Mechanism of copper transport and delivery in mammals: review and recent findings.
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Ammonium Sulfate↗
Biomedical subjects
M C Linder
Publications and source records attributed to M C Linder.
Dissociation of ferritins.
Apoferritins prepared from horse spleen and heart and rat heart and liver were dissociated by treatment with acetic acid (pH 1.3-3.0). Sedimentation velocity studies showed that apoferritins of spleen and liver (16-17 S) and heart (18-19 S) dissociated into material sedimenting near 3.2 S. Sedimentation equilibrium measurements determined that most of the material had a molecular weight of 38,000-43,000, corresponding to subunit dimers. Failure to dissociate into subunit monomers was confirmed by gel chromatography on Sephadex G-75 and G-150. With the exception of boiling in sodium dodecyl sulfate, further treatments with 0.1-0.4 M KCl, NaCl, 4-9 M urea, 0.01-0.5 M KSCN, 0.1-0.5% Triton X-100, 5-52% dimethylsulfoxide, 10% ethylene glycol, or 0.1% trifluoroacetic acid all failed to cause dissociation into individual subunits, as did exposure to 6 M guanidine-HCl or formic acid, or prior succinylation and/or nitration of the protein. Reassociation occurred between pH 4 and 7 but was not aided by the addition of Fe(II) or reducing agents. It is concluded that ferritins readily dissociate to subunit dimer units and that further dissociation does not occur without full denaturation of the protein.
Animals↗
Binding and uptake of copper from ceruloplasmin.
Specific binding of [67Cu]ceruloplasmin to plasma membrane containing preparations from rat tissues was shown in the presence of an excess of nonradioactive Cu(II) or ceruloplasmin. With Cu(II) there was positive cooperativity and an apparent KD of 10(-7) M. The effects of both "cold" ligands was partly additive. No "specific" binding was shown with Zn(II), unrelated proteins and after boiling the membranes. Total and specific binding of [67Cu]ceruloplasmin were 2-7 fold greater for heart and brain than for liver preparations, per g tissue or per mg protein, +/- correction for yield of 5'-nucleotidase. Cu(II) also inhibited uptake of [67Cu] from ceruloplasmin by CHO cells, but monensin did not, suggesting uptake of ceruloplasmin Cu occurs at the cell surface.
Animals↗
Non-ferritin, non-heme iron pools in rat tissues.
Concentrations of intracellular, low molecular weight (LMW) and desferrioxamine B (DF) chelatable Fe, in tissues of normal, Fe-deficient and Fe-loaded female rats, were determined. Ice cold, high speed supernatants were rapidly fractionated on Ultrogel AcA202 or by filter centrifugation. After correction for residual blood and DF effects on Fe proteins, liver, kidney, heart and spleen contained 3-8 micrograms/g LMW Fe, brain 20 micrograms/g, with DF; two-thirds of this was detected without DF. There was little variation with Fe status. MW standardization and fractionation on Sephadex G-25 indicated components of apparent MW 13,000, 1400 and 350; the latter two were rapidly labeled with in vivo 59Fe.
Animals↗
Turnover and excretion of copper in rats as measured with 67Cu.
We have examined the whole-body turnover and excretion of copper in rats, using 67Cu. Female Fischer rats, 3-4 mo of age, were injected intraperitoneally with tracer doses of 67CuCl2, and whole-body radioactivity was monitored over the next 3-6 days. The rate of loss was exponential and biphasic, with a half-life of 67 h for the first 70 h, followed by a much slower rate over the following days. A small percentage (2-7%) of the early loss was in the urine, the rest in the feces. In sexually immature animals, turnover was slower in the first and faster in the second phases. Pretreatment of rats with a large dose of copper had no significant effect on the initial turnover rate. Ligation of the common bile duct cut the rate in half, but did not block turnover. It is concluded that there are at least two pools of copper in the rat that turn over at different rates and that excretion of copper occurs via the bile and also by other intestinal routes.
Animals↗
Copper transport in rats involving a new plasma protein.
