Hepatic Copper Concentrations Research Articles

P wave dispersion is prolonged in patients with Wilson’s disease

To investigate the P wave dispersion as a non-invasive marker of intra-atrial conduction disturbances in patients with Wilson's disease. We compared Wilsonos disease patients (n = 18) with age matched healthy subjects (n = 15) as controls. The diagnosis was based on clinical symptoms, laboratory tests (ceruloplasmin, urinary and hepatic copper concentrations). P wave dispersion, a measurement of the heterogeneity of atrial depolarization, was measured as the difference between the duration of the longest and the shortest P-waves in 12 lead electrocardiography. All the patients were asymptomatic on cardiological examination and have sinusal rhythm in electrocardiography. Left ventricular and left atrial diameters, left ventricular ejection fraction and left ventricular mass index were similar in both groups. The Wilson's disease patients had a significantly higher P wave dispersion compared with the controls (44.7 +/- 5.8 vs 25.7 +/- 2.5, P < 0.01). There was an increase in P wave dispersion in cardiologically asymptomatic Wilson's disease patients which probably represents an early stage of cardiac involvement.

食品成分との相互作用によるミネラルの生体内利用性に関する研究

Both the lifestyle and nutrient intake have been improving in Japan; however, it is recognized that a high energy and high fat intake can lead to possible health problems. Furthermore, the intake of vitamins and minerals still tends to be low, this having resulted in a considerable number of vitamin and mineral supplements appearing on the market and being claimed to be functional nutrients. Although the physiological functions of vitamins and minerals have been primarily established by evidence-based nutrition, the interactions of minerals and vitamins with other food factors remain unclear. This present study examines the effects of several food factors on mineral bioavailability by using animal models.In order to determine the effect of dietary egg on iron deficiency, weanling male rats were fed on a normal diet, an iron-deficient diet, and an iron-deficient diet supplemented with 5% egg powder. The hemoglobin (Hb) level was decreased, and the heart weight, hepatic copper concentration, and the activities of metallo-enzymes were increased by iron deficiency. However, the supplementation of 5% egg powder improved the effects caused by this iron deficiency. In another experiment, egg iron also improved the decrease in Fib level due to iron deficiency, this observation being similar to that with rats fed on a meat diet which contained heme iron.The level of phytate in tempeh, a fermented soy product, is reduced during the fermentation process, so the iron bioavailability of tempeh was compared with that of unfermented soybean by using iron-deficient weanling male rats. The antioxidative efficacy of tempeh was also examined. The results of this study confirmed that, compared with unfermented soybean, the fermented soybean tempeh increased the level of hepatic iron, an indicator of iron storage, without promoting lipid peroxidation in the iron-deficient rats.The level of phosphatidylcholine hydroperoxide (PCOOH) is increased in the liver of iron-deficient rats. In order to determine the antioxidative effects of dietary β-carotene and α-tocopherol, and their metabolism, weanling male rats were fed iron-deficient diets containing four different levels of β-carotene and three different levels of α-tocopherol. The increased PCOOH level due to iron deficiency was suppressed by the supplementation of β-carotene, although no dose-dependence was apparent. In contrast, the antioxidative efficacy of α-tocopherol was reduced in the liver and plasma of the iron-deficient rats. The metabolism of both β-carotene and α-tocopherol was inhibited by this iron deficiency.Fructooligosaccharides (FOS), indigestible sugars, are well known enhancers of mineral absorption by the large intestine. It was found that FOS increased the bioavailability of daidzein, a major isoflavone found in soybean, and enhanced the transformation of daidzein to equol, a more biologically active metabolite than daidzein, by stimulating the intestinal microflora in rodents. Equol may have a beneficial effect on bone loss in rodents from osteoporosis and osteonenia models.Magnesium (Mg) deficiency induces kidney calcification, bone loss and hyperlipidemia in rodents. On the other hand, hesperidin, a citrus flavonoid, regulates hepatic cholesterol synthesis by inhibiting the activity of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase. It has been reported that statins, cholesterol-lowering agents, induce bone formation and inhibit bone resorption in vitro and in vivo. Thus, the effects of hesperidin and α-glucosyl (αG) -hesperidin, which is 10, 000 times more soluble in water than hesperidin, on bone and lipid metabolism was examined in Mg-deficient rats. Hesperidin administration not only reduced the cholesterol level in the serum and liver, but also inhibited bone loss.

