EPA-600/1-77-003
January 1977
Copper
By
Subcommittee on Copper
Committee on Medical and Biologic Effects of
Environmental Pollutants
National Research Council
National Academy of Sciences
Washington, D. C.
Contract No. 68-02-1226
Project Officer
Orin Stopinski
Criteria and Special Studies Office
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
A.
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
NOTICE
The project reported on here was approved by the Governing Board of- the
National Research Council, whose members are drawn from the Councils of the
National Academy of Sciences, the National Academy of Engineering, and the
Institute of Medicine. The members of the committee responsible for the
report were chosen for their special competences and with regard for
appropriate representation of experience and disciplines. The findings
and conclusions presented are entirely those of that committee.
This report has been critically reviewed according to procedures approved
by a Report Review Committee consisting of members of the National Academy of
Sciences, the National Academy of Engineering, and the Institute of Medicine.
Only after completion of the review process has it been released for publication.
The work on which this publication is based was performed pursuant to
Contract No. 68-02-1226 with the Environmental Protection Agency.
11
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
To aid the Health Effects Research Laboratory to fulfill the functions
listed above, the National Academy of Sciences (NAS) under EPA Contract
No. 68-02-1226 prepares evaluative reports of current knowledge of selected
atmospheric pollutants. These documents serve as background material for
the preparation or revision of criteria documents, scientific and technical
assessment reports, partial bases for EPA decisions and recommendations for
research needs. Copper is one of these reports.
Jtfhn H7
Knelson, M.D.
Director
Health Effects Research Laboratory
iii
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CARL A. PRICE, Department of Bioche
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-600/1-77-003
4. TITLE AND SUBTITLE
Copper
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSI ON-NO.
5 REPORT DATE
January 1977
7 AUTHOR(S)
Subcommittee on Copper
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Committee on Medical and Biologic Effects of
Environmental Pollutants
National Academy of Sciences
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1226
Washington, D.C.
12 SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report is a review of current knowledge of the distribution of copper
in the environment and living things. Metabolism and the effects of copper in
the biosphere are also considered. Copper compounds are common and widely distrv
buted in nature. They are also extensively mined, processed and redistributed
by man. Copper is an essential element in plant and animal nutrition. It is
closely related to iron, sulfur and molybdenum in animal metabolism.
Requirements differ in relation to the nutritional state of these other
elements. In plants copper toxicity is infrequent and usually results from
soil contamination due to human activities. Deficiency in plants is fairly
common, and may require supplementation for crops. In animals both deficiency
and toxicity are infrequent except in ruminants. Human copper poisoning occurs
rarely in industry, as a cause of food poisoning, resulting from some medical
treatments, and from genetic defects in metabolism. Copper levels found in
food, water and air have not been found to be injurious.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Copper
Air Pollution
Toxicity
Ecology
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
06 F, H, T
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(a) DESCRIPTORS - Select from the Thesaurus of Engineering and Scientific Terms the proper authorized terms that identify the major
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CONTENTS
1 Introduction 1
2 Copper in the Ecosystem 2
3 Copper in Plants 7
4 Copper in Animals 17
5 Human Copper Metabolism 39
6 Copper as an Industrial Health Hazard 82
7 Summary and Conclusions 87
8 Recommendations for Future Research 92
Appendix Copper Analysis in Environmental and 94
Biologic Samples
References 107
v
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CHAPTER 1
INTRODUCTION
Copper is essential to human life and health, and like all heavy metals,
it also is potentially toxic. In response to this duality, biochemical
mechanisms have evolved that control the absorption and excretion of cop-
per. These mechanisms operate to offset the effects of temporary
deficiency or excess of the metal in the diet.
Of the copper retained in the body, almost all plays a particular
physiologic role as the prosthetic element of more than a dozen specific
copper proteins, such as cytochrome c^ oxidase and tyrosinase. Thus
only extremely small concentrations of free copper ions are normally found in
body fluids. Because it is common for the toxicity of any heavy metal cation
to be sharply diminished when it is bound to proteins or other macromo-
lecules, the existence of these copper proteins—as well as of the homeo-
static mechanisms governing absorption and excretion—make toxicosis from
dietary copper extremely rare in man.
Only inordinately large amounts or concentrations of orally ingested
copper are toxic. For example, acidic foods or beverages that have been
in prolonged contact with copper metal may cause acute gastrointestinal
disturbance. However, when copper enters the body by a parenteral route,
for example, following inhalation or absorption from burned skin or a con-
traceptive device in the uterine cavity, there is a significant possibility
that toxicosis may result from amounts of copper that are innocuous when
eaten.
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CHAPTER 2
COPPER IN THE ECOSYSTEM
Copper metal and its compounds have been used by man since pre-
historic times and thus have been a part of the environment and ecosystem
in varying concentrations. An appreciation of the amount of copper
being removed from sites of its natural source and injected into the
ecosystem worldwide arid in the United States may be gained from the
following statistics on copper production. From 1955-1958, annual U.S.
124
production of recoverable copper was about 900,000 metric tons, but
575
by 1975 the production had risen to 1,260,000 metric tons.
Currently available statistics for world production of copper indicate
that the amount of copper entering the ecosystem annually has steadily
increased and now amounts to about 1,800,000 metric tons annually,
especially in the industrially developed nations. The world trade in
refined copper has been reported as amounting to 2,271,150 metric
620
tons in 1973.
Increased knowledge of the essential nature of copper in the
metabolism of plants arid animals, and of its versatility in many indus-
trial and agricultural operations, has led to recognition of the wide-
spread natural and man-made distribution of this element in concentrations
ranging from the severely deficient to the toxic.
Copper is an element with atomic number 29, and an atomic weight
of 63.546. In consists of two natural isotopes: copper-63, and -65,
which constitute 69.09% and 30.91%, respectively, of the whole. It
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occurs in nature as the metal in the 1+ and 2+ valence states with ionic
o o
radii of 0.96A and 0.72A (for sixfold coordination), respectively. In
+2 ° +2
the +2 state, it is isomorphous with Zn (ionic radius, 0.74A), Mg
o 605a
(ionic radius, 0.66A), and Fe+2 (ionic radius, 0.74A). Copper tends to
occur in sulfide deposits, particularly in igneous rocks.
The concentration of copper in the continental crust—generally
440
given as about 50 ppm— tends to be highest in the ferromagnesium
minerals, such as the basalts pyroxene and biotite, where it averages
140 ppm. Copper commonly tends to form organic complexes, although coal
is relatively low in copper. Sandstones contain 10—40 ppm, shales, 30-
150 ppm, and marine black shales, 20-300 ppm.
In the sedimentary cycle, copper is concentrated in the clay
mineral fractions with a slight tendency toward enrichment in those
clays rich in organic carbon, and it is notably concentrated in sedi-
mentary manganese oxides with values up to one-tenth of a percent.
Recovery of copper and other metals from ocean and lake floors may prove
more profitable than the recovery of the manganese nodules for which
such commercial ventures were originally designed. More than 99.9% of
the copper carried to the ocean is precipitated, mostly with the clays
and partially with manganese oxides. It is probable that much of the
copper reported in surface waters comes from contamination with metallur-
gic waste from industrial sources.
Factors influencing the relationship between copper in the parent
rock and the derivative soil include the degree of weathering, the
nature and intensity of the soil formation, drainage, pH, oxidation-
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16
reduction potential, and the amount of organic matter in the soil.
Since copper in rocks is likely to be more mobile under acid than
alkaline conditions, the relation of pH to copper mobility in the
environment has been of concern to agriculturalists and biologists.
Alkaline conditions in the soil and surface waters may cause deficiencies
in plants, and minimizie the effectiveness of copper-containing moLlus-
cicides. Conversely, acid conditions may promote availability of cop-
per, increase the concentration of ionic copper, and thereby change the
microorganism and other aquatic animal populations, depending upon
toleramce for various levels of copper in solution. The report of acid
rain occurring in various parts of the world is pertinent to this con-
312
sideration.
The amount of clay in the soil is a key factor in its capacity for
cation exchange and consequent movement or retention of copper. A more
71,152,300
acid pH favors copper availability. The moisture content of
the soil, a key factor in microorganism activity, also influences the
availability of copper. Because of these factors, the concentration
of copper in the soil is subject to considerable variation. The mean
is about 20 ppm and a range of 1-50 ppm occurs in agriculturally pro-
544
ductive soils. Much higher values may be encountered in soils derived
93
from mineralized parent: material.
Because of the variety of conditions that influence its availability,
total copper content of the soil is not an accurate indication of defi-
ciencies or excesses of copper in soil-rooted plants. Copper is much
more available to plants in soils with impeded drainage, because of. the
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microbial activities associated with these conditions. In highly organic
soils such as the peats or mucks, copper uptake by plants is usually low.
It is especially limited in those soils with pH values of 6 and above.
Appropriate weak extractant solutions such as a solution of ethylenediamine-
tetraacetic acid (EDTA; N2[CH2]2[CH COOH] ) may be used to obtain measures of
available copper in soils. Copper deficiency in plants is most marked
where the soils are acid (which permits leaching), low in clay, or high
in organic matter. Copper toxicosis is uncommon in plants, but may be
81
found in areas contaminated by mining or smelting activity.
Special problems, such as additions of copper salts to control
alternate hosts for parasites or algal growth, may exist where ionic
25,393
copper is present in the sea or fresh water. Surface and ground
waters are potential sources of copper imbalances, particularly when
14,147,153,157,310,606,619
added to soils through irrigation or flooding.
Nationally, the ecologic impact of water as a source of copper
is difficult to evaluate because the necessary information is lacking.
Above average levels of copper in water may be either natural or caused
278,287,340,432,447,507,557
by man. Most imbalances are localized near
their point sources, whereas readily available information about the
concentration of copper in water is for streams draining broad regions
2 287,553
of about 10,000 square miles (26,000 km ). Drinking water commonly is a
minor source of copper, contributing less than 2% of the daily dietary
218
intake. For continuous use in irrigation, 1 ppm copper in water
163
has been established as a maximum.
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Discussion of the significance of copper in soils and soil waters
follows in Chapter 3, "Copper in Plants," and Chapter 4, "Copper in Animals."
Such movement is of particular ecologic concern because of the varying
sensitivities of different species of fish. Considerable attention has also
been focused upon copper in animals and in animal wastes because of evalua-
tions made of the movement of copper through the ecosystem from source to
soil. Copper travels through water and to freshwater and marine organisms,
and to plants, animals, and humans. ' Although some attention has been
directtid to human tissue concentrations, ' most research has been
directed toward the effect of sewage and animal and industrial waste disposal.
Animal and industrial wastes (including sewage solids) commonly yield high
concentrations of copper and other trace elements. The current emphasis on
recycling these wastes may unintentionally supply excessive amounts of copper
j ..I. *.u i ^ . ,-u -T 130,310,418,619 _ , .. ..
and the other elements to the soil. Such recycling could
affect consumers indirectly if the yield of crops were reduced or copper were
increased in feed products.
-------
CHAPTER 3
COPPER IN PLANTS
56a
Since 1927 copper has been known to be essential for certain fungi,
and since 1931 for the normal growth and development of green plants. '
Its nutritional essentiality has since been demonstrated for scores of plants
154c
including all of the major agricultural crops. Although the quantitative
requirements of plants for copper are very low--only molybdenum is required
in smaller amounts—there are numerous instances of naturally occurring copper
deficiency. Correspondingly, copper toxicosis is almost never observed under
natural conditions, but may occur on mine spoils or where copper-rich manures
or fungicides have been used excessively.
COPPER PROTEINS
The Blue Oxidases
It is well established that the copper-containing blue oxidases--ascorbate
oxidase, the tree and fungal laccases, and the mammalian ceruloplasmin--con-
tain at least two kinds of cupric copper. Type I is associated with a sharp
electron paramagnetic resonance (EPR) spectrum and an intense absorption in the
visible spectrum around 600 nm, characteristics for which asymmetric coordina-
tion to the protein or charge transfer may be responsible. Type II copper is
associated with a broader EPR spectrum and has no detectable absorption in the
ultraviolet or visible region. Anion binding that inhibits enzymatic activity
will not affect the visible or EPR spectrum of Type I copper, but it will change
the EPR spectrum of Type II copper.
-------
Of the remaining copper atoms in the blue oxidases, some are possibly
cuprous and a third type of multivalent copper may exist. Anaerobic redox
titrations monitored by absorption at 330 nm (present in all of these proteins,
in addition to the absorption in the region of 600 nm) and by EPR have yielded
evidence that these proteins will accept one electron equivalent per copper
atom. The amount of EPR-detectable copper is not sufficient to account for the
number of electrons accepted. At least two alternative interpretations of this
i i i i
observation exist: Cu Cu couple in which the paired electrons preclude
detection by EPR spectroscopy or magnetic susceptibility; or, trivalent copper,
219a
i.e., Cu , which Hamilton has suggested exists in galactose oxidase.
Laccase
Laccase has been known for over 90 years. It was first isolated in 1883
from the milky secretion urushi, or latex, of the Japanese lac tree, Rhus
vernicifera. Laccases have since been found in many plants and fungi.
The animal protein ceruloplasmin shares with laccases the capacity to
catalyze the oxidation of a number of diamines ' "~ and diphenols, includ-
ing catechol (CJ3, [OH] ~) , hydroquinone (C,HfiO.) , and p_-phenylenediamine
(C,H0N') . Diphenols and monophenols, present in coniferyl alcohol, may constitute
DO/
179
the natural substrates of laccase.
In addition to Rhus laccase, laccase from Polyporus versicolor has been
. , , ,. , _ , _t 381,451 , . , 74,154,332,376
widely studied. Both Rhus and Polypqrus laccases contain
four atoms of copper per molecule of enzyme. Both enzymes are characteristically
blue in color, but will suffer loss of color, activity, and copper content by
dialysis against cyanide. The molecular weight of Rhus laccase is approxi-
mately 120,000; that of Polyporus laccase is about 60,000.
For ^ts forms of copper present and their roles in the catalytic activity
of the enzyme, Polyporus: laccase is perhaps the best understood copper enzyme.
-------
The copper system in Polyporus laccase is similar to that of other laccases
327
and serves as the model system for all the blue oxidases.
O Q O
Nakamura has proposed the following equations as representative of
the laccase reaction:
§ 2 4- -)-
2Cu + hydroquinone • - > 2Cu + p_-quinone + 2H
In this "valence shuttle" hypothesis, copper in the oxidized enzyme is reduced
first by one substrate, here hydroquinone, and the reduced enzymic copper then
is oxidized by the second substrate, molecular oxygen; each step requires a
two-electron transfer. Studies with ceruloplasmin have indicated a reaction
sequence in which the substrate is oxidized in a one-electron step. This one-
step reaction leads to a semiquinoid type of free-radical demonstrable by EPR
techniques. The same radical has been shown to be involved in oxidations with
_. i 380 625
Rhus laccase and ascorbate oxidase.
Along with pink cytochrome oxidase, the blue oxidases are the only
oxidases in which water is the product of the enzyme reaction in oxygen reduc-
tion. Enzymes which catalyze the reduction of oxygen to hydrogen peroxide in a
/•Q o c/I O Q/,
two-electron reduction are much more prevalent. ' ' Therefore, the
detailed mechanism for the four-electron transfer in the reduction of oxygen
to water, and the way in which this reaction may be coupled to the one-electron
oxidation of substrate remains an unsolved problem that has been discussed by
327
Malkin and Malmstrom.
On the basis of available data,164'328'331'332 it is highly probable that
the blue oxidases accomplish the four-electron reduction of oxygen to water in
multielectron steps, most likely by double-electron transfers. Such a mechanism
can only occur where cooperation exists between the different electron-accepting
sites present in a single molecule of enzyme.
-------
Ascorbate Oxidase
Ascorbate oxidase is another of the copper-containing blue oxidases.
„, . , . c , . n 4,18,104,183,205,216,217,221,281a,
This enzyme has been found in many plants.
320,343,344,388,412,413,415,448,469,489,589,596 _. . . .
' ' The richest sources are the yellc
crookneck squash (Cucurbita pepo condensa) and the green zucchini squash
(Cucurbita pepo medullosa), from which the most highly purified enzyme samples
have been obtained. ' The enzyme was first observed by Szent-Gyorgyi
in 1928, but it was not: sufficiently purified to justify its classification as
317
a copper protein until 1940. Ascorbate oxidase catalyzes the oxidation of
L-ascorbic acid (C^HgO,;) to dehydroascorbic acid (C^HgOg). It also catalyzes
624
the reduction of cytochrome £ in the presence of ascorbic acid and oxygen,
and is thought to play an important role in the electron transfer system of
plants.132"136'317'511
512
Ascorbate oxidase has a molecular weight of 140,000 and a copper content
132,306,383,534
corresponding to 8-10 atoms of copper per molecule of enzyme.
There are two identical subunits of molecular weight of approximately 70,000,
each of which is composed of two polypeptide chains of approximate molecular
534
weights of 30,000 and 40,000. Copper can be removed easily from ascorbate
424 293
oxidase by dialysis against cyanide to form the inactive, unstable,
copper-free apoenzytne. Copper and activity may be restored to the apoenzyme by
99a 292
forming the holoenzyme " with a molecular weight of about 285,000.
Plastocyanin
Plastocyanin, a ubiquitous protein that contains two atoms of copper per
molecule, plays a significant role in the electron transport system of plants
undergoing photosynthesis. ' ' Its concentration in chloroplasts is
271
comparable to that of cytochrome £. It is involved in the light-drivdi reduct-
ion of oxidized nicotinamide adenine dinucleotide phosphate (NADP ).
10
-------
Vernon et. al. have shown that plastocyanin increases the rate of the
dark-reduction of P700 (a small cytochrome-containing particle extracted from
chloroplasts by Triton X-100) in the presence of ascorbic acid and indophenol
dye.
234
Hind has demonstrated that plastocyanin increases the rate of oxidation
of endogenous cytochrome f_ through photosystem-I. There is an interaction
234 584
between P700 and plastocyanin in both instances. ' A plastocyanin-lacking
mutant of Chlamydomonas requires plastocyanin jLn vitro to mediate the electron
707
'
flow between cytochrome f_ and photosystem-I.
