Copper

X Xu, S Pin, J Shedlock and Z L Harris, Johns Hopkins Hospital and School of Medicine, Baltimore, MD, USA

© 2005 Elsevier Ltd. All rights reserved.

Introduction

Transition metals occupy a special niche in aerobic physiology: as facile electron donors and acceptors, they are essential participants in oxidation/ reduction reactions throughout the cell. These unique properties of transition metals are largely dependent on the electronic configuration of the electrons in the outer shell and in the penultimate outer shell. These metals can exist in different oxidation states, which is critical for their usefulness as catalysts. However, it is during these same committed reactions essential for aerobic metabolism that toxic reactive oxygen species can be generated. As such the transition metals are chaperoned as they traffic through the body and are regulated tightly. Subtle disruptions of metal homeostasis culminate in disease and death. Iron, copper, and zinc are the most abundant and well-studied transition metals. Copper is the oldest metal in use: copper artifacts dating back to 8700bc have been found. The physiology, requirements, and dietary sources of copper are described here with an emphasis on the role of copper in human health and disease.

Copper, as a trace metal, can be found in all living cells in either the oxidized Cu(II) or reduced Cu(I) state. Copper is an essential cofactor for many enzymes critical for cellular oxidation. These include: cytochrome c-oxidase, which is essential for mitochondrial respiration as the terminal enzyme in the electron transport chain; superoxide dismu-tase, a potent antioxidant defense mechanism; tyrosinase, which is critical for melanin production; dopamine B-hydroxylase, a prerequisite for catecho-lamine production; lysyl oxidase, which is responsible for collagen and elastin cross-linking; ceruloplasmin, a ferroxidase/metallo-oxidase; hephaestin, a ferroxidase/ metallo-oxidase; and peptidylglycine a-amidating monooxygenase, a peptide processor (Table 1). Mice that lack the copper transport protein Ctr1 are embryonic lethal, which confirms the importance of copper in enzyme function and normal cellular homeostasis.

Copper Homeostasis

Dietary intake of copper is approximately 5mgday-1 with an equivalent amount being excreted by bile in stool. Approximately 2 mg day-1 are directly absorbed across the gastrointestinal tract daily and incorporated into blood, serum, liver,

Table 1 Mammalian copper enzymes

Table 2 Copper content of various foods

Enzyme

Function

Cytochrome c-oxidase cu,zn-Superoxide dismutase Tyrosinase

Dopamine B-hydroxylase

Lysyl oxidase

Ceruloplasmin

Hephaestin

Mitochondrial respiration Antioxidant defense Melanin production Catecholamine production Collagen and elastin cross-linking Ferroxidase/metallo-oxidase Ferroxidase/metallo-oxidase Peptide processing brain, muscle, and kidney. An equal amount is excreted and maintains the sensitive copper balance (Figure 1). The main sources of copper are seeds, grains, nuts, beans, shellfish, and liver (Table 2). Drinking water no longer contributes significantly. When copper pipes were commonly used for plumbing, copper toxicity was a more recognized phenomenon.

It is difficult to define specific dietary copper requirements because of the lack of suitable indices to assess copper status. As such, knowledge of factors affecting the bioavailability of dietary copper is limited. Ceruloplasmin contains 95% of the copper found in serum and is frequently used as a marker of copper status. However, ceruloplasmin levels vary with pregnancy and inflammation and ceruloplasmin mRNA is regulated by estrogen, infection, and hypoxia among other factors. Currently, investigators are searching for genetic bio-markers in intestinal, liver, and lymphocyte cells that respond to copper levels and may serve as better markers of copper status. Whole-body

Dietary copper (5 mg day-1 )

Dietary copper (5 mg day-1 )

Figure 1 Mammalian copper metabolism: daily copper cycle including oral absorption, tissue distribution, and excretion. Values are for adult men (mgday-1). An equal amount of copper is absorbed and excreted to maintain copper balance.

