Copper binding components of blood plasma and organs,and their responses to influx of large doses of <Superscript>65</Superscript>Cu,in the mouse

To establish for the first time how mice might differ from rats and humans in terms of copper transport, excretion, and copper binding proteins, plasma and organ cytosols from adult female C57CL6 mice were fractionated and analyzed by directly coupled size exclusion HPLC-ICP-MS, before and after i.p. injection of large doses of ⁶⁵Cu. Plasma from untreated mice had different proportions of Cu associated with transcuprein/macroglobulin, ceruloplasmin and albumin than in humans and rats, and two previously undetected copper peaks (Mr 700 k and 15 k) were observed. Cytosols had Cu peaks seen previously in rat liver (Mr > 1000 k, 45 k and 11 k) plus one of 110 kDa. ⁶⁵Cu (141 μg) administered over 14 h, initially loaded plasma albumin and mainly entered liver and kidney (especially 28 kDa and 11 kDa components). Components of other organs were less (but still significantly) enriched. ⁶³Cu/⁶⁵Cu ratios returned almost to normal by 14 days, indicating a robust system for excreting excess copper. We conclude that there are significant differences but also strong similarities in copper metabolism between mice, rats and humans; that the liver is able to buffer enormous changes in copper status; and that a large number of mammalian copper proteins remain to be identified.     

Perspectives on copper biochemistry

The biochemistry of the essential trace element copper has been outlined. Following absorption, Cu(II) is transported by serum albumin and transcuprein to the liver where it is incorporated into the plasma Cu-protein, ceruloplasmin, or, possibly, stored as Cu-metallothionein or as superoxide dismutase. Ceruloplasmin is the long-term copper transporter and carries Cu(II) to the tissues for the biosynthesis of key Cu(II) enzymes, especially cytochrome c oxidase, lysyl oxidase and others. The production of copper enzymes raises many new questions about the metabolism of copper. Since ceruloplasmin is the centerpiece of copper metabolism and function, we conclude with more details on its chemistry and multifunctions. This Cu-protein of 132,000 daltons has now been totally sequenced and the copper-containing active sites located. Finally, we have proposed seven possible functions for ceruloplasmin, and there is now good evidence for the existence of ceruloplasmin receptors to expedite some of these functions.     

Copper transport to mammary gland and milk during lactation in rats

The delivery of copper to mammary gland and milk and the effects of lactation were examined in rats. Traces of (67)Cu/(64)Cu(II) were injected intraperitoneally or intravenously into virgin rats or lactating rats (2-5 days postpartum), and incorporation into blood, milk, and tissues was monitored. In virgin rats, most of the isotope first entered the liver and kidney. In lactating rats, almost 60% went directly to the mammary gland. Uptake rates and copper contents of the mammary gland were 20-fold higher in lactation. (67)Cu/(64)Cu appeared in milk and milk ceruloplasmin as rapidly as in mammary tissue and when there was no (67)Cu/(64)Cu-ceruloplasmin in the maternal plasma. Plasma (125)I-labeled albumin entered milk much more slowly. Milk ceruloplasmin (10 mg/l) had 25% of the (67)Cu/(64)Cu. Milk copper was 3.3 mg/l. Thus lactation markedly enhances the avidity of the mammary gland for copper, diverting most of it from liver and kidney to that tissue. Also, the primary source of milk ceruloplasmin is the mammary gland and not the maternal plasma.     

Transcuprein is a macroglobulin regulated by copper and iron availability

Transcuprein is a high-affinity copper carrier in the plasma that is involved in the initial distribution of copper entering the blood from the digestive tract. To identify and obtain cDNA for this protein, it was purified from rat plasma by size exclusion and copper-chelate affinity chromatography, and amino acid sequences were obtained. These revealed a 190-kDa glycosylated protein identified as the macroglobulin alpha(1)-inhibitor III, the main macroglobulin of rodent blood plasma. Albumin (65 kDa) copurified in variable amounts and was concluded to be a contaminant (although it can transiently bind the macroglobulin). The main macroglobulin in human blood plasma (alpha(2)-macroglobulin), which is homologous to alpha(1)-inhibitor III, also bound copper tightly. Expression of alpha(1)I3 (transcuprein) mRNA by the liver was examined in rats with and without copper deficiency, using quantitative polymerase chain reaction methodology and Northern blot analysis. Protein expression was examined by Western blotting. Deficient rats with 40% less ceruloplasmin oxidase activity and liver copper concentrations expressed about twice as much alpha(1)I3 mRNA, but circulating levels of transcuprein did not differ. Iron deficiency, which increased liver copper concentrations by threefold, reduced transcuprein mRNA expression and circulating levels of transcuprein relative to what occurred in rats with normal or excess iron. We conclude that transcupreins are specific macroglobulins that not only carry zinc but also carry transport copper in the blood, and that their expression can be modulated by copper and iron availability.     

