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Liver samples from 163 slaughtered animals were studied to provide data regarding normal hepatic Copper (Cu) concentration, and Iron (Fe). Results show the mean and median liver Cu concentration in majority samples were normal. Lambs had higher (P

OBJECTIVE To investigate disparities in hepatic copper concentrations determined by atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and digital image analysis of rhodanine-stained sections. ANIMALS 516 dogs. PROCEDURES Medical records of dogs for which hepatic biopsy specimens had been submitted between January 1999 and December 2019 for evaluation of copper content were reviewed. Paired hepatic copper concentrations obtained with digital image analysis and AAS or ICP-MS were compared, and Spearman rank correlation coefficients were calculated to test for correlations between qualitative copper accumulation scores and hepatic copper concentrations. For dogs for which ≥ 4 rhodanine-stained hepatic sections were available, intraindividual variation in copper distribution across hepatic sections was evaluated. RESULTS Median hepatic copper concentrations obtained with digital image analysis exceeded concentrations obtained with AAS or ICP-MS. Concentrations were also higher in older dogs (≥ 9 years vs < 9 years), dogs of breeds with a typical body weight ≥ 20 kg (44 lb), and dogs with necroinflammatory changes or uneven copper distribution. Qualitative copper accumulation scores were significantly associated with hepatic copper concentrations; however, the correlation between qualitative score and concentration obtained with digital image analysis (rs = 0.94) was higher than the correlation between qualitative score and concentration obtained with AAS (rs = 0.75) or ICP-MS (rs = 0.57). The coefficient of variation for hepatic copper concentrations obtained with digital image analysis was significantly higher for dogs with higher hepatic copper concentrations. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that spectroscopic-spectrometric analysis of hepatic biopsy specimens commonly underestimated the concentration obtained by digital image analysis of rhodanine-stained sections.

Objectives The aim of this study was to assess hepatic copper concentrations and zonal distribution in cat liver specimens. Methods For this study, 121 archived, formalin-fixed, paraffin-embedded liver specimens from cats were used. Tissue sections were stained for copper with rhodanine and scored from 0 (no copper accumulation) to 5 (panlobular copper accumulation). The tissue specimens were then deparaffinized and hepatic copper concentrations were measured using flame atomic absorption spectroscopy. Results Tissue samples were categorized into four groups based on histopathologic findings: (1) no significant histopathologic hepatic changes (n = 66); (2) hepatic steatosis (n = 18); (3) inflammatory or infectious disease (n = 24); and (4) neoplasia (n = 13). Of the 121 specimens, 13 (11%) stained positive for copper, with three having a score ⩾3. Thirty-seven specimens (31%) had copper concentrations above the reference interval ([RI] s = 0.46, P Conclusions and relevance Despite the fact that 31% of specimens had copper concentrations above the RI, only 11% showed positive copper staining and only 2.5% had a score ⩾3. Our findings suggest that hepatic copper concentrations greater than the upper limit of the RI are relatively common in cats. Further studies to determine the factors that influence hepatic copper staining in cats and to establish contemporary RIs for hepatic copper in healthy cats are warranted.

A 60‐year‐old man was referred to hospital for loss of consciousness. Her late‐onset Wilson disease was diagnosed with elevated serum bilirubin, hepatic copper concentration, and urinary copper excretion. Zinc sulfate administration decreased the majority of clinical symptoms.

BackgroundCopper associated hepatitis (CAH) has been increasingly recognized in dogs, and speculation exists that hereditary defects in copper metabolism have been exacerbated by increased environmental copper exposure. However, no broad epidemiological investigations have been performed to investigate quantitative hepatic copper concentrations ([Cu]H ) over time in both dogs that are (predisposed breed [PB]), and are not (non-predisposed breed [NPB]), considered at-risk for CAH.ObjectivesTo investigate [Cu]H in dogs and explore temporal, demographic, and histologic associations spanning 34 years.Animals546 archived liver specimens.MethodsRetrospective study. Searches of the Michigan State University Veterinary Diagnostic Laboratory database identified dogs that had undergone hepatic histopathologic assessment. Cases with archived tissue were reviewed and classified by breed, time period, and presence or absence of hepatitis. Inductively coupled plasma mass spectrometry was used to determine [Cu]H .ResultsIn time period 2009-2015, median [Cu]H were 101 μg/g and 313 μg/g greater than median [Cu]H in time period 1982-1988 for NPB and PB dogs, respectively (P < .001 for both comparisons). The proportion of dogs with [CU]H > 300 μg/g increased in NPB (28% to 49%) and PB dogs (48% to 71%) during these periods (P = .002 for both comparisons). Median [Cu]H in dogs with hepatitis increased 3-fold over time in both NPB (P = .004) and PB populations (P < .001).Conclusions and clinical importanceThe frequent recognition of CAH in recent years is likely due to the observed increases in [Cu]H over time. Importantly, effects are not limited to PB dogs.