Effect of dietary iron supplement on growth, haematology and microelements of juvenile grouper, Epinephelus coioides

An experiment was conducted to investigate the effect of dietary iron supplement on growth, haematology and microelements of juvenile grouper, Epinephelus coioides. Casein–gelatine-based diets supplemented with 0, 50, 100, 150, 200 and 250 mg kg−1 iron from ferrous sulphate were fed to grouper (mean initial weight: 21.0 ± 0.2 g) for 8 weeks. Weight gain was highest in fish fed the diet supplemented with 100 mg kg−1 iron, intermediate in fish fed diets with 50, 150, 200 and 250 mg kg−1 iron and lowest in fish fed the basal diet. Feed efficiency followed a similar trend except that the lowest value was in fish fed the basal diet and the diet supplemented with 250 mg kg−1 iron. Hepatic iron was highest in fish fed diets supplemented with iron ≥100 mg kg−1, followed by fish fed diet with 50 mg kg−1 iron and lowest in fish fed the basal diet. The whole-body iron was lowest in fish fed the basal diet but not significantly different from other groups, as judged by anova. Iron supplement to the basal diet had no significant effect on haematological parameters (red blood cell count, haematocrit and haemoglobin), hepatic copper concentration or manganese, zinc concentration in liver and whole body. Broken-line analysis of hepatic iron indicated that iron supplementation of 100 mg kg−1 satisfied the hepatic iron storage and that further supplementation did not expand the iron status.

Late-Onset Wilson’s Disease

The clinical symptoms and age at onset of Wilson's disease (WD) are highly variable. This study investigated patients who became symptomatic at >40 years of age. Clinical features, laboratory data, and mutation analysis were evaluated in 46 (3.8%) of 1223 patients who were investigated in a multinational study on genotype-phenotype correlations (1053 index patients, 170 siblings) who were >40 years of age at onset of symptoms and, in 2 asymptomatic siblings, diagnosed at >40 years of age. Thirty-one patients presented with neurologic symptoms (mean age, 44.5 years; range, 40-52; male/female, 14/17), 15 presented with liver disease (mean age, 47.1 years; range, 40-58; male/female, 6/9), and 2 were asymptomatic siblings. Hepatic copper content was measured in 17 patients and was above 250 microg/g dry weight in 13. One patient with hepatic presentation had "fulminant" WD, the remaining 14 abnormal liver function tests and/or hepatomegaly. Liver biopsy specimens were available in 13 patients presenting with liver disease (cirrhosis, 10; chronic hepatitis, 2; steatosis, 1; no abnormalities, 1) and in 14 neurologic patients (cirrhosis, 9; advanced fibrosis, 1; chronic hepatitis, 2; no abnormalities, 2). Twenty-seven of the 46 index cases had mutations on both chromosomes (including 13 H1069Q/H1069Q), 13 on just 1 chromosome. Late-onset WD is a frequently overlooked condition. The diagnostic features and the frequency of late-onset WD gene mutations were not different than in patients with an earlier onset of disease. Factors other than ATP7B mutations may modify the phenotypic presentation of WD.

Hepatic 64Cu excretion in Dobermanns with subclinical hepatitis

To investigate whether Dobermanns have impaired copper excretion an intravenous radioactive copper isotope ( 64Cu) was used as a tracer. Five patients and eight normal dogs (5 normal Dobermanns and 3 Beagles) were studied. The five female Dobermann patients had a subclinical hepatitis and an increased hepatic copper concentration (median 822 mg/kg, range 690–1380 mg/kg dry matter). The normal dogs, five Dobermanns and three Beagles, had no abnormal liver histopathology and hepatic copper concentrations were considered normal (Dobermanns; median 118 mg/kg, range 50–242 mg/kg dry matter; Beagles; median 82 mg/kg, range 50–88 mg/kg dry matter). Cholestasis was excluded in all dogs by means of a 99mTc-Bis-IDA hepatobiliary scintigraphy. Plasma clearance of 64Cu was comparable in all dogs with no statistically significant differences. The excretion of 64Cu into the bile, although not statistically significant, was less for the Dobermanns with subclinical hepatitis compared to the normal dogs. The findings suggest that impaired copper excretion may play a role in the aetiology of chronic hepatitis in the Dobermann.