Hemin-induced lysis of rat erythrocytes. Protective action of ceruloplasmin and different serum albumins

Hemin-induced lysis of rat erythrocytes is markedly reduced by ceruloplasmin (human) and serum albumins from different species, the order of effectiveness beings: bovine albumin approximately equal to ceruloplasmin greater than human albumin approximately equal to dog albumin greater than apotransferrin (human). Although the proteins studied had hemin binding capacity, the best protective agents, ceruloplasmin and bovine albumin, did bind hemin less strongly than human and dog albumin. The results suggest the existence of another protective mechanism, possibly involving an interaction between erythrocyte membranes and serum proteins.     

Copper transport and metabolism are normal in aceruloplasminemic mice.

Ceruloplasmin is an abundant serum glycoprotein containing greater than 95% of the copper found in the plasma of vertebrate species. Although this protein is known to function as an essential ferroxidase, the role of ceruloplasmin in copper transport and metabolism remains unclear. To elucidate the role of ceruloplasmin in copper metabolism, the kinetics of copper absorption, transport, distribution, and excretion were examined utilizing (64)Cu in wild-type and aceruloplasminemic mice. No differences in gastrointestinal absorption, hepatic uptake, or biliary excretion were observed in these animals. Furthermore, steady state measurements of tissue copper content utilizing (64)Cu and atomic absorption spectroscopy revealed no differences in the copper content of the brain, heart, spleen, and kidney. Consistent with these findings, the activity of copper-zinc superoxide dismutase in these tissues was equivalent in wild-type and ceruloplasmin-deficient mice. Hepatic iron was elevated 3.5-fold in aceruloplasminemic mice because of the loss of ferroxidase function. Hepatic copper content was markedly increased in aceruloplasminemic mice. As no differences were observed in copper absorption or biliary copper excretion, these data suggest that in these animals, hepatocyte copper intended for ceruloplasmin incorporation is trafficked into a compartment that is less available for biliary copper excretion. Taken together, these data reveal no essential role for ceruloplasmin in copper metabolism and suggest a previously unappreciated complexity to the subcellular distribution of this metal within the hepatocyte secretory pathway.     

Transport of silver in virgin and lactating rats and relation to copper

Virgin and lactating Sprague Dawley rats were used to determine whether the pathways of silver transport to tissues and milk resemble those for copper. Rats were injected i.p. with small amounts of ¹¹⁰AgNO₃. Blood and tissues were examined at various times thereafter for total radioactivity and for incorporation into copper binding proteins in plasma and milk. As with ⁶⁷Cu, much of the ¹¹⁰Ag was rapidly incorporated into the liver. Skeletal muscle, spleen, mammary gland, ovaries, uterus and adrenals also were significant initial accumulation sites, with or without lactation. Lactation enhanced uptake by the mammary gland, and radioactivity rapidly entered the milk and milk ceruloplasmin. In the plasma, most of the ¹¹⁰Ag bound to a single component of apparent molecular weight 800 k throughout the 52 h period examined. A small proportion was also incorporated into plasma ceruloplasmin, as determined by immunoprecipitation and native gel electrophoresis. There was little or no association of ¹¹⁰Ag with albumin or transcuprein. The binding of ¹¹⁰Ag to the 800 kDa protein was tight. Off rates during pH 7 dialysis were <2.5%/day even in the presence of 100 M histidine or Cu(II), but were accelarated by mercaptoethanol. Subunits of 145 and 45 kDa in virtually pure peak fractions were those of ₁-macroglobulin. We conclude that silver resembles copper in aspects of its tissue distribution, response to lactation, and incorporation into ceruloplasmin. However its main plasma carrier appears to be ₁-macroglobulin, a different macroglobulin than that involved in copper transport.     

