Copper Homeostasis in Infant Nutrition: Deficit and Excess : Journal of Pediatric Gastroenterology and Nutrition

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Copper Homeostasis in Infant Nutrition: Deficit and Excess

Olivares, Manuel; Araya, Magdalena; Uauy, Ricardo

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Journal of Pediatric Gastroenterology and Nutrition 31(2):p 102-111, August 2000.
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Copper is an interesting essential micronutrient. Deficiency and excess intake both induce a variety of clinical manifestations affecting mainly the hematopoietic system, the skeleton, the liver, and the brain. Copper deficiency was first identified in infants recovering from malnutrition in conjunction with protein energy deficit and chronic diarrheal disease. On the other side of the spectrum, toxic effects may occur when formulas are prepared in copper vessels, leading to high intakes. In recent years attention has focussed on the potential adverse or toxic effects that may appear in individuals who are exposed to moderately high copper intakes. It is currently proposed that at least some of these individuals carry genetic polymorphisms or specific traits that make them more susceptible to toxic reactions to normal copper exposures. Infancy is a critical period in life for copper nutrition, because rapid skeletal growth and the requirements for brain development increase the demand for copper. This is especially so in low-birth-weight infants who are born with diminished liver copper stores. In contrast, non–breast-fed babies depend on formula as their main source of nutrients, and thus, if the bottle contains copper-contaminated milk or water, copper exposure increases substantially.

This article reviews present knowledge of the molecular and cellular bases of copper homeostasis as they relate to the evaluation of copper status. The characteristics of copper deficiency and excess and the genetic and environmental factors that determine deficit and excess are described, stressing those aspects relevant to infant nutrition.


Copper is important for appropriate functional, structural, and catalytic activity of cuproproteins in various forms of life, from yeast to mammals. Most cuproproteins of interest are enzymes that display oxidative-reductase activity in which copper is essential to the electron transfer process. Copper has been shown to be an essential cofactor for catalytic activity of lysine 6-oxidase, catechol oxidase, superoxide dismutase-1 (SOD), cytochrome c oxidase (COX), and ceruloplasmin (Cp) (1–6). These redox enzymes are essential for normal cellular respiration, free radical defense, synthesis of melanin, connective tissue biosynthesis, and cellular iron metabolism. The biochemical and clinical manifestations of copper deficiency reflect abnormalities in functions directly related to these enzymes. For example the flag sign—loss of hair pigment in the form of a band that is typical in copper deficiency—is explained by the loss of tyrosine oxidase activity required for melanin synthesis. Fractured bones are explained by deficient collagen cross-linking due to insufficient lysine hydroxylation dependent on lysyl 6-oxidase.

At the molecular level, copper is essential for the expression of genes related to copper metabolism. The metal, bound to ligands, acts on specific DNA-responsive elements activating or repressing gene transcription (7,8). This information, obtained mainly in yeast, has been corroborated in higher eukaryotic organisms including mammals. The evolutionary conservation of the structure and function of copper transcription factors originally described in yeast has been well demonstrated. At the physiological level, transcription factors serve as sensors of intracellular copper levels or display a regulatory role in switching on or off gene expression associated with the processes responsible for copper homeostasis.

The transport of copper across cell membranes is fundamental to understanding the process of copper metabolism (9). Two homologous cation transporting P type adenosine triphosphatases (ATPases) are the main gene products related to cell copper homeostasis, the Menkes and the Wilson ATPases (MNK and WND, respectively) (10–14). They have similar structures and parallel roles in regulating the copper status of cells and tissues and in supplying copper to secreted cuproenzymes. In most cells, except in the liver, MNK is responsible for excreting copper when levels are high. In hepatocytes after birth, this role is performed by WND, which disposes of excess copper by secreting it into the biliary canalicular system and excreting it to intestine. Both proteins supply copper to the enzymes that are secreted by the cell by pumping the metal ion into the trans-Golgi network, where the metal is incorporated into the apoenzyme (9). The regulation of copper efflux by the cell is achieved by copper-induced relocalization from the trans-Golgi to the plasma membrane in the case of MNK or to an intracellular vesicular compartment in the case of WND.


Copper content in foods is highly variable, depending on inherent (genetics, age) and environmental conditions (soil, geographic location, season, water source, use of fertilizers, and methods of handling, processing, and cooking food) (15). For instance, the copper content in 16 varieties of wheat grown in the same soil under similar conditions ranges from 0.5 to 1.67 mg/100 g (16). Its concentration is higher in greener and younger portions of plants (15). Younger animals have a higher copper content in organs and muscles than older animals of the same species (15). Organ meats, shellfish, nuts, and some grains have higher copper concentrations, whereas milk and dairy products are poor sources of it (15). In cow's milk, the copper level is lower than in human milk. In human milk, copper concentrations range from approximately 0.4 to 0.6 mg/l in early lactation to 0.2 to 0.3 mg/l in mature milk, declining during the first 60 days of lactation (17–20). In women who give birth prematurely, copper in breast milk is somewhat higher than in milk of mothers of term neonates (20). In human milk, copper is associated mainly with whey proteins (56%) and lipids (15%) and a small proportion with casein (8%), whereas in cow's milk it is associated mainly with casein (44%) and a small proportion with lipids (2%) and whey (8%) (21).

Most infant formulas are supplemented with copper (22,23) to support the nutritional requirements of full-term or preterm infants. In powdered milk, the copper content of the formula also depends on the amount of water added to it. The United States Food and Drug Administration (FDA), the Codex Alimentarius (1976), and the American Academy of Pediatrics (AAP, 1985) recommend a minimum specification for infant formulas of 0.9 μmol of copper/418 kJ (60 μg of Cu/100 kcal) (24–26). The current recommendation of the ESPGAN Committee on Nutrition (1987) is 1.4 μmol of copper/418 kJ (90 μg of Cu/100 kcal) (27). Recommendations for premature formulas are 1.4 to 3.1 μmol/418 kJ (90–200 μg Cu/100 kcal) (26–28).

