Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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.0021 addition of a chemical modifier Mg(NO

) and an

emulsifying agent Triton X-100 (0.2% w/v), e.g., to

whole milk, nonfat milk, and whey milk;

0022 preparation of a slurry of finely pulverized sample

(slurry) in acidic diluents and subsequently treated

by sonication.

0023 The advantages of the slurries are a decrease in the

time needed for the analysis and a reduced risk of

external contamination due to reduced sample ma-

nipulation. Using slurries, copper detection limits

lower than 0.2 mgg

1

can be achieved in biological

samples.

0024 An alternative to slurry sampling and microwave-

assisted acid digestion is ultrasound-assisted extrac-

tion. The main advantage of this over slurry sampling

is a doubling of the tube lifetime, most likely because

the matrix is not introduced into the atomizer, thus

avoiding the buildup of carbonaceous residues or

silicates on the graphite platform. In comparison

with microwave-assisted digestion, it gives a twofold

better precision and better detection limits.

Determination

Molecular Absorption Spectroscopy

0025 A large number of reagents have been proposed for

the spectrophotometric determination of copper, but

only a few, such as pyridil-azo derivatives chloro-

azoantipyrine, 4-(2-pyridylazo) resorcinol, cuprizone

(oxalic acid bis(cyclohexylidene) hydrazine) and

diphenylcarbazone, diethyldithiocarbamate are of

any real practical use. Cuprizone, one of the reagents

used, reacts with Cu(II) to produce a blue, water-

soluble complex with a molar absorptivity of

1.6 10

l mol

1

at 600 nm.

0026 Visible spectrophotometric methods have been

applied mainly for determining the copper content of

water, a matrix that does not require prior digestion. In

general, these methods should only be considered as an

alternative when atomic absorption or plasma emission

techniques are not available. The main limitation of

molecular absorption spectroscopy methods is their

lack of sensitivity to determine copper at the low con-

tent levels of foods and beverages.

0027 A preconcentration using pretreated silica columns

or a chelation solvent extraction prior to applying

a visible spectrophotometric method will yield

sensitivities similar to those obtained with AAS

measurements. A concrete example of precolumn deri-

vatization uses a preconcentration column of

-bonded silica and pretreated with ammonium

1-pyrrolidinecarbodithioate – hexadecyltrimethylam-

monium bromide(I) ion-pair in mobile phase

buffer (50 mM Na acetate–20 mM NaNO

adjusted

to pH 6.0 with dilute HCl) (4:1). The metal–dithio-

carbamate complexes are then backflushed on to

an analytical column Spherisorb ODS2 (5 mm).

Elution with acetonitrile–buffer (17:8) containing I

(15 mM, respectively) and detection at 430 nm (Cu).

The detection limits are 0.5 mgl

1

, and the results

are comparable to those obtained by a conventional

method involving chelation, solvent extraction, and

AAS.

0028As an example of postcolumn derivatization,

there is an ion chromatographic method that uses

derivatization with 2-(2-benzoxazolylazo)-1-

naphthol (a-BOAN) detection at 565 nm in a nonio-

nic surfactant solution of Brij35 used for the separ-

ation and determination of copper(II). The detection

limit is 16 mg of Cu per liter. This method has been

used with good results in the separation and deter-

mination of Cu(II) in certified reference materials

(rice flour and bovine liver).

0029Moreover, some of the reactions between copper

and different organic reagents, such as cuprizone,

diethyldithiocarbamate, or 1,5-diphenylcarbazide,

have been adapted to flow injection analysis (FIA).

This analytical technique has become very popular

and has been used to determine the copper content

of water and different biological matrixes (cow milk,

milk-based infant formulas) or their digests (plants).

Detection limits of copper ranging from 0.02 to

0.3 mgml

1

have been reported in water and about

5 mgl

1

in plant digests after liquid–liquid extraction.

These values can be improved with purification

or separation after organic-matter destruction and

prior to determination.

Fluorescence

0030Several fluorescent reagents can be used to deter-

mine copper in food extracts and digests, the most

frequently reported being: 5-(2-carboxybenzenazo)

rhodanine and 5-(4-chloro-2-carboxyphenylazo) rho-

danine. The intensity of fluorescence is measured at

400–408 nm (excitation at 310 nm), with detection

limits ranging from 0.01 to 0.05 mg of Cu(II) per

milliliter.

Atomic Absorption Spectrometry

0031FAAS FAAS is the technique of choice for measur-

ing copper in food-sample digests. The most sensitive

line is 324.8 nm using a lean air acetylene flame, the

measurement is almost independent of the stoichiom-

etry of the flame, and the interferences are scarce,

though large amounts of some transition elements in

the presence of mineral acids can depress the re-

sponse; e.g., 10 000 mgml

1

depresses 10 mg of copper

per milliliter by 10%. At 324.8 nm, a detection limit

of 0.0015 mgml

1

, which is sufficient for many

1636 COPPER/Properties and Determination

routine determinations, can be achieved under opti-

mal conditions.

0032 Flame emission can be used at a wavelength of

324.8 nm with a lean nitrous oxide acetylene flame.

To improve the sensitivity of FAAS when trace

amounts of copper have to be measured, separation

and preconcentration/enrichment procedures based

on adsorption, chelation, or precipitation processes

can be used.

0033 ETAAS ETAAS is recommended when copper con-

tents are very low or the sample volume is very small.

The 324.8 nm line is used. Copper is easily atomized

from the wall or the platform of a graphite tube, and

only minor interferences have been reported, as a

result of which no chemical modifier is usually neces-

sary in the ashing step. Temperatures of 800 and

2000



C are used for ashing and atomization, respect-

ively. The rate-limiting step for the atomization of

copper is the reduction of Cu

O by graphite that

precedes atomization.