Uptake of copper from plasma proteins in cells where expression of CTR1 has been modulated

Plasma proteins rather than amino acid chelates are the direct sources of copper for mammalian cells. In continuing studies on the mechanisms by which albumin and transcuprein deliver copper and the potential involvement of CTR1, rates of uptake from these proteins and Cu-histidine were compared in cells with/without CTR1 knockdown or knockout. siRNA knocked down expression of CTR1 mRNA 60-85% in human mammary epithelial and hepatic cell models, but this had little or no effect on uptake of 1?μM Cu(II) attached to pure human albumin or alpha-2-macroglobulin. Mouse embryonic fibroblasts that did/did not express Ctr1 took up Cu(II) bound to albumin about as readily as from the histidine complex at physiological concentrations and by a single saturable process. Uptake from mouse albumin achieved a 2-4-fold higher Vmax (with a lower Km) than from heterologous human albumin. Maximum uptake rates from Cu(I)-histidine were >12-fold higher (with higher Km) than for Cu(II), suggesting mediation by a reductase. The presence of cell surface Cu(II) and Fe(III) reductase activity responding only slightly to dehydroascorbate was verified. Excess Fe(III) inhibited uptake from albumin-Cu(II). Ag(I) also inhibited, but kinetics were not or un-competitive. In general there was little difference in rates/kinetics of uptake in the Ctr1+/+ and -/- cells. Endocytosis was not involved. We conclude that plasma proteins deliver Cu(II) to homologous cells with greater efficiency than ionic copper at physiological concentrations, probably through the mediation of a Steap Cu(II)-reductase, and confirm the existence of an additional copper uptake system in mammalian cells.     

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein (less than 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin congruent to bovine albumin less than dog albumin less than ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.     

Sustained strenuous exercise in sled dogs depresses three blood copper enzyme activities

Studies show mixed conclusions about acute responses of copper status to strenuous exercise. Because copper function involves metalloenzyme activities, which might take days to change, the present study examined the response of three copper metalloenzyme activities to sustained strenuous exercise in sled dogs. A race lasting 12–15 d depressed activities for both plasma ceruloplasmin and erythrocyte superoxide dismutase in dogs consuming commercial dog foods and meats. A shorter, 3-d training run for dogs fed a commercial balanced diet also depressed ceruloplasmin activities but not superoxide dismutase activities. Dogs fed the same diet but that did not run showed no changes in either parameter. Activities of a third copper enzyme, plasma diamine oxidase, also decreased after a 3-d training run. In summary, blood activities of three copper enzymes were depressed by sustained strenuous exercise in sled dogs.     

The effect of 110mAg on ceruloplasmin oxidase activity in rats

The effect of single i.v. injection of 110mAgNO3 (0.183 mg Ag+ X kg-1 b.wt.) in rats on the ceruloplasmin oxidase activity (Cp) and copper serum concentration was studied. It was found that Cp activity in the serum decreased to 70% of the control value and simultaneously serum copper concentration decrease to 30% of the control level. In both cases the decrease was independent on the time elapsed after silver administration. Comparing these results with those reported recently in mice Cu deficit in the rat serum was approximately twice higher. This fact is considered to be an inter-species difference. The concentration of copper in the hepatic supernatant significantly decreased (to eight times from control value) after silver injection. Only less than 10% of the total amount of Ag found in whole liver was taken up to hepatic supernatant. GPC analysis of the supernatant (Sephadex G-75) revealed that no Ag-metallothionein fraction is present. On the basis of the results obtained it was concluded that the mechanism of silver inhibition of Cp oxidase activity remains still in question.     

Lack of ceruloplasmin expression alters aspects of copper transport to the fetus and newborn, as determined in mice

Copper transport and accumulation were studied in virgin and lactating C57BL/6 mice, with and without expression of ceruloplasmin (Cp), to assess the importance of Cp to these processes. One hour after i.p. injection of tracer ⁶⁴Cu, liver and kidney accounted for 80% of the radioactivity, and mammary gland 1%, while in lactating Cp+/+ mice 2–4 days post partum, uptake by mammary gland was 9-fold higher and that of liver and other organs was decreased, with ⁶⁴Cu rapidly appearing in milk. Parallel studies in Cp−/− mice (siblings from same colony) gave virtually identical results. However, their milk contained less ⁶⁴Cu, and actual copper contents determined by furnace atomic absorption were less than half those for milk from normal dams. Liver copper concentrations of pups born to Cp−/− dams also were half those of pups from wild type dams. Copper in pup brains was unaffected; but iron concentrations were reduced. We conclude that absence of Cp, while not affecting entry of exchangeable copper from the blood into the mammary gland, does have a significant effect on the availability of this metal to the newborn through the milk and in the form of stores accumulating in gestation.     