Animal studies indicate that the major site of copper absorption is the duodenum, although some is also absorbed in the stomach and some in the distal part of the small intestine (29). Approximately 15% of the copper excreted in bile is reabsorbed through the enterohepatic circulation. There is evidence suggesting that at low dietary copper levels, absorption occurs by a saturable, active transport mechanism, whereas at higher levels, passive diffusion plays a role (30). Variations due to age are unclear and may be relevant. Metallothionein (MT) in the intestinal epithelial cells is also involved in the regulation (30).

Several factors may modify copper absorption, some of which are not fully defined. Copper absorption is influenced by copper intake and by copper nutritional status (31,32). When dietary copper is low, the fraction absorbed increases, and endogenous losses decline. When dietary copper is high, a reduction of absorption and an increase of endogenous losses are observed. Probably the increase of copper absorption is the most important adaptive mechanism when copper intake is low. Furthermore, the evidence suggests that the upregulation of absorption in response to a dietary decrease is faster than the downregulation in response to an increase in dietary copper. Chemical compounds with higher solubility promote more efficient absorption (33). The stomach pH has an important role in facilitating copper solubility and modulating the interaction with ligands and other components of the diet. Absorption of 65CuCl2 administered as an aqueous solution is better than absorption of similar amounts provided in food (32). A study using stable isotopes of copper demonstrated that absorption is higher from an animal protein (41%) than from a plant protein diet (34%). However, because the copper content in the plant protein diet is higher, the absolute amount of copper absorbed is higher with this diet (31).

Tissue copper retention in rats is lower with a lactalbumin-based diet than with soy protein (34). It has been speculated that this could be related to competition with zinc absorption, because in this experiment the soy diets tested contained a significant amount of zinc. Animal studies suggest that phytate has a negative effect on copper availability, but its role in humans is less clear. Turnlund et al. (35), using stable isotopes, evaluated the effects of phytate and α-cellulose in young men and found no effect of either component on copper absorption, whereas zinc showed a marked decrease. However, serum copper declined with either phytate or α-cellulose added to the diet, suggesting that they may alter copper utilization or distribution. Some amino acids are known to form complexes with copper and influence copper absorption. For example, methionine increases copper absorption in humans (36), whereas cysteine decreases its absorption in chickens (37).

In animals, it is well known that l-ascorbic acid has a negative effect on absorption by reducing cupric to cuprous ions, which are less well absorbed. Whether animals used for these experiments are repleted or copper deficient seems to be significant. In young adult men no effect was observed after ingesting up to 600 mg of ascorbic acid per day (38). In low-birth-weight infants supplemented with ascorbic acid (50 mg/d) no negative effect was observed (39). However, these infants were largely in negative mineral balance, and it is possible that ascorbic acid under these conditions exerts no effect. Organic acids present in foods (citric, lactic, acetic, and malic acids) increase copper bioavailability, probably by a solubilization effect (40). Both animal and human studies suggest that fructose may have negative effects on copper metabolism. In humans, although fructose increases copper absorption (41), SOD activity is reduced (42).

Hill and Matrone (43) hypothesized that transitional elements that have the same configuration and form similar coordination complexes in water interact at the absorption level. Evidence obtained both in animals and humans indicates that this is the case. This is relevant to infant nutrition, because formula and other infant foods are often supplemented with zinc and iron, and therefore copper absorption may be impaired to some degree. Yet, the effect appears only with large doses of zinc. For instance, a 40% decrease in copper absorption was observed when rats received 900 mg/kg per day, but there was no effect when they ingested 450 mg/kg (44). In patients with Wilson disease, reduction of copper absorption is achieved by administering pharmacologic doses of zinc; however, lower doses have no effect (45,46).

This large body of information shows that copper homeostasis is a highly regulated phenomenon that depends on the amount of copper present in the intestinal lumen, the ratio between promoters and inhibitors, and copper nutritional status. Large dietary variations are compensated by these mechanisms so that homeostasis is maintained. Unfortunately, most of the regulatory mechanisms and interactions among the minerals involved are not fully understood.


Copper nutrition during fetal life represents the balance between the high requirements due to rapid growth and the placental carrier-mediated transport from maternal Cp and other copper-binding proteins (47–49). The fetus accumulates copper at a mean rate of 50 g/kg·day, mainly during the second half of gestation (50). Approximately 50% of it is stored in the liver, mostly bound to MT. The brain represents the second most important site of copper accumulation in the fetus. At the time of birth a full-term infant has close to 15 mg of copper, 9 mg of which is in the liver.

That copper is accumulated mostly during the third trimester of pregnancy explains why copper deficiency is more frequent in preterm infants, especially those with very low birth weight. Although copper transport to the fetus is high and liver storage is efficient, copper export from the hepatocytes to the bile and to blood Cp are reduced during this stage of life because of liver function immaturity. This leads to a high copper accumulation in the liver, in a magnitude similar to that observed in Wilson disease. It is not clear why the neonate can handle this large amount of copper without adverse consequences. The increased liver copper stores of the fetus have evolutionary advantages, because they may help to prevent copper deficiency during the first months of life.

After birth, liver copper concentration drops steadily, because early diet is insufficient to meet the requirements for rapid growth typical of this period. In addition, biliary function improves, augmenting fecal copper losses. Klein et al. (51) demonstrated that hepatic copper levels remain fairly constant at 51.0 ± 29.3 μg/g wet weight between 22 weeks of gestational age and 13 weeks after birth, declining thereafter to 5.7 ± 2.5 μg/g wet weight between 6 and 14 months after birth. In contrast, term infants achieve adult concentrations of serum copper and Cp after 6 months of age (in premature infants these increases are delayed). A slower increase in both serum copper and Cp continues up to 12 months of age (52–54).

