24b Copper

The adult human body contains 70–80mg of copper. Of this, approximately 25% is in the skeletal muscle, 15% in the skin, 15% in the bone marrow, 19% in the skeleton, 8.0 to 15% in the liver, and 8.0% in the brain. The liver and brain contain the highest concentrations of copper. Like iron, copper can be found in two valences, Cu⁺ and Cu²⁺, and it is this ability to gain and lose electrons that give the enzymes into which it is incorporated their function, catalyzing oxidation-reduction reactions. Examples of copper enzymes (cupro-enzymes) are given in (Table 24b.1).

Table 24b.1 Function of some copper enzymes with oxidation and reduction activity in humans. From Uauy et al., American Journal of Clinical Nutrition 67S: 952S–959S, 1998

Copper Enzyme	Function
Ceruloplasmin (Ferroxidase I)	Copper transport, iron mobilization, antioxidant
Cytochrome c oxidase	Electron transport, terminal oxidase
Superoxide dismutase	Free radical detoxication
Tyrosinase	Synthesis of melanin
Lysyl oxidase	Collagen and elastin cross-linking
Amine oxidases	Deamination of primary amines
Dopamine-β- monooxygenase	Dopamine→norepi- nephrine
Peptidylglycine monooxygenase	α-Amidation of neuro peptides

• Functions of copper

Many clinical features of copper deficiency can be explained by changes in the activities of cupro­enzymes. For instance, the ferr­oxidases, of which there are several examples, are involved in oxidising Fe²⁺ to Fe³⁺ to facilitate iron incorporation into trans­ferrin. Cerulo­plasmin, a serum protein, was the first to be identified. A reduction in cerulo­plasmin (ferr­oxidase I) (EC 1.16.3.1) impairs the transport of iron to the erythro­poietic sites, leading to hypo­chromic anemia. Hephaestin is located on the baso­lateral membrane of gut cells (Vulpe et al., 1999). The membrane bound form of cerulo­plasmin is pre­dominantly located in the brain (Jeong and David 2003), while zyklopen is central to trans-placental transfer of iron, though it is also located in retinal cells (Chen et al., 2010).

Deficiency of the cupro­enzyme lysyl oxidase (EC 1.4.3.13) may be responsible for the skeletal and vascular defects reported in copper deficiency. Lysyl oxidase is essential for the normal maturation of collagen, specifically in the steps involving the formation of lysine-derived cross-links (O'Dell, 1976). The latter are required for the formation of strong, flexible con­nective tissues (Rucker and Murray, 1978).

Depigment­ation is often associated with a deficiency of tyro­sinase (EC 1.14.18.1), an enzyme involved in the production of melanin. The latter is necessary for protection against excess ultra­violet exposure. The central nervous system disturb­ances in copper deficiency are the result of myelin­ization derange­ments or of abnormal catechol­amine levels and associated decreased activity of dopamine β mono­oxygen­ase (EC 1.14.17.1) or reduced activity of cyto­chrome c oxid­ase (EC 1.9.3.1) (Mason, 1979).

• Absorption and metabolism of copper

The major site of copper absorption is the small intestine, although some copper is also absorbed from the stomach. Absorption is inversely related to the amount of copper in the diet, decreasing the likelihood of deficiency and toxicity (Turnlund et al., 1989). For dietary intakes above 5mg/d, absorption is < 20%, whereas for intakes < 1mg/d, absorp­tion is as high as 50% (Figure 24b.1).

As noted above, on the apical membrane, facing the gut, copper is taken up by a carrier mediated process, involving a transporter named Ctr1 (Kuo et al., 2006). Following uptake, the Cu is transferred across the cell and excreted into the portal circulation using an energy-dependent process and a Cu-ATPase, known as ATP7A (Harris, 2000). This enzyme is deficient in Menkes disease patients, and its absence is responsible for the subsequent copper deficiency. It is mostly located in the gut, but it also plays an important role in trans-placental transport and may be involved in uptake across the blood brain barrier (Hardman et al., 2004).

Once absorbed, copper is trans­ported via the portal blood, bound to albumin and trans­cuprein, mainly to the liver. In the liver, copper is incorporated into cerulo­plasmin, and then dis­tributed to other tissues. For further details, see the review by Linder et al. (1998) and Harris (2000).

At present, the mechanisms of copper regulation are only beginning to be under­stood. Tissue copper con­cen­trations are main­tained within a narrow range, through homeo­static mechanisms that operate at the level of intestinal absorption and biliary excretion. Of the two levels, biliary excretion is the more important. As more copper is absorbed, turnover is faster, and, hence, more copper is excreted into the gastro­intes­tinal tract. Very little copper is excreted in the urine, and urinary losses do not help regulate copper stores. Relatively little copper is stored in the body, compared with other trace elements such as iron (Turnlund, 1998; McArdle et al. 1999).

Copper is taken into the gut cell through Ctr1. In the cell, and in fact in all other cells apart from red blood cells, copper is bound to one of a series of chaperones. The first to be identified was Atox1, followed by several others, each of which takes copper from the membrane transporter to specific target proteins (Palumaa, 2013). The copper is trans­ported across the cell, excreted into the portal circulation through the enzyme which is defective in Menkes disease, ATP7A and trans­ferred to the liver bound to albumin, trans­cuprein or as a  low-molecular weight complex with histidine or other amino acids (Gambling et al., 2011).

Within the liver, Cu is stored as metallo­thionein, re-excreted in the bile or incorp­orated into cerulo­plasmin. Excretion and Cp incorp­oration both involve a second Cu-ATPase, ATP7B. ATPB is a membrane protein, which changes location in the liver ER, depending on whether the copper being trans­ported is excreted or incorp­orated into cerulo­plasmin (McArdle et al.,1999).

• Copper deficiency in humans

Copper deficiency in humans is rare. It has been described in infants recovering from malnutrition (Graham and Cordano, 1969) and in premature and low-birth-weight infants fed cow's milk (al‑Rashid and Spangler, 1971; Griscom et al., 1971; Ashkenazi et al., 1973). Copper deficiency has also been reported in patients receiving prolonged total paren­teral nutrition (TPN) unsup­plemented with copper (Karpel and Peden, 1972; Dunlap et al., 1974; Vilter et al., 1974) and in a woman receiving a normal diet sup­plemented with antacids (Anonymous, 1984). Severely handicapped patients (n=60) receiving an enteral diet with only 15µg/1000kcal Cu for 12–66mo also developed copper deficiency (Higuchi et al., 1988).

