In dogs, chronic hepatitis is a clinical syndrome for which the causal factors remain poorly defined.1–4 Etiopathogenic mechanisms of chronic hepatitis include chronic exposure to infectious agents (eg, Leptospira spp and Adenovirus), hepatotoxins (eg, mycotoxins, drugs, and industrial agents), and pathological retention of transition metals, especially copper. Excessive accumulation of copper in the liver has been documented in various breeds of dogs, including Bedlington Terriers, in which a genetic mutation has been identified that predisposes affected dogs to accumulate excessive copper in the liver.1–9 A similar gene mutation has not been identified in Labrador Retrievers, although investigators of multiple studies6,7,9 have reported pathological accumulation of copper in the livers of Labrador Retrievers with chronic hepatitis, with 1 group suggesting that pathological copper accumulation may be a heritable condition of the breed. Generally, copper-storage hepatopathy in Bedlington Terriers is the result of an autosomal recessive deletion of exon 2 of COMMD1 (the gene responsible for binding cupric ions in 1:1 stoichiometry); however, copper-storage hepatopathy has also been diagnosed in Bedlington Terriers that do not have that genetic deletion, which suggests that additional causal factors are involved in the pathogenesis of the disease.10
Our clinical impression during an ongoing study at Cornell University involving dogs with chronic hepatitis has been that hepatic copper concentrations have increased in both purebred and mixed-breed dogs during the last decade (based on qualitative and quantitative tissue copper assessments). Also during that time, the number of Labrador Retrievers examined at the veterinary teaching hospital at Cornell University and on which liver biopsies have been performed has increased, perhaps because of the increased popularity of the breed as a family pet. However, during the period between 2006 and 2009, investigators of 3 studies6,7,9 performed at 3 different academic institutions reported an association between excessive hepatic copper concentration and chronic hepatitis in Labrador Retrievers. Whether the increased recognition of excessive hepatic copper concentrations in Labrador Retrievers reflects a genetic mutation or other factors, such as increased intake or bioavailability of copper in food or increased breed popularity, remains unclear.
Accumulation of copper in the liver, especially in its cupric form, is extremely toxic and increases oxidant stress. This compromises glutathione availability and damages nucleic acids, proteins, and lipids, which in turn promote hepatocyte apoptosis.11,12 Common histologic features of hepatic copper toxicosis include microvesicular steatosis in damaged hepatocytes and accelerated hepatocyte turnover associated with cytolytic necrosis and apoptosis. Copper bound to metallothionein within the cytoplasm or aggregated in lysosomes of hepatocytes can be identified via histochemical staining methods.13
Currently, the physiologic hepatic copper concentration reported for dogs is ≤ 400 μg/g of dry weight, and hepatic copper concentrations > 1,800 μg/g of dry weight are considered pathogenic.1,14–16 The physiologic reference limit for hepatic copper concentration (on a dry-weight basis) in dogs has progressively increased from 7 μg/g in 1929 to 80 μg/g in 1956 to 400 μg/g since the late 1970s.15,17 Some investigators1,15 have suggested that this increase in the physiologic reference limit for hepatic copper concentration reflects increased feeding of industrially produced dog food to pet dogs. In 1996, AAFCO recommended the elimination of the use of cupric oxide as supplemental copper in dog food premixes because of its poor absorption in favor of more bioavailable forms of copper such as copper sulfate or copper chelates.18,19 The purpose of the study reported here was to evaluate changes in the hepatic copper concentrations of Labrador Retrievers with and without chronic hepatitis over a 30-year period. Liver tissue specimens from Labrador Retrievers with and without chronic hepatitis obtained between 1980 and 2010 were evaluated for qualitative and quantitative copper concentrations. We hypothesized that there would be a significant increase in hepatic copper concentration in Labrador Retrievers with and without chronic hepatitis during that time period corresponding with the switch to more bioavailable forms of supplemental copper in commercially produced dog food. Moreover, on the basis of our observation that hepatic copper concentrations for all breeds of dogs has increased during the recent decade, we postulated that Labrador Retrievers might represent a risk sentinel for hepatic copper accumulation for all dogs.
Materials and Methods
Case selection—A sample size calculation was performed to determine how many dogs with and without chronic hepatitis would need to be evaluated to detect a clinically relevant (300 to 500 μg/g of dry weight) difference in hepatic copper concentration between the 2 groups. Historical quantitative hepatic copper concentrations from 5 Labrador Retrievers with and without chronic hepatitis within each of 2 study periods, 1980 to 1997 and 1998 to 2010, were used for that calculation. This analysis estimated that 18 dogs/group would be needed to detect at least a 500 μg/g difference in hepatic copper concentration between study periods for dogs with chronic hepatitis and at least a 300 μg/g difference in hepatic copper concentration between study periods for dogs without chronic hepatitis (0.99 power).
