Normal canine bile is composed primarily of water, mucin, conjugated bile acids, bile pigments, cholesterol, phospholipids, and inorganic salts.1 Bile salts emulsify fats, which facilitates their digestion and absorption. Gallbladder mucoceles are characterized by an insidious accumulation of thick, immobile, and viscous bile and mucus within the gallbladder.2 In dogs, several predisposing factors for gallbladder mucocele formation have been identified or proposed, such as concurrent endocrinopathies,2–5 concurrent hypercholesterolemia and hyperlipidemia,2,3 and poor gallbladder motility. However, the exact underlying cause of gallbladder mucocele formation remains unknown. In affected dogs, the gallbladder epithelium acquires a well-differentiated mucinsecretory phenotype that contributes to mucocele formation.6 Gallbladder mucoceles result from dysfunction and proliferation of mucus-secreting cells in the mucosal epithelium of the gallbladder.2 Hypersecretion of mucus leads to an accumulation of thick gelatinous bile within the gallbladder, which is attributed to cystic mucosal hyperplasia.
The gallbladder secretes mucin, a high–molecular-weight and densely glycosylated protein, which is the primary constituent of gallbladder mucus and biliary sludge in humans.7 Hypersecretion of that type of mucin and accumulation of bile in the gallbladder as a viscoelastic gel generally precede the formation of gallstones in prairie dogs8 and humans.9 Results of a retrospective analysis7 of the gallbladder contents of dogs affected by biliary sludge and gallbladder mucocele indicate that mucin is the main component of both biliary sludge and mucoceles.
Gallbladder mucoceles are often surgically removed by cholecystectomy owing to the high risk of extrahepatic biliary obstruction and bile peritonitis secondary to gallbladder rupture if left untreated. However, medical management, which consisted of the administration of various agents that systemically break up viscoelastic material, reportedly resulted in complete resolution of gallbladder mucoceles for 2 dogs.10
A condition similar to gallbladder mucoceles in dogs, termed bile-plug syndrome, is occasionally observed in human infants.9 Bile-plug syndrome is characterized as an obstruction of the common bile duct by bile sludge in full-term infants and generally requires surgical intervention. However, use of NAC with and without glucagon has been described for the treatment of patients with bile-plug syndrome.11–13 The patients in those studies11–13 received 2 mL of 20% NAC injected through a cholangiogram catheter; the NAC resulted in the immediate dissolution of the bile plug and resolution of clinical signs. N-acetylcysteine is a sulfhydryl donator and a thiol precursor of l-cysteine and reduced glutathione. It degrades mucin by reducing disulfide bonds to sulfhydryl bonds that no longer participate in cross-linking.14
The purpose of the study reported here was to evaluate the in vitro effect of NAC on the viscosity of normal canine bile over a period of 24 hours. Our hypothesis was that the addition of 20% NAC would significantly decrease the in vitro viscosity of normal canine bile.
Materials and Methods
Bile samples
Bile (minimum volume, 3 mL) was obtained during necropsy from 10 dogs that were euthanized for reasons unrelated to the biliary system and unrelated to this study. Samples were stored at 4°C until analysis, and all samples were analyzed within 72 hours after collection.
Sample processing and analysis
Immediately prior to analysis, all bile samples were centrifugeda at 3,000 × g for 5 minutes to allow for particle deposition. Each of the 10 samples was then divided into 3 aliquots (minimum volume/aliquot, 1 mL). One aliquot was designated to serve as an untreated control, 1 aliquot was designated for treatment with NAC,b and 1 aliquot was designated for treatment with sterile water.c Each aliquot was placed in an appropriately labeled glass container and stored at room temperature (approx 22°C) for the duration of the observation period.
The bile sample-to-diluent ratio was 1:4 for all NAC- and sterile water–treated samples. Treatment application was designated as hour 0 and represented the beginning of the observation period. The viscosity of the control samples and 20% NAC and sterile water diluents was measured at 0 hours. The viscosity of all control, NAC-treated, and sterile water–treated bile samples was measured at 1 and 24 hours after treatment application.