Diagnostic criteria for acute liver failure due to Wilson disease

To describe the diagnostic criteria for acute liver failure due to Wilson disease (WD), which is an uncommon cause of acute liver failure (ALF). We compared findings of patients presenting with ALF due to WD to those with ALF of other etiologies. Previously described criteria, such as low alkaline phosphatase activity, ratio of low alkaline phosphatase to total bilirubin or ratio of high aspartate aminotransferase (AST) to alanine aminotransferase (ALT), failed to identify patients with ALF due to WD. There were significant differences in low ALT and AST activities (53 +/- 43 vs 1982 +/- 938, P < 0.0001 and 87 +/- 44 vs 2756 +/- 2941, P = 0.037, respectively), low choline esterase activity (1.79 +/- 1.2 vs 4.30 +/- 1.2, P = 0.009), high urine copper concentrations (93.4 +/- 144.0 vs 3.5 +/- 1.8, P = 0.001) and low hemoglobin (7.0 +/- 2.2 vs 12.6 +/- 1.8, P < 0.0001) in patients with ALF caused by WD as compared with other etiologies. Interestingly, 4 of 7 patients with ALF due to WD survived without liver transplantation. In ALF, these criteria can help establish a diagnosis of WD. Where applicable, slit-lamp examination for presence of Kayser-Fleischer rings and liver biopsy for determination of hepatic copper concentration still remain important for the diagnosis of ALF due to WD. The need for liver transplantation should be evaluated carefully as the prognosis is not necessarily fatal.

Metal bioaccumulation in the greater white-toothed shrew, Crocidura russula, inhabiting an abandoned pyrite mine site

Hepatic and renal concentrations of iron, magnesium, zinc, lead, copper, manganese, mercury, cadmium, molybdenum, chromium, and nickel were quantified in shrews ( Crocidura russula) inhabiting a pyrite mine site in Portugal. Several morphological parameters (body weight, residual index, and relative weights) were also examined to clarify the physiological effects of pollution. Shrews from the mine showed increased bioavailability of Fe, Pb, Hg, Cd, Mo, and Ni in comparison with reference specimens. Adult shrews had the highest Cd levels while Cr and Ni concentrations diminished. Intersexual differences were found for Mo and Ni. As a consequence of metal pollution, the relative hepatic weight was higher in shrews from the mine site when compared with reference specimens. These data indicate that C. russula is a good bioindicator of metal pollution. We also evaluated the toxic effects of Pb, Hg, Cd, and Ni, because several shrews from the polluted site showed high concentrations of these metals. To approximate at the real biological impact of abandoned mines, after this first step it is necessary to associate the bioaccumulation levels and morphological effects with other physiological, ecological and genetical biomarkers.

Diagnosis and Management of Wilson's Disease

To report on the diagnostic features, management, and clinical outcome after different treatments of Wilson's disease patients followed over a mean period of 15 years. Thirty-five patients with Wilson's disease referred to the University of Padova's Department of Gastroenterology for diagnosis or treatment were observed for a mean 15 years. The diagnosis was based on clinical symptoms, laboratory tests (ceruloplasmin, urinary, and hepatic copper concentrations), and uptake of the radiostable isotope Cu into the plasma protein pool. Hepatic Cu content was measured by regular follow-up biopsies. Neurologic outcome after therapy was assessed using a newly developed scoring system. Twenty-three (65.7%) patients presented with liver disease; 12 (34.3%) had mixed neurologic and hepatic involvement. All patients had been initially treated with either penicillamine (23) or zinc sulfate (12). The neurologic symptoms became worse or remained stationary in 75% of those treated with penicillamine, whereas zinc treatment improved these symptoms in 90% of treated cases. Both treatments were effective in improving the hepatic symptoms. No differences in hepatic Cu content emerged between follow-up biopsies in either treatment group. Six patients (26%) had to abandon the penicillamine treatment due to side effects. In all, 4 patients underwent liver transplantation, which was successful in 3, with a mean survival after transplantation of 4.6 years; the fourth, who had a severe neurologic impairment, died of central pontine myelinolysis. Penicillamine and zinc can effectively treat Wilson's disease, though the side effects of penicillamine may be severe enough to prompt its suspension. Liver transplantation remains the treatment of choice for end-stage liver disease.

