conspectus of researchon copper metabolism and requirements
conspectus of researchon copper metabolism and requirements
conspectus of researchon copper metabolism and requirements
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A CONSPECTUS OF RESEARCH ON COPPER<br />
METABOLISM AND REQUIREMENTS OF MAN<br />
KARL E. MASON -<br />
Consultant—NIAMDD, National Institutes <strong>of</strong> Health,<br />
Bethesda, Maryl<strong>and</strong> 20014<br />
Pages 1979-2066<br />
THE JOURNAL OF NUTRITION<br />
VOLUME 109, NUMBER 11, NOVEMBER 1979<br />
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TABLE OF CONTENTS<br />
PAGE<br />
Introduction 1981<br />
Copper in the human body 1982<br />
Copper proteins 1983<br />
Ceruloplasmin (ferroxidase I ) 1984<br />
Superoxide dismutase 1986<br />
Cytochrome c oxidase 1987<br />
Lysyl oxidase 1987<br />
Tyrosinase (phenoloxidase ) 1987<br />
Dopamine /?-hydroxylase 1988<br />
Metallothionein 1988<br />
Other cuproproteins 1988<br />
Absorption <strong>of</strong> <strong>copper</strong> 1989<br />
Mechanisms 1990<br />
Amount 1991<br />
Transport <strong>of</strong> <strong>copper</strong> 1991<br />
Intestine to liver 1991<br />
Metabolism <strong>and</strong> distribution 1992<br />
Copper in blood 1993<br />
Excretion <strong>of</strong> <strong>copper</strong> 1994<br />
Biliary excretion 1995<br />
Salivary excretion 1996<br />
Gastrointestinal excretion 1996<br />
Urinary excretion 1997<br />
Sweat loss 1997<br />
Menstrual loss 1997<br />
Copper in the diet 1997<br />
Copper in foods 1997<br />
Dietary intake <strong>of</strong> <strong>copper</strong> 1998<br />
Copper <strong>metabolism</strong> in prenatal <strong>and</strong> postnatal life 2000<br />
Influence <strong>of</strong> pregnancy 2000<br />
Influence <strong>of</strong> oral contraceptives 2001<br />
Placental transfer 2002<br />
Infancy <strong>and</strong> childhood 2002<br />
Dietary <strong>copper</strong> deficiency 2004<br />
Menkes' disease 2005<br />
Manifestations 2005<br />
Metabolic abnormalities 2007<br />
Therapy 2008<br />
Animal models 2009<br />
Wilson's disease 2009<br />
Nature <strong>of</strong> the disease 2009<br />
Metabolic abnormalities 2010<br />
Therapy 2012<br />
Related disorders 2013<br />
Hypocupremia 2014<br />
Hypercupremia 2015<br />
Copper toxicity 2020<br />
Interrelationships between <strong>copper</strong> <strong>and</strong> other elements 2022<br />
Human <strong>requirements</strong> 2024<br />
Infants 2025<br />
Young children <strong>and</strong> adolescents 2030<br />
Adults 2032<br />
Resume 2036<br />
Acknowledgments 2039<br />
Literature cited . . 2039<br />
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INTRODUCTION<br />
The discovery <strong>of</strong> <strong>copper</strong>, following that<br />
<strong>of</strong> gold <strong>and</strong> silver, goes back to the post<br />
glacial epoch in southwestern Asia, espe<br />
cially the semi-arid regions <strong>of</strong> Central<br />
Anatolia <strong>and</strong> Iran, during the period 6000<br />
to 3000 B.C. (834). The later Bronze Age<br />
(3000-1000 B.C.) takes its name from the<br />
use during this period <strong>of</strong> bronze, an alloy<br />
<strong>of</strong> <strong>copper</strong> <strong>and</strong> tin. The word <strong>copper</strong> is de<br />
rived from the Latin cuprum, a corrup<br />
tion <strong>of</strong> cyprium, named after the isl<strong>and</strong> <strong>of</strong><br />
Cyprus which was an important source <strong>of</strong><br />
<strong>copper</strong> about 3000 B.C. Thus, aside from<br />
gold <strong>and</strong> silver, which were employed<br />
chiefly for ornamental purposes, <strong>copper</strong><br />
represents the first metal to be used by<br />
mankind for more practical purposes. The<br />
alloys <strong>of</strong> <strong>copper</strong> <strong>and</strong> tin (bronze), <strong>copper</strong><br />
<strong>and</strong> zinc (brass), <strong>and</strong> <strong>of</strong> <strong>copper</strong>, zinc <strong>and</strong><br />
nickel (nickel silver or German silver),<br />
have had a tremendous impact upon<br />
human development over many past<br />
centuries.<br />
Beginning about the time <strong>of</strong> Hippocrates<br />
(400 B.C.) <strong>copper</strong> compounds were com<br />
monly prescribed in the treatment <strong>of</strong> men<br />
tal, pulmonary <strong>and</strong> other diseases. During<br />
the 19th century many different <strong>copper</strong><br />
compounds came into use in the unsuc<br />
cessful treatment <strong>of</strong> a wide variety <strong>of</strong><br />
human diseases (447). Copper amulets<br />
were also in vogue. Not until about 150<br />
years ago was <strong>copper</strong> recognizd as a nor<br />
mal constituent <strong>of</strong> blood. And as early as<br />
1900, Abderhalden (1) recorded that ani<br />
mals kept on a whole milk diet developed<br />
an anemia that could not be prevented by<br />
additions <strong>of</strong> inorganic iron, <strong>and</strong> recog<br />
nized the fact that some other mysterious<br />
substance, probably organic, was wanting.<br />
But the thought that <strong>copper</strong> should ever<br />
be considered an important component <strong>of</strong><br />
the diet for either animals or man was<br />
quite remote until the second decade <strong>of</strong><br />
this century, following closely on the heels<br />
<strong>of</strong> the discovery <strong>of</strong> vitamins A, B, C, D<br />
<strong>and</strong> E. At this time there appeared the<br />
reports <strong>of</strong> Hart et al. (311, 312) <strong>and</strong> Waddell<br />
et al. (812) corroborating the obser<br />
vations <strong>of</strong> Abderhalden <strong>and</strong> indicating that<br />
1981<br />
either extracts or the ash <strong>of</strong> dried cabbage,<br />
corn meal <strong>and</strong> chlorophyll, all essentially<br />
iron-free, definitely favored assimilation<br />
<strong>and</strong> utilization <strong>of</strong> iron in hemoglobin build<br />
ing in rabbits fed a whole milk diet. Also,<br />
at about this same time, McHargue (507)<br />
recorded results <strong>of</strong> studies on rats fed puri<br />
fied diets deficient in <strong>copper</strong>, zinc <strong>and</strong>/or<br />
manganese in which he employed impro<br />
vised glass-lined cages. This represents the<br />
first known use <strong>of</strong> cages <strong>of</strong> this type. Mc<br />
Hargue concluded that manganese in par<br />
ticular, <strong>and</strong> possibly <strong>copper</strong> <strong>and</strong> zinc, have<br />
important functions in animal <strong>metabolism</strong>.<br />
There followed the classic studies <strong>of</strong><br />
Hart, Steenbock, Waddell <strong>and</strong> Elvehjem<br />
(313) demonstrating that rats fed ex<br />
clusively on milk developed an anemia<br />
which was responsive to iron only after the<br />
addition <strong>of</strong> <strong>copper</strong>; also, that the same<br />
relationships prevailed in chicks fed diets<br />
<strong>of</strong> milk <strong>and</strong> rice ( 187). Later came evi<br />
dence from experimental studies with pigs<br />
( 188) that whereas impure organic salts <strong>of</strong><br />
iron cured nutritional anemia, the pure<br />
salts failed to do so unless supplemented<br />
with small amounts <strong>of</strong> <strong>copper</strong>. Recognition<br />
<strong>of</strong> the clinical importance <strong>of</strong> these early<br />
observations was first given by Mills (520,<br />
521) <strong>and</strong> Josephs (389) who reported that<br />
<strong>copper</strong> supplements accelerated hemo<br />
globin synthesis in hypochromic anemia <strong>of</strong><br />
infants treated with iron salts. Although<br />
several investigators were not in agree<br />
ment (274, 275, 476), others confirmed<br />
these findings (186, 399, 455, 799) <strong>and</strong> ex<br />
tended them to hypochromic microcytic<br />
anemia <strong>of</strong> adults (521). The early history<br />
<strong>and</strong> later developments are detailed in<br />
several reviews (99, 540, 693). During this<br />
same period appeared Tompsett's report<br />
( 785 ) <strong>of</strong> the first <strong>copper</strong> balance study car<br />
ried out on 17 human subjects, indicating<br />
that the normal daily intake appeared to<br />
vary from 2.0 to 2.5 mg/day. In the same<br />
Received for publication October 16, 1978.<br />
1 Requests for reprints should be directed to Nutri<br />
tion Institute. Science <strong>and</strong> Education Administration,<br />
Building 307. Room 217, USDA. Beltsville. Maryl<strong>and</strong><br />
20705.<br />
- Deceased December S. 1978.<br />
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1982 KARL E. MASON<br />
year Daniels <strong>and</strong> Wright (142) reported<br />
the first balance study on young children<br />
(4-6 years old) indicating an average in<br />
take <strong>of</strong> 1.48 mg/day <strong>and</strong> a requirement<br />
not less than 0.10 mg/kg/day. These esti<br />
mates are in remarkably good agreement<br />
with those recorded in the later literature<br />
(see p. 2032). Thus, the stage was set<br />
for an extensive exploration <strong>of</strong> the metab<br />
olism, deficiencies, excesses <strong>and</strong> require<br />
ments <strong>of</strong> <strong>copper</strong> in man witnessed during<br />
the past half century.<br />
An overview <strong>of</strong> what has transpired dur<br />
ing this period should include: 1) a vast<br />
number <strong>of</strong> studies on <strong>copper</strong> depletion<br />
<strong>and</strong> effects <strong>of</strong> <strong>copper</strong> supplementation in<br />
laboratory animals; 2) recognition <strong>of</strong> natu<br />
rally occurring deficiency <strong>of</strong> <strong>copper</strong> in farm<br />
animals, especially cattle <strong>and</strong> sheep, in geo<br />
graphic areas where there exists a defi<br />
ciency <strong>of</strong> <strong>copper</strong> in the soil <strong>and</strong> vegeta<br />
tion; 3) exhaustive analyses <strong>of</strong> the <strong>copper</strong><br />
content <strong>of</strong> plant <strong>and</strong> animal tissues, in<br />
cluding those <strong>of</strong> man; 4) recognition <strong>of</strong><br />
states <strong>of</strong> <strong>copper</strong> deficiency in the human<br />
infant reared on diets similar to those em<br />
ployed in inducing <strong>copper</strong> deficiency states<br />
in experimental animals, combined with<br />
states <strong>of</strong> protein calorie malnutrition, in<br />
fection <strong>and</strong> other metabolic disorders;<br />
5) the effects <strong>of</strong> parenteral alimentation,<br />
especially in infants suffering from devel<br />
opmental anomalies <strong>of</strong> the alimentary tract<br />
<strong>and</strong> post-surgical stresses; 6) recognition<br />
<strong>of</strong> two genetically determined abnormali<br />
ties <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> in man ( Menkes'<br />
kinky-hair, or steely-hair, syndrome affect<br />
ing primarily young infants <strong>and</strong> Wilson's<br />
disease, affecting the adolescent <strong>and</strong> adult,<br />
together with therapeutic measures for the<br />
same; <strong>and</strong> 7) exhaustive studies <strong>of</strong> the<br />
many <strong>copper</strong>-containing proteins distrib<br />
uted throughout the blood <strong>and</strong> other tissues,<br />
<strong>and</strong> their role in metabolic processes <strong>of</strong><br />
man <strong>and</strong> lower animals.<br />
In the present review it is not possible<br />
to consider the first three categories <strong>of</strong> re<br />
search mentioned above, other than to<br />
make reference to certain observations<br />
which have particular relevance to the un<br />
derst<strong>and</strong>ing <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> <strong>and</strong> re<br />
quirements <strong>of</strong> man. For additional infor<br />
mation on the first areas <strong>of</strong> research men<br />
tioned the reader is referred to a rather<br />
extensive series <strong>of</strong> reviews (6, 7, 32, 71,<br />
99, 139, 140, 185, 211, 257, 319, 461, 493,<br />
779, 798).<br />
COPPER IN THE HUMAN BODY<br />
Many opinions have been expressed con<br />
cerning the total <strong>copper</strong> content <strong>of</strong> the<br />
human body. More than 40 years ago Chou<br />
<strong>and</strong> Adolph (115), in studies based upon<br />
analyses <strong>of</strong> nine organs from two adults at<br />
autopsy, in which they found an average <strong>of</strong><br />
about 116 mg, estimated that the human<br />
body contained between 100 <strong>and</strong> 150 mg<br />
<strong>of</strong> <strong>copper</strong>. Since then estimates have been<br />
reduced. Cartwright <strong>and</strong> Wintrobe (105)<br />
considered 80 mg <strong>of</strong> <strong>copper</strong> to be a more<br />
reasonable estimate for a 70-kg man. Sass-<br />
Kortsak (666), on the basis <strong>of</strong> data <strong>of</strong><br />
Tipton <strong>and</strong> Cook (781), calculated a mean<br />
<strong>of</strong> 75 mg, with a range <strong>of</strong> 50 to 120 mg.<br />
A slightly lower estimate <strong>of</strong> 70 mg has been<br />
given recently by Sumino et al. (762),<br />
based upon analyses <strong>of</strong> 18 different organs<br />
<strong>and</strong> tissues <strong>of</strong> 30 Japanese subjects, victims<br />
<strong>of</strong> accidental deaths, 28 <strong>of</strong> whom ranged<br />
in age from 20 to more than 60 years.<br />
These investigators also estimated that<br />
about one-third <strong>of</strong> body <strong>copper</strong> was in the<br />
liver <strong>and</strong> brain combined, one-third in the<br />
musculature, <strong>and</strong> the remaining third dis<br />
persed in other tissues. The mean content<br />
<strong>of</strong> <strong>copper</strong> in human liver is about \5% <strong>of</strong><br />
total body <strong>copper</strong> (668).<br />
It has long been recognized that the<br />
liver <strong>and</strong> brain contain a much higher con<br />
centration <strong>of</strong> <strong>copper</strong> than other organs <strong>and</strong><br />
tissues, amounting to about 8 mg in each<br />
<strong>of</strong> these organs ( 105). The liver content<br />
is, in large part, related to its function as<br />
a storage organ for <strong>copper</strong> <strong>and</strong> also as the<br />
only site <strong>of</strong> synthesis <strong>and</strong> release <strong>of</strong> ceruloplasmin.<br />
Copper is unevenly distributed<br />
in the brain. While some investigators re<br />
port very little difference in the <strong>copper</strong><br />
content <strong>of</strong> grey <strong>and</strong> white matter <strong>of</strong> the<br />
cerebral cortex (102, 138, 613), others have<br />
found a considerably higher content in the<br />
grey than in the white matter ( 134, 138,<br />
178, 780, 828). The substantia nigra <strong>and</strong><br />
locus ceruleus, both components <strong>of</strong> the<br />
grey matter, are exceptionally rich in cop<br />
per ( 134, 178). Possible relationships <strong>of</strong><br />
the pigmented nerve cells <strong>of</strong> the locus<br />
ceruleus to their content <strong>of</strong> melanin <strong>and</strong><br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1983<br />
to tyrosinase have been suggested <strong>and</strong> ex<br />
plored by in vitro studies with no positive<br />
results, possibly due to the use <strong>of</strong> necropsy<br />
material rather than fresh tissue (178).<br />
Gubler et al. (285) record mean <strong>copper</strong><br />
values (in terms <strong>of</strong> /*g/g wet weight) <strong>of</strong><br />
6.3, 6.5, 5.7, 4.2, 2.6 <strong>and</strong> 1.7, repsectively,<br />
for whole brain, cerebellum, basal ganglia,<br />
cerebral cortex, brain stem <strong>and</strong> cervical<br />
spinal cord. These data are based on five<br />
adult males who died following traumatic<br />
injuries.<br />
Compared to the liver <strong>and</strong> brain, lesser<br />
levels <strong>of</strong> <strong>copper</strong> are found in the heart,<br />
kidney, pancreas, spleen, lungs, bone <strong>and</strong><br />
skeletal muscle, diminishing generally in<br />
the order mentioned (102, 115, 182, 208,<br />
285, 397, 509, 691, 762, 786). Gubler et al.<br />
(285 ) give mean values (/¿g/gwet weight )<br />
<strong>of</strong> 5.3, 3.2, 2.2, 1.0 <strong>and</strong> 0.9, respectively, for<br />
liver, heart, kidney, spleen <strong>and</strong> skeletal<br />
muscle. The total <strong>copper</strong> content <strong>of</strong> the<br />
whole liver, brain, kidney, heart <strong>and</strong><br />
spleen, respectively, is estimated to be 8.0,<br />
8.0, 1.2, 0.9 <strong>and</strong> 0.1 mg (105).<br />
In the fetus <strong>and</strong> infant, the distribution<br />
<strong>of</strong> <strong>copper</strong> is quite different from that in the<br />
adult. During fetal life there is a progres<br />
sive increase in percentage <strong>of</strong> <strong>copper</strong> <strong>and</strong><br />
iron in the body, while that <strong>of</strong> zinc re<br />
mains relatively constant (704), such that<br />
at birth the liver <strong>and</strong> spleen contain about<br />
1/2 the <strong>copper</strong>, l/4th the zinc <strong>and</strong> l/8th<br />
the iron in the whole body (846). Liver <strong>of</strong><br />
the newborn has an exceptionally high con<br />
centration <strong>of</strong> <strong>copper</strong>, approximately 6 to<br />
10 times that <strong>of</strong> adult man (538, 844, 846).<br />
After the first few months <strong>of</strong> life these con<br />
centrations decrease rapidly to those <strong>of</strong> the<br />
adult (69, 565, 845). During the transition<br />
from infancy to the early years <strong>of</strong> life, there<br />
is a decrease in <strong>copper</strong> concentration in<br />
kidney, heart <strong>and</strong> spleen <strong>and</strong> an increase<br />
in brain (69, 845), which is relatively low<br />
in the newborn (780).<br />
On the basis <strong>of</strong> analyses <strong>of</strong> major organs<br />
<strong>of</strong> man in different geographic areas Forssen<br />
(222) states that the Finns have some<br />
what lower <strong>copper</strong> levels than Americans,<br />
<strong>and</strong> that peoples <strong>of</strong> Africa <strong>and</strong> the Near<br />
<strong>and</strong> Far East have levels 1.5 to 2 times<br />
those <strong>of</strong> the Finns. Whether these obser<br />
vations may or may not reflect differences<br />
in soils, dietary habits or ethnic factors is<br />
not clear. They are in accord with earlier<br />
observations <strong>of</strong> Schroeder et al. (691) who<br />
found that other races have larger mean<br />
amounts <strong>of</strong> <strong>copper</strong> in aorta, kidney, liver,<br />
lung <strong>and</strong> spleen than do Americans, Swiss<br />
<strong>and</strong> African Caucasoids, <strong>and</strong> that Orientals<br />
have especially high values.<br />
The levels <strong>of</strong> <strong>copper</strong> in human hair have<br />
been the subject <strong>of</strong> numerous studies in<br />
the hope that such information might pro<br />
vide some measure <strong>of</strong> the <strong>copper</strong> status <strong>of</strong><br />
the body, especially states <strong>of</strong> <strong>copper</strong> de<br />
ficiency. Wide individual variations with<br />
respect to age <strong>and</strong> sex (415), to hair pig<br />
mentation (357) <strong>and</strong> to exogenous con<br />
tamination (298) have indicated that cop<br />
per levels in hair have little meaningfulness<br />
in evaluating the status <strong>of</strong> <strong>copper</strong> in<br />
man (798). However, a recent report<br />
(378) states that determination <strong>of</strong> <strong>copper</strong><br />
in hair may be useful in evaluating total<br />
liver content <strong>of</strong> <strong>copper</strong>.<br />
COPPER PROTEINS<br />
Many proteins in tissues have the ca<br />
pacity to form <strong>copper</strong> complexes. Some <strong>of</strong><br />
these do not occur in mammalian species,<br />
but appear only in lower animal forms <strong>and</strong><br />
plants (e.g., hemocyanin, lacease, ascorbic<br />
acid oxidase, polyphenol oxidases, turacine).<br />
There have come to be recognized a<br />
large number <strong>of</strong> cuproproteins <strong>of</strong> mam<br />
malian species in which <strong>copper</strong> is part <strong>of</strong><br />
the molecular structure <strong>and</strong> in which there<br />
is a characteristic ratio between moles <strong>of</strong><br />
protein <strong>and</strong> atoms <strong>of</strong> associated <strong>copper</strong>.<br />
By virtue <strong>of</strong> these characteristics <strong>and</strong> the<br />
fact that the contained <strong>copper</strong> does not<br />
dissociate during isolation <strong>of</strong> the protein,<br />
these cuproproteins function as enzymes<br />
<strong>and</strong> are <strong>of</strong>ten grouped with other metalcontaining<br />
enzymes, all <strong>of</strong> which are re<br />
ferred to as "metalloproteins." Those ac<br />
cepted in this category include ceruloplasmin,<br />
Superoxide dismutase, cytochrome c<br />
oxidase, lysyl oxidase, tyrosinase <strong>and</strong> dopamine<br />
ß-hydroxylase. One other important<br />
metalloprotein, metallothionein, lacks enzymic<br />
properties but is capable <strong>of</strong> binding<br />
<strong>copper</strong> as well as certain other heavy<br />
metals. Several plasma <strong>and</strong> connective tis<br />
sue oxidases isolated from mammalian<br />
organs <strong>and</strong> tissues are at least <strong>copper</strong> de<br />
pendent (monoamine oxidase, spermine<br />
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1984 KARL E. MASON<br />
oxidase, diamine oxidase, benzylamineoxidase,<br />
etc.) but their significance in hu<br />
man <strong>metabolism</strong> is rather obscure. These<br />
proteins will be discussed in the general<br />
order mentioned. It may be noted that this<br />
panorama <strong>of</strong> cuproproteins has been sub<br />
ject to frequent changes in identification,<br />
description, terminology <strong>and</strong> functional at<br />
tributes over recent years. Hence, some<br />
statements <strong>and</strong> views expressed may be con<br />
sidered in a state <strong>of</strong> flux subject to consid<br />
erable revision as new advances are made.<br />
Ceruloplasmin (ferroxidase I)<br />
The classic studies <strong>of</strong> Holmberg <strong>and</strong><br />
Laurell (346, 347 ) described a blue plasma<br />
<strong>copper</strong>-containing protein which they<br />
named "ceruloplasmin." They reported that<br />
it was an «-globulinrepresenting almost all<br />
the <strong>copper</strong> present in mammalian plasma,<br />
<strong>and</strong> differed remarkably from all other<br />
such proteins in its molecular weight <strong>of</strong><br />
about 151,000 daltons, its <strong>copper</strong> content<br />
<strong>of</strong> about 0.32% <strong>and</strong> its content <strong>of</strong> eight<br />
atoms <strong>of</strong> <strong>copper</strong> per molecule. Human<br />
ceruloplasmin has been highly purified <strong>and</strong><br />
crystallized as tetragonal crystals (537).<br />
According to Scheinberg <strong>and</strong> Morell (676),<br />
who provide an excellent review <strong>of</strong> the sub<br />
ject, its molecular weight may vary from<br />
132,000 to 160,000, depending upon the<br />
method employed. More recently, it has<br />
been reported that its molecular weight is<br />
134,000 ±3,000, <strong>and</strong> that the number <strong>of</strong><br />
<strong>copper</strong> atoms varies from 6 to 6.6 (653).<br />
Of these, about one-half are in the cupric<br />
<strong>and</strong> the other half in the cuprous state<br />
(395). The nature <strong>of</strong> the <strong>copper</strong>-protein<br />
bond is not known. It is also recognized<br />
that ceruloplasmins from different animals<br />
show some cross-reactivity (395) <strong>and</strong> that<br />
they differ in p-phenylenediamine oxidase<br />
activity (502). Two other blue oxidases,<br />
ascorbate oxidase <strong>and</strong> lacease are enzymes<br />
found only in the plant world, where<br />
ceruloplasmin does not exist. An excellent<br />
comparison <strong>of</strong> the chemical structure <strong>and</strong><br />
physiological properties <strong>of</strong> these three ox<br />
idases is given by Dawson et al. (151).<br />
Ceruloplasmin contains about 8% car<br />
bohydrate, composed principally <strong>of</strong> glucosamine,<br />
mannose <strong>and</strong> galactose. Almost,<br />
if not all, <strong>of</strong> its oligosaccharide side chains<br />
are terminated by a sialic acid residue, ap<br />
parently essential for its survival in the cir<br />
culation (550). Copper is incorporated into<br />
ceruloplasmin only at the time <strong>of</strong> its syn<br />
thesis in the liver <strong>and</strong> the liver is its only<br />
site <strong>of</strong> synthesis (439, 742, 824). In the<br />
blood stream ceruloplasmin does not ex<br />
change its <strong>copper</strong> with other <strong>copper</strong> com<br />
plexes in the serum or blood cells. In vitro,<br />
<strong>copper</strong> is released from ceruloplasmin only<br />
after acidification, indicating strong protein<br />
binding <strong>of</strong> <strong>copper</strong>. Ceruloplasmin is hetero<br />
geneous, existing in several forms depend<br />
ing upon its prosthetic carbohydrate<br />
groups (65, 535).<br />
A moderate oxidase activity <strong>of</strong> cerulo<br />
plasmin toward a variety <strong>of</strong> substrates, <strong>of</strong><br />
which p-phenylenediamine is the best, was<br />
recognized by Holmberg <strong>and</strong> Laurell (348)<br />
in 1951. This enzyme reaction, as employed<br />
in the method <strong>of</strong> Ravin (629), has pro<br />
vided a useful means <strong>of</strong> qualitatively mea<br />
suring ceruloplasmin in body fluids. There<br />
has also been developed an immunological<br />
method using highly purified human ceru<br />
loplasmin as an antigen to incite specific<br />
antibody, usually in rabbits (339, 490, 674).<br />
Careful studies <strong>of</strong> Rosenberg et al. (644)<br />
demonstrate that both methods yield com<br />
parable results. Although ceruloplasmin is<br />
primarily a plasma protein, it is found also<br />
in synovial, ascitic <strong>and</strong> cerebrospinal fluids<br />
(744).<br />
Much interest has centered around this<br />
protein not only because <strong>of</strong> the mystery<br />
surrounding its true physiological functions<br />
but also because <strong>of</strong> impairment <strong>of</strong> its syn<br />
thesis <strong>and</strong> other possible derangements in<br />
Wilson's disease (p. 2009) <strong>and</strong> its low plasma<br />
levels associated with Menkes' kinky-hair<br />
(steely-hair) syndrome, a genetically de<br />
termined <strong>copper</strong>-deficiency disease in chil<br />
dren (p. 2005). It is <strong>of</strong> interest that in the<br />
early studies <strong>of</strong> Holmberg <strong>and</strong> Laurell,<br />
who were aware <strong>of</strong> recent evidence <strong>of</strong><br />
abnormalities <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> in<br />
Wilson's disease, ceruloplasmin plasma<br />
levels were found to be normal in a pa<br />
tient said to have Wilson's disease but who,<br />
several years later, was found not to be a<br />
victim <strong>of</strong> that disease. Hence, as stated by<br />
Scheinberg (671 ), "The capstone <strong>of</strong> physi<br />
ological significance to these discoveries<br />
ironically <strong>and</strong> undeservedly eluded them/'<br />
This same type <strong>of</strong> study carried out by<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1985<br />