Table 2 Copper content of various foods

Mitochondrial respiration Antioxidant defense Melanin production Catecholamine production Collagen and elastin cross-linking Ferroxidase/metallo-oxidase Ferroxidase/metallo-oxidase Peptide processing

Food

Copper

Size of typical

Copper/

concentration

serving (g)

serving

(mg wet wt)

(mgg-1)

Fish

0.61

120

0.070

Turkey

0.71

120

0.090

Chicken

0.34

120

0.040

Hamburger

0.95

120

0.110

Roast beef

0.82

120

0.100

Steak

1.2

120

0.140

Sheep liver

157.05

120

18.850

Pork liver

141.14

120

16.940

Egg

0.8

40

0.030

Single sliced

0.43

120

0.050

cheese

Whole wheat

1.07

30

0.030

Scallops

6.08

120

0.030

Clams

7.39

120

0.730

Crab

1.75

120

0.890

Shrimp

1.75

120

0.210

Oysters

2.89

120

0.350

Smoked

15

120

1.800

oysters

Mussels

4.75

120

0.570

Lobster

36.6

120

4.390

Candy bar

1.18

15

0.020

Milk

0.33

120

0.040

Peas

2.38

120

0.290

Soy beans

109

120

0.130

Applesauce

0.2

120

0.020

(can)

Avocado

1.68

120

0.200

Raisins

1.68

30

0.050

Peanut

8.53

30

0.260

butter

copper metabolism is difficult to study in human subjects. However, isotopic tracers and kinetic modeling have added a dimension to what can be learned in humans by direct measurement. These studies suggest that the efficiency of copper absorption varies greatly, depending on dietary intake. Mechanisms regulating total body copper seem to be strong, given the relatively small and constant body pool, but they are not yet well understood. Changes in efficiency of absorption help to regulate the amount of copper retained by the body. In addition, endogenous excretion of copper into the gastrointestinal tract depends heavily on the amount of copper absorbed. When dietary copper is high and an excess is absorbed, endogenous excretion increases, protecting against toxic accumulation of copper in the body. When intake is low, little endogenous copper is excreted, protecting against copper depletion. Regulation is not sufficient with very low amounts of dietary copper (0.38 mg day-1) and appears to be delayed when copper intake is high.

Recommended Intakes

The Tolerable Upper Intake Limit (UL) for adults is 10 mg daily, based on degree of liver damage associated with intake. UL for children vary with age: 1-3 years/1 mg daily, 4-8 years/3 mg daily, 9-13 years/ 5mg daily, 14-18 years/8 mg daily (irrespective of pregnancy or lactation status). UL for children under the age of 1 year are not possible to establish. There are no official recommended daily allowances (RDAs) for copper in children. The RDA for adult males and females is a daily intake of 0.9 mg. Measurements of the dietary requirements for copper in adult men have shown the requirement to range from about 1.0 to 1.6 mg daily. A review of nutrient intakes in the US from 1909 to 1994 confirms that intake varied between 1.5 mg day-1 (1965) to 2.1 mg day-1 (1909). These trends reflect a diet higher in copper-rich potatoes and grain predominating in 1909 versus a decline in potato popularity in 1965. Daily intake recommendations for children vary with age (see Table 3). Persons who consume diets high in zinc and low in protein are at risk of copper deficiency. High intakes of dietary fiber apparently increase the dietary requirement for copper. Diets in Western countries provide copper below or in the low range of the estimated safe and adequate daily dietary intake. Copper deficiency is usually a consequence of low copper stores at birth, inadequate dietary copper intake, poor absorption, elevated requirements induced by rapid growth, or increased copper losses.

Bioavailability

The issue of bioavailability from food sources and the interactions between food groups and copper availability remains a critical question. Lonnerdal et al. demonstrated that heat treatment of cows'

Table 3 Recommended dietary allowances for copper (mg day 1)

Age

RDA (daily)

Infants

<6 months

0.2 (30 mcg/kg)

6-12 months

0.2-0.3 (24 mcg/kg)

Children

1-3 years

0.34

4-8 years

0.44

9-13 years

0.7

14-18 years

0.89

Adult

19+ years

0.9

Pregnant women

1

Nursing women

1.3

milk formula decreases the copper bioavailability. Transitional complexes form in the milk upon heating that have a similar configuration to copper and thereby directly inhibit copper absorption. High doses of zinc also reduce copper bioavailability, as does combined iron and zinc supplementation. The dilemma is how to prepare an infant formula containing adequate copper, iron, and zinc that will meet the RDA for copper. Other nutrients dramatically affect copper absorption from foods. Soy protein-based diets promote less copper retention in tissues than lactalbumin-based diets. However, it is unclear if this effect is solely due to the soy protein composition or to the higher zinc in these soy-based formulas. In animals, phytate causes a drop in serum copper but human stable isotope studies reveal no effect on copper absorption in adult men. Patients with low copper indices need to be evaluated for the copper content of their diets, other foods ingested at the same time, and other mineral supplements that may be given.