Late Onset Wilson Disease Frequently Overlooked

Wilson disease (WD) is a rare autosomal recessive inherited disorder of copper metabolism and is characterized by excessive deposition of copper in the liver, brain, and other tissues. It’s frequency worldwide ranges from 1 case per 30,000 population in Japan to 1 case per 100,000 population in Australia. The increased frequency in certain countries is due to high rates of consanguinity. The genetic defect, localized to chromosome arm 13q, has been shown to affect the copper-transporting adenosine triphosphatase (ATPase) gene (ATP7B) in the liver. The major physiologic aberration is excessive absorption of copper from the small intestine and decreased excretion of copper by the liver. Patients with WD usually present with liver disease during the first decade of life or with neuropsychiatric illness during the third decade. In a study presented in Los Angeles, May 21 at Digestive Disease Week 2006, collaborators from Austria, Hungary, Germany, Turkey, and Poland point out that some people with WD may present much later in life and even may be in their seventies. The researchers state that late onset WD is “a frequently overlooked condition.” Of 1034 patients in this ongoing, multinational study on genotype-phenotype correlations, 85 (8.25) were age 35 years or older at onset of symptoms. Forty-six presented with neurologic symptoms (mean age, 40.8; range, 35–52; male/female, 21/25), 36 with liver disease (41.3 range, 35–58; male/female, 16/20). Three were asymptomatic siblings. Diagnosis was made at presentation only in 38 (45%), and in the remaining patients, there was delay in diagnosis of up to 15 years. The study found that Kayser-Fleischer (KF)-rings were present in 12 with hepatic presentation (33.3%) and in 34 (74%) patients with neurologic disease. Eleven patients had normal serum ceruloplasmin (13%). Hepatic copper content was measured in 31 patients and was above 250 μg in 24. Among the patients with hepatic presentation, 3 had fulminant hepatic failure and underwent liver transplantation, one had hemolytic anemia, 23 compensated or decompensated cirrhosis, and 7 just abnormal LFTs (liver biopsy showed chronic hepatitis in 2, steatosis in 3, and no abnormalities in 2). Liver biopsy was performed in 24 patients with neurologic presentation: 16 had cirrhosis, 3 advanced fibrosis, 2 chronic hepatitis, 1 mild steatosis, and 2 were without abnormalities. The study thus far found no significant phenotypic-genotypic correlations for late onset WD. “Surprisingly one third of the patients had only mild liver disease,” the researchers state, adding that the frequency of the observed WD gene mutations was not different than that of patients with an earlier onset of disease. “Apparently, factors other than ATP7B mutations may modify the phenotypic presentation of WD.”

Iron removal by phlebotomy for the prophylaxis of fulminant hepatitis in a Wilson disease model of Long-Evans Cinnamon Rats

Long-Evans Cinnamon rats are an animal model of Wilson disease with a compound load of copper and iron. However, the mechanisms of the high mortality rate from fulminant hepatitis in the rats remain undetermined. In this study, phlebotomy as an alternative for an iron-deficient diet was investigated to modify hepatitis in the rats. One group of female rats was treated with phlebotomy twice a week, and the other group received blood tests only every 2 weeks. Liver specimens were examined for biochemical non-heme iron and copper, and histochemical trivalent iron. Although all rats examined had icteric hepatitis around the age of 20 weeks, the mortality of fulminant hepatitis was lower in phlebotomized rats than in control rats (1/18 versus 8/20, p<0.05). Phlebotomy not only ameliorated the severity of hepatitis, but also reduced serum malondialdehyde. Non-heme iron in the liver was the greatest in victims of fulminant hepatitis in the control group. There was no difference in the hepatic copper content between the two groups. The prognosis of hepatitis in the rats with Wilson disease was hepatic non-heme iron-dependent, and iron removal by phlebotomy was an effective prophylaxis against fulminant hepatitis.

