Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human ⁶⁴Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the ⁶⁴Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The ⁶⁴Cu in ceruloplasmin purified from plasma of ⁶⁴Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived ⁶⁴Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent ⁶⁴Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of ⁶⁴Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.     

The response of serum ceruloplasmin to injections of walker 256 tumor cells or turpentine into rats

The subcutaneous injection of Walker 256 carcinosarcoma cells into a rat was followed by a pronounced increase in plasma ceruloplasmin activity. This lasted only about 3 weeks even though the tumors were still enlarging rapidly. Based upon studies with⁶⁷Cu, the increase in plasma ceruloplasmin seemed to reflect increased production of the enzyme by the livers of rats bearing the tumors. Injections of turpentine also raised plasma ceruloplasmin, but to a significantly lesser extent and normal values were reached within 2 weeks.     

Plasma ceruloplasmin Evidence for its presence in and uptake by heart and other organs of the rat

Evidence for the presence of the plasma protein, ceruloplasmin, in heart and other tissues of the rat was sought using various techniques. With p-phenylenediamine, ceruloplasmin-like oxidase activity was detected in heart postmitochondrial and 100 000 × g supernatants in amounts far exceeding those that could be accounted for by residual blood. Much lower levels were detected in kidney, brain and liver. Oxidase activity of heart purified on DEAE-cellulose in the same way as rat plasma ceruloplasmin and behaved identically also in disc gel electrophoresis. The presence of ceruloplasmin in heart extracts was confirmed immunologically by Ouchterlony diffusion, using rabbit antibody raised against pure rat ceruloplasmin. When pure [³H]leucin-labeled ceruloplasmin was infused intravenously into a copper-deficient rat, radioactivity was concentrated in the heart and brain within 2 h; radioactive counts per g attained 11 and 3 times those of plasma in the two organs, respectively. A lesser concentration occurred in the liver. The results suggest that circulating ceruloplasmin (made by the liver) finds its way into the cells of some organs, especially the heart, a phenomenon which may be related to the function of ceruloplasmin to provide copper to the cytochrome oxidase of various tissues.     

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.     

Studies of copper transport in cultured bovine chondrocytes

Copper serves as the cofactor for a number of important enzymes in cartillage, as well as in other tissues, including lysyl oxidase, superoxide dismutase, and cytochrome oxidase. Ceruloplasmin is resposible for the transport of approx. 95% of the copper in serum, but the mechanisms for intracellular copper transport are unknown. We have demonstrated recently that a high-molecular-weight cartilage glycoprotein, referred to as CMGP, has regions of sequence homology with ceruloplasmin. CMGP also binds copper and has at least some oxidase activity similar to that of ceruloplasmin. Other tissues synthesize intracellular ceruloplasmin-like proteins. The present report represents part of an effort examine the hypothesis the CMGP is a copper transport protein in chondrocytes and to characterize the enzymatic activities of CMGP. These studies demonstrate that CMGP is the principal chondrocyte protein labeled by ⁶⁷Cu in vitro and that the label is localized to the mitochondria, cytosol, and membrane fractions of sucrose gradients, suggesting copper transport through the cell. In parallel experiments, [³H]leucine was incorporated into proteins corresponding to the subunits and fragments of CMGP, as described previously, and in a similar distribution among the subcellular fractions as labeled copper. Additionally, CMGP has oxidase and ferroxidase activities similar to those of ceruloplasmin.     

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.     

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.     

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer’s disease. However, its physiological function remains elusive. Cu²⁺ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu²⁺ reduction and ⁶⁴Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu²⁺ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu²⁺ ions. Moreover, wild-type cells exposed to both Cu²⁺ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu²⁺ reductase activity and increased ⁶⁴Cu uptake. We conclude that Cu²⁺ reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.     

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.     

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.     

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M⁻¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.     