Trace mineral status is a critical component of bovine health. Impairment of physiological processes, caused by trace mineral toxicities or deficiencies, can be potential underlying factors of disease. Historically, the status of critical trace minerals, such as copper, manganese, selenium, and zinc, has been evaluated through the analysis of hepatic tissue. We assessed variation of these 4 elements between homogenized liver and samples of 0.02 g, 0.1 g, 0.5 g, and 1.0 g. We also evaluated concentration differences in copper, manganese, selenium, and zinc among samples stored under different durations. No differences in concentrations of copper, manganese, selenium, or zinc were observed among samples stored frozen for 3, 7, and 14 d post-collection. Statistical differences in concentrations of selenium and zinc were observed between 0.02-g biopsy samples and larger samples. Moisture content differed between 0.02-g biopsies and larger samples and over time. Results indicate that as little as 0.02 g of hepatic tissue dried to ~0.006 g is reliable for interpretation of trace mineral status and determination of toxicities and deficiencies in cattle pertaining to copper, manganese, selenium, and zinc, despite the small differences observed.

Introduction. Wilson’s Disease (WD) is an inherited disorder of impaired hepatic copper metabolism that leads to copper accumulation in organs such as the liver and brain. Using transcranial sonography (TCS), we investigated brain changes in WD patients during de-coppering treatment. Methods. Forty-one consecutive treatment-naive WD patients were classified as having hepatic (WDh; n = 20) or neurological WD (WDn; n = 21) based on symptoms at diagnosis; all patients received either D-penicillamine or zinc sulfate and were observed for 24 months. TCS was performed at regular intervals from study entry (month 0) to month 24. Results. At study entry, bilateral lenticular nucleus (LN) hyperechogenicity was found in 18 patients with WDn and in nine with WDh (p = 0.006). Substantia nigra (SN) hyperechogenicity was found in nine patients with WDn) and four with WDh (p = ns). After 24 months of treatment, bilateral LN hyperechogenicity was still present in 17 patients with WDn and 14 with WDh (p = ns). SN hyperechogenicity was present in one patient with WDn and two with WDh (p = ns). The decrease in the number of patients with SN hyperechogenicity was significant in the WDn group (p < 0.05). Conclusions. LN hyperechogenicity is the most common TCS abnormality in WD patients, and was observed despite two years of de-coppering treatment. SN hyperechogenicity was less common, and decreased after treatment introduction.

Copper toxicosis is a major cause of hepatitis in dogs. We have shown that variants in ATP7A and ATP7B modulate hepatic copper levels in Labrador retrievers and Dobermans. However, these variants cannot fully explain the observed variation in hepatic copper levels in these dog breeds. Homozygous deletion of exon 2 of COMMD1 causes copper toxicosis in Bedlington terriers. We investigated the possible involvement of COMMD1 in the multifactorial aetiology of copper toxicosis in Labrador retrievers and Dobermans. Thirty dogs of each breed with known hepatic copper status were selected for DNA sequence analysis of the three exons and flanking intronic regions of COMMD1. The observed variants were tested for association with hepatic copper levels by linear model analysis. Several variants were observed in the DNA sequence of COMMD1 in both Labrador retrievers (nine variants) and Dobermans (11 variants) but none of these was associated with variations of hepatic copper concentrations. We conclude that COMMD1 did not play a major role in the aetiology of copper associated hepatitis in Labrador retrievers and Dobermans.