0034 Chloride interferes in the determination of copper

by ETAAS, so chloride should be removed from the

sample before electrothermal atomization, a small

excess of an oxyacid (sulfuric or nitric) added before

the drying or ashing steps, or the method of standard

additions should be used for quantification. To sup-

press chloride interferences, NH

has been used

together with pyrolytically coated tubes, which are

more sensitive but less robust than uncoated tubes. A

1% ascorbic acid solution has been found to reduce

the interference of seawater upon the determination

of copper.

0035 Background corrections can be made by using a

deuterium lamp or a Zeeman correction, although

copper is one of the few elements to suffer from sensi-

tivity loss when the Zeeman correction is used. The

324.7-nm line has an anomalous Zeeman pattern,

and several isotopes split the line. The 327.4-nm line

may be preferable and should be chosen. The charac-

teristic mass for copper at the 327.4-nm line with

Zeeman background correction is about half that

with deuterium background correction. The reproduci-

bility of the results with Zeeman background correc-

tion is improved when a transverse-heated graphite

furnace is used instead of a longitudinal heated one.

0036 One of the advantages in using ETAAS in trace-

element analysis is that a complete digestion, minim-

izing residual carbon content, is not required. This

elimination or reduction of sample digestion reduces

the time needed for the analysis. Among the proposed

methods are direct injection of the sample, dissol-

ution of the sample in an adequate solvent, the use

of a chemical modifier together with an emulsifying

agent, and the use of slurries.

0037One of the facts to take into account in the direct

analysis of a solid sample by ETAAS graphite furnace,

is the sample weight. The use of too little or too

much sample may lead to erroneous results, even if

the analyte content lies within the linear dynamic

range. Masses smaller than 0.3 mg or larger than

1.1 mg lead to overestimated and underestimated

results, respectively.

0038Inductively coupled plasma-atomic emission spectro-

metry Inductively coupled plasma-atomic emission

spectrometry (ICP-AES) provides, under optimal con-

ditions, an approximate limit of detection of 1 mgl

1

Although the cost of ICP-AES instrumentation has

fallen considerably, AAS still remains the most widely

available atomic technique for most laboratories.

0039ICP-AES can be used to measure copper

(l ¼324.7 nm) in diluted biological fluids. In the

case of water, a prior preconcentration by chelate

formation and solvent extraction, or by adsorption

on a column may be necessary. Detection limits

of 0.07 mgl

1

can be achieved when an ultrasonic

nebulizer is used.

0040In the case of foods, samples have to be digested

and preconcentrated or purified, or, in the case of

solid samples such as cereals, samples are ground

and mixed with graphite powder as buffer in a ratio

of 2:3, after which an aliquot of the mixture is trans-

ferred to the powder sampler for introduction into the

sealed ICP-arc system. The limit of detection is

0.02 mgg

1

0041The destruction of the matrix with one or more

acids (e.g., HCl, HClO

, and H

) increases the

possibility of spectral overlap. Therefore, whenever

possible, only HNO

should be used in sample prep-

aration, because H, N, and O do not add any add-

itional spectral interferences to those already existing

in a pure water spectrum.

0042Inductively coupled plasma-mass spectrometry

Inductively coupled plasma-mass spectrometry

(ICP-MS) is a powerful technique for trace multiele-

ment and isotopic analysis, because of its high

sensitivity and ability to determine the isotope com-

position of a sample using less cumbersome pretreat-

ment procedures than other mass spectrometry

techniques.

0043However, ICP-MS is prone to interferences caused

by sample matrix components. The interferences may

be caused by polyatomic ions having the same nom-

inal mass as the analyte, signal suppression or en-

hancement due to nonspectroscopic matrix effects

and blockage of the nebulizer and sampler. Therefore,

in order to achieve accurate and reliable results,

matrix separation is needed when the matrix elements

COPPER/Properties and Determination 1637

in the prepared solution interfere with the determin-

ation. A detection limit of 4.8 mgl

1

can be obtained.

Chemiluminiscence

0044 FIA systems have been developed to determine

micro-amounts of copper in well, rain, lake, and

waste waters by using chemiluminiscence reac-

tions. The systems luminol–CN

–Cu(II) and lumi-

nol–KMnO

–Cu(II) permit detection limits ranging

from 10 ng l

1

to 8.5 mgl

1

to be achieved.

Potentiometric Methods

0045 Electroanalytical techniques can be considered as an

alternative to AAS or plasma techniques for

measuring elements at trace levels. The main advan-

tages of these techniques are an excellent sensitivity

and the possibility of the simultaneous determination

of different elements. Potentiometric methods also

permit the speciation of the analyte, an important

point from a nutritional and toxicological point of

view.

0046 The application of potentiometric techniques to

the determination of an element content in foods

requires complete organic-matter destruction. Once

the sample has been converted into a solution, a

sufficient amount of a base electrolyte should be

added, and the oxygen has to be removed, since

metals deposited on the electrode in the form of an

amalgam or a film are usually very sensitive to oxida-

tion, and oxygen interferes in the determination

through its double-wave reduction.

0047 The high sensitivity of the electrochemical methods

requires the use of high-purity reagents and the

adoption of measures to prevent sample contamin-

ation. Copper can be determined in food digests by

differential pulse polarography with a stationary Hg-

drop or a stick Au-base Hg film working electrode, a

Pt counter-electrode and an Ag–AgCl reference elec-

trode. The limits of detection of Cu are 0.45 mM,

and the corresponding peak potentials are 0.71 V/

0.74 V/0.65 V, depending on the supporting

electrolyte used.

0048 Anodic stripping voltammetry (ASV) is probably one

of most suitable methods for determining copper in

biological matrixes. In ASV, copper is selectively

deposited out of the sample matrix on to a working

electrode at a suitable working potential. After a selected

period of deposition, the analytes collected on the

working electrode are stripped back into the solution

phase, and the oxidation currents are measured.