Poor cognitive development and abdominal pain: Wilson's disease

An 8-year-old boy was referred to our hospital because of learning disabilities. His general cognitive functions were below the level for age, and he was diagnosed with dysphasia. The boy was transferred to a special class for children with learning problems. Three months later he was again referred to us because of acute epigastric pain. The only abnormal laboratory finding was a slightly elevated level of alanine aminotransferase. Although the symptoms disappeared in a few days, the transaminase levels remained above normal for the next 6 months. Further diagnostic work-up revealed low serum ceruloplasmin concentration and high 24-h urinary copper excretion. The hepatic copper concentration in liver biopsy was high (2900 µg/g dry weight), confirming the diagnosis of Wilson's disease. Brain MRI showed slight changes in white matter. The patient's asymptomatic sister was also diagnosed with Wilson's disease. Both siblings started penicillamine therapy and a copper-restricted diet. The copper content of the household water was found to be above average and a new plumbing system was installed. After 1 year from the initiation of the therapy, the transaminase concentrations normalized and both siblings were free of symptoms. After 2 years of therapy the patient was able to return to normal school. Wilson's disease must be borne in mind, when children are evaluated because of poor school performance, especially if they complain of abdominal symptoms

Accumulation of Copper Induces DNA Strand Breaks in Brain Cells of Long-Evans Cinnamon (LEC) Rats, An Animal Model for Human Wilson Disease

Copper accumulation and induction of DNA strand breaks were investigated in the brain of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease that is a heritable disease of copper accumulation and copper toxicity in the liver, kidney and brain. Copper contents in the brain of LEC rats increased from 20 weeks of age and were approximately 3.5 to 6 folds higher than those in the brain of WKAH rats at 24 weeks of age. Hepatic copper contents in LEC rats increased from 4 to 12 weeks of age in an age-dependent manner, and then decreased from 16 to 20 weeks of age. Thus, we consider that copper accumulated in the liver was released from severely damaged hepatocytes and deposited in the brain, although copper contents in the brain were 1/20-fold lower than those in the liver. We also evaluated the amounts of DNA single-strand breaks (SSBs) in the brain by comet analysis. The proportions of nuclei in the cerebrum and cerebellum without DNA damage decreased, and nuclei with severe DNA damage appeared in LEC rats at 24 weeks of age. The comet scores of cerebrum and cerebellum cells significantly increased in LEC rats and were significantly higher than those in WKAH rats at 24 weeks of age. The results show that SSBs in LEC rat brain cells are induced at a lower concentration of copper than are SSBs in hepatic cells.

Copper Metabolism and Oxidative Stress in Chronic Inflammatory and Cholestatic Liver Diseases in Dogs

Inherited defects of copper metabolism resulting in hepatic copper accumulation and oxidative-stress might cause breed-associated forms of hepatitis. Biliary excretion is the major elimination route of copper, therefore increased hepatic copper concentrations could also be caused by cholestasis. The aim of this study was to find criteria to determine whether copper-accumulation is primary or occurs secondary to hepatitis. Liver samples of Bedlington Terriers with copper toxicosis (CT), breeds with non-copper-associated chronic extrahepatic cholestasis (EC) or chronic hepatitis (CH), and healthy dogs were used. Copper metabolism was analyzed by means of histochemical staining (copper concentration) and quantitative reverse transcriptase polymerase chain reaction (Q-PCR) on copper excretion/storage (ATOX1, COX17, ATP7A, ATP7B, CP, MT1A, MURR1, XIAP). Oxidative stress was measured by determining GSH/GSSG ratios and gene-expression (SOD1, CAT, GSHS, GPX1, CCS, p27KIP, Bcl-2). Results revealed 5+ copper in CT, but no or 1-2+ copper in EC and CH. Most gene products for copper metabolism remained at concentrations similar to healthy dogs. Three clear exceptions were observed in CT: 3-fold mRNA increase of ATP7A and XIAP and complete absence of MURRI. The only quantitative differences between the diseased and the control groups were in oxidative stress, evidenced by reductions in all GSH/GSSG ratios. We conclude that 3+ or higher histochemical detection of copper indicates a primary copper storage disease. The expression profile of copper-associated genes can be used as a reference for future studies on copper-associated diseases. All 3 diseases have reduced protection against oxidative stress, opening a rationale to use antioxidants as possible therapy.