The aim of this study was to evaluate whether zinc (Zn) supplementation protects against hepatic copper (Cu) accumulation in copper-loaded sheep. Forty cross-bred lambs were assigned to five experimental groups. These included the control group (C) and four treatment groups that received Cu and/or Zn supplementation (dry matter (DM) basis) over 14 weeks, as follows: Cu (450 mg Cu/kg), Zn-35 (450 mg Cu + 35 mg Zn/kg), Zn-150 (450 mg Cu + 150 mg Zn/kg), and Zn-300 (450 mg Cu + 300 mg Zn/kg). Blood, liver, and bile samples were obtained for mineral determination by inductively coupled plasma optical emission spectrometry (ICP&ndash, OES). The hepatic metallothionein (MT) concentrations were also determined. At the end of the experiment, hepatic Cu concentrations were higher in all Cu-supplemented groups than in C. Hepatic Cu accumulation was lower in the groups receiving the Zn supplementation than in the Cu group, although the difference was only statistically significant (66%) in the Zn-300 group. The MT concentrations tended to be higher (almost two-fold) in the Zn groups (but were not dose related) than in the C and Cu groups, and they were related to hepatic Zn concentrations. Zn supplementation at 300 mg/kg DM is useful for preventing excessive hepatic Cu accumulation in sheep exposed to high dietary concentrations of Cu.

To test the hypothesis that the metabolism of Cu in dairy cows is affected by basal forage and added S and Mo, 56 dairy cows that were 35 (standard error ± 2.2) days postcalving and yielding 38.9 kg of milk/d (standard error ± 0.91) were offered 1 of 4 diets in a 2 × 2 factorial design for a 14-wk period. The 4 diets contained approximately 20 mg of Cu/kg of dry matter (DM), and had a corn silage-to-grass silage ratio of 0.75:0.25 (C) or 0.25:0.75 (G) and were either unsupplemented (-) or supplemented (+) with an additional 2 g of S/kg of DM and 6.5 mg of Mo/kg of DM. We found an interaction between forage source and added S and Mo on DM intake, with cows offered G+ having a 2.1 kg of DM lower intake than those offered G-, but no effect on the corn silage-based diets. Mean milk yield was 38.9 kg/d and we observed an interaction between basal forage and added S and Mo, with yield being decreased in cows offered G+ but increased on C+. No effect of dietary treatment on milk composition or live weight was noted, but body condition was lower in cows fed added S and Mo irrespective of forage source. We found an interaction between forage source and added S and Mo on milk somatic cell count, which was higher in cows offered G+ compared with G-, but not in cows fed the corn silage-based diets, although all values were low (mean values of 1.72, 1.50, 1.39, and 1.67 log10/mL for C-, C+, G-, and G+, respectively). Mean plasma Cu, Fe, and Mn concentrations were 13.8, 41.3, and 0.25 µmol/L, respectively, and were not affected by dietary treatment, whereas plasma Mo was 0.2 µmol/L higher in cows receiving added S and Mo. The addition of dietary S and Mo decreased liver Cu balance over the study period in cows fed either basal forage, but the decrease was considerably greater in cows receiving the grass silage-based diet. Similarly, hepatic Fe decreased more in cows receiving G than C when S and Mo were included in the diet. We concluded that added S and Mo reduces hepatic Cu reserves irrespective of basal forage source, but this decrease is considerably more pronounced in cows receiving grass silage- than corn silage-based rations and is associated with a decrease in intake and milk performance and an increase in milk somatic cell count.

This study focused on a flock of Dala sheep with recurrent cases of chronic copper (Cu) poisoning. The seasonal variation in hepatic Cu concentration was followed in individual sheep with repeated liver biopsies, four times per year, in two consecutive years. Thirty-six ewes were included, yielding a total of 279 biopsies. Cu concentrations were measured by atomic absorption spectroscopy. Hepatic Cu concentrations remained almost stable from December to March, fell substantially from March to June, and rose sharply during the summer pasture period from June to October. There were large individual differences in hepatic Cu levels. These differences remained stable through the two years. Treatment with ammonium tetrathiomolybdate (3 × 3.4 mg per kg bodyweight (bw) s.c.) in June had only weak and inconsistent effect on hepatic Cu levels in October. The results may partly explain why chronic Cu poisoning in sheep in Norway predominantly occur in the autumn and winter months.