Tyrosinase
Tyrosinase, or polyphenol oxidase, catalyzes the formation of melanin
pigments in many plants and animals through the oxidation of tyrosine
(CgH^NO.,). It also catalyzes the oxidation of several monophenolic substrates,
and p_-dihydric phenols or catechols. It was the first enzyme in which copper
295
was shown to be an essential part of the active molecule.
Tyrosinase displays two activities: catecholase activity, which involves
the dehydrogenation of catechols, and cresolase activity, which involves the
hydroxylation of monophenols. Whether these two activities both involve a
single molecule with different active sites, or two different molecular species,
or whether cresolase activity results from the hydroxylation of monophenols by
the orthoquinoid product of the catecholase activity is a subject of consider-
,, _ 76,280,342
able controversy.
Copper content and activity may be restored to the apoenzyme of tryosinases
• o c~i
by treatment with Cu . ' ' ' The cupric ion added during reconstitution
O Qf\
is reduced to the cuprous state; any excess copper remains cupric.
11
-------
Potato and mushroom tyrosinases (molecular weight 120,000) have been
57,295,329
found to contain four atoms of copper. All of the atoms are
279,281
thought to be in the cuprous, or univalent state.
Amine Oxidases
These enzymes are copper proteins that catalyze the oxidation of
amines, as indicated by the following equation:
0
RCH NH + 0 + HO fr R-C-H + NH + HO.
They generally are pink and are found in diverse plants, as well as in
animals and bacteria. Most of the copper in these enzymes is in the cupric
51,384,
state. A number of reviews have been published about amine oxidases.
630-632
S te1lacyanin
Stellacyanin (Rhus blue protein), like laccase, is found in the
Japanese lac tree, Rhus yernicifera. This copper protein has a molecular
weight of 16,800, and contains one atom of copper per molecule bound to a
399,400
highly asymmetrical site. Stellacyanin contains 20% carbohydrate
422
and 20% hexosamine, in addition to its 108 amino acid residues.
The biologic function of Stellacyanin is not known; it may function as
422
an intermediary electron carrier. It does not possess oxidase activity.
COPPER DEFICIENCY
Copper occurs in plant material at concentrations of 1-50 yg/g dry
weight of tissue; average concentrations are listed in Table 3-1. Except
for molybdenum^ this is the least abundant substance among the known essential plant
33,471a,531a
nutrients. Concentrations below 5 yg/g are a likely sign of
12
-------
TABLE 3-1
Copper Concentrations in Representative Agricultural and
Horticultural Plant Species—
Plant or Plant Part
Analyzed
Alfalfa, aboveground portion
cut for hay
Barley, grain
Beans, field; seed
Beets, root
Cabbage, edible portion
Carrots, roots
Clover, red, aboveground
portion
Corn, grain
Corn, stover
Kale, edible portion
Lettuce, edible portion
Oats, grain
Oats , straw
Onions , bulb
Orange, fruit
Peas, green, edible portion
Potatoes, tubers
Soybean, aboveground portion
cut for hay
Spinach, edible portion
Tomato, fruit
Wheat, grain
Wheat, straw
Number of
Analyses
8
12
12
15
26
15
41
6
16
6
45
29
26
11
3
9
143
32
34
51
108
24
Copper content, ppm
Maximum
15
41
16
27
28
18
20
17
9
56
33
51
54
24
22
15
24
12
24
34
24
5
Minimum
4
6
7
6
4
7
6
4
2
24
3
4
3
5
3
6
2
4
3
8
4
1
(dry wt)
Mean
9
16
11
10
14
11
10
8
5
36
19
11
11
12
10
9
8
9
9
14
9
3
a 33
-Data from Beeson.
13
-------
259a,451a
deficiency. The extremely sparse occurrence of copper corresponds
to low quantitative requirements for the metal, but deficiencies have been
295a,378b,451a
reported.
The limiting concentration of copper in the soil water is of the order
281b
of 0.5 mg/kg, or about 6 mg/kg of water and solids. High concentrations of
humus or heavy additions of lime result in complexing of copper with high-
stability constants and cause the limiting concentrations of copper to be
varied upwards to about: 30 mg/kg. High concentrations of phosphate, manganese or
zinc can also make for potential copper deficiency; the metals apparently
compete directly with copper for transport sites on the plant roots. Soils
containing a great deal of organic matter and brought into agricultural
production for the first time frequently trigger copper deficiency—hence
the term "reclamation disease."* Crops in deficient regions can be suc-
cessfully treated with cupric sulfate (CuSO ), copper ethylenediaminetetraacetic
acid (EDTA; C H NO), copper lignin sulfonates, or copper flavonoids, which
10 16 2 8
usually are incorporated into the soil by spraying. However, the leaves
378b
may be sprayed or the seed may be treated instead. Actual dosages are
378b
about 3 kg copper/hectare.
The amount of copper in plants also depends upon microbial activity
of the soil, pH, oxidation-reduction potential, moisture, rainfall, and of
10,33,165,545,560,574
course, the species of the plant.
Grasses, averaging 5 ppm, tend to be lower in copper than legumes,
which average 15 ppm. Grains (seeds) tend to be higher than leaves or
stems: animals whose diets consist principally of whole grains rarely
*A copper-deficiency disease to which many crops, especially cereals, are
susceptible, usually occurring on newly reclaimed peat land and characterized
by chlorotic leaf tips and failure to set seed.
14
-------
develop copper deficiencies. Regulatory mechanisms appear to limit the
concentration of copper found in plant tissues to about 20 ppm, although
78
higher levels occur under specialized conditions in some plant species
441
and in seeds.
The signs of copper deficiency in fruit trees are known as "exanthema"
or "dieback" and include death of the apical bud, resetting and multiple
bud formation, and chlorosis (yellowing) of the leaf margins. In cereals,
the younger leaves wither, show marginal chlorosis, and often fail to unroll.
The flower heads are dwarfed and the tips are chlorotic and underdeveloped, yet
the lower leaves remain green and bushy. Grain formation is disproportionately
295a,599b
affected.
The extreme susceptibility of bud development and the failure of an
otherwise fairly normal vegetative plant to set seed is symptomatic of
weak translocation of copper from various parts of the plant to the bud.
Similar patterns are observed with several plant nutrients, such as boron;
whereas others, notably nitrogen, are preferentially transported from older
to younger tissues. Nevertheless, the redistribution of copper from leaves
554a
to stem, bulbs, and other storage tissues has been measured.
Although there are no outstanding problems at the whole-organism
level concerning the requirements of higher plants for copper, our under-
standing of the physiologic and molecular bases for these requirements is
unsatisfactory. Nonetheless, numerous examples attest to the beneficial
36
effects of copper on a number of crops.
o
When fertilizer was supplemented with 0.3-0.8 g copper/m , eggplants
6
took longer to bloom, but their yield increased 23%. The application of
cupric sulfate and ammonium molybdate ([NH ] MoO.), (10 and 5 mg/kg soil,
424 404
respectively) to spring wheat increased the yield by 12-27%. Treating
15
-------
it with cupric sulfate before sowing increased the yield of grain, the
fullness of the wheat seedlings, and the percentage of wheat plants surviving
535
to harvest. Cotton seed treated with 0.01% cupric sulfate or soil
application of copper (10-20 rag/kg soil) accelerated the maturation and in-
628 445
creased the yield of cotton and cotton oil. The productive photo-
synthesis of the leaves increased markedly, growth was stimulated, and
289,491
carbohydrates accumulated when apple trees were sprayed with aqueous
copper solutions. Young corn plants increased their rates of photosynthetic
178
oxygen production when copper was added. Treating tobacco seeds with
0.01% cupric sulfate increased the activity of iron and copper proteins
(catalase, ascorbate oxidase, polyphenol oxidase, and peroxidase), and the
445 425 233
yield of tobacco. The yields of legumes and dorset marlgrass
similarly increased when their seeds were treated with 0.01% cupric sulfate.
COPPER TOXICITY
When swine and poultry are raised on diets rich in copper (250 ppm),*
the high level of copper in their manure may significantly increase copper
129,634
in soil. The level required to affect plant growth adversely will
depend upon the content of the clay and organic matter. Sandy soils may
452
reach such a level in five years, whereas silt, loam, or peat soils may
not show adverse effects for 50-100 years. Widespread and repeated usage
of copper compounds as fungicides also has resulted in soil accumulations
143
in certain areas. The application of sewage sludge also affects the cop-
308
per content of soils and of plants growing in them. Leeper has reviewed the
effects of copper upon the microorganisms active in sewage digestion, as well
as the effects of sewage sludge containing up to 8,000 ppm copper upon soils
and the crops grown in,them. The copper did depress microorganism activity,
but recovery was rapid.
*See Chapter 4.
16
-------
CHAPTER 4
COPPER IN ANIMALS
Copper has varied and numerous biologic effects in animals as an
essential element as well as a toxicant. Much of the knowledge of the
metabolism, physiology, and biochemistry of copper in man presented in
Chapter 5 applies to other animals, especially mammals, as well. This
chapter will treat principally those aspects of the animal biology of
copper which differ from the human.
Animal tissues show a wide range of copper concentration. To some
extent, this range reflects the level of copper in the diet, especially
when food with excessively high or low copper content succeeds in de-
feating the animal's homeostatic processes. Whole body copper contents
of most animals on average diets range around 2 ppm in the fat-free
tissues. Highest concentrations are found in liver and brain, with
98,99,118,319,501,
lesser amounts in heart, spleen, kidneys, and blood.
508,573,610
The brain appears to be the only tissue in which copper concen-
484
tration increases with age, approximately doubling from birth to maturity.
An exceptionally high concentration is found in the pigmented portions of
the eye, particularly the iris and choroid, where amounts up to 100 ppm
62,63
dry weight can occur. The amount of copper in serum can range from
5 to 130 wg/100 ml.
The occurrence of disorders related to either a deficiency or an
excess of copper in the United States is considered in detail in this
294
chapter and the next.
17
-------
COPPER DEFICIENCY
The actions of copper at the cellular level generally involve copper
proteins, many of which are enzymes with oxidative functions. Probably
no metal ion is more versatile than copper as a requirement of specific
enzymatic reactions. Tyrosinase, lactase, ascorbic acid oxidase, uricase,
monoamine oxidase, dopamine-$-hydroxylase, and cytochrome oxidase have
573
all been identified as; copper enzymes; indeed, diminished activity of
263
cytochrome oxidase is a sensitive indicator of copper deficiency.
185
Gallagher and Reeve have suggested that an uncomplicated copper
deficiency in the rat first causes the loss of cytochrome oxidase activity.
This loss leads to depressed hepatic mitochondrial synthesis of phospho-
lipids because it interferes with the provision of sufficient endogenous
adenosine triphosphate (ATP; ciQH16N5°12P3^ to maintain an optimal rate
of synthesis. Although copper is involved in many other biochemical
functions, depressed liver phospholipid synthesis is the primary result
of insufficient cytochrorae oxidase.
Severe anemia is a prominent manifestation of copper deficiency in
96,299,573
swine and other animalss. Copper deficiency is first manifested
by a slow depletion of body copper stores, including blood plasma. The
type of anemia associated with copper deficiency is identical to that
96,215a,299,305
caused by iron deficiency. Moreover, animals fed a diet
deficient in copper yet given adequate amounts of iron orally fail to
101,102,215a,215c,304,305
absorb iron and are iron-deficient. Indeed, such
96,304,305
animals even fail to respond to parenteral iron. Their raucosal
epithelial cells, hepatic parenchymal cells, and reticuloendothelial cells
are able to take up iron normally, but they are unable to release iron to
304,305,444,459
the plasma at the normal rate.
18
-------
In pigs, ceruloplasmin appears to be essential for the movement of
405
iron from cells to plasma; and lack of this copper protein accounts
for more than defects in iron metabolism. Retlculocytes from copper-
deficient animals can neither take up iron from transferrin normally nor
synthesize heme (C_,H330,N,FeOH) from Fe (III) and protoporphyrin
611a
(C-,H«,N,0,) at the normal rate. Mitochondria from copper-deficient
animals lack cytochrome oxidase, which apparently is required to reduce
Fe (III) to Fe (II) to provide a pool of Fe (II) as substrate for heme
synthesis. Thus, there are multiple defects in iron metabolism in copper-
deficient animals and the copper enzymes ceruloplasmin and cytochrome
oxidase are intimately associated with the movement of iron.
A variety of disorders in animals and man has been associated with
copper deficienty, but the concentration of copper relative to molybdenum,
zinc, iron, and sulfate (S0,~) is essential in defining the clinical sig-
nificance of its deficiency. Indeed, the ratio of copper to these dietary
components appears to be almost as important as the actual level of copper
in the diet. The pathogenesis of and susceptibility to copper deficiency
are different in ruminant and nonruminant animals, because in ruminants
the interactions of molybdenum and sulfate ion with copper are of primary
importance, whereas in nonruminants the interactions of iron and zinc
with copper are most important.
Ruminant Animals
Disorders associated with a relative copper deficiency in various
ruminants include anemia, depressed growth, bone disorders, depigmentation
of hair and wool (achromotrichia), abnormal wool growth, neonatal ataxia,
19
-------
impaired reproductive performance (fetal death and resorption in rats;
depressed estrus in cattle), heart failure, cardiovascular defects»and
573
gastrointestinal disturbances. Many factors influence the severity
of these dysfunctions, especially species, and even breed or strain charac-
teristics, age, dietary interrelationships, environment, and sex.
Bone abnormalities associated with copper deficiency have been
573
reported in rabbits, nice, chicks, dogs, pigs, foals, sheep, and cattle.
In ruminants, osteoporosis and spontaneous bone fractures are usually
associated with excess dietary molybdenum and a relative copper deficiency,
540
but Suttle et aj.. have presented evidence of the development of
osteoporosis in the offspring of ewes fed diets totally deficient in
copper.
Sheep suffering from simple copper deficiency and/or excess
molybdenum also develop depigmentation of dark wool as well as loss of
147a,573
crimp and quality of their fine wool. In Australia, a syndrome
called enzootic ataxia and in the United Kingdom a condition termed
swayback are probably caused by copper deficiency. Ewes with enzootic
ataxia become anemic. Their wool is stringy and their lambs develop
neurologic problems. Swayback lambs—particularly those less than, a
month old—are severely uncoordinated, ataxic, and usually blind, but
the ewes' wool is normal. Death comes from starvation, exposure, or
263,573 112
pneumonia. Cordy has reported that enzootic ataxia also occurs
in the United States.
Pathologic lesions associated with enzootic ataxia and swayback in
lambs include myelinolysis of the white matter of the cerebrum and degen-
eration of the motor tracts of the spinal cord. The destruction of the
20
-------
white matter may range from microscopic foci to massive subcortical
destruction. Neuronal degeneration as well as demyelination often
112,263
occurs.
The first evidence of cardiovascular disorders caused by copper
37-39
deficiency emerged from studies by Bennetts and co-workers of a
disorder in cattle known as falling disease. Sudden deaths characteristic
of the disease were attributed to heart failure, usually after exercise
or excitement. A similar condition, preventable by copper supplements, also
395,490
has occurred in pigs and chickens, but it has not been reported in
sheep or horses.
In the cardiovascular disorder, there is derangement of the elastic
tissue in major blood vessels, and spontaneous ruptures result. The tensile
strength of the aorta is markedly reduced and the myocardium becomes
friable. The primary biochemical lesion has been described by Hill
232
et^ al. as reduced activity of lysyl oxidase, a copper-containing
enzyme, in the aorta. This reduction in enzymic activity diminishes the
capacity for deaminating lysine (C.-H, ,N,,0-) in elastin and collagen.
Consequently, less of lysine's e-amino groups are converted to an aldehyde
function and cross-linkage in these proteins is diminished, and tensile
strength is reduced.
Apparently cattle are more susceptible than sheep to the combination
70,174a,573
of excess molybdenum and deficient copper in their diet.
When the ratio of copper to molybdenum in feed drops below 2:1, clinical
manifestations attributed to molybdenum poisoning, but just as logically
362
to copper deficiency, can be expected in cattle. This syndrome is
21
-------
manifested by emaciation, liquid diarrhea full of gas bubbles, swollen
genitalia, anemia, and achromotrichia. Prolonged purgation may inhibit
weight gain and cause death. About 80% of the cattle fed this diet
develop molybdenosis; if cases are not treated, the fatality rate
70
may be equally high. Osteoporosis and bone fractures have been reported
573
in prolonged cases of molybdenosis.
Feeding cattle forages and grains grown on soils naturally high in
554
molybdenum and/or low in copper also brings on this condition. In the
United States, such soils have been found in California, Oregon, Nevada,
70,174a,573
and Florida. Cattle grazing in pastures on muck or shale
soils in England, Ireland, New Zealand, and the Netherlands also have
573
suffered severe molybdenosis.*
362
Miltimore and Mason have made an extensive report of molybdenum
and copper concentrations and copper:molybdenum ratios in ruminant feeds.
The overall mean copper:molybdenum ratio of all feeds—legume hay, grass
hay, sedge hay, oat forage, corn silage, and grains—was 5.7:1 in British
Columbia. The copper:molybdenum ratio in sedge hays was 2.1:1, near the
critical ratio of 2:1; that is, a ratio of less than 2:1 copper:molybdenum
will bring on copper deficiency in cattle. The mean ratio of other hays
was 4.4:1, and the ratio for other feeds was over 5:1. Molybdenum levels
were generally low—often less than 1 ppm—and the highest concentration
reached 9.9 ppm. Most copper levels were below 10 ppm.
*See also Chapter 2.
22
-------
When the copper levels of feed or forages are normal (ranging from
8-11 ppm), cattle are generally resistant to molybdenum poisoning from
feed containing levels as high as 5-6 ppm. Sheep can resist levels up
to 10-12 ppm. But when the dietary copper level falls much below 8 ppm,
even 1-2 ppm molybdenum may be toxic to cattle. Increasing the copper
level in the diet to 13-16 ppm will protect cattle against 150 ppm
147a,573
molybdenum. As noted, the critical ratio of a normal diet—
one with 8-11 ppm copper and 1-2 ppm molybdenum—is 2tl. However, the
addition of as little as 5 ppm copper to the diet will protect cattle
from up to 150 ppm molybdenum if adequate sulfate ion is present. In
certain areas of the United States, such as Florida and states west of
the Rocky Mountains, it is not uncommon to find molybdenum-induced
39a,107a,112
copper deficiencies in cattle and occasionally sheep,
Copper deficiencies in plants and animals are unusual in most areas east
of the Rocky Mountains, Because there is no national program for reporting
cases of copper deficiency in animals, it is not possible to define the
extent of this problem.