The computer database for the veterinary teaching hospital at Cornell University was searched for medical records of Labrador Retrievers that had undergone liver biopsy or necropsy between 1980 and 2010. For a dog to be considered for study inclusion, there had to be sufficient archived paraffin-embedded liver tissue available for copper quantification (> 15 mg of tissue) and the acquisition of five 7-μm-thick tissue sections for special histologic stains that would contain at least 15 portal triads for regimented histologic evaluations.20–23 We mandated a minimum cutoff of 15 portal triads to ensure adequacy of architectural features as needed for accurate biopsy interpretation in human and canine patients.20–23 The database search yielded 65 Labrador Retrievers that were diagnosed with hepatitis between 1980 and 1997. Two reviewers (SAC and SPM) independently evaluated H&E-stained liver tissue specimens from those 65 dogs and selected 20 dogs that had histologic evidence of chronic hepatitis. The findings for those 20 dogs were reconciled, and 18 dogs with chronic hepatitis were selected for study inclusion such that sex was evenly distributed. The database search yielded 58 Labrador Retrievers that were diagnosed with hepatitis between 1998 and 2010, and a similar approach was used to enroll 18 dogs with chronic hepatitis into the study for that period.
One control dog was age- and sex-matched to each dog with chronic hepatitis enrolled in the study. All control dogs were considered free of hepatitis on the basis of a lack of histologic evidence of necroinflammatory lesions within liver tissue sections. Thus, liver samples from 72 dogs were evaluated in the study reported here.
Medical records review—For each of the study dogs, data regarding sex and age at the time the liver biopsy or necropsy was performed were obtained from the medical record. Also recorded was whether the dog had chronic hepatitis or was a control dog.
Qualitative histologic evaluation of liver tissue specimens—For each dog enrolled in the study, five 7-μm-thick sections were obtained from archived paraffin-embedded liver tissue specimens. Sections were stained with H&E, Masson trichrome, reticulin, rhodanine, or Prussian blue stain. A qualitative copper score was assigned to each section by a board-certified veterinary pathologist (SPM) and a board-certified veterinary internist with training in hepatic pathology (SAC) independently. Both evaluators used a modification of the Ishak-Knodell system24,25 developed at Cornell University used for evaluation of liver tissue (Appendix) in which liver tissue sections are scored on the basis of distinct histologic features (grade of inflammation or necrosis, extent of fibrosis, and presence of copper) and quantification of abnormal acinar and cell involvement and distribution. Histologic scores for each tissue section were assigned by the evaluators without knowledge of the measured copper concentration within that tissue.
The histologic scores assigned to liver tissue specimens by each evaluator were compared. The proportion of agreement between scores ranged from 0.82 to 0.95, and the K-statistic ranged from 0.75 to 0.89, which indicated very good agreement between assigned scores. The mean histologic scores for portal inflammation, interface hepatitis, lobular activity, and zonal necrosis were summed to provide a hepatic activity index for each dog. The histologic scores for the presence and severity of fibrosis had the least amount of agreement between the evaluators; therefore, both fibrosis scores were retained. The final histologic score for each tissue specimen was the summation of the hepatic activity index and the fibrosis scores.
Each liver tissue specimen also was assigned a qualitative copper score on the basis of the number of cells (hepatocytes, macrophages, or lipogranulomas) within the specimen that contained copper granules. The primary zonal localization of copper granules was recorded to determine whether the distribution differed between dogs with and without hepatitis and between the 2 study periods (1980 to 1997 vs 1998 to 2010).
Quantification of copper concentration in liver tissue specimens—For each study dog, copper concentrations were quantified via atomic absorption spectrometrya as described26 on fresh (n = 27) or archived deparaffinized (45) liver tissue specimens and were expressed as micrograms per gram on a dry weight basis. Liver specimens from some dogs throughout the study period were submitted as fresh tissue for copper quantification at the time of clinical disease characterization. Storage intervals for formalin-fixed paraffin-embedded liver specimens ranged from 6 months to 18 years (median, 15 years). Liver tissue copper concentrations < 400 μg/g were considered physiologically normal.
Statistical analysis—Data distributions were examined for normality via box-and-whisker plots, histograms, and the Kolmogorov-Smirnov test. For data that were not normally distributed, the medians and ranges were reported and nonparametric analyses were used for statistical comparisons. Wilcoxon rank sum tests were used to compare hepatic histologic scores, activity index, copper concentrations, and zonal distribution of copper between dogs with and without chronic hepatitis within each study period (1980 to 1997 and 1998 to 2010) and to compare age of dogs between the 2 study periods. Polynomial linear regression was used to evaluate change in hepatic copper concentration over time. Linear regression and Pearson and Spearman correlations were used to evaluate relationships between hepatic copper concentration and qualitative copper scores, histologic scores for portal inflammation, lobular activity, necrosis, fibrosis, hepatic activity index, and age. A Fisher exact test was used to determine whether the proportion of dogs with a hepatic copper concentration > 400 μg/g differed between the 2 study periods. All statistical analyses were performed with commercially available software programs,b,c and values of P < 0.05 were considered significant.