Scheinberg <strong>and</strong> Gitlin (674) laid the basis<br />
<strong>of</strong> the concept that Wilson's disease is<br />
usually characterized by a lifelong defi<br />
ciency or absence <strong>of</strong> ceruloplasmin, <strong>and</strong><br />
that this deficiency is autosomal recessive<br />
in nature.<br />
Advances in knowledge concerning ceru<br />
loplasmin, as well as the role <strong>of</strong> <strong>copper</strong> in<br />
human <strong>metabolism</strong>, have in large part been<br />
the consequence <strong>of</strong> intensive research on<br />
the nature, cause <strong>and</strong> treatment <strong>of</strong> Wilson's<br />
disease, resulting in truly voluminous lit<br />
erature. In contrast, investigations con<br />
cerning Menkes" disease have focused<br />
largely on the role <strong>of</strong> metallothionein-like<br />
cuproproteins <strong>and</strong> defects in intestinal ab<br />
sorption <strong>of</strong> <strong>copper</strong>. Here also, in the in<br />
terim since its first recognition in 1962<br />
(513), an extensive literature has evolved.<br />
On the other side <strong>of</strong> the ledger are ad<br />
vances made during the past 12 years in<br />
elucidating the role <strong>of</strong> <strong>copper</strong> in iron me<br />
tabolism. These have, in large part, re<br />
solved questions in the minds <strong>of</strong> those<br />
investigators <strong>of</strong> 50 years ago (311, 313)<br />
who first recognized the important role <strong>of</strong><br />
<strong>copper</strong> in nutrition.<br />
The role <strong>of</strong> ceruloplasmin in biological<br />
processes began to receive some rational<br />
explanation about 1960, with evidence that<br />
in vitro it catalyzed the oxidation <strong>of</strong> fer<br />
rous iron ( 141). Further investigation <strong>of</strong><br />
this oxidase activity <strong>of</strong> ceruloplasmin pro<br />
vided indications that it represented an<br />
enzyme in human plasma responsible for<br />
oxidation <strong>of</strong> ferrous iron, <strong>and</strong> that the latter<br />
was the substrate for its greatest activity<br />
(504, 583, 585). Based on these findings,<br />
Osaki et al. (583) proposed that the name<br />
ferroxidase may be more useful than desig<br />
nating this enzyme as a "sky-blue substance<br />
from plasma." Since then, ceruloplasmin<br />
<strong>and</strong> ferroxidase I have become synonymous<br />
terms. However, in the discussion to follow<br />
the more common designation "ceruloplas<br />
min" will be used.<br />
The hypothesis proposed was that by<br />
this mechanism ceruloplasmin may play an<br />
important biological role in the release <strong>and</strong><br />
transfer <strong>of</strong> iron from storage cells to plasma<br />
transferrin. This concept was promptly<br />
supported by studies on <strong>copper</strong>-deficient<br />
swine (445, 446, 625), <strong>and</strong> by liver per<br />
fusion studies on dogs <strong>and</strong> swine (584).<br />
The studies on <strong>copper</strong>-deficient swine<br />
clearly demonstrated the effectiveness <strong>of</strong><br />
ceruloplasmin in counteracting the defec<br />
tive movement <strong>of</strong> iron from hepatic cells,<br />
reticuloendothelial cells <strong>and</strong> the intestinal<br />
mucosa to the plasma. This general subject<br />
has been extensively discussed <strong>and</strong> re<br />
viewed elsewhere (211, 226-230, 390, 446).<br />
The current concept <strong>of</strong> ceruloplasmin<br />
function in iron <strong>metabolism</strong> appears to be<br />
as follows. For normal hemoglobin syn<br />
thesis iron must be transported from stor<br />
age sites in the liver, reticuloendothelial<br />
system <strong>and</strong> intestine to the bone marrow<br />
by transferrin. In the storage sites iron is<br />
present in the ferric state, as ferritin. This<br />
iron can be reduced by reduced rib<strong>of</strong>lavin<br />
<strong>and</strong> rib<strong>of</strong>lavin derivatives, liberating fer<br />
rous iron from the ferritin. This ferrous<br />
iron is then oxidized catalytically back to<br />
ferric iron by virtue <strong>of</strong> the ferroxidase ac<br />
tivity <strong>of</strong> ceruloplasmin, allowing the Fe3*<br />
to combine with apotransferrin, as the<br />
initial step in the mobilization <strong>of</strong> stored<br />
iron.<br />
The mechanism <strong>of</strong> iron transfer from the<br />
storage cell to transferrin has not been<br />
clearly established. It is possible that Fe2+<br />
<strong>and</strong> ceruloplasmin interact to form a ferric<br />
intermediate that transfers iron to apo<br />
transferrin by a specific lig<strong>and</strong> exchange<br />
reaction (850). In any case, Fe3* combines<br />
with transferrin <strong>and</strong> provides the progeni<br />
tors <strong>of</strong> the erythrocytes in the bone mar<br />
row with the necessary iron for hemoglobin<br />
synthesis. As has been expressed (230),<br />
the life cycle <strong>of</strong> the <strong>copper</strong> in ceruloplas<br />
min is a one-time journey to the tissues or<br />
a return to the liver for resynthesis.<br />
Ceruloplasmin is a multifunctional pro<br />
tein involved not only in the mobilization<br />
<strong>of</strong> plasma iron but also in <strong>copper</strong> transport<br />
<strong>and</strong> in regulation <strong>of</strong> biogenic amines. The<br />
suggestion <strong>of</strong> Broman (65) that it func<br />
tions as a <strong>copper</strong>-transport protein has been<br />
well substantiated by animal studies indi<br />
cating that its <strong>copper</strong> atoms are trans<br />
ferred to cytochrome c oxidase <strong>and</strong> prob<br />
ably to other <strong>copper</strong>-containing proteins <strong>of</strong><br />
body tissues (230, 365). Close correla<br />
tions between low serum ceruloplasmin<br />
<strong>and</strong> low cytochrome c oxidase in leuco<br />
cytes in Wilson's disease (715) suggest<br />
that the same is true in man. Ceruloplas-<br />
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1986 KARL E. MASON<br />
min also has the capacity to oxidize natu<br />
rally occurring substances such as sero<br />
tonin, melatonin, epinephrine <strong>and</strong> norepinephrine,<br />
<strong>and</strong> may possibly play an<br />
important role in the control <strong>of</strong> blood <strong>and</strong><br />
tissue levels <strong>of</strong> biogenic amines (585).<br />
There are also suggestions that abnormali<br />
ties in <strong>copper</strong> <strong>metabolism</strong> may be involved<br />
in Parkinson's disease (34). For further<br />
details concerning the multifunctional na<br />
ture <strong>of</strong> ceruloplasmin, its biological func<br />
tions <strong>and</strong> catalytic activity the reader is<br />
referred to the recent reviews <strong>of</strong> Frieden<br />
<strong>and</strong> Hsieh (230) <strong>and</strong> <strong>of</strong> Sass-Kortsak <strong>and</strong><br />
Beam (668).<br />
Ceruloplasmin is not the only compound<br />
with ferroxidase-like activity in human<br />
plasma. In 1969, Lee et al. (444) reported<br />
the presence <strong>of</strong> citrate, differing from<br />
ceruloplasmin in that it is not inhibited by<br />
azide <strong>and</strong> yet has the same capacity to<br />
accelerate oxidation <strong>of</strong> ferrous ion to ferric<br />
ion <strong>and</strong>, possibly, to accelerate the rate <strong>of</strong><br />
reaction <strong>of</strong> ferric ion with transferrin. Ad<br />
ditional mechanisms, perhaps not involving<br />
<strong>copper</strong> directly, have been implicated in<br />
iron translocation (56a). A deterrent to<br />
recognition <strong>of</strong> a role <strong>of</strong> ceruloplasmin in<br />
hematopoietic functions <strong>of</strong> man has been<br />
the finding that many subjects suffering<br />
from Wilson's disease may have no demon<br />
strable plasma ceruloplasmin <strong>and</strong> yet show<br />
no evidence <strong>of</strong> iron-deficiency anemia.<br />
This anomaly seems now to have a reason<br />
able explanation with the isolation <strong>of</strong> an<br />
other cuproprotein from human serum by<br />
Topham <strong>and</strong> Frieden (788) which nor<br />
mally accounts for about 1% <strong>of</strong> the total<br />
ferroxidase activity. It has a molecular<br />
weight <strong>of</strong> about 800,000 daltpns <strong>and</strong> con<br />
tains approximately 0.8% <strong>copper</strong>. Its desig<br />
nation as ferroxidase II seems quite appro<br />
priate. It is a lipoprotein with a cholesterol<br />
<strong>and</strong> phosphatidylcholine content <strong>of</strong> ap<br />
proximately 207o- It differs from cerulo<br />
plasmin also in its yellow color <strong>and</strong> its lack<br />
<strong>of</strong> p-phenylenediamine oxidase activity,<br />
<strong>and</strong> may be responsible for the mainte<br />
nance <strong>of</strong> near-normal iron <strong>metabolism</strong>,<br />
despite low-levels or absence <strong>of</strong> plasma<br />
ceruloplasmin, in Wilson's disease. Whether<br />
citrate plays a comparable role is still a<br />
question. Furthermore, most <strong>of</strong> the new<br />
postulations with respect to the roles <strong>of</strong> the<br />
ferroxidases <strong>and</strong> citrate are based upon in<br />
vitro studies on human blood <strong>and</strong> observa<br />
tions on experimental animals. Their appli<br />
cability to man seems reasonable but re<br />
mains to be established.<br />
Other intriguing aspects <strong>of</strong> ceruloplas<br />
min are: 1) its increased plasma levels in<br />
response to estrogenic hormones, as in<br />
users <strong>of</strong> oral contraceptives <strong>and</strong> pregnant<br />
women; 2) its very low levels in plasma<br />
<strong>of</strong> the fetus <strong>and</strong> newborn; 3) its rapid<br />
synthesis by the newborn infant through<br />
utilization <strong>of</strong> a special neonatal hepatomitochondrocuprein<br />
not found at any other<br />
stage <strong>of</strong> life; 4) its stabilization at adult<br />
plasma levels at or about puberty; 5) its<br />
low serum levels in the genetically deter<br />
mined Wilson's <strong>and</strong> Menkes' diseases;<br />
<strong>and</strong> 6) suggestions that <strong>copper</strong> is incor<br />
porated into cytochrome c oxidase only if<br />
it is presented to the cell as ceruloplasmin<br />
(3, 65). These topics will be considered<br />
later.<br />
Superoxide dismutase<br />
Almost 40 years ago, Mann <strong>and</strong> Keilin<br />
(486) isolated two blue <strong>copper</strong> proteins<br />
from bovine erythrocytes <strong>and</strong> liver which<br />
they designated hemocuprein <strong>and</strong> hepatocuprein,<br />
respectively. Both proteins had<br />
molecular weights <strong>of</strong> about 35,000 daltons<br />
<strong>and</strong> contained 0.34% <strong>copper</strong>. Similar pro<br />
teins were later isolated from human eryth<br />
rocytes (erythrocuprein) by Markowitz et<br />
al. (490), from human brain (cerebrocuprein)<br />
by Porter <strong>and</strong> Ainsworth (612)<br />
<strong>and</strong> from adult human liver (hepatocuprein)<br />
by Porter et al. (616).<br />
Subsequently, Carneo <strong>and</strong> Deutsch (95)<br />
clearly demonstrated that these <strong>copper</strong><br />
proteins were identical <strong>and</strong> proposed the<br />
term cytocuprein to encompass them. They<br />
later considered the term inappropriate,<br />
since the protein also contained 2-g atoms<br />
<strong>of</strong> zinc per mole (96). On the basis that<br />
these cuproproteins catalyze the dismutation<br />
<strong>of</strong> superoxide-free radical ions, <strong>and</strong><br />
thus have true enzymatic function, the<br />
designation "superoxide dismutase" was<br />
proposed by McCord <strong>and</strong> Fridovich (500,<br />
501), <strong>and</strong> is now in common usage. The<br />
primary function <strong>of</strong> superoxide dismutase<br />
appears to be that <strong>of</strong> scavenging the inter<br />
mediates <strong>of</strong> oxygen reduction in aerobic<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1987<br />
organisms, namely Superoxide aniónradical<br />
(500). Superoxide dismutase catalyzes the<br />
conversion <strong>of</strong> the Superoxide radical to<br />
hydrogen peroxide plus oxygen <strong>and</strong> the<br />
hydrogen peroxide is removed by catalyses<br />
<strong>and</strong> peroxidases. Thus, Superoxide dismu<br />
tase helps protect the cell from the damag<br />
ing effects <strong>of</strong> oxygen toxicity.<br />
Cytochrome c oxidase<br />
Although cytochrome c oxidase has been<br />
recognized as a <strong>copper</strong> <strong>and</strong> heme contain<br />
ing protein for almost 40 years, the state<br />
<strong>and</strong> function <strong>of</strong> its <strong>copper</strong> component have<br />
been very difficult to clarify. The history<br />
<strong>of</strong> these explorations, up to 1966, has been<br />
well reviewed by Beinert (43) <strong>and</strong> Wharton<br />
<strong>and</strong> Gibson (837). It has not yet been<br />
possible to clearly establish its molecular<br />
weight, or whether it contains one or two<br />
<strong>copper</strong> ions <strong>and</strong> one or two heme groups.<br />
This enzyme is found in all aerobic cells<br />
<strong>and</strong> is mainly responsible for the introduc<br />
tion <strong>of</strong> oxygen into the oxidative machinery<br />
that produces energy for biochemical syn<br />
thesis <strong>and</strong> for physical activity. As the<br />
terminal enzyme in the electron transport<br />
chain, it catalyzes the oxidation <strong>of</strong> reduced<br />
cytochrome c by molecular oxygen <strong>and</strong>, in<br />
the process, oxygen is reduced to water. It<br />
represents an enzyme vital to essentially<br />
all forms <strong>of</strong> life, by virtue <strong>of</strong> serving as the<br />
terminal enzyme in the oxidative phosphorylation<br />
process <strong>of</strong> living cells.<br />
A significant decrease in cytochrome c<br />
oxidase activity is considered a major cause<br />
<strong>of</strong> neural <strong>and</strong> cardiac abnormalities ob<br />
served in the <strong>of</strong>fspring <strong>of</strong> different animal<br />
species fed diets deficient in <strong>copper</strong>. Neural<br />
lesions varying from defective myelination<br />
to necrosis occur in lambs <strong>and</strong> goats (361,<br />
798), in the guinea pig (201, 202) <strong>and</strong> in<br />
the rat (92, 401 ). Myocardial abnormalities<br />
ranging from focal failure <strong>of</strong> tissue respi<br />
ration to myocardial hypertrophy <strong>and</strong><br />
acute cardiac failure occur in cattle (798),<br />
swine (707) <strong>and</strong> rats (3, 92, 241, 316).<br />
Lysyl oxidase<br />
This <strong>copper</strong>-containing enzyme, lysyl<br />
oxidase, is one <strong>of</strong> the amine oxidases (308 )<br />
but is given separate consideration here<br />
because <strong>of</strong> its well established status <strong>and</strong><br />
special importance. It has now been par<br />
tially purified from several sources (308,<br />
601, 648a, 650, 716). Its major function<br />
appears to be to catalyze the oxidative deamination<br />
<strong>of</strong> e-amino groups <strong>of</strong> peptidyl<br />
lysine or hydroxylysine to form «-aminoadipic-S-semialdehyde<br />
derivatives as a first<br />
step in the cross-linking <strong>of</strong> immature elastin<br />
<strong>and</strong> collagen into stabile fibrils. In collagen,<br />
the cross-links are derived from either ly<br />
sine or hydroxylysine. In elastin, however,<br />
hydroxylysine <strong>and</strong> hydroxylysine-derived<br />
cross-links are not present.<br />
The story <strong>of</strong> lysyl oxidase goes back to<br />
early studies <strong>of</strong> experimental <strong>copper</strong>-defi<br />
ciency in the chick (331, 571, 734) <strong>and</strong><br />
pigs (93, 94, 129, 707) in which dissecting<br />
aneurisms <strong>and</strong> sometimes rupture <strong>of</strong> the<br />
aorta <strong>and</strong> large vessels were noted. These<br />
vascular defects were ascribed to abnor<br />
malities <strong>of</strong> the elastic component <strong>of</strong> the<br />
vascular wall <strong>and</strong> to low tissue levels <strong>of</strong><br />
<strong>copper</strong>. These observations coincided in<br />
time with the demonstration by Partridge<br />
et al. (594) that the vital crosslinking<br />
groups in elastin, which they named "desmosine"<br />
<strong>and</strong> "isodesmosine," were formed<br />
from lysine. Other studies on <strong>copper</strong>-defi<br />
cient chicks (117) gave evidence <strong>of</strong> a role<br />
<strong>of</strong> <strong>copper</strong> in crosslinking <strong>of</strong> collagen. It is<br />
now clear that the basis for these observa<br />
tions was the role <strong>of</strong> <strong>copper</strong> as a c<strong>of</strong>actor<br />
for lysyl oxidase (648a).<br />
Further, a foundation was laid for the<br />
development <strong>of</strong> current concepts related<br />
to the role <strong>of</strong> <strong>copper</strong> with respect to bio<br />
chemical <strong>and</strong> structural abnormalities seen<br />
in collagen <strong>and</strong> elastin in experimental ani<br />
mals, livestock (798) <strong>and</strong> non-domestic<br />
animals (219). Furthermore, one can ob<br />
serve an example <strong>of</strong> application <strong>of</strong> knowl<br />
edge gained from experimentally induced<br />
deficiency in the chick <strong>and</strong> lower animals<br />
to a better underst<strong>and</strong>ing <strong>of</strong> human dis<br />
orders, since many <strong>of</strong> the manifestations<br />
<strong>of</strong> Menkes' kinky-hair syndrome (513), a<br />
congenital state <strong>of</strong> <strong>copper</strong>-deficiency in<br />
young children, are also characterized by<br />
vascular <strong>and</strong> skeletal defects. For further<br />
details, the reader is referred to several<br />
recent reviews (93, 130, 568-570, 648a,<br />
650) <strong>and</strong> top. 2009.<br />
Tyrosinase ( phenoloxidase )<br />
This cuproprotein enzyme contains about<br />
Q.2c/f <strong>copper</strong>, or 1 atom per molecule (68),<br />
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1988 KARL E. MASON<br />
<strong>and</strong> has a molecular weight <strong>of</strong> about 33,000<br />
daltons. Actually it may represent a series<br />
<strong>of</strong> cuproproteins which catalyze a series <strong>of</strong><br />
reactions that convert tyrosine to melanin<br />
(213, 479). The enzymatic activity is<br />
thought to be associated with the mitochondrial<br />
component <strong>of</strong> cells. The skin <strong>and</strong><br />
uveal tissues <strong>of</strong> the eye in human albinos<br />
have no demonstrable tyresinase activity;<br />
hence, the absence <strong>of</strong> melanin. Decreased<br />
production <strong>of</strong> tyrosinase is responsible for<br />
the loss <strong>of</strong> hair pigmentation in animals<br />
deficient in <strong>copper</strong> <strong>and</strong> in infants with<br />
Menkes' steely-hair syndrome.<br />
Dopamine ß-hijdroxylase<br />
This cuproprotein (3,4-dihydroxyphenylethylamine<br />
ß-hydroxylase) is an oxidase<br />
containing 4 to 7 <strong>copper</strong> atoms per mole<br />
cule <strong>and</strong> having a molecular weight <strong>of</strong><br />
about 290,000 daltons (232). It was origi<br />
nally isolated from beef adrenals <strong>and</strong> later<br />
from cattle brain <strong>and</strong> hearts. In experimen<br />
tal animals it appears to serve a function<br />
in catalyzing the conversion <strong>of</strong> dopamine<br />
to form norepinephrine (568, 570). A com<br />
parable role in man may be assumed but<br />
has not been proven.<br />
Metallothionein<br />
The rather tortuous history <strong>of</strong> identifica<br />
tion <strong>and</strong> nomenclature <strong>of</strong> <strong>copper</strong> proteins<br />
leading up to the proper recognition <strong>of</strong><br />
Superoxide dismutase has been somewhat<br />
paralleled by the history <strong>of</strong> metallothionein.<br />
This designation was given by Kagi<br />
<strong>and</strong> Vallee (392, 393) to a protein iso<br />
lated from equine <strong>and</strong> human kidney, with<br />
a molecular weight <strong>of</strong> about 10,000 daltons,<br />
a content <strong>of</strong> 26 sulfhydryl groups per mole,<br />
<strong>and</strong> a capacity to bind zinc <strong>and</strong> cadmium<br />
as well as <strong>copper</strong>. Metallothionein is not<br />
an enzyme. By virtue <strong>of</strong> its high content<br />
<strong>of</strong> sulfhydryl groups it binds <strong>copper</strong> by<br />
forming mercaptides. In fact, it may repre<br />
sent a family <strong>of</strong> metallothioneins specifi<br />
cally designed for the binding <strong>of</strong> either<br />
one metal or specific groups <strong>of</strong> metals such<br />
as <strong>copper</strong>, zinc <strong>and</strong> cadmium.<br />
Accumulating knowledge <strong>of</strong> metallothionein<br />
or metallothionein-like proteins, with<br />
variable affinities for <strong>copper</strong>, especially as<br />
they occur <strong>and</strong> function in the intestinal<br />
mucosa <strong>and</strong> liver, may well add greatly to<br />
our underst<strong>and</strong>ing <strong>of</strong> the two clinical dis<br />
orders <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> in man;<br />
namely, Wilson's disease <strong>and</strong> Menkes'<br />
steely-hair syndrome. For example, one<br />
recognized <strong>and</strong> basic defect in Menkes'<br />
disease is an inadequate transport <strong>of</strong> cop<br />
per across the intestinal mucosa (145,<br />
147). It is possible that the retention <strong>of</strong><br />
abnormal amounts <strong>of</strong> <strong>copper</strong> in the in<br />
testinal mucosa involves increased affinity<br />
<strong>of</strong> a mucosal metallothionein or <strong>of</strong> another<br />
protein not normally involved in <strong>copper</strong><br />
transport (351). Furthermore, there is evi<br />
dence that the usual metallothionein con<br />
cerned with storage <strong>and</strong> release <strong>of</strong> ab<br />
sorbed <strong>copper</strong> in the liver may, in Wilson's<br />
disease, be <strong>of</strong> an abnormal type with a<br />
binding constant about four times greater<br />
than normal ( 197).<br />
An unusual variant <strong>of</strong> metallothionein,<br />
referred to as neonatal hepatomitochondrocuprein,<br />
has been isolated from immature<br />
bovine liver <strong>and</strong> from human newborn<br />
liver (615). It contains 25% cystine <strong>and</strong><br />
about 49¿<strong>copper</strong>, which is at least 10 times<br />
that <strong>of</strong> adult hepatocuprein. It increases<br />
just before birth <strong>and</strong> decreases rapidly<br />
during the first few months <strong>of</strong> postnatal<br />
life, as it is released for synthesis <strong>of</strong> ceruloplasmin,<br />
which is present in plasma in<br />
small amounts at birth. It is thought to<br />
represent a special storage type <strong>of</strong> protein<br />
to tide the newborn infant over the early<br />
period <strong>of</strong> life when breast milk does not<br />
meet normal <strong>requirements</strong> (610 ). Although<br />
concentrated in the heavy mitochondrial<br />
fraction, it is not a true mitochondrial con<br />
stituent in that some <strong>of</strong> it may be localized<br />
in lysosomes <strong>of</strong> a "heavy" type, <strong>and</strong> prob<br />
ably represents a polymer <strong>of</strong> metallothio<br />
nein (614).<br />
Other cuproproteins<br />
Since the identification <strong>and</strong> characteriza<br />
tion <strong>of</strong> a sulfhydryl-rich cuproprotein in<br />
human liver with a molecular weight <strong>of</strong><br />
8,000 to 10,000 daltons, designated L-6D<br />
(536, 703), there have been described<br />
similar cuproproteins derived from chick<br />
intesane (733), rat liver (856), rat intes<br />
tine ( 198), adult human liver ( 74 ) <strong>and</strong><br />
human fetal liver (654). These proteins<br />
differ somewhat with respect to estimated<br />
molecular weight, <strong>copper</strong> content <strong>and</strong><br />
amino-acid components, especially cysteine<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1989<br />
<strong>and</strong> thionein, which may reflect differences<br />
in methods employed in isolation <strong>and</strong><br />
analysis as well as species differences in<br />
composition. There is good reason to be<br />
lieve that they have important functions<br />
in <strong>copper</strong>-homeostatic mechanisms such as<br />
storage, transport <strong>and</strong> detoxification, espe<br />
cially in the intestine <strong>and</strong> liver. Unques<br />
tionably, future research will clarify many<br />
<strong>of</strong> these differences <strong>and</strong> delineate more<br />
clearly the physiological <strong>and</strong> biochemical<br />
role(s) <strong>of</strong> these low-molecular weight cuproproteins.<br />
Monoamine oxidases. Aside from lysyl<br />
oxidase, there are other amine oxidases that<br />
are present in connective tissues (116, 330,<br />
334, 335). It is now presumed that these<br />
oxidases serve in deamination <strong>of</strong> norepinephrine,<br />
serotonin <strong>and</strong> histamine. Amine<br />
oxidases, presumably containing <strong>copper</strong>,<br />
derived from beef <strong>and</strong> swine plasma have<br />
been highly purified <strong>and</strong> crystallized (73,<br />
308, 649, 807, 867). A monoamine oxidase<br />
from human plasma has also been purified<br />
(505) <strong>and</strong> is said to increase in states <strong>of</strong><br />
congestive heart failure <strong>and</strong> parenchymal<br />
liver disease (506). However, whether or<br />
not this enzyme is <strong>copper</strong> dependent is not<br />
clear.<br />
Diamine oxidase. Purification <strong>of</strong> diamine<br />
oxidase from pig kidney has been carried<br />
out by many investigators since 1943. Its<br />
crystallization was first reported by<br />
Yamada et al. (866) who described it as a<br />
pink <strong>copper</strong>-protein containing 2.17 atoms<br />
<strong>of</strong> <strong>copper</strong> per molecule, capable <strong>of</strong> oxidiz<br />
ing histamine, cadaverine, putrescine <strong>and</strong><br />
other like substances. The identity <strong>of</strong> this<br />
enzyme with histiminase had previously<br />
been proposed by Mondovi et al. (534).<br />
Whether this enzyme plays a role in human<br />
<strong>metabolism</strong> remains to be determined.<br />
Albocuprein I <strong>and</strong> II. These two color<br />
less cuproproteins, neither <strong>of</strong> which possess<br />
enzyme activity or undergo alterations in<br />
pathological states, have been isolated<br />
from human brains (238). Both contain<br />
hexoses. Albocuprein II may be the pri<br />
mary <strong>copper</strong>-containing protein <strong>of</strong> the<br />
brain (550).<br />
Uncase (urate oxidase}. This cuproprotein,<br />
found in the kidney <strong>and</strong> liver <strong>of</strong> lower<br />
mammals, has a molecular weight <strong>of</strong> about<br />
110,000, a <strong>copper</strong> content <strong>of</strong> 0.6% <strong>and</strong> is<br />
involved in the catabolism <strong>of</strong> uric acid.<br />
There are no recognized effects <strong>of</strong> its de<br />
ficiency or excess in animals. It does not<br />
occur in primates (478).<br />
Tryptophan-2,3-dioxygenase. This heme<br />
protein with a molecular weight <strong>of</strong> 167,000<br />
daltons <strong>and</strong> 2 cuprous atoms per molecule,<br />
isolated from rat liver cytosol (694), is<br />
said to catalyze the insertion <strong>of</strong> molecular<br />
