors in various tissues, have been associated with gallbladder mucosal hyperplasia or mucinous dysplasia in dogs.8,9 Bile composition is altered in people with hyperadrenocorticism and in guinea pigs after long-term glucocorticoid administration.10,11 It is not currently known whether corticosteroids administered therapeutically or endogenous overproduction of cortisol affects specific bile components in dogs. Therefore, the purpose of the study reported here was to examine potential influences of twice-daily oral administration of hydrocortisone on the bile acids profile of gallbladder bile in dogs. The hypothesis was that HC of 12 weeks' duration would result in significant changes in the composition of the bile acids pool in dogs.
Materials and Methods
Dogs—Twelve 3-year-old Beagles, ranging in weight from 10.4 to 16.6 kg (median, 12.9 kg), were used in the study. There were 6 sexually intact males and 6 sexually intact females, which were considered to be healthy on the basis of the results of a physical examination, CBC, serum biochemical analysis, urinalysis, bacteriologic culture of urine, and assessment of the urine protein-to-creatinine ratio.
Procedures—The study was approved by the Committee for the Permission of Animal Experimentation, Canton of Zurich, Zurich, Switzerland. For purposes of the study, HC was induced via oral administration of hydrocortisone. The hydrocortisone dosage was chosen on the basis of previous studies12 in which HC was induced in dogs.
Both female and male dogs were randomly allocated to 1 of 2 groups: the control or HC group. Dogs in the control group received a placebo (gelatin capsule) orally every 12 hours for 84 days. Dogs in the HC group received hydrocortisonea (median dose, 8.5 mg/kg; range, 7.5 to 9.6 mg/kg) orally every 12 hours for 84 days. The caretaker and clinicians were unaware of which daily treatment each dog was receiving. All dogs were fed the same dietb throughout the study period. Dogs were examined before (day 0), during (days 28, 56, and 84), and after (days 28p, 56p, and 84p) treatment with either hydrocortisone or placebo. Each examination comprised a thorough physical examination, blood pressure measurement, and laboratory testing (CBC, serum biochemical analysis, and urinalysis). At those time points, dogs also underwent ultrasonographic examination of the gallbladder and PUC to obtain a sample of bile. During the study period, dogs were observed daily for typical cortisol-induced clinical abnormalities, which included hair loss, polyuria, and polydipsia. However, daily water intake and urine production were not quantified.
Ultrasonographic examination and PUC—At the predetermined time points, ultrasonographic examinations and PUCs were carried out by one of the authors (PHK). Food was withheld from the dogs for 18 to 20 hours prior to the procedures. Each dog was administered acepromazine maleatec (0.01 mg/kg, IM) and buprenorphined (0.05 μg/kg, IM) 1 hour before the procedures. The gallbladder of each sedated dog was examined ultrasonographically by use of an 8.5-MHz curved array transducer.e Ultrasonographic images were obtained with the dog in dorsal recumbency by use of a ventral subcostal approach. The thickness of the gallbladder wall was measured on longitudinal images on the side of the gallbladder body closest to the transducer and again in the fundus. Three images were obtained, and a mean value of gallbladder wall thickness was calculated. A bile sample was aspirated via PUC performed under aseptic conditions via a transhepatic approach by use of a 22-gauge, 3.5-inch needle attached to a 10-mL syringe, as described previously.13 Bile samples were stored for later analysis at (−20°C)14; storage time did not exceed 4 months.
Bile analyses—Differentiated quantification of gallbladder bile acids in bile samples collected before (day 0), during (days 28, 56, and 84), and after (days 28p, 56p, and 84p) treatment with either hydrocortisone or placebo was performed via high-performance liquid chromatography–tandem mass spectrometry at the Institute of Clinical Chemistry, University Hospital Zurich, as reported previously.15 Two dilutions of each bile sample were prepared with deionized water (1:100 and 1:1,000), and the following bile acids were measured in their unconjugated forms and as glycine- and taurine-conjugated derivatives: cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid. Coefficients of variation of bile acids measurements were 7.0%.
Statistical analysis—Data were analyzed by use of statistical softwaref and graphing software.g Differences within a group were tested by use of the Friedman repeated-measures test followed by Dunn posttests if the overall value of P was 0.05. Differences between the 2 groups of dogs were tested by use of the Mann-Whitney U test. For comparison of the 3 bile acid fractions (unconjugated-, glycine-, and taurine-conjugated bile acids), the concentrations of the individual bile acids within each fraction were totaled for each individual dog, and results were tested with a nonparametric 1-way ANOVA for differences within the group. Differences between the 2 groups were again tested by use of the Mann-Whitney U test. Values of P ≤ 0.05 were considered significant.