The time course of distribution of high-specific activity 67CuCl2 to tissues and plasma components was followed in adult, female rats. Immediately after intubation or injection, tracer 67Cu associated with two components of the blood plasma separable on columns of Sephadex G-150: albumin and another (larger) component, which was not ceruloplasmin. The latter, tentatively named transcuprein, had an apparent molecular weight of 270,000 and a high affinity for Cu2+, as judged by processing through Chelex-100, dilution, and exchange with albumin copper, in vitro and in vivo. It was capable of donating copper to tumor cells in serum-free medium. Analysis of "cold" plasma by furnace atomic absorption confirmed the presence of 10-15% of plasma copper in this peak. Plots of percent dose and 67Cu specific activity against time showed that copper followed a very specific pathway after binding to albumin and transcuprein, entering mainly the liver, then reappearing in the plasma on ceruloplasmin, and then achieving peak distribution in peripheral tissues (muscles, brain, etc.). 67Cu disappeared from liver and kidney with an apparent half-life of 4.5 days, the same exponential rate found for whole body turnover. Apparent turnover of ceruloplasmin copper was more rapid. Even after 7-12 days, tracer copper in plasma was still found exclusively with ceruloplasmin. The results indicate that copper follows a carefully prescribed path, on entering the blood and binding to a new transport protein.
Animals↗
Distribution of copper among components of human serum.
We examined the distribution of copper among four components of human serum separated by chromatography on Sephadex G-150 and Affi-gel blue. Analysis of copper by furnace atomic absorption indicated that normal adults have copper at an average of 600 ng/ml associated with ceruloplasmin; at 120 ng/ml with transcuprein, a new copper transport protein; at 150 ng/ml with albumin; and at 90 ng/ml with one to three components of low molecular weight (less than 30,000). Cancer patients had more total copper but similar proportions in the four serum fractions. In both groups, some individuals had very high levels of copper in transcuprein, albumin, and/or one or more components of the low-molecular-weight fraction. The results showed that, contrary to earlier conclusions, ceruloplasmin copper only comprised about 60% of the total in human serum; and not just ceruloplasmin but also other forms of serum copper may be elevated in cancer patients.
Adult↗
Cellular location of rat muscle ferritins and their preferential loss during cell isolation.
Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.
Animals↗
Other trace elements and the liver.
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Chromium↗
Effects of CO2 exposure on distribution of various forms of iron and copper in guinea-pig tissues.
The effects on iron and copper distribution and metabolism of exposure to high levels of CO2 were studied in the guinea-pig. Mature, male animals were placed in an atmosphere of 15% CO2, 21% O2 (balance N2), and sacrificed from 1 h to 1 week thereafter. Total iron and copper concentrations of blood, liver, spleen and bone, as well as concentrations of heme and ferritin iron, were measured together with blood hematocrit, reticulocytes, plasma hemoglobin, plasma ceruloplasmin and copper concentrations. The results show clearly that rapid and sustained red cell damage or hemolysis ensued several h from the start of CO2 treatment. This resulted in loss of iron and copper from the blood, an influx of both elements into liver, spleen and bone, and a rise in plasma ceruloplasmin. Influx of iron into liver and spleen caused an accumulation of ferritin, the main site for iron storage in cells. Following the effect on red cells, there was an accumulation of heme iron, and a decreased hematocrit, best explained by a depressed activity of the reticuloendothelial and erythropoietic systems. A period of adaptation succeeded these events, in which all blood parameters and most tissue values returned to normal, despite the continuing presence of high CO2. The only changes not reversed were the elevations in liver, spleen and bone iron stores. These remained high, with a net accumulation of greater than 2 mg iron, or 3-4 times more than originally present. The results indicate that at least in the guinea-pig, high CO2 exposure results in red cell damage and other events leading to an accumulation of additional iron in the body; also, that iron accumulated as ferritin and hemosiderin in liver and spleen may not be readily available to restore blood hemoglobin concentrations on an acute basis.
Animals↗
Interactions of pH and ascorbate in intestinal iron absorption.