Criteria used to define copper intake recommendations have differed over time; thus, recommended values have changed. The U.S. National Academy of Science Nutrition Research Council (NAS/NRC) (55) has recommended that infants from 0 to 6 months of age should receive a daily intake of 0.4 to 0.6 mg of copper, increasing progressively to 1 to 2 mg in children up to 10 years of age (recommended daily allowance [RDA]). For adolescents and adults this range is 1.5 to 2.5 mg and 1.5 to 3 mg, respectively. In 1996 the World Health Organization/Food and Agriculture Organization/International Atomic Energy Agency (WHO/FAO/IAEA) (56) defined the safe minimum mean copper intake at 0.33 to 0.55 mg/day for infants 0 to 3 months of age, increasing progressively to 1.15 and 1.35 mg/day for female and male adults, respectively.

Infants fed exclusively cow's milk–based diets are more prone to development of copper deficiency because of the low copper content of milk and limited absorption of this mineral from cow's milk (57,58). In contrast breast-fed infants absorb more copper, probably because of the lower casein content of human milk or because it contains factors that may enhance copper absorption (59,60). In developing countries, where infant feeding is often based on cow's milk with addition of high concentration of refined carbohydrates, copper deficit may be more prevalent, because fructose and other refined sugars influence copper utilization. Results in studies in which stable isotopes (65Cu) were used suggest that if sufficient copper is provided, infants can retain enough of it to meet their high requirements. However, absorption and its regulation during early life are still not well understood. Balance studies in preterm or term infants fed cow's milk–based diets or unfortified pasteurized human milk showed that most infants are in negative copper balance or at best in marginally positive balance (61,62). Infants fed human milk absorbed 67% to 80% of intake, whereas those receiving formula absorbed only 39% to 45%(58) (Olivares et al., unpublished observations, 1996). The most important mechanism of copper homeostasis may be dietary modification of absorption and/or excretion, but nutritional copper status also plays a role in adjusting the degree of intestinal absorption. Whether neonates and small infants can fully adjust is unclear.


Menkes and Wilson diseases represent the classic examples of genetic conditions that induce severe tissue copper deficiency and severe toxic effects, respectively. Because animal models are available for both conditions (the mottled mouse and Long-Evans cinnamon rat or Bedlington terrier models, respectively) these two extreme conditions have been extensively studied. Knowledge of the molecular basis of copper metabolism has advanced primarily based on Wilson and Menkes diseases models (63,64). Menkes disease is an X-linked recessive disorder characterized by a defective ATP7A or MNK protein, which leads to abnormal absorption, transport, and distribution of copper in the body, at both the organ and cellular levels. The incidence of this disorder is 1:50,000 to 1:100,000 live births (65). Affected patients manifest severe copper deficiency, including profound mental retardation, growth failure, hypothermia, skin and hair depigmentation and abnormal spiral twisting of the hair (pili torti), lax skin and articulations, tortuosity and dilatation of major arteries, varicosities of veins, osteoporosis, flaring of metaphyses, bone fractures, excessive wormian bone formation, severe central nervous system damage, and retinal dystrophy, and premature death (66). Results of early treatment with copper-histidine of milder phenotypes of the disease suggest that some improvement may be achieved when copper is provided soon after birth, the molecular abnormality is not universal, and there is some residual activity of the ATPase (65). Designing new strategies to deliver copper directly to the cells is a major medical challenge today.

Wilson disease, an autosomal recessive disorder characterized by the presence of a dysfunctional ATP7B or WD gene, represents the main cause of hepatic copper accumulation in humans and severe toxic effects in nature (12–14). Ample variety and severity of clinical manifestations are in accordance with the genetic heterogeneity of the alteration located in chromosome 13 (67). Its incidence is 1:30,000 live births, whereas carrier frequency is 1:90 (67). Symptoms rarely appear before 5 years of age. Clinical manifestations depend on copper deposition in specific organs, mainly liver, brain, and cornea (Kayser-Fleischer ring). Chronic liver disease and/or neurologic or psychiatric impairment, frequently with kidney malfunction, is the most frequent presentation, but ophthalmologic, hematologic, and skeletal manifestations are also common in some series (67). Despite high levels of copper in the liver, Cp and copper levels in blood are low, whereas urinary copper excretion is increased (67). Restriction of dietary copper has little impact on the course of the disease. Current therapeutic strategy consists of decreasing copper absorption by using pharmacologic doses of oral zinc (40–50 mg/d) and/or increasing copper excretion by means of chelating agents such as d-penicillamine or British anti-Lewisite (BAL) (67). Whether heterozygotes for ATP7B have a higher of risk of development of manifestations of copper overload when copper exposure increases remains to be determined.


Food normally accounts for more than 90% of copper intake in adults and children if water has low copper content (<0.1 mg/l). If drinking water copper content is higher than 1 to 2 mg/l, it may account for up to 50% or more of total intake (68). In infants, the contribution of water to copper intake is high, because they consume proportionally more water than do adults (69).

In acquired copper deficiency, low dietary intake (or poor bioavailability) is only one of the factors to be considered. Rapid growth rate in infants, reduced liver stores in premature babies, presence of malabsorption syndromes, and increased copper losses are also determinants of risk when dietary intake is borderline (70,71). Anemia refractory to iron and low copper plasma levels are the main manifestations of nutritional copper inadequacy. This was first reported in 1956 (72), but it was not until 1964 when Cordano (71) reported controlled clinical studies in infants recovering from malnutrition that copper deficiency in humans was fully recognized.

Other frequent clinical manifestations accompanying anemia are neutropenia and bone abnormalities (73,74). Anemia is hypochromic, normocytic, or macrocytic, with a reduced reticulocyte count, hypoferremia, neutropenia, and thrombocytopenia (73–75). Although unresponsive to iron, these changes are readily corrected by copper supplementation. The current prevailing view is that anemia in copper deficiency is caused by defective iron mobilization resulting from reduced Cp (ferroxidase I) activity (73,74). Less frequent manifestations of copper deficiency are hypopigmentation of the hair and hypotonia (73,74), impaired growth (76), increased incidence of infections (77), alterations of phagocytic capacity of the neutrophils, and impairment of cell immunity (78). In addition, abnormalities of cholesterol and glucose metabolism have been reported, but they are less well established.

