- Top of page
- Materials and Methods
Copper-associated hepatitis in the Labrador Retriever is a complex hereditary disease recently characterized in the Dutch and American Labrador Retriever populations.[2, 3] Copper accumulation in the canine liver progresses over several years without apparent clinical signs. Eventually, chronic hepatitis and cirrhosis result in liver failure at middle or old age. When diagnosed in the clinical phase, the disease often has a fatal course within a few months.
The National Research Council (NRC) provides recommendations for pet food nutrient composition with the aim to meet requirements of dogs with minimal risk of deficiency or toxicity. Recommendations are often based on values that are either extrapolated from data in growing puppies or composition of commercial maintenance diets that have not been associated with signs of deficiencies. There is no reference in these guidelines to appropriate dietary intake for animals with metal metabolism abnormalities.
Labrador Retrievers with an increased hepatic copper concentration after D-penicillamine treatment that were fed a diet with a copper content of 1.23 mg/1,000 kcal metabolizable energy (ME) had a significant decrease in their hepatic copper concentration.
Zinc salts are often used in the treatment of Wilson's disease, the best described human form of copper toxicosis. Zinc does not promote cupriuresis, like the copper chelators D-penicillamine or trientene, but creates a negative copper balance by blocking copper uptake in the enterocytes. Therefore, zinc is often used in presymptomatic patients and for maintenance therapy. Zinc induces the endogenous copper chelator metallothionein when it is absorbed into the enterocytes. Here it forms a complex with metallothionein in the cytosol. When copper enters the enterocyte, zinc is displaced by copper from the metallothionein binding site, forming a copper-metallothionein complex, which remains in the enterocyte and does not pass to the portal circulation. The enterocyte including copper will be shed into the feces. The effect of high-dose zinc salts to decrease hepatic copper in dogs has been described previously.
Currently, no data are available on the potential influence of dietary copper and zinc concentrations that are regularly present in commercial dry diets on hepatic copper and zinc concentration in Labrador Retrievers.
In this study, we collected pedigree information and analyzed liver biopsy specimens and diet samples from 55 Labrador Retrievers to investigate whether dietary copper and zinc content at levels present in commercially available dry diets were associated with hepatic copper and zinc concentration and could therefore be a potential risk factor for copper-associated hepatitis in Labrador Retrievers with a genetic predisposition for this disease.
- Top of page
- Materials and Methods
The relationship between copper and zinc in commercially available dry diet and hepatic copper concentrations was studied in Labrador Retrievers. Despite the fact that all but 4 dogs in this study population were clinically healthy, 75% had an abnormally high hepatic copper concentration. The majority of dogs in our dataset were first-generation relatives of Labrador Retrievers affected with copper toxicosis and thus might be at risk for inherited copper toxicosis.
A positive association between dietary copper levels and hepatic copper concentration and a negative association between dietary zinc levels and hepatic copper concentration were identified in this study. Dogs that were fed diet nr. 28 had a significantly lower hepatic copper concentration than dogs fed any of the other diets. This study indicates that the level of copper in pet food, as well as this level relative to zinc content, may be a factor in the accumulation of hepatic copper in Labrador Retrievers with a family history of copper-associated hepatitis.
Hepatic copper concentration in this study was measured in Tru-cut needle biopsy specimens taken with a 14G needle and an automated spring-triggered device. Biopsy specimens collected in this way are less invasive compared with biopsy specimens obtained during laporoscopy or laparotomy because they can be collected under local anesthesia causing little distress to the dog. Previous studies that compared the reliability of hepatic needle biopsies in dogs and cats to wedge biopsy specimens concluded that results from needle biopsies must be interpreted with caution.[16, 17] In those studies, 18G needle biopsies were used, that result in a much smaller biopsy specimen volume compared with the 14G biopsy needles that are used in this study. Copper accumulation in primary metabolic copper toxicosis is present in the centrolobular areas of the canine liver, but can extend to portal areas in severe cases. This distribution pattern is consistent throughout the canine liver. For a reliable estimate of copper, both centrolobular and portal areas should be present in a biopsy specimen, and a typical 14G needle biopsy specimen contains on average 15 centrolobular and portal areas. For quantitative copper measurement by instrumental neutron activation analysis we used biopsy specimens of approximately 5 mg dry weight, which meets the standard requirements when performing metal analysis of needle-core biopsy specimens.[18, 19] In advanced stages of copper-associated hepatitis, when liver cirrhosis is present, results from hepatic copper determination can become less reliable because there can be a heterogeneous distribution of tissue with fibrotic regions and regenerative nodules. Fibrotic tissue does not contain copper, and regenerative nodules usually contain less copper, depending when the biopsy sample was taken in relation to the formation of the nodule. Indeed, copper accumulation in newly formed hepatocytes takes time. Overall, in cases with chronic copper-associated hepatitis, hepatic copper concentrations are generally lower than in cases of acute copper-associated hepatitis. In this study population, 9 of 55 dogs were diagnosed with chronic hepatitis. Six of these cases had increased hepatic copper concentrations and were independently histologically classified as copper-associated chronic hepatitis by evaluation of H&E and rubeanic acid staining. The rubeanic acid staining scores correlated well with the measured hepatic copper concentrations in these cases. However, in these 6 cases, measurements of hepatic copper concentrations may have been less reliable owing to an increased chance for sampling error.