Iron absorption is frequently studied experimentally in animals by placing iron directly into tied-off intestinal segments in vivo, usually in acid solutions. We have monitored the changes in pH that occur in the intestinal lumen when acid iron solutions are administered and have related this to the time course of iron absorption in iron-deficient rats. Within 5 minutes of giving of 5.6 micrograms Fe (FeCl3), in 0.50 ml 0.01 M HCl, 0.9% NaCl, the pH of the lumen had climbed above pH 4, where Fe3+ is insoluble; reached 6.8 by 10-15 minutes. In parallel with rising pH, mucosal Fe uptake ceased by 5 minutes after its administration. Addition of 2 mM ascorbate to the same solution prevented the cessation of iron uptake despite the usual pH rise. Intestinal fluid of fasted rats had a limited buffering capacity from pH 7 to 3. When added to this fluid, 59FeCl3 largely precipitated, but microgram quantities bound to components with apparent molecular weights greater than 25,000. This iron was available to desferrioxamine B. The results demonstrate the dramatic effects of pH on iron bioavailability, in the absence of chelators such as ascorbate, and the importance of considering iron solubility in measurements of iron absorption.
Animals↗
Concentration, structure and iron saturation of ferritins from normal human lung and lung tumors with graded histopathology.
Samples of graded, human lung cancer and of normal lung were assayed for total iron, ferritin, and ferritin iron saturation. Both kinds of tissues contained highly variable amounts of total and ferritin iron and had a range of ferritin iron:protein ratios. No quantitative correlations were found between cancer histopathology and these parameters, in contrast to previous findings for transplantable rat hepatomas. Examination of pooled ferritins isolated from normal lung and lung tumors by quantitative polyacrylamide gel electrophoresis, isoelectric focusing before and after acid-urea dissociation, and SDS electrophoresis, revealed no structural differences. It is concluded that at least for the human lung, malignancy of the kind examined causes no change in ferritin gene expression, and that ferritin assays would not be useful in the grading or detection of human lung cancer.
Ferritins↗
Circulating ceruloplasmin is an important source of copper for normal and malignant animal cells.
The relative uptake of copper from ceruloplasmin and non-ceruloplasmin plasma pools, by normal and malignant cells, was investigated in vivo and in vitro, using 64Cu and 67Cu. 1. Most of the copper administered intravenously to normal and tumor-bearing rats was removed within 1 h, a substantial portion entering the liver. There were differences in the apparent avidity of individual tissue for ceruloplasmin vs. ionic copper, but when calculated on the basis of actual microgram absorbed, all showed a preference for ceruloplasmin. 2. Appreciable amount of copper from either source were also absorbed by the tumors, and cultured Ehrlich ascites tumor cells showed a rapid uptake and marked preference for ceruloplasmin over non-ceruloplasmin copper, as did primary rat muscle cell cultures. 3. Ceruloplasmin protein was also absorbed by normal and neoplastic rat tissues, but less rapidly than ceruloplasmin copper, as determined by administration of pure [3H]leucine- or [125I]ceruloplasmin. Copper deficiency did not accelerate this process. 4. It is concluded that, at least in rat, ceruloplasmin is the preferred plasma source of copper for normal and malignant cells, and that the copper on ceruloplasmin turns over more rapidly than the protein moiety, a finding consistent with its role as a copper transport protein.
Animals↗
The size and shape of heart and muscle ferritins analyzed by sedimentation, gel filtration, and electrophoresis.
We have compared the size and shape of ferritins from human heart, rat heart, and skeletal muscle with those of rat liver and horse spleen ferritins, using sedimentation and gel filtration techniques. The electrophoretically "fast" form of heart ferritin was partially separated from both the "slow" heart form and from rat liver ferritin by gel filtration (Stokes radii 72 A versus 69 and 68.5 A). Sedimentation velocity after iron removal showed an 18.5 S boundary even for mixtures of the two heart species, versus 17.3 S for rat liver and horse spleen apoferritins. Holoferritins gave a broad boundary, with coefficients from 66-80 S depending on iron content. Variable amounts of disaggregated ferritin (2.6 S) were also present in the heart ferritin preparations. Removal of iron significantly increased the electrophoretic migration of the fast but not the slow heart ferritin species. By sedimentation equilibrium, the molecular weights of all apoferritins save the fast heart form were about 490,000; that for the latter was near 750,000. Since electrophoresis revealed no major differences in subunit size, it is concluded that the larger, more asymmetric form of muscle ferritin contains 34 to 38 rather than 24 subunits.