0049 Different electrodes are available:

0050 Working electrode: hanging mercury drop elec-

trode or a Hg film deposited on a 3-mm-diameter

glassy-carbon rotating electrode;

0051Reference electrodes Ag–AgCl or saturated calomel

electrode (SCE); and

0052Auxiliary/counter-electrode electrode:platinum(Pt)

electrode.

0053The reference and auxiliary electrodes are con-

nected with the sample solution via a liquid bridge

filled with a solution of suprapure potassium chlor-

ide (KCl). The supporting electrolytes are: 1 M KCl,

2M NH

Ac/HAc, 1 M NH

/NH

Cl, 0.075 M

HClO

0054To determine copper by ASV, copper can be elec-

trodeposited on a hanging/stationary mercury drop

working electrode at 1.1 and 1.2 V vs. Ag–AgCl

or SCE (with a platinum auxiliary electrode) for times

ranging from 100 s to 4 min. The deposited metal can

be determined by differential pulse ASV at þ0.04 V.

Detection limits at the ng l

1

or ng g

1

level have been

reported.

0055The use of a selective electrode allows copper to

be measured over a wide range of concentrations with

an approximate practical detection limit of 10

8

mol l

1

. Copper ions in aqueous solution can be

determined with an ion-selective membrane electrode

containing a mixture of copper and silver sulfides.

A supporting electrolyte, such as 2.5 M NaNO

needed. The electrode response is linear from 1 mM

to 1 mM Cu. The presence of Ag, Hg, Fe, and

strong reducing agents (hydrazine 20–200 mgl

1

does not affect the potential but reduces the life of

the electrode.

Neutron Activation Analysis

0056Neutron activation analysis (NAA) is a nondestruc-

tive method that has been used for copper deter-

mination in biological material and bioavailability

studies. The values obtained applying NAA correlate

well with those obtained by AAS or ICP. NAA is

not in common use, being restricted to specialized

laboratories.

X-ray Fluorescence

0057X-ray fluorescence (XRF) techniques allow the

determination of copper in very small samples

(microsamples) of biological matrixes (e.g., blood

serum, a hair, a cell). Samples have to be dried,

milled, homogenized and compacted into disks.

The destruction of the organic matter is not

required. Copper content can be determined by

direct XRF using a wavelength dispersive XRF

spectrophotometer and a synthetic standard prepared

by incorporating the analyte into cellulose. The detec-

tion limit is 5 mg of Cu per gram. There is a good

agreement between the results obtained by XRF

and NAA.

1638 COPPER/Properties and Determination

Speciation

0058 ICP-AES and ICP-MS are element-specific detect-

ors for high-performance liquid chromatography

(HPLC) that have been used for copper speciation.

With this aim, they can be coupled to size-exclusion

chromatography for simultaneous monitoring of

molecular weight fractions and their associated

elements. HPLC coupled with ICP-AES has been

used in copper speciation in foods such as soy bean

flour extracts. Using size-exclusion chromatography

coupled to ICP-AES in human milk, the distribution

of copper between the different protein and non-

protein fractions can be estimated.

Conclusions

0059 AAS techniques are still the most widely used tech-

niques to determine the copper content of foods

and beverages. The most difficult and risky (because

of contamination or analyte losses) step in copper

determination in foods is sample preparation prior

to the element measurement, a fact that is reflected

in the numerous sample treatments found in the lit-

erature. ETAAS offers the advantage of not requiring

complete organic-matter destruction, thus permitting

the direct introduction of a sample, slurry, or acid

extract.

0060 However, ICP-AES offers the advantage of simul-

taneous element determination in a sample, as do

potentiometric techniques, which also permit the

differentiation between oxidation states, although,

in the latter, complete organic-matter destruction is

required. The use of appropriate reference materials

is essential to check analytical accuracy. Currently,

NAA remains useful as a reference method. The hy-

phenated techniques based on the coupling of a sep-

aration technique such as HPLC with an element

detection will contribute to copper speciation in

foods and beverages.

See also: Mass Spectrometry: Principles and

Instrumentation; Applications; Spectroscopy:

Fluorescence; Atomic Emission and Absorption; Visible

Spectroscopy and Colorimetry

Further Reading

Belarra MA, Crespo C, Martı

´nez

Garbayo MP and Castillo

JR (1997) Direct determination of metals in solid

samples by graphite-furnace atomic absorption spec-

trometry: Does sample mass influence the analytical

results? Spectrochimica Acta 52B: 1855–1860.

Bermejo-Barrera P, Domı

nguez-Gonzalez R and Bermejo-

Barrera A (1997) Direct copper determination in whole

milk, non-fat milk and whey milk by electrothermal

atomic absorption spectrometry. Fresenius Journal of

Analytical Chemistry 357: 457–461.

Boumans PWJM (ed.) (1987) Inductively Coupled Plasma

Emission Spectroscopy. Part II: Applications and

Fundamentals. New York: John Wiley.

Cao QE, Zhao Y, Cheng X, Hu Z and Xu Q (1999) Sequen-

tial fluorescent determination of copper (II) and cobalt

(II) in food samples by flow injection analysis. Food

Chemistry 65: 405–409.

Capelo JL, Filgueiras AV, Lavilla I and Bendicho C (1999)

Solid–liquid extraction of copper from slurried

samples using high intensity probe sonication for elec-

trothermal atomic absorption spectrometry. Talanta 50:

905–911.

Cotton A and Wilkinson G (1989) Advanced Inorganic

Chemistry, 5th edn. pp. 755–757. NewYork: John Wiley.

Dracheva LV (1990) Ionometric copper determination in

aqueous media with use of a copper-selective electrode.

Gigiena i Sanitariya 8: 81–83.