The earliest clinical manifestation of copper deficiency in humans is persistent neutropenia, usually followed by hypochromic microcytic anemia, scurvy-like bone changes, and osteoporosis (in infants) (Cordano et al., 1964; al-Rashid and Spangler, 1971; Karpel and Peden, 1972; Higuchi et al., 1988). The anemia probably results from defective iron mobilization and hemoglobin formation related to lower cerulo­plasmin activity (Danks, 1988), or to increased susceptibility to oxidative damage (Johnson and Kramer, 1987).

Less frequent features of copper deficiency include hypo­pigmen­tation of the hair and hypo­tonia (low muscle tone), as well as impaired growth, an increased incidence of infections, and changes in the phago­cytic capacity of the neutro­phils (Uauy et al., 1998). In some experimental depletion-repletion studies, changes in blood clotting factors (Milne and Nielsen, 1996) and in markers of bone meta­bolism (Baker et al., 1999) have also been reported. Low copper intakes have been implicated as a risk factor in the develop­ment of cardio­vascular disease (Lukaski et al., 1988; Klevay, 2000).

A few cases of adults with sickle cell disease who were receiving prolonged zinc therapy have devel­oped hema­tological signs of copper deficiency (Prasad et al., 1978). Clinical features of copper deficiency, with the exception of anemia, occur in infants with Menkes kinky hair syndrome (KHS), an X-linked, recessive, hereditary disorder of copper metabolism with an intra­cellular defect of copper utilization (Danks et al., 1972).

Most of the reports of copper deficiency in humans have resulted from inadequate dietary intakes of copper, often associated with pro­longed diarrhea, which prevents reabsorp­tion of copper from the bile. Hence, it is not surprising that subjects with mal­absorp­tion syn­dromes — including tropical sprue, celiac disease, cystic fibrosis, and short-bowel syndrome resulting from intestinal resection — are at risk of copper deficiency (Rodriguez et al., 1985).

Diseases associated with chronic loss of proteins, such as nephritic syndrome and protein-losing enter­opathy, may also cause copper deficiency as a result of loss of cerulo­plasmin and of copper bound to albumin. High intakes of fructose and sucrose exacer­bate the effects of copper deficiency, but the importance of this effect in humans is unclear (O'Dell, 1990).

• Food sources and dietary intakes

Copper is widely distributed in foods; the richest food sources are oysters, other shellfish, organ meats (e.g., liver, kidney), nuts, and dried legumes. Milk and dairy products are low in copper. The copper level in plants is not influenced by the copper content of the soil in which they grow (Institute of Medicine, 2001). Drinking water may contribute a relatively large amount of copper to the daily intake, especially if the water is slightly acidic or very soft and is supplied from copper pipes (Lönnerdal, 1996).

Copper deficiency among the general population has not been described. Dietary intakes of copper for adult European populations range from 0.9 to 2.3mg/d (van Dokkum, 1995). Data are available on the copper intakes of the U.S. population based on the Continuing Survey of Food Intakes by Individuals (CSFII 1994–1996) and NHANES III (IOM, 2001) and for the U.K. population, based on the National Diet and Nutrition Surveys (Gregory et al., 1990; Gregory et al., 1995; Gregory et al., 2000; Finch et al., 1998; Whitton et al., 2011). Copper intakes of infants fed breast milk or cow's milk are often low. More recently, the European Food Safety Authority has published data on dietary intake values in the human population (EFSA Panel on Dietetic Products 2015).

The composition of mixed western diets has little effect on the bioavailability of copper in humans. Copper deficiency has been observed in some cases of excessively high intakes of iron or zinc supplements (Fischer et al., 1984; Barclay et al., 1991; Botash et al., 1992) induced by their adverse effect on copper absorption. There is some evidence that the adverse effect of high dietary iron intake on copper absorption may occur only when copper status is low or marginal (Cohen et al., 1985).

• Effect of high intakes of copper

Under normal circumstances, copper levels in the body are tightly regulated. However, several cases of accidental copper toxicity in humans have been described, arising from ingestion of copper sulfate (Chuttani et al., 1965), acidic drinks in prolonged contact with copper (Paine, 1968), or drinking water with an unusually high copper concentration (800µg/L) (Salmon and Wright, 1971). Adverse effects include nausea, gastrointestinal effects (nausea, diarrhea, cramps, abdominal pain), and, in some cases, liver damage (O'Donohue et al., 1993; Pizarro et al., 1999).

Wilson disease results from defects in ATP7B, the Cu-ATPase responsible for trans­ferring copper from the liver to the bile or to the sites of ceruloplasmin synthesis. The defect results in copper overload in the liver and the brain. Patients suffering from Wilson's disease exhibit symptoms of copper toxicity such as nausea, vomiting, diarrhea, acute hemolytic anemia, hepatic necrosis, hepatic central vein dilation, and jaundice (Zelkowitz et al., 1980). In Wilson's disease, copper accumulates in the liver because the copper in the liver cells can neither enter the bile nor participate in cerulo­plasmin synthesis (Linder and Hazegh-Azam, 1996; Suzuki and Gitlin 1999). See Czlonkowska et al., 2019 for a com­pre­hensive review. Idio­pathic copper toxicosis has been described in an Austrian population and termed Tyrolean copper toxicosis (Muller et al., 1998). It is hypoth­esized that the disorder arises as a combination of an inherited genetic mutation and high coper intake. A similar disease has been described by Tanner and colleagues in Indian children (Tanner 1998), although the situation is probably more complex than a simple copper overload (Pankit and Bhave 2002). There is no evidence that high dietary copper intakes are a public health problem. The U.S Tolerable Upper Intake Level (UL) for copper for adults > 19y is 10mg/d. For pregnant and lactating women and adolescents 14–18y, the UL is set at 8mg/d (IOM, 2001). Currently in Europe (2020), EFSA is examining upper levels of copper to harmonise permitted pesticide levels with nutritional guidelines.

• Indices of copper status

The methods used to assess copper status in humans include static tests, as well as the measurement of the activities of certain copper enzymes. These methods are discussed below. Other approaches use cate­gorical regression to determine the optimal levels of copper in the diet (Chambers et al., 2010). There is still no consensus on the best bio­marker to use, in part, because of the efficiency of the homeo­static control that maintains tissue copper concen­trations within a narrow range (Harvey and McArdle 2008; Harvey et al., 2009; Danzeisen et al., 2007). As a result, measures of copper status tend to be resistant to change, except when dietary intakes are very low or very high. Serum copper or serum cerulo­plasmin are most often used to assess copper status, but the sensitivity and specificity of both these measures are poor. Hence, they should be combined with a more sensitive and specific functional measure such as cyto­chrome c oxi­dase in platelets or super­oxide dismu­tase in erythro­cytes. It is likely that in the future functional indicators, perhaps including anti­oxidant status, changes in immune function, and the lysyl oxidase activity in skin, will be used in conjunction with other biochemical markers.