A rotational viscometerd was used to measure the viscosity of all samples. A rotational viscometer is a high-precision rheometer for accurate measurement of viscosity. Steady state shear sweep for shear rates between 100 and 1,000 seconds−1 was used to determine the mean viscosity of the samples. The upper geometry used for analysis was a 60-mm 1° titanium cone and plate, whereas the lower geometry was a Peltier plate, which was used to control sample temperature (25°C). The sample was injected between the 2 geometries, and steady torque to cover the entire range of shear rates was applied to the upper geometry. For each sample, the rheometer ran three 10-second iterations to ensure that the viscosity was within the 5% tolerance range defined for a valid measurement. The geometries were cleaned with isopropyl alcohol between samples to avoid contamination.
Data analysis
Viscosity data were assessed with the D'Agostino and Pearson test and found to be normally distributed. Descriptive data for the 3 treatment groups were generated, and results were reported as the mean ± SD. A 2-way repeated-measures ANOVA was used to assess viscosity as a function of treatment and time. The Mauchly test of sphericity was performed on each main effect (time and treatment) as well as the interaction between time and treatment. Results indicated that treatment violated the assumption of sphericity, and the Greenhouse-Geisser correction was performed. The Bonferroni correction was used when pairwise comparisons were necessary. All analyses were performed with data analysis software,e and values of P < 0.05 were considered significant.
Results
Dogs from which bile samples were obtained
The 10 dogs from which bile samples were obtained had a mean ± SD age of 6.60 ± 2.98 years and body weight of 24.79 ± 12.45 kg. There were 3 mixed-breed dogs, 2 Boxers, 2 German Shepherd Dogs, 1 Great Dane, 1 Great Pyrenees, and 1 Border Collie.
Viscosity
The viscosity of 20% NAC was 1.82 mPa·s, and the viscosity of the sterile water was 0.9 mPa·s. The viscosity of the untreated control bile samples immediately prior to treatment application ranged from 2.96 to 10.61 mPa·s. The mean viscosity of the 3 treatments at 1 and 24 hours after treatment application was summarized (Table 1). Viscosity was significantly associated with treatment (P < 0.001) but not time (P = 0.763) or the interaction between treatment and time (P = 0.413). Post hoc analyses indicated that the addition of NAC to bile significantly (P = 0.005) decreased its viscosity by approximately 3.35 mPa·s (95% CI, 1.58 to 5.12 mPa·s). Likewise, the addition of sterile water to bile significantly (P = 0.001) decreased its velocity by approximately 2.74 mPa·s (95% CI, 1.33 to 4.14 mPa·s). The viscosity of the NAC-treated bile samples was significantly (P = 0.005) less than that of the water-treated bile samples by approximately 0.61 mPa·s (95% CI, 0.21 to 1.01 mPa·s).
Mean ± SD viscosity (mPa·s) at 1 and 24 hours after treatment application for normal canine bile samples that were diluted 1:4 with 20% NAC or sterile water or left untreated (control).
| Time after treatment application | ||
|---|---|---|
| Treatment group | 1 hour | 24 hours |
| NAC | 3.583 ± 0.921 | 3.417 ± 0.599 |
| Sterile water | 4.140 ± 1.204 | 4.087 ± 1.150 |
| Control | 6.803 ± 2.752 | 6.895 ± 2.61 |
Bile samples were obtained from 10 dogs that were euthanized for reasons unrelated to biliary disease. Each sample was centrifuged to remove particulates, then divided into 3 aliquots (treatment groups). Thus, each value represents the mean ± SD for 10 samples. Mean viscosity differed significantly (P < 0.05) among the 3 treatment groups at each time.
Discussion
Results of the present study indicated that the addition of 20% NAC to normal canine bile (bile sample-to-NAC dilution ratio, 1:4) significantly decreased its in vitro viscosity relative to the viscosity of untreated (control) bile samples and bile samples treated with sterile water. Thus, we accepted (ie, failed to reject) our hypothesis. To our knowledge, the present study was the first that was conducted to evaluate the rheological behavior of normal canine bile in the presence of a mucolytic agent.