Effect of dietary copper deficiency on the distribution of dopamine and norepinephrine in mice and rats

Dietary copper deficiency was produced in Swiss albino mice and Sprague Dawley rats to determine the organ specificity of alterations in norepinephrine (NE) and dopamine (DA) concentrations and the relationship with organ copper levels. A 5-week dietary treatment was used, which started 1 week after birth for mice, initially via dams, and 3 weeks after birth for rats. Mice offspring (6 weeks of age) and rats (8 weeks of age) maintained on a copper-deficient (-Cu) treatment were compared with copper-adequate (+Cu) controls. Compared with +Cu animals, -Cu mice and rats were anemic and had low (<1% of +Cu) ceruloplasmin activities but normal body weights. The -Cu mice had organ copper concentrations ranging between 30% and 65% of +Cu values for eight organs studied, with the thymus being the least depleted. For -Cu rats, the range was 15% to 65%. Significant reductions in NE concentration were observed in the heart, pancreas, and spleen of -Cu mice. Elevated DA levels were observed in all organs except the brain. For -Cu rats, the NE level was lower in the heart and the DA level was higher in both the heart and spleen compared with +Cu rats. Dopamine elevation in the heart and spleen for both -Cu mice and rats was four- and fivefold higher, respectively. Adrenal catecholamine levels were only slightly changed by copper deficiency in mice or rats. Urinary levels of both NE and DA were higher in -Cu rats and mice. Plasma and heart tyrosine levels were not altered in -Cu mice. Elevated DA in -Cu rodents may be due to limiting dopamine-beta-monooxygenase. Higher urinary NE and lower organ NE may be due to a combination of decreased synthesis and enhanced turnover. The magnitude of decreased organ copper was not predictive of altered catecholamine pool size.     

Plasma peptidylglycine alpha-amidating monooxygenase (PAM) and ceruloplasmin are affected by age and copper status in rats and mice

In an attempt to identify a sensitive and improved marker of mammalian copper status during neonatal development experiments compared two plasma cuproenzymes, peptidylglycine alpha-amidating monooxygenase (PAM ), an enzyme involved in peptide posttranslational activation, to ceruloplasmin (Cp), a ferroxidase involved in iron mobilization. Dietary Cu deficiency (Cu-) was studied in dams and offspring at postnatal age 3 (P3), P12, and P28. Rodent Cp activity rose during lactation whereas PAM activity fell. Reduction in Cp activity was more severe than reduction in PAM activity in Cu- offspring and dams. Cp activity was greater in rats than mice whereas PAM activity was similar in adults but greater in mouse than rat pups. Both cuproenzymes changed during neonatal development and when dietary copper was limiting. With proper controls, each enzyme can be used to assess copper status.     

Studies on the allergenic significance and structure of rat serum albumin

This study evaluated the capacity of rat serum albumin and of its proteolytic fragments to activate human basophils for IgE-mediated histamine release. The leukocytes from 8 out of 33 patients allergic to rats released histamine with rat serum albumin. Two proteolytic fragments of rat serum albumin, each constituting half of the molecule, were used to study the IgE-reactive antigenic sites. These fragments released histamine with the cells of some of the donors, thus demonstrating the presence of at least 2 antigenic determinants on each fragment for a total minimum of 4 sites on the intact rat serum albumin molecule. Most of the allergenic activity, however, was not recovered in the 2 fragments (total recovery mean = 6.4%, range between 0.1 and 31%). This loss could be due to cleavage of the rat albumin molecule in the middle of the third domain with loss of antigenic sites and/or due to minor conformational changes in the fragments as compared with the intact molecule. There was up to a 500-fold difference in the percent of activity recovered in the fragments when tested on cells from different patients. Therefore, there is no single immunodominant site on the molecule equally important for all patients. The cells of all 8 patients also reacted with mouse serum albumin but only 2 with bovine serum albumin. At least 1 determinant on mouse and rat serum albumin is cross-reactive with IgE.     

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 tissues for ceruplasmin vs. ionic copper, but when calculated on the basis of actual μg absorbed, all showed a preference for ceruplasmin. 2. Appreciable amounts 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 ceruplasmin over non-ceruplasmin copper, as did primary rat muscle cell cultures. 3. Ceruplasmin protein was also absorbed by normal and neoplastic rat tissues, but less rapidly than ceruplasmin 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 ceruplasmin turns over more rapidly than the protein moiety, a finding consistent with its role as a copper transport protein.     

Metals in biochemistry : P.M. Harrison and R.J. Hoare,Chapman & Hall,New York, 1980, 80 pp. $5.95 (paper)

An ultrafiltration technique was used to study stripping by glycine of the first copper and zinc ion equivalents bound by bovine, dog, and rat serum albumins at pH 7.5. Affinity of dog serum albumin for copper was poorer than for the other albumins, consistent with the absence in the former albumin of the copper binding site present at the amino terminus of the latter albumins. Affinities of all three proteins for zinc were similar, suggesting that the albumin amino terminus is not the primary zinc ion binding site.     