Objective—To evaluate the accuracy of digitally scanned rhodanine-stained liver biopsy specimens for determination of hepatic copper concentration and compare results with qualitatively assigned histologic copper scores in dogs. Sample—353 liver biopsy specimens from dogs. Procedures—Specimens (n = 139) with quantified copper concentration ranging from 93 to 6,900 μg/g were allocated to group 1 (< 400 μg/g [37]), group 2 (401 to 1,000 μg/g [27]), group 3 (1,001 to 2,000 μg/g [34]), and group 4 (> 2,001 μg/g [41]); stained with rhodanine; and digitally scanned and analyzed with a proprietary positive pixel algorithm. Measured versus calculated copper concentrations were compared, and limits of agreement determined. Influence of nodular remodeling, fibrosis, or parenchymal loss on copper concentration was determined by digitally analyzing selected regions in 17 specimens. After method validation, 214 additional liver specimens underwent digital scanning for copper concentration determination. All sections (n = 353) were then independently scored by 2 naive evaluators with a qualitative scoring schema. Agreement between assigned scores and between assigned scores and tissue copper concentrations was determined. Results—Linear regression was used to develop a formula for calculating hepatic copper concentration ≥ 400 μg/g from scanned sections. Copper concentrations in unremodeled specimens were significantly higher than in remodeled specimens. Qualitative scores widely overlapped among quantitative copper concentration groups. Conclusions and Clinical Relevance—Calculated copper concentrations determined by means of digital scanning of rhodanine-stained liver sections were highly correlated with measured values and more accurate than qualitative copper scores, which should improve diagnostic usefulness of hepatic copper concentrations and assessments in sequential biopsy specimens.

BACKGROUND AND AIMS. Determination of hepatic copper (Cu) concentration is important in Wilson's disease (WD) diagnosis. The aim of this study was to evaluate uneven distribution of liver Cu concentration and the utility of double-sample biopsy in WD diagnosis. METHODS. Thirty-five WD patients (20 male; mean age 41 ± 9 years) were enrolled in the study and double-liver samples for biopsy were obtained. A further 30 WD patients, in whom Cu determination was performed using single-liver samples, were also enrolled as controls. RESULTS. A marked difference in hepatic Cu concentration was observed between the two sample groups (p0.0001). This difference is statistically significant for all levels of liver fibrosis (p0.001) and for the comparison of hepatic and neurological phenotypes (p0.01). The sensitivity of the Cu concentrations obtained from the double-sample biopsies for the conventional cut-off value of 250 mg/g dry weight of tissue was 85.7% compared to 80% in the single-sample biopsies. By lowering the cut-off value from 250 to 50 µg/g of dry weight of tissue, the sensitivity of Cu content to diagnose WD increased to 97% for double-sample liver biopsy compared to 93% for single-sample liver biopsy. CONCLUSIONS. Liver Cu content was unevenly distributed in the WD subjects, irrespective of fibrosis levels and disease phenotypes; hence WD can be misdiagnosed using single-sample liver Cu measurement. Double-sample biopsy sensitivity is greater than that obtained with single-sample biopsy and should therefore be considered to evaluate liver Cu concentration at initial diagnosis in all patients.

In contrast to dogs, the role of copper and iron accumulation in feline hepatic disease remains poorly characterized. Therefore, the objective of the current study was to compare the amount and distribution of copper and iron accumulation for different disease processes in feline liver biopsies. Liver biopsies (from 104 privately owned cats) were categorized by primary histopathologic lesion. Copper (by rubeanic acid) and iron (by Prussian blue) accumulation were graded by amounts (0-3) and location (centrilobular, midzonal, periportal, random). The Kruskal-Wallis test and Pearson chi-square test were used to assess differences in metal grade and location, respectively, between diagnostic categories. Histologic diagnoses were normal (n = 12), congenital (n = 6), neoplastic (n = 16), infectious and/or inflammatory (n = 39), and other (n = 31). Hepatocellular iron staining was negative in 18 samples; remaining samples had grade 1 (n = 38), 2 (n = 40), and 3 (n = 8) accumulation. Ninety-two samples were negative for copper; remaining samples had grade 1 (n = 5), 2 (n = 6), and 3 (n = 1) accumulation. No significant differences were found in the amount of iron or copper accumulation between the different diagnostic categories. Diagnostic category and the location of copper or iron accumulation were not associated. Hepatic iron accumulation was common and not associated with histologic diagnosis. Hepatocellular copper accumulation was more common in cats than previously reported, had a similar pattern of distribution to fibrotic changes, and was not present in histologically normal liver biopsies.