Nonruminant Animals
Copper deficiency in nonruminant animals will result in anemia,
aortic rupture, bone deformation and reduced calcification, cerebral
573
edema, cortical necrosis, achromotrichia, and fetal absorption. The
levels of ceruloplasmin and copper in serum and of cytochrome oxidase
and copper in tissues all decrease in animals fed a copper-deficient
152b,251,409,444
diet.
Nonruminants are also more tolerant of excessive levels of molybdenum
than ruminants. Swine appear to be the most tolerant of the nonruminants.
130
Davis reported that a diet containing 1,000 ppm molybdenum fed to swine
23
-------
for 3 mo had no ill effects on them. In pigs, the storage of copper in
the liver does not appear to be influenced by the level of molybdenum in
227,284,285
the diet.
However, excess molybdenum in rats may cause symptoms similar to
those produced by a copper deficiency, and, as in ruminants, the level
of molybdenum required to produce toxicosis depends upon the amount of
391 211
copper in the rat's diet. Neilands et al. and Gray and Daniel
have demonstrated that growth and hemoglobin ([C7«,,H-. 1 (if.^e^2Ci^26^2^ 4 ^
levels of rats on low copper diets can be reduced by feeding them 100
ppm molybdenum. When the diet contains adequate amounts of copper,
500-1,000 ppm molybdenum are required to cause such effects. In rats,
copper levels in the blood and liver tend to increase when molybdenum
108
is added to their diet,,
Supplemental L-ascorbic acid (C HQ0 ) has been demonstrated to
6 8 6 192a 251
aggravate copper deficiency in chicks, swine, and rabbits. Hunt et al.
found that 0.5% dietary ascorbic acid reduced hepatic copper levels and
590
increased deaths caused by ruptured aortas. Voelker and Carlton have
reported that 2.5% ascorbic acid in the diet of swine intensified symptoms
of copper deficiency.
18a,385a
Numerous reports have indicated that swine fed diets high in copper
(up to 250 ppm) during the 8 wk of the early post-weaning period increased
their daily weight gains. However, continuous feeding at these levels
did not affect overall rate of gain, or feed-gain ratios. Copper stores
in the liver increased linearly with increasing dietary copper, but re-
moval of the added dietary copper reduced hepatic copper content.
24
-------
Significant differences were discovered from location to location,
suggesting that genetic and environmental factors may contribute to the
differences observed in swine. The action of the high level of copper
in promoting early rather than later growth in the animals fed it, resembles
the action of broad spectrum antibiotics. Perhaps the copper is exerting
87,88
a selective antimicrobial action in the intestine.
The difficulty in evaluating the effects of feeding swine high
levels of copper was revealed by North Central Regional Committee Re-
385a
port. Feeding 125-250 ppm copper for up to 8 wk after weaning caused
an average increase in daily gain over no added copper, although wide
variations did occur from state to state.
Such variations resemble those reported for the use of wide spectrum
antibiotics like Aureomycin (C00H00ClN000.HC1). When pathogen levels
2.2. 2-j 2. o
were significant in the environment, addition of the antibiotic signif-
icantly improved the daily rate of gain. When the environment was
117a
relatively free of pathogens, response to antibiotics was minimal.
Although tests by the Food and Drug Administration (FDA) failed to
demonstrate its effectiveness, widespread use of poultry rations sup-
plemented with 250 ppm copper sulfate (CuSO,) is generally believed to
574a
prevent or cure crop mycosis (moniliasis caused by Candida albicans).
When used in conjunction with antibiotics, the net effect of copper
59a 498a
sulfate is to reduce poultry growth rate. '
As with swine, results vary when high levels of copper are fed to
poultry. A level of 240 ppm copper given turkey poults encouraged
5a
growth in Iowa, 60 ppm copper in the diet of chicks in Ontario was
498a
without effect, and 125 ppm in the diet of young turkeys depressed
223b
their growth.
25
-------
COPPER TOXICITY
Copper toxlcity and interactions of copper with other trace elements
present complex and significant consequences for animal husbandry in the
United States. Dieta.ry imbalances of copper and molybdenum may result
from either ad libitum consumption of mineral mixtures, or of conventional
58,82,
feeds that have been fortified with inappropriate mineral mixtures.
85,290,291,543
Up to 15 ppm copper is generally recognized as safe (GRAS)
as a livestock feed ingredient by the FDA, whereas molybdenum is not.
Therefore, copper is routinely and ubiquitously added to commercial trace
element mixtures used in livestock feeds, whereas molybdenum is prohibited.
Unfortunately, these regulations do not recognize species differences
between cattle and sheep in their requirements for a balance between cop-
per and molybdenum. Cattle can tolerate mineral mixtures and feeds with
added copper and without molybdenum, even when their natural grain and
forages contain adequate levels of copper. In contrast, sheep are suscep-
tible to the toxic effects of added copper, especially when the natural
forage contains adequate levels of copper and low levels of molybdenum.
Since the cattle-feeding industry is of major economic importance, and
the sheep-feeding industry is not, it has not been economically feasible
for manufacturers of livestock mineral mixtures to provide special formu-
lations for sheep with the proper balance between copper and molybdenum
(6-10 parts copper/1 part molybdenum).
Copper toxicoses in sheep are not rare in the Midwest and Great
Plains states. They extend northward well into Canada. Because the
levels o. copper found in plants vary greatly and depend upon many factors,*
*See Chapters 2 and 3.
26
-------
no general geographic distribution of copper and molybdenum levels in
plants has been mapped. However, it appears that grains and forages
grown in the upper Midwest and Great Plains states contain sufficient
copper and are low enough in molybdenum content to make the addition of
the GRAS 15 ppm copper to the total diet of sheep responsible for ex-
cessive accumulation of copper in the liver. In these areas^ 1-5% of
sheep consuming such feed develop hemolytic crises. Sheep may develop
copper toxicosis on a diet containing a normal concentration of copper
84,238
(8-10 ppm) if the molybdenum levels are below 0.5 ppm. When a
vitamin-mineral preparation containing copper but not molybdenum is
added to a ration, the copper concentration of the ration may be elevated
to 25-30 ppm or more. Since the natural molybdenum concentration in
most feedstuffs is usually below 2 ppm, the copper:molybdenum ratio in
the resulting diet is greater than 10:1. Over 20 episodes of chronic
82
copper toxicosis in sheep were found in Iowa from June 1968^June 1970,
especially in feeder lambs, show lambs, and ram lambs being tested for
weight gain and feed efficiency (unpublished data, W.B. Buck).
387a
These problems could be solved economically by computerized on-
line feed-forward control of feed mills. For example, on-line analysis
of incoming ingredients for their copper, molybdenum, sulfate, iron and
zinc contents would produce data from which to calculate the trace elements
necessary to provide the proper levels and balance in feeds for each species.
Adding excess copper to sheep, swine, and poultry feeds may create
a hazard to the consuming public because the metal accumulates in the
animal liver.* Sheep fed a diet with copper and molybdenum imbalances
*See Chapter 5.
27
-------
have accumulated an average of 1,600 ppm copper in the liver tissue on a
5
wet-weight basis. In some instances, accumulations have run as high as
1,564
3,000 ppm. Pigs fed rations containing 250 ppm copper had a mean
hepatic concentration of 220 ppm on a dry-weight basis, as compared to a
mean of 24 ppm in animals not receiving the added copper.* Even higher
levels of copper may accumulate in livers of swine if their dietary levels
of iron and zinc are inadequate. Using sheep, swine, and poultry livers
from animals fed such diets in human consumption could be deleterious,
especially in the preparation of baby food. Baby foods made from liver
containing 550 ppm copper (wet wt) would contain 15 mg copper/1 oz (28 g)
serving. This level of copper is 5-10 times the daily dietary requirement
15 4b
for girls aged 6-10.
Other causes of copper toxicosis in ruminants include:
• consumption of plants contaminanted by copper-containing pesticides
used to spray orchards, such as a Bordeaux mixture with 1-3% cop-
per sulfate;
• use of copper sulfate to control helminthiasis and infectious
pododermatitis in cattle and sheep;
• contamination of soils and vegetation in the vicinity of mining
and refining operations;
» use of calcium-copper ethylenediaminetetraacetic acid (EDTA;
N2[CH2]2[CH2COOH]^) as an injectable source of copper in countries
where sheep frequently are subject to copper deficiency problems;2"5^'255 >2~
and
1,65,558
• confining sheep that have no access to green forage con-
taining sufficient molybdenum to prevent excessive accumulation
560
of copper in the liver.
*See Chapter 5.
28
-------
In western Australia, sheep grazing on pastures containing various
species of Lupinus develop hepatic toxicosis from lupine alkaloids more
186,187
readily in the presence of excess copper. Susceptibility to cop-
per poisoning in sheep may be enhanced by the forage. Thus, in Australia
and New Zealand, plants of the Heliotropium, Echium, and Senecio genera
contain pyrrolizidine alkaloids that cause hepatic necrosis; animals
grazing on these plants will be unable to metabolize and excrete normal
32,86,465,573
dietary levels of copper.
Toxic Interactions
Before discussing the pathologic physiology of copper toxicosis, it
is essential to review the mechanism of interaction of copper with
molybdenum, sulfate, iron, and zinc. Evidence exists that copper and
152c,152d,249
molybdenum form an in vivo complex with a molar ratio of 4:3,
which may not prevent intestinal absorption of copper, but does inhibit
copper accumulation in the ruminant liver.
Copper and molybdenum, especially in ruminants, appear to interact
120,148,149,238,337,559
with inorganic sulfate in the diet, affecting
149
biliary and urinary excretion of copper and molybdenum. Dick reported
that increased urinary excretion of molybdenum occurs with increased con-
337,338
tent of inorganic sulfate in the diet, and Marcilese et^ a^. found
that increased dietary levels of molybdenum and sulfate result in more
249
urinary and biliary excretion of copper. Huisingh and Matrone found that
molybdate inhibited the reduction of sulfate to sulfite, and that copper
reduced this inhibition greatly. Molybdate inhibition of sulfate reduction
increase.! as the concentration of sulfate decreased.
29
-------
Copper added to sheep diets in the. form of the sulfate may he less toxic
than copper added as the acetate (C,HgCuO, .I^O), oxide (Ci^O), carbonate
(CuCO,..Cu[OH]0.H00), gluconate (Cn 0H99Cu01x .H90), iodide (Cul) , chloride
J 2. 2 \l L2. LH I 82,84,56
(CuCl2), orthophosphate (Cu [PO,]„.SH^O), or pyrophosphate (H^P207.2Cu).
The first clinical evidence of the relation between copper and
molybdenum metabolism was obtained when it was learned that teart, the
drastic scouring disease of cattle—thought to be a manifestation of
chronic molybdenum poisoning—could be controlled by treating the cattle
169a,573 149a
with large amounts of copper. Then Dick and Bull reported
that molybdenum was effective treatment for copper poisoning in sheep.
Subsequent investigations have characterized these interactions in
1,120,147a,148,1*9»152c,152d,204,247,290,291,337,430,462,558,559,573
ruminants.
The effects of interactions of copper, molybdenum, and sulfate are
m 193 227
much less marked in the nonruminant. Gipp et al. and Hays and Kline
were unable to demonstrate any effect of molybdenum and sulfate on the
storage of copper in the liver by pigs fed varying levels of copper.
122
Dale observed similar results, although he found that ceruloplasmin
levels were depressed when sulfate was added to swine diets containing
about 10 ppm copper.
Zinc and iron affect copper metabolism in nonruminant animals,
especially swine. Both elements protect swine from the adverse effects
223,456,541,542
of high levels (250-750 ppm) of dietary copper, and
542
deficiency of zinc and iron tends to intensify copper toxicosis in swine.
In rats, copper was a prophylactic against the anemia and reduced activities
of catalase and cytochrome oxidase in the liver associated with zinc
210,580
toxicosis.
30
-------
PATHOLOGIC PHYSIOLOGY OF COPPER TOXICOSIS
Ruminant Animals
Copper bichloride is 2-4 times more toxic than copper sulfate and
sheep are poisoned by 20-100 mg/kg single dose. Signs of acute poisoning
by large oral doses of a copper formulation are vomiting, excessive saliva-
tion, abdominal pain, and diarrhea (fluid, greenish-tinged feces). Collapse
and death follow within 24-48 h.
When sheep consume small but excessive amounts of copper over a
period of weeks to months, particularly when the copn^r "nolybdenum ratio
is greater than 10:1, no toxic signs will be manifested until a critical
level of copper—3-15 times the normal level, or about 150 ppm (wet wt)—
is reached in the liver. Suddenly, the animal becomes weak, trembles, and
loses its appetite. It usually develops hemoglobinuria, hemoglobinemia,
and icterus. Occasionally, an animal will only show pale mucous membranes,
539
and not icterus and hemoglobinuria. Although morbidity is usually
less than 5%, the mortality of the affected animals is usually over 75%.
The hepatocytes may exhibit cytoplasmic vacuolation and necrosis.
All lobules may contain clusters of dead cells with fragmented nuclei and
263
acidophilic cytoplasm. Fibrosis begins early and is distributed portally.
The kidney tubules are clogged with hemoglobin; accompanying degeneration
and necrosis of the tubular and glomerular cells occurs. The spleen is
crowded with fragmented erythrocytes, and status spongiosus in the white
257
matter of the central nervous system has been reported.
Morphologic and histochemical changes occur in sheep when copper
257
accumulates in their livers. In biopsies taken 6 mo before the hemolytic
31
-------
crisis, swelling and necrosis of isolated hepatic parenchymal cells have
been noted, together with swollen Kupffer cells rich in acid phosphatase
and containing para-aminosalicylic acid (PAS; C.,H,NO.,)-positive, diastase-
resistant material and copper. Various increases in liver-related serum
enzyme activities have been recorded 6-8 wk before the hemolytic crisis.
These enzymes include serum glutamic oxaloacetic transaminase, lactic
dehydrogenase, sorbitol dehydrogenase, arginase, and glutamic dehydrogenase.
256,257,322,461,562,564,578
The increased serum activities of these
enzymes often subside to nearly normal levels 1-2 wk before the hemolytic
crisis, but very high levels of activity occur shortly before or during
the crisis. It is important to note that these elevations are not cor-
related with increases of copper levels in the blood, which only occur
shortly before and during the hemolytic crisis and therefore are of no
347
diagnostic value before the animals fall sick.
During the hemolytic crisis, the activities of hydrolytic adenosine-
triphosphatase, nonspecific esterase, and succinic dehydrogenase are
256,257,428
reduced.
562,563
Todd and Thompson, working with sheep, reported a marked
reduction of glutathione (C H N 0 S) concentration and an accumulation
of methemoglobin in the blood in the hemolytic crisis of copper toxicosis
resulting from sudden release of copper from the liver. Death may re-
sult from blockage of the kidneys by hemoglobin and subsequent kidney
failure.
32
-------
Nonruminant Animals
Toxic levels for ruminants (20-50 ppm) are well tolerated by non-
ruminants. Dietary levels in excess of 250 ppm are required to produce toxi-
64,541,542,599
cosis in swine and rats. Copper toxicosis in nonruminants may not
cause rapid destruction of red blood cells—the hemolytic crisis—although
jaundice has been observed in pigs fed toxic levels (250-500 ppm) of
573,599 361
copper. Milne and Weswig have shown that sheep accumulate cop-
per in the liver in proportion to the dietary intake, whereas rats maintain
normal hepatic copper levels until a diet extremely high in copper is
reached (1,000 ppm).
Studies with rats and mice injected with copper compounds have shown
22,196,302
that copper accumulates in liver lysosomes. Some researchers
have postulated that acid hydrolases capable of producing cellular injury
313,314,582
are thereby released, thus causing hepatic damage. Conversely,
it has been demonstrated that high concentrations of copper in the toad,
Bufo marinus, are localized to liver lysosomes and are made innocuous
198
because of this localization.
Copper levels of 125-250 ppm in the diet of swine increase the un-
138,154a,549
saturation of depot fat so that it turns soft.
355a 503
Poultry resist copper toxicosis better than most mammals. Smith
fed copper sulfate to day-old chicks for 25 days at zero, 100, 200, or
350 ppm copper concentrations in a basal ration containing 10 ppm copper.
Chicks on the 100 ppm copper diet increased slightly in daily gains,
whereas those on the 350 ppm diet showed a slight but statistically signif-
195
icant reduced weight gain. Goldberg et al. gave copper acetate in
33
-------
capsules to adult chickens (weighing 1.8 + 0.25 kg) at a rate of 50 mg
copper per chicken/day for 1 wk, 75 mg/day for a second week and 100 mg/
day until anemia or toxicosis appeared or death occurred. After 2-6 wk
of being dosed with copper, the birds became weak, anorectic and lethargic.
Eight out of 23 developed anemia concomitant with toxicosis, perhaps
because erythrocytes were destroyed in the liver by exposure to copper.
Turkey poults have been reported to tolerate up to 676 ppm dietary copper
sulfate for 21 days, but growth was depressed when fed 910 or 1,000 ppm
594 611
copper, and signs of toxicosis appeared at 1,620 ppm. Wiederanders
tried to produce copper toxicosis in turkeys by injecting copper sulfate
subcutaneously. He injected 0.5 mg per bird for 84 days, and elevated
the dose to 5.0 mg per bird for an additional 17 days, yet copper toxicosis
was not produced. He concluded that turkeys and perhaps other fowl have
metabolic and excretory pathways for copper different from those of mammals
and pointed out that ceruloplasmin did not increase in the turkeys injected
with copper.
Extensive studies of acute and chronic copper toxicosis in chickens,
442,443
pigeons, and ducks were conducted by Pullar. He found the minimum
lethal dose (MLD) of copper for these species to vary from 300-1,550 on
a mg/kg body weight basis, depending on the form of copper fed. The
maximum daily intake of copper carbonate tolerated by chickens was 60
mg/kg body weight; mallards tolerated 29 mg/kg body weight. It was not
possible to produce poisoning in chickens given copper sulfate in drinking
water at 250 ppm (1:4,000 dilution of copper sulfate in drinking water),
and no obvious signs of copper poisoning were observed in mallards con-
suming 250 ppm copper sulfate in their feed.