Results
Evaluation of the descriptive data for the study dogs revealed that the protocol used to match dogs that had chronic hepatitis with control dogs was effective. The median age for all study dogs was 8.5 years (range, 1.8 to 15 years). The median age (8.4 years [range, 1.8 to 15.0 years]) of dogs with chronic hepatitis did not differ significantly from that (8.7 years [range, 2.0 to 15.0 years]) of the control dogs. Also, age did not differ significantly for dogs with chronic hepatitis or control dogs between the 2 study periods (chronic hepatitis dogs, 8.5 years [range, 1.8 to 15.0 years] vs 8.3 years [range, 4.3 to 12.8 years]; control dogs, 8.8 years [range, 2.0 to 15.0 years] vs 8.5 years [range, 4 to 14 years] for the 1980–1997 and 1998–2010 study periods, respectively). There were 18 female and 18 male dogs enrolled in each of the 2 study periods. The dogs with chronic hepatitis included 6 sexually intact males, 12 castrated males, 5 sexually intact females, and 13 spayed females. The control dogs included 8 sexually intact males, 10 castrated males, 4 sexually intact females, and 14 spayed females.
For each of the 2 study periods, hepatic copper concentrations from dogs with and without chronic hepatitis were plotted (Figure 1). When all study dogs were considered, the mean hepatic copper concentration for dogs with chronic hepatitis (614 μg/g of dry weight [range, 104 to 4,234 μg/g of dry weight]) was significantly (P = 0.01) higher, compared with the mean hepatic copper concentration for the control dogs (299 μg/g of dry weight [range, 93 to 3,810 μg/g of dry weight]). During polynomial regression analysis, the hepatic copper concentrations for 2 dogs with chronic hepatitis in the 1980–1997 study period were classified as statistical outliers and removed from the analysis. The final results of the polynomial regression analysis revealed that hepatic copper concentrations increased significantly (P < 0.001) over time for all dogs (Figure 2). The proportion (22/36) of dogs with and without chronic hepatitis that had a hepatic copper concentration > 400 μg/g of dry weight (upper reference limit for physiologic hepatic copper concentration in dogs) during the 1998–2010 study period was significantly greater, compared with that (14/36) during the 1980–1997 study period.
Box-and-whisker plots of hepatic copper concentrations for Labrador Retrievers with and without (control dogs) chronic hepatitis during each of 2 study periods, 1980 to 1997 (dogs with chronic hepatitis, n = 18; control dogs, 18) and 1998 to 2010 (dogs with chronic hepatitis, 18; control dogs, 18). All study dogs were examined at a veterinary teaching hospital between 1980 and 2010. For each study period, a control dog was matched with each dog with chronic hepatitis on the basis of similar age and sex. Each box represents the interquartile range (25th to 75th percentiles), the horizontal line in each box represents the median, and the vertical lines (whiskers) represent the 95% confidence interval. Circles represent copper concentrations for individual dogs, crosses represent outlier values > 1.5 but ≤ 3.0 times the interquartile range, and triangles represent outlier values > 3.0 times the interquartile range.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Box-and-whisker plots of hepatic copper concentrations for Labrador Retrievers with and without (control dogs) chronic hepatitis during each of 2 study periods, 1980 to 1997 (dogs with chronic hepatitis, n = 18; control dogs, 18) and 1998 to 2010 (dogs with chronic hepatitis, 18; control dogs, 18). All study dogs were examined at a veterinary teaching hospital between 1980 and 2010. For each study period, a control dog was matched with each dog with chronic hepatitis on the basis of similar age and sex. Each box represents the interquartile range (25th to 75th percentiles), the horizontal line in each box represents the median, and the vertical lines (whiskers) represent the 95% confidence interval. Circles represent copper concentrations for individual dogs, crosses represent outlier values > 1.5 but ≤ 3.0 times the interquartile range, and triangles represent outlier values > 3.0 times the interquartile range.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Box-and-whisker plots of hepatic copper concentrations for Labrador Retrievers with and without (control dogs) chronic hepatitis during each of 2 study periods, 1980 to 1997 (dogs with chronic hepatitis, n = 18; control dogs, 18) and 1998 to 2010 (dogs with chronic hepatitis, 18; control dogs, 18). All study dogs were examined at a veterinary teaching hospital between 1980 and 2010. For each study period, a control dog was matched with each dog with chronic hepatitis on the basis of similar age and sex. Each box represents the interquartile range (25th to 75th percentiles), the horizontal line in each box represents the median, and the vertical lines (whiskers) represent the 95% confidence interval. Circles represent copper concentrations for individual dogs, crosses represent outlier values > 1.5 but ≤ 3.0 times the interquartile range, and triangles represent outlier values > 3.0 times the interquartile range.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Scatterplot of copper concentations in liver tissue specimens obtained from Labrador Retrievers with (black circles; n = 36) and without (white circles; 36) chronic hepatitis by year during which the specimens were obtained. Dashed line represents the polynomial regression line; the equation for the regression line was 4,804,437 − 4,858x + 1.228x2. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Scatterplot of copper concentations in liver tissue specimens obtained from Labrador Retrievers with (black circles; n = 36) and without (white circles; 36) chronic hepatitis by year during which the specimens were obtained. Dashed line represents the polynomial regression line; the equation for the regression line was 4,804,437 − 4,858x + 1.228x2. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Scatterplot of copper concentations in liver tissue specimens obtained from Labrador Retrievers with (black circles; n = 36) and without (white circles; 36) chronic hepatitis by year during which the specimens were obtained. Dashed line represents the polynomial regression line; the equation for the regression line was 4,804,437 − 4,858x + 1.228x2. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Copper granules were observed in liver tissue of 45 of 72 (63%) dogs, and lobular distribution of copper granules varied among individual dogs. For dogs assigned qualitative copper scores of 3 to 5 (ie, moderate to severe copper accumulation; n = 18), copper granules were generally distributed within zone 3 (periacinar or centrilobular region) hepatocytes and macrophages. There was a significant (P = 0.001) positive correlation (r = 0.66) between qualitative copper scores ≥ 1.0 and the deposition of copper granules in zone 3 cells. Although hepatic copper concentration was significantly (P < 0.001) associated with the qualitative copper score, the correlation coefficient (r = 0.66) suggested only a modest positive correlation between the 2 variables (Figure 3). For 24 of 72 (33%) study dogs, the qualitative copper score did not accurately predict the hepatic copper concentration (eg, a liver tissue specimen with a high [≥ 3] qualitative copper score had a low [≤ 400 μg/g of dry weight] copper concentration or vice versa), and the data points for 22 of those 24 dogs were outside of the 95% confidence interval that surrounded the regression line.
Scatterplot of hepatic copper concentration versus qualitative copper score (as determined via histologic evaluation of rhodanine-stained liver tissue specimens) for liver tissue specimens obtained from Labrador Retrievers with (n = 36) and without (36) chronic hepatitis. For each liver tissue specimen, the presence of copper granules was qualitatively scored on a 6-point scale (0 = no copper granules detected, 1 = a few copper granules in an occasional cell, 2 = obvious copper granules in some cells, 3 = numerous copper granules in < 50% of cells, 4 = numerous copper granules in ≥ 50% but < 75% of cells, 5 = numerous copper granules in ≥ 75% of cells). The solid black line represents the regression line, and the dotted lines represent the 95% confidence limits for the regression line. The regression line equation was 186.4 + 426.2x, and the coefficient of determination for the regression line was 0.43. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Scatterplot of hepatic copper concentration versus qualitative copper score (as determined via histologic evaluation of rhodanine-stained liver tissue specimens) for liver tissue specimens obtained from Labrador Retrievers with (n = 36) and without (36) chronic hepatitis. For each liver tissue specimen, the presence of copper granules was qualitatively scored on a 6-point scale (0 = no copper granules detected, 1 = a few copper granules in an occasional cell, 2 = obvious copper granules in some cells, 3 = numerous copper granules in < 50% of cells, 4 = numerous copper granules in ≥ 50% but < 75% of cells, 5 = numerous copper granules in ≥ 75% of cells). The solid black line represents the regression line, and the dotted lines represent the 95% confidence limits for the regression line. The regression line equation was 186.4 + 426.2x, and the coefficient of determination for the regression line was 0.43. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Scatterplot of hepatic copper concentration versus qualitative copper score (as determined via histologic evaluation of rhodanine-stained liver tissue specimens) for liver tissue specimens obtained from Labrador Retrievers with (n = 36) and without (36) chronic hepatitis. For each liver tissue specimen, the presence of copper granules was qualitatively scored on a 6-point scale (0 = no copper granules detected, 1 = a few copper granules in an occasional cell, 2 = obvious copper granules in some cells, 3 = numerous copper granules in < 50% of cells, 4 = numerous copper granules in ≥ 50% but < 75% of cells, 5 = numerous copper granules in ≥ 75% of cells). The solid black line represents the regression line, and the dotted lines represent the 95% confidence limits for the regression line. The regression line equation was 186.4 + 426.2x, and the coefficient of determination for the regression line was 0.43. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 242, 3; 10.2460/javma.242.3.372
Within each study period, dogs with chronic hepatitis generally had significantly (P < 0.002) higher histologic scores, compared with the histologic scores for the control dogs (Table 1). The median qualitative copper score for the dogs with chronic hepatitis in the 1998–2010 study period was significantly (P < 0.001) higher, compared with that for dogs with chronic hepatitis in the 1980–1997 study period. Similarly, the median qualitative copper score for the control dogs in the 1998–2010 study period was significantly (P < 0.001) higher, compared with that for control dogs in the 1980–1997 study period. Also, the liver biopsy specimens obtained from dogs with chronic hepatitis during the 1998–2010 study period had a significantly (P = 0.002) greater amount of copper accumulation within zone 3 cells, compared with that for liver biopsy specimens obtained from dogs with chronic hepatitis during the 1980–1997 study period.