Absorption and Excretion

Dietary copper is absorbed across the small intestine. It diffuses through the mucous layer that covers the wall of the bowel via the divalent metal transporter DMT1. Copper is thus released into the serum and presumably is transported bound to either albumin or histidine to the multiple sites that require copper or to storage tissues. The liver is the primary storage organ for copper followed by muscle and bone. Not all of the copper ingested is absorbed and gastrointestinal cells that hold on to the excess copper are 'sloughed' when the lining of the gut is turned over every 24-48 hs. Copper bound to albumin or histidine enters the hepatocyte via the high-affinity mammalian copper transporter, hCtr1. Initially identified in yeast by functional complementation studies, this protein has subsequently been cloned in mice and humans. Human Ctr1 has a high homology to the yeast proteins Ctr1 and Ctr3 involved in high-affinity copper uptake. The N-terminus of the protein is rich in histidine and methionine residues, which presumably bind the copper and move it into the cell. Characterization of hCtr1 confirms its localization on the plasma membrane consistent with its role as a copper transporter. In vitro work has also identified a vesicular perinuclear distribution for hCtr1 that is copper concentration dependent. Redistribution of the hCtr1 suggests that under different copper states, copper moves through the membrane transporter and into a vesicular compartment for further 'assignment' within the cell.

hCtr2, a low-affinity copper uptake transporter, has also been identified. This low-affinity copper transporter is unable to complement the respiratory defect seen in yeast strains lacking copper transport capabilities. Once inside the cell, copper has one of four fates: (1) bind to and be stored within a glu-tathione/metallothionein pool; (2) bind to CCS, the copper chaperone for Cu, Zn - SOD; (3) bind to cox 17 for delivery to mitochondrial cytochrome c-oxidase; or (4) bind to HAH1 (human Atox1 homolog) for subsequent copper delivery to either the Wilson disease P-type ATPase or the Menkes' P-type ATPase. Copper from HAH1 is incorporated into ceruloplasmin, the most abundant serum cuproprotein, within the trans golgi network (TGN). How the protein unfolds within the TGN to accept copper and how the copper is incorporated into ceruloplasmin is still under study. Ceru-loplasmin is then secreted into the serum, and any excess copper not incorporated into ceruloplasmin is recycled in vesicles containing either the Wilson disease P-type ATPase or Menkes' P-type ATPase, and excreted into bile or stored in the liver. Recent characterization of a new protein, Murr1, suggests that this protein regulates copper excretion into bile such that mutations in the Murr1 gene are associated with normal copper uptake but severe defects in exporting copper from hepatocytes.

Approximately 15% of the total copper absorbed is actually transported to tissues while the remaining 85% is excreted. Of that copper pool, 98% is excreted in bile with the remaining 2% eliminated in the urine. The liver is the predominant organ responsible for regulating copper homeostasis at the level of excretion. Whereas copper import is highly conserved between yeast and humans, copper export in vertebrates involves a complex vesicular system that culminates in a lysosomal excretion pathway 'dumping' copper into the bile for elimination. At steady state, the amount of copper excreted into the biliary system is directly proportional to the hepatic copper load. In response to an increasing copper concentration within the hepatocyte, biliary copper excretion increases. There is no enter-ohepatic recirculation of copper and once the unabsorbable copper complex is in bile it is excreted in stool. Localization studies reveal redistribution of the ATP7b from the TGN to a vesicular compartment that migrates out to the biliary epithelium in response to increasing copper concentrations. Alternatively, under conditions of copper deficiency, the ATP7b remains tightly incorporated with the TGN for maximal copper incorporation into ceruloplasmin.

The highly homologous Wilson disease P-type ATPase (ATP7b) and the Menkes's P-type ATPase (ATP7a) differ only in their tissue expression and both function to move copper from one intracellular compartment to another. The ATP7a is predominantly located in the placenta, blood-brain barrier, and gastrointestinal tract and hence any mutation in the Menkes's P-type ATPase results in a copper deficiency in the fetus, brain, and tissues. In contrast, the Wilson's disease P-type ATPase is expressed in the liver and mutations in this culminate in profound copper overload of the liver because of the inability to shuttle copper into the trans golgi network for incorporation into ceruloplas-min. The excess copper is stored in the liver and eventually leaks out in the serum where it is deposited within sensitive tissues: the eye and brain. The psychiatric illnesses ascribed to Wilson's disease are a result of hepa-tocyte-derived copper 'leaking' out of the liver and accumulating within the basal ganglia. Similarly, Kayser-Fleischer rings arise from copper deposition in the cornea. The toxic copper in the liver eventually results in cirrhosis and hepatic fibrosis as a result of oxyradical damage. Menkes's syndrome has an incidence of 1:300000 while Wilson's disease has an incidence of 1:30000. Expression of these diseases may differ considerably among affected family members.