34
-------
Numerous studies in rats and mice have been conducted in an effort
to learn more about hepatolenticular degeneration (Wilson's disease) in
22,301,302,313,314,355,582,591,617
humans. Prolonged daily intraperitoneal
injections of as little as 0.3 mg copper/kg body weight will elevate hepatic
301,302
copper levels. There is indication that an increase in hepatic cop-
per occurs without saturating the rat's excretory capacity. Copper levels
in the kidney also increase with copper exposure, but this seems to be
301,302
unrelated to liver storage. Both hepatic and renal necrosis
observed in rats and mice are linked to increased copper levels. '
However, there is no apparent deposition of copper in the brain, skeletal
and cardiac muscle, or skin, and only transient elevations of copper are
301,302
found in bone following copper exposure.
Very recently, Hardy et al. have described a form of hepatic cirrhosis
223a
in Bedlington terriers that has striking similarities to Wilson's disease.
The disorder appears to be hereditary and autosomal, and the histologic and
functional abnormalities of the liver are extraordinarily like those seen
in man. The concentrations of hepatic copper found in these terriers
exceed 10 mg/g (10,000 ppm) dry liver, to be compared to 0.25-3.0 mg/g in
patients with Wilson's disease and normal levels of less than 0.1 mg/g in
humans and dogs. Although this disorder is fatal to the dogs, Kayser-
Fleischer rings and neurologic dysfunction have not been observed so far.
Aquatic Organisms
Copper is poisonous to many aquatic organisms. It may reach toxic
levels either from mining or industrial operations, influxes of copper-
containing fertilizers, or use of copper salts to control aquatic vegetation
35
-------
or mollusks. Desalinization plants may cause local excessive concen-
trations of copper in the ambient salt water because their effluent is
hot, hypersaline, and of low pH—all of which are conditions that will dis-
solve the metal in the copper pipes or vessels through which the waste
flows. Toxic concentrations are also functions of the species, the age
of the individual organism, the concentrations of mineral and organic
material, temperature of the water, and whether the copper is ionic or not.
In fresh water, acute toxicosis in fish is unusual if the concen-
tration is below 0.025 ppm. (The accepted standard for drinking water
153
is 1.0 ppm.) In soft fresh water, however, 0.01-0.02 ppm has been
260,261,429,438
found to be toxic.
As exposure time is lengthened, the minimal toxic concentration
diminishes. The 48-h LC,-n (lethal concentration for 50% of the animals)
79
in rainbow trout (Salmo gairdnerii) has been found to be 0.67-0.84 ppm.
The 96-h TL (median threshold limit, killing 50% of test animals in 96 h)
m 396
of copper for blue gills (Lepomis macrochirus) is reported as 0.24 ppm,
although levels over 0.01 ppm alter oxygen consumption. The 10-day LCcQ
of copper for brook trout (Salvelinus fontinalis) is about 0.05 ppm.
228
Chinook salmon eggs can withstand 0.08 ppm, but the fry exhibit: acute
toxicosis at Q.04 ppm, and even 0.02 ppm copper inhibits their growth and
increases mortality.
Relatively few data are available on more chronic exposure of fish
377
to copper. Mount found that fathead minnows (Pimiphales promelas)
exposed to copper for 11 mo did not show impaired growth or reproduction
at 3-7% of the 96-h TL]n of 0.43 ppm. That is, they were unaffected by
0.02 ppm. Minnows are unaffected by 3 times this concentration of copper
36
-------
378
if the water has an EDTA hardness of 30 mg/1 as calcium carbonate (CaC03).
14
In surprising contrast, Arthur and Leonard found that 8 to 14.8 ppm cop-
per had no effect on fish after 6 wk in soft water, but this tolerance
might have been a phenomenon of species difference.
Copper pollution of waters has significant effect on marine inver-
447
tebrates. Copper concentrations of 0.1 ppm are acutely toxic to nereis.
Perhaps most important is the sensitivity of some species of phytoplankton
14,25,
whose photosynthesis can be inhibited by as little as 0.006 ppm copper.
157,212,225,334,392
In certain species of algae, copper appears to be as
toxic as mercury (personal communication, L. Kamp-Nielsen).
The cupric ion appears to be the toxic agent for marine invertebrates,
and fish may be protected from copper toxicity by chelating agents like
447,506
EDTA and nitrilotriacetic acid (NTA; N[CH2COOH]3). To protect fish,
NTA must be used in a molar concentration at least three times that of
the copper, and EDTA must be at least six times the amount of the element.
Since these agents become biodegradable quickly in natural waters, they
would be used chiefly as temporary treatments in unusual circumstances.
For example, chelating agents could render a slug of toxic copper innocuous
as it passed through a critical section of river.
The effectiveness of copper as a molluscicide to control schisto-
somiasis depends upon the lethal dose (LD). The LD is the product of con-
centration and time, usually expressed as parts per million-hours (ppm-h).
As the time of exposure increases, the lethal concentration of the com-
pound decreases disproportionately, so that the lethal dose also decreases.
For example, the LDg^ is 80 ppm-h when an organism is exposed to copper
sulfate for 1 h, and only 14 ppm-h when exposed for 24 h.
37
-------
Various copper compounds have been used as molluscicides since the
1920's, when copper sulfate was introduced for this purpose. Other com-
pounds include copper pentachlorophenate ([Cx-HCl,-0]2Cu) >
copper tartrate (CuC^H^Og), copper carbonate (Cu2C03), copper ricinoleate
if
([C,gH3,03]2.Cu), copper resinate, cuprous oxide (Cu20), copper
dimethyldithiocarbamate ([CoH6NS2]2Cu), cuprous chloride (CuCl), copper
3-phenylsalicylate ([C13H1()03]2.Cu), copper (II) acetylacetonate
([C5H702]2Cu), and copper acetoarsenite (Paris green) (3Cu[A502]2.Cu[C2H302
Most of these compounds are effective in two quite different ranges of
concentration, depending on whether the treated water is clear—when they
are effective at concentrations as low as 1 ppm—or turbid or muddy. In
the latter case, mud seems to bind the copper or its compounds, and reduce
its toxicity to the snail, except when the animal actually ingests the
copper-mud complex.
Attempts have been made to erect a "chemical barrier" molluscicide
by continuously injecting very small concentrations of copper (0.1-0.3 ppm)
147,157
into the river or lake. Results have been varied and unpredictable.
Although other molluscicides have been developed to control schis-
tosomiasis, copper—either in the ionic form or in one of the organic
326
compounds listed above—remains a primary agent for this purpose.
*0f indeterminate composition.
+Copper pentachlorophenate is now known as bis(pentachlorophenolato) copper,
copper ricinoleate as ricinoleic acid, copper (II) salt, copper
dimethyldithiocarbamate as bjL§(dimethyldithiocarbamato) copper, copper
3-phenylsalicylate as 3-phenylsalicylic acid, copper II salt, and copper
(II) acetylacetonate as bi,s (2,4-pentanedionato) copper.
38
-------
CHAPTER 5
HUMAN COPPER METABOLISM
DIETARY SOURCES
Copper is essential and generally considered a trace element, and
almost every diet supplies a relatively large amount of the metal for
476
body needs. Indeed, it is difficult to prepare an acceptable diet
which contains less than 2 rag of copper daily, and a single day's diet
may contain 10 mg or more. The copper content of selected foods and
beverages is listed in Table 5-1.
Oysters, liver, mushrooms, nuts, and chocolate are particularly
rich in copper, but their copper content, like that of other foods,
varies with the copper content of the soil or water in which they or
574
their animal origins are grown. Consequently, quantitative data
differ widely from one tabulation to another. For example, one source
indicates that kidney beans have almost no copper per 100 g edible
150 318
portion, whereas another reports a copper content of 0.11 mg/100 g.
The copper content of raw lobster in one tabulation is given as 2.2 mg/100 g,
yet the next entry indicates that canned lobster has less than 1 mg/100 g.
39
-------
TABLE 5-1
Copper Content of Selected Foods and Beverages-
Food
Fruits, Fruit Juices
Apples, sweet, fresh
Apricots, fresh
Dried
Avocados, fresh
Bananas, fresh
Blackberries, fresh
Blueberries, fresh
Cantaloupe, fresh
Cherries, fresh
Cranberries, fresh
Currants (red), fresh
Dates, dried
Figs, fresh
Dried
Gooseberries, fresh
Grapes, fresh
Grape juice
Grapefruit, fresh
Lemons, fresh
Olives, green
Oranges, fresh
Copper, mg/100 g edible portion—
0.08
0.12
0.4
0.4
0.2
0.12
0.11
0.04
0.07
0.09
0.12
0.21
0.06
0.4
0.08
0.1
0.02
0.02
0.26 (0.04)
0.46
0.07
40
-------
TABLE 5-1 - continued
Food Copper, mg/100 g edible portion—
Orange juice, fresh 0.08
Peaches, fresh 0.01
Dried 0.3
Pears, fresh 0.13
Pineapple, fresh 0.07
Plums, fresh 0.3
Raisins, dried 0.2
Raspberries, fresh 0.13
Strawberries, fresh 0.13 (0.02)
Tangerines 0.1
Watermelon 0.07
Vegetables
Asparagus, fresh 0.14
Beans
Kidney, fresh — (0.11)
Lima, fresh 0.86
Beets (beet roots), peeled, fresh 0.19
Cabbage (red or white), fresh 0.06
Carrots, fresh 0.08
Cauliflower, fresh 0.14
Corn (sweet), fresh 0.06
Cucumbers, fresh 0.06
Dandelion greens, fresh 0.15
Eggplant, fresh 0.08
Kale, fresh 0.09
Kohlrabi tubers 0.14
41
-------
TABLE 5-1 - continued
Food
Lentils, dried
Lettuce, fresh
Onions, fresh
Parsley, fresh
Parsnips, fresh
Peas, green, fresh, unripe
Dried, split
Peppers (green), fresh
Potatoes, raw
Pumpkins, fresh
Radishes, fresh
Rhubarb, fresh
Soybeans, dried
Spinach, fresh
Sweet potatoes, fresh
Tomatoes, fresh
Turnips, fresh
Greens
Watercress
Nuts
Almonds, dried
Brazil
Chestnuts, fresh
Coconuts, fresh
Copper, mg/100 g edible portion~
0.7
0,07
0.13
0.21
0.10
0.23
0.80
0.11
0.16
0.08 (0.03)
0.13
0.05
0.11
0.20
0.15
0.10
0.07
0.09
0.04
0.14
1.1
0.06
0.32
42
-------
TABLE 5-1 - continued
Food
Hazelnuts
Peanuts, roasted
Pecans
Walnuts
Cereals, Cereal products
Barley, pearled
Cornflakes
Flour, buckwheat
Rye, dark
Wheat, whole
White, unenriched
Oatflakes
Rice, polished, raw
Wheat germ
Sugar
Dextrose, anhydrous
Honey
Sugar cane or beet, white
Fats
Lard
Olive oil
Dairy products, Eggs
Eggs, whole, raw
Egg yolk, raw
Copper, mg/100 g edible portion
1.35
0.27
0.31
0,4
0.17
0,7
0.2
0.74
0.06-0.19
1.3
(0,12)
0.2
0.02
0.07
0.03
0.02
43
-------
TABLE 5-1 - continued
b_
Food Copper, mg/100 g edible portion
Milk (cow's) pasteurized, whole 0.01
Nonfat
Human breast milk 0.05
Goat's milk 0.04
Meat, Poultry (raw unless otherwise stated)
Bacon, fat, salted
Beef, brain 0.2
Kidneys 0.35
Liver 2.1
Calf, liver 4,4
Duck, medium fat 0.4
Goose, medium fat 0.3
Pork, loin or chops, cooked — (0.09)
Turkey, medium fat 0.2
Fish, Seafood (raw unless otherwise stated)
Cod 0.5 (0.10)
Flounder 0.18
Haddock 0.23
Halibut 0.23
Lobster, 2.2
Canned
Mackerel 0.16
Oysters 3.6
Salmon (Atlantic) 0,2
Scallops
Shrimp 0.4
44
-------
TABLE 5-1 - continued
a 150 318
Derived from Geigy Scientific Tables and Low Copper Diet.
b
In instances where copper content given by Geigy Scientific Tables
differs by more than 50% from the value supplied by Low Copper Diet,
the value from Low Copper Diet is included in parentheses.
45
-------
PATHWAYS FOLLOWED BY DIETARY COPPER
Studies on man using radioactive copper-64 (physical half-life, 12.8 h)
or copper-67 (half-life, 61.8 h) indicate that about half of dietary copper
514,532
is not absorbed, but excreted directly in the feces. When the
amounts of separately administered oral and intravenous 2 mg doses of
radioactive copper incorporated into the plasma copper protein, ceruloplasmin,
were compared, the average absorption in 49 normal subjects was found to be
514
40%. In another study, average absorption was 56% (40-70%) when the
retention of 0.4-4.5 mg orally administered copper-64 was compared to the
532
retention of simultaneously injected copper-67. When 100 mg nonradio-
active copper was administered an an emetic to children, about 30 mg was
243
absorbed, suggesting that the fraction absorbed from an oral dose of
copper decreases little1 as the dose increases.
The form and mechanism by which copper is absorbed and transported
by the intestine are unknown. After entering the epithelial cells, it is
160,510 158
taken up by a cytosol protein similar to the metallothionein of
liver. Recent studies of copper absorption in infants suffering from an
126
inherited, X-linked defect (Menkesfe disease, discussed below) suggest
that the transfer of copper from intestinal cells to plasma involves an
125
active process.
Impaired copper absorption occurs in severe, diffuse diseases of the
91,518
small bowel, including sprue, lymphosarcoma, and scleroderma. Copper
deficiency in plasma generally follows, but is of little, if any, clinical
significance in the face of the multiple nutritional abnormalities symptomatic
of these illnesses. The low serum concentrations of ceruloplasmin and copper
are easily corrected with successful treatment of the underlying disease.
46
-------
Immediately after it is absorbed, copper is transported in plasma bound
30 387,470
to albumin and perhaps to amino acids. Almost all of the copper
401,406,519
is soon deposited in the liver where about 80% of it is found in the
335
cytosol bound to three proteins—hepatocuprein, copper-chelatin or
370 158
L-6-D and metallothionein (L-6-D). The remaining 20% is incorporated
into other specific copper proteins like cytochrome c^ oxidase, or is se-
199,528
questered by lysosomes.
Despite continued deposits of dietary copper, the hepatic concentration
of the metal does not increase with age in man. The secretion of some copper
into the blood following its incorporation into ceruloplasmin during hepatic
519 528
synthesis and the excretion of copper from the lysosomes into the bile
maintain this constant concentration. About 150-300 mg of ceruloplasmin
520
containing 0.5-1.0 mg of copper is catabolized daily in the adult's liver
and about 30 mg ceruloplasmin, containing 0.1 mg copper, is excreted into
598
the intestine. Biliary copper may not be available for reabsorption
201
because of its binding to a protein. The rest of fecal copper comes
477
from copper in salivary, gastric, pancreatic, and jejunal secretions,
along with any portion of dietary copper that has not been absorbed. Table
5-2 sets forth the copper content of various human tissues and body fluids.
47
-------
TABLE 5-2
Copper Content of Human Tissues and Body Fluids—
mg Copper/Whole Organ
Tissues
Adrenal
Aorta
Bone
Brain
Breast
Erythrocytes
(per 100 ml
packed red
blood cells)
Hair
Heart
Kidney
Leukocytes (per
109 cells)
Liver
Lung
Muscle
Nails
Ovary
Pancreas
363
Placenta
Prostate
Mg/g
Mean
7.4
6.7
4.2
23.9
4.6
89.1
23.1
16.5
14.9
0.9
25.5
9.5
5.4
18. L
8.L
7.4
13.5
6.5
Dry Weight"
Range
1.1-28.9
2.4-21.9
0.9-11.8
13.1-39.4
1.4-8.4
63.0-107.0
7.4-54.5
10.1-22.9
5.1-35.7
0-1.4
9.2-46.8
4.2-15.9
2.0-13.8
3.2-58.2
3.1-16.5
2.4-20.0
11.8-16.6
1.8-11.0
or Tissue^-
Median 80% Range
0.02 O.OL-0.02
8.1 5.4-11.3
1.2 0.8-1.5
0.9 0.8-1.1
11.3 7.1-28.7
1.3 1.0-2.0
26.7 18.0-43.2
0.009 0.007-0.0:
0.1 0.08-0.2
0.02 0.01-0.03
48
-------
TABLE 5-2 - continued
mg Copper/Whole Organ
u
yg/g Dry Weight"
Tissues Mean
Skin 2 . 0
Spleen 6.8
Stomach and 12.6
intestines
Thymus 6 . 7
Thyroid 6.1
Uterus 8.4
Body Fluids
189
Aqueous humor
477
Bile
264
Cerebrospinal fluid
477
Gastric juice
477
Pancreatic juice
97
Plasma, Wilson's disease
141
Saliva
98
Serum, female
98
Serum, male
473
Serum, newborn
239
Sweat, female
239
Sweat, male
Range
0.3-5.4
3.1-16.1
4.5-36.6
3.3-11.5
1.6-17.5
3.5-25.2
yg/100 ml
Mean
12.4
27.8
28.1
28.4
50.0
31.7
120.0
109.0
36.0
148.0
55.0
Q
or Tissue-
Median 80% Range
1.4 1.1-2.1
0.2 0.1-0.3
3.3 1.1-2.6
0.02 0.009-0.05
Range
24.0-538.0
10.0-70.0
0.0-200.0
0.0-69.0
33.0-65.0
5.0-76.0
87.0-153.0
81.0-137.0
12 . 0-67 . 0
59.0-228.0
3.0-144.0
49
-------
TABLE 5-2 - continued
yig/100 ml
Body Fluids Mean Range
390
Synovial fluid 21.0 4.0-64.0
90
Urine (24 h ) 18.0 3.9-29.6
477
—Derived from Scheinberg and Sternlieb.