Gawalko EJ, Nowicki TW, Babb J, Tkachuk R and Wu SL

(1997) Comparison of closed-vessel and focused open-

vessel microwave dissolution for determination of

cadmium, copper, lead, and selenium in wheat, wheat

products, corn bran, and rice flour by transverse-heated

graphite furnace atomic absorption spectrometry.

Journal of AOAC International 80: 379–387.

Giglio L, Bocca AP and Caroli S (2000) Determination of

the total content and binding pattern of elements in

human milk by high performance liquid chromatog-

raphy-inductively coupled plasma atomic emission

spectrometry. Talanta 53: 295–303.

Herna

ndez Co

rdoba M (1995) Copper. In: Townshend

A (ed.) Encyclopedia of Analytical Science, vol. 2,

pp. 852–858. London: Academic Press.

Hwang TJ and Jiang SJ (1996) Determination of copper,

cadmium, and lead in biological samples by isotope

dilution inductively coupled plasma mass spectrometry

after on-line pre-treatment by anodic stripping voltam-

metry. Journal Analytical Atomic Spectrometry 11:

353–357.

Lavilla I, Filgueiras AV and Bendicho C (1999) Comparison

of digestion methods for determination of trace minor

metals in plant samples. Journal of Agricultural and

Food Chemistry 47: 5072–5077.

Li GH, Zhang WG and Meng Z (1993) Determination

of micro-amounts of copper in waste water by flow-

injection analysis using the checking effect of the chemi-

luminescence reaction. Lihua-Jianyan-Huaxue-Fenxi

29: 290–291.

Lima EC, Barbosa F, Krug FJ, Silva MM and Vale MGR

(2000) Comparison of ultrasound-assisted extraction

slurry sampling and microwave-assisted digestion for

cadmium, copper and lead determination in biological

and sediment samples by electrothermal atomic

absorption spectrometry. Journal of Analytical Atomic

Spectrometry 15: 995–1000.

Price WJ (1983) Spectrochemical Analysis by Atomic

Absorption. New York: John Wiley.

Slavin W (1984) Graphite Furnace AAS: A Source Book.

Norwalk, USA: Perkin Elmer.

COPPER/Properties and Determination 1639

Physiology

M A Johnson, The University of Georgia, Athens, GA,

USA

Introduction

0001 Copper was identified as an essential nutrient early in

the twentieth century. Today, research focuses on the

molecular biology of copper metabolism and the role

of copper in the heart, blood vessels, nervous system,

immune function, formation of red blood cells, and

bone health. A major challenge is the lack of a copper

protein in blood that can reliably assess the copper

status of humans. Although copper has been added to

infant formulas for decades, copper is rarely added

to enriched or fortified cereals consumed by older

children and adults. An easily absorbed form of

copper, such as copper sulfate, is added to only a

few multiple vitamin/mineral supplements.

Body Content

0002 Adult humans contain about 50–120 mg of copper,

with the highest concentrations found in the liver,

brain, heart, skeleton, hair, and nails (Table 1).

Because of its size, muscle contains the largest per-

centage of body copper. Copper in liver, kidney, and

intestine is strongly related to copper intake.

0003Serum is the most widely used indicator of copper

status, but it is not strongly related to the body stores

of copper and becomes low only when the stores of

copper are nearly exhausted. Similarly, copper in hair

is not strongly related to the body levels of copper.

Although sweat contains copper, it is difficult to

collect and the relationship between body stores and

sweat levels of copper has not been established. Sweat

is not considered a major excretory route, unless

sweat loss is extremely high. Losses of copper in the

feces are proportional to intake, while urinary excre-

tion is less than 5% of intake. Unlike for iron, men-

strual losses do not deplete the body of copper.

Absorption, Transport, Storage, Excretion

0004Copper absorption in humans is usually estimated

from copper intake minus copper excretion in the

urine and feces. However, more accurate estimates

of copper absorption and excretion are made by

using a stable isotope of copper.

0005The primary site of copper absorption in humans

and other animals is in the intestine in the duodenum.

When fed a typical diet, human adults generally absorb

about 50–75% of the copper ingested from food. The

percentage absorption decreases as the amount of

copper ingested increases. In adults, advanced age and

gender do not influence copper absorption.

0006At the lower levels of copper intake, absorption is

probably regulated by a saturable active transport

mechanism, while at high levels of copper intake

passive diffusion plays a role. Metallothionein is a

metal-binding protein found in the intestine and

other tissues that may also regulate copper absorp-

tion. Copper bound to metallothionein in the mucosal

cell will be lost when these cells slough off the intes-

tine. As will be discussed in more detail, copper ab-

sorption is negatively influenced by numerous dietary

factors, including zinc, iron, molybdenum, ascorbic

acid, fructose, and sucrose.

0007Copper is transported in the circulation bound

primarily to albumin and to a lesser extent to trans-

cuprein and low-molecular-weight compounds.

Newly absorbed copper is transported to the liver

and rapidly incorporated into ceruloplasmin. Cerulo-

plasmin is the transport protein from the liver to other

organs. Some liver copper will also be incorporated

into metallothionein, especially when copper intake is

high. Metallothionein may help detoxify copper when

copper exposure is high.

0008Gestation is a very important period of life for

storage of copper. At birth the human liver has 5–10

times the adult concentration of copper. Liver copper

gradually decreases during the first year of life as the

infant uses liver copper for other functions in the

tbl0001 Table 1 Copper in human fluids, excretions, and tissues

Fluids and excretions Level

Serum 0.5–1.5 mg l

1

Sweat 20–100 mgl

1

Urine 4–66 mgl

1

5–50 mg day

1

Feces 0.5–2.5 mg day

1

20–100 mg kg

1

dry weight

Menstruation 0.5 mg per period

Hair 10–50 mg kg

1

dry weight

Tissues Concentration

(mgkg

1

wet weight)

Estimated % of

totalbodycopper

Bone marrow 5–7 15

Liver 5–7 8

Brain 5–7 8

Bone 2–3 19

Skin 2–3 15

Lung, heart, kidney 2–3 <5

Muscle < 2 25

Blood < 2 5

Spleen < 2 <1

Reproduced from Johnson MA (1998) Copper: physiology, dietary sources

and requirements. In: Encyclopedia of Human Nutrition. Academic Press,

with permission.