Effects of copper and ceruloplasmin on iron transport in the Caco 2 cell intestinal model

Previous studies have implicated copper proteins, including ceruloplasmin, in intestinal iron transport. Polarized Caco2 cells with tight junctions were used to examine the possibilities that (a) ceruloplasmin promotes iron absorption by enhancing release at the basolateral cell surface and (b) copper deficiency reduces intestinal iron transport. Iron uptake and overall transport were followed for 90 min with 1 &mgr;M 59Fe(II) applied to the apical surface of Caco2 cell monolayers. Apotransferrin (38 &mgr;M) was in the basolateral chamber. Induction of iron deficiency with desferrioxamine (100 &mgr;M; 18 h) markedly increased uptake and overall transport of iron. Uptake increased from about 20% to about 65% of dose, and overall 59Fe transport from <1% to 60% of dose. On the basis of actual iron released into the basal chamber (measured with bathophenanthroline), transport increased 8-fold. Desferrioxamine pretreatment reduced cellular Fe by 55%. The addition of freshly isolated, enzymatically active human ceruloplasmin to the basolateral chamber during absorption had no effect on uptake or transport of iron by the cells. Unexpectedly, pretreatment with three different chelators of copper (18 h), which reduced cellular levels about 40%, more than doubled iron uptake and raised overall transport to 20%. This was so, whether or not cells were also made iron deficient with desferrioxamine. Acute addition of 1 &mgr;M Cu(II) to the apical chamber had no significant effect upon iron uptake, retention, or transport in iron deficient or normal cells, in the presence of absence of ascorbate. We conclude that intestinal absorption of Fe(II) is unlikely to depend upon plasma ceruloplasmin, and that cuproproteins involved in this form of iron transport must be binding copper tightly.     

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.     

Mechanism of copper transport from plasma to hepatocytes

The effects of plasma components on the kinetics of copper transport by rat hepatocytes were examined in an attempt to determine how copper is mobilized from plasma for uptake by the liver. Specific protein-facilitated transport was indicated by saturation kinetics, competition by related substrates, and similar kinetic parameters for uptake and efflux. For copper uptake, Km = 11 +/- 0.6 microM and Vmax = 2.7 +/- 0.6 nmol Cu/(min X mg protein). Zinc is a competitive inhibitor of copper uptake, and copper competes for zinc uptake. Copper efflux from preloaded cells is biphasic. The kinetic parameters for the initial rapid phase are similar to the parameters for uptake. Copper transport by hepatocytes is strictly passive. A variety of metabolic inhibitors have no effect on uptake and initial rates are solely dependent on extracellular-intracellular concentration gradients. Albumin markedly inhibits copper uptake by a substrate removal mechanism, and histidine facilitates albumin-inhibited copper uptake. The active species that delivers copper to hepatocytes under conditions of excess albumin and excess histidine is the His2Cu complex. Experiments with [3H]His2 64Cu showed that the transported species is free ionic copper. The kinetic parameters of copper transport by hepatocytes isolated from the brindled mouse model of Menkes' disease are normal. However, these cells show a decreased capacity to accumulate copper on prolonged incubation. An intracellular metabolic defect seems to be involved.     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

α-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of α-tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR-α-tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Ceruloplasmin gene expression in human cancer cells

The copper transport protein, ceruloplasmin, is suggested to have a role in cancer since it is involved in angiogenesis and neovascularization. In order to understand the role of ceruloplasmin in malignant cells, we have recently isolated and sequenced a human ceruloplasmin cDNA clone. In the present study, we have investigated the ceruloplasmin gene expression in human colon and breast cancer cell lines. The poly (A) RNA from human colon (WiDr) and human breast (MCF-7) cancer cell lines was analyzed for the presence of ceruloplasmin mRNA. The Northern blot analysis revealed the presence of a 3.7 kb band of ceruloplasmin mRNA in these cell lines. Dot blot analysis revealed that ceruloplasmin mRNA is at least three fold more abundant in tumor cells as compared to normal rat liver.     

