Copper Handling Research Articles

Localization of the Wilson's disease protein in human liver

Background & Aims: Wilson's disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilson's disease protein in hepatic copper handling, its localization in human liver was investigated. Methods: By use of a specific antibody, localization of the Wilson's disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. Results: The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. Conclusions: These results show that in human liver the Wilson's disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion. GASTROENTEROLOGY 1999;117:1380-1385

Expression of the Menkes disease homolog in rodent neuroglial cells

We surveyed qualitative expression of the Menkes disease homolog in a variety of brain regions, and cultured neurons and glia from rat and mouse. Cultured astrocytes from adult rat cerebral cortex, corpus striatum and cerebellum all expressed the gene, as did cerebellar granule cell neurons isolated from whole cerebellum. Pulse-chase experiments with radiolabeled copper demonstrated normal copper handling by cultured adult astrocytes. We also assessed expression of the Menkes homolog in astrocytes from various earlier developmental stages (embryonic day 20, postnatal days 3 and 8), and detected expression on D3 and D8 but not in E20 astrocytes. Our data provide evidence that this copper transport gene is expressed in specific glial cells and neurons, findings that may be pertinent to treatment considerations in human patients with Menkes disease.

Retrorsine in breast milk influences copper handling in suckling rat pups

Aims: To explore the hypothesis that a second xenobiotic agent is required with excess copper to produce Indian Childhood Cirrhosis, this study investigated the effect of the pyrrolizidine alkaloid retrorsine fed to the mother during the suckling period upon the serial changes in neonatal copper status. Methods: Female Wistar rats with new-born litters were fed either a control or a retrorsine (50 mg/kg) diet. At 0, 4, 8, 11, 15, 18 and 21 days, pups from each litter were weighed, sacrificed and their livers removed for copper, DNA and metallothionein analysis. Serum samples were assayed for caeruloplasmin oxidase activity and albumin. Results: 1) Higher than adult level of hepatic copper in normal rats which rose post-natally before declining from day 11 after birth, 2) raised hepatic copper concentrations and total copper in the retrorsine group from day 15; levels were higher than adult at birth, 3) reduced serum caeruloplasmin oxidase activity and albumin levels in retrorsine group, but both groups lower than adult, 4) lower hepatic metallothionein levels in retrorsine group, but both groups higher than adult, and 5) reduced liver DNA in the retrorsine group when expressed as total DNA and per gram of tissue. These changes were not secondary to under-nutrition as a small study on under-nourished rat neonates showed that copper handling is not significantly altered when compared to well-nourished rats. Conclusions: Retrorsine passing to rat neonates via breast milk causes: 1) the accumulation of hepatic copper, 2) impairment of the rise in serum caeruloplasmin, which could indicate a decline in synthesis or failure of copper incorporation into the apo-protein, 3) a decrease in hepatic metallothionein and serum albumin levels, again suggesting diminished protein synthesis, and 4) reduced hepatic DNA indicative of decreased cell number but increased cell size. Accumulation of liver copper but reduction of copper-binding proteins could result in free copper and explain the synergistic hepatotoxicity of copper and retrorsine.

Glycogen depletion and altered copper and manganese handling in Nephrops norvegicns (L.) following starvation and exposure to hypoxia

Glycogen depletion and altered copper and manganese handling in Nephrops norvegicns (L.) following starvation and exposure to hypoxia

Glycogen depletion and altered copper and manganese handling in Nephrops norvegicus following starvation and exposure to hypoxia

Glycogen depletion and altered copper and manganese handling in Nephrops norvegicus following starvation and exposure to hypoxia

Copper-64 metabolism in two patients with non-Wilsonian movement disorders and copper deficiency

Copper-64 studies are presented of 2 patients with non-Wilsonian movement disorder and with abnormal copper handling. Both patients differred from the usual phenotype of non-Wilsonian low copper movement disorder as they had choreiform movement disorders with an onset in the first decade; one patient lacked significant intellectual impairment. Both patients had reduced serum total copper and marginal free copper and caeruloplasmin levels, and both patients were capable of incorporating 64Cu 2+ into caeruloplasmin but the second case did so at markedly reduced level. Both showed slightly increased basal and stimulated urinary copper loss compared to normal controls with the rate in patient 1 being capable of leading to copper depletion. Liver copper content was normal in both cases. These 2 patients add to the reports of cases with copper deficiency and movement disorder in whom copper chelation therapy is unlikely to be beneficial.