—Values for most tissues in these two columns derived from Fell, Smith,
169
and Howie, except where other references are indicated.
c 471
""Values in these two columns are from Sass-Kortsak.
50
-------
COPPER PROTEINS
A protein containing copper may be a specific copper protein, like
69
ceruloplasmin; or like albumin, it may bind the metal more loosely.
That a protein is a specific copper protein requires two proofs. First,
progressive purification must result in a ratio of copper to protein in
which copper increases to an asymptotic value corresponding to an integral
number of copper atoms per molecule of protein. Second, some properties of
the metalloprotein should disappear when copper is removed, and reappear
when a reversible recombination of copper and protein is effected. By
applying these criteria, butyryl coenzyme A dehydrogenase, 6-aminolevulinic
acid dehydrase, and 3-mercaptotranssulfurase, all originally described as
253,296,324
copper proteins, subsequently have been shown only to be con-
529,579,614
taminated with the metal.
Twenty mammalian copper proteins (Table 5-3) have been isolated but
at least three of them—erythrocuprein, hepatocuprein, and cerebrocuprein—
94a
are identical, and several have more than one name.
51
-------
TABLE 5-3
Mammalian Copper Proteins
Isolated from
Protein
Albocuprein I
Albocuprein II
Ceruloplasmin
Copper-chelatin (L-6-D)
Cytochrome £ oxidase
3,4-dihydroxyphenylethylamine
3-hydroxylase
Dopamine 3-hydroxylase
Ferroxidase II
Hepatomit ochondrocuprein
Lysyl oxidase
Metallothionein
Mitochondrial monoamine oxidase
Pink copper protein
Plasma/serum monoamine oxidase
Superoxide dismutase (cytocuprein)
Cerebrocuprein
Erythrocuprein
Hemocuprein
Hepatocuprein
Tryptophan-2,3-dioxygenase
Tyrosinase
Species
Man
Man
Man, rat
Numerous
Cattle
Cattle
Man
Man,
cattle
Chicken
Man,
cattle
Man, rat,
cattle
Man
Man,
rabbit,
Pig
Man
Man
Man
Man
Rat
Man
Organ or Tissue
Brain
Brain
Numerous, Plasma
including
man
Liver
Heart, liver, etc.
Adrenals
Adrenals
Serum
Liver
Cartilage
Liver
Liver, brain
Erythrocytes
Plasma/serum
Brain
Erythrocytes
Blood
Liver
Liver
Skin, eye
52
-------
Albocuprein I and II. Albocuprein I and II are two pale yellow
184
proteins recently isolated from human brains. Their molecular weights
are 14,000 and 72,000, Neither has any detectable enzymatic activity
nor close similarities to cytocuprein (superoxide dismutase) or
ceruloplasmin. Albocuprein I contains 0.25% copper, and Albocuprein II
holds 1.4% copper in its molecule. Both proteins also contain hexoses.
Albocuprein II may be the primary copper -containing protein of the
brain. The relation of these proteins to cerebral pathology has not
yet been investigated.
Ceruloplasmin . Ceruloplasmin (polyamine oxidase, ferroxidase I; EC
1.12.3.1) is a blue plasma glycoprotein containing 0.3% copper and 8%
73-75,368,475,493,494
carbohydrate. Depending on the analytic method
323,464 266
employed, its molecular weight is between 132,000 and 160,000.
To a moderate degree, it catalyzes the oxidation of several polyamines,
catecholamines and polyphenols, particularly paraphenylenediamine
121,240,266,405,434,464
(PPD; C H [NH ] ), and Fe(II) to Fe(III).
o 4 22.
The last reaction may be essential in some species for the uptake of
ferric iron by apotransferrin. Recently histaminase activity has also been
220 459
attributed to ceruloplasmin. Except for ferroxidase activity, all of
these enzymatic activities only have been observed in vitro, and their
physiologic significance is obscure. The purpose of the reversible dissociation
.
of the molecule's copper is similarly enigmatic, although small amounts of
94c 242
apoceruloplasmin have been reported to be present in human and rat serum.
The 8-10 oligosaccharide chains of ceruloplasmin are composed principally
of glucosamine (HOayCHOH^CHtNH^CHO^ mannose (CH OH[CHOH] CHO) , and galac-
tose (HOCH2[CHOH]4CHO); almost all chains are terminated by a sialic acid
residue. The presence of this residue appears to be essential to the survival
213,367
of the protein in the circulation.
53
-------
Copper-chelatin (L-6-D). Copper-chelatin, a cytoplasmic copper-
binding protein of about 8,000 daltons, has been isolated from livers of
615a
rats. It is characterized by a high sulfydryl (-SH) concentration
and a content of six atoms of copper per molecule. In the human liver,
370
a similar protein (L-6-D) is found in concentrations of about 5 mg/g
wet tissue, although its copper content may vary.
Cytochrome c oxidase. Cytochrome £ oxidase (EC 1.9.3.1), present in
the mitochrondria of many animal and plant tissues, contains one heme
(C.,AH.joO,N,FeOH) molecule, one iron atom, and apparently two copper atoms
per molecule, and weighs about 270,000 daltons. Studies of the paramagnetic
resonance and oxidation-reduction characteristics of beef heart cytochrome
oxidase indicate its copper is of two different species. ' ' Copper
379
is essential to the protein's enzymatic activity: severe copper defi-
ciency, whether acquired or hereditary, is generally associated with reduced
.. , ,j ... .„ 123,176,177
cytochrome oxidase activity.
Dopamine B-hydroxylase. Dopamine 3-hydroxylase (3,4-dihydroxyphenyl-
ethylamine 3-hydroxylase; EC 1.14.2.1) is an oxidase that weighs about
180
290,000 daltons and contains 4-7 copper atoms per molecule. It
catalyzes the conversion of dopamine (CoH-,-iN02) to norepinephrine
505
(CgH,-|NOo). In preparations isolated from beef adrenal glands, about
one-third of the copper atoms are cuprous and the enzymatic hydroxylation
appears to require reduction of about another third to the cuprous state.
Hydroxylation of the substrate is accompanied by a stoichiometrically
equivalent oxidation of one-third of the enzyme's cuprous atoms to
180
cupric. The enzyme is also found in cattle brains and hearts.
54
-------
Ferroxidase II. Ferroxidase II, thought to be another copper protein
that can catalyze the oxidation of ferrous iron, has been isolated from the
serums of normal individuals and patients with Wilson's disease. Ferroxidase
II is yellow, contains 0.1% copper, and displays no oxidase activity toward
566
PPD.
Hepatomitochondrocuprein. Hepatomitochondrocuprein contains about 3%
copper and has been isolated from crude mitochondrial fractions of bovine
435,436
and neonatal human liver. Despite its name, the actual subcellular
437
locus of this protein is probably lysosomal.
Lysyl oxidase. Lysyl oxidase, an enzyme that has been isolated from
chick cartilage, converts certain lysyl residues in collagen and elastin
into the corresponding adipic semialdehydes. The enzyme is almost certainly
a copper protein: extracts of cartilage from chicks raised on a copper-
deficient diet have no lysyl oxidase activity. Dialysis of the purified
enzyme against the chelating agent a, a'-dipyridyl (CinHRN9) abolishes its
enzymatic activity; activity is restored when the apoenzyme is dialyzed
against 0.001 M copper bichloride (CuCl0). Deficient activity of this
z
enzyme may be related to the loss of structure in collagen, elastin, and
495
keratin seen in natural and experimental copper deficiencies.
Metallothionein. Metallothionein is a colorless protein, first described
577c
by Vallee and co-workers; it has a molecular weight of about 10,000
daltons, a high sulfhydryl content and the capacity for binding copper,
159
zinc, and cadmium. It has since been isolated from the cytosol of human,
607a 68a 263a 338a
rat, chicken, cow, and horse liver; equine renal cortex; and
55
-------
160
chick, cow, and rat duodenal mucosa. The equine liver and kidney protein
263a
appears to have a monomeric molecular weight of 6,000 daltons.
Mitochondrial monoamine oxidase. Mitochondrial monoamine oxidase (EC 1.4.3.4),
present in the mitochondria of liver and brain, is enzymatically active towards
the same substrates as the plasma enzyme, and it also oxidizes epinephrine
(CQH-,oNOo) and serotonin (C1 nH-, 9N90) . Copper is essential for its enzymatic
y 1J J 10 1^ i 384,394
activity. Mitochondrial monoamine oxidase has not been solubilized.
Plasma/serum monoamine oxidase. Plasma/serum monoamine oxidase (EC
1.4.3.4) is a copper protein weighing 255,000 daltons. It has been purified
348,623
from human and rabbit serum, and steer and hog plasma. In vitro, the
enzyme catalyzes the oxidative deamination of several monoamines to form hydro-
349
gen peroxide (H^O ), ammonia (NH ) , and the corresponding aldehydes. Its
physiologic and pathologic significance is obscure, although its concentration
350
increases in the prsence of congestive heart failure or parenchymal liver disease.
Superoxide dismutase. Superoxide dismutase (cytocuprein) is also called
erythrocuprein (or hemocuprein), hepatocuprein or cerebrocuprein, according
to whether it has been isolated from the cytosol of erythrocytes, liver, or
94a,179a
brain. A light blue-green metalloprotein with a molecular weight of
33,600 daltons, it catalyzes the dismutation of the Superoxide anion (02) into
282,346,490,509
hydrogen peroxide (^02) and oxygen (02) and may also scavenge oxygen
atoms in certain reactions. For optimal enzymatic activity, at least two
94b,259,607
of the protein's four metal ions must be cupric. About 30 mg of the
protein are present in 100 ml of packed normal human erythrocytes, accounting
339
for more than 60% of total erythrocyte copper. This protein—or a very
similar one—is also present in human kidney, thyroid, pituitary, and adrenal
56
-------
224
glands. Recently a protein containing one copper atom and exhibiting
superoxide dismutase activity has been isolated from the marine bacterium
442a
Photobacterium leiognathi.
Tryptophan-2,3-dioxygenase. Tryptophan-2,3-dioxygenase (EC 1,13.1.12)
486
has been isolated from rat liver cytosol. It is a heme protein with a
molecular weight of 167,000 daltons and two cuprous atoms per molecule.
This enzyme catalyzes the insertion of molecular oxygen into the pyrrole
66
ring of L-tryptophan.
Tyrosinase. Tyrosinase, found principally in the melanocytes of skin
77
and eye, contains about 0.2% copper. Actually, it may be a series of
similar proteins, each of which catalyzes one of the sequential reactions
172-174
that convert tyrosine (CqH-i-iNOo) to melanin. A genetic abnormality in
tyrosinase may cause the tyrosinase-negative, oculocutaneous albinism
171a
inherited as an autosomal recessive trait.
A pink copper protein, still unnamed, has been isolated from erythrocytes.
Though its molecular weight is close to that of superoxide dismutase, it
differs from the latter in spectral, electrophoretic, chromatographic, and
450
enzymatic characteristics. Its biologic role is unknown.
COPPER DEFICIENCY
Copper deficiency is a rare condition, because the normal infant is
372,487a,613
born with a generous store of copper in the liver. However, Josephs
suggested in 1931 that copper deficiency might account for a resistant
262
anemia in milk-fed infants, because cow's milk is one of the few major
foods deficient in copper (Table 5-1). Indeed, during the past several
57
-------
years, numerous instances of copper deficiency associated with anemia,
neutropenia, and severe demineralization of the bone in both premature and
8,15,110,111,209,487
full-term infants have been reported. Induction of
clinically significant copper deficiency seems to require that either severe
malnutrition or intestinal malabsorption be present. Fortification of the
feeding formula with iron might impair the absorptive mechanisms for copper
487
and lead to copper deficiency. Copper deficiency has been observed in
265
an infant: with ileal atresia and in adults maintained on parenteral
588
nutrition for prolonged p>eriods. The addition to the diet or infusions
of 1 mg copper/day (however, the premature infant should receive only
109a
100 yg/kg) is more than adequate supplementation in all these instances,
and is capable of reversing the anemia, neutropenia, and bone lesions that
110,111
are the most significant clinical effects of copper deficiency in infants.
Menkes's Disease (Trichopoliodystrophy)
Steely- or kinky-hair disease, first described as a syndrome by Menkes
356a
et^ a^. in 1962, is an X-linked fatal disorder. Affected male infants
exhibit kinky, depigmented hair; physical and mental retardation, with
widespread degeneration of the brain; hypothermia; and death within the
356a
first several years of life.
126
In 1972 Danks et al. demonstrated copper deficiency in infants with
markedly low serum concentrations of copper and ceruloplasmin. They pointed
out that the kinky or steely depigmented hair was similar to the abnormal
wool seen in copper-deficient sheep, and believed to be caused by defective
cross-linking of keratin. Similar defective cross-linking in collagen and
elastin—probably a consequence of abnormally low concentrations of the
58
-------
copper-containing enzyme, lysyl oxidase—also was thought to lead to degen-
eration of the internal elastic lamina of arterial walls, in turn causing
126
brain degeneration. Brain degeneration may also be related to a deficiency
176,177
of cytochrome c^ oxidase. A generalized defect in transport of copper
across membranes, rather than defective absorption from the gastrointestinal
125
tract alone, may underlie the disorder.
126
Unfortunately, therapeutic administration of copper, whether oral
215e
or parenteral, has done little more than raise concentrations of
ceruloplasmin and copper in serum. No definite clinical improvement or
decrease of mortality has occurred.
An X-linked inherited defect in mottled mice is associated with
pathologic findings somewhat similar to Menkes's syndrome; it is also asso-
251a
ciated with defective copper transport and deficiency. These abnormal
mice and the affected Bedlington terriers mentioned below may provide animal
models of two serious inherited disorders of human copper metabolism.
In adults^ copper deficiency can occur when intake or absorption of the
91,110,518
metal is drastically reduced, when disease causes prolonged,
449,598
excessive urinary or intestinal loss of ceruloplasmin, or when the
copper balance becomes negative during the prolonged administration of
penicillamine (^-mercaptovaline; C5H,,NO?S) to individuals with normal copper
229
stores. All of these conditions are associated with lowered blood con-
centrations of ceruloplasmin and copper.
Occasionally, administration of D-penicillamine may cause copper
deficiency which is accompanied by impaired taste acuity that will return
229
to normal if a few milligrams of copper salt are added to the daily diet.
59
-------
TOXICITY
A survey of 969 water systems located in nine geographic areas in the
344b
United States showed average copper concentrations of 134 yg/1. The
highest concentration was 8,350 Pg/1, and 1.6% of the 2,595 samples exceeded
the drinking water standard of 1 mg/1. This limit on copper in drinking
water was established not because toxicosis was of concern, but because ex-
cessive concentrations impart an undesirable taste to water. Higher con-
centrations are more frequent in acidic, soft waters; at a pH below 7.0,
5% of the samples exceeded the standard. The concentration of copper in
drinking water is rarely high enough to affect its taste or produce toxicosis.
Acute Toxicosis
Acute copper poisoning occurs in man when at least several grams of copper
sulfate (CuSO ) are ingested or when acidic food or drink—vinegar, carbonated
4 56,244,354,411,
beverages, or citrus juices—have had prolonged contact with the metal.
When carbonated water remains in copper check-valves of drink dispensing
machines overnight, the copper content of the first drink of the day may be
increased, enough to cause metallic taste, ptyalism, nausea, vomiting and
244
epigastric burning, and diarrhea. Whiskey sours and fruit punches mixed
or stored in copper-lined cocktail shakers or vessels have had the same
354,575a
effects. A whiskey sour that contained 120-135 ppm copper, or approx-
imately 10 mg of cupric ions in a 60-90 ml drink, produced abdominal cramps,
575a
vomiting, and diarrhea within 10-90 min of ingestion. Eight children
and one of two adults who drank an orange-flavored beverage refrigerated
overnight in a brass pot became nauseated, and several of the children
vomited. The drink contained 34 ppm copper, so that 240 ml would have
60
-------
supplied 8.5 mg copper but, since the children were aged 1-4, it is likely
575b
that smaller amounts of the beverage and less copper were ingested.
In eight other instances of acute copper poisoning involving over two dozen
575c
individuals and reported by the Center for Disease Control since 1968,
copper plumbing or vessels, with which acidic (generally carbonated) water
575d-575f
had prolonged contact, led to the toxicosis.
The vomiting and diarrhea induced by ingesting milligram quantities
of ionic copper generally protect the patient from its serious systemic
toxic effects: hemolysis, hepatic necrosis, gastrointestinal bleeding,
oliguria, azotemia, hemoglobinuria, hematuria, proteinuria, hypotension,
106,128
tachycardia, convulsions, coma or death. When more than gram quanti-
ties of a salt such as copper sulfate are ingested—generally with suicidal
intent—gastrointestinal mucosal ulcerations, hemolysis, hepatic necrosis,
and renal damage from deposition of hemoglobin and/or copper constitute the
106,597
pathogenetic factors underlying these effects.
Hemolysis has also been reported after applying solutions of copper
241
salts to large areas of burned skin, or after introducing copper into the
319a
circulation during hemodialysis. The source of this copper may come from
the semipermeable membranes (generally fabricated with copper) and copper
tubing or heating coils of the dialysis equipment. Copper in the membrane
appears to be transferred to the patient; in one instance a Cuprophan mem-
575b
brane introduced 632 yg copper into a patient. Copper from tubing or
coils seems hazardous only when the dialysate becomes acidic; the pH of the
circulating fluid can drop to 2.5 when a deionizer in the circuit is
20,53,54,283,336
exhausted. Therefore, even 1-2 yr of twice weekly dialysis
20,53
can raise hepatic copper concentration to abnormally high levels. Copper
introduced into the circulation by hemodialysis can produce febrile reactions
61
-------
remarkably similar to classical metal fume fever experienced by workers in
319a
copper smelters and refineries. Similarly, copper stopcocks in circuits
used for exchange transfusions have been reported as the source of potentially
52
hazardous infusions of copper for neonates.
Chronic Toxicosis
Presence of a fragment of metallic copper in the eye (chalcosis
bulbi) may result in loss of the eye, sunflower cataracts, or visible
222,460
deposits of copper in the cornea known as Kayser-Fleischer rings.
Prolonged administration of D-penicillamine may be the. only available
nonsurgical therapy.