oxygen into the pyrrole ring <strong>of</strong> L-tryptophan<br />
(58).<br />
Pink <strong>copper</strong> protein. A pink <strong>copper</strong> pro<br />
tein with a molecular weight <strong>of</strong> about<br />
32,000 has been isolated from human erythrocytes<br />
(631). The biological function <strong>of</strong><br />
this protein still remains to be demon<br />
strated.<br />
Mitochondrial monoamine oxidase. This<br />
enzyme, previously obtained in highly puri<br />
fied form from many sources (beef <strong>and</strong> hog<br />
plasma, human plasma, rabbit serum, liver<br />
<strong>and</strong> kidney <strong>of</strong> several animal species, <strong>and</strong><br />
human placenta) has been isolated from<br />
human liver, <strong>and</strong> identified as a flavoprotein<br />
with a molecular weight <strong>of</strong> 64,000<br />
(563). It is said to have the ability to<br />
oxidize epinephrine <strong>and</strong> serotonin. On the<br />
other h<strong>and</strong>, its status as a cuproenzyme has<br />
since been questioned (730).<br />
The large number <strong>of</strong> cuproproteins <strong>and</strong><br />
the multiplicity <strong>of</strong> their enzymatic func<br />
tions, as well as the homeostatic mecha<br />
nisms involved in normal <strong>metabolism</strong> <strong>of</strong><br />
<strong>copper</strong>, emphasize its great importance in<br />
mammalian nutrition. For further aspects<br />
<strong>of</strong> cuproproteins, the reader is referred to a<br />
series <strong>of</strong> reviews (191-195, 211, 308, 309,<br />
350, 458, 550, 568-570, 634, 650, 663, 671,<br />
676).<br />
ABSORPTION OF COPPER<br />
As is true <strong>of</strong> most metals, absorption <strong>of</strong><br />
<strong>copper</strong> is regulated at the level <strong>of</strong> the in<br />
testinal mucosa <strong>and</strong> excretion is predomi<br />
nantly through the intestinal tract, either<br />
via the bile or as nonabsorbed <strong>copper</strong>.<br />
Urinary excretion is negligible in normal<br />
healthy man, amounting to about 1 to 2%<br />
<strong>of</strong> the intake.<br />
Although the site <strong>of</strong> maximal absorption<br />
<strong>of</strong> <strong>copper</strong> varies among different mam<br />
malian species, in man absorption occurs<br />
primarily in the stomach <strong>and</strong> duodenum.<br />
This conclusion is based upon observations<br />
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1990 KARL E. MASON<br />
that after oral administration <strong>of</strong> radio<br />
active <strong>copper</strong> the isotope appears rapidly<br />
in the blood, reaching maximum levels<br />
within 1 to 3 hours, as further discussed<br />
below. Concepts <strong>of</strong> the processes <strong>of</strong> absorp<br />
tion <strong>of</strong> <strong>copper</strong> from dietary sources in man<br />
are based in large part upon studies <strong>of</strong><br />
experimental animals.<br />
Animal studies indicate that at least two<br />
mechanisms are concerned in <strong>copper</strong> ab<br />
sorption ( 132, 256 ). One <strong>of</strong> these, pre<br />
sumably an energy-dependent one, in<br />
volves the absorption <strong>of</strong> complexes <strong>of</strong> cop<br />
per <strong>and</strong> amino acids. There is also evidence<br />
that L-amino acids facilitate the absorption<br />
<strong>of</strong> <strong>copper</strong>, <strong>and</strong> that absorption progres<br />
sively decreases with increasing molecular<br />
size <strong>of</strong> <strong>copper</strong>-amino acid complexes (408 ).<br />
The other mechanism is an enzymatic one<br />
involving the binding <strong>of</strong> <strong>copper</strong> to, <strong>and</strong><br />
successive release from, macromolecular<br />
proteins.<br />
Studies <strong>of</strong> experimental animals indicate<br />
that mechanisms <strong>of</strong> <strong>copper</strong> storage <strong>and</strong><br />
transfer involve not only metallothionein as<br />
first identified in the chick intestinal mu<br />
cosa (733), but that there also may exist<br />
a variety <strong>of</strong> metallothioneins, differing<br />
slightly in amino-acid content <strong>and</strong> metalbinding<br />
characteristics (518, 562). There<br />
is real need for better determination <strong>of</strong> the<br />
extent to which findings in experimental<br />
animals have application to the problem <strong>of</strong><br />
<strong>copper</strong> absorption <strong>and</strong> <strong>metabolism</strong> in man.<br />
This applies also to the many factors in<br />
animals which are known to interfere with<br />
<strong>copper</strong> absorption through various mecha<br />
nisms (competition for binding sites by<br />
zinc, perhaps to a much lesser extent by<br />
cadmium; interactions between molyb<br />
denum, sulphates <strong>and</strong> <strong>copper</strong>; the effects <strong>of</strong><br />
dietary phytates, <strong>and</strong> the influence <strong>of</strong><br />
ascorbic acid intake). In the case <strong>of</strong> the<br />
latter, dietary deficiency results in in<br />
creased liver <strong>and</strong> plasma <strong>copper</strong> (337),<br />
whereas increased oral intake decreases<br />
the absorption <strong>and</strong> retention <strong>of</strong> <strong>copper</strong> in<br />
the chick (333), rabbit (370), pig (254)<br />
<strong>and</strong> rat (199). Moreover, in the pig, high<br />
intake <strong>of</strong> ascorbic acid can overcome the<br />
effects <strong>of</strong> excess <strong>copper</strong> either by inter<br />
fering with <strong>copper</strong> absorption or by in<br />
creasing the absorption <strong>and</strong> utilization <strong>of</strong><br />
iron (254). Such evidence raises questions<br />
as to whether due consideration has been<br />
given to the secondary effects that may be<br />
related to the somewhat astronomical hu<br />
man intakes <strong>of</strong> ascorbic acid currently in<br />
vogue. It is also important to note that the<br />
type, configuration <strong>and</strong> degree <strong>of</strong> polymeri<br />
zation <strong>of</strong> amino acids present in the in<br />
testine can influence the absorption <strong>of</strong> cop<br />
per (798). Moreover, little is known re<br />
garding the chemical forms <strong>of</strong> <strong>copper</strong> in<br />
foods, <strong>and</strong> the influence <strong>of</strong> different meth<br />
ods <strong>of</strong> cooking upon its availability.<br />
The great difficulty in determining that<br />
portion <strong>of</strong> dietary <strong>copper</strong> which is ab<br />
sorbed becomes apparent when one con<br />
siders the many variable factors affecting<br />
<strong>copper</strong> absorption such as: competition for<br />
protein-binding sites in the intestinal lumen<br />
<strong>and</strong> mucosa; inhibition <strong>of</strong> binding at these<br />
sites; difficulties in measuring the amount<br />
<strong>of</strong> <strong>copper</strong> secreted by the bile, by accessory<br />
gl<strong>and</strong>s <strong>of</strong> the digestive tract <strong>and</strong> by the in<br />
testinal mucosa; <strong>and</strong> possible reabsorption<br />
by the intestinal mucosa <strong>of</strong> some <strong>of</strong> the<br />
secreted <strong>copper</strong>. Excellent reviews <strong>of</strong> this<br />
subject have been presented by Dowdy<br />
(169), Evans (192), Hambidge <strong>and</strong> Wairavens<br />
(301) <strong>and</strong> Van Campen (804).<br />
Mechanisms<br />
The general concept <strong>of</strong> the mechanism<br />
involved in <strong>copper</strong> absorption is as follows:<br />
from ingested foodstuff <strong>copper</strong> is released<br />
either as ionic <strong>copper</strong> or as a <strong>copper</strong>amino<br />
acid complex. In the intestinal<br />
lumen there exists a high molecular weight<br />
protein which binds <strong>copper</strong> <strong>and</strong> preferen<br />
tially releases it to the plasma membranes<br />
on the luminal side <strong>of</strong> the absorptive cells<br />
<strong>of</strong> the mucosa. Within the absorptive cells<br />
is metallothionein (or metallothionein-like<br />
proteins) rich in sulfhydryl groups, which<br />
binds <strong>copper</strong> through formation <strong>of</strong> mercaptide<br />
bonds. Since this cuproprotein<br />
serves as a storage depot <strong>and</strong> also releases<br />
<strong>copper</strong> to the plasma cell membrane on the<br />
serosal side, it is considered to provide one<br />
<strong>of</strong> the protective <strong>and</strong> regulatory mecha<br />
nisms in <strong>copper</strong> homeostasis. However, it<br />
is not quite that simple, since its binding to<br />
<strong>copper</strong> is constantly in competition with<br />
other trace elements <strong>and</strong> can also be in<br />
fluenced by other dietary components such<br />
as the sulphate radical, phytates, fiber <strong>and</strong><br />
ascorbic acid.<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1991<br />
Dynamic studies with radioactive cop<br />
per have yielded important information. In<br />
man, oral administration <strong>of</strong> 64Cu or 67Cu is<br />
followed by a prompt appearance <strong>of</strong> the<br />
isotope in the blood serum, indicating at<br />
least major absorption from the stomach<br />
<strong>and</strong> duodenum (40, 42, 80, 179, 387, 832).<br />
Within 1 or 2 hours the isotope is bound<br />
to serum albumin <strong>and</strong> amino acids (41,<br />
80). There follows a sharp decline as the<br />
isotope is taken up by the liver. Subse<br />
quently, there occurs increased activity <strong>of</strong><br />
the serum for 48 to 72 hours as the liver<br />
incorporates the radioisotope into newly<br />
synthesized ceruloplasmin <strong>and</strong> releases it<br />
into the blood (40, 41, 80, 179). That not<br />
immediately extracted by the liver remains<br />
in the serum attached to albumin or amino<br />
acids, or is used to maintain erythrocyte<br />
<strong>copper</strong> levels.<br />
Amount<br />
The proportion <strong>of</strong> dietary <strong>copper</strong> that is<br />
actually absorbed is very important in the<br />
making <strong>of</strong> judgments on balance studies<br />
<strong>and</strong> their bearing upon normal human re<br />
quirements for <strong>copper</strong>. Unfortunately the<br />
information currently available is both<br />
meager <strong>and</strong> somewhat inconclusive. Van<br />
Ravensteyn (805) estimated that about<br />
25% <strong>of</strong> <strong>copper</strong> (as CuSO4) added to the<br />
diet <strong>of</strong> normal men, is absorbed. Later,<br />
Cartwright <strong>and</strong> Wintrobe ( 105) stated that<br />
about 32% <strong>of</strong> ingested <strong>copper</strong> is absorbed.<br />
Early studies employing oral <strong>and</strong> intra<br />
venous administration <strong>of</strong> 64Cu to small<br />
numbers <strong>of</strong> control subjects in investiga<br />
tions primarily designed to determine<br />
whether or not there is an absorption de<br />
fect in Wilson's disease gave quite variable<br />
results, in terms <strong>of</strong> the percentage <strong>of</strong> the<br />
dose recovered in the feces over periods<br />
<strong>of</strong> 3 to 4 days (41, 80, 498). Furthermore,<br />
since radio-<strong>copper</strong> is both excreted into<br />
<strong>and</strong> absorbed by the gastrointestinal tract,<br />
fecal excretion provides a measure <strong>of</strong> re<br />
tention but not <strong>of</strong> true absorption. Assum<br />
ing that after 48 hours the serum concen<br />
tration <strong>of</strong> 64Cu is proportional to either an<br />
intravenous dose or to a certain fraction <strong>of</strong><br />
an oral dose absorbed, Sternlieb (736)<br />
estimates that on the basis <strong>of</strong> studies on<br />
49 normal subjects the mean absorption <strong>of</strong><br />
an oral dose <strong>of</strong> 2 mg <strong>of</strong> <strong>copper</strong> daily, is<br />
0.8 mg, or 40%. Weber et al. (832), em<br />
ploying similar methodology, concluded<br />
that the net absorption <strong>of</strong> orally administerred<br />
<strong>copper</strong> varies from 15 to 97%, with<br />
a mean <strong>of</strong> about 60%. A more sophisticated<br />
approach by Strickl<strong>and</strong> et al. (752), in<br />
volving four normal adults given 64Cu<br />
orally <strong>and</strong> ti7Cu intravenously, with <strong>copper</strong><br />
absorption calculated by whole body<br />
counting <strong>and</strong> by plasma 64Cu <strong>and</strong> 07Cu<br />
concentrations, gave a mean <strong>copper</strong> ab<br />
sorption value <strong>of</strong> 56% (range 40-70%). It<br />
was their opinion that if Sternlieb (736)<br />
had made allowance for a 20%) fecal ex<br />
cretion <strong>of</strong> <strong>copper</strong> (753), the three studies<br />
mentioned would have been in close agree<br />
ment. Quite recently King et al. (407) re<br />
ported an overall <strong>copper</strong> absorption <strong>of</strong> 57%<br />
based upon balance studies with the stable<br />
isotope 65Cu. On the basis <strong>of</strong> the evidence<br />
presented above, it seems reasonable to<br />
assume an absorption <strong>of</strong> 40 to 60% <strong>of</strong> the<br />
oral intake <strong>of</strong> <strong>copper</strong>, accepting the fact<br />
that there is wide individual variation.<br />
Copper absorption may be significantly<br />
impaired in states <strong>of</strong> severe, diffuse disease<br />
<strong>of</strong> the small bowel produced by sprue,<br />
lymphosarcoma, or scleroderma (739), or<br />
protein calorie malnutrition (474). In<br />
Menkes' steely-hair syndrome defects in<br />
intestinal transport <strong>and</strong> release constitute<br />
one <strong>of</strong> the primary bases for this disorder<br />
(p. 2007). Information concerning the role <strong>of</strong><br />
the lymphatics in the absorption <strong>of</strong> <strong>copper</strong><br />
is sadly lacking. Sternleib et al. (748) re<br />
port that in the dog <strong>and</strong> man the amount<br />
<strong>of</strong> an oral dose <strong>of</strong> 64Cu absorbed by the<br />
intestinal lymphatics is negligible. On the<br />
other h<strong>and</strong>, Trip et al. (789) found con<br />
centrations <strong>of</strong> <strong>copper</strong> ( non-ceruloplasmic )<br />
in the thoracic duct <strong>of</strong> three patients<br />
equivalent to or higher than in serum. It<br />
must be noted that these subjects suffered<br />
from carcinoma <strong>and</strong> Hodgkin's disease <strong>and</strong><br />
cannot be considered normal. Regrettably,<br />
the impossibility <strong>of</strong> obtaining thoracic duct<br />
lymph from normal healthy subjects <strong>of</strong>fers<br />
little hope <strong>of</strong> determining the role <strong>of</strong> the<br />
intestinal lymphatics in absorption <strong>of</strong><br />
<strong>copper</strong>.<br />
TRANSPORTOF COPPER<br />
Intestine to liver<br />
Following release from the intestinal<br />
mucosa, <strong>copper</strong> becomes bound to albumin<br />
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1992 KARL E. MASON<br />
<strong>and</strong> to amino acids in the portal blood. In<br />
the form <strong>of</strong> these complexes it can be mea<br />
sured colorimetrically following reaction<br />
with diethyldithiocarbamate, a <strong>copper</strong><br />
chelator. It is referred to as "direct react<br />
ing" or "labile" <strong>copper</strong>. By virtue <strong>of</strong> its<br />
homeostatic mechanisms the liver allows a<br />
portion <strong>of</strong> these loosely bound <strong>copper</strong> com<br />
plexes to pass directly to the systemic cir<br />
culation, where they constitute about 1%<br />
<strong>of</strong> plasma <strong>copper</strong>. Upon arrival at the liver,<br />
<strong>copper</strong> is released from albumin to hepatocyte<br />
cell membrane receptors from which it<br />
is transferred to the cytosol where it is<br />
bound to metallothionein (or metallothionein-like<br />
cuproproteins ).<br />
Metabolism <strong>and</strong> distribution<br />
Copper apparently binds also to pro<br />
teins other than metallothionein in the hepatocytes,<br />
as revealed by analyses <strong>of</strong> subcellular<br />
fractions <strong>of</strong> the liver <strong>of</strong> laboratory<br />
animals. How applicable these findings are<br />
to man is not clear. In a review <strong>of</strong> the sub<br />
ject Evans (192) lists four different frac<br />
tions as follows: 1) microsomal fraction<br />
containing about 10% <strong>of</strong> liver <strong>copper</strong>, most<br />
<strong>of</strong> which is probably located in newly syn<br />
thesized cuproproteins being prepared for<br />
transport to other sites; 2) nuclear frac<br />
tion, with about 20c/( <strong>of</strong> total liver <strong>copper</strong>,<br />
which may represent a temporary site <strong>of</strong><br />
storage; 3) large granule fraction, with<br />
about the same amount <strong>of</strong> <strong>copper</strong>, contain<br />
ing both mitochondria <strong>and</strong> lysosomes, the<br />
latter being especially involved in seques<br />
tering <strong>copper</strong> prior to biliary excretion; <strong>and</strong><br />
4) the cytosol, containing about one-half<br />
<strong>of</strong> total liver <strong>copper</strong>, in which a small per<br />
centage is in <strong>copper</strong>-dependent enzymes<br />
<strong>and</strong> the predominant portion in the form <strong>of</strong><br />
a <strong>copper</strong>-binding protein similar to metal<br />
lothionein. Evans (192) also describes in<br />
teresting differences in the <strong>copper</strong> content<br />
<strong>of</strong> these cell fractions in the newborn <strong>and</strong><br />
during postnatal development, <strong>and</strong> also<br />
changes observed in animals given dietary<br />
excess <strong>of</strong> <strong>copper</strong>. The latter studies indi<br />
cate that in metallothionein there is prefer<br />
ential binding, after which excess <strong>copper</strong><br />
is distributed to other fractions. He notes<br />
that the binding capacity <strong>of</strong> metallothio<br />
nein is limited, <strong>and</strong> that the lysosomes <strong>and</strong><br />
nuclear proteins assist in maintaining cop<br />
per homeostasis.<br />
Marceau <strong>and</strong> Aspin (488, 489) have<br />
shown that when rats are given [07Cu]ceruloplasmin<br />
intravenously the 67Cu is<br />
taken up by all tissues, but primarily by<br />
the liver where it appears in a specific<br />
protein fraction <strong>of</strong> the hepatocyte cytosol<br />
having a molecular weight <strong>of</strong> 30,000 to<br />
40,000 daltons <strong>and</strong> exhibiting Superoxide<br />
dismutase activity. It also becomes tightly<br />
bound to cytochrome c oxidase in the mito<br />
chondria. Since no [G7Cu]ceruloplasmin is<br />
demonstrable in the cell, it is assumed that<br />
<strong>copper</strong> is released at or within the cell<br />
membrane. In contrast, the G7Cu<strong>of</strong> [n7Cu]albumin<br />
complexes with proteins <strong>of</strong> about<br />
10,000 daltons in the soluble cell fraction<br />
<strong>and</strong> becomes loosely bound to cytochrome<br />
c oxidase. Similar distribution <strong>of</strong> the two<br />
plasma proteins is observed in the rat brain<br />
<strong>and</strong> spleen.<br />
In addition to serving as the major path<br />
way <strong>of</strong> <strong>copper</strong> excretion via the biliary<br />
tract, the liver releases <strong>copper</strong> to maintain<br />
the labile pool <strong>of</strong> <strong>copper</strong> in the serum <strong>and</strong><br />
blood cells. This pool provides <strong>copper</strong> for<br />
incorporation into Superoxide dismutase<br />
<strong>and</strong> into the many other <strong>copper</strong>-containing<br />
enzymes <strong>of</strong> body tissues, some <strong>of</strong> which<br />
may be synthesized in the liver itself. How<br />
ever, a major function <strong>of</strong> the liver is the<br />
synthesis <strong>of</strong> ceruloplasmin. This form <strong>of</strong><br />
tightly bound <strong>copper</strong> is referred to as "in<br />
direct reacting" <strong>copper</strong>, since it requires<br />
acidification to release its 0.3% <strong>copper</strong><br />
which can then be measured, as in the case<br />
<strong>of</strong> "direct reacting" <strong>copper</strong>, by the diethyl<br />
dithiocarbamate reaction. Its more reliable<br />
measurement by enzymatic <strong>and</strong> immunological<br />
methods has been discussed earlier<br />
(p. 1984). Once synthesized, ceruloplasmin<br />
is released by the liver such that it com<br />
prises approximately 93r/f <strong>of</strong> plasma cop<br />
per. In healthy adult humans this level is<br />
remarkably constant. There is no inter<br />
change in the blood stream between ce<br />
ruloplasmin <strong>copper</strong> <strong>and</strong> other forms <strong>of</strong><br />
<strong>copper</strong> (742). Animal studies (741) indi<br />
cate that the liver microsomes are the site<br />
<strong>of</strong> ceruloplasmin synthesis. Despite evi<br />
dence that almost 0.5 mg <strong>of</strong> <strong>copper</strong> is in<br />
corporated into ceruloplasmin daily, close<br />
to the estimated daily absorption from the<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1993<br />
diet (742), the physiological role <strong>of</strong> ceruloplasmin<br />
has only recently begun to be clari<br />
fied (pp. 1984-1986).<br />
Copper in blood<br />
The first evidence <strong>of</strong> the presence <strong>of</strong><br />
<strong>copper</strong> in blood, that <strong>of</strong> the ox, was re<br />
corded in 1830 by Sarzeau (665). The first<br />
demonstration <strong>of</strong> <strong>copper</strong> in human serum<br />
was reported almost 100 years later by<br />
Warburg <strong>and</strong> Krebs (827) <strong>and</strong> Krebs<br />
(426), who employed a catalytic method<br />
developed by Warburg. Although the<br />
values obtained were somewhat below<br />
those currently accepted, they did recog<br />
nize lower levels in normal males (aver.<br />
0.82 /xg/100 ml) than in females (aver.<br />
0.98 ^ig/100 ml), <strong>and</strong> also increased levels<br />
in pulmonary tuberculosis (aver. 1.55 /¿g/<br />
100 ml) <strong>and</strong> in the later stages <strong>of</strong> preg<br />
nancy (aver. 2.07 /¿g/100ml). Quite com<br />
parable values were reported by Locke et<br />
al. (464 ), who were among the first to em<br />
ploy diethyldithiocarbamate as a reagent<br />
for the detection <strong>and</strong> estimation <strong>of</strong> <strong>copper</strong>.<br />
While not recognized at the time, the rou<br />
tine acidification <strong>of</strong> samples necessary for<br />
release <strong>and</strong> measurement <strong>of</strong> <strong>copper</strong> in<br />
ceruloplasmin did not make it possible to<br />
recognize that the increased levels in preg<br />
nancy <strong>and</strong> infectious states were due pri<br />
marily to increases in ceruloplasmin.<br />
Because <strong>of</strong> the diversity <strong>of</strong> chemical, bio<br />
physical <strong>and</strong> immunological methods for<br />
the estimation <strong>of</strong> <strong>copper</strong> <strong>and</strong> ceruloplas<br />
min in blood <strong>and</strong> other tissues for over<br />
more than 50 years, the values presented<br />
in the literature for whole blood, plasma,<br />
serum <strong>and</strong> red cells show considerable<br />
variation. An excellent description <strong>and</strong> ap<br />
praisal <strong>of</strong> methods used up to 1965 has<br />
been presented by Sass-Kortsak (666).<br />
There has not come to the author's atten<br />
tion a comparable review <strong>and</strong> critique <strong>of</strong><br />
methods developed since that time. Even<br />
with the employment <strong>of</strong> a single method<br />
such as atomic absorption spectrometry<br />
values obtained for <strong>copper</strong> levels in serum,<br />
plasma <strong>and</strong> urine vary considerably, due<br />
in large part to differences in preparation<br />
<strong>of</strong> the sample (763).<br />
Blood levels <strong>of</strong> <strong>copper</strong> are commonly<br />
expressed either as serum or plasma levels,<br />
with little or no distinction made be<br />
tween the two. Cartwright ( 100) states<br />
that since the ratio <strong>of</strong> the volume <strong>of</strong> erythrocytes<br />
to leukocytes <strong>and</strong> platelets in nor<br />
mal blood is about 47/0.7, failure to sepa<br />
rate the latter from erythrocytes results in<br />
an insignificant difference in <strong>copper</strong> values<br />
obtained. It must be recognized, however,<br />
that white blood cells do contain a small<br />
amount <strong>of</strong> <strong>copper</strong> (about l/4th the con<br />
centration in erythrocytes) even though<br />
they represent a rather small component<br />
<strong>of</strong> total blood cells. A recent report (646)<br />
records significant differences between<br />
serum <strong>and</strong> plasma <strong>copper</strong> levels in 28 adult<br />
subjects studied on the same day, mean<br />
values being 119 <strong>and</strong> 127 /ug/100 ml, re<br />
spectively. These findings require con<br />
firmation. Investigators suggest that cop<br />
per might be released from platelets,<br />
leukocytes or erythrocytes during coagu<br />
lation <strong>and</strong> clot reaction.<br />
Heilmeyer et al. (319) summarized 10<br />
prior studies on adult humans employing<br />
six different methods <strong>and</strong> proposing nor<br />
mal values ranging from 65 to 200 /¿g/lOO<br />
ml in blood serum. Their own studies<br />
yielded mean values <strong>of</strong> 106.2 /¿g/100ml for<br />
15 males <strong>and</strong> 106.9 /¿g/100ml for 15 fe<br />
males, thus failing to reveal the sex differ<br />
ences reported in later studies. In a review<br />
<strong>of</strong> the literature up to 1950, Cartwright<br />
( 100) gives data from seven different<br />
studies involving a total <strong>of</strong> 184 males <strong>and</strong><br />
274 females from which can be calculated<br />
average mean values <strong>of</strong> 106 <strong>and</strong> 114 /¿g/<br />
100 ml for plasma <strong>of</strong> males <strong>and</strong> females,<br />
respectively. Neale et al. (551 ) report cor<br />
responding mean serum levels <strong>of</strong> 100 <strong>and</strong><br />
108 jug/100 ml for 53 normal subjects <strong>of</strong><br />
each sex. Wintrobe et al. (857) record<br />
plasma <strong>copper</strong> values, <strong>of</strong> 105 ±16 <strong>and</strong><br />
116 ±16 /xg/100 ml for males <strong>and</strong> females,<br />
respectively. An increase in serum <strong>copper</strong><br />
with age is said to occur in males but not<br />
in females (871), but no adequate ex<br />
planation is <strong>of</strong>fered.<br />
In plasma (or serum) most <strong>of</strong> the cop<br />
per is bound to ceruloplasmin as indirect<br />
reacting <strong>copper</strong> (119, 286). In man it was<br />
first estimated that this represents 96% <strong>of</strong><br />
total plasma <strong>copper</strong> (286). There are later<br />
estimates <strong>of</strong> 93% (105) <strong>and</strong> <strong>of</strong> 90% (75,<br />
321). Most investigators now accept 93%.<br />
The remaining <strong>copper</strong>, constituting the<br />
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1994 KARL E. MASON<br />
labile pool, is less firmly bound in large<br />
part to albumin <strong>and</strong> in smaller part to<br />
amino acids (553), especially to histidine,<br />
threonine <strong>and</strong> glutamine (554). The<br />
smaller portion <strong>of</strong> "free" <strong>copper</strong> bound to<br />
amino acids, the existence <strong>of</strong> which was<br />
first demonstrated by Neumann <strong>and</strong> Sass-<br />