Copper deficient rats and mice both develop anemia but only rats have lower plasma and brain iron levels

Iron homeostasis depends on adequate dietary copper but the mechanisms are unknown. Mice (Mus musculus) and rat (Rattus norvegicus) offspring were compared to determine the effect of dietary copper deficiency (Cu-) on iron status of plasma, liver, brain and intestine. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 13 and 32. Cu- rat and mouse pups were both anemic, but only rat pups had lower plasma and brain iron levels. Plasma iron was lower throughout the suckling period in Cu- rats but not Cu- mice. Cu- mice derived from dams restricted of Cu only during lactation were also severely anemic without hypoferremia. Intestinal metal analysis confirmed that Cu- pups had major reductions in intestinal concentration of Cu, increased Fe, and normal Zn. However, whole mouse (less the intestine) analysis demonstrated normal content of Fe indicating that the limitation in iron transport by intestinal hephaestin had no consequence to total iron reserves of the mouse. Further research will be needed to determine the reason Cu- mice were anemic since the "ferroxidase" hypothesis does not explain this phenotype.     

Changes in copper and zinc status and response to dietary copper deficiency in metallothionein-overexpressing transgenic mouse heart

Previous studies have shown that cardiac-specific overexpression of metallothionein (MT) inhibits progression of dietary copper restriction-induced cardiac hypertrophy. Because copper and zinc are critically involved in myocardial response to dietary copper restriction, the present study was undertaken to understand the effect of MT on the status of copper and zinc in the heart and the subsequent response to dietary copper restriction. Dams of cardiac-specific MT-transgenic (MT-TG) mouse pups and wild-type (WT) littermates were fed copper-adequate (CuA) or copper-deficient (CuD) diet starting on the fourth day post delivery, and the weanling mice were continued on the same diet until they were sacrificed. Zinc and copper concentrations were significantly elevated in MT-TG mouse heart, but the extent of zinc elevation was much more than that of copper. Dietary copper restriction significantly decreased copper concentrations to the same extent in both MT-TG and WT mouse hearts, and decreased zinc concentrations along with a decrease in MT concentrations in the MT-TG mouse heart. Copper deficiency-induced heart hypertrophy was significantly inhibited, but copper deficiency-induced suppression of serum ceruloplasmin or hepatic Cu,Zn-SOD activities was not inhibited in the MT-TG mice. These results suggest that elevation in zinc but not in copper in the heart may be involved in the MT inhibition of copper deficiency-induced cardiac hypertrophy.     

Interactions of copper with glycated proteins: possible involvement in the etiology of diabetic neuropathy

Humans and animals with diabetes frequently develop peripheral vascular dysfunction and peripheral neuropathies. There is accumulating evidence that impaired peripheral nerve function may derive from diminished endoneural blood flow. The decrements in nerve blood flow may, in turn, be due to diminished endothelium-dependent vasodilation. Although a number of possible causes of this defective vasodilation have been suggested, none has been definitely proven. Regardless of the precise cause, the impaired vasodilatory activity may reflect diminished availability of endothelium-derived relaxing factor (EDRF), variously thought to be nitric oxide or thiol adducts of nitric oxide. Other investigators have reported that administration of transition metal chelators to diabetic rats corrects EDRF-mediated arterial relaxation and restores both neural blood flow and nerve conduction velocity, suggesting the involvement of transition metals. Our investigations center about the hypothesis that glycated proteins bind transition metals such as copper and iron, and that such 'glycochelates' accumulate within the vasculature in diabetes and catalytically inactivate EDRF. In partial support of this hypothesis: (1) Glycated albumin binds approximately 3-fold greater amounts of both copper and iron. (2) Copper bound to glycated albumin remains redox active (e.g. capable of supporting the oxidation of ascorbic acid). (3) Copper and copper-containing glycochelates cause the rapid decomposition of one putative form of EDRF, nitrosocysteine. (4) The amount of exchangeable (i.e. chelatable) copper in the plasma of diabetic rats is approximately twice that in normal rat plasma. (5) Similarly, tail tendons of diabetic animals have about twice as much bound copper as do tendons of normal rats. (6) Implants bearing adsorbed glycated albumin placed in the peritonea of normal mice for 48 h accumulate approximately 5 times as much bound copper as do implants coated with control albumin. Overall, these observations support--but do not conclusively prove - the hypothesis that transition metals such as copper, bound to glycated proteins, may blunt normal EDRF-dependent relaxation of diabetic arteries and provide a rationale for the use of transition metal chelators in the therapy of diabetic vasculopathy and neuropathy.