Dogs with incidentally discovered, or subclinical, gallbladder mucoceles are a conundrum for clinicians because controlled studies and guidelines for determining when surgical intervention is warranted for such dogs are lacking. The decision to proceed with cholecystectomy should not be made lightly. In 1 study,15 the perioperative mortality rate was 21.7% (5/23) for dogs with gallbladder mucoceles that underwent cholecystectomy. Postoperative complications associated with the procedure include leakage of bile from the surgery site, pancreatitis, and reobstruction of the common bile duct with gelatinous bile.15,16 Successful medical management of gallbladder mucocele has been reported for 2 dogs10; drugs administered to those dogs included ursodiol, S-adenosyl-l-methionine, and famotidine. In dogs, mucin is the primary component in both biliary sludge and gallbladder mucoceles.7 Specifically, dogs with biliary sludge or gallbladder mucoceles have an abnormal increase in MUC5AC, which has properties similar to that of mucus formed in animals with experimentally induced cystic fibrosis.17,18 N-acetylcysteine is used in the mucinous sputum of human patients with cystic fibrosis to facilitate airway penetration of nanoparticles carrying drugs and other therapeutics.19,20 It is also used to successfully treat human infants with bile-plug syndrome,11–13 a condition with characteristics similar to those of gallbladder mucoceles in dogs. Therefore, it seems likely that a mucolytic agent such as NAC could facilitate the breakup of inspissated bile in dogs with gallbladder mucoceles and represent a viable alternative or complement to traditional medical and surgical treatments.
For human infants with bile-plug syndrome, the condition resolved almost instantaneously following the infusion of 2 mL of 20% NAC through a cholangiogram catheter.11–13 In one of those studies,13 the cholangiogram catheter was initially flushed with sterile saline (0.9% NaCl) solution, which failed to dissolve the bile plug and suggested it was the mucolytic properties of NAC that were responsible for plug dissolution. Results of the present study supported that supposition because, although the addition of sterile water to normal canine bile decreased its viscosity, it was not decreased to the same extent as that when NAC was added to the samples.
The commercial 20% NAC solution we used as a diluent in the present study consisted of NAC powder dissolved in sterile water; therefore, we chose to use sterile water as the NAC-negative control diluent. We used a 1:4 bile sample-to-diluent ratio on the basis of extrapolation of the volume of NAC used (2 mL) in the studies11–13 involving human infants with bile-plug syndrome. In human infants prior to feeding, the mean ± SD gallbladder volume is 2.4 ± 1.8 mL, or 0.31 ± 0.18 mL/kg.21 Dilution of canine bile samples with NAC at a ratio > 1:4 may further decrease sample viscosity. Moreover, in the present study, the viscosity of all samples was measured at 25°C instead of at the normal rectal temperature for dogs1 (38° to 39.2°C). Thus, injection of NAC into the gallbladder of a live dog in a volume sufficient to achieve an estimated 1:4 bile-to-NAC ratio will likely decrease viscosity to a greater extent than observed in this study because viscosity decreases as temperature increases.
The viscosity of the untreated control bile samples was fairly low (range, 2.96 to 10.61 mPa·s), which was expected because the samples were obtained from dogs with clinically normal biliary systems. Because the bile samples evaluated in the present study had a low initial viscosity, a large decrease in viscosity following the addition of NAC was not expected. The lowest possible viscosity of a solution is dictated by the size of the smallest possible particle within that solution.22 In the case of bile, the addition of a mucolytic agent (eg, NAC) initially results in the breakdown of macromolecules such as mucin and other proteins into peptides. Then, the peptides are broken down into amino acids (the smallest particles), at which point the lowest possible viscosity is achieved. The viscosity of a solution will actually increase slightly during the breakdown of macromolecules owing to an increase in the number of particles within the solution.22 The exact contents of mucin and other proteins in the bile samples evaluated in the present study were unknown because component analyses were not performed. Some of the variability in the viscosity of the untreated control samples was likely caused by varying concentrations of proteins and other macromolecules. The bottom line is that the low viscosity of the bile samples evaluated in the present study likely muted the magnitude of the mucolytic effects of NAC. In clinical practice, mucolytics would be used in dogs with biliary disease to decrease the viscosity of biliary sludge or help dissolve a gallbladder mucocele, and we believe the viscosity-decreasing effect of NAC relative to sterile water in such samples would be more pronounced than that observed for the normal bile samples evaluated in this study.