Acute copper toxicity is infrequent in humans and is usually the consequence of consuming contaminated foodstuffs or beverages (including water), or accidental or deliberate ingestion of high quantities of copper salts. Acute symptoms include excessive salivation, epigastric pain, nausea, vomiting, and diarrhea (79). When acute ingestion exceeds 100 g of copper salts, severe copper poisoning ensues, resulting in multisystemic failure, shock, coma, and eventual death.

Recently, various experimental clinical assays have demonstrated that a concentration of 2 mg Cu/l of potable water does not produce an increase in gastrointestinal symptoms in infants (80). Pettersson in Sweden reported a higher frequency of diarrhea in infants receiving an average copper intake from water of 0.27 mg/day compared with those receiving an average intake of 0.48 mg/day (81). This result is discordant with the response observed in experimental subjects consuming water with a copper content of 4 to 6 mg/l, suggesting that the antimicrobial properties of copper may contribute to diarrhea prevention. The analysis of current publications supports the safety of 2 mg Cu/l, set as a provisional level, by the World Health Organization (WHO) for copper content of drinking water.

Toxic effects associated with increased chronic exposure are rare and occur as clusters in specific geographical areas. In India, cases of infantile cirrhosis (Indian childhood cirrhosis [ICC]) have been associated with increased copper intake (82). The cases reported were in families living in areas where milk is warmed, boiled, and stored in brass or copper containers. Tanner calculated that these children may receive up to 930 ± 36 μg/kg per day, an amount high enough to explain by itself the appearance of liver damage in the absence of genetic abnormalities of copper metabolism (83). Between 1900 and 1974 in rural areas of Austrian Tyrol, where copper utensils were also used to prepare infant foods, 138 infants and young children died from liver cirrhosis attributed to a high chronic copper exposure (84,85). In this case, inheritance followed the typical pattern of a Mendelian recessive trait. No further cases were observed after copper utensils where abandoned.

Sporadic cases have also been reported in several countries. In some of these cases high levels of copper in drinking water have been demonstrated post facto (86). However, that some of these cases occurred in consanguineous marriages, that cases were more frequent among boys, and that some patients had not received high amounts of copper in the diet (including water) suggests that these cases may be of genetic origin. One or more traits may underlie the genetic susceptibility to toxicity at normal or mildly elevated levels of copper intake (82,85,87). Moreover, that there were other children living in the same geographical areas receiving similar levels of exposure who did not have liver abnormalities supports the view of increased genetic susceptibility. A more thorough understanding of copper absorption and excretion in early infancy and the infants' response to high intakes is crucial to unraveling the origin of ICC and idiopathic copper toxicosis (ICT) and its relationship to copper.

In summary, the mechanisms that explain the consequences of severe deficits or toxicity are better understood based on the genetic disorders of copper homeostasis. The factors that explain individual susceptibility to deficiency or toxicity in the “normal” population or in specific population groups are poorly understood. A normal gaussian distribution is frequently assumed in the estimation of population nutrient requirements; based on this, risk from deficit or excess follows a sigmoid-shaped curve as shown in Figure 1. Recommended nutritional intake (RNI) and upper limit of intakes should prevent deficiency and toxic effects, respectively. It is also possible that ICT can be explained by a specific gene or a given genetic polymorphism. In this case, a subset of the population may be susceptible to development of detrimental effects at lower intakes. If this group is a significant proportion of the population, the upper limit should be based on the need to protect them from excess. If the genetic trait is extremely rare, however, it is debatable whether the recommendations for the general population should be based on a small genetically defined subgroup. For example, an upper limit for copper intake to protect subjects who are homozygous for Wilson disease (1:10,000 may have the genetic trait but 1:30,000 have the clinical syndrome) or an RNI to protect persons with Menkes (1:25,000 may have the genetic trait but 1:100,000 have manifestations) from copper deficit would place most of the normal population at a risk of deficit or excess, respectively.

FIG. 1.:
Risk assessment model proposed to define the risk areas for deficit and excess. This example is based on data obtained from the literature from infants and small children. Striped area shows a range based on different criteria to define deficiency and toxicity risks. In individuals with idiopathic copper toxicosis, the toxicity risk curve shifts to the left (dotted line).


Serum copper and Cp concentrations are currently used to evaluate copper status. These indicators are sensitive to moderate or severe copper deficiency but are not responsive to marginal deficiency, especially if low intake is recent (88). Their low sensitivity and the fact that they change with age and sex (89) and during inflammatory or infectious processes are major limitations in their use as markers of copper status (88). A study of both Cp enzymatic activity and Cp protein concentration showed that copper deficiency induces a reduction of enzymatic activity, whereas the concentration of the apoprotein remains unchanged (89). This has served to support the notion that the ratio of enzymatic activity to mass protein concentration of Cp may be a better indicator of copper status; in addition, this index is not influenced by factors such as hormonal changes or gender (89). Hair copper content measurement has not been useful, mainly because it changes only in prolonged copper deficiency and may be modified by external agents, including environmental contamination with copper.

Reduced erythrocyte SOD activity has been demonstrated in copper-deficient patients or in subjects receiving a low copper intake (90). The activity of SOD does not seem to vary with age, gender, or hormonal therapy (89). It is currently under evaluation as an indicator of marginal deficiency. The COX activity of leukocyte and platelets is reduced in copper deficiency (89). Further, this decrease occurs before the appearance of a reduction of SOD activity (89) suggesting this may be a more sensitive marker of tissue status.

Measurement of diamine oxidase (DAO) has also been used to measure copper nutritional status. Animal and human studies have demonstrated that plasma DAO activity is reduced in marginal copper deficiency (91). However, tissue injury of organs such as kidney or intestine may lead to elevation of plasma DAO activity, confounding its interpretation (91). Table 1 summarizes the change in these indicators at each stage in the progression of copper deficiency. Unfortunately, none of these laboratory parameters by themselves or as a group is sensitive in the evaluation of copper excess.