Copper-Associated Chronic Hepatitis in Labrador Retrievers

This study summarizes the clinical and pathologic findings in 15 Labrador Retrievers with copper-associated chronic hepatitis (CACH). Our hypothesis was that this form of hepatitis is caused by a defect in hepatic copper metabolism, which most likely originates from a genetic defect. Affected Labradors consisted of 11 female and 4 male Labrador Retrievers. Eight family members of 2 of these patients were examined prospectively, as were 6 unrelated healthy Labrador Retrievers. All dogs were registered at the breed club. The average age at clinical presentation was 7 years (range, 2.5-10.5 years). All dogs were presented for anorexia, which was associated with vomiting in 8 patients. The diagnosis of CACH was based on histologic examination of liver biopsy specimens in all dogs, including semiquantitation of copper. A disproportionate increase in alanine aminotransferase (ALT) activity relative to alkaline phosphatase (ALP) activity, as well as the centrolobular localization of copper and the association of copper accumulation with hepatic lesions, suggested a primary copper storage disease rather than primary cholestatic liver disease causing copper accumulation. Mean hepatic copper concentration measured in related Labradors was 1,317 microg/g dry weight liver (range, 402-2,576 microg/g). Mean hepatic copper concentration of unrelated normal Labradors was 233 microg/g dry weight liver (range, 120-304 microg/g). Our findings support the hypothesis that a hereditary form of hepatitis occurs in Labrador retrievers and is caused by a defect in hepatic copper metabolism.

Diagnostic Value of Quantitative Hepatic Copper Determination in Patients With Wilson’s Disease

A 5-fold increase of hepatic copper concentration is considered as the best available test for diagnosis of hepatic Wilson's disease (WD). However, the sensitivity and specificity of this test have never been fully investigated. Copper content was measured by flame atomic absorption spectroscopy in 114 liver biopsies obtained at diagnosis of WD, in 219 patients with noncholestatic liver diseases (including 144 with chronic hepatitis C and 44 with nonalcoholic fatty liver disease), and in 26 without evidence of liver disease. Liver copper content was >250 microg/g in 95 WD patients (83.3%), between 50 and 250 microg/g in 15, and below 50 microg/g in 4. It did not correlate with age (r(2) = .003), the grade of fibrosis, or the presence of stainable copper. Liver copper content was >250 or between 50 and 250 microg/g in 3 (1.4%) and 20 (9.1%) of 219 patients with noncholestatic liver diseases, respectively. By lowering the cutoff from 250 to 75 microg/g, the sensitivity of liver copper content to diagnose WD increased from 83.3% (95% confidence interval, 75.2%-89.6%) to 96.5% (91.3%-99.1%), but the specificity decreased from 98.6% (96.0%-99.7%) to 95.4% (91.8%-97.8%). There is no gold standard for the diagnosis of WD. Liver copper content is a useful parameter, but a value below 250 microg/g does not exclude WD. Diagnosis requires the combination of a variety of clinical and biochemical tests.