Bordeaux mixture, a 1-2% solution of copper sulfate neutralized
with hydrated lime, is used widely to prevent mildew on grape vines,
particularly in France:, Portugal, and southern Italy. Pulmonary copper
deposition and fibrosis occur in the lungs of some vineyard workers after
431
years of exposure to such solutions. Their lungs may be blue, sug-
gesting the presence of excess copper. More recently, granulomas and
431a,587a
malignant tumors have appeared in these laborers' livers and lungs.
In contrast, studies of Chilean copper miners show that liver and serum
477
concentrations of copper are normal, despite years of exposure to
copper sulfide and oxide dusts, which are, of course, insoluble.
Drinking water with an unusually high copper concentration (800
466
yg/1) may have caused acrodynia (pink disease) in a 15-month-old infant.
567 182 24
Gingivitis, lichen planus, and eczematous dermatitis have been
attributed to the copper alloys used in some dental and other prostheses.
Wearing copper bracelets as apocryphal treatment for arthralgias only
leads to green-stained skin.
From the rarity with which either human copper deficiency or toxicosis
occurs, it appears that the evolutionary process has produced an effective
62
-------
pair of buffering mechanisms: humans seem able to avoid both deficiency
and toxicosis despite wide variations in dietary supply.
Wilson's Disease (Hepatolenticular or Hepatocerebral Degeneration)
Genetics. Wilson's disease has been found in every racial group where
29,286,479,603,615
it has been sought. The illness is inherited as an autosomal
recessive trait with a general prevalence of about 1 in 200,000. This
figure is consonant with the possession of 1 "Wilson's disease gene" by
1 in about 200 people.* The heterozygotes remain free of pathologic
manifestations of Wilson's disease. As with any recessive disorder, the
incidence will be higher in locales where a significant amount of inbreeding
occurs.
Pathogenesis and pathology. Almost all patients with Wilson's disease
474
exhibit a lifelong deficiency of the plasma copper-protein ceruloplasmin,
199,479,513,524,526
and an excess of hepatic copper. This excess copper in
the liver may be caused, in part, by impairment of lysosomal excretion of
181,402,407,528,533
hepatic copper into bile, and is associated with diminished
520
or absent hepatic synthesis of ceruloplasmin. It is remarkable that re-
taining only 1% of the dietary intake of copper (10-20 mg/yr) is sufficient
to cause Wilson's disease.
*Since 1 In 40,000 nonconsanguineous marriages will be between
such heterozygotes, and since one-quarter of their children will inherit a
pair of abnormal alleles, Wilson's disease will develop in about 1 in
160,000 people.
63
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During the early stages of Wilson's disease, the liver is capable of
binding as much as 30-50 times its normal concentration of copper with
524
little, if any, overt clinical disorder. Ultimately, hepatic copper
is released into the bloodstream, and in the face of massive necrosis of
hepatic parenchyma, that action may suddenly infuse large amounts of copper
458,513,526
into the plasma, inducing severe hemolysis and jaundice. In
most patients, however, the metal diffuses into the circulation gradually,
causing the plasma concentration of free copper to rise 5-10 times to about
25-50 p.g/100 ml. This copper diffuses out of the vascular compartment into
extracellular fluids and tissues with toxic effects in susceptible cells.
Characteristic ultrastructural changes, fatty degeneration of hepatocytes
eventuating in necrosis, collapse of parenchyma, and postnecrotic cirrhosis
168,513,526
occur in the liver. Later, the excess hepatocellular copper is
sequestered by lysosomes, a process that seems to render the metal innocuous
for other cytoplasmic organelles.
Unless the patient with Wilson's disease succumbs to hepatic necrosis,
the toxic effects of copper ultimately are manifested primarily in the
central nervous system and kidneys. In the corneas, copper deposits are
29,116,192,286,
visible as pathognomonic Kayser-Fleischer rings or crescents
397,453,480,485,618
seen best with the slit lamp as golden or greenish-brown
516,538
pigment grains in the periphery of Descement's membrane. In some
patients, copper is also deposited on the capsular surfaces of the lens as
92
a sunflower cataract.
Diagnosis. In about half of all patients, the first clinical evidence
of Wilson's disease represents dysfunction of the liver. Ascites, esophageal
66
-------
527
variceal hemorrhage, a syndrome mimicking toxic or infectious hepatitis,
139
hemolysis caused by sudden release of sequestered copper, deficiency of
clotting components, hypersplenism, or gonadal dysfunction may be mani-
478,479,526
fested.
In almost all other patients, neurologic or psychiatric disorders are
the initial clinical manifestation. The neurologic picture may resemble
parkinsonism, multiple sclerosis, chorea, dystonia, or any combination of
29,144,286,479,603
these diseases; or it may be sui generis. The usual
onset is insidious. Dysarthria is a frequent sign in children, and often
subtle incoordination, resting or intentional tremors, athetoid movements,
rigidity, or dystonic posturing and distortion can occur at all ages.
Excessive salivation and drooling are often troublesome. Epileptiform
416,500
seizures have been reported but are unusual. Specific disturbances
in reflexes, sensation, or muscular strength are so rare in Wilson's disease
that their presence should lead the physician to search for another diagnosis.
Psychiatric disorders may accompany the neurologic symptoms or precede
28,200,297
any other evidence of disease. In young adults, the spectrum
ranges from mild behavorial disturbance difficult to differentiate from the
normal vicissitudes of adolescence through marked deterioration of school
work and neurosis, to the manic-depressive or schizophrenia-like psychosis,
which may appear in all age groups. The emotional disturbance may be partly
a reaction to the somatic dysfunction. Yet it is hard not to conclude that
cerebral deposits of copper (Table 5-4) must also exert a direct toxic
effect on higher brain centers, although no specific psychiatric syndrome
has been attributed to Wilson's disease as yet. However, very few sophisticated
psychiatric studies have been made.
67
-------
In rare instances, hematuria has been cited as the first evidence of
169
the toxic effects of copper.
Relatives, particularly siblings, of patients with Wilson's disease
must be examined even if they appear perfectly healthy, because of the
524
autosomal recessive transmission of the illness. Fortunately, bio-
chemical findings in patients with the manifest disease have made it possible
to confirm the diagnosis in an asymptomatic individual when there are less
than 20 mg ceruloplasmin/100 ml serum and more than 250 yg copper/g dry
513
liver.
Treatment. From Wilson's treatise in 1911 until 1948, this disease was
considered progressive and fatal. The recognition of the etiologic role of
copper and Cumings's suggestion that British antilewisite (2,3-dimercapto-l-
propanol; CoHgOS^) might halt that progression led to successful specific
therapy. ' In 1957, an effective oral treatment to remove copper with
penicillamine was introduced by Walshe, and now it is generally recognized
that specific treatment of the disease produces dramatic results. u>->15>->2.3,
600 603
Even prevention of all overt manifestations and an apparently normal
life span are feasible for asymptomatic patients with Wilson's disease; they
require only the regular, lifelong administration of penicillamine.
Recently Walshe has introduced triethylene tetramine dihydrochloride as a new
f\f\ 0
chelating agent for the treatment of Wilson's disease.
Biochemical Basis of Copper Toxicosis
The effects of copper in organs, tissues, cells and subcellular
organelles from patients with Wilson's disease are compared here with data
derived from in vitro studies and natural or experimental copper toxicosis
in animals. The Dominican toad, Bufo marinus, normally accumulates concen-
trations of hepatic copper comparable to those seen in humans with Wilson's
68
-------
198
disease, but copper is sequestered in the toad's lysosomes where, as in
197
the newborn baby, it does not seem to cause pathologic changes. Experi-
mental copper poisoning of animals has clarified only a few biochemical,
physiologic and pathologic aspects of copper metabolism in the liver, brain,
and kidney. Very recently, a form of chronic copper toxicosis (probably
223a
inherited) has been reported to occur in certain Bedlington terriers.
Liver. Chronic administration of copper to rabbits, rats, mice, pigs,
and sheep forces deposition of the metal in the liver, as well as in other
7,21,22,151,167,196,301,313,330,361,582,617,621
organs. Copper accumulates
when the excretory capacity of the liver cell is exceeded. Fractionation
of liver homogenates (Table 5-5) and morphologic studies on such copper-
loaded animals show:
• an increase in hepatic copper concentration, which is even more
621
marked if there is bile duct obstruction;
• a change in the relative subcellular distribution of copper with
a 200-300% increase in the proportion in the mitochondria and
lysosomes, and a marked decrease in the proportion in cytosol of
167,582
hepatocytes, and
22,196
• increased numbers and prominence of copper-containing lysosomes.
This induction of lysosomes is not evident in young patients with Wilson's
disease, because their organelles are inconspicuous despite high cytoplasmic
513,524
concentrations of the metal. In older patients, however, the numbers
199
of lysosomes increase and the metal is sequestered as the disease progresses.
This phenomenon seems to protect the liver cells from the cytotoxic effects
observed in the younger patients. In contrast, rats experimentally poisoned
69
-------
by copper release acid phosphatase and other hydrolases from lysosomes
314,355
to cytosol and microsomes, which does not occur in copper-poisoned
582 528
mice or in patients with Wilson's disease.
• Human, rat. and cow hepatic cytosol contain two or three copper-
55,158,370
binding proteins weighing about 8,000, 10,000, and 40,000 daltons.
528,615a
The lower molecular weight protein isolated from patients with
370
Wilson's disease appears identical to that from control subjects. A
recent study concluded that metallothionein samples from Wilson's disease
159,472
patients have a greater than normal affinity for copper.
70
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Erythrocytes. In Wilson's disease and in chronic copper toxicosis in
sheep, massive or submassive necrosis of liver sometimes can free large
enough amounts of copper in a sufficiently short time to cause considerable
139,352,417,458,565,612
hemolysis. Since copper analyses are rarely per-
formed in blood at the height of the hemolytic process, it is difficult to
pinpoint the maximal concentration of free copper in the plasma, which is
the probable cause of hemolysis. Estimation of this value is also unreliable,
because free plasma copper is the difference between total copper and
ceruloplasmin-bound copper, and usually both measurements are not given in
case reports. In one of the published reports, the value of free plasma
139
copper given during the hemolytic crisis was about 0.7 yg/ml, or about
10 times the normal free copper concentration. In another patient, 1.8
Pg/ml was present temporarily in the plasma at the peak of hemolysis. A
qualitative estimate of the concentration of free plasma copper may be
made from studies of daily urinary copper excretion. Excreted copper
rarely exceeds 0.5 mg/day in nonhemolyzing, untreated Wilson's disease
patients, because free plasma copper is the only source of urinary copper.
In each of five patients not receiving penicillamine, the highest 24-h
565 139
urinary excretion measured during hemolysis was 1.2, 3.0, 2.4, 2.5, and 4.7
352
mg. Table 5-6 lists the results of studies showing biochemical effects
of ionic copper on several intraerythrocytic compounds.
73
-------
TABLE 5-6
Experimental Biochemical Effects of Copper on Human Erythrocytes
Study No.
161
Preparation
Concentration
of Copper, pg/ml
Effect
417
139
357
612
166
59
Nicotinamide adenine 63.0
dirmcleotide phosphate,
reduced (NADPH)
Glucose-6i-phosphate 63.0
dehydrogenase
(G-6-PD)
Glutathione (C^EL-NoO^S) 63.0
G-6-PD 6.3
Glutathione 6.3-63.0
G-6-PD 6.3-63.0
Glutathione reductase 6.3-63.0
NADPH 6.3-63.0
Glutathione 2.5
Catalase 5.0
Glutathione reductase 5.0
Pyruvate kinase 0.3-1.89
Adenosine 63.0
triphosphate (ATP;
C10H16N5°1-2P3^
Hexokinase 0.95-6.3
Phosphofructokinase 0.95-6.3
Phosphoglyceric kinase 0.95-6.3
Pyruvate k.inase 0.95-6.3
6-Phosphogluconate
dehydrogenase 0.95-6.3
G-6-PD 3.15-6.3
Oxidation enhanced
Inhibited
Oxidized
Inhibited
Content and stability
diminished
Inhibited
Inhibited
Oxidation unchanged
Content diminished
Diminished
Diminished
Inhibited
Utilization
diminished
Inhibited
Inhibited
Inhibited
Inhibited
Inhibited
Inhibited
74
-------
59
The data show that only six of the enzymes studied by Boulard et al. and
612
the pyruvate kinase investigated by Willms et al. were affected by the
concentrations of free copper attained in patients. An hereditary defect
in any one of these enzymes is generally associated with nonspherocytic
hemolytic anemia; thus it is attractive to speculate that such copper
inhibition in vivo is the direct cause of hemolysis. However,
measurement of the activities of these six enzymes in erythrocytes from
three patients with Wilson's disease during a hemolytic crisis, and from
one patient two years after hemolysis, showed increased activity in every
612
instance. In another patient with Wilson's disease, normal values for
glutathione reductase*, glucose-6-phosphate dehydrogenase (G-6-PD)r and
pyruvate kinase were noted during and after the hemolytic crisis. There-
fore, the cause of hemolysis in Wilson's disease remains unknown.
Brain. A number of studies on the effects of copper on the respiration
of preparations of neural tissue have shown that either facilitation or
480
inhibition results, depending on experimental conditions. The data do
not significantly contribute to an understanding of the mechanisms underlying
17,60,89a,105,155,426,427,502,
the neurophysiology or neuropathology of copper.
555,592,593
Cholestasis
Copper frequently accumulates in the livers of patients with chronic
250,499,622
extra- or intrahepatic cholestasis. Two- or threefold elevations
of hepatic copper concentration have been reported in these disorders.
612
*Values were above normal for four patients studied by Willms et al.
59
and not measured by Boulard et al.
75
-------
More marked elevations (even as high as 1,200 yg/g dry weight), in the range
seen in patients with Wilson's disease, occur in children with biliary
517 622
atresia and in some patients with biliary cirrhosis. The etiologic
role of copper in these disorders certainly is not primary, but it is pos-
sible that copper toxicosis may aggravate the severity of their degenerative
process. Nevertheless, a role for copper-chelating therapy has not. been
established in treatment of biliary cirrhosis.
ADDITIONAL CLINICAL ASPECTS OF COPPER METABOLISM
Diagnostic Value of Measuring Serum Copper and Ceruloplasmin Concentrations
The normal range of concentration of ceruloplasmin in healthy adults
is 20-45 Mg/100 ml of serum or plasma, corresponding to a copper concentration
of about 80-140 yg/100 ml. Normally, 95% or more of serum copper is integrally
bound to ceruloplasmin; the rest is loosely bound to albumin. Consequently,
a measurement of the concentration of either serum copper or ceruloplasmin
usually serves as an accurate measure of the other. The only important
exception occurs in patients with Wilson's disease, who may have little or
no ceruloplasmin, and yet exhibit appreciable concentrations of serum
copper (Table 5-2).
433
Serum ceruloplasmin is reduced in all normal neonates and is
474,479
diagnostically lowered Ln patients with Wilson's disease and Menkes's
126
syndrome. There is little diagnostic significance in the lowered con-
centrations found in association with severe malnutrition and malabsorption
518,598 604 449
syndromes, massive hepatic necrosis, the nephrotic syndrome,
598
and protein-losing enteropathies.
Late in pregnancy, the serum concentration of ceruloplasmin increases
213
2-3 times. The administration of estrogens or their analogues also
95
brings on similar elevations.
76
-------
Increased concentrations of ceruloplasmin and copper are seen in
rheumatoid arthritis, rheumatic fever, lupus erythematosus, myocardial
infarction, lymphoma, leukemia, carcinoma, various liver diseases, and many
325,454,476,477,521,522,547,551,552
infections.
Deviations from normal ranges of serum copper concentrations have been
found to be useful indicators for monitoring patients with acute leukemia
550,552
and other lymphomas. Tessmer et al. considered the highly significant
relationship between the serum copper level and the percentage of blast
cells in the marrow, generally a useful guide for leukemia therapy.
In Hodgkin's disease, a concentration of serum copper higher than 150
yg/100 ml strongly suggests active disease, except in the presence of pregnancy,
605
estrogen administration, or chronic inflammation. Similar observations
were made on reticulum cell sarcoma, lymphosarcoma, and multiple myeloma.
Twenty-three of 24 patients with generalized disease had increased serum
copper concentrations, whereas abnormally high serum copper concentrations
374
were less often seen when the process was localized. The concentration
of serum copper in one patient with multiple myeloma was an astounding
3,350 pg/100 ml, the highest ever reported, and the administration of
penicillamine did not significantly increase urinary copper. The mechanism
203
of this hypercupremia remained obscure.
The differentiation of several types of liver disease sometimes can
525
be achieved through the diagnostic use of radiocopper.
Copper in Neoplasms
The copper content of benign tumors has been shown to be lower than
190
of carcinomas of the esophagus, bronchus, intestinal tract and breast.
77
-------
Sandberg noted large accumulations of copper and iron in liver and spleen
of patients with cancer of the respiratory system, genito-urinary tract,
468
or breast. Pedrero reported that metastatic tumors in the liver,
reticulum cell sarcoma not accompanied by hepatic disease, and diabetes
420
mellitus were often associated with a low hepatic copper content, but
hepatic copper was found to be normal in patients with bronchogenic
371
carcinoma.
A possible relation between environmental zinc and copper and the
occurrence of neoplasms was considered by Stocks and Davies, who examined
these metals in garden soils in Wales, Cheshire, and Devonshire. They
found the zinc-copper ratio consistently higher in gardens of persons
530
who had died of cancer of the stomach. In contrast, the experimental
administration of 0.5% cupric oxyacetate (CuC20.,H/) in the diet of albino
rats given maize with 0.09% 4-dimethylaminoazobenzene (C,,H,,N.,) for 7 mo
^ lb J 162,246
or longer afforded striking protection against the development of tumors.
Antimicrobial Effects
The effects of the combination of copper ions with proteins probably
account for the low-grade antimicrobial activity of cupric sulfate (blue-
stone). A solution of about 0.05% has been used as a retention enema to
treat typhoid fever and amebiasis. Cupric sulfate also has been applied
408
topically for treatment of trachoma. Metallic copper displays a
gonococcicidal effect _in vitro that may provide a degree of prophylaxis
170
for women employing a copper-containing intrauterine device. Copper
sieves that form traps in an inhalation therapy circuit partially sterilize
137
the vapor and reduce the incidence of pulmonary infection.