This study was set up in a cross-sectional way, which may not be the most appropriate study design to investigate a causal relationship between dietary content and hepatic copper concentrations. We optimized the reliability of the data by including only dogs that were fed commercially available dry diet for at least 1 year because hepatic copper concentrations change gradually over time and will take several months to become measurable.
In this set-up, we were able to obtain an unbiased sample of a range of dog foods and to investigate the dietary content of copper and zinc. In dogs, NRC 2006 recommended allowance for copper and zinc are 1.5 and 15 mg/1,000 kcal, respectively. NRC 2006 recommended allowance is the minimum level of an essential nutrient that should be present in commercial dog food to take into account nutrient bioavailability as well as ingredient and individual animal variabilities.
For this study we made the assumption that the dogs were fed according to their caloric needs and did not gain or lose weight in the period of 1 year preceding collection of the liver and dietary sample. To be able to compare the different types of diet in this study, we used the copper and zinc content expressed as mg/1,000 kcal energy density of the diet. Pet food companies may provide data about the copper and zinc content of their diet on the labels. This data are usually expressed as mg/kg diet as fed. The range of copper and zinc content in our dataset was 5–23 mg copper and 104–265 mg zinc/kg diet as fed, respectively. Owners should be aware of the fact that when copper and zinc content is stated on the package label, this is often the amount of copper and zinc that is added to the diet as a premix and does not include the copper or zinc that was already present in the raw foodstuff.
We choose to analyze diet composition instead of relying on the package information as this is often not accurate. The sample size of 250 g that was analyzed may not always be representative for the different production batches of diet. However, when more samples of a diet from different participants in the study were available, there appeared to be little variation (Table 2). This indicates that the conclusions drawn are likely valid.
Here we show that Labrador Retrievers fed a diet with a relatively high level of zinc and low level of copper did have significantly lower hepatic copper concentrations than dogs fed any of the other diets. The results from this study are consistent with results from a previous randomized double-blind placebo-controlled clinical trial that showed a decrease in hepatic copper concentration in dogs fed a low copper diet. Zinc supplementation did not have any additional effect on the reduction in hepatic copper concentrations. In this study, dietary zinc showed a significant negative correlation with copper concentrations in the canine liver. This is in agreement with results from studies in dogs and humans in which zinc supplementation is used in treatment of copper toxicosis.[7, 8] However, zinc concentrations used in treatment regimes are far above the supplemented amount present in canine diets. Investigation of the relative influence of dietary copper or zinc concentration or the ratio of their combination on hepatic copper and zinc level requires a prospective clinical trial.
Increased hepatic copper concentrations caused by high copper intake has been described before in several animal species, including dogs and ruminants.[22, 23] Also a positive association between dietary copper and hepatic copper concentration in people with or without a genetic predisposition is well documented.[24-28] Overall hepatic copper concentration in this study population was almost twice the upper limit for normal hepatic copper concentration in dogs. This may indicate that dietary copper content as currently present in commercial dry diets is at the high range of what dogs predisposed to hepatic copper accumulation can tolerate to keep their body copper composition within a reference range. The majority of dogs in this study population were family members of Labrador Retrievers with histologically diagnosed copper toxicosis, implying that our results may not be translatable to the general Labrador Retriever or canine population. The effect of dietary copper on hepatic copper concentration may be restricted to dogs with a genetic predisposition.
The dogs fed diet nr. 28 had significantly lower hepatic copper concentrations compared with dogs fed the other diets. Two of these dogs had normal histology, 3 showed nonspecific reactive hepatitis, 1 was a clinical case diagnosed with acute hepatitis, and 2 had subclinical chronic hepatitis. These dogs did not show histologic signs of copper-associated hepatitis. Like any other dog, the Labrador Retrievers can suffer from infectious hepatitis and there may be immune-related factors involved in some forms of hepatitis in the Labrador. This observation indicates that dogs that were fed diet nr. 28 were not in general protected from liver inflammation, but were rather protected from liver inflammation that is caused by high hepatic copper concentrations.
It was striking that despite the fact that 51 of 55 dogs in this population appeared clinically healthy, there was serious copper accumulation, which was associated with hepatitis, in the majority of the dogs. These results may indicate that copper-associated hepatitis is an underestimated health problem in the Labrador Retriever and requires more awareness of owners and veterinarians.
From pedigree analysis it is clear that copper-associated hepatitis in Labrador Retrievers is not a monogenic recessive trait as it is in the Bedlington Terrier. In Labrador Retrievers there is a complex pattern of inheritance, with a female predisposition. We believe that multiple gene variations, each with a small effect determine the phenotype in conjunction with environmental factors, like diet. Currently it is impossible to determine the risk in clinically healthy Labrador Retrievers because there are no genetic markers or biomarkers available. Further prospective evaluation of the role of dietary copper and zinc in the Labrador Retriever population requires elucidation of the underlying genotypic variation so that well-defined study groups with regard to genetic susceptibility can be formed.