One hundred and eight Texel sired lambs were weaned at 37 days old and allocated three days later to one of two treatments, using two pens per treatment, in order to evaluate lamb hepatic copper (Cu) values. In Treatment 1 (T1) the lambs were confined to pens having recycled plastic slatted flooring, while in Treatment 2 (T2) the lambs were confined to pens having straw bedded floors, with fresh straw added on alternate days. The lambs had ad libitum access to a coarse lamb concentrate. The crude protein (g/kg DM), crude fibre (g/kg DM), total zinc (mg/kg) and background levels of copper (Cu) (mg/kg) and molybdenum (Mo) (mg/kg) of the diet were 17.2, 10.9, 152, 9.7 and 0.92, respectively. The lambs were slaughtered when they reached about 39 kg live weight. Following slaughter, the liver was removed and weighed and the caudal lobe frozen for subsequent Cu analysis. The livers of lambs on the plastic floor treatment had higher liver weights (843 vs. 804 g, S.E.M. 11.0; P < 0.05) and higher hepatic copper (396 vs. 315 mg/kg DM, S.E.M. 18.2; P < 0.05) than those from lambs that were bedded with straw. The Cu accumulation per kg of liver DM per week was estimated to be 8.4 and 13.7 mg for lambs on the straw bedding and plastic slated treatments, respectively. It is concluded that when lambs are housed on plastic slatted flooring, relative to straw bedding, there is a higher accumulation of hepatic copper levels likely to result in a greater risk of Cu toxicity when lambs are reared on all concentrate diets.

The present study was conducted as a preliminary evaluation of toxicologic and carcinogenic risks of copper gluconate and to determine its optimal dose for use in an upcoming study utilizing the medium-term multi-organ carcinogenesis protocol. Male BrlHan:WIST@Jcl (GALAS) rats were initially treated with N-nitrosodiethylamine, N-methylnitrosourea, 1,2-dimethylhydrazine, N-butyl-N-(4-hydroxybutyl)-nitrosamine and 2,2'-dihydroxy-di-n-propylnitrosamine for a total multiple initiation period of 4 weeks. In addition, rats were fed a diet containing copper gluconate at 0, 1000, 3000, 4800, 6000, or 12000 ppm from the commencement for 13 weeks. While no systemic toxicity was detected, hepatic granulomas were observed at copper gluconate doses of 6000 ppm, or greater. Hepatic copper accumulation and metallothionein induction were demonstrated at doses of 3000 ppm and 1000 ppm, respectively, or greater. Putative preneoplastic lesions in the liver appeared to increase at a dose of 12000 ppm, and 8-hydroxydeoxyguanosine formation was enhanced at doses of 6000 ppm, or greater. These results suggest that copper gluconate exerts effects on the liver at the relatively low dietary dose of 1000 ppm and causes hepatic injury at 6000 ppm. The hepatotoxicity and possible hepatocarcinogenicity of copper gluconate may be attributable to oxidative stress due to the impairment of hepatic copper metabolism.

Copper toxicity has been described in numerous domestic species. The characteristic lesions include hemoglobinuric nephrosis and piecemeal hepatic necrosis with bile ductular hyperplasia and portal fibrosis. Certain species, such as sheep, are prone to toxicity when exposed to copper in feed, whereas an inherent genetic defect of copper storage is present in some breeds of dogs (Bedlington Terriers, West Highland White Terriers, Doberman Pinschers). In nondomestic species, reference ranges have not been established for copper in internal organs, so the establishment of copper toxicity as a diagnosis is difficult. A case of copper toxicity in a captive Mexican fruit bat is presented. Hepatic copper levels in 16 additional bats, of at least 3 different species, were measured. To the authors' knowledge, this is the first reported case of copper toxicity in a chiropteran.

BACKGROUND: Non-Wilsonian hepatic copper toxicosis includes Indian childhood cirrhosis (ICC), endemic Tyrolean infantile cirrhosis (ETIC) and the non-Indian disease known as idiopathic copper toxicosis (ICT). These entities resemble the hepatic copper overload observed in livers of Bedlington terriers with respect to their clinical presentation and biochemical and histological findings. We recently cloned the gene causing copper toxicosis in Bedlington terriers, MURR1, as well as the orthologous human gene on chromosome 2p13-p16.AIM: To study the human orthologue of the canine copper toxicosis gene as a candidate gene for ICC, ETIC, and ICT.METHODS: We sequenced the exons and the intron-exon boundaries of the human MURR1 gene in 12 patients with classical ICC, one patient with ETIC, and 10 patients with ICT to see whether these patients display any mutations in the human orthologue of the canine copper toxicosis gene.RESULTS: No mutations in the MURR1 gene, including the intron-exon boundaries, were identified in a total of 23 patients with non-Wilsonian hepatic copper toxicosis.CONCLUSIONS: Our results demonstrate that copper toxicosis in Bedlington terriers is not an animal model for the non-Wilsonian hepatic copper toxicosis described in this study.