Median (range) histologic scores by variable evaluated for liver tissue specimens obtained from Labrador Retrievers with and without (control dogs) chronic hepatitis during each of 2 study periods, 1980 to 1997 (dogs with chronic hepatitis, n = 18; control dogs, 18) and 1998 to 2010 (dogs with chronic hepatitis, 18; control dogs, 18).
| Study period | ||||
|---|---|---|---|---|
| 1980–1997 | 1998–2010 | |||
| Variable | Dogs with chronic hepatitis | Control dogs | Dogs with chronic hepatitis | Control dogs |
| Portal inflammation | 1 (0–4)* | 0 (0–1) | 2 (0–4)* | 0 (0–1) |
| Interface hepatitis | 0 (0–4) | 0 | 1.5 (0–4)* | 0 |
| Zonal necrosis | 0 (0–4) | 0 (0–5) | 2 (0–4)* | 0 (0–2) |
| Lobular activity | 2 (0–4)* | 0 (0–1) | 2 (0–4)* | 0 (0–2) |
| Fibrosis | 5.5 (0–18)* | 1 (0–5) | 11 (4–21)* | 1.5 (0–5) |
| Activity index | 4 (1–15)* | 0 (0–5) | 7 (2–17)* | 0 (0–2) |
| Activity index + fibrosis | 6 (3–18)* | 2 (0–5) | 11 (4–20)* | 0.5 (0–5) |
| Zone of copper distribution | 0 (0–5) | 0 | 3 (0–5)*† | 0 |
| Qualitative copper | 1 (0–4)* | 0 (0–1) | 3 (0–5)*† | 1.5 (1–4)† |
For each study period, a control dog was matched with each dog with chronic hepatitis on the basis of similar age and sex. For variables with no range reported, all data points were equal to the median. For each liver tissue specimen evaluated, histologic scores were independently assigned by 2 experienced evaluators (SPM and SAC) in accordance with a scoring system modified from the Ishak-Knodell system that was developed at Cornell University to rank histologic features of and assign qualitative copper scores for liver tissue sections. Because the κ-test statistic indicated very good agreement between the scores assigned by the evaluators, the mean scores for portal inflammation, interface hepatitis, zonal necrosis, and lobular activity were recorded for each liver tissue specimen. The histologic scores for fibrosis had the least amount of agreement between the evaluators; therefore, both fibrosis scores were recorded for each liver tissue specimen. The activity index score was calculated as the sum of the mean scores for portal inflammation, interface hepatitis, zonal necrosis, and lobular activity. The activity index + fibrosis score was calculated as the sum of the scores for activity index and fibrosis.
Within a study period, the values for dogs with chronic hepatitis significantly (P < 0.002) differed from that for control dogs.
Within a variable and dog classification (dogs with chronic hepatitis or control dogs), value for the 1998–2010 study period differed significantly (P < 0.002) from that for the 1980–1997 study period.
For dogs with chronic hepatitis, hepatic copper concentration was not significantly associated with age (P = 0.75), histologic scores for fibrosis (P = 0.31), or portal inflammation (P = 0.40) but was associated with the histologic scores for interface hepatitis (P = 0.017), lobular activity (P = 0.017), zonal necrosis (P = 0.02), and hepatic activity index (P = 0.003). The histologic score for fibrosis was significantly associated with the histologic scores for activity index (P < 0.001) and zonal necrosis (P = 0.008).
Discussion
Results of the present study indicated that the prevalence of Labrador Retrievers with increased hepatic copper concentrations (> 400 μg/g of dry weight) has increased over time. The cause for this increased prevalence is unknown. However, in the present study, polynomial regression results indicated that hepatic copper concentrations began to increase exponentially in the mid- to late 1990s, the time during which AAFCO recommended that dog food manufacturers discontinue the use of cupric oxide as a source of supplemental copper in commercial dog food in favor of more bioavailable forms, such as cupric sulfate or various forms of chelated copper. Thus, the results of the present study support our hypothesis that the increased hepatic copper concentrations in dogs over time reflect increased ingestion of more bioavailable forms of copper.
Excessive copper accumulation in the liver can be caused by genetic mutations that affect complex protein interactions that regulate cellular uptake, intracellular transport, chaperone linkage, or biliary excretion of copper; hepatocyte dysfunction that compromises physiologic copper homeostasis; or chronic copper ingestion that exceeds the body's capability to excrete copper.11–13,27,28 Excess copper is exported from the liver across the canalicular membrane into bile, where it is eliminated from the body in feces. In Bedlington Terriers with the COMMD1 gene deletion and humans with Wilson disease, inadequate copper excretion results in excessive hepatic copper accumulation such that concentrations may exceed the physiological concentration by 50-fold.1,14 We believe that the increased hepatic copper concentrations for dogs of the present study reflected increased copper ingestion that resulted in a 10-fold increase from the currently accepted physiologic copper concentration.