1640 COPPER/Physiology

body. Premature infants are at high risk for copper

deficiency unless they receive breast milk or copper-

fortified formula.

0009 Copper is lost from the body primarily through the

bile and the feces, and retention of copper in the body

is controlled primarily through regulation of copper

absorption at the intestinal cells and excretion

through the bile. Very little of the copper excreted

into the bile is reabsorbed. Copper loss from the

urine, skin, nails, and hair is very small, and, unlike

some other minerals, the kidney does not regulate

copper homeostasis.

Functions and Essentiality

0010 Most of what we know about copper comes from

livestock or from experimental animals fed diets defi-

cient in copper. Some information is available from

infants fed diets low in copper, healthy adults fed low

amounts of copper, and adults given total parenteral

nutrition without copper. Copper clearly is required

for the immune system, the nervous system, the

cardiovascular system, skeletal health, for iron me-

tabolism, and for formation of red blood cells. How-

ever, the roles of copper in other functions such as the

regulation of cholesterol, glucose, and blood clotting

are not well understood. Some disorders of the vascu-

lar and nervous systems induced by copper deficiency

during prenatal development in animals cannot

be reversed by copper supplementation after birth.

Thus, copper must be provided during pregnancy.

Copper’s role in cardiovascular and skeletal health

also has important implications for major public

health problems seen in industrialized nations,

namely heart disease and osteoporosis.

0011Copper functions in several enzymes and proteins

in the human body. Table 2 lists copper enzymes

found in mammals. Cytochrome c oxidase is essential

tbl0002 Table 2 Human enzymes and proteins that contain copper

Enzyme orprotein Function

Cytochrome c oxidase Mitochondrial enzyme involved in the electron transport chain; reduces oxygen to water and

allows formation of adenosine triphosphate; activity is highest in the heart and also high in

the brain, liver, and kidney

Ceruloplasmin (ferroxidase I) Glycoprotein with 6–7 copper atoms; 4 copper atoms involved in oxidation/reduction

reactions; role of other copper atoms not fully known; scavenges free radicals; oxidizes

some aromatic amines and phenols; catalyzes oxidation of ferrous iron to ferric iron;

assists with iron transport from storage to sites of hemoglobin synthesis; about 60% of

plasma copper bound to ceruloplasmin; primarily extracellular; activity will be low during

severe copper restriction

Ferroxidase II Catalyzes oxidation of iron; no other functions known; in human plasma is only about 5% of

ferroxidase activity

Monoamine oxidase Inactivates catecholamines; reacts with serotonin, norepinephrine (noradrenaline),

tyramine, and dopamine; activity inhibited by some antidepressant medications

Diamine oxidase Inactivates histamine and polyamines; highest activity in small intestine; also high activity in

kidney and placenta

Lysyl oxidase Acts on lysine and hydroxylysine found in immature collagen and elastin; important for

integrity of skeletal and vascular tissue; use of estrogen increases activity

Dopamine beta-hydroxylase Catalyzes conversion of dopamine to norepinephrine, a neurotransmitter; contains 2–8

copper atoms; important in brain and adrenal glands

Copper, zinc superoxide dismutase Contains 2 copper atoms; primarily in cytosol; protects against oxidative damage by

converting superoxide ion to hydrogen peroxide; erythrocyte levels are somewhat

responsive to changes in copper intake

Extracellular superoxide dismutase Protects against oxidative damage by scavenging superoxide ion radicals and converting

them to hydrogen peroxide; small amounts in plasma; larger amounts in lungs, thyroid,

and uterus

Tyrosinase Involved in melanin synthesis; deficiency of this enzyme in skin leads to albinism; catalyzes

conversion of tyrosine to dopamine and oxidation of dopamine to dopaquinone. Present in

eye and skin and forms color in hair, skin, and eyes

Metallothionein Cysteine-rich protein that binds zinc, cadmium, and copper; important for sequestering metal

ions and preventing toxicity

Albumin Binds and transports copper in plasma and interstitial fluids; about 10–15% of copper in

plasma is bound to albumin

Transcuprein Binds copper in human plasma; may transport copper

Blood-clotting factors V and VIII Role in clotting and thrombogenesis not well understood; part of structure homologous with

ceruloplasmin

Reproduced from Johnson MA (1998) Copper: physiology, dietary sources and requirements. In: Encyclopedia of Human Nutrition. Academic Press, with

permission.

COPPER/Physiology 1641

for energy production in mitochondria. Ferroxidases

such as ceruloplasmin (ferroxidase I) and ferroxidase

II are copper enzymes. Many amine and diamine

oxidases contain copper. Lysyl oxidase catalyzes the

cross-linking of collagen and elastin; thus, copper

deficiency causes bone and vascular abnormalities

in many species. Superoxide dismutases are anti-

oxidants. Copper deficiency in animals with dark

hair can cause dark hair to lighten because of de-

creased activity of tyrosinase. Copper-binding pro-

teins include metallothionein, albumin, transcuprein,

and blood-clotting factors V and VIII. Copper also

binds to low-molecular-weight ligands such as amino

acids and small peptides.

Assessment of Copper Status

0012 Severe copper deficiency is verified by low serum or

plasma copper or ceruloplasmin, low red cell super-

oxide dismutase activity, anemia, and neutropenia. A

major problem with using low ceruloplasmin as an

index of copper status in people with chronic diseases

such as cardiovascular disease, cancer, or inflamma-

tory diseases is that ceruloplasmin synthesis is ele-

vated during disease states and hence reflects disease

state rather than copper status. Therefore, patients at

risk for copper deficiency, such as those receiving

total parenteral nutrition without copper, should have

their serum copper routinely assessed.