78
-------
Phosphorus Burns
Copper sulfate, applied in solution to the skin, is used to treat
phosphorus burns, because the formation of copper phosphide (Cu.jP) renders
the phosphorus innocuous. If copper sulfate concentrations of 3% or more
241,536
are used, hemolysis and death may occur.
Ernesis
Oral doses of 100-300 mg copper sulfate in water bring about especially
243,408
effective emesis after the ingestion of phosphorus. However, cupric
sulfate is not a safe emetic. If vomiting does not occur after its use,
gastrointestinal irritation, hemolysis or other effects of acute toxicosis
may supervene. Recently a patient given 2 g cupric sulfate as an emetic
died from copper poisoning.
Other Conditions
There is no evidence that copper metal or salts are of value in the
408
treatment of arthritis or epidermophytosis.
SITUATIONS OF POTENTIAL COPPER TOXICOSIS
Copper-containing Intrauterine Contraceptive Devices
Winding several hundred square millimeters of copper wire around a
plastic intrauterine device (IUD) improves its contraceptive efficiency
from 18.3 pregnancies to less than 1.0 pregnancy/100 woman-years of
114,219,315,548,633
experience. Analysis of such luT/s that have been in utero
from months to years shows that about 25-50 mg copper is lost per year.
Some of the metal is excreted with endometrial secretions, but studies in
398
rats suggest that as much as 10-20 mg may be absorbed. There is at
least a possibility that such retention could lead to chronic toxicosis over
the years or decades that a woman is likely to use an IUD. The amount of
copper absorbed from the uterus is of the same order as that retained from
79
-------
dietary copper by the tissues of patients with Wilson's disease. Although
in both cases, the amount in question may only be a small fraction of
dietary copper usually ingested, the parenterally absorbed copper from the
IUD may not be metabolized and excreted by the same homeostatic mechanisms
operating on orally ingested copper. Unfortunately, neither periodic
determinations of blood or urinary copper, nor of clinical liver function
tests, can indicate whether copper is accumulating in the liver. Therefore,
quantitative analyses for copper as well as light and electron microscopic
examinations of hepatic biopsy tissues from women who have used these devices
for varying durations would be necessary to determine if systemic toxicosis
is occurring.
Copper-supplemented Animal Feeds
Porcine liver is a. principal constituent of some prepared meats, and
much is eaten fresh. Pigs fed rations containing 250 ppm copper to acceler-
ate growth (cf. Chapter 4) increase their hepatic copper from a normal
67
mean of 24 ug to a mean of 220 yg/g dry tissue. One-quarter Ib (112 g) of livei
from swine on such diets may contain 10 mg of copper—an amount capable of
causing acute toxicosis—or 2-3 times the average daily supply of the metal
in a Western diet. Liver proteins bind at least a portion of the metal,
mitigating the acute toxicosis, but no data on the effects of eating this
amount of copper for long periods are available.
Manure from pigs raised on copper-supplemented feeds also constitutes
a potential problem if it is used to fertilize land on which human food
crops are grown: the copper content of vegetation and water
run-offs from dressed fields may be increased.
80
-------
Because of its antibiotic effect (see Chapter 4), copper has also been
added to poultry feeds. Few data are available on the concentrations of
tissue copper in chickens and turkeys.
Because of the possible ill effects on the environment and humans of
adding 250 ppm copper to pig and poultry feed, the Food and Drug Administration
currently limits the amount of copper for finished feeds to no more than
385,576
15 ppm.
81
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CHAPTER 6
COPPER AS AN INDUSTRIAL HEALTH HAZARD
OCCUPATIONAL EXPOSURE
The paucity of literature on ill effects caused by exposure to copper
and its compounds in industry suggests that copper is not a particularly
hazardous industrial substance. However, if workers are exposed to excess
concentrations of the metal in any of its forms, undesirable health effects
can occur. Copper melts and boils at high temperatures and does not give
off metal fumes as readily as do more volatile metals like lead, cadmium, and
zinc. Dusts and fumes from copper and its compounds usually have an objection-
able taste--a warning that tends to limit exposures before serious toxic intake
can occur. However, metal fume fever from exposure to copper can occur.
319a 345
Typical metal fume fever, ' a 24-48 h illness characterized by
chills, fever, aching muscles, dryness in the mouth and throat, and head-
ache, was found in a copper refinery worker riveting heavy copper bus bars
by a shielded-arc welding technique (personal communication, K. W. Nelson).
171
Another worker contracted metal fume fever when he welded a copper tank.
Copper fever has been discovered among men handling copper oxide powder in
481
a paint factory, and copper acetate dusts have caused complaints of
72
sneezing, coughing, digestive disorders, and fever. Workmen handling
"jewelry sweeps," a dusty scrap from jewelry manufacturing, experienced a
bitter taste and nasal irritation traced to the verdigris formed from cop-
per in the jewelry alloys (personal communication, K. W. Nelson). Apparent
metal fume fever has also been reported in three men who were exposed to
194
dust produced during the polishing of copper plates.
82
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466a
Contact dermatitis associated with copper has been reported,
but few cases of dermatitis caused by copper metal or compounds occur in
418a 80
industry. Neither Patty nor Browning in their comprehensive reviews
of industrial toxicology mention skin complaints, other than a green color-
ation noted more than a century ago among copper workers. A similar localized
coloration is caused today from wearing jewelry made of copper or high cop-
per alloys.
Observations of scores of copper smelter and refinery workers over
the last 25 years have not revealed any significant incidence of dermatitis
that could be traced to exposure to either copper or many of its inorganic
compounds (personal communication, S. S. Pinto). Nor have chronic
systemic effects from copper exposure been significant. The existence of
such effects has been a subject of speculation, but no solid supporting
evidence has been advanced.
107b
In a recent review of health hazards from copper exposure, Cohen
observed that copper was ordinarily a benign agent. The combination of
conditions in industry which would produce excessive concentrations of
copper as a dust, fume, or mist, or in particle sizes and chemical forms
such that toxic effects would be generated from the copper absorbed, are
relatively rare.
The U.S. Occupational Safety and Health Administration (OSHA) has
adopted standards for exposure to airborne copper at work. The time-
weighted average for 8-h daily exposures to copper dust is limited to
3 3
1.0 mg/m air. The standard for copper fume was changed in 1975 to 0.2 mg/m .
No particle
/size or solubility specifications are included in the standards, which
were derived from threshold limit values (TLV) adopted by the American
Conference of Governmental Industrial Hygienists. Documentation for the
194
TLV's consists only of Gleason's research and a personal communication.
83
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Four studies have: found increased incidences of lung cancer among
298,303a,359,564a
workers in copper smelters. The authors have suggested that
the cancer was caused by exposure to arsenic trioxide (As_CL) in dust and
fumes produced by the various pyrometallurgic processes. They did not
suggest, that copper itself played any etiologic role in the cancer deaths.
COMMUNITY EXPOSURES
Water
A 1969 Public Health Service study of 969 urban water supply systems
revealed that 11 supplies contained copper in concentrations above the
577
drinking water standard of 1 ppm, a standard based on taste. The maximum
concentration found was 8.35 ppm. Copper in public water supplies has not
been treated by regulatory agencies as a significant problem. Indeed, cop-
per is intentionally added to the New York City water supply to maintain a
282a
concentration of 0.059 ppm, which controls algal growths.
Air
The National Air Sampling Network's (NASN) 1966 data indicate a range
3
of airborne copper concentrations from 0.01 to 0.257 pg/m in rural and
577b
urban communities. Continuous monitoring of air near copper smelters
•j
for over 10 years usually has shown fractional pg/m concentrations.
Occasionally weekly averages of 1-2 pg are reported (personal communi-
cation, K. W. Nelson). Even when airborne copper does reach this level,
the dose of the metal would be about 1% of the normal daily ingested dose,
given a 15 m daily intake of air and a total penetration, retention and
absorption of all airborne copper. Schroeder reached the same quantitative
483
conclusion in reviewing NASN data.
84
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It should be noted that the validity of all airborne copper measure-
ments derived from samples collected with conventional high-volume sampling
236
equipment has been questioned. Copper abraded from motor commutators
may have contaminated the air around the sampling units.
Most copper emissions in the United States are produced by
131
the metallurgic processing of ores and concentrates. Sources of copper-
bearing dust and fume in smelters are roasters, reverberatory furnaces,
and converters. The typical particulate collection systems are made up of
large balloon flues for gravity separation of the coarser dusts and fume
agglomerates, and electrostatic precipitators with collection efficiencies
of 95-99%.
The second most important source of copper emissions is the
131
iron and steel industry. Trace quantities of copper enter the steel-
making process in raw materials. Emissions are generated mostly from blast
furnaces and open hearth furnaces; the emitted dusts and fumes contain
0.1-0.5% copper. Controls are a combination of cyclone spearators and
electrostatic precipitators.
Power plants that burn coal are the third most important
source of copper emissions. Based on measurements by Cuffe, the average
concentration of copper in particulates in power plant stack gasses (with
3
electrostatic precipitators) is 230 yg/m . Emissions from plants without
emission controls would be about 7 times higher.
Other significant emission sources are brass and bronze foundries,
secondary smelting of copper and its alloys, burning of insulation from
copper wire, and miscellaneous fabricating operations. Because of the
low magnitude of emissions from these sources and the minimal environmental
85
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impact of copper, not much general data have been accumulated on such
emissions.
Copper Emission and Ambient Air Standards
Because the economic value of copper encourages its capture from
process gases, general air pollution controls are used to prevent significant
mass emissions of copper. Atmospheric levels of copper have not been proven
to pose a risk to human health; hence, no emission or ambient air standards
for copper have been established or proposed.
86
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CHAPTER 7
SUMMARY AND CONCLUSIONS
SUMMARY
Copper in the Ecosystem
Almost 2 million metric tons of copper are removed from the sites of
their natural sources and injected into the world ecosystem annually. The concen-
tration of copper in the continental crust is about 50 ppm. Most soils,
plants, and many surface and ground waters contain 1 or more ppm copper.
The total body content of copper in adult mammals is about 2 ppm wet
weight.
Supplementing the copper in an animal's feed with appropriate levels
of iron and zinc has been thought to increase growth rates. Because such
supplements also will produce manure containing as much as 8,000 ppm cop-
per, potentially harmful to soils, crops, and animals, care is required
in their disposal.
Copper in Plants
Several specific copper proteins have been isolated from plant tissues
and characterized chemically.
Copper is essential to the normal growth and development of almost
all plants. Plants grown in soils that contain less than about 5 ppm are
likely to show adverse effects. Copper toxicosis in plants rarely is
observed under natural conditions but may occur where large amounts of
copper have been added to the soil. The absolute concentrations of
copper that result in pathologic deficiency or excess depend upon the species
87
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of plant and the physlcochemical characteristics of the soil. For a number
of food species, supplementation of soil, seeds, or the whole plant with
copper can enhance crop yields.
Copper in Animals
Copper deficiency can be produced experimentally in many animal
species, but naturally occurring, clinically significant deficiency mostly
is limited to cattle a.nd sheep. Cattle are more susceptible than sheep,
and monogastric animals rarely are subject to copper deficiency. Cop-
per toxicosis also can be induced in many species, but naturally occurring
toxicosis,like deficiency, commonly occurs only in sheep and cattle.
Sheep are more susceptible than cattle to copper toxicosis. Again,, non-
ruminant animals are much more resistant to copper toxicosis. It should
be understood that the amounts of copper required to prevent deficiency
or cause toxicosis in animals may vary significantly with the amounts of
zinc, iron, molybdenum, and sulfate in the diet.
The differences between cattle and sheep make it highly advisable
that feeds and mineral supplements be differently formulated for each
species. It has been reported that supplementing feeds with high levels
of copper may quicken the rate of weight gain in young pigs and chickens,
but the evidence is inconclusive.
Some aquatic organisms, including edible fish, are susceptible to
toxicosis by copper concentrations two orders of magnitude lower than the
accepted standard for drinking water (1.0 ppm). Copper is an effective
molluscicide, and is useful in the control of schistosomiasis.
88
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Human Copper Metabolism
Copper is essential to normal health and longevity in man. It is
the prosthetic element of more than a dozen specific copper proteins.
In relation to strict metabolic requirements, copper is overabundant
in almost all human diets. Therefore, clinically significant copper
deficiency is extremely unusual and is virtually limited to instances of
severe gastrointestinal malabsorption, drastically reduced dietary intake
(and even this condition is significant only in newborn infants), or to
the presence of a rare X-linked disorder of copper absorption and trans-
port known as Menkes's steely- or kinky-hair disease. Human copper
toxicosis is also extremely rare, and appears in clinically significant
form only when suicide is attempted by the ingestion of large quantities
of a copper salt, or where a genetic defect in copper metabolism is
inherited in an autosomal recessive fashion (Wilson's disease). In
patients with Wilson's disease, copper steadily accumulates, first in
the liver and then in other parts of the body. Damage, particularly
evident in the liver and central nervous system, is ultimately fatal.
Successful treatment and prophylaxis is effected by a chelating agent,
D-penicillamine, which promotes the urinary excretion of copper.
Despite the efficiency of genetic mechanisms in regulating the
balance of dietary copper, there is little knowledge of whether parenter-
ally introduced copper is subject to these controls. Copper introduced
into the uterus as a contraceptive or during hemodialysis is absorbed
systemically to some degree. There is evidence that copper also may be
absorbed parenterally through the skin, lungs, and uterine mucosa. It
89
-------
is not known whether this copper accumulates or is excreted. Hepatic
and pulmonary granuloraas and neoplasms have been observed in vineyard
workers exposed to sprays of copper sulfate solutions.
Alterations in copper metabolism, reflected in the concentrations
of copper and ceruloplasmin in the serum, are associated with pregnancy
and the administration of estrogens. Changes in these concentrations
also accompany many acute and chronic disorders.
Copper in Drinking Water
Despite the widespread use of copper and brass plumbing, copper con-
centrations in drinking water rarely exceed the accepted standards of 1 ppm,
unless acidic liquids or water of low pH are allowed to stand for a long
time in such plumbing.
Copper as an Industrial Health Hazard
Although copper can act as a toxic agent in an occupational setting,
it is benign under ordinary circumstances. However, if workers are exposed
to excessive concentrations of the metal in any of its forms, there may be
undesirable health effects. Because of the absence of reports on signi-
ficant environmental effects from airborne copper, copper and its compounds
as dusts or fumes dispersed into the atmosphere have not been considered
hazardous.
CONCLUSIONS
Copper in the Ecosystem
Copper should be used with awareness of its ultimate distribution and
effects on the ecosystem.
90
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Copper in Plants
Judicious use of copper may aid in obtaining optimal yields of
crops, and inhibiting the growth of undesirable plants, particularly fungi.
Copper in Animals
Deficient and excessive copper in soil and water is significant for
agriculture, animal husbandry, and the economic and medical aspects of the
biology of certain aquatic organisms.
Human Copper Metabolism
Copper is essential to the life and health of human beings, and the
interaction of its environmental supply with genetic mechanisms
controlling its absorption, transport, and excretion is so finely tuned
that significant clinical manifestations of deficiency or toxicosis are
very rare.
91
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CHAPTER 8
RECOMMENDATIONS FOR FUTURE RESEARCH
1. Research into the mechanisms of interaction between copper and
molybdenum, sulfate, iron, and zinc in plant and animal metabolism
is desirable.
2. The optimal dietary requirements of copper, molybdenum, sulfate,
iron, and zinc for the various species of animals that are sources
of human food should be determined.
3. A system for verifying and tabulating incidents of deficiency and
excess of copper and interrelated trace elements in animals should
be initiated on a national basis.
4. Copper should not be generally recognized as safe for livestock feeds
without qualification.
5. Copper should continue to be added to livestock and poultry feeds
only in the concentration (15 ppm) generally regarded as safe. How-
ever, because of widespread use of high level (250 ppm) copper
supplementation in animal feeds in the United States and elsewhere,
the beneficial and harmful effects of such supplementation should
be further investigated. This inquiry should include a careful
monitoring of the disposal of animal wastes.
92
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6. Research directed at understanding better the biochemistry and
physiology of copper proteins should be encouraged and supported.
7. Because copper is absorbed by the lungs, skin, and uterus, as well
as the gastrointestinal tract, a nationwide clinical investigation
should be carried out to determine whether any long-term hazard of
human copper toxicosis is possible from the added burden of body
copper introducted parenterally through chronic hemodialysis, inha-
lation, or from the skin or copper-containing intrauterine contra-
ceptive devices.
8. The role, if any, of copper in producing granulomas or malignant
tumors, particularly in liver and lungs, should be defined.
9. Studies defining the role of copper as an etiologic agent in metal
fume fever should be carried out.
93
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APPENDIX
COPPER ANALYSIS IN ENVIRONMENTAL AND BIOLOGIC SAMPLES
Selected methods for the quantitative analysis of copper in the environ-
ment (water and air), in biologic materials and animal feeds, and of cerulo-
plasmin in human serum are described in detail in this appendix. No attempt
has been made to describe and compare all the analytic procedures available.
Instead, methods known to be feasible and accurate by the members of the
Subcommittee are presented.
Techniques most commonly used to analyze environmental samples for
copper are noted. The analysis of water is emphasized, because airborne
particulate, as well as other environmental samples may often be assayed
by analysis of aqueous solutions or suspensions.
A lengthy description of atomic absorption is presented because this
method is accepted as standard for copper by the American Society for
Testing and Materials (ASTM), the Environmental Protection Agency (EPA),
and other organizations that have presented standard methods. The method
has a sensitivity of .04^tg/l as presented, but the optimal concentration
range may be varied by changing the analytic line used to suit a particular
sample. Most atomic absorption measurements have an accuracy of approximately
2%.
WATER
Atomic absorption spectroscopy is similar to flame emission photometry
in that a sample is atomized and aspirated into a flame. Flame photometry,
however, measures the amount of light emitted, whereas in atomic absorption
spectrophotometry, a light beam is directed through the flame into a mono-
chromator, and then onto a detector that measures the amount of light
94
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absorbed. In many instances, absorption is more sensitive because it depends
upon the presence of free unexcited atoms and even at flame temperatures the
ratio of unexcited to excited atoms is very high. Since the wavelength of the
light passed by the monochromator is selected to be characteristic of the
difference between two energy levels of the metal being determined, the light
energy absorbed by the flame is a measure of the concentration of that metal
in the sample. This principle forms the basis of atomic absorption
spectroscopy.