The loss, uptake and tissue distribution of copper and zinc during the moult cycle in an ecological series of talitrid amphipods (Crustacea:Amphipoda)

The exuvial loss, subsequent uptake, and tissue and body distributions of the essential trace metals copper and zinc were investigated in an ecological series of talitrid amphipods, immediately after moult and throughout a post-moult period. Negligible quantities of copper or zinc were detected in cast exuvia of individual talitrids. Neither tissue nor whole body copper and zinc concentrations underwent significant changes during the moult cycle and all species showed extreme conservatism in their handling of body copper. There appeared to be no differential treatment of copper between species at the time of moult throughout the ecological series, with no obvious increase in copper retention by the more terrestrial species. The impact of moult cycle stage therefore is unlikely to affect the use of littoral talitrid amphipods as biomonitors for ambient copper and zinc concentrations.

Menkes disease: an X-linked neurological disorder of the copper metabolism.

Menkes disease is an X-linked, recessive disturbance of copper metabolism associated with a progressive clinical course and abnormal hair. The disease is dominated by neurological symptoms combined with connective tissue manifestations, most of which can be explained by the lack of important copper enzymes. Despite excessive accumulation of the metal in various tissues, a functional copper deficiency is evident, probably caused by a defective intracellular copper transport protein of unknown nature. The molecular basis of the copper disturbance has proven difficult to define and will most likely have to await cloning of the gene. The chromosomal region of interest has now been narrowed down to a sub-band on the long arm of the chromosome (Xq13.3), and positional cloning is in progress in a number of laboratories including our own. Identification of the Menkes gene will be of importance for our understanding of the cellular handling of copper and other trace elements.

Markedly Reduced Activity of Lysyl Oxidase in Skin and Aorta from a Patient with Menkes' Disease Showing Unusually Severe Connective Tissue Manifestations

In Menkes' disease, a severe disturbance of copper handling appears to render copper unavailable for copper-requiring processes. We have measured the activity of lysyl oxidase, the copper-dependent enzyme that initiates the cross-linking of collagen and elastin, in extracts of skin and aorta obtained at autopsy from a patient with unusually marked connective tissue manifestations, and found it to be only 6-12% of normal, thus suggesting a basis for these alterations.

Two pathways for biliary copper excretion in the rat: The role of glutathione

To evaluate the role of glutathione in biliary copper excretion, we studied this process in control Wistar rats and in mutant Wistar rats (GY rats), in which the secretion of glutathione into bile is deficient. For comparison, biliary zinc excretion was determined simultaneously. In spite of the markedly reduced bile flow (−45%) in GY rats, biliary output rates of endogenous copper were virtually identical in GY and control rats. In contrast, zinc output was drastically reduced in GY rats compared to controls (−80%). Biliary excretion patterns after intravenous administration of copper, in doses ranging from 65 to 2265 nmol/100 g/body wt, showed a distinct rapid and slow phase in control rats. In GY rats, on the other hand, the rapid phase in copper excretion was absent but the slow phase appeared to be unaffected. Pretreatment of rats with diethylmaleate to deplete hepatic and biliary glutathione abolished the rapid phase of copper excretion in control rats, while the slow phase remained unaffected. No significant effect of diethylmaleate on the hepatic handling of exogenous copper was observed in GY rats. The maximal capacity of the slow copper excretion pathway was 40–45 nmol/hr/100 g body wt, both in control and GY rats; the capacity of rapid excretion pathway depended on the administered copper load. Intravenous injection of copper induced the biliary excretion of a substantial amount of zinc in control rats; but not in GY rats. These results indicate the existence of at least two distinct biliary excretory pathways for copper in the rat. i.e. a slow and a rapid pathway, with a glutathione dependency of the latter only. The basal excretion of (endogenous) copper, in contrast to that of zinc, can proceed independently of glutathione excretion. However, glutathione appears to be involved in the rapid secretion of excess copper.

Abnormalities of copper in gilles de la tourette syndrome

The Gilles de la Tourette syndrome is a disorder whose etiology and pathogenesis are little understood. The number of biochemical abnormalities described in this disorder is minimal. Ten of a total of 80 patients were found to have an abnormally low serum copper. A report is presented on two patients who consented to further detailed investigation and in whom copper radioisotope studies were carried out. Both exhibited abnormalities of copper handling, in that we observed an abnormally fast disappearance of copper from the plasma and an abnormally slow uptake by the liver. The rates of intestinal absorption and urinary excretion were normal. We did not identify an abnormal site of sequestration of the metal in the body.