The recognition of a novel disorder of iron metabolism associated with mutations in the copper-containing protein ceruloplasmin revealed an essential role for ceruloplasmin as a ferroxidase and regulator of iron homeostasis. Patients and mice lacking the serum protein ceruloplasmin have normal copper kinetics: normal absorption, distribution, and copper-dependent activity. These data suggest that although under experimental conditions ceruloplasmin may donate copper, ceruloplasmin is not a copper transport protein. The six atoms of copper are incorporated into three type 1 coppers, one type 2 copper, and a type 3 copper. The type 1 coppers provide the electron shuttle necessary for the concomitant reduction of oxygen to water that occurs within the trinuclear copper cluster comprised of the type 2 and type 3 copper. This reaction is coupled with the oxidation of a variety of substrates: amines, peroxidases, iron, NO, and possibly copper. The recent observation that Fet3, the yeast ceruloplasmin homolog, also has critical cuprous oxidase activity in addition to ferroxidase activity has prompted renaming some of the multicopper oxidases (ceruloplasmin, Fet3, hephaestin) as 'metallo-oxidases' rather than ferroxidases.

Copper Deficiency

Reports of human copper deficiency are limited and suggest that severe nutrient deficiency coupled with malabsorption is required for this disease state to occur. Infants fed an exclusive cows' milk diet are at risk for copper deficiency. Cows' milk not only has substantially less copper than human milk but the bioavailability is also reduced. High oral intake of iron or zinc decrease copper absorption and may predispose an individual to copper deficiency. Other infants at risk include those with: (1) prematurity secondary to a lack of hepatic copper stores; (2) prolonged diarrhea; and (3) intestinal malabsorption syndromes. Even the premature liver is capable of impressive copper storage. By 26 weeks' gesta-tional age the liver already has 3 mg of copper stored. By 40 weeks' gestational age, the hepatic liver has 10-12 mg copper stored with the majority being deposited in the third trimester. Iron and zinc have been shown to interfere with copper absorption and further complicate the picture of copper deficiency. The most frequent clinical manifestations of copper deficiency are anemia refractory to iron treatment, neutropenia, and bone demineralization presenting as fractures.

The anemia is characterized as hypochromic and normocytic with a reduced reticulocyte count, hypo-ferremia, and thrombocytopenia. Bone marrow aspirate reveals megaloblastic changes and vacuolization of both erythroid and myeloid progenitor lineages. It is believed that a profound copper deficiency results in a multicopper oxidase deficient state and as such bone marrow demands are unmet by the lack of ferroxidase activity. Bone abnormalities are common and manifest as osteoporosis, fractures, and epiphyseal separation. Other manifestations of copper deficiency include hypopigmentation, hypotonia, growth arrest, abnormal cholesterol and glucose metabolism, and increased rate of infections.

Multiple factors associated with copper deficiency are responsible for the increased rate of infection seen. Most copper-deficient patients are malnourished and suffer from impaired weight gain. The immune system requires copper to perform several functions. Recent research showed that interleukin 2 is reduced in copper deficiency and is probably the mechanism by which T-cell proliferation is reduced. These results were extended to show that even in marginal deficiency, when common indexes of copper are not affected by the diet, the proliferative response and interleukin concentrations are reduced. The number of neutrophils in human peripheral blood is reduced in cases of severe copper deficiency. Not only are they reduced in number, but their ability to generate superoxide anion and kill ingested microorganisms is also reduced in both overt and marginal copper deficiency. This mechanism is not yet understood.