Stages in the progression of copper deficiency

Noninvasive laboratory indicators to measure status of tissue reserves, its changes within the homeostatic range of exposure, and early overload are not available. Ideally, measurement of liver copper content would serve as a sensitive and specific indicator of excess, as long as the various compartments were well characterized. For example total copper content of the liver of full-term infants is similar to that found in patients with Wilson disease or in biliary cirrhosis (92). Thus, evaluation of discrete copper storage domains is necessary to define risk of excess intake. Data obtained by Klein et al. (51) demonstrate that most of the copper in the liver of human infants is MT bound. With advancing postnatal age, liver copper content decreases, and MT copper saturation decreases significantly, reaching 10% to 20% by 6 months of age. Thus, MT saturation may be a critical marker; moreover, it may serve as an early sign of copper-induced damage because if copper exceeds binding capacity, free copper–induced oxidative damage may ensue (63,93). Results from in vitro and in vivo studies suggest that this event may be the final step, triggering a cascade of further protein oxidative damage leading to a progressive loss of the copper-binding capacity of MT and an increase in free copper. These steps conclude in the activation of Kupfer cells with release of cytokine leading to necrosis, progressive liver damage, and fibrosis (63).

The Long-Evans cinnamon rat, a model for Wilson disease demonstrates copper-induced fulminant hepatitis leading to death 4 months after birth. Pretreatment with antioxidants or corticoesteroids ameliorates its course. This suggests that oxidant damage and cytokine release may be important mediators for copper-induced cirrhosis of the liver. However, because of obvious ethical concerns, liver biopsies cannot be performed in normal persons to evaluate the upper limit of the safe range of human copper intake. Noninvasive methods to evaluate copper excess are urgently needed.

In an effort to measure the effect of a higher copper burden in infants, we explored the use of erythrocyte MT as a potential indicator of copper load. As described in the preceding section, most of the copper in tissues is stored bound to MT, and copper overload induces MT gene expression (94). One hundred twenty-eight healthy infants were randomly assigned at 3 months of age to 150 μg/kg or 50 μg/kg body weight copper intakes by providing them with water of different copper content to dilute the formula powder during 9 months. The copper intake values were within WHO recommendations for age. Blood indicators of copper status (serum copper, Cp, SOD, and erythrocyte MT) were monitored at 3-month intervals and found no changes between groups (95). These results may be interpreted as evidence for adaptive responses to the higher or lower copper intake studied or, alternatively, that biochemical indices used were not sensitive to detect changes within the ranges evaluated.

Approximately 90% of copper in blood is bound to ceruloplasmin. Elevation of the non–ceruloplasmin-bound copper level has been observed in patients with Wilson disease and ICT (96,97) and human neonates supported by TPN (98). We have calculated from our published data the concentration of non–ceruloplasmin-bound copper in the 128 well-nourished infants receiving copper within a range of 50 to 150 μg/kg per day (95). Figure 2 depicts the correlation between non–ceruloplasmin-bound copper and serum copper. A strong correlation suggests that when serum copper exceeds the lower normal limit (14 μmol/l = 90 μg/dl), ceruloplasmin would be fully saturated if no other binding compound were available. The non–ceruloplasmin-bound copper increased linearly within the normal range, suggesting tight binding by other compounds, because free copper is not physiologically present in the serum.

FIG. 2.:
Correlation between serum copper and non–ceruloplasmin-bound copper in normal infants given copper at daily intake of 50 to 150 μg/kg (95).


The need to develop specific approaches for the assessment of risk associated with the exposure to essential elements has now been well established. Present efforts to establish health risks for essential elements are being spearheaded by WHO as part of its program for chemical safety (99,100). Considerations for risk assessment are being defined based on the following considerations:

  • Health risk assessment for essential elements should take into account risks associated with chronically low intakes as well as high intakes (101). The curve describing the relationship between intake or exposure and chronic effects (risk of deficiency and toxicity) has a U shape. This approach requires the definition of an adequate range of oral copper intake (AROI) to prevent toxicity and to minimize the risk of deficiency.
  • Risk of deficiency and excess should be defined based on equivalent indicators (biochemical markers, functional endpoints with or without health significance, subclinical signs, mild symptoms, clinical disease). For example, if toxicity is defined based on a level of intake that induces a subclinical, sign so should the definition of deficient intake.
  • For some essential elements (such as copper when present in water) risk assessment should include potential acute effects, if the element can cause acute gastrointestinal manifestations or other acute effects of health significance.
  • The lower level of AROI, independent of how the value was derived, should be within the range of observed intakes in healthy human populations.
  • The AROI should include appropriate correction factors for bioavailability and utilization based on how the element is actually ingested and to account for the possible interaction with other dietary factors.
  • The lower and upper limits of AROI should be protective of deficit and excess for the majority of the population. The definition of majority rests within the national authorities.

Implementation of this risk assessment approach requires valid indices to detect homeostatic regulation across a range of intakes and the capacity to assess where the limit of physiologic regulation ends and when the adaptation detected may have adverse consequences with public health relevance. Figure 1 represents an integrated summarized view of the AROI model for copper in infants, based on data available from the literature. On the deficiency side, the curve on the extreme left is based on copper intake associated with low hemoglobin secondary to copper deficit, a marker for anemia with known health consequences; the right side of the range is based on copper retention required for normal growth. This curve would move further to the right if the indicator used were an enzyme activity with unknown health consequences (i.e., SOD or COX). On the toxicity side there are no early markers, unfortunately, although there is general agreement that measurement of copper liver content should be the reference standard, it is not possible to make these measurements in “healthy” individuals. The dotted curve in Figure 1 is based on a hypothetical response of sensitive population based on genetic traits, the solid curve is the suggested response assuming that there may be early indicators of liver injury. The extreme right curve is based on copper intake associated with liver damage in infants affected with ICC, as reported by Tanner et al. (83) We propose that markers such as copper saturation of MT in selected tissues or unbound serum copper (that is the difference between total serum copper and that bound to Cp and small molecular weight compounds) be explored as potential early indices of excess. The use of new microarray technology to evaluate the effect of copper on gene expression in selected tissues may provide novel means to assess the range of adaptation and the evaluation of consequences from exposure to high and low copper intakes. A better definition of biomarkers for excess represents a major challenge in providing appropriate copper nutrition for infants.