Studies on the uptake of radioactive copper by sheep erythrocytes in vitro

From previous studies different mechanisms for uptake of Cu by mammalian tissues from buffer and media containing proteins have been proposed. The interpretation of some of these investigations may have been complicated by the binding of Cu to proteins in the media.The uptake of ⁶⁴Cu from both buffer and plasma has therefore been studied using sheep erythrocytes in order to determine the mechanism and the effects of protein. Cu uptake was proportional to concentration of added Cu and the kinetics were those of a first order reaction for both media. There was no evidence for the participation of a membrane carrier and studies with inhibitors indicated that active transport was not involveed. For a given concentration of added Cu the rate of uptake was much slower in plasma and the effective concentration was calculated to be about 13% of that added. Efflux from labelled cells was much faster into plasma than into buffer. The addition of histidine to the medium increased uptake from dialysed plasma but not from buffer.It is concluded that Cu is taken up by the erythrocyte by simple diffusion, and the reduced rate in plasma is due to the binding of Cu to plasma protein thus reducing the effective concentration. The effect of histidine is attributed to the formation of a Cu-histidine complex which exists in equilibrium with Cu bound to albumin and facilitates removal of Cu from the latter.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Effects of Different Copper Sources and Levels on Plasma Superoxide Dismutase, Lipid Peroxidation, and Copper Status of Lambs

This study was performed to determine the effects of different copper (Cu) sources and levels on plasma superoxide dismutase (SOD), lipid peroxidation, and Cu status of lambs. Fifty Dorper × Mongolia wether lambs (approximately 3?month of age; average BW?=?23.8?±?0.6?kg) were divided into five equal groups each with ten animals according to their weight. Treatments consisted of (1) control (no supplemental Cu), (2) 10?mg Cu/kg DM from Cu-lysine, (3) 20?mg Cu/kg DM from Cu-lysine, (4) 10?mg Cu/kg DM from tribasic copper chloride (Cu(2)(OH)(3)Cl; TBCC), and (5) 20?mg Cu/kg DM from TBCC. The Cu concentration was 6.74?mg/kg DM in the basal diet. Plasma copper concentrations and ceruloplasmin activities were not affected on day?30 by Cu supplementation. Copper supplementation increased plasma and liver copper concentrations and ceruloplasmin activities on day?60. Muscle Cu concentrations were not affected by Cu supplementation. There were no differences in plasma, liver, and muscle Cu concentrations and ceruloplasmin activities between Cu-lysine and TBCC. Liver copper concentrations and plasma ceruloplasmin activities were increased in lambs supplemented with 20?mg Cu/kg DM than in those supplemented with 10?mg Cu/kg DM on day?60. However, copper levels had no effects on Cu concentrations in plasma and muscle. Malondialdehyde (MDA) concentrations were decreased in plasma and liver tissues, but not affected in muscle by Cu supplementation. Plasma SOD activities were increased by Cu supplementation. There were no differences in plasma, liver, and muscle MDA concentrations and plasma SOD activities between Cu sources and levels. These results indicated that Cu supplementation increased plasma SOD activity, lipid oxidative stability, and copper status of lambs, but did not influence lipid oxidative stability in sheep muscle. Cu-lysine and TBCC were of similar availability when offered to finishing sheep.     

A kinetic study on the copper-albumin catalyzed oxidation of ascorbate

Serum albumin can specifically bind one Cu(II)-ion, and is proposed to function as a copper transport protein in vivo. Cu(II)-albumin is rapidly reduced by ascorbate. A second order rate constant of 0.54 mM^–1 min^–1 was estimated for the reaction. The oxidation process is catalytic, the Cu(I)-albumin molecule being reoxidized by molecular oxygen. The reaction was found to follow Michaelis-Menten kinetics, characterized by an apparent K_m-value of 0.89 mM, and a catalytic constant of 0.066 M O₂/min. An apparent inhibition of oxygen uptake was obtained with catalase (but not with superoxide dismutase), suggesting the formation of H₂O₂ in the system. Wilson's disease patients usually have increased amounts of non-ceruloplasmin copper in plasma. The low level of plasma ascorbate observed in such patients could possibly be due, at least in part, to an oxidation by Cu(II)-albumin.