Dogs can generally tolerate a greater hepatic copper concentration than can most other species,15,28–30 although the reason for this is unknown. Despite having a similar hepatic metallothionein concentration, humans are unable to tolerate hepatic copper concentrations as high as those tolerated by dogs.30 Dogs have lower concentrations of serum ceruloplasmin, a copper transport protein, than do many other species (eg, rats, mice, and humans).29–32 In contrast to humans with Wilson disease, in which a genetic mutation suppresses formation of ceruloplasmin from apoceruloplasmin, serum copper and ceruloplasmin concentration do not accurately reflect excessive hepatic copper concentration in dogs.29,30 A rare exception would be a dog with copper-associated hepatopathy that has severe hepatocellular necrosis, in which large concentrations of copper are released from the liver into the systemic circulation.14,15
Despite the fact that dogs can tolerate relatively higher hepatic copper concentrations than can most other species, dogs with hepatic copper concentrations > 1,000 μg/g of dry weight usually have liver damage or at least increased alanine aminotransferase activity. Copper may directly induce oxidant injury via accumulation of its transition (cupric) state in hepatocytes, or its presence and transition state reactivity may augment cell damage initiated by other primary hepatic disorders.1,33 Thus, excessive hepatic copper accumulation can be a primary or secondary cause of liver damage, which can progress to chronic hepatitis and cirrhosis. Therefore, understanding the pathogenesis of increased hepatic copper concentrations in individual dogs is important for clinical management.
In human patients, increased hepatic copper concentration often develops concurrently with primary necroinflammatory and cholestatic liver disorders.34,35 In contrast, dogs appear to be less susceptible to secondary hepatic copper accumulation unless copper ingestion is increased.15,36 Furthermore, when copper accumulates within the liver secondary to another hepatic disorder, it generally integrates within or adjacent to previous pathological lesions instead of primarily within zone 3 hepatocytes. Results of the present and other studies1,15,16 suggest that some dogs with chronic hepatitis do not accumulate excessive hepatic copper concentrations and some dogs with increased hepatic copper concentrations do not develop lesions consistent with hepatitis. Thus, it is difficult to know when to initiate copper chelation treatment for some dogs with subclinical hepatitis that have histologic lesions of the disease and increased hepatic copper concentrations.
In the present study, we attempted to eliminate sampling error as a variable that could influence the assignment of histologic scores by the preferential enrollment of dogs from which liver tissue samples were obtained via surgical wedge or laparoscopic biopsy or necropsy to ensure that adequate tissue would be available for evaluation.21–23 Given that the etiopathogenesis of chronic hepatitis is often undetermined in veterinary patients, we developed a standardized scoring system to objectively characterize the histologic features of each liver tissue section evaluated in an attempt to identify histologic patterns of disease.37 The data obtained from the scoring system were used to calculate a hepatic activity index and a qualitative copper score, which were in turn used to define disease activity, stage, and severity for each study dog. Thus, comparisons could be made between dogs with and without chronic hepatitis over time, and this system could be easily adapted to make longitudinal comparisons within an individual dog. Scoring bias was minimized in the present study via independent scoring of each liver tissue specimen by 2 evaluators who had no knowledge regarding the dog's history or hepatic copper concentration.
The range of values for the variables histologically evaluated in the present study varied substantially, which suggested that the study dogs were at various stages of liver disease or that the disease was caused by various factors. Although the qualitative copper score was positively correlated with hepatic copper concentration, the qualitative copper score did not accurately predict the hepatic copper concentration for 33% of the study dogs. This apparent discordance between the qualitative copper score and hepatic copper concentration may be the result of copper evaluation on unrepresentative liver tissue sections (ie, regenerative nodules or regions of parenchymal extinction that have low copper concentration).16,34,35 Regardless, the findings of the present study suggested that the qualitative copper score and the hepatic copper concentration be adjunctively considered.
In the present study, dogs with chronic hepatitis during the 1998–2010 study period had higher hepatic copper concentrations, compared with those for dogs with chronic hepatitis during the 1980–1997 study period. For the liver tissue sections obtained during the 1998–2010 study period, copper was distributed primarily within zone 3. Zone 3 is the region of the liver where copper first accumulates in Bedlington Terriers with the COMMD1 gene deletion as well as most other dogs with copper-associated hepatopathy (our clinical impression after evaluation of > 500 canine liver tissue specimens with increased copper concentrations that were submitted to the Cornell University Diagnostic Pathology Service). Although hepatic copper accumulation within zone 3 is not definitive for a genetic mutation or a primary hepatic copper-storage disorder in dogs, we propose that hepatic copper accumulation within zone 3 is an indication of copper-associated liver injury, especially if there is a concurrent increase in alanine aminotransferase activity. However, primary distribution of copper within zone 3 is not consistent for copper-associated liver damage among all species (eg, humans with Wilson disease primarily accumulate copper in zone 1 [periportal region] of the liver).33,35,38 Furthermore, the concentration at which copper granules bound to metallothionein or glutathione and aggregated within lysosomes become microscopically visible varies among species.39,40 On the basis of the results of the present study, copper granules may become microscopically visible in dogs with hepatic copper concentrations as low as 200 μg/g dry weight.