In determining copper concentrations, contamination and loss are of prime
concern. Dust in the laboratory environment, impurities in reagents and im-
purities on laboratory apparatus which the sample touches are all sources of
potential contamination. For liquid samples, containers can introduce either
positive or negative errors in the measurement of trace metals by contributing
contaminants through leaching or surface desorption and by depleting them
through adsorption. Thus the collection and treatment of the sample prior to
analysis requires particular attention. The sample bottle should be thoroughly
washed with detergent and tap water; next it should be successively rinsed
with 107o hydrochloric (HC1) or nitric (HNC^) acid, and three times with
distilled or demineralized water. Before collecting the sample, it should be
decided what type of data is desirable, that is, dissolved, suspended, total, or
extractable.
For the determination of dissolved copper, the sample should be filtered
through a 0.45-jim membrane filters as soon as practical after collection. Use
the first 50-100 ml of filtrate to rinse the filter flask. Discard this
portion and collect the required amount of filtrate. Acidify the filtrate
with 1:1 redistilled nitric acid (3 ml/1). Normally, this amount of acid
will lower the pH to 2 or 3 and should be sufficient to preserve the sample
indefinitely. Analyses performed on a sample so treated should be reported
as "dissolved" concentrations.
95
-------
To determine suspended copper, a representative volume of sample should
be filtered through a 0,,45-jum membrane filter. When considerable sediment is
present, as little as 100 ml of a well-shaken sample is filtered. Record the
volume filtered and transfer the membrane filter containing the sediment to a
250 ml Griffin beaker and add 3 ml distilled nitric acid. Cover the beaker
with a watch glass and heat gently. The warm acid will soon dissolve the
membrane. Increase the temperature of the hotplate and digest the material.
When the acid has evaporated, cool the beaker and watch glass and add another
3 ml distilled nitric acid. Cover and continue heating until the digestion
is complete, generally indicated by a light-colored residue. Add 2 ml dis-
tilled L:l hydrochloric acid to the dry residue and warm the beaker again
gently to dissolve the material. Wash down the vatch glass and beaker walls
with distilled water and filter the sample to remove silicates and other in-
soluble material that could clog the atomizer. Adjust the volume to some
predetermined value based on the expected concentrations of the metal present.
This volume will vary according to the metal being determined. The sample
is now ready for analysis. Concentrations so determined should be reported
as "suspended." Quantities of copper determined on unused membrane filters
should be deducted from the total quantity found. Ordinarily such amounts
are insignificant.
To determine total copper, the sample is not filtered before processing.
Choose an amount of sample appropriate for the expected level of the metal. If
much suspended material is present, as little as 50-100 ml of well-mixed sample
will most probably be sufficient. (The sample volume required may vary pro-
portionally with the number of metals to be determined.)
Transfer a representative aliquot of the well-mixed sample to a Griffin
beaker and add 3 ml concentrated distilled nitric acid. Place the beaker
on a hotplate and evaporate to dryness, making certain that the sample does
96
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not boil. Cool the beaker and add another 3 ml portion of distilled con-
centrated nitric acid. Cover the beaker with a watch glass and return to
the hotplate. Increase the temperature of the hotplate so that a gentle re-
flux action occurs. Continue heating, adding additional acid as necessary
until the digestion is complete, generally indicated by a light-colored
residue. Add sufficient distilled 1: 1 hydrochloric acid and warm the beaker
again to dissolve the residue. Wash down the beaker walls and watch glass
with distilled water and filter the sample to remove silicates and other in-
soluble material that could clog the atomizer. Adjust the volume to some
predetermined value based on the expected metal concentrations. The sample
is now ready for analysis. Concentrations so determined should be reported
as "total."
Optimal Concentration Range. 0.1-10 mg/1
Wavelength. 324.7 nm
Sensitivity. 0.04 mg/1
Detection Limit. 0.005 mg/1
Preparation of Standard Solution.
1. Stock solution: Carefully weigh 1.0 g electrolytic copper(analytic
reagent grade). Dissolve in 5 ml redistilled nitric acid and make up to 1 liter
with distilled water. Final concentration is 1 mg copper/ml (1,000 mg/1).
2. Prepare dilutions of the stock solution to be used as calibration
standards at the time of analysis. Maintain an acid strength of 0.15% nitric
acid in all calibration standards.
577a
General Instrument Requirements.
1. Copper hollow cathode lamp
2. Wavelength: 324.7 nm
3. Type of burner: Boling
97
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4. Fuel: acetylene
5. Oxidant: air
6. Type of flame: oxidizing
7. Photomultiplier tube: IP-28
229a
Herrman and Lang first described atomic absorption analysis for
40a
copper in 1963, and Barman has reviewed copper analysis by atomic ab-
sorption in tissues and biologic samples. Dispersion of copper in methyl
isobutyl ketone (CgHj^O) will increase the test sensitivity about four times.
The high temperature at which copper is volatilized (600 C) permits ashing of
samples,an action that will remove several interfering substances adequately.
Tissue, feed, grain, forage, and other materials have been analyzed easily
40a,508a
using atomic absorption spectrophotometry.
Other techniques may be employed for determining copper in aqueous
solution or suspension, including spectrophotometry., applied below to
biologic samples, polarography, and anodic stripping voltammetry.
AIR
Samples of airborne particulates preferably are collected on glass fiber
or membrane filters. Ideally, particle size distributions and identifications
or chemical compounds should be attempted, but this is impractical for any
routine monitoring. However, a rough separation of respirable and irrespirable
particles is practical and provides useful information.
The analytic method of choice is atomic absorption after acid digestion
of the filter and appropriate dilution. Spectrographic, poLarographic,
spectrophotometric, neutron activation analysis, and anodic stripping volt-
ammetry may also be used. High sensitivity, accuracy, and precision are
easily attainable.
98
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Microanalysis of Biologic Materials
This procedure, a combination and modification of two procedures
153a,370b,428a
described in the literature, is used to determine quantitatively
the total copper content of organic material by using dicyclohexanone-
oxalyldihydrazone (DCO; CeHioiNNHCOCONHNiCgHlo).
Reagents
1. Concentrated sulfuric acid (H2S04), reagent grade of the American
Chemical Society (ACS).
2. Perchloric acid (HClO^), 60%, reagent ACS.
3. Ammonium hydroxide (NH^O/), reagent ACS.
4. Phosphate-citrate buffer, pH 7.4, made of 900 ml 0.4 M anhydrous
dibasic sodium phosphate (Na2HPO.) and 100 ml 0.2 M monohydrate citric acid
(C6H8Oy .H20).
5. DCO reagent, prepared by dissolving 0.17o DCO (Eastman Organic
7175)* in hot 50% (vol/vol) ethanol (C2H OH). Do not heat reagent and
ethanol together.
6. Standard copper solutions containing 1.0 and 2.0>ig copper/ml are
made in 0.10 M sulfuric acid.
Equipment
1. Spectrophotometer: Zeiss PMQ II, Beckman DU, or equivalent instru-
ment with attachments for use of cuvettes of 40 or 50 mm path length.
2. Digestion apparatus: Microdigestion shelf, gas heated, 6-unit,
with Pyrex glass fume duct (Fisher Scientific 21-130).*
"Specific products have been listed solely to help readers who desire more
information. Mention of these products does not constitute an endorsement
by the National Academy of Sciences.
99
-------
3. Digestion tubes: Tubes, similar in form to blood sugar tubes,are
*u
made by Robert C. Ewald, Inc., Middle Village, N.Y. Volume of the bulb,
approximately 6.5 ml; constricted part of the tube graduated at 7.0 ml (just
above the bulb), 8.0 ml,and 9.0 ml; total height of the tube, 21 cm; diameter
of the upper portion of the tube, 2 cm; diameter and length of the constricted
portion of the tube, 1 cm and 5.5 cm, respectively.
All glassware must be washed free of copper with 107o hydrochloric acid
and rinsed with a large volume of distilled or de-ionized water.
Procedure. Blanks, standards,and unknown samples of biologic
materials are run in triplicate where possible. Pipet the following
solutions into digestion tubes which contain three glass beads.
1. Blank: 1.0 ml of 0.10 M sulfuric acid.
2. Standards: 1.0 ml of 1.0 and 2.0 iig/ml copper solutions.
3. Unknown samples:
• Serum or plasma: 1.0 ml.
• Urine: 1.0-3.0 ml, depending on expected concentration of
copper. For urine specimens of patients not on chelation
therapy, 3.0 ml is appropriate.
• Tissue: For this assay, 50-100 mg of dried tissue are usually
sufficient. If much less material is available, the procedure
can be modified (see below). Add 1.0 ml of de-ionized water.
Add 1.0 ml of concentrated sulfuric acid to all tubes and mix.
Add 1.0 ml of 60% perchloric acid and mix.
/v
Specific products have been listed solely to help readers who desire more
information. Mention of these products does not constitute an endorsement by
the National Academy of Sciences.
100
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Tubes are heated in the digestion apparatus and shaken gently until the
onset of boiling. Particles of carbon ascend to about the middle of the tube,
eventually to be washed down by the refluxing liquid. The digest turns
colorless, then yellow, then colorless again. At this point, sulfuric acid
starts to reflux, and should be continued for 15 min. Total time of digestion
is approximately 30 min.
After cooling, 1.0 ml distilled water is added and the solution is mixed
and cooled again. From a burette, 3.5 ml concentrated ammonium hydroxide are
added slowly while the tube is cooled in ice water. Tubes are placed in a
water bath maintained at 65-70 C for 17-18 h. Under these conditions prac-
tically all the free ammonia, but no ammonium ion^is removed from the
solution.
To each tube, add 2.0 ml phosphate-citrate buffer and dilute with water
to the 8.0 ml mark. This addition brings the contents of the tube within the
pH limits of 7-8 necessary for color to develop.
Add 0.8 ml DCO reagent to each tube, cover with Parafilm* and mix
thoroughly.
After 1 h at room temperature, the optical density of each solution is
read against water at 600 nm in the spectrophotometer using 40 or 50 mm light-
path cuvettes.
If the optical density of a solution is greater than 0.600, the sample
should be diluted with phosphate-citrate buffer and more DCO reagent, equal
to 10% of the volume of sample and buffer, then should be added. Alternatively,
the unknown sample should be redigested, using a smaller amount.
^Specific products have been listed solely to help readers who desire more
information. Mention of these products does not constitute an endorsement
by the National Academy of Sciences.
101
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Calculations and sources of error. The copper content of each unknown
is calculated by comparing its net optical density to the net optical density
of the standard.
The standard deviation (SD) of analyses performed by this method over the
range of 1.4-3.7 /ig copper/pil was estimated to be 0.0392 ug copper/ml, according
to data from seven sets of triplicate measurements.
There are three common sources of error in this procedure: specimens
have been contaminated with copper; copper may be lost if bumping occurs
during digestion; or, the pH of the final solution may be outside the 7-8
range.
Modifications for Applications of Method for Needle Biopsy Specimens of Liver
1. Standards: 1.0 ml of 0.25^ig/ml and l.O^ig/ml of copper.
2. Digestion: Add to blank, standard, and unknown tubes:
0.3 ml concentrated sulfuric acid
0.3 ml 607o perchloric acid
Digest until sulfuric acid refluxes for 10 min.
3. Neutralization: To the cooled digested sample, add 0.5 ml distilled
water and 1.1 ml concentrated ammonium hydroxide (NH/OH). Place in
90 C water bath until odor of the ammonium hydroxide disappears
(about l%-2 h). Wash the contents of the digestion tube into a
test tube calibrated at 2.7 ml, using a total of 1.0 ml of phosphate-
citrate buffer which has been diluted 1:1 with distilled water. Add
water to the 2.7 mark.
4. Development of color: Add Q.3 ml DCO reagent and mix. Read optical
density after 1 h.
Range of Values
Serum or plasma. The plasma of Americans and Europeans contains
102
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476
approximately 100 ug copper/100 ml, most of which is tightly bound to
ceruloplasmin. The range of copper concentration in the plasma of normal
98
men is 81-137, and in normal women, 87-153 ^ig/100 ml.
Concentrations of serum copper below the lower limit of the normal range
are found in patients with hereditary Wilson's or Menkes's diseases, as well as
in five acquired pathologic conditions: the nephrotic syndrome, kwashiorkor,
sprue, scleroderma of the intestine, and protein-losing enteropathy.
Physiologic hypocupremia is present during the first four to six months of
life in almost all infants.
Concentrations of serum copper above the upper limit of normal ranges are
common in late pregnancy, and following the ingestion of estrogens or contra-
ceptive pills; high concentrations are also found in many inflammatory,
476.
necrotizing,and neoplastic diseases.
Urine. The normal 24-h excretion of copper in the urine is less than
30/ig. Patients with Wilson's disease who have received no treatment
476
usually excrete considerably more than 100 /ig/24 h. Treatment with
appropriate pharmacologic agents will greatly increase the amount of copper
excreted.
Hepatic copper. The normal mean + SD concentration of hepatic copper
is 31.5 + 6.8^ug/g dry liver. Hepatic copper concentrations of untreated
patients with Wilson's disease measure more than 250 ug/g dry liver.
Measurement of the Concentration of Ceruloplasmin in Human Serum
Ceruloplasmin is a copper-containing globulin of plasma which can
239,240,370a
catalyze the oxidation of paraphenylenediamine (PPD; C H [NH ] ).
64 22
The rate at which PPD is oxidized is proportional to the concentration of
ceruloplasmin in serum. When determined under precisely defined conditions
of composition of the medium, and at a given temperature, the rate of
103
-------
oxidation allows the calculation of the concentration of enzyme Ln the
serum. In the method described below, the rate of PPD oxidation is
measured by quantitatively determining the rate of darkening of its
solution in a spectrophotometer.
The major diagnostic value in measuring the concentration of. serum
ceruloplasmin is in suspecting, confirming, or ruling out the diagnosis of
479,522
Wilson's disease (hepatolenticular degeneration).
As described here, this method has been calibrated only for human
serum.
Reagents.
1. Acetate buffer. Dissolve 10.05 g sodium chloride (NaCl) and 49.20 g
anhydrous sodium acetate (C H NaCU) in about 1,900 ml distilled water.
Adjust pH to 5.12 with about 10 ml glacial acetic acid (02^02). Bring
volume to 2,000 ml with distilled water. Buffer is stable indefinitely at 4 C.
2. PPD reagent. A 0.57o solution of paraphenylenediamine dihydrochloride
(CgH^ /NH2 Jr2-2HC1) in acetate buffer which has been
warmed to 30 C is prepared immediately before its addition to the cuvette.
Equipment
1. Zeiss Spectrophotometer PMQ II.
2. Water bath circulator (Bronwill Circulator, Will Corp., N.Y.)"
JU
3. Electronic therometer with flexible probe (Tri-R Instruments).
4. Electric timer or stopwatch.
5. Hotplate.
6. Cuvettes, 1 cm path length.
Procedure. Water is circulated through the cell compartment of the
spectrophotometer and the temperature is adjusted so that the reading taken
Specific products have been listed solely to help readers who desire more
information. Mention of these products does not constitute an endorsement
by the National Academy of Sciences.
104
-------
in a reference cuvette containing water is 30+0.1 C. The enzymatic
activity of ceruloplasmin in this system is increased or decreased
respectively by about 1% for each 0.1 C rise or fall in temperature.
1. Place 1.0 ml distilled water in reference cuvette and 1.0 ml
fasting, nonhemolyzed serum in the sample cuvette.
2. Dissolve weighed PPD in an appropriate volume of warm buffer, and
add 2.0 ml of this warm PPD reagent to the serum in the sample cuvette.
3. Add 2.0 ml of warmed buffer to the reference cuvette.
4. Cover cuvettes with Parafilm' and mix by inverting them.
5. Warm unknown sample quickly to 30 C on hotplate, and begin taking
readings about 3 min after mixing.
At 530 nm, readings of optical density of the unknown are made against
the reference cuvette at intervals of 30 sec-2 min, depending on the number
of samples and the concentration of ceruloplasmin. A sufficient number
of readings is made so that the last 5 or 6 points fall on a straight line.
When plotted against time, that is, the change in optical density/min/ml,
the slope of this line is proportional to the ceruloplasmin content of the
serum. Be sure to check the temperature of sample at end of run.
JL.
Specific products have been listed solely to help readers who desire more
information. Mention of these products does not constitute an endorsement
by the National Academy of Sciences.
105
-------
Calculations and sources of error. If x is the ceruloplasmin concentration
, *
in mg/100 ml of serum, and % is LOD/min/ml - 0.0012, then x = 900y_.
This method has been calibrated with human serum of known ceruloplasmin
267
copper content; the copper content of ceruloplasmin was assumed to be 0.31%.
Serum copper was determined by wet digestion of a sample of serum from which
nonceruloplasmin copper (approximately 57» of total serum copper) was removed
by addition of sodium diethyldithiocarbamate (C C
520
and passage through a column of activated charcoal. Hemolyzed, icteric,
lipemic, aged, or frozen and thawed specimens may yield unsatisfactory assays.
Changes in temperature during assays will also produce errors.
Range of values. In a series of 185 unselected normal adult subjects, the
mean + SD ceruloplasmin concentration was 30.5 + 3.5 mg/100 ml of serum.
Almost all patients with Wilson's disease exhibit ceruloplasmin concentra-
tions of 0-20 mg/100 ml of serum. Decreased serum concentrations also have
been found in about 20% of healthy heterozygous carriers of 1 abnormal
479
"Wilson's disease gene" and in all newborns during the first 6 months of
life.
Pathologic deficiency of serum ceruloplasmin may occur in the nephrotic
syndrome, kwashiorkor, sprue, scleroderma of the intestine, protein-losing
enteropathy, Menkes's disease, and in rare instances of severe hepatitis.
Increased concentrations of serum ceruloplasmin have little diagnostic
significance, since they are encountered late in pregnancy and following the
ingestion of estrogens or contraceptive pills, and in many inflammatory,
522
necrotizingj and neoplastic diseases.
^Change in optical density.
106
-------
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185
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