0013 Marginal copper deficiency is very difficult to detect

and limits our ability to identify the role of copper

in human health. Many copper proteins are easily

measured in the blood, but their concentrations

do not change very much when copper intake is

increased or decreased. Studies at the US Department

of Agriculture Human Nutrition Research Centers

have identified the dietary intakes associated with

decreases in copper proteins and other indices. Elderly

women fed 0.57 mg copper per day for 105 days had

low red cell superoxide dismutase activity, low platelet

cytochrome c oxidase activity, low red cell glutathione

peroxidase activity, and elevated plasma factor VII,

but had no changes in plasma copper or ceruloplas-

min. In contrast, young men fed 0.38 mg copper day

1

for 42 days had decreased plasma copper and cerulo-

plasmin concentration and activity. Thus, it appears

that copper intakes must be below 0.6 mg copper

day

1

for copper proteins to be decreased. Also, the

magnitude of these decreases is not large enough to be

used for routine assessment of healthy adults.

0014 Ceruloplasmin and serum and plasma copper are

increased by cigarette smoking, oral contraceptives,

estrogen status, pregnancy, infectious disease, hema-

tological diseases, diabetes, coronary and cardio-

vascular diseases, uremia, cancer, inflammation, and

after surgery. Ceruloplasmin is an acute-phase react-

ant which accounts for its rise during numerous dis-

orders and therefore limits the use of ceruloplasmin as

a copper status marker, especially during illness.

Copper Deficiency in Infants

0015In infants, severe copper deficiency can be diagnosed by

assessing plasma or serum copper, ceruloplasmin, and

neutrophils. Sometimes copper deficiency is mistaken

for iron deficiency because both mineral deficiencies

are associated with anemia and low hemoglobin

concentrations. However, only the anemia of copper

deficiency responds to iron supplements.

0016Infants are more susceptible to severe copper defi-

ciency than are children and adults. Risk factors

for copper deficiency were identified in 52 copper-

deficient infants (Table 3). Copper accumulates in the

liver late during gestation. These liver stores are used

in the first few months of life, because milk contains

little copper. The highest risk for copper deficiency is

in infants who are malnourished, premature, or of

low birth weight. When these physiological condi-

tions are combined with feeding practices such as

cows’ milk or total parenteral nutrition, the risk for

copper deficiency can increase. Breast-feeding helps

protect against copper deficiency because the copper

in human milk is well utilized by infants.

0017Copper deficiency is rare among infants in developed

countries because infant formulas are supplemented

with copper. Also, it is recommended that copper be

added to total parenteral nutrition solutions for infants

as well as adults. However, where malnutrition is

prevalent, copper deficiency may continue to be a

tbl0003Table 3 Copper deficiency in infants

%ofcases

Predisposing factors

<18 months of age 96

Low birth weight (<2500 g) 40

Fed exclusively or predominantly cows’ milk 54

Received total parenteral nutrition 23

Formula-fed 12

Antecedent malnutrition 25

Physical features

Hypotonia (loss of muscle tone) 43

Hypopigmentation 29

Psychomotor retardation 21

Skin rash 21

Dilated veins 15

Hepatosplenomegaly (complication of anemia) 15

Bone fractures 11

Median age at presentation was 8.3 months for full-term infants and 3.0

months for low-birth-weight infants. Reproduced from Johnson MA (1998)

Copper: physiology, dietary sources and requirements. In: Encyclopedia of

Human Nutrition. Academic Press, with permission.

1642 COPPER/Physiology

problem. For example, clinicians in Chile reported that

copper deficiency was more common than zinc defi-

ciency and that supplements of copper improved the

growth of 11 infants with malnutrition complicated by

copper deficiency. It is believed that the cows’ milk-

based diet used during recovery from malnutrition in

many developing countries does not provide enough

copper for optimal growth and development.

Nutrient Interactions Influencing Copper

Bioavailability

0018 The bioavailability of copper is decreased by high

intakes of several nutrients, including zinc, iron,

molybdenum, ascorbic acid, sucrose, and fructose. It

is well documented that high intakes of zinc interfere

with copper absorption in humans and animals. The

mechanism appears to involve the induction by zinc

of metallothionein synthesis in the intestine and

copper becomes ‘trapped’ in this protein and is not

available for transport into the circulation. Zinc

probably has little influence on copper status when

the zinc to copper ratio is 15:1 or less. However,

50 mg of zinc day

1

has decreased some indices of

blood copper status in humans. It is recommended

that healthy adults limit their supplemental intake

of zinc to less than 20 mg day

1

. The current recom-

mended dietary allowance in the USA for zinc is 8 mg

for women and 11 mg for men.

0019 Although studies in animals have shown that high

iron intakes interfere with copper status, there are not

enough studies to determine if supplements of iron

impair the copper status of people. Interactions

between copper and molybdenum are well docu-

mented in ruminant animals, but little information

is available for humans. High intakes of ascorbic

acid induce copper deficiency in many species, but

there is very little information in humans. In one

study, 600 mg ascorbic acid day

1

did not decrease

copper absorption, but in another study, 1500 mg

ascorbic acid day

1

decreased the activity of cerulo-

plasmin. Supplemental intakes of ascorbic acid in

excess of 1000 mg day

1

are common in North Amer-

ica, but the long-term impact of excess ascorbic acid

on copper status is unknown.

0020Rats fed copper-deficient diets that contain 50% or

more by weight fructose or sucrose develop more

severe copper deficiency than when they are fed

starch as the source of dietary carbohydrate. It is

believed that in other species, including humans, the

type of carbohydrate may not markedly influence the

copper status of people. For example, in one human

study, high intakes of fructose caused an increase in

copper absorption but a decrease in red blood cell

copper, zinc superoxide dismutase.