Wilson's disease is an inherited disorder of copper metabolism in which copper accumulates and damages particularly the liver and the nervous system. Initial partial descriptions of the disorder date from the 1880s, but the first detailed clinical and pathological description was given by the British neurologist Samuel Alexander Kinnier Wilson in 1912. The associated Kayser–Fleischer (KF) rings had been described in 1902 by the German ophthalmologist Bernhard Kayser and reinforced in 1903 by Bruno Fleischer. In 1913, Rumpel reported excess hepatic copper in a patient dying of Wilson's disease, but it was not until the 1930s and 1940s that copper deposition was established in KF rings and the brain. A genetic basis for Wilson's disease was suggested by Byrom Bramwell in 1916 and by Hans Christian Hall in 1921 (who also coined the term ‘hepatolenticular degeneration’), but it was not until 1960 that the autosomal recessive mode of transmission was established unequivocally. In 1948, Cumings, on the basis of his demonstration of copper deposition in brain and liver, suggested that British anti-Lewisite (BAL) might be of value in removing copper from the body and might thereby improve the prognosis of Wilson's disease. BAL was the first effective therapy for Wilson's disease, but has subsequently been superseded by less toxic alternatives.

Objective—To determine signalments, clinical features, clinicopathologic variables, imaging findings, treatments, and survival time of cats with presumed primary copper-associated hepatopathy (PCH) and to determine quantitative measures and histologic characteristics of the accumulation and distribution of copper in liver samples of cats with presumed PCH, extrahepatic bile duct obstruction, chronic nonsuppurative cholangitis-cholangiohepatitis, and miscellaneous other hepatobiliary disorders and liver samples of cats without hepatobiliary disease. Design—Retrospective cross-sectional study. Animals—100 cats with hepatobiliary disease (PCH [n = 11], extrahepatic bile duct obstruction [14], cholangitis-cholangiohepatitis [37], and miscellaneous hepatobiliary disorders [38]) and 14 cats without hepatobiliary disease. Procedures—From 1980 to 2013, cats with and without hepatobiliary disease confirmed by liver biopsy and measurement of hepatic copper concentrations were identified. Clinical, clinicopathologic, and imaging data were compared between cats with and without PCH. Results—Cats with PCH were typically young (median age, 2.0 years); clinicopathologic and imaging characteristics were similar to those of cats with other liver disorders. Copper-specific staining patterns and quantification of copper in liver samples confirmed PCH (on the basis of detection of > 700 μg/g of liver sample dry weight). Six cats with PCH underwent successful treatment with chelation (penicillamine; n = 5), antioxidants (5), low doses of elemental zinc (2), and feeding of hepatic support or high-protein, low-carbohydrate diets, and other hepatic support treatments. One cat that received penicillamine developed hemolytic anemia, which resolved after discontinuation of administration. Three cats with high hepatic copper concentrations developed hepatocellular neoplasia. Conclusions and Clinical Relevance—Results suggested that copper accumulates in livers of cats as primary and secondary processes. Long-term management of cats with PCH was possible.

Liver tissue samples were reviewed from 35 Doberman Pinschers with chronic active hepatitis in the precirrhotic stage. Thirty dogs had elevated hepatic copper concentrations, and five had normal liver copper concentrations. The earliest changes were inflammation and scar tissue deposition around the small hepatic vein branches. There was also apoptosis of scattered hepatocytes in zone 3. Inflammation consisted of macrophages, lymphocytes, and plasma cells. As the disease progressed, collagen deposition increased around the hepatic veins; in some liver specimens, thin scar tissue septa radiated from the hepatic vein branches, and inflammation spread to include the portal tracts. The sinusoids adjacent to the scar tissue were converted to endothelial-lined, thin-walled vessels. Chronic active hepatitis (commonly referred to as Doberman hepatitis or chronic active hepatitis of Dobermans) is a progressive fibrosis, inflammation and hepatocyte loss beginning among zone 3 hepatocytes around the terminal hepatic vein branches. The histomorphologic changes were the same among those Dobermans with elevated hepatic copper and those with normal hepatic copper. The cause was not determined, but these morphologic studies support the idea of immune-mediated disease.