24b.1 Serum copper

There are two main forms of copper in serum; one is firmly bound to cerulo­plasmin, the other is reversibly bound to serum albumin. and to amino acids and other, poorly defined components (Linder 2016). Serum also contains the two copper enzymes cyto­chrome c oxi­dase and mono­amine oxidase.

Serum copper con­cen­trations are a reliable bio­marker in cases of severe copper deficiency, when they are almost always low. However, they are not sensitive enough to be used as a measure of marginal copper status, as noted earlier, and do not reflect dietary intake, except when it is low. Normally, sup­plemen­tation with copper does not increase serum copper because con­cent­rations are under strict homeo­static control. Levels are only reduced after copper stores are signif­icantly depleted. In healthy adults consuming diets adequate in copper, the lower end of the normal range for serum copper is 10µmol/L.

Experimental studies have also confirmed that serum copper is not a sensitive index of marginal copper status in adults (Reiser et al., 1985; Turnlund et al., 1989; Milne and Nielsen, 1996). In a depletion–repletion study of post­meno­pausal women fed a diet with only 0.57mg/d Cu for 105d, serum copper levels did not change significantly during the copper-depletion period (Figure 24b.2),

despite the presence of other bio­chem­ical changes (Milne and Nielsen, 1996).

Eventually serum copper concen­trations do fall. In patients receiving prolonged TPN unsup­ple­mented with copper, falls of about 1.7µmol/L per week may occur (Solomons et al., 1976). Values < 9.5–11µmol/L have been reported in infants with copper deficiency (Cordano et al., 1964; Castillo-Duran and Uauy, 1988). Low serum copper concen­trations also occur with the two genetic disorders of copper meta­bolism: Menkes kinky hair syndrome and Wilson's disease. In Menkes kinky hair syndrome, low serum copper concen­trations arise from an intra­cellular defect of copper utilization, as discussed earlier (Danks et al., 1972). In Wilson's disease, the low serum copper levels are induced by defects in the hepatic storage of copper and in cerulo­plasmin meta­bolism.

Plasma copper concen­trations appear to be consistently lower than corres­ponding values in serum (Table 24b.2)

Table 24b.2 Copper concentrations of serum or citrated plasma prepared from single venipuncture samples of eight subjects. Plasma was prepared by adding 0.15mL of 30% sodium citrate per 15mL of whole blood. Percentage difference = [(serum − plasma)/serum] × 100. Data from Smith et  al., Journal of the American College of Nutrition 4: 627–638, 1985

Subject no.	Serum (μmol/L)	Plasma (μmol/L)	Difference
1	20.0	19.4	3%
2	18.7	17.8	5%
3	19.0	17.3	9%
4	19.2	16.7	13%
5	19.5	16.7	15%
6	19.4	16.5	15%
7	18.1	16.5	9%
8	16.5	15.4	7%
Mean	18.9±1.1	17.0±1.1	9±4

(Smith et al., 1985). In contrast, concentrations of copper in serum and erythrocytes are comparable, so slight hemolysis does not affect serum copper concentrations.

More recently, examination of the non-cerulo­plasmin pool of copper, the exchange­able fraction, has suggested that this might be a good indicator of copper overload, as in diseases such as Wilson's disease (Woimant et al., 2019).

• Factors affecting serum copper

It is important to remember that most assays do not differentiate between the different forms of copper in serum and usually, changes in “serum copper" are generally caused by alterations in the concen­tration of cerulo­plasmin. The vast majority of coper in serum is bound to cerulo­plasmin. Estimates vary from 60% to 95% with the remainder being bound to a poorly characterised series of chelators (Linder 2016). As well as being a ferr­oxidase, involved in iron metabolism, cerulo­plasmin is an acute phase protein, and concen­trations can be altered by other factors than copper status. This makes interpretation of the changes described below complex.

Age-related changes in serum copper concen­trations are well documen­ted. Newborn infants have low values, which rise to adult levels by 6 to 12mo of age (Salmenperä et al., 1986). In men, and in women not taking estrogen-containing prep­arations, serum copper levels increase with age until 55–64y, after which they decline (Table 24b.3)

Table 24b.3 Effect of age in men and women who were not taking estrogen-containing prepar­ations on serum copper concentration and ceruloplasmin activity. Data from Fischer et al., Nutrition Research 10: 1081–1090, 1990.

Age group (years)	Men	Women
	Serum copper (μmol/L)
25–34	14.7 ± 0.6	14.8 ± 0.5
35–44	15.0 ± 0.3	16.7 ± 0.4
45–54	16.2 ± 0.4	17.8 ± 0.6
55–64	16.3 ± 0.4	18.8 ± 0.7
65+	15.3 ± 1.0	16.8 ± 0.5
	Ceruloplasmin (U/dL)
25–34	9.9 ± 0.7	11.0 ± 0.6
35–44	10.8 ± 0.3	12.8 ± 0.4
45–54	11.9 ± 0.4	15.2 ± 1.1
55–64	11.8 ± 0.5	14.1 ± 0.8
65+	11.3 ± 1.2	12.3 ± 1.1

(Fischer et al., 1990; Milne and Johnson, 1993).

Gender influences serum copper. Levels in adult females tend to be higher than in males (Table 24b.3).

Pregnancy influences serum copper concen­trations. Higher serum copper concen­trations are evident after the third month of pregnancy (Halsted et al., 1968; Hambidge and Droegemueller, 1974). Whether this reflects changes in cerulo­plasmin, which is part of the acute phase response, or an actual change in serum copper concen­trations, is not always clear.

Estrogen-containing preparations, such as oral contra­ceptive agents and estrogen replace­ment therapy, elevate serum copper concen­trations (hyper­cupremia) (Fischer et al., 1990; Nielsen et al., 1992). This effect is due to an estrogen-mediated rise in serum ceruloplasmin concentration No differences in copper absorption or copper balance have been reported between users and non-users of oral contra­ceptive agents (King et al., 1978; Crews et al., 1980). (Table 24b.4).

Table 24b.4 Effect of oral contraceptive agents (OCA) and estrogen replacement therapy on serum copper concentration and ceruloplasmin activity. Data from Fischer et al., Nutrition Research 10: 1081–1090, 1990.

	Premenopausal women
	Non-users of OCA	Users of OCA
Serum copper (µmol/L)	16.2±0.3	26.8±1.3
Ceruloplasmin (U/dL)	12.2±0.4	20.4±0.9
	Postmenopausal women
	Non-users of estrogens	Users of estrogens
Serum copper (µmol/L)	18.1±0.4	25.9±1.2
Ceruloplasmin (U/dL)	14.5±0.7	19.7±1.0

Diurnal variation in serum copper concen­trations has been noted (Cartwright, 1950; Guillard et al., 1979) with the highest levels occur in the morning (Lifschitz and Henkin, 1971; Cartwright et al., 1954; Yokoyama et al., 2000).