Results
Dogs—All dogs in the HC group developed clinical signs (ie, polyuria, polydipsia, polyphagia, no regrowth of hair within 1 month after clipping, and thinning of the skin with prominent subcutaneous veins in the ventral portion of the abdomen) and clinicopathologic abnormalities (stress leukogram, high alkaline phosphatase activity, isosthenuria, and mild proteinuria) consistent with cortisol excess.13,16,h Nevertheless, during the entire study period, all dogs remained bright, alert, and afebrile. They were released from the study in good health.
Ultrasonography and PUC—No complications developed during or after repetitive PUC in any of the dogs. At each sample collection, the gallbladder was emptied completely by use of a 22-gauge needle. No differences in gallbladder wall thickness were detected between the HC and control groups or among time points. At baseline, the gallbladder wall thickness in all dogs ranged from 0.8 to 1.6 mm (median, 1.3 mm).
Bile acid fractions—Bile acid fractions in the gallbladder bile samples obtained from all 12 dogs on day 0 were assessed. The overall distribution of the 3 bile acid fractions was as follows: taurine-conjugated bile acids, 99.04%; glycine-conjugated bile acids, 0.15%; and unconjugated bile acids, 0.81%.
Unconjugated bile acids—The median baseline bile concentration of cholic acid in the HC and control groups was 2.39 mmol/L (range, 0.17 to 3.09 mmol/L) and 1.29 mmol/L (range, 0.12 to 5.12 mmol/L), respectively (Figure 1). Cholic acid concentration in bile increased as a result of hydrocortisone treatment. In the HC group, the concentration was 21.5 mmol/L (range, 0.91 to 2.8 mmol/L) at day 84 of treatment; this value was significantly (P = 0.028) greater than the baseline value. After discontinuation of hydrocortisone administration, the cholic acid concentration was significantly (all P = 0.028) decreased in the HC group at all 3 time points (ie, days 28p, 56p, and 84p), compared with the value at day 84 during treatment. The concentration of cholic acid in bile in the HC and control groups differed significantly on days 56 (P = 0.015) and 84 (P = 0.004) during treatment.
The median baseline bile concentration of chenodeoxycholic acid in the HC and control groups was 0.03 mmol/L (range, 0.02 to 0.04 mmol/L) and 0.03 mmol/L (range, 0.02 to 0.04 mmol/L), respectively (Figure 2). Chenodeoxycholic acid concentration in bile increased as a result of hydrocortisone treatment. In the HC group, the concentration was 0.09 mmol/L (range, 0.04 to 0.24) on day 84 of treatment; this value was significantly (P = 0.028) greater than the baseline value. After discontinuation of hydrocortisone administration, the chenodeoxycholic acid concentration was significantly decreased in the HC group at days 28p (P = 0.028) and 56p (P = 0.043), compared with the value at day 84 during treatment. The concentration of chenodeoxycholic acid in bile in the HC and control groups differed significantly (P = 0.002) on day 84 during treatment.
The median baseline bile concentration of deoxycholic acid in the HC and control groups was 0.16 mmol/L (range, 0.02 to 0.19 mmol/L) and 0.065 mmol/L (range, 0.02 to 0.13 mmol/L), respectively (Figure 3). Deoxycholic acid concentration in bile increased as a result of hydrocortisone treatment. In the HC group, the concentration was 0.53 mmol/L (range, 0.05 to 1.09 mmol/L) on day 84 of treatment; this value was significantly (P = 0.028) greater than the baseline value. After discontinuation of hydrocortisone administration, the deoxycholic acid concentration was significantly decreased in the HC group at all 3 time points (ie, days 28p [P = 0.027], 56p [P = 0.028], and 84p [P = 0.027]), compared with the value at day 84 during treatment. The concentration of deoxycholic acid in bile in the HC and control groups differed significantly on days 56 (P = 0.009) and 84 (P = 0.004) during treatment.
Lithocholic acid was not detected in bile samples collected at any time point. Ursodeoxycholic acid was quantified in 2 of 84 (2.4%) samples (0.29 and 0.06 mmol/L); both specimens were from dogs in the HC group at day 28p (ie, 28 days after discontinuation of hydrocortisone administration).