The Long-Evans Cinnamon (LEC) rat, an inbred mutant rat derived from the Long-Evans strain, is characterized by spontaneous hepatitis due to gross accumulation of hepatic Cu. The accumulation, accompanied by marked induction of metallothionein (MT), is believed to be due to the inherent lack of output of Cu into the bile duct and blood vessels. In this study, the acute effect of tetrathiomolybdate (TTM), a chelator for output of hepatic Cu and Cd in LEC rats treated with Cd, was investigated. Female LEC rats were injected subcutaneously with Cd (Cd; 1.0 mg/kg) to induce Cd, Cu-MT. Fischer rats were treated with Cd (Cd; 1.0 mg/kg) and Cu (Cu; 3.0 mg/kg). Forty-eight hours after the injections of metals, TTM (5 mg/kg bw) was injected intravenously under anesthesia. The TTM injection rapidly stimulated biliary excretions of Cu (at a microgram/ml level) and Cd (at a ng/ml level). Furthermore, Cu and Cd concentrations were increased in serum sampled 60 min after the TTM injection. The increase of biliary Cu excretion was not accompanied by increased biliary excretion of MT. The TTM injection caused the hepatic Cu concentrations to decrease from 306 +/- 2 to 262 +/- 12 and from 43 +/- 6 to 20 +/- 5 micrograms/g in LEC and Fischer rats, respectively. The hepatic Cd concentration was not decreased by TTM treatment. Hepatic MT and Cu, but not Cd, concentrations in the MT fraction were also reduced by TTM injection. Our results showed that TTM can rapidly remove Cu from MT to increase bile and blood Cu levels. The output of Cd stimulated by TTM injection may be related to MT reduction resulting from removal of MT-bound Cu. Our results indicate that to avoid the toxic effect of Cu, TTM injection is an effective initial treatment, although it remains to be established how metals, including Cu, are finally metabolized.

Objective—To evaluate differences in hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis. Design—Retrospective case-control study. Sample—Liver tissue specimens from 36 Labrador Retrievers with chronic hepatitis and 36 age- and sex-matched Labrador Retrievers without chronic hepatitis (control dogs). Procedures—Liver tissue specimens were obtained during 2 study periods (1980 to 1997 and 1998 to 2010). For each tissue specimen, a histologic score was assigned independently by each of 2 interpreters, and the hepatic copper concentration was qualitatively determined via rhodanine staining and quantitatively determined via atomic absorption spectroscopy. Results—Mean hepatic copper concentration was significantly higher in dogs with chronic hepatitis (614 μg/g of dry weight [range, 104 to 4,234 μg/g of dry weight]), compared with that in control dogs (299 μg/g of dry weight [range, 93 to 3,810 μg/g of dry weight]), and increased significantly over time. A higher proportion of liver tissue specimens collected during the 1998–2010 study period had hepatic copper concentrations > 400 μg/g of dry weight (the upper limit of the reference range), compared with the proportion of liver tissue specimens collected during the 1980–1997 study period. The qualitative copper score did not accurately predict quantitative hepatic copper concentration in 33% of study dogs. Conclusions and Clinical Relevance—Results suggested that the increase in hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis over time may be the result of increased exposure of dogs to environmental copper, most likely via the diet.

Summary Earlier studies with ruminants point to a depressant effect of dietary iron on the copper status. To verify this we determined hepatic copper concentrations in dry, non‐pregnant goats subjected to a 56×56‐days cross‐over trial with adequate copper rations containing either 269 or 2380 mg iron/kg dry matter. High iron intake reduced the group mean plasma copper (by 18%) and caerulo‐plasmin activity (by 13%) and produced a significant decrease (27%) in hepatic copper concentrations. Hepatic iron concentrations were raised (by 56%) after feeding the high iron ration. It is concluded that high dietary levels of iron, within the range of their fluctuation in silage and forage, can impair the copper status of ruminants, especially when concurrent intakes of copper are low.