Kortsak (553, 554) <strong>and</strong> Sakar <strong>and</strong> Kruck<br />
(658), may be important as a transport<br />
form <strong>of</strong> <strong>copper</strong> in the blood, capable <strong>of</strong><br />
actively diffusing across cell membranes<br />
such as those <strong>of</strong> erythrocytes, whereas al<br />
bumin-bound <strong>copper</strong> preferentially releases<br />
its <strong>copper</strong> to receptor proteins <strong>of</strong> the<br />
plasma membrane <strong>of</strong> hepatocytes <strong>and</strong> other<br />
cells. After exposure <strong>of</strong> human serum to a<br />
centrifugal force greater than necessary to<br />
sediment albumin, <strong>copper</strong> may be demon<br />
strated in serum in the form <strong>of</strong> mixed<br />
amino acid-complexes consisting <strong>of</strong> one<br />
atom <strong>of</strong> <strong>copper</strong> <strong>and</strong> two different amino<br />
acids, predominantly complexes <strong>of</strong> cop<br />
per wittìhistidine, threonine <strong>and</strong> glutamine<br />
(553, 554). Moreover, in ultrafiltrates <strong>of</strong><br />
human serum there can be identified not<br />
only these complexes but also a mixed com<br />
plex <strong>of</strong> histidine-<strong>copper</strong>-threonine (554).<br />
In the red blood cells <strong>copper</strong> exists both<br />
in a labile pool, much like that in the<br />
plasma but proportionately much larger,<br />
<strong>and</strong> also in a firmly bound form, almost<br />
entirely as erythrocuprein ( Superoxide dismutase).<br />
Total erythrocyte <strong>copper</strong> in nor<br />
mal man is about 89 ±11.4 /¿g/100ml <strong>of</strong><br />
packed red cells (492). The labile pool<br />
represents <strong>copper</strong> complexed with amino<br />
acids, freely dialyzable <strong>and</strong> probably in<br />
volved in providing <strong>copper</strong> for Superoxide<br />
dismutase. It contains about 4Q% <strong>of</strong> the<br />
erythrocyte <strong>copper</strong>. The remaining 60% is<br />
almost entirely bouYid to erythrocuprein, a<br />
cuproprotein first isolated by Markowitz et<br />
al. (490), described more fully by Kimmel<br />
et al. (406), <strong>and</strong> later identified as superoxide<br />
dismutase by McCord <strong>and</strong> Fridovich<br />
(501). In addition, a small amount <strong>of</strong> cop<br />
per is thought to be bound to a pink cop<br />
per-binding protein isolated by Reed et al.<br />
(631). This may correspond to the nonerythrocuprein<br />
<strong>copper</strong> fraction observed in<br />
human red blood cells by Shields, et al.<br />
(708). This protein has no amine oxidase<br />
or Superoxide dismutase activity, <strong>and</strong> its<br />
biological function remains to be demon<br />
strated.<br />
It is apparent that neither whole blood,<br />
blood cell nor blood plasma levels <strong>of</strong> cop<br />
per provide useful information regarding<br />
nutritional <strong>copper</strong> status in man. The<br />
erythrocyte <strong>copper</strong> <strong>of</strong> both compartments<br />
is remarkably stable regardless <strong>of</strong> dietary<br />
intake, <strong>and</strong> is not involved in transport <strong>of</strong><br />
<strong>copper</strong> to tissues. The latter function is<br />
mainly ensured by the small compartment<br />
<strong>of</strong> plasma <strong>copper</strong> (about 7c/f <strong>of</strong> the total)<br />
bound largely to albumin <strong>and</strong> to a lesser<br />
degree to amino acids, the latter com<br />
plexes being essential for active transport<br />
<strong>of</strong> <strong>copper</strong> across cell membranes. The al<br />
bumin <strong>and</strong> amino acid-bound forms <strong>of</strong><br />
plasma <strong>copper</strong>, together with minute<br />
amounts <strong>of</strong> free ionic <strong>copper</strong>, represent the<br />
direct reacting <strong>copper</strong> <strong>of</strong> serum which may<br />
increase with intake only temporarily be<br />
fore liver homeostasis comes into play. The<br />
much larger compartment <strong>of</strong> ceruloplasmin<br />
<strong>copper</strong> is generally not influenced by di<br />
etary intake <strong>of</strong> <strong>copper</strong> but does react to a<br />
great variety <strong>of</strong> conditions responsible for<br />
states <strong>of</strong> hypocupremia <strong>and</strong> hypercupremia,<br />
as discussed later (pp. 2014-2020).<br />
A small diurnal variation in plasma <strong>copper</strong><br />
has been reported (434, 460).<br />
EXCRETION OF COPPER<br />
As previously stated (p. 1991) in normal<br />
man perhaps up to 40 to 60% <strong>of</strong> dietary<br />
<strong>copper</strong> is actually absorbed, with the gas<br />
tric <strong>and</strong> duodenal mucosa playing the<br />
major role. Such estimates are, in large<br />
part, based upon differences between oral<br />
<strong>and</strong> intravenous intake <strong>and</strong> fecal excretion,<br />
since urinary excretion <strong>of</strong> <strong>copper</strong> plays a<br />
very minor role. Therefore, the real prob<br />
lem in evaluating these differences lies in<br />
determining what fecal excretion truly<br />
represents. Presumably, it represents unabsorbed<br />
dietary <strong>copper</strong> plus <strong>copper</strong> ex<br />
creted via the biliary tract (a major fac<br />
tor), salivary gl<strong>and</strong>s <strong>and</strong> gastric <strong>and</strong> in<br />
testinal mucosae, minus <strong>copper</strong> which may<br />
be reabsorbed by the gastrointestinal tract<br />
in the course <strong>of</strong> transit. Aside from these<br />
considerations are losses <strong>of</strong> <strong>copper</strong> via<br />
sweat <strong>and</strong> the menses, which are measur<br />
able with a limited degree <strong>of</strong> accuracy. It<br />
is hoped that the discussion to follow may<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1995<br />
delineate the state <strong>and</strong> lack <strong>of</strong> current<br />
knowledge concerning many <strong>of</strong> the physi<br />
ological mechanisms mentioned.<br />
Biliary excretion<br />
Sheldon <strong>and</strong> Ramage (705) were the<br />
first to note the presence <strong>of</strong> <strong>copper</strong> in bile<br />
<strong>and</strong> to suggest that this represents a chan<br />
nel <strong>of</strong> excretion. They also hypothesized,<br />
on the basis <strong>of</strong> the high values obtained<br />
from gall bladder bile, that the body may<br />
attempt to conserve its supply <strong>of</strong> <strong>copper</strong><br />
by reabsorption through the highly vascu<br />
lar wall <strong>of</strong> the gall bladder. The latter re<br />
mains an unsettled question. An isolated<br />
report <strong>of</strong> phenomenally high concentra<br />
tion in pigment gall stones (689 ) seems not<br />
to have been confirmed.<br />
In 1944 Van Ravensteyn (805) stated<br />
that "We could not find in the medical lit<br />
erature any data on <strong>copper</strong> excretion with<br />
the bile or via the intestinal wall in man<br />
after administration <strong>of</strong> <strong>copper</strong> by mouth<br />
or by intravenous injection <strong>of</strong> <strong>copper</strong> com<br />
pounds." In his studies he found that, un<br />
like iron, oral <strong>copper</strong> had little effect upon<br />
blood levels, but caused a marked increase<br />
in bile <strong>and</strong> feces. He also discussed the<br />
possible reabsorption <strong>of</strong> biliary <strong>copper</strong> by<br />
the small intestine <strong>and</strong> <strong>of</strong> fecal <strong>copper</strong> by<br />
the colon. For the latter there is little or no<br />
evidence. This suggestion was based upon<br />
his observations that biliary excretion <strong>and</strong><br />
fecal excretion did not run parallel, follow<br />
ing intravenous injections <strong>of</strong> a non-toxic<br />
organic salt <strong>of</strong> <strong>copper</strong>. For the duodenal<br />
bile <strong>of</strong> eight normal subjects, Van Raven<br />
steyn found the average <strong>copper</strong> content to<br />
be 0.118 mg/100 ml (range 0.03-0.20 mg/<br />
100 ml). These may be compared to an<br />
average content <strong>of</strong> 0.2 mg/100 ml (range<br />
0.06-0.32 mg/100 ml) for common duct<br />
bile in three subjects, <strong>and</strong> <strong>of</strong> 0.48 mg/100<br />
ml (range 0.09-1.07 mg/100 ml) in gall<br />
bladder bile from 19 cases <strong>of</strong> chronic chole<br />
cystitis, obtained at the time <strong>of</strong> operation<br />
for bladder stones, as reported the same<br />
year by Judd <strong>and</strong> Dry ( 391 ).<br />
As noted by the next investigators to<br />
contribute to this subject ( 105), a number<br />
<strong>of</strong> factors makes it very difficult to deter<br />
mine with a reasonable degree <strong>of</strong> accuracy<br />
the amount <strong>of</strong> <strong>copper</strong> excreted via the<br />
biliary tract. The daily outflow is inter<br />
mittent <strong>and</strong> may vary from 250 to 1,100<br />
ml/day. Bile cannot be collected quanti<br />
tatively from normal subjects, even those<br />
with exterioration <strong>of</strong> the bile duct or in<br />
dwelling T-tube, since in these situations<br />
the volume <strong>of</strong> bile flow is not normal.<br />
Moreover, <strong>copper</strong> in bile aspirated from<br />
the gall bladder during surgery or post<br />
mortem is considerably concentrated as<br />
compared to liver bile or duodenal bile,<br />
<strong>and</strong> the latter is subject to contamination<br />
by duodenal contents <strong>and</strong> the tubes used<br />
for the collection. It is reported ( 105) that<br />
<strong>copper</strong> in gall bladder bile obtained post<br />
mortem from six normal subjects ranged<br />
from 0.024 to 0.54 mg/100 ml, with an<br />
average value <strong>of</strong> 0.329 mg/100 ml. One<br />
subject with a cutaneous bile fistula follow<br />
ing complete obstruction <strong>of</strong> the common<br />
duct excreted an average <strong>of</strong> 0.46 mg/100<br />
ml <strong>copper</strong> per day. Another subject with<br />
primary biliary cirrhosis <strong>and</strong> T-tube drain<br />
age, gave an average value <strong>of</strong> 0.05 mg/100<br />
ml (range 0.03-0.95 mg/100 ml) in bile<br />
collected daily for 17 days. No data on<br />
total daily output <strong>of</strong> <strong>copper</strong> were obtained.<br />
In their conclusions, Cartwright <strong>and</strong><br />
Wintrobe ( 105) state that assuming a daily<br />
intake <strong>of</strong> 2.0 to 5.0 mg <strong>of</strong> <strong>copper</strong>, 0.6 to 1.6<br />
mg (32%) is absorbed. From 0.01 to 0.06<br />
mg is excreted in urine, 0.1 to 0.3 mg<br />
passes directly into the bowel, <strong>and</strong> 0.5 to<br />
1.3 mg is excreted in the bile. The latter<br />
estimate is, in reasonable accord with a<br />
later report <strong>of</strong> Frommer (235) indicating<br />
that in 10 control subjects biliary excretion<br />
approximated 1.2 mg/day, based upon bile<br />
aspirated from the duodenum after over<br />
night fasting. Walshe (822) states that in<br />
subjects with external biliary drainage up<br />
to 107' <strong>of</strong> ingested labeled <strong>copper</strong> can be<br />
recovered in the bile within 24 hours.<br />
Human bile is said to contain <strong>copper</strong>binding<br />
complexes <strong>of</strong> low <strong>and</strong> high molecu<br />
lar weight, the former predominating in<br />
hepatic bile <strong>and</strong> the latter in gall bladder<br />
bile (263, 266). However, the high molecu<br />
lar weight fractions may represent artifacts<br />
in Chromatographie procedures, <strong>and</strong> essen<br />
tially all <strong>of</strong> the <strong>copper</strong> may be bound to<br />
complexes <strong>of</strong> low molecular weight (4,000-<br />
8,000 daltons) (236), as observed in bile<br />
<strong>of</strong> the rat ( 196) which, by the way, pos<br />
sesses no gall bladder. The question <strong>of</strong> an<br />
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1996 KARL E. MASON<br />
enterohepatic circulation <strong>of</strong> <strong>copper</strong>, con<br />
sidered unlikely on the basis <strong>of</strong> the macromolecular<br />
complexes <strong>of</strong> <strong>copper</strong> predomi<br />
nating in the gall bladder bile (266), now<br />
justifies more critical study.<br />
More recently evidence has been found<br />
that the mechanism <strong>of</strong> <strong>copper</strong> excretion<br />
may involve its complexing with taurochenodeoxycholate<br />
in the liver, thereby<br />
preventing its reabsorption from the upper<br />
intestine, with subsequent splitting <strong>of</strong> the<br />
complex in the lower intestine where reabsorption<br />
<strong>of</strong> the bile acid but not <strong>of</strong> cop<br />
per may take place (454). Other evidence,<br />
also based upon bile from T-tubes, sug<br />
gests that orally administered 04Cu be<br />
comes combined with conjugated bilirubins<br />
(503 ). It is obvious that there is still much<br />
more to be learned about the binding <strong>of</strong><br />
<strong>copper</strong> in bile <strong>and</strong> its possible reabsorbability.<br />
Salivary excretion<br />
Little or no attention had been given to<br />
the presence <strong>of</strong> <strong>copper</strong> in saliva until 1952<br />
when Dreizen et al. ( 170) reported finding<br />
in the whole saliva <strong>of</strong> 14 normal humans<br />
(48 samples) a mean <strong>copper</strong> concentration<br />
<strong>of</strong> 25.6 /Ag/100 ml. In general agreement<br />
with these findings are those <strong>of</strong> De Jorge<br />
et al. (157) based on 40 normal, fasting<br />
subjects, giving a mean value <strong>of</strong> 31.7 /¿g/<br />
100 ml, <strong>and</strong> <strong>of</strong> Gollan et al. (264) based on<br />
18 normal subjects <strong>and</strong> providing a mean<br />
value <strong>of</strong> 29 /¿g/100ml. The ' <strong>copper</strong> <strong>of</strong><br />
saliva is in the labile form, since reactions<br />
for ceruloplasmin are negative (157). If<br />
one considers the daily output <strong>of</strong> saliva to<br />
be 1.5 liters (range 1-2 liters), the total<br />
daily excretion <strong>of</strong> <strong>copper</strong> would amount to<br />
from 0.38 to 0.47 mg, on the basis <strong>of</strong> the<br />
data recorded above. There is also evi<br />
dence that the <strong>copper</strong>-binding substances<br />
in saliva retain their activity after transit<br />
through the stomach to the site <strong>of</strong> absorp<br />
tion in the small intestine. De Jorge et al.<br />
(157) also report mean <strong>copper</strong> values <strong>of</strong><br />
submaxillary, parotid <strong>and</strong> pancreas re<br />
moved from 10 postmortem cases as 1.43,<br />
0.55 <strong>and</strong> 0.85 mg/100 g dry weight <strong>of</strong> tis<br />
sue, respectively.<br />
Gastrointestinal excretion<br />
In his pioneer studies on the <strong>metabolism</strong><br />
<strong>of</strong> <strong>copper</strong> in man, Van Ravensteyn (805)<br />
found an average <strong>copper</strong> concentration <strong>of</strong><br />
0.023 mg/100 ml (range 0.01-0.04 mg/100<br />
ml) in gastric juice <strong>of</strong> eight normal sub<br />
jects. Some 27 years later, other data were<br />
provided by Gollan et al. (265) who, in<br />
four normal subjects, found a mean con<br />
centration <strong>of</strong> 0.034 mg/100 ml (range 0.02-<br />
0.96 mg/100 ml). In the latter studies<br />
gastric juice free <strong>of</strong> swallowed saliva <strong>and</strong><br />
nasopharyngeal secretions was aspirated<br />
from fasting subjects, <strong>and</strong> particulate mat<br />
ter was removed by centrifugation at 2,000<br />
X g for 20 minutes. Accepting the values<br />
reported by Gollan et al. (265), employing<br />
exacting collecting <strong>and</strong> analytical pro<br />
cedures, <strong>and</strong> considering current estimates<br />
that the daily volume <strong>of</strong> gastric secretions<br />
approximates 3 liters (range 2-4 liters), it<br />
can be calculated that the gastric mucosa<br />
secretes approximately 1.0 mg/day.<br />
The only recorded study <strong>of</strong> duodenal<br />
secretion <strong>of</strong> <strong>copper</strong> is that <strong>of</strong> Gollan (263 ).<br />
Normal <strong>and</strong> secretin-stimulated aspirates<br />
<strong>of</strong> the duodenum, obtained from fasting<br />
normal subjects through a tube positioned<br />
such as to exclude gastric contents (<strong>and</strong><br />
presumably to also exclude bile <strong>and</strong> pan<br />
creatic secretions) indicates the presence<br />
<strong>of</strong> <strong>copper</strong>-binding substances <strong>of</strong> low molec<br />
ular weight such as also found in saliva<br />
<strong>and</strong> gastric juice. Gollan presents evidence<br />
that the latter binding substances retain<br />
their activity after transit through the<br />
stomach to the site <strong>of</strong> absorption in the<br />
small intestine. He also postulates that<br />
these secretions, through their capacity to<br />
solubilize the metal at an alkaline pH, may<br />
enhance the availability <strong>of</strong> <strong>copper</strong> for ab<br />
sorption.<br />
Relatively little attention has been given<br />
to, or estimates made <strong>of</strong>, the amount <strong>of</strong><br />
<strong>copper</strong> released into the feces via the ex<br />
tensive dehiscence <strong>of</strong> epithelial cells <strong>of</strong> the<br />
intestinal mucosa. In these cells there is a<br />
considerable amount <strong>of</strong> <strong>copper</strong> bound to<br />
metallothionein or metallothionein-like pro<br />
teins, functioning in <strong>copper</strong> storage <strong>and</strong> in<br />
protection against excess <strong>copper</strong> intake.<br />
Considering that the absorptive cells lining<br />
intestinal villi are replaced every 5 to 6<br />
days in man (475), this contribution <strong>of</strong><br />
<strong>copper</strong> to the intestinal contents <strong>and</strong> to<br />
the fecal output may be quite significant,<br />
since it is thought to be nonabsorbable<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1997<br />
(192). It may represent 17% <strong>of</strong> the daily<br />
fecal excretion (301). It certainly justifies<br />
more consideration as a factor in evaluat<br />
ing <strong>copper</strong> excretion <strong>and</strong> maintenance <strong>of</strong><br />
<strong>copper</strong> balance in the human organism.<br />
Questions arise concerning the fate <strong>of</strong><br />
the large amount <strong>of</strong> <strong>copper</strong> bound to sub<br />
stances <strong>of</strong> low molecular weight (amino<br />
acids <strong>and</strong> peptides) which is excreted via<br />
the saliva, gastric juice <strong>and</strong> duodenal se<br />
cretions. Unfortunately, studies presented<br />
record many data but make few predic<br />
tions as to what the findings may mean<br />
with regard to <strong>copper</strong> absorption or metab<br />
olism. Some <strong>of</strong> this <strong>copper</strong> may be reabsorbed<br />
<strong>and</strong> some may be added to the<br />
fèces, but the relative amounts are un<br />
known. And, in addition, the physiological<br />
catabolism <strong>of</strong> ceruloplasmin may add<br />
about 0.1 mg to fecal excretion <strong>of</strong> <strong>copper</strong><br />
per day (814).<br />
Urinary excretion<br />
Estimates <strong>of</strong> <strong>copper</strong> lost in the urine are<br />
somewhat variable, apparently due to dif<br />
ferences in sensitivity <strong>and</strong> accuracy <strong>of</strong><br />
methods used, <strong>and</strong> possible contamination<br />
from extraneous sources. Early reports are<br />
summarized by Butler <strong>and</strong> Newman (83)<br />
who, in a study <strong>of</strong> 12 healthy adults, re<br />
ported mean excretion values <strong>of</strong> 18.0 jug/<br />
day, (range 3.9-29.6 fig/day). Values re<br />
ported since that time are in reasonable<br />
accord with these results. If one selects<br />
from the literature those reports based<br />
upon 10 or more adult subjects, chrono<br />
logically recording results in terms <strong>of</strong><br />
/«.g/dayexcreted in the urine, the values<br />
are: 18 (113); 18 (765); 20 (253); 37<br />
(398) <strong>and</strong> 52 (153). These values are all<br />
within the range <strong>of</strong> 10 to 60 /¿g/daypro<br />
posed by Cartwright <strong>and</strong> Wintrobe ( 106).<br />
Thus, urinary excretion <strong>of</strong> <strong>copper</strong>, amount<br />
ing to approximately 0.5 to 3.0$ <strong>of</strong> the<br />
daily intake, places the main excretory re<br />
sponsibility on fecal excretion. There still<br />
remains the possibility that the human<br />
kidney possesses the capacity for tubular<br />
reabsorption <strong>of</strong> <strong>copper</strong>.<br />
Sweat loss<br />
The loss <strong>of</strong> <strong>copper</strong> through sweat has<br />
received limited consideration. The pioneer<br />
studies <strong>of</strong> Consolazio et al. ( 122) record<br />
that on a constant dietary intake <strong>of</strong> <strong>copper</strong><br />
(3.5 mg) <strong>and</strong> in an environment <strong>of</strong> 37.8°<br />
<strong>and</strong> humidity <strong>of</strong> 50$;, three normal sub<br />
jects showed a significant negative <strong>copper</strong><br />
balance. During 10 days <strong>of</strong> observation the<br />
sweat loss from the men averaged 1.6 mg/<br />
day, or about 45% <strong>of</strong> their total dietary in<br />
take. Consequently, the negative balance<br />
averaged 1.1 mg/day. Mitchell <strong>and</strong> Hamil<br />
ton (528) found an average <strong>of</strong> 58 /¿g/li ter<br />
in the sweat <strong>of</strong> four adult males under hot,<br />
humid conditions. Another report (343)<br />
records for 33 males after sauna bathing<br />
an average excretion <strong>of</strong> 550 ±350 /^g/liter<br />
in the sweat. In view <strong>of</strong> these data it ap<br />
pears that the loss <strong>of</strong> <strong>copper</strong> through sweat<br />
is much greater than heret<strong>of</strong>ore recog<br />
nized. In fact, Hohnadel et al. (343) <strong>and</strong><br />
Sunderman et al. (764) extol the possible<br />
virtues <strong>of</strong> the sauna bath as a therapeutic<br />
method for increasing the excretion <strong>of</strong> toxic<br />
metals, such as <strong>copper</strong> in Wilson's disease.<br />
Technical methods defy measurements <strong>of</strong><br />
<strong>copper</strong> loss through insensible perspiration.<br />
Menstrual loss<br />
Data on the loss <strong>of</strong> <strong>copper</strong> via menstrual<br />
flow is likewise meager. For four menstrual<br />
periods in three subjects values <strong>of</strong> 0.19,<br />
0.24, 0.39 <strong>and</strong> 0.61 mg per period (aver.<br />
0.47 mg) are given by Ohlson <strong>and</strong> Daum<br />
(572). Comparable average values <strong>of</strong> 0.32,<br />
0.48, 0.65 <strong>and</strong> 0.74 mg <strong>copper</strong> for four<br />
consecutive periods in four different sub<br />
jects are recorded by Leverton <strong>and</strong> Binkley<br />
(452), <strong>and</strong> a mean <strong>of</strong> 0.11 ±0.07 mg<br />
per period for 12 adolescent girls is re<br />
ported by Greger <strong>and</strong> Buckley (278 ).<br />
COPPER IN THE DIET<br />
Copper in foods<br />
Copper is ubiquitous in plants <strong>and</strong> ani<br />
mals. Its widespread occurrence in food<br />
was demonstrated in the early reports <strong>of</strong><br />
Lindow et al. (462) <strong>and</strong> <strong>of</strong> Hodges <strong>and</strong><br />
Peterson (341) on the <strong>copper</strong>-content <strong>of</strong><br />
samples <strong>of</strong> commonly used food in the<br />
USA, <strong>and</strong> that <strong>of</strong> Adolph <strong>and</strong> Chou (8) on<br />
Chinese foods. It is well recognized that<br />
the <strong>copper</strong> in foods varies greatly, depend<br />
ing upon the soils from which they have<br />
been obtained, <strong>and</strong> on contamination be<br />
fore <strong>and</strong> after reaching the market place.<br />
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1998 KARL E. MASON<br />
The richest sources in human dietaries are<br />
liver (especially calf, lamb <strong>and</strong> beef),<br />
crustaceans <strong>and</strong> shell fish (especially<br />
oysters). Of somewhat lesser content, <strong>and</strong><br />
roughly in descending order, are nuts <strong>and</strong><br />
seeds, high-protein cereals, dried fruits,<br />
poultry, fish, meats, legumes, root vege<br />
tables, leafy vegetables, fresh fruits <strong>and</strong><br />
non-leafy vegetables. One <strong>of</strong> the lowest <strong>of</strong><br />
commonly used foods is cow's milk. The<br />
exceptionally high <strong>copper</strong> content <strong>of</strong> the<br />
Atlantic coast oyster, which may vary<br />
widely with the season <strong>and</strong> with degrees<br />
<strong>of</strong> contamination <strong>of</strong> environmental waters,<br />
is not true <strong>of</strong> the Pacific or the Australian<br />
oyster (290).<br />
Throughout the literature one repeatedly<br />
finds the statement that the average North<br />
American diet provides 3 to 5 mg <strong>of</strong> <strong>copper</strong><br />
per day. Because <strong>of</strong> the widespread pres<br />
ence <strong>of</strong> <strong>copper</strong> in foods <strong>and</strong> in drinking wa<br />
ter, especially that obtained via <strong>copper</strong><br />
pipes, it is difficult to devise a balanced diet<br />
composed <strong>of</strong> natural foodstuffs that contains<br />
less than 1 mg per day (50 /¿g/cal/day),<br />
according to Schroeder et al. (691) who<br />
give extensive data on <strong>copper</strong> in foods,<br />
beverages <strong>and</strong> water <strong>of</strong> many types. They<br />
also state that to provide such a diet the<br />
following foods must be avoided; most<br />
meats, shellfish, vegetables, phospholipids,<br />
legumes, fruits, nuts, some grains, gelatin,<br />
tea, c<strong>of</strong>fee, s<strong>of</strong>t drinks, beer <strong>and</strong> distilled<br />
liquors. Such a diet would be low in pro<br />
tein, monotonous <strong>and</strong> marginally deficient<br />
in several essential trace metals. In con<br />
trast, they estimate that a diet <strong>of</strong> only 1,200<br />
calories, with 100 calories from each <strong>of</strong> the<br />
high <strong>copper</strong> foods (oysters, clams, pork,<br />
margarine, turnips, carrots, mushrooms,<br />
rhubarb, papaya, nuts, grapenuts <strong>and</strong><br />
orange juice) would contain approximately<br />
34 mg <strong>of</strong> <strong>copper</strong>.<br />
There have been many other published<br />
lists giving the <strong>copper</strong> content <strong>of</strong> com<br />
monly used foods. A rather extensive list<br />
<strong>of</strong> <strong>copper</strong> <strong>and</strong> other inorganic elements in<br />
foods used in hospital menus has been<br />
presented by Gormican (273). A recent<br />
<strong>and</strong> complete compilation <strong>of</strong> data pertain<br />
ing to the <strong>copper</strong> content <strong>of</strong> foods, as re<br />
corded by investigators worldwide, is that<br />
<strong>of</strong> Pennington <strong>and</strong> Galloway (595). Of the<br />
222 references used in their literature sur<br />
vey only 104 specified the methods used,<br />
many <strong>of</strong> which represented individual<br />
modification <strong>of</strong> other methods. Methods <strong>of</strong><br />
expressing values obtained were indeed<br />
numerous <strong>and</strong> <strong>of</strong>ten difficult to reduce to<br />