Regular strenuous exercise may also affect serum copper concen­trations. Lukaski et al. (1983) showed values to be higher in male university athletes relative to controls.

Smoking raises serum copper concen­trations (Kocyigit et al., 2001).

Infection, inflam­mation, and stress all elevate serum copper concen­trations (Honkanen et al., 1991; Brown et al., 1993). This is a result of an increase in cerulo­plasmin (an acute phase protein), effected by leuko­cytic endo­genous mediators (Pekarek et al., 1972).

Malabsorption syndromes such as celiac disease, cystic fibrosis, and ulcerative colitis (Sternlieb and Janowitz, 1964) are often associated with low serum copper concen­trations, arising from pro­longed diarrhea which prevents reabsorp­tion of copper from the bile (Rodriguez et al., 1985).

Certain disease states lead to alterations in serum copper concen­trations. Levels increase in leukemia, Hodgkin's disease, various anemias, collagen disorders, hemo­chrom­atosis, and myo­cardial infarction and in patients with dilated cardio­myopathy (Mason, 1979; Oster, 1993). In other diseases (e.g., the nephrotic syndrome), serum copper concen­trations are low (Cartwright et al., 1954).

• Interpretive criteria

Table 24b.5 Plasma copper concentrations in children, grouped according to age (mean±SD). Data from Rükgauer et al., Journal of Trace Elements in Medicine and Biology 11: 92–98, 1997.

Age group (years)	n	Serum Cu (µmol/L)
1 to < 2	15	21.1±4.6
2 to < 4	23	21.5±3.9
4 to < 6	19	19.4±5.3
6 to < 10	25	23.4±2.5
10 to < 14	21	21.1±3.7
14 to < 18	17	20.5±4.4

Interpretive criteria used for serum copper concentrations for adults are 8.8–17.5µmol/L for men and 10.7–26.6µmol/L for women who are not taking oral contraceptive agents. A slightly higher range (15.7–31.5µmol/L) has been proposed for women taking estrogen-containing prepar­ations (Milne and Johnson, 1993). Rükgauer et al. (1997) have published reference values for plasma copper for children ranging from 1mo to 18y, although the number of children studied in some of the age groups was small. These reference values are based on self-selected samples of apparently healthy subjects who were not taking any vitamin or mineral supplements (Table 24b.5). Salmenperä et al. (1986) have compiled reference values for serum copper in exclusively breast­fed infants followed longitudinally for the first year of life. These reference values are shown in (Figure 24b.3). Serum copper levels were measured in subjects from 3 to 74y in the Canada Health Survey (Health and Welfare Canada, 1981). The National Health and Nutrition Examination Survey (NHANES II) also collected data on serum copper concentrations,

but were not measured in the more recent U.K. national surveys.

• Measurement of serum copper

The most widely used method for measuring serum copper is AAS, although inductively coupled mass spectrometry is becoming more common (McMillen et al., 2009). Generally, a direct technique is used, involving sample dilution with deionized water (1 part plasma or serum to 1 part deionized water) (Osheim, 1983; Smith et al., 1985): a signal-enhancing mixture such as butanol and water is also some­times used (Meret and Henkin, 1971). In some instances, use of a “high solids” burner head may be necessary (Boling, 1966). Sometimes, the protein in the blood sample is removed using an acid such as trichloro­acetic acid (Kelson and Shamberger, 1978), but this procedure may introduce volume errors during the deprotein­ization step and adventitious contamin­ation from the acid and is not recommended. Analytical variation for serum copper by AAS is usually small; Gallagher et al. (1989) reported it to be < 2.6%. For very small pediatric samples, graphite furnace AAS, with Zeeman-effect background correction, may be used (Wang and Demshar, 1993). Increasingly inductively coupled plasma spectrometry (ICP) is being used because it is a multi-element technique with a detection limit and sensitivity better than flame AAS (Nixon et al., 1986). Standard reference materials for the analysis of copper in serum are available from NIST, Gaithersburg, MD.

24b.2 Serum ceruloplasmin

Ceruloplasmin is the major copper-containing protein in the α2-globulin fraction of human serum. More than 60% to 95% of serum copper is associated with cerulo­plasmin (Wirth and Linder, 1985), so changes in serum copper generally parallel the level of cerulo­plasmin in the blood.

Cerulo­plasmin (EC 1.16.3.1) is a copper transport protein synthesized primarily in the liver. It is a single-chain glyco­protein with six copper atoms per molecule. A ferr­oxidase, it assists in iron transport by oxidizing intra­cellular Fe²⁺ to Fe³⁺, which can then combine with trans­ferrin. Cerulo­plasmin is also among those enzymes involved in the acute-phase reaction of inflam­mation and in the scavenging of oxygen radicals to protect cells against oxidative damage (Linder and Hazegh-Azam, 1996).

Cerulo­plasmin levels in serum, like copper, are significantly reduced in cases of severe copper deficiency that can occur with the hereditary diseases associated with disturb­ances in copper meta­bolism: Menkes kinky hair syndrome and Wilson's disease. However, the response of ceruloplasmin to marginal copper deficiency or short-term copper depletion is variable (Milne and Nielsen, 1993; 1996). In most short-term studies of copper depletion (e.g., Milne and Nielsen, 1996) or sup­plement­ation (e.g., Kehoe et al., 2000), there has been little or no response by cerulo­plasmin (Figure 24b.2).

Many of the factors that influence serum copper also affect serum cerulo­plasmin and limit its usefulness as a marker of copper status, except in cases of severe copper deficiency. Furthermore, the compli­cations and variations in measuring cerulo­plasmin and copper in disorders such as Wilson's disease mean that their value as biomarkers need to be carefully assessed (Walshe, 2010).

• Factors affecting ceruloplasmin

Age and gender influence serum cerulo­plasmin activity in the same manner as serum copper as shown in a Table 24b.3 (Fischer et al., 1990).

Estrogen-containing preparations tend to increase the serum cerulo­plasmin activity, as shown in Table 24b.4 (Fischer et al., 1990). Cerulo­plasmin synthesis and secretion by the liver is regulated in the long term by estrogen, probably through stabil­ization of mRNA (Middleton and Linder, 1993).

Pregnancy can increase serum cerulo­plasmin activity (Fischer et al., 1990) again via the effect of estrogen on cerulo­plasmin synthesis. This effect may be related to the copper needs of the growing fetus during pregnancy (Lee et al., 1993).