Animals↗
Ceruloplasmin assays in diagnosis and treatment of human lung, breast, and gastrointestinal cancers.
Ceruloplasmin was assayed as enzyme activity, as antigen, and as total copper in serum samples from 150 male lung cancer patients and comparable numbers of male controls. By all three assays, ceruloplasmin was significantly increased above the normal before treatment, and the degree of elevation was related to TNM stage [i.e., the International Union Against Cancer classification system based on extent of primary tumor (T), condition of lymph nodes (N), and absence of presence of metastases (M)]. Surgery had no immediate effects, but in patients who evidence of disease for longer periods, ceruloplasmin returned to nearly normal values. High levels of ceruloplasmin was elevated in 6 of 9 patients before tumor recurrence; 2 of 3 smokers (in the first panel of sera) with elevated ceruloplasmin levels subsequently developed lung cancer. The relative merits of the three assays were compared. Some sex- and age-related differences among normal controls were apparent. The results of pilot studies on men with gastrointestinal cancer and women with breast cancer are presented. It is concluded that only in limited situations will assays of ceruloplasmin aid in diagnosis, prognosis, and long-term monitoring of cancer patients.
Adult↗
Synthesis of rat muscle ferritins and function in iron metabolism of heart and diaphragm.
Two major species of ferritin are found in heart and other muscles of the rat, which may be separated in polyacrylamide gel electrophoresis after preliminary purification. We have examined the time-course of incorporation of [3H]leucine and 59Fe into these slow- and fast-migrating ferritins in heart and diaphragm, in vivo, following intraperitoneal or intravenous injection. The results show: 1. Both species of ferritin are synthesized from amino acids in parallel with total tissue protein and receive iron in concert; consequently, there is no structural precursor/product or iron-donor/receiver relationship between them. 2. Under iron stimulation, both ferritins are synthesized at different rates within the same tissue and between tissues, and there is a correlation between tissue concentration and its rate of synthesis. In the diaphragm, the concentration and rate of synthesis of the slow-migrating species predominate, and in the heart the opposite pertains. 3. In both tissues iron is preferentially absorbed by the first species upon entry into the tissue, and this is independent of the relative concentration of the two ferritins or their iron saturation. 4. From the data on 59Fe uptake it appears that myoglobin synthesis and metabolism are considerably more active in the heart than in the diaphragm. However, neither ferritin species is a direct iron donor for myoglobin synthesis. Instead, the ferritins and myoglobin all draw their iron from a common pool, which may be of the small molecular weight type reported for intestinal epithelium and some other cells.
Animals↗
Altered copper absorption in tumor-bearing and estrogen-treated rats.
Intestinal copper absorption was studied in normal, tumor-bearing and estrogen-treated rats, using 64Cu(NO3)2. Rats bearing Dunning mammary tumors (DMBA 5A) of more than 2 g absorbed 70-100% more of an intragastric copper dose than controls, and a much larger percentage was found in the plasma, with less in liver and kidney. The distribution of copper measured chemically mimicked these findings, and the enhanced capacity for absorption was also demonstrated in vitro with everted duodenal segments. Further examination of the intestinal mucosa revealed that binding of radioactive copper to a 10,000-dalton component of the cytosol was inversely proportional to the amount absorbed into the body, tumor-bearing rats retaining much less in, and transferring much more from, the intestine than controls. Daily injection of 17 beta-estradiol over 2 wk had the reverse effect. It is concluded that the absorption of copper is an endogenously regulated process that may be altered by cancer and estrogen treatment and mediated by the extent of its binding to a component (possibly metallothionein) within the mucosal cell.
Animals↗
Copper regulation of ceruloplasmin in copper-deficient rats.
In copper-deficient rats, oral intubation of copper increases the rate of ceruloplasmin synthesis without affecting general synthesis of plasma or liver proteins. It also restores the enzyme from half to full activity. Copper given by injection at doses commonly employed has additional nonspecific effects on protein synthesis and in some strains of rats produces severe hemolysis. In contrast to deficient rats, in normal rats copper does not elevate plasma ceruloplasmin unless hemolysis also occurs. Thus, at least in deficiency, copper availability controls the rate of synthesis, acitvation, and plasma concentration of ceruloplasmin.
Animals↗