a common denominator. Aside from vari<br />
able contamination <strong>of</strong> water, reagents <strong>and</strong><br />
glassware in analytical procedures, factors<br />
such as <strong>copper</strong> content <strong>of</strong> soil, water source,<br />
season, <strong>and</strong> use <strong>of</strong> fertilizers, insecticides,<br />
pesticides <strong>and</strong> fungicides raised serious<br />
questions concerning the validity <strong>of</strong> values<br />
reported for the <strong>copper</strong> content <strong>of</strong> the vast<br />
list <strong>of</strong> food items recorded.<br />
Hughes et al. (366) give analyses for<br />
<strong>copper</strong> in a great variety <strong>of</strong> commercially<br />
prepared baby foods. Highest levels were<br />
found in those containing beef liver <strong>and</strong><br />
high protein cereals (2.64 <strong>and</strong> 1.85 mg/100<br />
g, respectively). Next in order were other<br />
precooked cereal foods (range 0.78-0.26<br />
mg/100 g), as compared to 0.04 to 0.30<br />
mg/100 ml in human milk (pp. 2025-2026).<br />
Vegetables, fruits <strong>and</strong> desserts were vari<br />
ably lower. Cooked cereals, which fre<br />
quently are the first non-milk foods <strong>of</strong>fered<br />
to infants, if they are <strong>of</strong> high protein quality,<br />
will provide a large part <strong>of</strong> their require<br />
ment. It was their opinion that by the time<br />
infants reach 6 months <strong>of</strong> age the variety<br />
<strong>of</strong> supplementary foods normally <strong>of</strong>fered<br />
should, in most cases, meet or exceed the<br />
generally accepted requirement <strong>of</strong> 0.05<br />
mg/kg/day.<br />
From what has been said, it is apparent<br />
that most populations appear to have an<br />
adequate dietary intake <strong>of</strong> <strong>copper</strong>, which<br />
well justifies previous opinions that a recog<br />
nizable state <strong>of</strong> <strong>copper</strong> deficiency in adult<br />
man is not likely to be recognized. But<br />
this does not imply that some diets may<br />
not be decidedly marginal, as discussed in<br />
the following section. Such matters are con<br />
stantly <strong>of</strong> concern in any review <strong>of</strong> reports<br />
on dietary intake <strong>and</strong> on balance studies<br />
which represent the basic information<br />
necessary for the determination <strong>of</strong> human<br />
<strong>copper</strong> <strong>requirements</strong>.<br />
Dietary intake <strong>of</strong> <strong>copper</strong><br />
Before progressing to a discussion <strong>of</strong><br />
human dietary needs <strong>of</strong> <strong>copper</strong> for adult<br />
man it may be pertinent to review what<br />
has been recorded concerning the usual<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 1999<br />
dietary intake in various countries <strong>of</strong> the<br />
world. At the beginning it should be recog<br />
nized that in the vast majority <strong>of</strong> such<br />
analyses <strong>copper</strong> has <strong>of</strong>ten been only one <strong>of</strong><br />
many trace elements under investigation<br />
<strong>and</strong> <strong>of</strong>ten has been secondary compared to<br />
iron, zinc, <strong>and</strong> other elements. Unfortu<br />
nately, there have been almost no studies<br />
in which <strong>copper</strong> has been the only, or<br />
even the primary, focus <strong>of</strong> attention.<br />
On the basis <strong>of</strong> studies conducted in<br />
Engl<strong>and</strong>, New Zeal<strong>and</strong> <strong>and</strong> the United<br />
States, Underwood (798) estimates that<br />
most western-style mixed diets provide<br />
adults with 2 to 4 mg/day. Guthrie <strong>and</strong><br />
Robinson (292) think that the <strong>copper</strong><br />
status <strong>of</strong> some New Zeal<strong>and</strong>ers may be<br />
inadequate. In India, in adults consuming<br />
rice <strong>and</strong> wheat diets, the <strong>copper</strong> intake<br />
may be as much as 4.5 to 5.8 mg/day<br />
(154). Estimated daily intakes for adults<br />
in other countries have been reported as:<br />
2.26 to 7.3 mg (mean 4.10) in Kiev <strong>and</strong> 11<br />
other cities <strong>of</strong> the Ukraine (239); 1.29 to<br />
6.39 mg for inmates <strong>of</strong> old people's homes<br />
in Switzerl<strong>and</strong> (688); about 2.0 mg for<br />
New Zeal<strong>and</strong>ers (511); 1.5 mg for inhabi<br />
tants <strong>of</strong> an isolated Polynesian isl<strong>and</strong> (510);<br />
2 to 4 mg for Japanese (543) <strong>and</strong> about<br />
2.0 mg for Taiwanese (793). In the latter<br />
study, a control subject maintained on a<br />
low <strong>copper</strong> diet for 2 weeks, providing a<br />
daily intake <strong>of</strong> 1.34 mg, maintained a posi<br />
tive <strong>copper</strong> balance <strong>of</strong> 0.12 mg. Unfortu<br />
nately, the methods employed for analysis<br />
<strong>of</strong> <strong>copper</strong> were not stated. On the other<br />
h<strong>and</strong>, it is estimated that in 20 USDA diets<br />
examined the mean <strong>copper</strong> intake would<br />
provide 1.05 mg/day (419).<br />
Recent <strong>and</strong> well planned studies based<br />
upon analyses <strong>of</strong> <strong>copper</strong> <strong>and</strong> zinc content<br />
<strong>of</strong> duplicate samples <strong>of</strong> daily diets <strong>of</strong> 22<br />
subjects ( 14-64 years <strong>of</strong> age ) over a 14-day<br />
period, provide valuable information (344,<br />
860). Considering a 6-day average (after<br />
8 days <strong>of</strong> adjustment) for each subject,<br />
the mean daily intake <strong>of</strong> <strong>copper</strong> was 1.01<br />
mg day ±0.4. This is considerably below<br />
estimates <strong>of</strong> 1.62 mg/day given by Hartley<br />
et al. (314), <strong>and</strong> 1.34 mg/day by Tu et al.<br />
(793), but in good agreement with the<br />
estimates <strong>of</strong> 1.05 mg by Klevay et al. (419).<br />
There has also been an increasing aware<br />
ness <strong>of</strong> an inadequacy <strong>of</strong> not only <strong>copper</strong><br />
but also other trace elements <strong>and</strong> certain<br />
vitamins in the usual hospital diets (67,<br />
273, 419). It is true that most patients are<br />
hospitalized over a sufficiently short period<br />
such that they can rely on liver <strong>and</strong> other<br />
tissue storage to compensate for inade<br />
quate intake. The problem <strong>of</strong> long term<br />
parenteral nutrition is a separate issue<br />
which will be discussed later (pp. 2028,<br />
2034). However, it may be noted that a<br />
recent study <strong>of</strong> regular, vegetarian <strong>and</strong><br />
renal diets in a hospital situation, based on<br />
diets collected over 7 consecutive days, in<br />
dicate a mean daily <strong>copper</strong> intake <strong>of</strong> 0.90,<br />
1.10 <strong>and</strong> 0.51 mg (67), respectively. For<br />
subjects with Wilson's disease such diets<br />
would be most desirable. For others, they<br />
may be marginal or inadequate.<br />
Analyses <strong>of</strong> school lunches have given<br />
but rather fragmentary evidence <strong>of</strong> the<br />
<strong>copper</strong> content <strong>of</strong> the American diet for<br />
children <strong>and</strong> adolescents. A survey <strong>of</strong> such<br />
lunches served 6th grade children in 300<br />
schools in 19 states <strong>of</strong> the USA (544, 545)<br />
indicates an average content <strong>of</strong> 0.34 mg/<br />
day (range 0.06-2.19 mg). Considering<br />
one-third <strong>of</strong> the daily intake represented,<br />
the average intake would amount to about<br />
1.0 mg/day. These values are somewhat<br />
less than those reported in metabolic<br />
studies <strong>of</strong> 7 to 9 year-old children fed diets<br />
typical <strong>of</strong> low income groups in the south<br />
eastern USA, recording mean daily intakes<br />
<strong>of</strong> 1.67 mg/day (618). On the basis <strong>of</strong> mg/<br />
kg/food consumed, values given for insti<br />
tutionalized children in Samark<strong>and</strong> are<br />
0.46 to 1.62 (620), <strong>and</strong> for the same in 28<br />
USA cities are 0.44 to 0.87 (548).<br />
Waslein (830) reports that data col<br />
lected on the <strong>copper</strong> content <strong>of</strong> the diet <strong>of</strong><br />
377 babies from the USA indicated a wide<br />
range <strong>of</strong> from 7 to 170 /*g/kg body weight,<br />
or 0.18 to 0.92 mg/day. Total daily intakes<br />
<strong>of</strong> <strong>copper</strong> ranged from an average <strong>of</strong> 0.16<br />
for 1-month old infants to 0.38 mg for<br />
6-month old infants, 50 to 607o <strong>of</strong> the in<br />
take coming from milk. Furthermore, in<br />
takes <strong>of</strong> children participating in balance<br />
studies or living in institutions in the USA<br />
or USSR had mean <strong>copper</strong> intakes <strong>of</strong> 0.9<br />
to 2.2 mg/day, <strong>and</strong> similar studies on<br />
adults in the United Kingdom <strong>and</strong> New<br />
Zeal<strong>and</strong> gave values <strong>of</strong> 1.7 <strong>and</strong> 2.4 mg/day,<br />
respectively. However, the average <strong>copper</strong><br />
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2000 KARL E. MASON<br />
intake <strong>of</strong> 5.8 mg/ day from diets in India,<br />
determined on composites made from selfselected<br />
diets in a dozen locations through<br />
out the country ( 154), is more than double<br />
the mean found for diets in developed<br />
countries, for which no explanation is<br />
given. In evaluating such reported data<br />
one must consider not only the differences<br />
in methods <strong>of</strong> assay <strong>and</strong> <strong>of</strong> care against<br />
contamination but also differences in sam<br />
ple preparation <strong>and</strong> differences in food<br />
preparation in different countries. There<br />
is also need to consider phytate <strong>and</strong> fiber<br />
content <strong>of</strong> diets, which may influence ab<br />
sorption or utilization <strong>of</strong> dietary <strong>copper</strong>.<br />
From what has been said, it is quite ap<br />
parent that data based upon the daily in<br />
take <strong>of</strong> <strong>copper</strong> by peoples in different parts<br />
<strong>of</strong> the globe give relatively little guidance<br />
as to what the minimal or optimal level <strong>of</strong><br />
intake may be.<br />
COPPER METABOLISM IN PRENATAL<br />
AND POSTNATAL LIFE<br />
A preceding section has dealt with the<br />
labile <strong>and</strong> the more firmly protein-bound<br />
types <strong>of</strong> <strong>copper</strong> in the blood cells <strong>and</strong><br />
blood serum or plasma <strong>of</strong> normal adult<br />
man. Briefly stated, the labile pools repre<br />
sent about 40 <strong>and</strong> 1%, <strong>and</strong> the proteinbound<br />
pools (superoxide dismutase <strong>and</strong><br />
ceruloplasmin ) approximately 60 <strong>and</strong> 93$,<br />
<strong>of</strong> the <strong>copper</strong> present in blood cells <strong>and</strong><br />
plasma, respectively. The ratio <strong>of</strong> cell to<br />
plasma <strong>copper</strong> is about 0.70 ( 100). The<br />
<strong>copper</strong> content <strong>of</strong> the red blood cells re<br />
mains remarkably constant, little influenced<br />
by dietary intake or metabolic stresses<br />
(286, 435). That <strong>of</strong> the plasma is subject<br />
to rather remarkable changes during preg<br />
nancy, reflecting the influence <strong>of</strong> hormones,<br />
particularly estrogens, upon the synthesis<br />
<strong>and</strong> release <strong>of</strong> ceruloplasmin.<br />
There exists a vast literature dealing<br />
with 1) the increase <strong>of</strong> maternal blood<br />
<strong>copper</strong> levels during pregnancy <strong>and</strong> the<br />
influence <strong>of</strong> estrogenic hormones; 2) the<br />
role <strong>of</strong> the placenta in transfer <strong>of</strong> <strong>copper</strong><br />
to the fetus; 3) the role <strong>of</strong> fetal liver in<br />
storage <strong>of</strong> <strong>copper</strong> to meet inadequacies <strong>of</strong><br />
mammary transfer during early lactation<br />
<strong>and</strong> 4) postnatal changes in blood <strong>copper</strong><br />
levels in the infant <strong>and</strong> adolescent. Knowl<br />
edge <strong>and</strong> interpretation <strong>of</strong> these processes<br />
are <strong>of</strong> importance not only in determining<br />
the role <strong>of</strong> <strong>copper</strong> in reproductive physi<br />
ology <strong>and</strong> neonatal development in man,<br />
but also in obtaining a better underst<strong>and</strong><br />
ing <strong>of</strong> human <strong>requirements</strong> <strong>of</strong> <strong>copper</strong> for<br />
the infant <strong>and</strong> adolescent. It is the pur<br />
pose <strong>of</strong> this section to review briefly what<br />
has been learned concerning the rather<br />
complex changes, not yet clearly under<br />
stood, which occur in the pregnant mother,<br />
fetus <strong>and</strong> young infant.<br />
Influence <strong>of</strong> pregnancy<br />
It seems remarkable that the first investi<br />
gators to demonstrate the presence <strong>of</strong> cop<br />
per in human blood (827) should also<br />
have been the first to recognize not only<br />
normal sex differences but also increased<br />
levels <strong>of</strong> <strong>copper</strong> in the blood <strong>of</strong> pregnant<br />
women (426). These observations were<br />
soon verified by many other investigators<br />
( 181, 184, 206, 294, 318, 345, 348, 434, 464,<br />
556, 561, 577-579, 655, 660, 787). However,<br />
the significance <strong>of</strong> these findings remained<br />
obscure until evidence was presented that<br />
similar increases occur in infants receiving<br />
diethylstilbestrol therapeutically for treat<br />
ment <strong>of</strong> hemophilia (794), <strong>and</strong> in adults <strong>of</strong><br />
either sex receiving estrogens (183, 245,<br />
368, 651) but not in those receiving pro<br />
gesterone or <strong>and</strong>rogens (183). These in<br />
vestigations suggest that increased plasma<br />
<strong>copper</strong> levels in pregnancy could be ex<br />
plained by increased levels <strong>of</strong> estrogens,<br />
but this may not be the total story.<br />
Beginning during the first trimester <strong>of</strong><br />
pregnancy, there occurs a progressive in<br />
crease in maternal plasma <strong>copper</strong> levels.<br />
In groups <strong>of</strong> pregnant women at successive<br />
lunar months <strong>of</strong> gestation, values have<br />
been reported to increase progressively<br />
from 146.1 to 277.6 jig/100 ml (181), <strong>and</strong><br />
131 to 213 Mg/100 ml (757), <strong>and</strong> from 172<br />
to 273 /ig/100 ml (54). Similar data are<br />
presented by others (158, 524). The in<br />
creased plasma <strong>copper</strong> levels <strong>of</strong> pregnancy<br />
have been well documented (23, 54, 77,<br />
152, 231, 255, 318, 320, 324, 527, 574, 578,<br />
686, 687, 847, 858, 878). The <strong>copper</strong> con<br />
tent <strong>of</strong> erythrocytes <strong>of</strong> mother <strong>and</strong> fetus<br />
remains remarkably constant ( 181, 345 ).<br />
Hence, the striking rise in plasma <strong>copper</strong><br />
during pregnancy to about 2 to 3 X normal<br />
is attributable almost entirely to increased<br />
synthesis <strong>of</strong> ceruloplasmin (324, 491, 673).<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2001<br />
Since there are no known alterations in<br />
absorption or excretion, the major source<br />
<strong>of</strong> the non-dietary <strong>copper</strong> for this synthesis<br />
is presumably the maternal liver. Direct<br />
evidence in support <strong>of</strong> this assumption is<br />
the much lower concentration <strong>of</strong> <strong>copper</strong><br />
in the liver (as compared to other organs<br />
examined) in pregnant as compared to<br />
nonpregnant women, all victims <strong>of</strong> acci<br />
dental death (524), <strong>and</strong> very low liver<br />
<strong>copper</strong> levels in women succumbing to<br />
late toxemia <strong>of</strong> pregnancy associated with<br />
unusually high serum ceruloplasmin levels<br />
(628). Assuming also increments <strong>of</strong> about<br />
25% in plasma volume during gestation<br />
(166), maintenance <strong>of</strong> high plasma <strong>copper</strong><br />
levels places special dem<strong>and</strong>s upon ma<br />
ternal tissue stores. Associated with in<br />
creased plasma ceruloplasmin levels <strong>of</strong><br />
<strong>copper</strong> there occurs a reciprocal decrease<br />
in plasma zinc levels both in states <strong>of</strong><br />
pregnancy (152, 300, 324, 388, 843) <strong>and</strong><br />
following use <strong>of</strong> oral contraceptives (297),<br />
although the latter statement has been<br />
questioned (579). Differences in competi<br />
tive binding <strong>of</strong> these two elements under<br />
special circumstances may explain these<br />
inverse relationships <strong>of</strong> zinc <strong>and</strong> <strong>copper</strong><br />
during gestation.<br />
Richterich et al. (638), comparing their<br />
data with that <strong>of</strong> prior investigators (338,<br />
491, 673), indicate general agreement that<br />
1) ceruloplasmin blood levels in pregnant<br />
women are two to three times those <strong>of</strong><br />
nonpregnant women, <strong>and</strong> that 2) the<br />
levels in mothers at term are approximately<br />
eight times those in umbilical cord blood.<br />
Ceruloplasmin values recorded are quite<br />
comparable, whether obtained by the en<br />
zymatic method <strong>of</strong> Ravin (629) or the<br />
immunological method <strong>of</strong> Hitzig (339).<br />
Maternal serum <strong>copper</strong> levels return to<br />
non-pregnant levels over a period <strong>of</strong> 4<br />
weeks or more following legal abortion<br />
(54) <strong>and</strong> normal delivery (388, 561, 777).<br />
Since estrogen production <strong>and</strong> serum<br />
ceruloplasmin levels are not proportional<br />
during pregnancy, <strong>and</strong> since blood estro<br />
gens decrease rapidly after abortion or<br />
delivery compared to ceruloplasmin levels,<br />
questions are raised as to whether estrogenie<br />
stimuli alone are responsible for the<br />
increase <strong>of</strong> serum <strong>copper</strong> in pregnancy. A<br />
similar dilemma relates to the increase in<br />
serum <strong>copper</strong> levels, over <strong>and</strong> above those<br />
<strong>of</strong> normal pregnancy, observed in pre<br />
eclampsia <strong>and</strong> eclampsia (205, 527, 552,<br />
578, 628, 777, 847, 858, 868), <strong>and</strong> in a case<br />
<strong>of</strong> hydatidiform mole (318). Evidence that<br />
maternal serum <strong>copper</strong> levels show even<br />
greater increases with intervening infec<br />
tious diseases <strong>and</strong> malignant processes has<br />
led to suggestions that increased serum<br />
<strong>copper</strong> levels in pregnancy reflect a re<br />
sistance reaction <strong>of</strong> the maternal organism<br />
to continuous invasion <strong>of</strong> the fetus into the<br />
maternal circulation (181). Another con<br />
cept relates these changes to a conse<br />
quence <strong>of</strong> hormonal adaptation <strong>of</strong> the<br />
maternal organism to the increased meta<br />
bolic <strong>and</strong> hormonal dem<strong>and</strong>s <strong>of</strong> pregnancy<br />
(167).<br />
Influence <strong>of</strong> oral contraceptives<br />
Reference has been made to early ob<br />
servations (183, 651, 794) <strong>of</strong> the thera<br />
peutic use <strong>of</strong> estrogens <strong>and</strong> the ensuing in<br />
crease in plasma levels <strong>of</strong> ceruloplasmin<br />
which, in turn, suggested an explanation<br />
for the comparable phenomenon observed<br />
previously in pregnant women. To this<br />
may be added evidence (98) that in nor<br />
mal individuals oral contraceptives cause<br />
marked increases in serum <strong>copper</strong> levels<br />
involving increased synthesis <strong>of</strong> ceruloplas<br />
min, <strong>of</strong>ten greater than that observed in<br />
the state <strong>of</strong> pregnancy. Since that time<br />
there has arisen a rather extensive litera<br />
ture on the effect <strong>of</strong> oral contraceptives<br />
<strong>and</strong> intrauterine <strong>copper</strong> devices in the<br />
human female.<br />
Since this topic has been well reviewed<br />
in recent years (589, 723, 771), it will not<br />
be subject to further consideration here.<br />
However, it must be emphasized that the<br />
continued use <strong>of</strong> oral contraceptives has<br />
been, <strong>and</strong> will continue to be, an impor<br />
tant factor in influencing <strong>copper</strong> homeostasis<br />
in women. Estrogens, whether en<br />
dogenous or exogenous, have a remark<br />
able capacity to stimulate synthesis <strong>of</strong><br />
ceruloplasmin in the liver <strong>and</strong> to increase<br />
urinary excretion <strong>of</strong> <strong>copper</strong>. The extent to<br />
which prolonged use <strong>of</strong> oral contraceptives<br />
may decrease body <strong>copper</strong> storage <strong>and</strong><br />
modify the daily <strong>copper</strong> requirement has<br />
not been examined. It does justify special<br />
study.<br />
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2002 KARL E. MASON<br />
Placental transfer<br />
Nonceruloplasmin <strong>copper</strong> readily crosses<br />
the placenta by passive diffusion, <strong>and</strong> its<br />
concentration in erythrocytes <strong>and</strong> plasma<br />
<strong>of</strong> mother <strong>and</strong> fetus is relatively constant<br />
throughout gestation (181, 673). Total<br />
<strong>copper</strong> in cord blood is about 1/4 to 1/5<br />
that in maternal blood (181, 464, 552, 556,<br />
561, 660, 673, 686). Studies in which blood<br />
<strong>of</strong> the umbilical vein (placenta to fetus)<br />
<strong>and</strong> umbilical artery (fetus to placenta)<br />
have been analyzed for <strong>copper</strong> give values<br />
<strong>of</strong> 53.4 to 40.0 fig/100 ml (556), 54.9 <strong>and</strong><br />
40.2 Mg/100 ml (167) <strong>and</strong> 74.4 <strong>and</strong> 41.8<br />
/ig/100 ml (770), respectively. Such find<br />
ings clearly indicate an important role <strong>of</strong><br />
the placenta in transfer <strong>of</strong> <strong>copper</strong> from<br />
mother to fetus. The high <strong>copper</strong> concen<br />
tration in the placenta (525, 605), liver<br />
<strong>and</strong> other fetal organs <strong>and</strong> tissues, referred<br />
to later, indicates the efficiency <strong>of</strong> this<br />
transfer.<br />
Questions <strong>of</strong> placental transfer <strong>and</strong> <strong>of</strong><br />
fetal synthesis <strong>of</strong> ceruloplasmin have never<br />
been satisfactorily clarified. It has been<br />
assumed that its large molecular size pre<br />
cludes placental transfer (673), although<br />
this may not be a valid conclusion (339).<br />
There are possibilities that with pro<br />
nounced thinning <strong>of</strong> the hemoendothelial<br />
membrane <strong>of</strong> placental villi, <strong>and</strong>/or tiny<br />
breaks therein during late phases <strong>of</strong> ges<br />
tation, some ceruloplasmin may be trans<br />
ferred to the fetal circulation (552). Also,<br />
one cannot exclude the possibility that<br />
ceruloplasmin may actually cross the pla<br />
centa, <strong>and</strong> that its rate <strong>of</strong> utilization <strong>and</strong><br />
breakdown may be equivalent to or greater<br />
than its rate <strong>of</strong> transfer (666). Although<br />
apoceruloplasmin can be identified immunologically<br />
in plasma <strong>of</strong> the newborn<br />
(496, 714), there is no evidence that ce<br />
ruloplasmin can be synthesized by the<br />
fetus. It is assumed that synthesis by the<br />
fetal liver does not begin until shortly<br />
after birth. In the domestic pig neither<br />
apoceruloplasmin nor ceruloplasmin ap<br />
pear in die piglet serum until about 15<br />
hours after birth (108, 109). In view <strong>of</strong><br />
these facts, the presence <strong>of</strong> small amounts<br />
<strong>of</strong> ceruloplasmin in cord blood <strong>of</strong> the new<br />
born (338, 606, 639, 673) suggests its<br />
transport from placenta to fetus. If this be<br />
so, the time over which this transfer may<br />
take place <strong>and</strong> its magnitude is unknown,<br />
<strong>and</strong> almost impossible to determine. A<br />
question which naturally arises is whether<br />
the fetus has or needs a ferroxidase type<br />
<strong>of</strong> enzyme, as a substitute for lack <strong>of</strong><br />
ceruloplasmin, to provide for the intensive<br />
hemopoietic activities <strong>of</strong> the fetal liver,<br />
spleen <strong>and</strong> bone marrow during fetal life.<br />
Somewhat ancillary to this discussion are<br />
observations <strong>of</strong> Widdowson et al. (842)<br />
that <strong>copper</strong> concentration in the liver <strong>of</strong> 30<br />
fetuses representing the 20th to 41st weeks<br />
<strong>of</strong> gestation were consistently high (aver<br />
age 6.4, range 3.5-9.3 mg/100 g fresh<br />
tissue), as compared to values <strong>of</strong> 0.5 mg/<br />
100 g fresh tissue for adult human liver,<br />
lyengar <strong>and</strong> Apte (377) give values <strong>of</strong><br />
4.76, 4.37, 4.38 <strong>and</strong> 4.23 mg/100 g fresh<br />
tissue for livers <strong>of</strong> 38 fetuses <strong>of</strong> gestational<br />
ages less than 28, 28 to 32, 33 to 36 <strong>and</strong><br />
37 to 40 weeks, respectively. On the other<br />
h<strong>and</strong>, Sultanova (761) reports that the<br />
more premature the infant the lower are<br />
the fetal liver reserves, while Butt et al.<br />
(85) find lower hepatic <strong>copper</strong> values in<br />
full term infants than in prematures.<br />
Neither study provides the firm data char<br />
acterizing the first two studies (377, 842)<br />
mentioned. Significantly lower levels <strong>of</strong><br />
total <strong>copper</strong> <strong>and</strong> ceruloplasmin in cord<br />
blood <strong>of</strong> neonates <strong>of</strong> undernourished<br />
mothers compared to those <strong>of</strong> well nour<br />
ished mothers suggest that poor nutri<br />
tional states <strong>of</strong> the mother are reflected in<br />
reduced capacity <strong>of</strong> the fetal liver to syn<br />
thesize proteins in general, <strong>and</strong> cerulo<br />
plasmin in particular (429).<br />
According to one investigator (526),<br />
amniotic fluid contains <strong>copper</strong> <strong>and</strong> other<br />
bioelements in about the same concen<br />
tration as in the maternal plasma <strong>and</strong>, by<br />
virtue <strong>of</strong> its being swallowed in appre<br />
ciable amounts, provides an additional<br />
supply to the developing fetus. However,<br />
other investigators report the presence <strong>of</strong><br />
very small amounts or only traces <strong>of</strong> cop<br />
per in this fluid (303, 321, 324, 564),<br />
which is in accord with the concept <strong>of</strong> the<br />
amniotic fluid as a protein-poor dialysate<br />
diluted with fetal urine.<br />
Infanctj <strong>and</strong> childhood<br />
Following normal delivery a series <strong>of</strong> in<br />
teresting <strong>and</strong> not fully explained events<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2003<br />
occur in mother <strong>and</strong> infant. Maternal levels<br />