Infection and inflammatory stress can lead to markedly elevated cerulo­plasmin activity levels in serum (Brown, 1998). Cerulo­plasmin is an acute-phase protein and the inflammatory hormones (interleukin-1 [IL-1], IL-6, and tissue necrosis factor) regulate its acute synthesis and secretion by the liver (Linder and Hazegh-Azam, 1996). Hence, it is not surprising that serum cerulopla­smin activity is also elevated in rheum­atoid arthritis (DiSilvestro et al.,1992).

• Interpretive criteria

Reference data for serum ceruloplasmin are limited. Salmenperä et al. (1986) have published reference values for exclusively breastfed infants followed during the first year of life, and they are shown in Figure 24b.3. Levels increase steadily during the first year of life, paralleling the trend for serum copper.

Milne (1994). suggests that a ratio of enzyme activity (cerulo­plasmin oxidase activity) to cerulo­plasmin protein concen­tration may be a useful index of copper status. The ratio has several advantages. Effects of age, sex, or hormone use are small (Table 24b.6)

Table 24b.6 Effect of age, gender, and hormone use on mean ceruloplasmin concentrations, measured both enzymatically (ENZ) and by radial immunodiffusion (RID). The ratio of ENZ/RID is also shown. FOC, females taking oral contraceptives; FE,females using estrogen therapy. Significance levels from analysis of variance (ANOVA) are also shown (interactions between gender and decade were nonsignificant in all three cases). Data from Milne and Johnson, Clinical Chemistry: 39, 883–887, 1993

Age (years)	Group	Enzyme activity (mg/L)	RID (mg/L)	ENZ/RID
20–29	M (n = 10) F (n = 10) FOC (n = 10)	408 503 612	266 330 371	1.56 1.53 1.71
30–39	M (n = 10) F (n = 10) FOC (n = 11)	416 516 744	296 314 525	1.49 1.66 1.42
40–49	M (n = 11) F (n = 11)	442 519	337 359	1.31 1.48
50–59	M (n = 9) F (n = 13) FE (n = 4)	441 516 593	310 370 384	1.43 1.44 1.57
60–69	M (n = 8) F (n = 10)	470 559	284 354	1.70 1.60
70+	M (n = 7) F (n = 7)	485 470	332 368	1.51 1.29
ANOVA	Gender Decade	0.0001 NS	0.0002 0.03	NS NS

(Milne and Johnson, 1993), and it appears to be more sensitive to changes in copper status than either the enzymatic activity or immuno­reactive protein alone (Milne et al., 1988). Indeed, in some indiv­iduals, cerulo­plasmin oxidase activity (but not cerulo­plasmin protein concentration) was depressed during experi­mental copper depletion (Milne et al., 1988). Further, in young women, cerulo­plasmin oxidase activity has been reported as inversely related to physio­logical changes due to copper depletion (e.g., auto­nomic cardio­vas­cular function) (Lukaski et al., 1988).

Table 24b.6 presents mean cerulo­plasmin values measured both enzym­atically (ENZ) and by radial immuno­diffusion, and the ratio of the enzyme activity to the immuno­reactive protein for males and females by age and hormone use (Milne and Johnson, 1993). Values for cerulo­plasmin for the two assays were standard­ized to mg/L by using purified human ceruloplasmin as a standard.

• Measurement of serum ceruloplasmin

Serum ceruloplasmin can be assayed enzym­atically by measuring p-phenyl­enedia­mine oxidase activity (Sunderman and Nomoto, 1970). Cerulo­plasmin protein levels can be measured immuno­chemically by radial immuno­diffusion techniques (Mancini et al., 1965; Buffone et al., 1979). Alternatively, nephel­ometry (Buffone et al., 1979) or immuno­electo­phoresis techniques (Gibbs and Walshe, 1979) and ELISA (various commercial kits are available) can be used. Some discrepancies have been reported between radial immuno­diffusion and nephel­ometry, which may be attributed to different sources of anti­bodies used for the assays (Buffone et al., 1979). Analytical variation for serum cerulo­plasmin performed by radial immuno­diffusion ranges up to 6% (Gallagher et al., 1989) .

24b.3 Erythrocyte superoxide dismutase

Approximately 60% of the copper found in erythro­cytes is present in the cytosol as super­oxide dismutase (Cu,Zn-SOD, EC 1.15.1.1). This enzyme, which contains both divalent copper and divalent zinc, has a molecular weight of approximately 32,500. It is important as a scavenger of the free radical O–
2 that causes damage to membranes and biological structures. It catalyzes the dismutation reaction, converting the highly reactive super­oxide anion O–
2, to less reactive hydrogen peroxide and oxygen:

Table 24b.7 Effect of copper supplementation on mean (±SD) serum copper, ceruloplasmin, and erythrocyte superoxide dismutase in infants recovering from malnutrition. Data from Uauy et al., Journal of Nutrition 115: 1650–1655, 1985.

Measure	Time	Study (n=8)	Control (n=9)
Plasma copper (µmol/L)	Before After	15.4±10.5 26.0±5.4	22.4±4.1
Ceruloplasmin (mg/L)	Before After	330±260 500±140	540±120
SOD (U/g Hb)	Before After	1073±312 1371±207	1461±451

         O–
2 + O–
2 + 2H⁺ →SOD→ H₂O₂ + O₂

Studies with both animal models and humans have shown that the activity of erythro­cyte Cu,Zn-SOD is depressed in copper deficiency and that the activity is often, but not always, restored during copper repletion (Bettger et al., 1979; Okahata et al., 1980; Uauy et al., 1985; Milne et al., 1990; Milne and Nielsen, 1996). Table 24b.7 presents data on erythro­cyte Cu,Zn-SOD activity in infants recovering from malnutrition and receiving marginal copper intakes: levels returned to normal after copper sup­plemen­tation (Uauy et al., 1985) and there was a significant positive correlation between eryth­ro­cyte Cu,ZnSOD and plasma copper concen­trations in these mal­nourished infants (Figure 24b.4). Such a correlation was not reported by Fischer et al., (1990). in their study of healthy adults, probably because the individuals studied had a copper status within the normal range.

Positive correlations between erythro­cyte Cu,Zn-SOD activity and other measures of copper status (e.g., liver cyto­chrome c oxi­dase) have also been observed in some animal studies (Bettger et al., 1979; Andrewartha and Caple, 1980).

Erythro­cyte Cu,Zn-SOD activity appears to be a more sensitive index of copper depletion than serum copper or cerulo­plasmin. In several experi­mental copper depletion-repletion studies in humans (Reiser et al., 1985; Milne and Nielsen, 1996), erythro­cyte Cu,ZnSOD activity declined signif­icantly during the depletion phase, despite no detectable decrease in serum copper or cerulo­plasmin. This trend is shown in Figure 24b.2.