N-acetylcysteine is a sulfhydryl donator and a thiol precursor of l-cysteine and reduced glutathione.23 It is approved for use in humans for the treatment of acute acetaminophen toxicosis and as a mucolytic in patients with respiratory tract disease14,23 and has been effectively used for similar conditions in veterinary species. In veterinary medicine, NAC is most commonly used for the treatment of cats with acetaminophen toxicosis.24 It has fairly low toxicity and is associated with mild adverse effects such as signs of nausea, vomiting, pruritus, and tachycardia.24
N-acetylcysteine reduces disulfide bonds of proteins, thereby disrupting ligand bonding and altering the structure of those proteins.23 Reduction of disulfide bonds in cross-linked mucous proteins is the primary mechanism responsible for the mucolytic activity of NAC.23
The powerful mucolytic activity of NAC makes it a viable alternative for many veterinary purposes.24–26 In foals, NAC has been used as a retention enema to successfully treat meconium impactions with no adverse effects reported.26 Meconium is a mucilaginous material that contains a mixture of cellular debris, intestinal gland secretions, bile, and amniotic fluid found in the intestine of full-term neonates.25 N-acetylcysteine is also recommended for the treatment of respiratory tract diseases characterized by excessive mucus production in young foals.26 Meconium, respiratory tract mucus, and bile have similar compositions and consist primarily of mucin, which suggests that NAC may be effective for the dissolution of biliary sludge.
Limitations of the present study included its in vitro nature and the use of normal canine bile. It is likely that the viscosity of biliary secretions is dependent on the concentration of mucins and other proteins.7,22 Component analysis of bile samples was beyond the scope of this study but would have provided important information that may have helped elucidate the NAC mechanism of activity in bile. The efficacy of NAC for decreasing the viscosity of biliary sludge and dissolving gallbladder mucoceles warrants further investigation. On the basis of the results of this study, we theorize that injection of NAC into the gallbladder of dogs would decrease the viscosity of mucin-rich biliary secretions in a manner similar to that observed in human infants with bile-plug syndrome.11–13 However, the safety and efficacy of such a practice must be evaluated in vivo before it can be recommended for clinical patients.
In the present study, dilution of normal canine bile 1:4 with NAC significantly decreased the in vitro viscosity of the resulting mixture. This suggested that NAC might represent a less invasive method for the treatment of dogs with persistent ultrasonographic evidence of biliary sludge because decreasing the viscosity of the sludge would allow it to flow more readily into the gastrointestinal tract. N-acetylcysteine might also help dissolve biliary sludge and gallbladder mucoceles sufficiently so as to avoid the need for cholecystectomy. It could also be used to flush the biliary tree to ensure patency of the common bile duct during cholecystectomy. Randomized prospective studies are warranted to evaluate the effect of NAC on highly viscous biliary secretions (sludge or mucoceles) and to assess the in vivo safety and feasibility of various NAC delivery methods before it can be recommended for use in clinical practice.
Acknowledgments
This study was performed at the College of Veterinary Medicine, Oregon State University.
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript.
The authors thank Dr. Willie Rochefort for technical assistance and guidance and Dr. Laura Kelly for statistical consultation.
ABBREVIATIONS
| CI | Confidence interval |
| NAC | N-acetylcysteine |
Footnotes
Centrifuge 5702, Eppendorf AG, Hamburg, Germany.
Fresenius-Kabi, Bad Homburg vor der Höhe, Germany.
Hospira Inc, Lake Forest, Ill.
DHR rheometer, TA Instruments, New Castle, Del.
STATA, version 14.0, Stata Corp, College Station, Tex.
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