Effect of sodium phytate supplementation on fat digestion and cholesterol metabolism in female rats

The effects of sodium phytate supplementation on fat digestion and cholesterol metabolism were investigated in female rats. On the basis of an in vitro experiment showing that phytate raised the solubility of bile acids, it was predicted that phytate feeding would depress faecal bile acid excretion, raise apparent fat digestibility and elevate serum cholesterol concentrations. The experimental diets with or without sodium phytate were either cholesterol-free or cholesterol-rich and had a normal calcium concentration. Rats fed on the cholesterol-rich diet with sodium phytate showed enhanced faecal bile acid excretion, but there was no effect on fat digestibility. In rats fed the cholesterol-free diets, phytate did neither affect fat digestion nor bile acid excretion. Sodium phytate inclusion in the cholesterol-rich diet raised serum cholesterol concentrations, but reduced liver cholesterol concentration. Thus, the in vivo data do not agree with the in vitro observations. Both phytate and cholesterol feeding influenced mineral and trace element metabolism. Liver zinc concentrations were raised by phytate feeding. Cholesterol consumption reduced hepatic concentrations of copper, iron and zinc. Both phytate and cholesterol feeding reduced the apparent absorption of calcium, magnesium and phosphorus.

Differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in Doberman pinschers.

BackgroundThe role of copper accumulation in the onset of hepatitis is still unclear. Therefore, we investigated a spontaneous disease model of primary copper-toxicosis in Doberman pinschers so to gain insights into the pathophysiology of copper toxicosis, namely on genes involved in copper metabolism and reactive oxygen species (ROS) defences.ResultsWe used quantitative real-time PCR to determine differentially expressed genes within a target panel, investigating different groups ranging from copper-associated subclinical hepatitis (CASH) to a clinical chronic hepatitis with high hepatic copper concentrations (Doberman hepatitis, DH). Furthermore, a non-copper associated subclinical hepatitis group (N-CASH) with normal hepatic copper concentrations was added as a control. Most mRNA levels of proteins involved in copper binding, transport, and excretion were around control values in the N-CASH and CASH group. In contrast, many of these (including ATP7A, ATP7B, ceruloplasmin, and metallothionein) were significantly reduced in the DH group. Measurements on defences against oxidative stress showed a decrease in gene-expression of superoxide dismutase 1 and catalase in both groups with high copper. Moreover, the anti-oxidative glutathione molecule was clearly reduced in the DH group.ConclusionIn the DH group the expression of gene products involved in copper efflux was significantly reduced, which might explain the high hepatic copper levels in this disease. ROS defences were most likely impaired in the CASH and DH group. Overall, this study describes a new variant of primary copper toxicosis and could provide a molecular basis for equating future treatments in dog and in man.

Treatment of Wilson’s disease with zinc from the time of diagnosis in pediatric patients: A single-hospital, 10-year follow-up study

Wilson's disease (WD) is an inherited disorder of copper metabolism characterized by a failure of the liver to excrete copper, leading to its accumulation in the liver, brain, cornea, and kidney, with resulting chronic degenerative changes. It is generally accepted that "presymptomatic" patients--in whom WD is diagnosed in childhood and who are defined as those who, although still asymptomatic, do have liver disease, as indicated by increased serum concentrations of transaminases--should be treated prophylactically. Here we report our results in 22 children treated with continuous oral zinc therapy for 10 years. Zinc sulfate was administered at a dosage of 25 mg elemental zinc twice a day until the age of 6 years, 25 mg three times a day between the ages of 7 and 16 years or until the child attained a body weight of 125 lb, and 50 mg three times a day thereafter. Five years after the start of zinc treatment, we noted highly significant decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and urinary copper excretion, but white blood cell counts did not vary significantly. Six of 22 patients continued to demonstrate greater-than-normal ALT concentrations and only 1 patient demonstrated an ALT concentration more than 1.5 times the upper normal limit. Further decreases in ALT, AST, and urinary copper excretion were observed at the end of the 10-year follow-up, but these decreases were not statistically significant. Only 1 patient continued to demonstrate abnormal ALT levels. Again, white blood cells showed no significant variations. All histologic scores (steatosis, inflammation, and fibrosis) were significantly decreased after treatment. Hepatic copper content was also significantly decreased, although it remained higher than normal in all patients. The removal of toxic copper was confirmed by disappearance of Kayser-Fleischer rings in 3 patients. Zinc did not have adverse effects on growth. The efficacy of zinc in WD in presymptomatic pediatric patients has been established in previous studies, and our study adds considerably to the earlier findings because it includes a large number of very young children, as many as 11 younger than 6 years and 20 younger than 10. The excellent clinical results in all patients, coupled with the improvement in hepatic histologic findings in the vast majority, indicate convincingly that zinc treatment can control the disease effectively and safely, preventing its progression over the course of 10 years. Histologic findings reportedly improved in 3 patients treated in an earlier study, but our data are numerically much more relevant. Notably, histologic study of the liver revealed that copper concentration was reduced by treatment, suggesting that oral zinc was able not only to prevent further accumulation of copper but also to promote, at least in part, the depletion of its stores. The lack of adverse effects of zinc on growth suggests that our patients received enough anticopper therapy to prevent damage resulting from copper toxicity but an adequate amount of copper for proper growth and development. In conclusion, our findings indicate that zinc is the treatment of choice in presymptomatic pediatric patients with WD.