1. Rucker RB, Kosonen T, Clegg MS, et al. Copper, lysyl oxidase, and extracellular matrix protein cross-linking. Am J Clin Nutr 1998; 67:996S–1002S.
2. Linder MC, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutr 1996; 63:797S–811S.
3. Sánchez-Ferrer A, Rodríguez-López JN, García-Cánovas F, García-Carmona F. Tyrosinase: A comprehensive review of its mechanism. Biochim Biophys Acta 1995; 1247:1–11.
4. Tainer JA, Getzoff ED, Richardson JS, Richardson DC. Structure and mechanism of copper, zinc superoxide dismutase. Nature 1983; 306:284–7.
5. Yoshikawa S, Mochizuki M, Zhao XJ, Caughey WS. Effects of overall oxidation state on infrared spectra of heme a3 cyanide in bovine heart cytochrome c oxidase: Evidence of novel mechanistic roles for CuB. J Biol Chem 1995; 270:4270–9.
6. Kaplan J, O`Halloran TV. Iron metabolism in eukaryotes: Mars and Venus at it again. Science 1996; 271:1510–2.
7. Thiele DJ. Metal-regulated transcription in eukaryotes. Nucleic Acids Res 1992; 20:1183–91.
8. O'Halloran TV. Transition metals in the control of gene expression. Science 1993; 261:715–25.
9. Peña MMO, Lee J, Thiele DJ. A delicate balance: Homeostatic control of copper uptake and distribution. J Nutr 1999; 129:1251–60.
10. Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. J Nat Genet 1993; 3:7–13.
11. Mercer JFB, Livingston J, Hall B, et al. Isolation of a partial candidate gene for Menkes disease by positional cloning. J Nat Genet 1993; 3:20–5.
12. Yamaguchi Y, Heiny ME, Gitlin JD. Isolation and characterization of a human liver cDNA as a candidate gene for Wilson's disease. Biochem Biophys Res Commun 1993; 197:271–7.
13. Petrukhin KE, Lutsenko S, Chernov I, Ross BM, Kaplan JH, Gillian TC. Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: Genomic organization, alternative splicing, and structure/function predictions. Hum Mol Genet 1994; 3:1647–56.
14. Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. J Nat Genet 1993; 5:327–36.
15. Pennington JT, Calloway DH. Copper content of foods. J Am Diet Assoc 1973; 63:143–53.
16. Greaves JE, Andersen A. Influence of soil and variety on the copper content of grains. J Nutr 1936; 11:111–8.
17. Casey CE, Hambidge KM, Neville MC. Studies in human lactation: Zinc, copper, manganese and chromium in human milk in the first month of lactation. Am J Clin Nutr 1985; 41:1193–200.
18. Feeley RM, Eitenmiller RR, Jones Jr JB Barnhart H. Copper, iron, and zinc contents of human milk at early stages of lactation. Am J Clin Nutr 1983; 37:443–8.
19. Fransson GB, Lönnerdal B. Zinc, copper, calcium, and magnesium in human milk. J Pediatr 1982; 101:504–8.
20. Mendelson RA, Anderson GH, Bryan MH. Zinc, copper and iron content of milk from mothers of preterm and fullterm infants. Early Hum Dev 1982; 6:145–51.
21. Lönnerdal B. Copper nutrition during infancy and childhood. Am J Clin Nutr 1998; 67:1046S–53S.
22. Lönnerdal B, Keen KL, Ohtake M, Tamura T. Iron, zinc, copper, and manganese in infant formulas. Am J Dis Child 1983; 137:433–7.
23. Johnson MA, Smith MS, Edmonds JT. Copper, iron, zinc, and manganese in dietary supplements, infant formulas, and ready-to-eat breakfast cereals. Am J Clin Nutr 1998; 67:1035S–40S.
24. Food and Drug Administration. Rules and regulations: Nutrient requirements for infant formulas. Federal Register 1985;50:45106–8.
25. Codex Alimentarius Commission. Joint FAO/WHO Food standards programme: Recommended international standards for foods for infants and children. CAC/RS 72/74-1976. Rome: Secretariat of the joint FAO/WHO food standards programme, 1976.
26. American Academy of Pediatrics. Recommended ranges of nutrients in formulas. Appendix I. In:Pediatric nutrition handbook. 2nd ed. Elk Grove Village, IL: American Academy of Pediatrics, 1985:356–7.
27. European Society of Paediatric Gastroenterology and Nutrition. Committee on Nutrition of the Preterm Infant. Nutrition and feeding of the preterm infant. Oxford: Blackwell Scientific Publications, 1987.
28. Lönnerdal B. Trace element absorption in infants as a foundation to setting upper limits for trace elements in infants formulas. J Nutr 1989; 119:1839–45.
29. van Campen DR, Mitchell EA. Absorption of Cu64, Zn65, Mo99, and Fe59 from ligated segments of the rat gastrointestinal tract. J Nutr 1965; 86:120–4.
30. Varada KR, Harper RG, Wapnir RA. Development of copper intestinal absorption in the rat. Biochem Med Metab Biol 1993; 50:277–83.
31. Turnlund JR, Keyes WR, Anderson HL, Acord LL. Copper absorption and retention in young men at three levels of dietary copper by use of the stable isotope 65Cu. Am J Clin Nutr 1989; 49:870–8.
32. Milne DB, Johnson PE, Klevay LM, Sandstead H. Effect of copper intake on balance, absorption, and status indices of copper in man. Nutr Res 1990; 10:975–86.
33. Mills CF. Dietary interactions involving the trace elements. Ann Rev Nutr 1985; 5:173–93.
34. Greger JL, Mulvaney J. Absorption and tissue distribution of zinc, iron and copper by rats fed diets containing lactalbumin, soy and supplemental sulfur-containing amino acids. J Nutr 1985; 115:200–10.