Never­theless, the response of erythro­cyte Cu,Zn-SOD activity to copper repletion in these exper­imental studies has not been consistent. Several inves­tigators have failed to show a response by erythro­cyte Cu,Zn-SOD to copper supple­mentation (Turnlund et al., 1990; Medeiros et al., 1991). Even in a later study by Kehoe et al. (2000) involving two different copper compounds and two levels of copper supple­mentation, the activity of Cu,Zn-SOD in erythro­cytes (and leuko­cytes) did not respond to the copper supple­ments.

It appears that the study duration, amount of copper fed, and probably rates of cell turnover all influence the response of erythro­cyte Cu,Zn-SOD activity to copper repletion (Klevay et al., 1984; Milne et al., 1988; Turnlund et al., 1990; Medeiros et al., 1991; Milne and Nielsen, 1996; Kehoe et al., 2000). In Figure 24b.2 the erythrocyte Cu,Zn-SOD activity remained at a plateau during copper repletion, perhaps assoc­iated with the rate at which new erythro­cytes were being syn­thesized. In contrast, in the study of mal­nourished copper-deficient infants (Table 24b.7), erythro­cyte Cu,Zn-SOD activity increased to normal levels after sup­plement­ation with copper (80µg/kg/d) for 120d. Furthermore, Cu,Zn-SOD levels increase in a variety of diseases, including pancreatitis (Ściskalska et al. 2020). In some conditions that produce oxi­dative stress (Lukaski et al., 1990), including alcohol­ism and Down's syndrome (Del Villano et al., 1980; Porstmann et al., 1990), erythro­cyte Cu,Zn-SOD activity may be elevated. In rheumatoid arthritis patients, lower levels than those in controls have been observed (DiSilvestro et al., 1992). Activity levels, but not total concen­tration, appears to be reduced in hyper­glycemic, but not normal, diabetic patients possibly by glyco­sylation of the active site of the enzyme (Kotake et al., 1998).

Very few invest­igators have used erythro­cyte Cu,Zn-SOD activity as an index of copper status in community-based studies. Fischer et al. (Fischer et al., 1990) measured erythro­cyte Cu,Zn-SOD activity (and serum copper and cerulo­plasmin) in 384 free-living adults and examined the relationships with age, sex, estrogen use, smoking, alcohol use, and exercise, as noted earlier. Table 24b.8

Table 24b.8 Effect of age in men and women (not taking estrogen-containing preparations) on erythrocyte Cu,Zn-superoxide dismutase activity (U/mg Hb). The nonsignificant differences shown in the lower part of the table demonstrate the minimal impact of oral contraceptive use and estrogen replacement therapy on Cu,Zn-superoxide dismutase activity. Data from Fischer et al., Nutrition Research 10: 1081–1090, 1990.

Age group (years)	Men (Mean±SEM)	Women (Mean±SEM)
25–34	82.1±6.3	74.7±2.9
35–44	76.7±1.4	75.3±2.7
45–54	76.2±2.1	79.0±5.7
55–64	77.8±1.8	75.5±3.9
65+	77.4±2.9	68.9±3.9
Women Pre- menopausal	Non-users of oral contr. 75.1±2.1	Users of oral contr. 77.2±6.1
Women Post- menopausal	Non-users of estrogens 76.7±3.5	Users of estrogens 73.8±7.9

presents the mean ±SEM for erythro­cyte Cu,Zn-SOD activity for males and females by age. Unlike serum copper levels and cerulo­plasmin activity, erythro­cyte Cu,Zn-SOD activity appears to be unaf­fected by age, gender (Table 24b.8), hormone use, or the acute phase status (Solomons, 1985; Fischer et al., 1990; Milne and Johnson, 1993).

• Interpretive criteria

Interpretive criteria for erythro­cyte Cu,ZnSOD activity are limited. Labor­atories should establish their own reference range for erythro­cyte Cu,Zn-SOD activity. Milne and Johnson (1993) have compiled ranges for healthy adults based on the assay of Winterbourne et al. (1975). However, there were relatively small numbers of subjects in each age group. Activity levels are unaf­fected by age, gender, and hormone use. The U.K. National Diet and Nutrition Survey of young people aged 4–18y measured erythro­cyte Cu,Zn-SOD activity; levels decreased for both boys and girls with increasing age. Mean, median, and the lower and upper 2.5 percentile values by age and sex are presented (Gregory et al., 2000). However, work is still required to confirm the utility of erythro­cyte Cu,Zn-SOD activity in studies of the copper status of the general population.

• Measuring erythrocyte Cu,Zn-SOD

There is no standard assay for the measurement of erythro­cyte Cu,Zn-SOD activity. Several methods are available; some are very time consuming, require large samples, neces­sitate the removal of hemo­globin immediately after sample collection, and are subject to inter­ferences.

The assay generally involves the inhibition of oxidation-reduction reactions which are catalyzed by the superoxide anion. The latter may be gener­ated enzym­atically by, for example, xanthine plus xanthine oxidase, which, in turn, reduces cyto­chrome c. The reduction of cyto­chrome c is followed spectro­photom­etrically (Marklund and Marklund, 1974).

Several of these procedures are now automated. L'Abbé and Fischer (1986) developed a method for determining Cu,Zn-SOD activity that can be used with small samples. Commercial kits based on either an enzyme-linked immuno­sorbent assay or on a spectro­photometric assay are now available for measuring Cu,Zn-SOD activity in human serum and urine.

The analytical coefficient of variation for the erythro­cyte super­oxide dismutase assay when determined using an automated method ranges from 1.9% to 11.9%. The within subject variation for erythro­cyte Cu,Zn-SOD activity is generally high (Gallagher et al., 1989).

24b.4 Cytochrome c oxidase

Cytochrome c oxidase (EC 1.9.3.1) is present in most tissues of living organisms and is the terminal enzyme of the electron transport chain. It reacts directly with molecular oxygen in cellular respiration; it catalyzes the electron transfer from cyto­chrome c to O².

Cyto­chrome c oxi­dase activity in erythro­cytes, platelets, or mononuclear leukocytes (lympho­cytes and mon­ocytes) has potential as a sensitive measure of copper status. Animal studies have generally reported reduced tissue cyto­chrome c oxidase activity as an early sign of copper deficiency (Underwood, 1971). in association with a reduction in the respiratory capacity of mito­chondria, especially in the liver, heart, and brain. Coupling of respiration to phos­phoryl­ation is generally not affected (Linder and Hazegh-Azam, 1996). In some of these animal studies, cyto­chrome c oxidase activity in platelets correlated very strongly with liver copper levels (r = 0.99; p < 0.0001) (Johnson et al., 1993).