0021Combinations of copper antagonists interfere with

copper status in animals. For example, high concentra-

tions of sucrose exacerbate the effects of iron, zinc, and

ascorbic acid on copper deficiency in rats. These type of

relationships have not been evaluated in humans.

Genetic Diseases

0022Menkes disease, Wilson disease, and aceruloplasmi-

nemia are genetic disorders involving the regulation

of copper and/or ceruloplasmin (Table 4). These

tbl0004 Table 4 Genetic disorders of copper metabolism

Menkes disease Wilson disease Aceruloplasminemia

Genetics Xq13.3 13q14.3 3q 23–35 (ceruloplasmin),

X-linked Autosomal recessive, Autosomal recessive

1 in 300 000 1 in 30 000 Frequency unknown

Clinical Onset before birth, gray-matter

degeneration, abnormal hair,

hypothermia, hypopigmentation,

tortuous arteries, early death

(< 3 years)

Onset late in childhood, abnormal

basal ganglia, liver disease,

Kayser–Fleischer rings in eyes,

psychiatric disorders

Adult onset, dementia, dystonia,

diabetes, retinal degeneration

Laboratory Low serum copper and ceruloplasmin,

low liver copper

Low serum copper and

ceruloplasmin, elevated liver

copper

Low serum copper and

ceruloplasmin, normal liver

copper, elevated liver iron

Defect Impaired copper transport in intestine

and placenta

Impaired biliary excretion of copper Impaired export of iron from

reticuloendotheilial cells

Treatment No effective treatment Chelation very effective to remove

body copper; zinc supplements

help prevent further copper

accumulation

Unknown

Animal models Mottled mouse LEC rat None

Reproduced from Johnson MA (1998) Copper: physiology, dietary sources and requirements. In: Encyclopedia of Human Nutrition. Academic Press, with

permission.

COPPER/Physiology 1643

disorders are advancing the understanding of the cel-

lular and molecular regulation of copper homeo-

stasis. All three disorders are characterized by low

serum copper and low serum ceruloplasmin; however,

Menkes disease results in copper deficiency, Wilson

disease leads to copper overload, and aceruloplasmi-

nemia causes iron overload in certain tissues.

0023 Menkes disease is an X-linked disorder that causes

mental deterioration, hypothermia, failure to thrive,

connective tissue disorders, and death in early child-

hood. The advanced neuronal degeneration at birth

suggests that the disease begins in utero and that

copper is needed for fetal development of the nervous

system. Copper uptake into cells is normal but copper-

dependent enzyme activity is deficient. Placental and

intestinal transport of copper is decreased. Neither

oral supplements nor injections of copper will reverse

this copper-deficient state. There may also be a milder

type of Menkes disease that was formerly known as

X-linked cutix laxa. Most patients reach adulthood,

but have impaired physical and mental function.

0024 Wilson disease is an autosomal recessive disorder

that usually presents in adolescence with liver cirrhosis

and neuronal degeneration. Copper accumulation in the

liver and basal ganglia appears to account for tissue

damage but the mechanism of toxicity is unknown.

Copper chelation therapy is beneficial if the disease is

diagnosed early. Supplements of zinc help limit copper

absorption and subsequent accumulation. However, the

neurologic symptoms are often irreversible and liver

disease may be quite advanced at the time of diagnosis.

The metabolic defect appears to be decreased biliary

copper excretion and impaired incorporation of copper

into newly synthesized ceruloplasmin.

0025 Aceruloplasminemia is an autosomal recessive dis-

order of iron metabolism that was discovered in some

people who were thought to have Wilson disease but

did not follow the strict clinical criteria for the disease

and did not have mutations in the Wilson P-type

adenosine triphosphatase (ATPase). Their symptoms

were somewhat similar to the dementia of Parkinson

disease, combined with retinal degeneration and

elevated liver iron, but normal liver copper. Basal

ganglia had high iron concentrations as shown during

magnetic resonance imaging of the brain. Mutations

in the ceruloplasmin gene resulting in truncations of

the open reading frame were found in these patients.

Thus, it appears that abnormalities in the ceruloplas-

min gene and its protein products cause disordered

iron homeostasis.

0026 The genes responsible for Menkes and Wilson dis-

ease do not involve ceruloplasmin. Rather, the genetic

disorder appears to involve a transmembrane protein

with homology to cation-transport P-type ATPases.

There is 65% homology between the Wilson and

Menkes P-type ATPases. Both contain an area pro-

posed to bind copper. The Wilson gene appears to

be expressed predominantly in the liver while the

Menkes gene is expressed in most tissues other than

the liver. The phenotypic differences in Wilson and

Menkes disease are probably due to the tissue-specific

expression of the genes. Evolutionary conservation of

these types of proteins in bacteria, yeast, plants, and

mammals indicates that a unique protein structure

necessary for copper transport is preserved through-

out the animal and plant kingdom.

Requirements and Recommended Intakes

0027The lack of a biochemical index of marginal copper

status has made it very difficult to determine copper

requirements in humans. When recommended dietary

allowances in the USA were revised in 1989, there

were insufficient data to make a firm recommenda-

tion for copper. However, in 2001 Dietary Reference

Intakes in the USA were estabilished (Table 5). For

adults, the Recommended Dietary Allowance for

adults is 0.9 mg day

1

and the upper level is

10 mg day

1

. Liver damage was the endpoint used

to estimate the upper level. The European Commis-

sion has also established population references for

several nutrients, including copper.