35. Turnlund JR, King JC, Gong B, Keyes WR, Michel MC. A stable isotope study of copper absorption in young men: Effect of phytate and -cellulose. Am J Clin Nutr 1985; 42:18–23.
36. Kies C, Chuang JH, Fox HM. Copper utilization in humans as affected by amino acids supplements (abstract). FASEB J 1989; 3:A360.
37. Baker DH, Czarnecki-Maulden GL. Pharmacologic role of cysteine in ameliorating or exacerbating mineral toxicities. J Nutr 1987; 117:1003–10.
38. Jacob RA, Skala JR, Omaye ST, Turnlund JR. Effect of varying ascorbic acid intakes on copper absorption and ceruloplasmin levels of young men. J Nutr 1987; 117:2109–15.
39. Stack T, Aggett PJ, Aitken E, Lloyd DJ. Routine L-ascorbic acid supplementation does not alter iron, copper, and zinc balance in low-birth-weight infants fed a cow's milk formula. J Pediatr Gastroenterol Nutr 1990; 10:351–6.
40. Wapnir R. Copper absorption and bioavailability. Am J Clin Nutr 1998; 67:1054S–60S.
41. Holbrook JT, Smith JC, Reiser S. Dietary fructose or starch: Effects on copper, zinc, iron, manganese, calcium, and magnesium balances in humans. Am J Clin Nutr 1989; 49:1290–4.
42. Reiser S, Smith Jr JC Mertz W, et al. Indices of copper status in humans consuming a typical American diet containing either fructose or starch. Am J Clin Nutr 1985; 42:242–51.
43. Hill CH, Matrone G. Chemical parameters in the study of in vivo and in vitro interactions of transition elements. Federal Proc 1970; 29:1474–81.
44. Hall AC, Young BW, Bremner I. Intestinal metallothionein and the mutual antagonism between copper and zinc in the rat. J Inorg Biochem 1979; 11:66–7.
45. August D, Janghorbani M, Young VR. Determination of zinc and copper absorption at three dietary ZnCu ratios by using stable isotope methods in young adult and elderly subjects. Am J Clin Nutr 1989; 50:1457–63.
46. Turnlund JR, Wada L, King JC, Keyes WR, Accord LL. Copper absorption in young men fed adequate and low zinc diets. Biol Trace Element Res 1988; 17:31–41.
47. Lee SH, Lancey R, Montaser A, Madani N, Linder MC. Ceruloplasmin and copper transport during the latter part of gestation in the rat. Proc Soc Exp Biol Med 1993; 203:428–39.
48. McArdle HJ, Erlich R. Copper uptake and transfer to the mouse fetus during pregnancy. J Nutr 1991; 121:208–14.
49. Wirth PL, Linder MC. Distribution of copper among multiple components of human serum. J Natl Cancer Inst 1985; 75:277–84.
50. Widdowson EM, Dauncey J, Shaw JCL. Trace elements in foetal and early post-natal development. Proc Nutr Soc 1974; 33:275–84.
51. Klein D, Scholz P, Drash GA, Müller-Höcker J, Summer KH. Metallothionein, copper and zinc in fetal and neonatal human liver: Changes during development. Toxicol Lett 1991; 56:61–7.
52. Henkin RI, Schulman JD, Schulman CD, Bronzert DA. Changes in total, nondiffusible, and diffusible plasma zinc and copper during infancy. J Pediatr 1973; 82:831–7.
53. McMaster D, Lappin TR, Halliday HL, Patterson CC. Serum copper and zinc levels in the preterm infant. A longitudinal study of the first year of life. Biol Neonate 1983; 44:108–13.
54. L'Abbé MR, Friel JK. Copper status of very low birth weight infants during the first 12 months of infancy. Pediatr Res 1992; 32:183–8.
55. National Research Council. Recommended dietary allowances. 10th ed. Washington, DC: National Academy of Sciences, 1989:224–30.
56. WHO/FAO/IAEA. Trace elements in human nutrition and health. Geneva: World Health Organization, 1996:123–43.
57. Dörner K, Dziadzka S, Hohn A, et al. Longitudinal manganese and copper balances in young infants and preterm infants fed on breastmilk and adapted cow's milk formulas. Br J Nutr 1989; 61:559–72.
58. Ehrenkranz RA, Gettner PA, Nelli CM, et al. Zinc and copper nutritional studies in very low birth weight infants: Comparison of stable isotopic extrinsic tag and chemical balance methods. Pediatr Res 1989; 26:298–307.
59. Naveh Y, Haqzani A, Berant M. Copper deficiency with cow's milk diet. Pediatrics 1981; 68:397–400.
60. Lönnerdal B, Bell JG, Keen CL. Copper absorption from human milk, cow's milk, and infant formulas using suckling rat model. Am J Clin Nutr 1985; 42:836–44.
61. Cavell PA, Widdowson EM. Intakes and excretion of iron, copper, and zinc in the neonatal period. Arch Dis Child 1964; 39:496–501.
62. Dauncey MJ, Shaw JCL, Urman J. The absorption and retention of magnesium, zinc, and copper by low birth weight infants fed pasteurized human breast milk. Pediatr Res 1977; 11:991–7.
63. Klein D, Lichtmannegger J, Heinzmann U, Müller-Höcker J, Michaelsen S, Summer KH. Association of copper to metallothionein in hepatic lysosomes of Long-Evans cinnamon (LEC) rats during the development of hepatitis. Eur J Clin Invest 1998; 28:302–10.
64. Mercer JFB. Menkes syndrome and animal models. Am J Clin Nutr 1998; 67:1022S–8S.
65. Kaler SG. Diagnosis and therapy of Menkes syndrome, a genetic form of copper deficiency. Am J Clin Nutr 1998; 67:1029S–34S.
66. Danks DM. Disorders of copper transport. In: Scriver CL, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill, 1995:2211–35.
67. Scheinberg IH, Sternlieb I. Wilson disease and idiopathic copper toxicosis. Am J Clin Nutr 1996; 63:842S–55S.