Except for Bedlington Terriers with the COMMD1 gene deletion, it is unknown whether accumulation of copper within zone 3 of the liver is indicative of a primary (ie, genetic), secondary (ie, environmental or dietary), or ecogenetic (eg, presumed for human patients with endemic Tyrolean infantile cirrhosis or Indian childhood cirrhosis, both of which are associated with increased copper ingestion) disorder.39 Therefore, use of the term primary copper or copper-storage hepatopathy to describe necroinflammatory liver damage associated with histologic evidence and quantification of excessive copper concentration must be done with caution. We also propose that the term copper-associated hepatopathy be used alternatively. In the present study, excessive hepatic copper concentration was not associated with age; therefore, it is unclear whether Labrador Retrievers accumulate copper within the liver throughout their lifetimes (in a manner similar to Bedlington Terriers), achieve a plateaued hepatic copper concentration (similar to that described in some West Highland White Terriers), or merely accumulate copper within the liver in accordance with dietary intake.1,3
The fact that hepatic copper concentrations increased significantly over time for all dogs, including the control dogs, in the present study supports our supposition that increased hepatic copper concentrations in dogs were caused by the increased bioavailability of supplemental copper in dog food that corresponded with the AAFCO recommendation to ban the use of a less bioavailable form (ie, cupric oxide) of supplemental copper in dog food in the mid- to late 1990s.18,19 This supposition was not refuted by the 2 dogs with chronic hepatitis that had extremely high hepatic copper concentrations during the 1980–1997 study period because those 2 dogs may have represented rare dogs that were fed diets with high concentrations of bioavailable copper or may have had excessive copper accumulation secondary to chronic hepatitis. Results of 3 studies6,7,9 suggest that copper-associated hepatopathy in Labrador Retrievers may be a heritable condition. On the basis of the results of the present study, we believe that it was unlikely that the significant increase in hepatic copper concentrations in Labrador Retrievers was caused only by a genetic mutation. Moreover, although pedigrees for the dogs of the present study were not available for evaluation, it is unlikely that the study dogs represented 1 kindred because of the diverse geographic origins of the dogs. It seems more probable that the increased incidence of copper-associated hepatopathy in Labrador Retrievers reflects a breed-related intolerance to increased ingestion of more bioavailable forms of copper and that this breed may be serving as a risk sentinel for other dogs.
Currently, the most common forms of supplemental copper used in dog foods are cupric sulfate and various forms of chelated copper, which are substantially more bioavailable than cupric oxide.40,41 The relative bioavailability of copper from feed grade cupric oxide is low (–5%), compared with the bioavailability of copper from reagent grade copper acetate (100%), copper carbonate (107%), and copper sulfate (60%).40 Most data regarding the copper requirements for adult dogs have been extrapolated from studies42–47 that involved other species and allometric assumptions of copper requirements for growth, gestation, and lactation. Current dietary recommendations for copper in adult dogs were made on the basis of results of a study48 that involved puppies, in which ingestion of < 8 mg of copper/kg of dry matter fed/d reduced serum ceruloplasmin concentration. In dogs, serum ceruloplasmin concentration does not accurately reflect copper bioavailability; therefore, it is an invalid measure for the determination of copper adequacy and formulation of dietary recommendations.1,14,15,36 The National Research Council's recommendation for the dietary copper requirement for adult dogs is 6.0 mg of copper/kg of dry matter fed/d (2.7 mg of copper/lb of dry matter fed/d; 4.0 kcal metabolizable energy/g) or 0.06 mg/kg of body weight/d (0.03 mg/lb of body weight/d).47 The AAFCO recommendation for the dietary copper requirement for dogs (regardless of age) is 7.3 mg of copper/kg of dry matter fed/d (3.3 mg of copper/lb of dry matter fed/d; 3.5 kcal metabolizable energy/g).49 With the increasing number of niche pet foods on the market and a general lack of regulation of the trace mineral content in those foods, it is common for companies that make premix formulations for those foods to add enough trace mineral to the premix to comply with AAFCO's recommendation. That premix is then added to the dog food, often without consideration of the copper concentration of the other ingredients in the formula. Unpublished data obtained by our laboratory group suggests that the copper concentration in some dog foods may exceed > 2 times the AAFCO recommendation. It is possible that some dogs may consume as much as 10 mg of copper daily, which is equivalent to the safe upper limit for chronic copper consumption in humans.50
The results of the present study indicated that the hepatic copper concentration in Labrador Retrievers with and without chronic hepatitis has increased significantly since 1980. These results provide provocative evidence that the increased hepatic copper concentration in Labrador Retrievers was associated with the switch to the use of more bioavailable forms of supplemental copper in commercial dog food in the mid- to late 1990s, although a direct cause-and-effect relationship cannot be proven. Whether copper-associated hepatopathy in Labrador Retrievers is caused by a genetic mutation remains to be determined. However, a genetic mutation that promotes hepatic copper accumulation would be exacerbated by excessive bioavailability of copper in the diet, as evidenced by humans with Indian childhood cirrhosis and endemic Tyrolean infantile cirrhosis.33 Although the diets of dogs in the present study were unknown, they were likely diverse and representative of commercial dog food formulations. We believe that the amount of copper in many commercial dog foods is excessive and that Labrador Retrievers, because of their popularity as family pets, may be functioning as sentinels for trends in hepatic copper concentrations in the general pet dog population.