Background/Aims: Determination of hepatic copper concentration is important in the diagnosis of Wilson's disease. We studied copper distribution in the cirrhotic liver of a patient who died of Wilson's disease. Methods: A liver slice extending from the left to the right lobe was divided into 38 samples. Each sample was analyzed for copper content by Induced Coupled Plasma Atomic Emission Spectroscopy. Results: The mean copper concentration in the liver was 1370 μg/g dt. A striking variability, up to 2–3-fold, in copper levels was observed between the samples: the copper concentration ranged from 880 to 2100 μg/g dt, with significant differences even between adjacent samples. Lobar differences were also observed, with a tendency of the right lobe to accumulate more copper than the left lobe. Histochemical analyses confirmed the uneven distribution of copper even at the acinar level. Copper was mainly stored in periportal hepatocytes (zone 1) and at the periphery of the regenerating nodules. Moreover, we observed some nodules with the majority of hepatocytes full of copper granules, adjacent to areas of parenchyma negative for copper stains. Conclusions: Our data show that: 1) copper is unevenly distributed in Wilson's disease in the cirrhotic stage; 2) a lobar pattern of copper distribution is evident in this case, characterized by a higher copper concentration in the right lobe; 3) the observed lobal pattern is different from that described in the newborn liver, characterized by a higher copper content in the left compartment of the liver; 4) copper content determined in a small liver sample cannot be considered as absolutely representative of the mean hepatic copper concentration. From a practical point of view, our data show that sampling variability deserves more consideration in the diagnosis and in the monitoring of Wilson's disease. The use of hepatic copper concentration in monitoring the efficacy of the copper-chelating therapy may be unreliable, particularly in the cirrhotic stage, because of the patchy distribution of copper, as demonstrated in this study.

原発性胆汁性肝硬変(PBC)における銅の肝組織内分布および半定量を,組織化学および今回初めてカラー表示を用いて銅の蓄積量の高低を明示する波長分散型電子線マイクロアナライザー法(EMA)を用いて,Scheuerの病期別に検討し,また他の肝疾患と比較検討した.PBC117例中orcein陽性顆粒は黄疸例(血清総ビリルビン2.0mg/dl以上)29例中全例で,無黄疸例(血清総ビリルビン2.0mg/dl未満)88例中28例に陽性であった.EMA法による肝組織内銅の分布は,PBCでは門脈周囲肝細胞が主体で,病期進展例では中間帯にも認められた.組織内銅の電顕像との対比では,核内に分布はほとんどみられず細胞質内に認められた.以上よりEMA法は従来の銅結合蛋白染色や銅染色法に比し,銅の検出に鋭敏であり,分布および半定量性に優れていることが明らかであった.また組織化学的変化との関連から,肝細胞内の銅はPBCの病変の増悪,進展に関与するものと推測された.

J Vet Intern Med. 2012 Nov;26(6):1274-80. doi: 10.1111/j.1939-1676.2012.01001.x. Epub 2012 Sep 24. Association of dietary copper and zinc levels with hepatic copper and zinc concentration in labrador retrievers. Fieten H, Hooijer-Nouwens BD, Biourge VC, Leegwater PA, Watson AL, van den Ingh TS, Rothuizen J. Source Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Abstract BACKGROUND: Copper-associated hepatitis is an inherited disease in the Labrador Retriever. Apart from genetic factors, dietary intake of copper and zinc are suspected to play a role in the pathogenesis. OBJECTIVES: To investigate whether dietary copper and zinc levels of commercially available dry diets are associated with hepatic copper and zinc concentrations in Labrador Retrievers. ANIMALS: Fifty-five Labrador Retrievers that were fed a single brand and type of commercial dry food for at least 1 year. Of these, 44 dogs were family members of Labrador Retrievers with copper-associated hepatitis. METHODS: Liver biopsies, blood samples, and diet samples were obtained. Liver specimens were scored histologically and copper and zinc concentrations were quantified. Dietary concentrations of copper and zinc were measured. The association between dietary intake of copper and zinc and hepatic copper and zinc concentrations was investigated by linear regression analysis. RESULTS: High dietary copper and low dietary zinc levels were significantly associated with high hepatic copper levels. No association between dietary intake and hepatic zinc was present. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary copper and zinc at current levels in commercially available dry dog food can influence hepatic copper and can be a risk factor for the development of copper-associated hepatitis in Labrador Retrievers with a genetic susceptibility to copper. Copyright © 2012 by the American College of Veterinary Internal Medicine. PMID: 22998127 [PubMed - in process]