68. Uauy R, Olivares M. Health significance of copper. In: Moore MR, Imray P, Dameron C, Callan P, Langley A, Mangas S, eds. Copper: Report of an International Meeting. Brisbane, Australia: Openbook Publishers, 1997:7–15.
69. Pettersson R, Rasmussen F. Daily intake of copper from drinking water among young children in Sweden. Environ Health Perspect 1999; 107:441–6.
70. Olivares M, Uauy R. Copper as an essential nutrient. Am J Clin Nutr 1996; 63:791S–6S.
71. Cordano A. Clinical manifestations of nutritional copper deficiency in infants and children. Am J Clin Nutr 1998; 67:1012S–6S.
72. Sturgeon P, Brubaker C. Copper deficiency in infants. Am J Dis Child 1956; 92:254–65.
73. Shaw JCL. Copper deficiency in term and preterm infants. In: Fomon SJ, Zlotkin S, eds. Nutritional Anemias. Nestlé Nutrition Workshop Series Vol 30. New York/Vevey: Raven Press/Nestec, 1992:105–119.
74. Danks DM. Copper deficiency in humans. Ann Rev Nutr 1988; 8:235–57.
75. Ashkenazi A, Levine S, Dialjetti M, Fishel E, Benvenisty O. The syndrome of neonatal copper deficiency. Pediatrics 1973; 52:525–33.
76. Castillo-Duran C, Uauy R. Copper deficiency impairs growth of infants recovering from malnutrition. Am J Clin Nutr 1988; 47:710–4.
77. Castillo-Durán C, Fisberg M, Valenzuela A, Egaña JI, Uauy R. Controlled trial of copper supplementation during the recovery of marasmus. Am J Clin Nutr 1983; 37:898–903.
78. Percival SS. Copper and immunity. Am J Clin Nutr 1998: 67:1064S–8S.
79. Pizarro F, Olivares M, Gidi V, Araya M. The gastrointestinal tract and acute effects of copper in drinking water and beverages. Rev Environ Health 1999; 14:231–8.
80. Pizarro F, Olivares M, Uauy R, Contreras P, Rebelo A, Gidi V. Acute gastrointestinal effects of graded levels of copper in drinking water. Environ Health Perspect 1999; 107:117–21.
81. Pettersson R, Sandström B. Copper. Oskarsson A. Risk evaluation of essential trace elements: Essential versus toxic levels of intake. Copenhagen: Nordic Council of Ministers, 1995:149–167.
82. Tanner MS. Role of copper in Indian childhood cirrhosis. Am J Clin Nutr 1998; 67:1074S–81S.
83. Tanner MS, Kantarjian AH, Bhave SA, Pandit AN. Early introduction of copper-contaminated animal milk as possible cause of Indian childhood cirrhosis. Lancet 1983; 2:992–5.
84. Müller T, Feichtinger H, Berger H, Müller W. Endemic Tyrolean Cirrhosis: An ecogenetic disorder. Lancet 1996; 347:877–80.
85. Müller T, Müller W, Feichtinger H. Idiopathic copper toxicosis. Am J Clin Nutr 1998; 67:1082S–6S.
86. Müller-Höcker J, Meyer U, Wiebeche B, Hübner G. Copper storage disease of the liver and chronic dietary copper intoxication in two further German infants mimicking Indian childhood cirrhosis. Pathol Res Pract 1988; 183:39–45.
87. Olivares M, Uauy R. Limits of metabolic tolerance to copper and biological basis for present recommendations and regulations. Am J Clin Nutr 1996; 63:846S–52S.
88. Milne DB. Copper intake and assessment of copper status. Am J Clin Nutr 1998; 67:1041S–5S.
89. Milne DB, Johnson PE. Assessment of copper status: Effect of age and gender on reference ranges in healthy adults. Clin Chem 1993; 39:883–7.
90. Uauy R, Castillo-Duran C, Fisberg M, Fernandez N, Valenzuela A. Red cell superoxide dismutase activity as an index of human copper nutrition. J Nutr 1985; 115:1650–5.
91. DiSilvestro RA, Jones AA, Smith D, Wildman R. Plasma diamine oxidase activities in renal dialysis patients, a human with spontaneous copper deficiency and marginally copper deficient rats. Clin Biochem 1997; 7:559–63.
92. Epstein O. liver copper in health and disease. Postgrad Med J 1983;59 (suppl 4):88–94.
93. Jiménez I, Gotteland M, Zarzuelo A, Uauy R, Speisky, H. Loss of the metal binding properties of metallothionein induced by hydrogen peroxide and free radicals. Toxicology 1997; 120:37–46.
94. Cousins RJ. Absorption, transport, and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol Rev 1985; 65:238–309.
95. Olivares M, Pizarro F, Speisky H, Lönnerdal B, Uauy R. Copper in infant nutrition: Safety of WHO provisional guideline value for copper content of drinking water. J Pediatr Gastroenterol Nutr 1998; 26:251–7.
96. Ogihara H, Ogihara T, Miki M, Yasuda H, Mino M. Plasma copper and antioxidant status in Wilson's disease. Pediatr Res 1995; 37:219–26.
97. Eife R, Weiss M, Müller-Höcker J, et al. Chroning poisoning by copper in tap water: Copper intoxication with predominant systemic symptoms. Eur J Med Res 1999; 4:224–8.
98. Soo TL, Simmer K, Carlson L, McDonald L. Copper and very low birthweight babies. Arch Dis Child 1988; 63:79–81.
99. International Programme on Chemical Safety (IPCS). Copper. environmental health criteria 200. Geneva: World Health Organization, 1998.
100. International Programme on Chemical Safety (IPCS). Principles and methods for the assessment of risk from essential trace elements. First draft. Geneva: World Health Organization, 1999.
101. Olivares M, Uauy R. Models to evaluate health risks derived from copper exposure/intake in humans. Adv Exp Med Biol 1999; 448:17–28.
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