ABBREVIATION
| AAFCO | Association of American Feed Control Officials |
Varian AA-1275, Varian Inc, Palo Alto, Calif.
Statistix, version 9.0, Analytical Software, Tallahassee, Fla.
Analyse-It, 2.22 Excel +, Analyse-It Software Ltd, Leeds, West Yorkshire, England.
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Appendix
Scoring system for grading and staging chronic hepatitis in dogs that was developed at Cornell University to rank histologic features of and assign qualitative copper scores for liver tissue sections.
| Grading | |
| Interface hepatitis—inflammatory cells within the portal tract extending through the limiting plate and associated with individual hepatocyte necrosis | |
| Normal (no inflammation) | 0 |
| Mild (focal, in < 50% of all portal areas) | 1 |
| Mild to moderate (focal, in ≥ 50% of all portal regions) | 2 |
| Moderate (continuous around < 50% of portal regions or septa) | 4 |
| Severe (continuous around ≥ 50% of portal regions or septa) | 5 |
| Portal inflammation—inflammatory cells in the portal tract | |
| Absent | 0 |
| Mild (< 5 inflammatory cells in some or all portal regions) | 1 |
| Moderate (≥ 5 but < 20 inflammatory cells in some or all portal regions) | 2 |
| Moderate to severe (≥ 20 but < 75 inflammatory cells in > 50% of all portal regions) | 3 |
| Severe (≥ 75 inflammatory cells in > 50% of all portal regions) | 4 |
| Lobular activity—intermittent necrosis, apoptosis, focal inflammation and hydropic degeneration (excluding fatty change and vacuolar hepatopathy) | |
| Absent | 0 |
| < 2 foci/10 × field | 1 |
| 2 4 foci/10 × field | 2 |
| 5 10 foci/10 × field | 3 |
| > 10 foci/10 × field | 4 |
| Zonal necrosis | |
| Absent | 0 |
| Zonal necrosis in < 50% of lobules | 1 |
| Zonal necrosis in ≥ 50% of lobules | 2 |
| Zonal necrosis with occasional bridging | 3 |
| Zonal necrosis with frequent bridging | 4 |
| Panlobular or multilobular necrosis | 5 |
| Fibrosis | |
| No fibrosis | 0 |
| Fibrous expansion of < 50% of portal regions with no fibrous septaa | 1 |
| Fibrous expansion of ≥ 50% of portal regions with no fibrous septaa | 2 |
| Fibrous expansion of ≥ 50% of portal regions with fibrous septaa | 3 |
| Fibrous expansion of ≥ 50% of portal regions with < 50% bridging septab | 4 |
| Fibrous expansion of ≥ 50% of portal regions with ≥ 50% bridging septab | 5 |
| Cirrhosisc | 6 |
| Copper in hepatocytes, macrophages, and lipogranuloma cells within each zone (1, 2, and 3) | |
| Not detected | 0 |
| A few copper granules in an occasional cell | 1 |
| Obvious copper granules in some cells | 2 |
| Numerous copper granules in < 50% of cells | 3 |
| Numerous copper granules in ≥ 50% but < 75% of cells | 4 |
| Numerous copper granules in ≥ 75% of cells | 5 |
The scoring system is a modification of the Ishak-Knodell scoring system.
Consists of bands of fibrosis that distort hepatic architecture but do not bridge between zones.
Consists of bands of fibrosis that extend between portal regions (zone 1 to zone 1), portal regions and terminal hepatic venules (zone 1 to zone 3), or terminal hepatic venules (zone 3 to zone 3).
Architecture of the entire liver is disrupted by fibrous septa.
Because focal damage can mimic true cirrhosis, information obtained via physical examination, diagnostic imaging, or a biopsy specimen from another region of the liver is critical. Fibrous septa may bridge or may occur as broad fibrous tracts that obliterate multiple adjacent lobules (extinction of parenchyma). Regenerative nodules may not be present if the fibrosis has developed rapidly.