Diminution of toxic copper accumulation in toxic milk mice modeling Wilson disease by embryonic hepatocyte intrasplenic transplantation

To observe the therapeutic effect of intrasplenic transplantation with embryonic hepatocytes on amelioration of hereditary copper accumulation in toxic milk (TX) mouse modeling Wilson disease. Donor hepatocytes were harvested from 14-d fetal liver of a pregnant homogeneous DL mouse. These cells were successively cultured, labeled with fluorescein dye Hoechst 33342 for 24 h, and sequentially infused into the spleen parenchyma of the recipient TX mice. No host immunosuppression measures were taken. Two and four weeks after transplantation, the recipients were killed for routine histologic investigation and immunohistochemistry study up to 4 wk after transplantation. The serum copper and ceruloplasmin concentrations of the recipient mice were determined by graphite furnace atomic absorption spectroscopy. In the following 2nd and 4th wk after transplantation, the donor hepatocytes could be visualized in the livers of 47.3% recipients. The serum ceruloplasmin and copper concentrations increased by 1.6-fold after 2 wk and 2.0-fold times after 4 wk respectively, which ultimately rose from about 30% of the normal level to nearly 60% (P<0.01). The hepatic copper concentration decreased 7.2%, 4 wk after transplantation. Pathologic examination showed that there were many actively proliferative hepatocyte precursor cells with specific embryonic hepatocyte marker AFP migrated into hepatic sinusoids of the recipients. A large number of cells carrying hepatocytes marker and albumin were observed in the recipient spleen tissues. Embryonic hepatocytes are capable of differentiating into mature hepatocytes in vivo. After transplantation, the hereditary abnormalities of copper metabolism in TX mice could be corrected partially by intrasplenic transplantation of homogeneous embryonic hepatocytes.

Improvement in Liver Pathology after 4 Months of D‐Penicillamine in 5 Doberman Pinschers with Subclinical Hepatitis

Five female Doberman Pinschers with increased hepatic copper concentrations and persistent (3–4 years) subclinical hepatitis were treated with D‐penicillamine for 4 months. Before and after treatment, the dogs underwent clinical, hematologic (red blood cell, white blood cell, and differential and thrombocyte counts), and clinical chemistry (creatinine, alkaline phosphatase, alanine aminotransferase, and total bile acid concentrations) examinations, and liver biopsies were examined histologically and their copper content measured quantitatively. No adverse effects were observed during treatment, and CBC and serum chemistry test results did not change. The mean liver copper concentration was 1,036 mg/kg dry matter before treatment and decreased to 407 mg/kg after treatment (P= .03). The copper concentrations had decreased (by between 134 and 1,135 mg/kg dry matter) in all of the dogs. The histopathologic appearance had improved or returned to normal in all 5 dogs. We conclude that D‐penicillamine effectively reduced copper retention in these dogs and improved the histopathologic appearance of the lesions. However, because D‐penicillamine has both copper‐chelating and anti‐inflammatory properties, it is not possible to draw conclusions on the etiology of this disease.