The median baseline bile concentration of total unconjugated bile acids in the HC and control groups was 2.6 mmol/L (range, 0.23 to 3.3 mmol/L) and 1.38 mmol/L (range, 0.16 to 5.29 mmol/L), respectively (Figure 4). Total unconjugated bile acids concentration in bile increased as a result of hydrocortisone treatment. In the HC group, the concentration was 22.1 mmol/L (range, 1 to 44.13 mmol/L) on day 84 of treatment; this value was significantly (P = 0.041) greater than the baseline value. After discontinuation of hydrocortisone administration, the total unconjugated bile acids concentration in the HC group decreased to 2.94 mmol/L (range, 0.37 to 8.34) on day 84p. The concentration of total unconjugated bile acids in the HC and control groups differed significantly on days 56 (P = 0.015) and 84 (P = 0.004) during treatment.
Glycine-conjugated bile acids—The concentrations of glycocholic acid and glycodeoxycholic acid in bile samples did not differ significantly between or within groups during the study. In the HC group, the median baseline concentration of glycochenodeoxycholic acid (0.04 mmol/L; range, 0.03 to 0.3 mmol/L) differed significantly (P = 0.02) from that determined on day 84 of treatment (0.03 mmol/L; range, 0.02 to 0.04 mmol/L). This finding was attributed to 1 outlier baseline measurement (glycochenodeoxycholic acid, 0.3 mmol/L; Figure 5). Glycolithocholic acid and glycoursodeoxycholic acid were not detected in bile samples from any of the dogs. The concentrations of total glycine-conjugated bile acids (glycocholic acid, glycochenodeoxycholic acid, and glycodeoxycholic acid) did not differ between groups or among time points within each group.
Taurine-conjugated bile acids—The concentrations of taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, taurolithocholic acid, and tauroursodeoxycholic acid did not differ significantly between the HC and control groups or among time points within each group. For concentrations of the total taurine-conjugated bile acids, the overall ANOVA-Friedman analysis results were 0.048 mmol/L for the HC group and 0.07 mmol/L for the control group. The median baseline concentration of total taurine-conjugated bile acids in the HC and control groups was 255.74 mmol/L (range, 226.79 to 335.09 mmol/L) and 232.5 mmol/L (range, 174 to 346 mmol/L), respectively. In the HC group, the concentration was 164 mmol/L (range, 97 to 304 mmol/L) on day 84 of treatment (significant decrease from baseline [P = 0.031]) but was 333 mmol/L (range, 273 to 421 mmol/L) on day 84 after discontinuation of hydrocortisone administration. Compared with the control group, the total taurine-conjugated bile acids concentration in the HC group differed significantly on day 28 (P = 0.002) and 84 of treatment (P = 0.026; Figure 6).
Discussion
An association between gallbladder disease and concurrent hyperadrenocorticism in dogs has been suggested from results of several recent studies,1,3–6 which prompted us to investigate the relationship between glucocorticoid excess and bile acid composition. For the dogs receiving hydrocortisone in the present study, the concentration of total unconjugated bile acids (cholic acid, chenodeoxycholic acid, and deoxycholic acid) significantly increased and the concentration of total taurine-conjugated bile acids significantly decreased from the baseline (day 0) value as a result of treatment. Even though the concentration of total taurine-conjugated bile acids in the HC and placebo-treated control groups differed significantly on day 28 of treatment, this appeared to be attributable to concentration increases in the control group, and the data obtained for both groups during the course of the study supported an evident effect of the administered hydrocortisone.
Bile acids differ primarily in the number and orientation of hydroxyl groups on the sterol ring. Primary bile acids are those synthesized de novo by the liver and include cholic acid and chenodeoxycholic acid. Dehydroxylation of primary bile acids by intestinal bacteria produces the more hydrophobic secondary bile acids deoxycholic acid and lithocholic acid. Deoxycholic acid and chenodeoxycholic acid are more hydrophobic because they contain only 2 hydroxyl groups and thus are less polar. In general, cytotoxic effects on biomembranes increase with increasing hydrophobicity. In order of decreasing cytotoxicity, bile acids rank as follows: unconjugated, taurine-conjugated, and glycine-conjugated bile acids and monohydroxy, dihydroxy, and trihydroxy bile acids. The position of hydroxy groups is also very important because the 7β-dihydroxy bile acid ursodeoxycholic acid has no cytotoxic effects and is instead protective.