Dietary Sources

0028The amount of copper in various foods varies by

about 100-fold and depends on where a given food

was produced and how it was processed (Table 6).

tbl0005Table 5 Dietary reference intakes for copper

Age (years) Recommended dietary

allowance (mg day

1

)

Upper intake

level (mg day

1

)

0–0.5 0.20

–

>0.5–1 0.22

–

1–3 0.34 1

4–8 0.44 3

9–13 0.70 5

14–18 0.89 8

19–50 0.90 10

 51 0.90 10

Pregnancy

14–18 1.0 8

19–50 1.0 10

Lactation

14–18 1.3 8

19–50 1.3 10

Adequate intake.

From: Food and Nutrition Board (2001) Copper. In: Dietary Reference

Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper,

Iodine, Iron, Mangnese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc,

pp. 224–257. National Academy of Sciences. Washington DC: National

Academy Press.

1644 COPPER/Physiology

Variation in copper content may be the result of

soil conditions, type of fertilizer or other agricultural

chemicals, weather, time of harvesting, and process-

ing. The amount of copper in a typical serving can be

approximated by using portion sizes of 250 g for fluid

milk and juices, 120 g for small servings of fish or

meat, 100 g for fruits, vegetables, and legumes and

30 g for a slice of bread, small bowl of cereal, or

slice of cheese. Copper concentrations are highest in

legumes, nuts, and shellfish, and lowest in dairy foods

such as cows’ milk. Removal of the germ and bran

during refining of flour decreases the copper content

of grain foods. Copper in drinking water is quite

variable and depends on the natural mineral content,

the pH of the water, and the use of copper water

pipes. Acidic water that flows through copper pipes

will pick up some copper. Estimates of the contribu-

tion of water to copper intake range from less than

10% to over 50% of the recommended intakes.

0029In the USA, copper is added to some, but not all,

multiple-vitamin/mineral supplements. Cupric oxide is

one of the most common sources of copper used in

multiple-vitamin/mineral supplements; however, its

solubility and bioavailability are probably very poor

for humans (but quite good for ruminants). It is

recommended that copper sulfate be used in these mul-

tinutrient preparations. Most infant formulas are sup-

plemented with copper in the form of copper sulfate.

Copper is not routinely added to ready-to-eat breakfast

cereals intended for older children and adults.

Copper Toxicity

0030Tolerance to high intakes of copper varies considerably

among species. For example, rats tolerate high amounts

of copper, while sheep are intolerant of high intakes of

copper. Soluble salts of copper (carbonates, sulfates,

and chlorides) are more toxic than the less soluble

forms (nitrates, acetates, and oxides). High intake of

zinc protects again copper toxicity in many species.

0031Acute toxicity from copper is rare in humans and

usually occurs from accidental or intentional inges-

tion of copper salts. Acute symptoms include saliva-

tion, nausea, vomiting, epigastric pain, and diarrhea.

Ingestion of about 100 g or more of copper sulfate can

produce hemolytic anemia, liver failure, renal failure,

shock, coma, or death.

0032Data are quite limited to make a scientifically

based safe upper limit of copper intake for humans.

tbl0006 Table 6 Copper in foods

Food Copper content

(mgkg

1

)

Dairy

Cheese 0.4–0.8

Chocolate milk 0.2–0.3

Cottage cheese 0.1–0.2

Cows’ milk (skim, 2%, whole, buttermilk) 0.02–0.08

Human milk 0.02–0.08

Yogurt 0.01–0.09

Eggs (fried, scrambled, or soft-boiled) 0.4–0.8

Fish and shellfish (cooked)

Cod, fish sticks, haddock, salmon,

sardines, tuna

0.3–0.8

Shrimp 2–3

Oysters 0.3–16

Fruits

Apples (red, with peel, raw) 0.1–0.4

Apple juice (canned) 0.02–0.2

Banana (raw) 1–2

Grapes (raw, purple, or green) 0.4–1.4

Grape juice (canned) 0.01–0.13

Orange (raw, all varieties) 0.1–1

Orange juice 0.1–0.3

Dried fruits (currants, dates, figs,

prunes, or raisins)

1–5

Grains and cereals (cooked or processed)

Barley, pearl 0.4

Bread

White (loaf, rolls, biscuits) 1–2.6

Whole wheat 2–3

Rye 1.6–2.8

Corn (fresh, frozen, cream-style, grits) 0.1–0.4

Flour

Whole grain 2–8

White 1–3

Macaroni (cooked) 0.6–1

Oatmeal (cooked) 0.3–1.2

Wheat bran 10–20

Wheat cereals (shredded, bran flakes,

puffed wheat, raisin bran)

4.5–5.5

Legumes

Beans (boiled or baked; cowpeas,

kidney, lima, navy or pinto)

1–4

Beans (dry; cowpeas, kidney or lima) 5–10

Peanuts (fresh, roasted or butter) 3–10

Meats (cooked: beef, pork and poultry)

Muscle meat 0.7–1.4

Liver 20–180

Nuts and seeds (almonds, brazil nuts,

hazelnuts, pecans, pistachios, sesame

seeds, sunflower seeds, or walnuts)

8–18

Vegetables

Broccoli (fresh, frozen, or boiled) 0.1–0.9

Cabbage (fresh, boiled, or coleslaw) 0.1–0.2

Carrots (raw or boiled) 0.5–1

Cauliflower (fresh, frozen, or boiled) 0.2–1

Onions (raw or cooked) 0.2–1

Peas (green, canned, cooked) 1–1.5

Potatoes

Baked (with peel) 0.6–1.8

Boiled (without peel) 0.3–1

Spinach (canned, fresh, frozen, or

boiled)

0.6–1.2

Sweet potatoes (boiled or baked) 1.6–2.0

Tomatoes (raw, canned or juice) 0.3–1.4

Reproduced from Johnson MA (1998) Copper: physiology, dietary sources

and requirements. In: Encyclopedia of Human Nutrition. Academic Press,

with permission.

COPPER/Physiology 1645