Genetic defects in the activity of cyto­chrome c oxidase, whereby activity is only about 50% of normal, may lead to neurological, cardiac, and muscle disease in children (DiMauro et al., 1985; Tulinius et al., 1991; Van Coster et al., 1991). Reduced activity of cyto­chrome c oxidase has also been reported in leukocytes in infants and children with Menkes kinky hair syndrome, a genetic defect in copper meta­bolism (Garnica et al., 1977).

More recently, results of copper depletion-repletion studies have reported that platelet cyto­chrome c oxidase activity may be more sensitive than either serum copper or cerulo­plasmin to changes in copper intake. In Figure 24b.2 for example, the activity of cyto­chrome c oxidase in platelets was significantly lower (p < 0.0001) at the end of the copper-depletion period than at baseline Milne and Nielsen, 1996); levels for many women were below the reference range for comparable healthy women (Table 24b.9)

Table 24b.9 Effect of age, gender, and hormone use on mean cytochrome-c oxidase concentrations, measured in both platelets and mononuclear blood cells. FOC, females taking oral contraceptives; FE, females using estrogen therapy. Significance levels from analysis of variance (ANOVA) are also shown (interactions between gender and decade were nonsignificant in both cases). Data from Milne and Johnson, Clinical Chemistry 39: 883–887, 1993

Age (years)	Group	Platelets U/109 cells	Mononuclear blood cells U/106 cells
20–29	M F FOC	2.75 (10) 2.36 (9) 2.87 (9)	0.33 (9) 0.27 (7) 0.32 (7)
30–39	M F FOC	2.94 (9) 2.75 (9 ) 3.87(10)	0.31 (8) 0.36 (10) 0.39 (11)
40–49	M F	3.52 (9) 3.08 (10 )	0.40 (10) 0.35 (9)
50–59	M F FE	3.89 (9) 3.88 (12) 3.54 (3)	0.35 (9) 0.38 (10) 0.42 (3)
60–69	M F	4.92 (7) 4.56 (9)	0.43 (6) 0.37 (9)
70+	M F	4.51 (6) 3.65 (5)	0.45 (4) 0.39 (7)
ANOVA	Gender Decade	NS 0.0001	NS 0.004

(Milne and Johnson, 1993). Moreover, unlike erythro­cyte Zn,Cu-SOD activity or levels of serum copper and cerulo­plasmin, the activity of platelet cyto­chrome c oxidase showed a positive response to copper repletion, with activity levels returning to about 60% of control values (Milne and Nielsen, 1996).

Cyto­chrome c oxidase activity responded similarly in both platelets and mono­nuclear leukocytes to copper repletion in young women fed a copper-depleted diet. However, baseline values were not measured, so that the response of these indices to depletion cannot be evaluated (Milne et al., 1988).

The activity of cyto­chrome c oxidase in both platelets and mono­nucleated leuko­cytes tends to be higher in older adults, and varies markedly between subjects (Table 24b.9, Milne and Johnson, 1993). The activity in mono­nuclear cells, but not platelets, was affected by oral contra­ceptive use, and only in the 30–39y age group. Adolescents with cystic fibrosis also have lower cyto­chrome c oxidase activity in mono­nucleated leuko­cytes, compared with age and sex matched controls (Percival et al., 1995). The postulated mechanism relates to the alterations in chloride transport in cystic fibrosis, which, in turn, impairs copper transport (Alda and Garay, 1990).

The interaction between copper meta­bolism and cyto­chrome c oxidase activity is complex and mediated by transporters, chaperones and affected by their metals and anions. For a recent review see Cobine et al. (2021).

• Interpretive criteria

Table 24b.9 presents mean cyto­chrome c oxidase activity in platelets and mono­nuclear­ blood cells (lymphocytes and mono­cytes) by age and sex for adults 20 to > 70y. Subjects were healthy, non­pregnant, and non­smokers and were not taking any vitamin or mineral sup­plements, or prescribed medications, with the exception of estrogen.

• Measurement of cytochrome-c oxidase

Platelets and mono­nuclear cells have a shorter lifespan (≈10d) than erythrocytes (≈120d) and therefore activities in these shorter-lived cell types better reflect the metabolically active pool of endog­enous copper. However, the isolation of such specific cell types is time consuming and complex, as noted in Section 15.1.3. Moreover, the enzyme cyto­chrome c oxidase is labile, and between subject variation is large. Hence, before the test can be used in community studies, the assay method must be rigorously standardized. An assay developed by Prohaska and Wells (1974) is often used, as are kits that measure ferro­cyto­chrome to ferri­cyto­chrome colour­imetrically.

24b.5 Skin lysyl oxidase

Lysyl oxidase (EC 1.4.3.13) is another extra­cellular copper-containing enzyme that is essential for the normal maturation and function of collagen, specifically in the steps involving the formation of lysine-derived cross-links. The latter are required for the formation of strong, flexible connective tissue (Rucker and Murray, 1978).

Copper deficiency in animals results in low lysyl oxidase activity in heart, aorta, connective tissue, lung, and skin, which is reversed with copper supplementation (O'Dell, 1976). Decreased lysyl oxidase activity may be responsible for the skeletal and cardiac abnormalities reported in copper deficiency (Kagan and Trackman, 1991). In rats, despite no differences in bone weight or femoral ash content, torsional strength of the femur was markedly reduced in copper deficiency (Jonas et al., 1993).

To date, very few experimental copper depletion-repletion studies have examined the effect of low dietary copper on the activity of skin lysyl oxidase. Results in Figure 24b.5

show the changes in mean lysyl oxidase activity in the skin of healthy males receiving diets containing four levels of dietary copper. There was a significant fall in activity when the subjects were fed a low copper diet (0.38mg/d), which returned to baseline values after copper repletion. No such decline was observed when the marginal copper diet (0.66mg/d) was fed. Plasma copper, cerulo­plasmin, and urinary copper were also measured in this study; levels fell significantly on feeding the low-copper diet (Werman et al., 1997). The results of this initial study suggest that lysyl oxidase activity declines when dietary copper intake is inadequate, indicating that the cross-linking of collagen may be modulated by copper deficiency. Skin lysyl oxidase could be used as a biomarker of copper status in young men. Further studies are needed to assess whether the test can also be used in women and older subjects. Skin biopsies are invasive procedures, so that the test probably will have limited use in community-based studies.

24b.6 Diamine oxidase

Diamine oxidase (EC 1.4.3.6) is a copper metallo­enzyme that is responsible for the deamination of putre­scine, cadaverine, and hist­amine (Wolvekamp and de Bruin, 1994). The activity of this enzyme has been reported to be low in some studies in rats (Kehoe et al., 2000) and renal dialysis patients with copper deficiency (DiSilvestro et al., 1997).