Is copper hepatotoxic in primary biliary cirrhosis?

In primary biliary cirrhosis (PBC) liver copper retention occurs as a complication of cholestasis. By analogy with Wilson's disease, it has been suggested that copper retention is hepatotoxic in PBC, and this has been the rationale for the use of D-penicillamine in this disease. The hypothesis that copper is hepatotoxic in PBC has not been tested and in this study we have evaluated the role of liver copper retention in the pathogenesis of PBC. Sixty-four patients with PBC have been studied. Fifty-four had increased liver copper concentrations. Liver cell synthetic function was well preserved. All the patients had normal prothrombin times, and only two had subnormal serum albumin concentrations. There was no correlation between liver copper concentrations and the degree of liver cell damage assessed biochemically (aspartate transaminase), and histologically. Electron microscopy was performed on liver biopsies from five patients with markedly increased liver copper concentrations. The liver cell ultrastructure was compatible with cholestasis. Liver cells contained electron dense lysosomes, which were shown to contain copper and sulphur by x-ray probe microanalysis. The characteristic organelle changes associated with copper toxicity in Wilson's disease were not observed. The biochemical, histological, and histochemical differences between PBC complicated by liver copper retention, and Wilson's disease, indicates that there are differences in the handling of copper in these disease. In this study we could find no evidence to suggest that copper plays an important role in the pathogenesis of liver dysfunction in PBC.

A kinetic model of copper metabolism in sheep

A three-compartment model was developed to provide a simple kinetic description of the metabolism of copper in six sheep with liver copper concentrations of 433�51 ppm ( mean � SD dry weight). This was achieved by measuring the radioactivity in samples of blood, liver, urine and bile as a function of time after the intravenous administration of c. 1.4 mCi of 64Cu. Sizes of compartments, flow rates and rate constants were evaluated and validated experimentally by measuring the excretion of copper in urine and bile and its accumulation in the liver. The model postulates two separate mechanisms for the handling of copper by the liver, and biliary copper excretion was not related to total liver copper content. The model was tested in sheep with a wider range of liver copper concentrations, and the copper in the two compartments (C2 and C3), attributed to liver, corresponded to the actual liver copper content when this was between 30 and 70 mg. The rate constants also responded consistently to increased liver copper status. Within the 'normal' liver copper range of 50-70 mg, the three-compartment model was closed but outside this range, the undefined parameter K03 was no longer zero. This response suggests that copper is moving from C2 and C3 to either supply tissue requirements or be redistributed in an additional storage compartment.

Studies with radioactive copper ( 64 Cu and 67 Cu); the incorporation of radioactive copper into caeruloplasmin in Wilson's disease and in primary biliary cirrhosis.

1. A comparison has been made of the handling of radioactive copper by patients with Wilson's disease, patients with primary biliary cirrhosis and a small control group. The object of this study has been to assess the influence of an expanded hepatic pool of copper on the incorporation of radioactive copper into caeruloplasmin. 2. Studies with 67Cu have shown that radioactivity disappears steadily from the plasma of patients with Wilson's disease for periods up to 296 h. Both in patients with primary biliary cirrhosis and in controls, a well-marked secondary rise occurs as radioactive copper is incorporated into caeruloplasmin. 3. Caeruloplasmin has been isolated from the serum of one control subject, one patient with primary biliary cirrhosis and one with Wilson's disease. Of the plasma radioactivity 90% was present in the caeruloplasmin of the control subject and the patient with primary biliary cirrhosis; only 27% was found in the caeruloplasmin of the patient with Wilson's disease. 4. Turnover time for liver copper has been determined; in the control subjects it was of the order of 20–30 days, in the patients with primary biliary cirrhosis it was between 600 and 700 days, in Wilson's disease the time exceeded 1800 days. In all cases the turnover time for radioactive copper was significantly less. 5. We conclude (a) that newly arrived radioactive copper is preferentially handled in the presence of copper overload and (b) dilution of radioactive copper in an expanded hepatic pool of the metal cannot alone account for the delayed incorporation found in patients with Wilson's disease.