<strong>of</strong> serum <strong>copper</strong> decrease to non-pregnant<br />
levels during the first 2 weeks postpartum<br />
(100, 206, 293, 294, 561). This is ascribed<br />
to the abrupt cessation <strong>of</strong> estrogenic<br />
stimuli for ceruloplasmin synthesis. At the<br />
same time, infant levels <strong>of</strong> serum <strong>copper</strong>,<br />
which are lower at birth than at any pe<br />
riod <strong>of</strong> life, promptly increase until adult<br />
levels are attained between the 2nd <strong>and</strong><br />
3rd months <strong>of</strong> life (91, 293, 325, 690).<br />
This is due almost entirely to increased<br />
synthesis <strong>of</strong> ceruloplasmin by the infant's<br />
liver. In other studies, estimates <strong>of</strong> the age<br />
at which adult values are reached vary<br />
from about 3 to 6 months (338, 494, 638),<br />
to 9 to 12 months (411, 606). Several in<br />
vestigators report that after adult serum<br />
<strong>copper</strong> levels are attained during the first<br />
2 or 3 months <strong>of</strong> life, these levels rise sig<br />
nificantly above normal after the 4th<br />
month (758 ) or during the 2nd year <strong>of</strong> life<br />
(131, 362, 656), <strong>and</strong> then gradually de<br />
cline to adult levels at puberty. Sass-<br />
Kortsak (666) gives mean values <strong>of</strong> 140,<br />
129 <strong>and</strong> 117 /¿g/100ml for 2-, 6- <strong>and</strong> 10year<br />
old children. Hrgovic <strong>and</strong> Hrgovic<br />
(362) record mean values <strong>of</strong> 179, 151, 133<br />
<strong>and</strong> 111 Mg/100 ml for age groups 0 to 5,<br />
5 to 10, 10 to 15 <strong>and</strong> 15 to 18 years. No<br />
sex differences are apparent until puberty,<br />
after which the effect <strong>of</strong> female estrogens<br />
on increased serum <strong>copper</strong> levels becomes<br />
manifest. Similar observations have been<br />
reported by other investigators (573, 776).<br />
Neither the reason for, nor the significance<br />
<strong>of</strong> these fluctuations in early life has been<br />
elucidated.<br />
Immunological methods have identified<br />
an apoceruloplasmin in newborn infant<br />
plasma in concentrations similar to that<br />
<strong>of</strong> ceruloplasmin in the adult, thus indi<br />
cating that only the inability to charge the<br />
apoprotein with its normal complement <strong>of</strong><br />
<strong>copper</strong> is underdeveloped at birth (714).<br />
Similar observations have been made in<br />
studies with piglets (108, 109, 523).<br />
On the other h<strong>and</strong>, remarkable changes<br />
occur in the liver <strong>of</strong> the newborn, which<br />
contains about one-half the <strong>copper</strong> in the<br />
body <strong>and</strong> a concentration, in terms <strong>of</strong> cop<br />
per per unit weight, 5 to 10 times that <strong>of</strong><br />
the adult liver (843, 846). A large com<br />
ponent <strong>of</strong> this <strong>copper</strong> is bound to hepatic<br />
initochondrocuprein, first isolated <strong>and</strong> de<br />
scribed by Porter et al. (615). This cop<br />
per-storage protein, found only in the fetus<br />
<strong>and</strong> newborn, is thought to represent a<br />
<strong>copper</strong>-rich polymerized form <strong>of</strong> metallothionein<br />
which sequesters <strong>copper</strong> prior to<br />
birth (611, 614). Liver <strong>copper</strong> rapidly<br />
disappears during the first few months <strong>of</strong><br />
life (69, 627, 846), releasing <strong>copper</strong> for<br />
ceruloplasmin synthesis <strong>and</strong> the general<br />
needs <strong>of</strong> tissues <strong>of</strong> the rapidly growing in<br />
fant. The ceruloplasmin synthesized by the<br />
neonate is identical to that <strong>of</strong> the adult<br />
(870). Thus there is a logical explanation<br />
for the early reports <strong>of</strong> Kleinman <strong>and</strong><br />
Klinke (414) <strong>and</strong> Morrison <strong>and</strong> Nash<br />
(538) that the concentration <strong>of</strong> <strong>copper</strong> in<br />
the liver <strong>of</strong> newborn <strong>and</strong> young infants is<br />
6- to 18-fold that <strong>of</strong> adults.<br />
Although <strong>copper</strong> <strong>and</strong> ceruloplasmin<br />
blood levels are lower in the newborn than<br />
at any other period <strong>of</strong> life, the <strong>copper</strong> con<br />
centration in fetal <strong>and</strong> neonatal organs <strong>and</strong><br />
tissues is much higher than in the adult<br />
(207, 248, 414, 565, 781). The studies <strong>of</strong><br />
Fazekas (207), based upon analysis <strong>of</strong> 29<br />
different organs <strong>and</strong> tissues <strong>of</strong> 109 fetuses<br />
<strong>and</strong> full-term infants <strong>of</strong> varied gestational<br />
age, indicate that in addition to liver, the<br />
concentration <strong>of</strong> <strong>copper</strong> in muscle, skin,<br />
adrenal gl<strong>and</strong>s <strong>and</strong> thyroid is particularly<br />
high compared to that <strong>of</strong> adults. The cop<br />
per content <strong>of</strong> the placenta is also rather<br />
high (525, 605). The high concentrations<br />
<strong>of</strong> <strong>copper</strong> in organs <strong>and</strong> tissues <strong>of</strong> the<br />
newborn decrease gradually to normal<br />
levels during the first year <strong>of</strong> postnatal life<br />
(69, 248, 565).<br />
The unusually high concentrations <strong>of</strong><br />
<strong>copper</strong> in liver <strong>and</strong> other tissues <strong>of</strong> the<br />
neonate appear to represent a reserve to<br />
assure an adequacy <strong>of</strong> <strong>copper</strong> for syn<br />
thesis <strong>of</strong> ceruloplasmin <strong>and</strong> other <strong>copper</strong>containing<br />
proteins to meet metabolic<br />
needs for hematopoietic, maturational <strong>and</strong><br />
other functions in the rapidly growing in<br />
fant <strong>and</strong> adolescent prior to puberty. These<br />
changes naturally create difficulties in<br />
reaching definitive conclusions concerning<br />
the dietary <strong>requirements</strong> during these early<br />
years <strong>of</strong> human development. For other<br />
details concerning the role <strong>of</strong> <strong>copper</strong> in<br />
pregnancy, <strong>and</strong> in prenatal <strong>and</strong> postnatal<br />
development, the reader is referred to a<br />
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2004 KARL E. MASON<br />
number <strong>of</strong> informative reviews (461, 533,<br />
666, 843).<br />
DIETARY COPPER DEFICIENCY<br />
There appear repeated statements in the<br />
literature that in view <strong>of</strong> the ubiquitous<br />
occurrence <strong>of</strong> <strong>copper</strong> in foods <strong>of</strong> every<br />
type, <strong>and</strong> lack <strong>of</strong> evidence <strong>of</strong> any recog<br />
nized manifestations <strong>of</strong> <strong>copper</strong> deficiency<br />
such as commonly observed after dietary<br />
depletion <strong>of</strong> <strong>copper</strong> in experimental ani<br />
mals or under natural conditions in farm<br />
animals, man appears to be free <strong>of</strong> hazards<br />
<strong>of</strong> a state <strong>of</strong> <strong>copper</strong> deficiency. However,<br />
there has developed convincing evidence<br />
that a <strong>copper</strong> deficiency state can occur in<br />
man, even though it may be <strong>of</strong> rare oc<br />
currence <strong>and</strong> as the result <strong>of</strong> rather special<br />
types <strong>of</strong> situations.<br />
A syndrome characterized by hypocupremia,<br />
hyp<strong>of</strong>erremia, hypoproteinemia,<br />
edema <strong>and</strong> hypochromic anemia, respon<br />
sive to oral <strong>copper</strong> but not to iron, has<br />
been observed in infants fed diets limited<br />
largely to milk (432, 437, 758, 796, 797,<br />
874). In certain cases, especially those <strong>of</strong><br />
Ulstrom et al. (796, 797), a fundamental<br />
defect in protein <strong>metabolism</strong> at the cellular<br />
level may have been a primary factor,<br />
rather than exhaustion <strong>of</strong> neonatal <strong>copper</strong><br />
stores (874). The fact that infants 6 to 18<br />
months <strong>of</strong> age usually have been involved<br />
suggests a relation to periods <strong>of</strong> life when<br />
initial liver storage <strong>of</strong> <strong>copper</strong> has been<br />
depleted, combined with prolonged main<br />
tenance on milk diets <strong>and</strong> increased de<br />
m<strong>and</strong>s for <strong>copper</strong> during a period <strong>of</strong> rapid<br />
growth. However, the possibility <strong>of</strong> de<br />
grees <strong>of</strong> protein depletion sufficient to im<br />
pair retention <strong>of</strong> dietary <strong>copper</strong> deserved<br />
consideration.<br />
Maintenance <strong>of</strong> two infants (one 8 days<br />
old with multiple congenital anomalies,<br />
<strong>and</strong> the other 10 months old) for 4 to 5<br />
months on a milk diet identical to one<br />
which produced <strong>copper</strong> deficiency in pig<br />
lets, caused neither anemia nor hypocupremia<br />
(101). Comparable results were<br />
obtained in the studies <strong>of</strong> Wilson <strong>and</strong><br />
Lahey (854) involving seven premature<br />
infants with mean body weight <strong>of</strong> 1.24 kg<br />
fed a similar milk diet for 7 to 10 weeks.<br />
It was concluded that small premature in<br />
fants fed a diet providing approximately<br />
15 /xg/kg/day <strong>of</strong> elemental <strong>copper</strong> over a<br />
2 to 3-month period do not differ, by any<br />
<strong>of</strong> the criteria used, from prematures fed<br />
five or more times this amount. However, it<br />
should be recognized that at this period<br />
such infants could be utilizing liver stores<br />
<strong>of</strong> <strong>copper</strong>, <strong>and</strong> that the depletion period<br />
was much shorter than that required for<br />
production <strong>of</strong> a deficiency state in piglets<br />
with a relatively more rapid rate <strong>of</strong> growth.<br />
Not until 1964 was a state <strong>of</strong> dietary<br />
<strong>copper</strong> deficiency documented in humans<br />
when Cordano et al. (126, 128) reported<br />
finding in infants, recovering from maras<br />
mus on exclusive milk diets, deficiency<br />
manifestations (anemia, decreased plasma<br />
<strong>copper</strong> <strong>and</strong> ceruloplasmin levels, intermit<br />
tent neutropenia, severe osteoporosis <strong>and</strong><br />
pathological fractures) quite comparable<br />
to those observed after experimental cop<br />
per deficiency in pigs (436). Similar find<br />
ings were observed in a 6-year old child<br />
with severe chronic intestinal malabsorption,<br />
who gave a dramatic response to cop<br />
per therapy (127). In a later report,<br />
Graham <strong>and</strong> Cordano (276) state that in a<br />
series <strong>of</strong> 173 infants suffering from severe<br />
malnutrition <strong>and</strong> chronic diarrhea, admit<br />
ted to the British American Hospital, Lima,<br />
Peru, over a period <strong>of</strong> 6 years, 62 instances<br />
<strong>of</strong> <strong>copper</strong> deficiency were identified, <strong>of</strong><br />
which 44 were judged to have been de<br />
pleted <strong>of</strong> <strong>copper</strong> prior to admission. The<br />
peak incidence was at 7 to 9 months <strong>of</strong><br />
age. Responses to oral <strong>copper</strong> therapy in<br />
dicated that repletion <strong>of</strong> total serum cop<br />
per was more important than restoration <strong>of</strong><br />
ceruloplasmin in correction <strong>of</strong> the defi<br />
ciency state (352). Instances <strong>of</strong> <strong>copper</strong> de<br />
pletion have also been observed in infants<br />
with chronic diarrhea in the United States<br />
(354).<br />
There have also appeared more recent<br />
reports <strong>of</strong> neonatal <strong>copper</strong> deficiency in a<br />
premature infant 3 months old (17), in<br />
three very small premature infants during<br />
their third month <strong>of</strong> life (280), in a pre<br />
mature infant 3 months <strong>of</strong> age (700), <strong>and</strong><br />
in a premature infant at 6 months <strong>of</strong> age<br />
(25). Neutropenia, low plasma ceruloplas<br />
min <strong>and</strong> osteoporosis have been among the<br />
manifestations regularly observed. Favor<br />
able response to oral <strong>copper</strong> has usually<br />
been reported. Although none <strong>of</strong> the in-<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2005<br />
vestigators make reference to it, the clini<br />
cal <strong>and</strong> pathological findings reported bear<br />
striking resemblance to many <strong>of</strong> those<br />
characteristic <strong>of</strong> Menkes' steely-hair syn<br />
drome (p. 2007). A comparable picture<br />
has been observed in an infant maintained<br />
for some months on total parenteral nutri<br />
tion following surgery for ileal atresia<br />
(394) <strong>and</strong> in others treated likewise for<br />
protracted diarrhea (463). Also, a state<br />
<strong>of</strong> <strong>copper</strong> deficiency has been described<br />
in a 12-year old child <strong>and</strong> an adult after<br />
prolonged total parenteral therapy follow<br />
ing extensive bowel resection ( 177). In<br />
all cases there was a good response to cop<br />
per therapy.<br />
It is worthy <strong>of</strong> note that in all instances<br />
where a naturally occurring <strong>copper</strong> de<br />
ficiency has been observed, as described<br />
above, only premature infants have been<br />
involved. This is in accord with the fact<br />
that premature infants do not benefit from<br />
the additional <strong>copper</strong> storage in the liver<br />
<strong>and</strong> other tissues acquired by the fullterm<br />
infant, <strong>and</strong> are customarily main<br />
tained for longer periods on natural milk<br />
or milk formulae before having access to<br />
cereals <strong>and</strong> other foods. Furthermore, pre<br />
matures have much lower <strong>copper</strong> reserves<br />
in the liver <strong>and</strong> spleen than do full-term<br />
infants, <strong>and</strong> show a negative <strong>copper</strong> bal<br />
ance during the first month <strong>of</strong> life which<br />
tends to become positive only after the<br />
second month <strong>of</strong> life (761). Suggestions<br />
that consideration should be given to<br />
special supplementation <strong>of</strong> the premature<br />
infant formula with <strong>copper</strong> (843) appears<br />
to be well justified.<br />
The major manifestations <strong>of</strong> <strong>copper</strong> de<br />
ficiency in infancy, <strong>and</strong> their relationship<br />
to decreased activity <strong>of</strong> <strong>copper</strong>-containing<br />
proteins, justify brief summarization.<br />
1) Neutropenia <strong>and</strong> hypochromic anemia<br />
responsive to oral <strong>copper</strong> but not to oral<br />
iron are early manifestations <strong>of</strong> deficiency,<br />
in large part the result <strong>of</strong> lowered levels <strong>of</strong><br />
ceruloplasmin <strong>and</strong> impaired release <strong>and</strong><br />
transport <strong>of</strong> iron from body stores.<br />
2) Osteoporosis, with enlargement <strong>of</strong><br />
costochondral cartilages, is also an early<br />
phenomenon, followed by cupping <strong>and</strong><br />
flaring <strong>of</strong> metaphyses <strong>of</strong> long bones with<br />
spur formation <strong>and</strong> submetaphyseal frac<br />
ture, periosteal reactions <strong>and</strong> spontaneous<br />
fractures, especially <strong>of</strong> the ribs. These are<br />
usually referred to as "scurvy-like" changes,<br />
<strong>and</strong> may also suggest the "battered child<br />
syndrome." Deficiency <strong>of</strong> <strong>copper</strong>-contain<br />
ing oxidases, essential for the cross-linking<br />
<strong>of</strong> bone collagen, adequately explains these<br />
manifestations.<br />
3) Decreased pigmentation <strong>of</strong> the skin<br />
<strong>and</strong> general pallor <strong>of</strong> <strong>copper</strong>-deficient in<br />
fants, can be attributed to decreased activ<br />
ity <strong>of</strong> tyrosinase, necessary for the produc<br />
tion <strong>of</strong> melanin.<br />
4) In later stages <strong>of</strong> deficiency there<br />
may be neurological abnormalities such<br />
as hypotonia, episodes <strong>of</strong> apnea <strong>and</strong> pos<br />
sible psychomotor retardation, generally<br />
attributed to decreased levels <strong>of</strong> cytochrome<br />
c oxidase.<br />
MENKES' DISEASE<br />
This progressive brain disease inherited<br />
as a sex-linked recessive trait was first<br />
recognized in five young boys (siblings),<br />
<strong>and</strong> its major clinical <strong>and</strong> pathological<br />
manifestations described in 1962 by Menkes<br />
et al. (513). Other cases were soon re<br />
ported by Bray (59) <strong>and</strong> Aguilar et al.<br />
(11). While subsequently referred to as<br />
"kinky-hair" disease <strong>and</strong> "trichopoliodystrophy"<br />
(225), the currently accepted des<br />
ignation is "steely-hair" disease (or syn<br />
drome) proposed by Danks et al. who<br />
( 145) in 1972, first recognized the disease<br />
as an inherited defect in <strong>copper</strong> absorp<br />
tion; in fact, a congenital <strong>copper</strong> deficiency.<br />
Since the term "kinky-hair" implied<br />
crimped hair like that <strong>of</strong> the black races,<br />
whereas that <strong>of</strong> affected infants more<br />
closely resembled depigmentation <strong>and</strong><br />
loss <strong>of</strong> crimp in wool observed in <strong>copper</strong>deficient<br />
sheep (252), "steely-hair" appears<br />
to be more appropriate ( 146). It is said to<br />
occur in 1 <strong>of</strong> 35,000 live births (145). As <strong>of</strong><br />
1977, Ahlgren <strong>and</strong> Vestermark (12) list 42<br />
cases reported in the literature.<br />
Manifestations<br />
Symptoms <strong>of</strong> Menkes' disease usually ap<br />
pear between birth <strong>and</strong> 3 months <strong>of</strong> age,<br />
followed by death prior to the 4th or 5th<br />
year <strong>of</strong> life. The age <strong>of</strong> onset is somewhat<br />
earlier in prematures than in full-term in<br />
fants. Occurrence as late as the 6th year<br />
has been reported (28). Characteristics <strong>of</strong><br />
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2006 KARL E. MASON<br />
the disease, as originally recorded by<br />
Menkes et al. (513), are: short, broken,<br />
spirally twisted scalp hair (pili torti) with<br />
loss <strong>of</strong> pigment; frequent convulsive sei<br />
zures, failure to thrive or poor weight gain,<br />
mental retardation <strong>and</strong>, at necropsy, wide<br />
spread degenerative changes in the cere<br />
brum <strong>and</strong> cerebellum. To these manifesta<br />
tions have since been added: hypothermia<br />
(56, 72, 145, 148, 168, 225, 530); marked<br />
tortuosity, associated with defects <strong>of</strong> the<br />
internal elastic lamina <strong>and</strong> hyperplasia <strong>of</strong><br />
the overlying intima, <strong>of</strong> large muscular<br />
arteries, particularly those supplying the<br />
brain <strong>and</strong> its appendages (4, 12, 114, 145,<br />
146, 422, 582, 713, 717, 808, 817); excessive<br />
Wormian bone formation (283, 645, 732,<br />
835) <strong>and</strong> pronounced changes in certain<br />
long bones similar to those <strong>of</strong> scurvy <strong>and</strong>/<br />
or the battered child syndrome (56, 145,<br />
168, 669, 713, 717, 732). Biochemically,<br />
there is increased <strong>copper</strong> content <strong>of</strong> the<br />
intestinal mucosa ( 145, 148, 470 ) ; greatly<br />
reduced levels <strong>of</strong> serum <strong>copper</strong> <strong>and</strong> ceruloplasmin<br />
(55, 72, 148, 168, 310, 530, 713,<br />
817) despite normal <strong>copper</strong> levels in<br />
erythrocytes, <strong>and</strong> much reduced levels <strong>of</strong><br />
<strong>copper</strong> in the liver ( 145, 148, 329, 817) <strong>and</strong><br />
brain (145, 817).<br />
Other reports have made reference to<br />
deficient visual functions <strong>and</strong> ocular ab<br />
normalities (49, 310, 453, 697, 717, 863)<br />
<strong>and</strong> to neurogenic bladders with diverticulum<br />
formation (306, 838), presumably due<br />
to defective innervation or vascular abnor<br />
malities. In the only known case <strong>of</strong> Menkes'<br />
disease in a black infant, Volpintesta (809 )<br />
observed a remarkable light skin in con<br />
trast to the dark-skinned parents, an un<br />
usual mottled skin pigmentation in a 7-year<br />
old female sibling, <strong>and</strong> a small degree <strong>of</strong><br />
pili torti in the mother <strong>and</strong> two female<br />
siblings. Manifestations typical <strong>of</strong> Menkes'<br />
syndrome, except for the absence <strong>of</strong> steely<br />
hair, have been reported in a Japanese in<br />
fant by Osaka et al. (582) who question<br />
whether some cases <strong>of</strong> the disease may go<br />
unrecognized because <strong>of</strong> too great a re<br />
liance on the hair abnormality as a diag<br />
nostic feature.<br />
Several new observations have special<br />
relevance to early diagnosis <strong>of</strong> Menkes'<br />
disease. A recently described method for<br />
measuring ceruloplasmin using a dried<br />
blood clot (21) may have value in screen<br />
ing for early diagnosis, but only when ap<br />
plied at 3 months <strong>of</strong> age or later (495).<br />
An intense metachromasia<br />
sue cultures <strong>of</strong> fibroblasts<br />
in primary tis<br />
has interesting<br />
possibilities as a genetic marker in early<br />
diagnosis <strong>and</strong> in identification <strong>of</strong> homozy<br />
gotes <strong>and</strong> hétérozygotes in affected fam<br />
ilies (145, 147). This defective cellular<br />
<strong>metabolism</strong><br />
dicated by<br />
<strong>of</strong> <strong>copper</strong> in fibroblasts is in<br />
other observations that cul<br />
tured skin fibroblasts from subjects<br />
Menkes' disease have an abnormally<br />
with<br />
high<br />
<strong>copper</strong> content (259) <strong>and</strong> also can incor<br />
porate much greater amounts <strong>of</strong> 84Cu from<br />
the medium than do fibroblasts <strong>of</strong> un<br />
affected subjects (358). The <strong>copper</strong> con<br />
tent <strong>of</strong> the culture medium may be critical<br />
in such evaluations. Regrettably, there<br />
exist rather limited prospects that im<br />
provements in early diagnosis will be paral<br />
lelled by more effective therapeutic mea<br />
sures. Widespread degeneration <strong>of</strong> cerebral<br />
grey matter, secondary to degeneration <strong>of</strong><br />
cerebral white matter <strong>and</strong> diffuse atrophy<br />
<strong>of</strong> the cerebral cortex, associated with<br />
bizarre changes in shape <strong>and</strong> arrangement<br />
<strong>of</strong> Purkinje cells, as first described by<br />
Menkes et al. (513), has been confirmed by<br />
later neuropathologic studies ( 11, 251, 336,<br />
624, 802, 810). Nerve tracts <strong>of</strong> the spinal<br />
cord may sometimes be involved (11, 251).<br />
Peripheral nerves are not affected. It is<br />
proposed that these lesions reflect two<br />
types <strong>of</strong> change; viz., cerebral necrosis due<br />
to abnormalities <strong>of</strong> the extracranial arteries,<br />
<strong>and</strong> typical dystrophic lesions in the<br />
cerebellar cortex (810). Confirming <strong>and</strong><br />
extending the original descriptions <strong>of</strong> the<br />
cerebellar lesions by Menkes et al. (513)<br />
<strong>and</strong> Aguilar et al. (Il), ultrastructural<br />
studies <strong>of</strong> the bizarre elaboration <strong>of</strong> perisomatic<br />
dendrites <strong>of</strong> Purkinje cells sug<br />
gest retarded development <strong>of</strong> the somal<br />
membrane <strong>of</strong> these cells (336, 624). Such<br />
evidence is cited in support <strong>of</strong> the concept<br />
that the disease process is operative in<br />
utero (340).<br />
A careful light <strong>and</strong> electron microscopic<br />
study <strong>of</strong> the eye has revealed degeneration<br />
<strong>of</strong> retinal ganglion cells, loss <strong>of</strong> nerve fibers,<br />
optic atrophy, abnormal pigment epithe<br />
lium <strong>and</strong> abnormal elastin in Bruch's mem<br />
brane (863). A similar study <strong>of</strong> aorta <strong>and</strong><br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2007<br />
skin in Menkes' disease has demonstrated<br />
abnormalities <strong>of</strong> elastic fibers similar to<br />
those observed in animal studies on <strong>copper</strong><br />
deficiency (566). Reported ultrastructural<br />
changes in skeletal muscle (251) have not<br />
been confirmed (717). Neurochemical<br />
studies on two infants whose neuropathology<br />
was described by Aguilar et al. (Il)<br />
record abnormally low levels <strong>of</strong> polyunsaturated<br />
glycerophosphates, especially in<br />
the frontal gray matter, <strong>and</strong> accumulation<br />
<strong>of</strong> oxidized lipid products in the neurones,<br />
suggesting an interference with the molec<br />
ular machinery <strong>of</strong> the cells (567). These<br />
observations have been both challenged<br />
(469) <strong>and</strong> confirmed (645). French et al.<br />
(224, 225) who proposed the term "trichopoliodystrophy"<br />
report much reduced<br />
levels <strong>of</strong> cytochrome a <strong>and</strong> a3 in mitochon<br />
dria <strong>of</strong> brain, muscle <strong>and</strong> liver, <strong>and</strong> con<br />
clude that deficient terminal respiration<br />
<strong>and</strong> failure <strong>of</strong> tissue energetics, secondary<br />
to diminished body <strong>copper</strong> content, may<br />
explain some <strong>of</strong> the neuropathology <strong>of</strong><br />
Menkes' disease. Significantly reduced<br />
levels <strong>of</strong> erythrocyte Superoxide dismutase<br />
<strong>and</strong> <strong>of</strong> dopamine ß-hydroxylasemay have<br />
bearing upon manifestations <strong>of</strong> the disease<br />
(645).<br />
Metabolic abnormalities<br />
Danks et al. (145, 147) propose that the<br />
primary defect in Menkes' disease is dimin<br />
ished ability to transfer <strong>copper</strong> across ab<br />
sorptive cells <strong>of</strong> the intestinal mucosa to<br />
the serosal side <strong>and</strong> the portal circulation.<br />
While considering this an important factor,<br />
Bucknall et al. (72), based upon studies<br />
on a 3-month old infant, speculate that<br />
neurological damage may occur in utero,<br />
perhaps as a result <strong>of</strong> defective placental<br />
transport <strong>of</strong> <strong>copper</strong>. This is supported by<br />
reports <strong>of</strong> the occurrence <strong>of</strong> one or more<br />
manifestations <strong>of</strong> Menkes' disease at term<br />
or within the week thereafter (283, 513,<br />
530). However, defective placental trans<br />
port may not provide the total answer.<br />
Heydorn et al. (329) <strong>and</strong> Horn et al.<br />
(359) consider that defective binding <strong>of</strong><br />
<strong>copper</strong> in the fetal liver or atypical distri<br />
bution in fetal tissues may be involved.<br />
The conclusions <strong>of</strong> Heydorn et al. (329)<br />
are based upon tissue analyses <strong>of</strong> a fetus<br />