In an experi­mental copper sup­plement­ation study of healthy adults, the activity of diamine oxidase in serum increased significantly during all three Cu-supplement­ation regimens in both males and females, as shown in (Table 24b.10,

Table 24b.10 Response of serum diamine oxidase (U/L) in men and women to three different regimens of copper supplementation. In all three cases and for both sexes, activity in the treatment group differed significantly from placebo (p<0.01). CuGC: copper gluconate. Abstracted from Kehoe et al., British Journal of Nutrition 84: 151–156, 2000

Group (n)	Mean (SE)	Mean (SE)
Men (7) Women (9)	3mg CuSO4/d 0.87 (0.27) 1.64 (0.47)	Placebo 0.27 (0.17) 0.33 (0.17)
Men (9) Women (11)	3mg CuGC/d 1.21 (0.36 ) 1.92 (0.41)	Placebo 0.47 (0.01) 0.42 (0.23)
Men (7) Women (10)	6mg CuGC/d 1.55 (0.49) 2.37 (0.79)	Placebo 0.44 (0.28) 1.16 (0.32)

Kehoe et al., 2000). These results suggest that the activity of diamine oxidase in serum has the potential to be a sensitive indicator of copper status in adults and warrants further investigation. Whether there are any adverse health conse­quences associated with low serum diamine oxidase activity is unknown.

The activity of this enzyme is, however, modified by conditions other than copper status, including pregnancy (Kusche et al., 1974), uremia (Tam et al., 1979), malab­sorption syn­dromes or gastro­intestinal damage (Bayless et al., 1981), and kidney dialysis (DiSilvestro et al., 1997). Further studies are needed with other population groups to establish the specificity of this enzyme to changes in copper status. A sensitive auto­mated color­imetric assay developed by Takagi et al. (1994) can be used for the assay of serum diamine oxidase.

24b.7 Copper chaperones

Copper, once inside the cell, is bound to one of a series of chaperones, depending on its final target. The first to be identified was in yeast, and was termed ATX1 (Lin and Culotta 1995). Subsequently, several others have been identified and the best candidate for an indicator of status is CCS. In rat feeding studies, CCS in erythrocytes is responsive to both marginal and deficient diets (Bertinato et al., 2003) and also showed sensitivity to secondary copper deficeincy induced by Zn overload (Iskandar et al., 2005). Other labs confirmed these results in both rats and mice and in copper deficiency and excess (Prohaska et al., 2003). Prohaska and colleagues went one step further, suggesting the CCS:SOD1 ratio may be a more sensitive marker of status (Prohaska et al. 2003).

Araya and colleagues tested the hypothesis that CCS would not be affected by inflam­matory processes, unlike cerulo­plasmin, by measuring CCS mRNA and CCS protein in serum in volunteers who had inflam­matory responses, as demon­strated by clinical history and C-reactive protein. They showed no change in either CCS protein or mRNA, supporting the idea that CCS could be used as a copper marker, and not as an indicator of ceruloplasmin (Araya et al., 2014).

24b.8 Other indices of copper status

• Hair copper concentrations

Concentrations of copper in hair appear to correlate with levels in the liver (Jacob et al., 1978), heart, and kidney (Klevay, 1981). of rats. However, the validity of hair copper concentrations as a biomarker of copper status in humans is less certain. In a detailed human study of therosclerosis patients, hair copper concen­trations did not correlate with the copper content of heart, muscle, liver, kidney, aorta, or rib (Aalbers and Houtman, 1985).

Many studies have confirmed that gender (in adults), pregnancy, lactation, prematurity, hair color, race, and age all affect hair copper concen­trations (Petering et al., 1971; Creason et al., 1975; Gibson and DeWolfe, 1980; Taylor, 1986). The marked rise in hair (and serum) copper concen­trations during early infancy is presumably associated with the redistribution of tissue copper which occurs at this time, and not with changes in dietary copper intake (Gibson and DeWolfe, 1980) (Figure 24b.6).

Normal hair copper levels are found in two conditions in which copper accumulates in the liver — primary biliary cirrhosis (Epstein et al., 1980) and Wilson's disease (Rice and Goldstein, 1961; Gibbs and Walshe, 1965). Additionally, infants with copper deficiency, character­ized clinically by neutro­penia and biochem­ically by concen­trations of serum copper and cerulo­plasmin below the detection limit, did not have lower hair copper levels than age and sex-matched controls (Bradfield et al., 1980). Similarly, the copper content of the hair of children with Menkes kinky hair syndrome is normal (Danks, 1980). It appears, therefore, that hair copper cannot be used as a biomarker of copper status in humans.

• Urinary copper concentrations

Urinary copper is seldom used as an index of copper status; levels are very low in healthy subjects (10–60µg/d) because copper is efficiently reabsorbed by the renal tubules. The main excretory route for copper is the biliary system. Urinary copper concentrations do decrease in persons receiving TPN copper-deficient solutions (Solomons, 1979), conserving body copper. However, in a 13wk experimental depletion-repletion study in which men were fed three levels of dietary copper, urinary copper levels were unaffected by dietary copper levels (Turnlund et al., 1990).

• Other indices

Very limited data are available on copper concentrations in erythro­cytes (Vitoux et al., 1999), and finger­nails (Martin, 1964). Elevated values for erythro­cyte copper have been reported in patients with the two inherited metabolic disorders associated with defects in copper metabolism (Wilson's disease and Menkes syndrome), as well as in children with Down's syndrome and cystic fibrosis. Further studies are needed to ascertain the validity of these measures of copper status.

Changes in some biochemical markers of bone metabolism have been noted during experimental copper depletion-repletion (Baker et al. 1999). and also in some supplementation trials (Baker et al., 1999; Cashman et al., 2001). Results suggest that changes in both urinary pyridin­oline and deoxypyridin­oline (biomarkers of bone resorption) may serve as additional functional markers of copper status in populations that consume diets low in copper; details of these biomarkers of bone resorption are given in Section 23.1.5.

Peptidylglycine α-amidating monooxygenase (PAM) is a cupro­enzyme required for neuro­peptide synthesis. Prohaska and colleagues have suggested its enzyme activity, but not protein levels, may be modulated by mild copper deficiency (Bousquet-Moore et al. 2010; Prohaska et al. 1995). .

• Multiple indices

Serum copper is frequently used to assess copper status in both clinical and community studies, but it is not very sensitive or specific (Harvey and McArdle 2008). Increasingly, because of its higher sensitivity and specificity, the activity of Cu,Zn-SOD in erythro­cytes is used in combin­ation with serum copper. In the future, the activity of cyto­chrome c oxidase in platelets or diamine oxidase in serum may be used in combination with serum copper, instead of erythro­cyte Cu,Zn-SOD, if their validity as bio­markers of copper status in a community setting is confirmed.