For dogs, data regarding quantification of gallbladder bile acids is scarce, and to our knowledge, profiles of unconjugated gallbladder bile acids have not been published to date. Washizu et al17,18 have published results for 6 dogs wherein the taurine-conjugated bile acids constituted > 99% of the total bile acids pool. In the present study, the gallbladder bile acids profile for all 12 dogs on day 0 (ie, taurine-conjugated bile acids, 99.04%; glycine-conjugated bile acids, 0.15%; and unconjugated bile acids, 0.81%) supported those findings.
In isolated bile ductule fragments and isolated livers from rats, direct application of unconjugated bile acids to the biliary epithelium resulted in epithelial damage.19 Epithelial cells were not damaged by taurine- or glycine-conjugated bile acids, but they were very sensitive to the cytotoxic effects of hydrophobic unconjugated bile acids in concentrations of 10 to 50 μmol/L.19 However, little information is available on the concentration of free bile acids required to cause epithelial damage in the gallbladder.
Similarly, a study20 of canine gallbladder epithelial cell cultures revealed significantly greater mucin secretion in cultures treated with low concentrations (0.5 mmol/L) of the more hydrophobic bile acids. These acids can act as intraluminal secretagogues of mucin even in amounts less than their critical micellar concentration.21 Considering that the median deoxycholic acid concentration was 0.53 mmol/L on day 84 of treatment in the hydrocortisone-treated dogs in the present study, shifts in bile acid composition that lead to dysfunction of mucus-secreting cells in the gallbladder mucosa could serve as an inciting factor in hypercortisolemic dogs with GBM. Because quantitative changes were most pronounced for the taurine-conjugated bile acids, an alternate hypothesis would be that decreased concentrations of taurine-conjugated bile acids predispose the gallbladder to mucosal lesions because of the loss of the taurine-conjugated bile acids–associated amphipathic effect.22 In addition, results of many studies23 have also suggested an antioxidant role for taurine. Explanations for the increase in unconjugated bile acids concentration and decrease in taurine-conjugated bile acids concentration in the HC group in the present study might include a combination of direct effects of glucocorticoids on hepatic bile acid conjugation together with increased intestinal deconjugation secondary to altered intestinal propulsion or bacterial proliferation. A direct choleretic and therefore dilutional effect of hydrocortisone that is not mediated by bile acids would be another explanation for the decrease in total taurine-conjugated bile acids concentration. In fact, this mechanism has been shown experimentally in dogs24 and stresses the importance of our findings regarding the increased concentration of unconjugated bile acids in hydrocortisone-treated dogs.
The present study had several limitations. The duration of induced HC may not have been sufficient to mimic spontaneous hyperadrenocorticism, which typically has an insidious development and chronic course. Prolonged glucocorticoid excess possibly amplifies the toxic actions of hydrophobic bile acids via biliary sphincter dysmotility.11 Evaluation of bile acids kinetics could lead to a better understanding of the pathogenesis of the HC-induced shift in bile acids composition; however, the required techniques were not available. Another limitation of the study was the relatively small number of dogs that were evaluated. More definitive results might be achieved by increasing the number of dogs in both groups. The findings in hypercortisolemic dogs in the present study should therefore be regarded as somewhat preliminary.
The results of the present study indicated that a shift in the bile acids composition of gallbladder bile in favor of unconjugated and more hydrophobic bile acids may be an important part in the pathophysiology of gallbladder disease in dogs with high serum cortisol concentrations. Further studies involving dogs with GBM with and without concurrent hyperadrenocorticism are needed to corroborate the findings. Also, because existing data on the effect of corticosteroids on gallbladder motility are ambiguous,11,12 motility studies together with evaluation of the rate of production of mucus glycoproteins should ideally be examined to assess the influence of corticosteroids on bile solubility in dogs with GBM with and without concurrent hyperadrenocorticism.
Abbreviations
GBM | Gallbladder mucocele |
HC | Hypercortisolemia |
PUC | Percutaneous ultrasound-guided cholecystocentesis |
Hydrocortisone tablets, Hotz Pharmacy, Kusnacht, Switzerland.
Provimi Kliba SA, Penthalaz, Switzerland.
Prequilan, Fatro SPA, Ozzano Emilia, Italy
Temgesic, Essex Pharma GmbH, Munich, Germany.
Acuson, Sequoia, Siemens, Munich, Germany.
SPSS, version 11, SPSS Inc, Chicago, Ill.
GraphPad PRISM, version 3.0, San Diego, Calif.
Schellenberg S, Wenger M, Reusch CE, et al. Course of hematological and biochemical changes during and after long-term hydrocortisone treatment in healthy Beagles (abstr). J Vet Int Med 2008;22:1476.
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