Copper Excess

Excess copper is the result of either excessive copper absorption or ineffective copper excretion. The most common diseases associated with copper excess are: (1) Wilson's disease, a genetic disease resulting in mutations in the Wilson's disease P-type ATPase and excessive hepatocyte copper accumulation; (2) renal disease, in patients on hemodialysis due to kidney failure when dialysate solutions become contaminated with excess copper; and (3) biliary obstruction. Excessive use of copper supplements may also contribute to copper toxicity and is clinically manifested by severe anemia, nausea and vomiting, abdominal pain, and diarrhea. Copper toxicosis can rapidly progress to coma and death if not recognized. Current management of most diseases associated with copper toxicity includes a low-copper diet, a high-zinc diet (competitively interferes with copper absorption), and use of copper chelators such as penicillamine and trientine. Affected individuals should have their tap water analyzed for copper content and drink demi-neralized water if their water contains more than 100 mg/liter. Given that the liver is the most significant copper storage organ, any activity that can affect hepatic cellular metabolism needs to be monitored. Hence, alcohol consumption is strongly discouraged.

There are reports of chronic copper exposure resulting in toxic accumulation. Fortunately, these events appear to be geographically restricted. Indian childhood cirrhosis (ICC), also known as Indian infantile cirrhosis or idiopathic copper toxicosis, has been associated with increased copper intake from contaminated pots used to heat up infant milk. The milk is stored and warmed in brass (a copper alloy) or copper containers. It is interesting to note that the increased copper absorption alone is not critical for disease formation but rather this occurs in infants that already have prenatal liver copper stores in excess of adult values. How the neonatal liver is able to compartmentalize this toxic metal so effectively is unknown. Perhaps in ICC, this delicate balance is disrupted. Tyrolean liver disease, occurring in the Austrian Tyrol, despite having a Mendelian pattern of inheritance suggestive of an autosomal recessive trait, appears related to use of copper cooking utensils. However, recent reports describe how a persistent percentage of the German population remains susceptible to copper toxicosis despite adjustments in cooking utensils. Perhaps a genetic susceptibility exists in this population that has yet to be determined.

Conclusion

Adult copper homeostasis rests on the foundation of an adequate copper balance in early life. Copper deficiency, either due to inadequate intake or abnormal absorption, may result. While the clinical stigmata of severe copper deficiency are easy to identify, the subtle changes in neurobehavioral development associated with mild copper deficiency are unknown. Given the high copper concentration in the brain, one could postulate that critical copper deficiency during development could lead to significant central nervous system deficits. Recent evidence suggesting that copper metabolism may be involved as an epige-netic factor in the development of Alzheimer's disease (AD) highlights the importance of balance. In this scenario, elevated central nervous system copper, as seen in AD, may initiate increased oxyradical formation and hasten damage. In fact, some are advocating that serum copper might be a good biomarker for AD. Copper is an essential trace metal critical for normal development. The goal of future studies will be to develop sensitive biomarkers for copper status. Only with these tools can we adequately assess copper status and treat copper-deficient and copper excess states appropriately.

See also: Bioavailability. Zinc: Physiology.

Further Reading

Araya M, Koletzko B, and Uauy R (2003) Copper deficiency and excess in infancy: developing a research agenda. Journal of Pediatric Gastroenterology and Nutrition 37: 422-429.

Bush Al and Strozyk D (2004) Serum copper: A biomarker for Alzheimer disease. Archives of Neurology 61: 631-632.

Gitlin JD (2003) Wilson disease. Gastroenterology 125: 1868-1877.

Klein CJ (2002) Nutrient requirements for preterm infant formulas. Journal of Nutrition 132: 1395S-1577S.

Lutter CK and Dewey KG (2003) Proposed nutrient composition for fortified complimentary foods. Journal of Nutrition 133: 3011S-3020S.

Prohaska JR and Gybina AA (2004) Intracellular copper transport in mammals. Journal of Nutrition 134: 1003-1006.

Rees EM and Thiele DJ (2004) From aging to virulence: forging connections through the study of copper homeostasis in eukaryotic microorganisms. Current Opinion in Microbiology 7: 175-184.

Schulpis KH, Karakonstantakis T, Gavrili S et al. (2004) Maternal-neonatal serum selenium and copper levels in Greeks and Albanians. European Journal of Clinical Nutrition 1: 1-5.

Shim H and Harris ZL (2003) Genetic defects in copper metabolism. Journal of Nutrition 133: 1527S-1531S.

Tapiero H, Townsend DM, and Tew KD (2003) Trace elements in human physiology. Copper. Biomedicine & Pharmacother-apy 57: 386-398.

Uauy R, Olivares M, and Gonzalez M (1998) Essentiality of copper in humans. American Journal of Clinical Nutrition 67(S): 952S-959S.

Wijmenga C and Klomp LWJ (2004) Molecular regulation of copper excretion in the liver. Proceedings of the Nutrition Society 63: 31-39.

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