suspected <strong>of</strong> Menkes' disease, obtained by<br />
therapeutic abortion at 18 weeks gesta<br />
tion. Compared to four normal fetuses <strong>of</strong><br />
15 to 21 weeks gestation, <strong>copper</strong> concen<br />
trations in brain, lung, spleen, kidney, pan<br />
creas, muscle, skin <strong>and</strong> placenta were<br />
several times greater than, but liver cop<br />
per was only about one-third, that <strong>of</strong> con<br />
trols. Yet, the estimated total <strong>copper</strong> con<br />
tent <strong>of</strong> all fetuses was essentially the same.<br />
These findings, until substantiated, do not<br />
eliminate possibilities <strong>of</strong> defective placental<br />
transfer. However, they do raise questions<br />
regarding <strong>copper</strong> storage in fetal liver <strong>and</strong><br />
atypical distribution <strong>of</strong> <strong>copper</strong> in other<br />
fetal tissues <strong>and</strong> organs in Menkes' disease<br />
(329, 635).<br />
Menkes' disease <strong>and</strong> nutritional <strong>copper</strong><br />
deficiency have certain features in com<br />
mon; 1) usual occurrence in infancy;<br />
2) subnormal plasma levels <strong>of</strong> <strong>copper</strong> <strong>and</strong><br />
ceruloplasmin; 3) tortuosity <strong>and</strong> defects in<br />
elastin <strong>of</strong> the aorta due to lack <strong>of</strong> lysyl<br />
oxidase; 4) scorbutic-like changes in costochondral<br />
junctions <strong>and</strong> epiphyses <strong>of</strong> long<br />
bones; <strong>and</strong> 5) decreased pigmentation <strong>of</strong><br />
skin or hair. Menkes' disease differs from<br />
the state <strong>of</strong> dietary <strong>copper</strong> deficiency in<br />
the following respects: 1) alterations <strong>of</strong><br />
hair structure <strong>and</strong> decreased pigmentation<br />
<strong>of</strong> hair, the latter attributable to lack <strong>of</strong><br />
tyrosinase; 2) highly variable <strong>and</strong> <strong>of</strong>ten<br />
extensive lesions involving both white <strong>and</strong><br />
gray matter <strong>of</strong> the cerebrum <strong>and</strong> cerebel<br />
lum, usually ascribed to lack <strong>of</strong> cytochrome<br />
oxidase <strong>and</strong> Superoxide dismutase which,<br />
in turn, may also be factors involved in<br />
3) hypothermia, a frequently observed<br />
phenomenon; <strong>and</strong> 4) the almost routine<br />
occurrence <strong>of</strong> convulsive seizures <strong>and</strong><br />
mental retardation. To these may be added<br />
5) absence <strong>of</strong> anemia <strong>and</strong> neutropenia <strong>and</strong><br />
6) unresponsiveness to orally administered<br />
<strong>copper</strong> other than significant increases in<br />
plasma levels <strong>of</strong> <strong>copper</strong> <strong>and</strong> ceruloplasmin.<br />
Separate oral <strong>and</strong> intravenous adminis<br />
tration <strong>of</strong> '"Cu, permitting calculation <strong>of</strong><br />
the percentage <strong>of</strong> dose absorbed, indicates<br />
that children with Menkes' disease absorb<br />
only 11 to I3c/c <strong>of</strong> oral <strong>copper</strong> as com<br />
pared to 46^ by unaffected controls, sug<br />
gesting a reduced absorption <strong>of</strong> <strong>copper</strong> as<br />
an important factor in the disorder ( 159).<br />
Also, most <strong>of</strong> the <strong>copper</strong> absorbed is re<br />
tained by the liver for extended periods <strong>of</strong><br />
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2008 KARL E. MASON<br />
time <strong>and</strong> excretory loss is reduced, thus in<br />
creasing the biological half-life in the body<br />
by two to three times, as compared to nor<br />
mal controls (159, 160). Under similar<br />
circumstances subjects with Wilson's dis<br />
ease show normal absorption <strong>of</strong> <strong>copper</strong><br />
but, because <strong>of</strong> reduced biliary excretion,<br />
the half-life is increased to about the same<br />
degree as in Menkes' disease ( 160 ). Hence,<br />
these two diseases have dissimilarities re<br />
lating to intestinal absorption but similari<br />
ties in inability to release <strong>copper</strong> acquired<br />
by the liver.<br />
Although a defect in the intestinal trans<br />
port <strong>of</strong> <strong>copper</strong> undoubtedly plays an im<br />
portant role in postnatal life, it does not<br />
provide an adequate explanation for the<br />
diverse manifestations <strong>of</strong> Menkes' disease.<br />
With impressive evidence that this disease<br />
begins in utero, it would appear that vary<br />
ing degrees <strong>of</strong> genetic expression may ex<br />
plain differences in postnatal age when<br />
manifestations, such as frequent seizures,<br />
make their appearance. There remain many<br />
important questions the answers to which<br />
will be difficult to obtain. For example,<br />
is there defective placental transfer <strong>of</strong> cop<br />
per comparable to that proposed in the in<br />
testine? Is placental transfer normal, but<br />
the types <strong>of</strong> fetal protein to which <strong>copper</strong><br />
becomes bound abnormal? For this or for<br />
other reasons, is the concentration <strong>of</strong> nor<br />
mally or abnormally bound <strong>copper</strong> in cer<br />
tain tissues <strong>and</strong> organs significantly de<br />
ranged? Are there abnormalities in the<br />
production <strong>of</strong> <strong>copper</strong>-containing enzymes<br />
or in membrane receptors in certain cells<br />
<strong>and</strong> tissues? Obviously, knowledge <strong>of</strong> the<br />
underlying metabolic disturbances in Men<br />
kes' disease is in a state <strong>of</strong> immaturity, but<br />
<strong>of</strong>fers many challenges for future research.<br />
For further details the reader is referred to<br />
some recent reviews (75, 143, 144, 272,<br />
301, 351, 818).<br />
Therapy<br />
Oral administration <strong>of</strong> <strong>copper</strong> salts to<br />
infants with Menkes' disease has been<br />
reported to cause slight clinical improve<br />
ment, such as reduced hypothermia <strong>and</strong><br />
improved hair color, <strong>and</strong> slight increase in<br />
serum <strong>copper</strong> levels (148, 467, 468), but<br />
other investigators find no beneficial effect<br />
(72, 243, 566, 833, 849). Intramuscular<br />
injections <strong>of</strong> <strong>copper</strong> complexed with EDTA<br />
can cause a significant increase in serum<br />
<strong>copper</strong> <strong>and</strong> serum ceruloplasmin (146,<br />
817, 838), as also can a slow subcutaneous<br />
drip <strong>of</strong> <strong>copper</strong> sulphate over a period <strong>of</strong><br />
2 hours every 3 to 4 days (161). In one<br />
case so treated for 5 months a moderately<br />
encouraging clinical response was obtained<br />
( 161). However, Wheeler <strong>and</strong> Roberts<br />
(838) report that while intramuscular in<br />
jections <strong>of</strong> a <strong>copper</strong>-EDTA complex main<br />
tained reasonably normal serum <strong>copper</strong><br />
<strong>and</strong> ceruloplasmin levels for 8 months in<br />
one infant, no clinical improvement was<br />
apparent.<br />
Intravenously administered <strong>copper</strong> in<br />
various forms (<strong>copper</strong> sulphate, <strong>copper</strong><br />
acetate, <strong>copper</strong>-albumin, <strong>copper</strong>-EDTA<br />
complexes <strong>and</strong> human ceruloplasmin) has<br />
produced increases in serum <strong>copper</strong> <strong>and</strong><br />
ceruloplasmin to values approaching nor<br />
mal (72) or essentially normal (146, 161,<br />
282, 283, 833, 849), or no significant change<br />
(243, 244). Usually the period <strong>of</strong> treat<br />
ment has been short (7-10 days) or inter<br />
mittent over somewhat longer periods. In<br />
one instance normal serum <strong>copper</strong> levels<br />
<strong>and</strong> subnormal ceruloplasmin levels were<br />
maintained for more than 9 months by<br />
weekly intravenous infusions (833). A<br />
similar experience with 427 days <strong>of</strong> subcutaneously<br />
administered <strong>copper</strong> as a<br />
CuClo + L-histidine complex, has also been<br />
reported (849). However, in all cases the<br />
disease has pursued its relentless course.<br />
But a faint gleam <strong>of</strong> hope comes from a<br />
report <strong>of</strong> Grover <strong>and</strong> Scrutton (282, 283)<br />
who, employing repeated infusions <strong>of</strong> cop<br />
per sulphate once or twice weekly in an<br />
infant diagnosed as having Menkes' disease<br />
at 3 days <strong>of</strong> age, obtained mental func<br />
tional levels equivalent to 4 months at 6<br />
months <strong>of</strong> age. However, similar treatment<br />
<strong>of</strong> another infant beginning at 4 months <strong>of</strong><br />
age <strong>and</strong> continued for 9 months provided<br />
no improvement. Hence, the answer is<br />
equivocal. Excessive urinary excretion <strong>of</strong><br />
<strong>copper</strong> observed after parenteral therapy<br />
(242, 243, 849) is attributed to decreased<br />
hepatic uptake <strong>of</strong> <strong>copper</strong>, reflecting an ab<br />
normality <strong>of</strong> transport comparable to that<br />
in the intestinal mucosa. These observa<br />
tions suggest that a defect in membrane<br />
transport could explain both the in utero<br />
<strong>and</strong> postnatal deprivation <strong>of</strong> <strong>copper</strong> at<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2009<br />
multiple organ levels including placenta,<br />
intestine <strong>and</strong> liver; however, a favored<br />
alternative is that <strong>of</strong> a defect in intracellular<br />
transport in intestine, liver <strong>and</strong><br />
kidney due to abnormalities in the trans<br />
port <strong>and</strong> storage protein, metallothionein<br />
(242, 244).<br />
From the information currently available,<br />
it seems questionable whether institution<br />
<strong>of</strong> any type <strong>of</strong> therapeutic measure during<br />
the period <strong>of</strong> gestation, if such were pos<br />
sible, would significantly alter the course <strong>of</strong><br />
this genetically determined abnormality <strong>of</strong><br />
<strong>copper</strong> <strong>metabolism</strong>.<br />
Animal models<br />
It seems obvious that there is still much<br />
to be learned regarding the metabolic de<br />
fect in Menkes' disease. Challenging op<br />
portunities for future research are pro<br />
vided by several genetic animal models <strong>of</strong><br />
this disease. One model is a recessive gene<br />
mutation (crinkled, CR) in mice. Such<br />
mice have a smooth coat with thin skin,<br />
delayed pigmentation, early mortality, <strong>and</strong><br />
hair changes closely resembling those <strong>of</strong><br />
Menkes' disease (374). In fact, all three<br />
types <strong>of</strong> hair abnormalities found in<br />
Menkes' disease are demonstrable (373).<br />
The observation that increased dietary in<br />
take <strong>of</strong> <strong>copper</strong> during pregnancy <strong>and</strong> lacta<br />
tion favorably altered the expression <strong>of</strong> the<br />
mutant gene (374) is certainly worthy <strong>of</strong><br />
further exploration. Another model, a sexlinked<br />
"brindled" or "mottled" (MO"r)<br />
mutant in the mouse, is characterized by<br />
subnormal levels <strong>of</strong> lysyl oxidase (648),<br />
plasma ceruloplasmin, decreased <strong>copper</strong><br />
levels in brain <strong>and</strong> liver, increased <strong>copper</strong><br />
in the intestinal wall <strong>and</strong> virtual absence<br />
<strong>of</strong> hair pigmentation (371). Moreover, a<br />
recent study (200) indicates that in the<br />
affected homozygous male both intestinal<br />
absorption <strong>and</strong> hepatic uptake are im<br />
paired, <strong>and</strong> that in the heterozygous fe<br />
male they are intermediate between the<br />
affected male <strong>and</strong> normal mice. Danks<br />
(143, 144) <strong>and</strong> Holtzman (351) present<br />
excellent reviews <strong>of</strong> the literature <strong>and</strong> in<br />
teresting comparisons <strong>of</strong> altered <strong>copper</strong><br />
<strong>metabolism</strong> in the mottled mutant mouse<br />
<strong>and</strong> infants with Menkes' disease. A third<br />
mouse mutant called "quaking" manifests<br />
some <strong>of</strong> the neurological signs <strong>of</strong> <strong>copper</strong>-<br />
deficient animals. Dietary <strong>copper</strong> decreases<br />
their tremors, indicating that <strong>copper</strong> me<br />
tabolism is involved in expression <strong>of</strong> the<br />
gene (396).<br />
Furthermore, Prohaska <strong>and</strong> Wells (621)<br />
describe striking similarities between bio<br />
chemical abnormalities in the brain <strong>of</strong> suck<br />
ling young <strong>of</strong> <strong>copper</strong>-deficient rats <strong>and</strong><br />
those observed in Menkes' disease. These<br />
involve slow growth, abnormal behavior,<br />
decrease in myelin, reduction in cerebellar<br />
cytochrome c oxidase <strong>and</strong> Superoxide dismutase<br />
<strong>and</strong> a 5-fold reduction in brain<br />
<strong>copper</strong>. Extensive lesions <strong>of</strong> the cerebral<br />
cortex <strong>and</strong> mid-brain have also been de<br />
scribed in <strong>copper</strong>-deficient rats (92). In<br />
the guinea pig, which undergoes consid<br />
erable myelination in utero, <strong>copper</strong> de<br />
ficiency causes gross brain abnormalities,<br />
aneurisms, agenesis <strong>of</strong> the cerebellum,<br />
ataxia, wiry nature <strong>and</strong> depigmentation <strong>of</strong><br />
hair, abnormal behavior patterns, <strong>and</strong> de<br />
creased liver <strong>copper</strong> (201). Morphologi<br />
cally, there is underdevelopment <strong>of</strong> myelin<br />
<strong>and</strong> cellular derangement <strong>and</strong> loss <strong>of</strong> neural<br />
elements in the cerebellum (202). Further<br />
study <strong>of</strong> the <strong>copper</strong>-deficient rat <strong>and</strong><br />
guinea pig might well shed further light on<br />
the nature <strong>of</strong> Menkes' disease.<br />
WILSON'S DISEASE<br />
A vast literature has dealt with the na<br />
ture, diagnosis <strong>and</strong> treatment <strong>of</strong> Wilson's<br />
disease. The findings have been well re<br />
corded in a number <strong>of</strong> reports <strong>and</strong> reviews<br />
(37, 38, 46, 137, 262, 267, 555, 666-668,<br />
672, 678, 679, 683, 684, 737, 740, 744, 747,<br />
756, 791, 821, 822). What follows is largely<br />
a summation <strong>of</strong> early observations concern<br />
ing the nature <strong>of</strong> the disease, current con<br />
cepts concerning the metabolic abnormali<br />
ties involved <strong>and</strong> therapeutic measures.<br />
Nature <strong>of</strong> the disease<br />
The history <strong>of</strong> this disease, as well out<br />
lined by Goldstein <strong>and</strong> Owen (262), goes<br />
back to 1912 when Wilson (855) de<br />
scribed a familial disease associated with<br />
cirrhosis <strong>of</strong> the liver <strong>and</strong> neurological mani<br />
festations, occurring predominantly during<br />
the first few decades <strong>of</strong> life. The term<br />
"hepatolenticular degeneration" was coined<br />
in 1921 by Hall (296), who also recognized<br />
the recessive mode <strong>of</strong> inheritance <strong>of</strong> the<br />
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2010 KARL E. MASON<br />
disease. This designation has since been<br />
largely replaced by the term "Wilson's dis<br />
ease." Not until 1953, through the more<br />
extensive studies <strong>of</strong> Beam (36), was the<br />
autosomal recessive nature <strong>of</strong> this inborn<br />
error <strong>of</strong> <strong>metabolism</strong><br />
i.e., that both parents<br />
clearly established;<br />
<strong>of</strong> an affected sub<br />
ject must be hétérozygote carriers <strong>of</strong> the<br />
abnormal gene, <strong>and</strong> that their siblings have<br />
a one to four chance <strong>of</strong> receiving both<br />
genes; i.e., in being homozygous abnormal.<br />
For these reasons, the number <strong>of</strong> affected<br />
individuals is maintained at a relatively<br />
low level in the population, accentuated<br />
only by consanguinity. Other studies over<br />
the period (1912-1954) provided valid<br />
evidence that this disease represented a<br />
state <strong>of</strong> <strong>copper</strong> toxicosis characterized by<br />
1) abnormally high levels <strong>of</strong> <strong>copper</strong> in the<br />
liver <strong>and</strong> brain (102, 134, 258, 316); 2) in<br />
creased urinary excretion <strong>of</strong> <strong>copper</strong> (163,<br />
485, 499, 609, 731, 873); 3) aminoaciduria<br />
(123,<br />
serum<br />
136, 163, 499, 608, 735);<br />
levels <strong>of</strong> ceruloplasmin<br />
4) low<br />
(674);<br />
5) decreased fecal excretion <strong>of</strong> <strong>copper</strong> (41,<br />
80, 581, 753, 873) <strong>and</strong> 6) the occurrence<br />
<strong>of</strong> Kayser-Fleischer rings (134, 258) which<br />
had been shown as early as 1934 to repre<br />
sent excessive accumulations<br />
around the cornea (249).<br />
<strong>of</strong> <strong>copper</strong><br />
Symptoms <strong>of</strong> the disease are decidedly<br />
variable in nature, time <strong>of</strong> onset <strong>and</strong> degree<br />
<strong>of</strong> severity. In the experience <strong>of</strong> some in<br />
vestigators the predominance <strong>of</strong> hepatic<br />
<strong>and</strong> neurological manifestations is about<br />
equally divided. In that <strong>of</strong> others, one or<br />
the other has been predominant. An excel<br />
lent discussion <strong>of</strong> laboratory findings <strong>and</strong><br />
clinical manifestations<br />
Strickl<strong>and</strong> <strong>and</strong> Lev<br />
has been given by<br />
(756), Sass-Kortsak<br />
<strong>and</strong> Beam (668) <strong>and</strong> Tu (791). How<br />
genetic determinants hold in check the<br />
metabolic <strong>and</strong> clinical expression <strong>of</strong> the<br />
disease for such variable periods <strong>of</strong> post<br />
natal life, <strong>and</strong> <strong>of</strong>ten for many decades, is<br />
unexplained.<br />
Wilson's disease<br />
Heterogeneity<br />
may be an<br />
<strong>of</strong><br />
important<br />
the gene<br />
fac<br />
for<br />
tor. One may consider the fact that while<br />
hétérozygote carriers (parents <strong>of</strong> patients<br />
with Wilson's disease ) cannot be identified<br />
clinically, they do differ from normal indi<br />
viduals in showing a prolonged biological<br />
turnover <strong>of</strong> 67Cu (580, 753), reduced<br />
biliary excretion <strong>of</strong> <strong>copper</strong> (581), hyper-<br />
cupriuresis after penicillamine loading<br />
(792) <strong>and</strong> certain renal dysfunctions (448).<br />
Metabolic abnonnalities<br />
The classic form <strong>of</strong> this relatively rare<br />
inborn error <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> is char<br />
acterized by: 1) usual, but not universal,<br />
low serum levels <strong>of</strong> <strong>copper</strong>, primarily <strong>of</strong><br />
ceruloplasmin, suggesting defective syn<br />
thesis <strong>of</strong> this cuproprotein by the liver;<br />
2) abnormally high storage <strong>of</strong> <strong>copper</strong> in<br />
the liver, associated with decreased fecal<br />
excretion <strong>and</strong> chronic liver disease, reflect<br />
ing impaired biliary excretion <strong>of</strong> <strong>copper</strong><br />
<strong>and</strong>/or abnormal <strong>copper</strong> protein binding<br />
by the liver; 3) progressive accumulation<br />
<strong>of</strong> <strong>copper</strong> in the brain, leading to a wide<br />
variety <strong>of</strong> neurological disorders; 4) ac<br />
cumulation <strong>of</strong> <strong>copper</strong> in the kidney, asso<br />
ciated with renal damage, cupruresis <strong>and</strong><br />
aminoaciduria; 5) deposition <strong>of</strong> <strong>copper</strong> in<br />
the cornea, leading to the formation <strong>of</strong><br />
Kayser-Fleischer rings <strong>and</strong>, occasionally,<br />
sunflower-type cataracts (87); <strong>and</strong> 6) epi<br />
sodes <strong>of</strong> hemolysis reflecting a rather sud<br />
den release <strong>of</strong> <strong>copper</strong> from a supersatu<br />
rated <strong>and</strong> cirrhotic liver. Since in normal<br />
man concentration <strong>of</strong> <strong>copper</strong> is higher in<br />
the liver, central nervous system <strong>and</strong> kid<br />
ney than in other organs <strong>and</strong> tissues<br />
(p. 1982) it might be expected that these<br />
levels would be significantly increased in a<br />
state <strong>of</strong> <strong>copper</strong> toxicosis. The report <strong>of</strong> a<br />
high concentration <strong>of</strong> <strong>copper</strong> in the skin <strong>of</strong><br />
two patients with Wilson's disease (113)<br />
warrants verification.<br />
Of particular interest is recent evidence<br />
that in subjects with this disease there is<br />
in the liver an abnormal metallothionein<br />
having a binding constant for <strong>copper</strong> about<br />
4-fold that in normal liver (197). It is<br />
felt that the increased binding affinity <strong>of</strong><br />
this protein alters normal homeostasis such<br />
that decreased biliary <strong>copper</strong> excretion <strong>and</strong><br />
decreased ceruloplasmin synthesis result,<br />
<strong>and</strong> with saturation <strong>of</strong> binding sites in<br />
hepatocytes non-ceruloplasmin <strong>copper</strong> is<br />
released to the serum. Whether this pro<br />
tein, or the presence <strong>of</strong> an abnormal pro<br />
tein <strong>of</strong> similar nature, may explain <strong>copper</strong><br />
accumulation in non-hepatic organs <strong>and</strong><br />
tissues is an unresolved question.<br />
In view <strong>of</strong> the fact that low serum ceru<br />
loplasmin levels are characteristic <strong>of</strong> young<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2011<br />
infants <strong>and</strong> subjects with Wilson's disease,<br />
it is not possible to diagnose this disease<br />
(other than perhaps by liver biopsy) <strong>and</strong><br />
to institute therapeutic measures, prior to<br />
the 3rd month <strong>of</strong> life (638). Largely for<br />
this reason little has been learned about<br />
the presymptomatic manifestations <strong>of</strong> the<br />
disease during early <strong>and</strong> late infancy.<br />
However, the report <strong>of</strong> Scheinberg <strong>and</strong><br />
Sternlieb (682) that <strong>copper</strong> concentrations<br />
in the liver <strong>of</strong> a 3M-year old child with<br />
Wilson's disease were at least 40-fold nor<br />
mal adult levels, does indicate progressive<br />
liver storage prior to clinical manifestation<br />
<strong>of</strong> the disease <strong>and</strong> supports the concept<br />
that the liver is the primary site <strong>of</strong> the dis<br />
order. The latter is characterized by a dis<br />
ruption <strong>of</strong> the normal homeostatic mech<br />
anisms for utilization <strong>and</strong> excretion <strong>of</strong><br />
<strong>copper</strong>. Possibly at fault is the presence <strong>of</strong><br />
either an abnormal protein with high<br />
avidity for <strong>copper</strong>, as proposed by Uzman<br />
et al. in 1956 (801), a similar protein un<br />
usually rich in sulfhydryl groups <strong>and</strong> given<br />
the designation L-6D (536) or a metallothionein-like<br />
protein with a protein-binding<br />
constant about 4-fold that <strong>of</strong> normal man<br />
(197). The concept that the metabolic<br />
defect in Wilson's disease is liver-based is<br />
supported by observations that in two<br />
teen-age boys with Wilson's disease treated<br />
with orthoptic liver transplantation the<br />
extrahepatic manifestations <strong>of</strong> the disease<br />
were significantly reduced (281 ). A similar<br />
response to the same procedure is reported<br />
in a 11-year old boy in whom there was<br />
strong but not incontrovertible evidence <strong>of</strong><br />
Wilson's disease (172).<br />
In the normal infant liver stores <strong>of</strong> cop<br />
per are gradually decreased <strong>and</strong> serum<br />
<strong>copper</strong> levels increased during the first 3<br />
or more months <strong>of</strong> life, until a close to zero<br />
<strong>copper</strong> balance is maintained. According to<br />
a recent evaluation <strong>of</strong> Wilson's disease<br />
(672), there may be an early arrest <strong>of</strong><br />
these postnatal processes, especially abili<br />
ties to synthesize normal amounts <strong>of</strong> ceruloplasmin<br />
<strong>and</strong> to excrete the normal frac<br />
tion <strong>of</strong> dietary <strong>copper</strong> through hepatic<br />
lysosomes (normally an important func<br />
tion <strong>of</strong> lysosomes) into the biliary system.<br />
As a result, <strong>copper</strong> progressively accumu<br />
lates in the liver, leading to inflammatory<br />
reactions, hepatic cell necrosis <strong>and</strong> post-<br />
necrotic cirrhosis. Meanwhile, excessive<br />
amounts <strong>of</strong> non-ceruloplasmin <strong>copper</strong><br />
cause, in an unpredictable manner, ac<br />
cumulation <strong>and</strong> injury to different regions<br />
<strong>of</strong> the brain, the kidney <strong>and</strong> the cornea.<br />
It is somewhat academic to ask whether<br />
administered estrogens or those <strong>of</strong> preg<br />
nancy can significantly influence the low<br />
serum ceruloplasmin levels in Wilson's<br />
disease. Beam (37) finds that synthetic<br />
estrogens may have a significant effect in<br />
some patients but not in others, with no<br />
influence on urinary <strong>copper</strong> excretion. With<br />
somewhat larger oral doses <strong>of</strong> a different<br />
estrogen, German (250) reports improve<br />
ment in some cases <strong>and</strong> accentuation <strong>of</strong><br />
symptoms in others, <strong>and</strong> also notes a cupriuresis<br />
in some cases correlated with in<br />
creased serum levels <strong>of</strong> direct reacting cop<br />
per but not with ceruloplasmin. Subjects<br />
with Wilson's disease have been able to<br />
complete gestation with delivery <strong>of</strong> normal<br />
infants (14, 33, 47, 104, 155, 680). There<br />
is a report <strong>of</strong> one untreated case in which<br />
there was an appreciable increase in ceru<br />
loplasmin, reaching a maximum at delivery<br />
( 104). Effects <strong>of</strong> pregnancy upon the clini<br />
cal status <strong>of</strong> the mothers have been<br />
equivocal. In instances where penicillamine<br />
treatment was discontinued prior to gesta<br />
tion (155) <strong>and</strong> after the first trimester<br />
(680), neither ceruloplasmin levels nor<br />
clinical symptoms were improved. In fact,<br />
in one case occurrence <strong>of</strong> hemolytic anemia<br />
during the 5th month required restoration<br />
<strong>of</strong> therapy (155). In three other instances,<br />
where therapy was maintained throughout<br />
pregnancy, Aere is reported definite ameli<br />
oration <strong>of</strong> clinical manifestations which<br />
continued postpartum for periods <strong>of</strong> a few<br />
weeks (14), 3 months (47) <strong>and</strong> 6 months<br />
(706). Data on serum ceruloplasmin are<br />
fragmentary. The ocurrence <strong>of</strong> several<br />
spontaneous abortions during therapy, both<br />
prior to (47) <strong>and</strong> following (14) preg<br />
nancies, raises questions concerning pos<br />
sible deleterious effects <strong>of</strong> penicillamine<br />
upon the fetus.<br />
Although the <strong>copper</strong>-binding capacity <strong>of</strong><br />
bile is not altered in Wilson's disease<br />
(233), there is increasing evidence that<br />
decreased biliary excretion <strong>of</strong> <strong>copper</strong> repre<br />
sents a major metabolic defect (234, 235,<br />
581, 752, 753), <strong>and</strong> also that this defect<br />
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2012 KARL E. MASON<br />
may reside in the hepatic cell lysosomes<br />
(260, 261, 749, 755) whose catabolic func<br />
tions <strong>and</strong> importance in transfer <strong>of</strong> <strong>copper</strong><br />
to the bile canaliculi are well recognized.<br />
It appears that early in the disease <strong>copper</strong><br />
is diffusely distributed in hepatocytes, later<br />
as more discrete granules, <strong>and</strong> that when<br />
hepatic damage is more widespread it be<br />
comes more localized in the lysosomes,<br />
where it may be less toxic ( 261 ). Delay in<br />
this uptake by lysosomes could be a key<br />
factor in the defective liver transport <strong>of</strong><br />
<strong>copper</strong> in Wilson's disease (721). Ques<br />
tions still remain as to whether abnormal<br />
<strong>copper</strong>-binding proteins or lack <strong>of</strong> un<br />
known cell enzymes involved in transfer<br />
<strong>of</strong> <strong>copper</strong> to, or in release <strong>of</strong> <strong>copper</strong> from,<br />
the lysosomes are responsible. The inter<br />
esting observations <strong>of</strong> Goldfisher <strong>and</strong> Sternlieb<br />
(161) have raised the hypothesis that<br />
Wilson's disease may prove to be a "lysosomal<br />
disease," as discussed in some detail<br />
by Sternlieb et al. (749) <strong>and</strong> Strickl<strong>and</strong> et<br />
al. (755). f<br />
Major results <strong>of</strong> reduced biliary excre<br />
tion are increased <strong>copper</strong> accumulation in<br />
hepatocytes, varying degrees <strong>of</strong> pathology,<br />
jaundice <strong>and</strong> episodes <strong>of</strong> hemolytic anemia,<br />
the latter being secondary to release <strong>of</strong><br />
<strong>copper</strong> from an overloaded <strong>and</strong> damaged<br />
liver into the blood stream. If this release<br />
is sudden, there can be severe damage to<br />
circulating erythrocytes, resulting in repet<br />
itive or fatal episodes <strong>of</strong> hemolytic anemia.<br />
A recent report (516) tabulates pertinent<br />
data on 18 reports involving 28 subjects.<br />
Of these, six presented hemolysis prior to<br />
any diagnosis <strong>of</strong> Wilson's disease <strong>and</strong> 20<br />
showed evidence <strong>of</strong> hemolysis at the time<br />
<strong>of</strong> diagnosis. Evidence <strong>of</strong> hepatic dysfunc<br />
tion was noted in at least 22, whereas<br />
neurological dysfunction was recognized in<br />
only three or four. A more recent report <strong>of</strong><br />
two cases <strong>of</strong> fulminating hemolysis in<br />
Wilson's disease calls attention to acute<br />
renal failure as well as hepatic failure<br />
(302). Hence, this hemolytic manifestation<br />
<strong>of</strong> Wilson's disease has become a more<br />
common phenomenon than previously rec<br />
ognized. In view <strong>of</strong> the fact that there is<br />
an associated marked increase in the cop<br />
per content <strong>of</strong> erythrocytes <strong>and</strong> in the<br />
number <strong>of</strong> Heinz bodies during periods <strong>of</strong><br />
crisis, hemolysis is attributed to increased<br />
oxidative stress due to an excessive ac<br />
cumulation <strong>of</strong> <strong>copper</strong> in the cells (155,<br />
508). Whether this is primarily a mem<br />
brane defect is still an open question<br />
(516). It has been considered (102, 155,<br />
508) a counterpart <strong>of</strong> the well known<br />
"enzootic jaundice" in sheep, the history<br />
<strong>and</strong> nature <strong>of</strong> which has been presented<br />
by Underwood (798). This concept is<br />
strongly supported by a recent study <strong>of</strong><br />
controlled, experimental <strong>copper</strong> poisoning<br />
in sheep demonstrating extensive forma<br />
tion <strong>of</strong> Heinz bodies, predominantly mem<br />
brane-attached, as the first morphological<br />
alteration observed (725). Hepatocellular<br />
<strong>and</strong> renal tubular necrosis were also noted.<br />
Therapy<br />
The chance observation <strong>of</strong> M<strong>and</strong>elbrote<br />
et al. (485), in a study <strong>of</strong> <strong>copper</strong> mobiliza<br />
tion in multiple sclerosis, that one <strong>of</strong> the<br />
control subjects who was later found to<br />
have Wilson's disease was greatly benefited<br />
by treatment with BAL (/3,/3-dimercaptopropanol),<br />
known to have properties <strong>of</strong> a<br />
chelator, provided the first therapeutic<br />
measure, introduced by Cumings in 1948<br />
( 135). While daily intramuscular injections<br />
proved effective in increasing the urinary<br />
output <strong>of</strong> <strong>copper</strong> (39, 46, 135, 163), there<br />
was no effect upon the aminoaciduria or<br />
other clinical manifestations. The same was<br />
true when BAL treatment was combined<br />
with intravenous casein hydrolysate <strong>and</strong><br />
oral potassium sulfide, the latter forming<br />
an insoluble <strong>copper</strong> compound in the di<br />
gestive tract (102); <strong>and</strong> also when EDTA<br />
( ethylenediamine-tetra-acetic acid, or "ver<br />
sene") was extensively tested (46). Nine<br />
weeks <strong>of</strong> estrogen treatment <strong>of</strong> an adult<br />
male with Wilson's disease failed to im<br />
prove serum <strong>copper</strong> or ceruloplasmin<br />
levels (652). Intravenous use <strong>of</strong> a purified<br />
concentrate <strong>of</strong> human ceruloplasmin also<br />
proved ineffective (681). These discourag<br />
ing results, together with the adverse side<br />
effects <strong>of</strong> BAL therapy, stimulated search<br />
for better measures.<br />
In 1956 Walshe (820) described the re<br />
markable effectiveness <strong>of</strong> oral DL-penicillamine<br />
as a chelating agent capable <strong>of</strong> mark<br />
edly increasing the urinary output <strong>of</strong><br />
<strong>copper</strong>. A few years later die less toxic<br />
D-penicillamine (ß,/3-dimethylcysteine) be-<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2013<br />
came available <strong>and</strong> has since been ex<br />
tensively used, <strong>of</strong>ten in combination with<br />
low-<strong>copper</strong> diets, in the treatment <strong>of</strong> Wil<br />
son's disease. If instituted during early<br />
phases <strong>of</strong> the disease, especially in asymp<br />
tomatic patients, it can gradually reduce<br />
excessive tissue levels to reasonably nor<br />
mal levels <strong>and</strong> provide assurance <strong>of</strong> a nor<br />
mal life expectancy provided no adverse<br />
reactions occur (24, 156, 745, 746, 821). In<br />
such instances, which are rare, Walshe<br />
(823) proposes the use <strong>of</strong> tetraethylene<br />
tetramine dihydrochloride. This compound,<br />
which is cheap <strong>and</strong> easy to prepare, has<br />
not been found to be associated with toxic<br />
reactions. It is very effective as a chelating<br />
agent, <strong>and</strong> its mobilization <strong>of</strong> <strong>copper</strong> may<br />
differ from the action <strong>of</strong> penicillamine<br />
(823). It has not been produced commer<br />
cially, but would seem to justify more<br />
thorough testing as an inexpensive thera<br />
peutic agent. Beneficial effects <strong>of</strong> L-dopa<br />
as an adjunct to a <strong>copper</strong>-deficient diet<br />
<strong>and</strong> oral penicillamine are reported (246)<br />
but not verified.<br />
Despite disappearance <strong>of</strong> disease symp<br />
toms <strong>and</strong> remarkable improvement in liver<br />
function following penicillamine therapy<br />
for 2 to 7 years (277) <strong>and</strong> 9 to 13 years<br />
(156), hypocupremia, hypoceruloplasminenia<br />
<strong>and</strong> hypercupruria have persisted<br />
( 156) <strong>and</strong> no more than limited improve<br />
ment in liver morphology has been ob<br />
served in most cases (277). One exception<br />
is that <strong>of</strong> a 10-year old girl in whom 27<br />
months <strong>of</strong> penicillamine treatment not only<br />
abolished clinical symptoms but greatly<br />
improved liver morphology (204). Mitochondrial<br />
abnormalities <strong>of</strong> hepatocytes<br />
characteristic <strong>of</strong> Wilson's disease are less<br />
pronounced or absent after 3 to 5 years <strong>of</strong><br />
therapy, which may have relevance to im<br />
proved liver function, but liver structure<br />
is not significantly influenced (738).<br />
Penicillamine has the properties <strong>of</strong> a<br />
lathyrogen, with ability to not only chelate<br />
<strong>copper</strong> but also to inhibit cross-linking in<br />
collagen (381, 560). Gr<strong>and</strong> <strong>and</strong> Vawter<br />
(277) suggest that penicillamine may re<br />
tard the formation <strong>of</strong> permanent scars if<br />
begun prior to the onset <strong>of</strong> the cirrhotic<br />
process, as it appears to do in the case <strong>of</strong><br />
chronic active hepatitis (440). Once cir<br />
rhosis is established one might expect some<br />
thinning <strong>of</strong> fibrous scars with prolonged<br />
therapy (277). It is disappointing that<br />
morphological <strong>and</strong> ultrastructural studies<br />
on biopsies <strong>of</strong> liver exposed to many years<br />
<strong>of</strong> penicillamine therapy make no com<br />
ment on changes observed in liver col<br />
lagen (204, 738 ). Another feature <strong>of</strong> peni<br />
cillamine action that justifies further ex<br />
ploration is the generalized loss <strong>of</strong> taste<br />
acuity in a variable number <strong>of</strong> subjects<br />
with scleroderma, rheumatoid arthritis,<br />
cystinuria <strong>and</strong> idiopathic pulmonary fibrosis<br />
given penicillamine treatment, <strong>and</strong><br />
the restoration to normal after oral admin<br />
istration <strong>of</strong> <strong>copper</strong> (323). That only 4% <strong>of</strong><br />
Wilson's disease subjects under the same<br />
treatment show hypogeusia is attributed to<br />
the fact that only rarely are their tissue<br />
stores <strong>of</strong> <strong>copper</strong> sufficiently reduced (323 ).<br />
A further complexity is presented by a<br />
case <strong>of</strong> hypogeusia in a patient with mul<br />
tiple myeloma which responded effectively<br />
to either oral <strong>copper</strong> or oral zinc (322).<br />
The role <strong>of</strong> <strong>copper</strong> in taste acuity is still<br />
questionable.<br />
Low-<strong>copper</strong> diets <strong>of</strong>ten used in addition<br />
to penicillamine treatment <strong>of</strong> patients with<br />
Wilson's disease, usually providing 1.0 to<br />
1.5 mg <strong>copper</strong>, not only exclude a number<br />
<strong>of</strong> generally consumed foods but also are<br />
monotonous <strong>and</strong> <strong>of</strong> low nutritional value<br />
(90). In predominantly rice-eating coun<br />
tries, such as Taiwan, preparation <strong>and</strong><br />
acceptance <strong>of</strong> such diets present no great<br />
problem (754, 755, 793). A vegetarian diet<br />
is said to be highly effective in decreasing<br />
positive <strong>copper</strong> balance <strong>and</strong> in increasing<br />
fecal output, due perhaps to <strong>copper</strong> bind<br />
ing to some unabsorbed component <strong>of</strong> the<br />
diet (90). There appears to be no confir<br />
mation <strong>of</strong> these observations.<br />
Related disorders<br />
Two other abnormalities <strong>of</strong> <strong>copper</strong> me<br />
tabolism, both associated with low serum<br />
levels <strong>of</strong> ceruloplasmin justify brief men<br />
tion. Gahlot et al. (240) describe 15 cases<br />
<strong>of</strong> primary retinitis pigmentosa unrespon<br />
sive to conventional treatment. Serum<br />
ceruloplasmin levels were very low <strong>and</strong><br />
urinary <strong>copper</strong> excretion very high, al<br />
though serum <strong>copper</strong> levels were normal<br />
or nearly normal. The investigators sug<br />
gest that this retinal pigmentary distur-<br />
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2014 KARL E. MASON<br />
bance, in certain cases at least, may not be<br />
an abiotrophy but a condition <strong>of</strong> chronic<br />
<strong>copper</strong> toxicity reflecting an inborn error<br />
in <strong>copper</strong> <strong>metabolism</strong>. The results <strong>of</strong><br />
penicillamine treatment, said to be in<br />
progress, <strong>and</strong> further exploration <strong>of</strong> these<br />
observations by others, will be anticipated<br />
with much interest.<br />
There has also been described, in three<br />
brothers, a hereditary disorder character<br />
ized by dementia, spastic dysarthria, verti<br />
cal eye movement paresis, gait disturbance,<br />
splenomegaly <strong>and</strong> an abnormal <strong>metabolism</strong><br />
<strong>of</strong> <strong>copper</strong> (851). The disorder is prepubertal<br />
in onset <strong>and</strong> progresses slowly<br />
over many years. Copper kinetic studies<br />
the unique combination <strong>of</strong> dementia,<br />
splenomegaly, <strong>and</strong> abnormalities <strong>of</strong> speech,<br />
vision <strong>and</strong> gait favor the view that this<br />
condition represents a new syndrome dis<br />
tinct from Wilson's disease. As far as can<br />
be determined, no comparable cases have<br />
since been reported. Both abnormalities<br />
may possibly prove to be variants <strong>of</strong> Wil<br />
son's disease.<br />
Wilson's disease is not the only disorder<br />
in which high liver levels <strong>of</strong> <strong>copper</strong> occur.<br />
Chronic active liver disease closely resem<br />
bles Wilson's disease in changes in hepatic<br />
function <strong>and</strong> morphology, but can be dif<br />
ferentiated from the latter in that ceruloplasmin<br />
levels are elevated in about 50 %<br />
<strong>of</strong> the cases <strong>and</strong> not below normal levels<br />
in others (442), <strong>and</strong> the cupriuria after<br />
penicillamine treatment is comparable to<br />
that <strong>of</strong> normal individuals (473).<br />
One other liver disease requires special<br />
consideration. Levels <strong>of</strong> liver <strong>copper</strong> quite<br />
comparable to <strong>and</strong> even greater than those<br />
found in Wilson's disease occur in pri<br />
marily biliary cirrhosis (215, 369, 721, 722,<br />
862), a chronic, slowly progressive disease<br />
with evidence <strong>of</strong> extrahepatic biliary ob<br />
struction (223). Unlike the situation in<br />
Wilson's disease, plasma clearance <strong>and</strong><br />
liver uptake <strong>of</strong> intravenous 84Cu are normal<br />
(721). Similar conclusions were reached<br />
by Fleming et al. (215) on the basis <strong>of</strong><br />
other evidence, including a new observa<br />
tion that patients with primary biliary cir<br />
rhosis also had significantly increased levels<br />
<strong>of</strong> <strong>copper</strong> in the renal cortex <strong>and</strong> spleen.<br />
In a study involving an extensive evalua<br />
tion <strong>of</strong> 81 patients with primary biliary<br />
cirrhosis, Kayser-Fleischer rings were found<br />
in three cases, <strong>and</strong> also in another patient<br />
with chronic active liver disease (216). In<br />
the three patients mentioned, <strong>copper</strong> in<br />
serum, urine <strong>and</strong> liver were significantly<br />
elevated, resembling conditions seen in<br />
Wilson's disease except for the high serum<br />
<strong>copper</strong> <strong>and</strong> capacity to incorporate radio<strong>copper</strong><br />
into ceruloplasmin. Concentration<br />
<strong>of</strong> liver <strong>copper</strong> above a specified level <strong>of</strong><br />
250 /Ag/g <strong>of</strong> dry tissue, previously consid<br />
ered as one <strong>of</strong> the four or five criteria for<br />
diagnosis <strong>of</strong> Wilson's disease, now appears<br />
to have limited value with accumulated<br />
indicate similarities to those <strong>of</strong> hétérozyevidence<br />
that such elevated concentrations<br />
gote carriers <strong>of</strong> Wilson's disease. However, occur in the two types <strong>of</strong> liver disease just<br />
mentioned. Furthermore, the presence <strong>of</strong><br />
Kayser-Fleischer rings can no longer be<br />
considered pathognomonic <strong>of</strong> Wilson's<br />
disease.<br />
HYPOCUPREMIA<br />
Previous sections have dealt with<br />
Menkes' syndrome <strong>and</strong> states <strong>of</strong> <strong>copper</strong> de<br />
ficiency in premature infants in which low<br />
blood levels <strong>of</strong> <strong>copper</strong>, especially <strong>of</strong> cerulo<br />
plasmin, are associated with various other<br />
manifestations <strong>of</strong> <strong>copper</strong> deficiency. States<br />
<strong>of</strong> hypocupremia without any evidence <strong>of</strong><br />
dietary <strong>copper</strong> deficiency characterize Wil<br />
son's disease <strong>and</strong> occur, somewhat in<br />
frequently, in a variety <strong>of</strong> other metabolic<br />
<strong>and</strong> disease situations. Certain <strong>of</strong> these<br />
justify recording.<br />
The terms "hypocupremia" <strong>and</strong> "hypercupremia"<br />
were introduced by Sachs et al.<br />
(655) whose excellent review <strong>of</strong> early<br />
studies on <strong>copper</strong> <strong>and</strong> iron in human blood,<br />
<strong>and</strong> their newer contributions, are worthy<br />
<strong>of</strong> note. Hypocupremia is defined as a<br />
serum <strong>copper</strong> level <strong>of</strong> 80 ¿ig/100ml or less<br />
(106). Since 93% <strong>of</strong> serum <strong>copper</strong> is nor<br />
mally bound to ceruloplasmin, hypocu<br />
premia must <strong>of</strong> necessity be synonymous<br />
with hypoceruloplasminemia, except in un<br />
usual circumstances. A syndrome charac<br />
terized by hypocupremia, hyp<strong>of</strong>erremia,<br />
hypoproteinemia, edema <strong>and</strong> hypochromatic<br />
anemia has been described in infants<br />
<strong>and</strong> children <strong>and</strong> attributed to either a<br />
dietary deficiency <strong>of</strong> <strong>copper</strong> <strong>and</strong> iron, with<br />
hypoproteinemia considered a secondary<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2015<br />
effect <strong>of</strong> iron depletion (432, 437, 759,<br />
874), to a transient dysproteinemia (796,<br />
797), or to inability to synthesize the apoenzyme<br />
<strong>of</strong> ceruloplasmin (412). Other<br />
studies by Schubert <strong>and</strong> Lahey (692),<br />
based upon 14 infants with the above men<br />
tioned syndrome <strong>and</strong> 54 infants with irondeficiency<br />
anemia but no hypocupremia,<br />
led to the hypothesis that an initial severe<br />
deficiency <strong>of</strong> iron results in marked anemia<br />
<strong>and</strong> consequent protein depletion which,<br />
in turn, causes impaired <strong>copper</strong> retention<br />
<strong>and</strong> resultant development <strong>of</strong> the complete<br />
syndrome.<br />
Reduced serum levels <strong>of</strong> <strong>copper</strong>, cerulo<br />
plasmin, iron <strong>and</strong> protein are also charac<br />
teristic <strong>of</strong> kwashiorkor (76, 180, 269, 305,<br />
402, 433, 632, 664) <strong>and</strong> marasmus (305,<br />
402, 532). Only one investigator reports<br />
no significant change in marasmus (269).<br />
There are other findings that in kwashior<br />
kor <strong>and</strong> marasmus both plasma <strong>and</strong> erythrocyte<br />
<strong>copper</strong> levels are significantly re<br />
duced (402). There is general accord that<br />
the hypocupremia is not due to dietary in<br />
sufficiency <strong>of</strong> <strong>copper</strong> but is secondary to a<br />
state <strong>of</strong> hypoproteinemia <strong>and</strong> inability to<br />
provide adequate amounts <strong>of</strong> the apoprotein<br />
for ceruloplasmin synthesis. Opinions<br />
differ as to whether in kwashiorkor the<br />
<strong>copper</strong> content <strong>of</strong> hair is significantly de<br />
creased (269, 474) or unaffected (76,<br />
443). A report describing marked hypercupremia<br />
in Filipino children <strong>and</strong> attrib<br />
uted to states <strong>of</strong> malnutrition (750) may<br />
well be ascribed to faulty methodology<br />
<strong>and</strong> inadequate controls.<br />
Hypocupremia has also been described<br />
in subjects with non-tropical sprue (78,<br />
101, 284, 739), tropical sprue <strong>and</strong> macrocytic<br />
anemia (86, 106), malabsorption due<br />
to small bowel disease (739), <strong>and</strong> with<br />
both hyperchromic <strong>and</strong> hypochromic<br />
anemia (315). There are also isolated re<br />
ports <strong>of</strong> hypocupremia associated with ex<br />
cessive gastrointestinal loss <strong>of</strong> protein<br />
(814, 869), celiac disease (27), cystic fibrosis<br />
<strong>of</strong> the pancreas (702) <strong>and</strong> the<br />
nephrotic syndrome (78, 101, 106, 491).<br />
In the latter disorder hypoceruloplasminemia<br />
comparable to that in Wilson's dis<br />
ease may occur, attributable primarily to<br />
high urinary loss <strong>of</strong> ceruloplasmin. In the<br />
other states <strong>of</strong> hypocupremia mentioned<br />
above decreased levels <strong>of</strong> serum cerulo<br />
plasmin are characteristic, suggesting qual<br />
itative or quantitative abnormalities <strong>of</strong><br />
protein <strong>metabolism</strong> rather than insufficient<br />
intake or absorption <strong>of</strong> <strong>copper</strong>.<br />
HYPERCUPREMIA<br />
Since the early observations <strong>of</strong> Krebs<br />
(426 ) that hypercupremia is associated not<br />
only with the state <strong>of</strong> pregnancy but also<br />
with many acute <strong>and</strong> chronic infections,<br />
there has accumulated an extensive litera<br />
ture on a wide variety <strong>of</strong> diseases <strong>and</strong> ab<br />
normal physiological states in which hyper<br />
cupremia, predominantly due to hyperceruloplasminemia<br />
occurs. It is beyond the<br />
scope <strong>of</strong> this review to discuss these obser<br />
vations in detail, especially since there<br />
have been provided no well accepted hy<br />
potheses or explanations <strong>of</strong> the mechanisms<br />
involved in this apparent stimulus for in<br />
creased synthesis <strong>and</strong> release <strong>of</strong> ceruloplas<br />
min. However, comments will be made on<br />
certain disease states involving hypercu<br />
premia which appear relevant to the pur<br />
pose <strong>of</strong> this review.<br />
Infectious diseases. Hypercupremia has<br />
been recorded as a phenomenon commonly<br />
associated with chronic <strong>and</strong> acute infec<br />
tious diseases <strong>of</strong> man. The lists recorded<br />
by various investigators are bewildering.<br />
There is general agreement that it is cerulo<br />
plasmin which is primarily increased <strong>and</strong><br />
that during the recovery period, whether<br />
spontaneous or the result <strong>of</strong> therapy, nor<br />
mal levels are restored. This has been well<br />
demonstrated, for example, in cases <strong>of</strong><br />
tuberculosis (61, 319, 542, 593, 655), lep<br />
rosy (403), viral hepatitis, <strong>and</strong> pneumonia<br />
<strong>and</strong> chickenpox (413). In many instances<br />
there has also been recorded a decreased<br />
serum level <strong>of</strong> iron (61, 63, 103) <strong>and</strong> <strong>of</strong><br />
zinc (718), reflecting differences in the<br />
proportions <strong>of</strong> circulating albumins <strong>and</strong><br />
globulins to which these metals may be<br />
bound.<br />
Hématologie disorders. Hypercupremia<br />
is commonly associated with iron deficiency<br />
anemia (100, 103, 319, 879) hemorrhagic,<br />
aplastic <strong>and</strong> pernicious anemias ( 100, 103,<br />
237, 319) <strong>and</strong> sickle cell anemia (576). In<br />
most anemias there is an inverse relation<br />
ship between serum <strong>copper</strong> <strong>and</strong> iron ( 100,<br />
655, 657), but both may be increased in<br />
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2016 KARL E. MASON<br />
pernicious anemia, aplastic anemia <strong>and</strong><br />
thalassemia major ( 103). It should be kept<br />
in mind that in view <strong>of</strong> decreased blood<br />
iron levels in anemia, the increased <strong>copper</strong><br />
levels may be more apparent than real. In<br />
iron deficiency anemia, pernicious anemia<br />
<strong>and</strong> leukemia, as well as in chronic infec<br />
tions, there is an increased <strong>copper</strong> level in<br />
whole blood, red blood cells <strong>and</strong> plasma,<br />
<strong>and</strong> only in iron deficiency anemia is there<br />
an increase in the ratio <strong>of</strong> erythrocyte to<br />
plasma <strong>copper</strong> (100, 592). Aside from<br />
states <strong>of</strong> <strong>copper</strong> toxicity, blood cell <strong>copper</strong><br />
remains quite constant. Since direct react<br />
ing <strong>copper</strong> <strong>of</strong> serum is only about 1% <strong>of</strong><br />
the total, both hypo- <strong>and</strong> hypercupremic<br />
states reflect changes primarily in ceruloplasmin<br />
<strong>copper</strong>. To the present there have<br />
been proposed no acceptable explanations<br />
<strong>of</strong> the basic mechanisms involved, <strong>of</strong> the<br />
tissues from which the <strong>copper</strong> is mobilized,<br />
or the function(s) that hypercupremia<br />
serves. An extensive literature on this sub<br />
ject has been well reviewed elsewhere (7,<br />
44, 100, 211, 319, 666).<br />
Neoplasms. Hypercupremia is a common<br />
feature <strong>of</strong> acute <strong>and</strong> chronic leukemia<br />
(100, 162, 630, 773), lymphatic leukemia<br />
(255), Hodgkin's disease (100, 328, 363,<br />
364, 386, 774, 775), malignant tumors<br />
(255) <strong>and</strong> <strong>of</strong> multiple <strong>and</strong> acute myeloma<br />
(255, 268, 456, 457). In leukemia <strong>of</strong> chil<br />
dren plasma zinc levels are low, <strong>and</strong> the<br />
ratio <strong>of</strong> zinc to <strong>copper</strong> may prove useful<br />
in monitoring the response to treatment<br />
( 162 ). In Hodgkin's disease blood cop<br />
per levels are valuable in evaluating the<br />
disorder itself <strong>and</strong> the effects <strong>of</strong> therapy<br />
(363, 364, 774, 775, 778, 829).<br />
Cases <strong>of</strong> multiple myeloma appear to<br />
present special abnormalities <strong>of</strong> <strong>copper</strong><br />
<strong>metabolism</strong>. Goodman et al. (268) report<br />
a case in a 69-year old woman whose<br />
serum <strong>copper</strong> levels ranged from 20- to 40fold<br />
normal, due entirely to a phenomenal<br />
increase in nonceruloplasmin <strong>copper</strong>. Evi<br />
dence indicated its association with an<br />
abnormal monoclonal immunoglobulin.<br />
More recently, Lewis et al. (457 ) recorded<br />
a quite similar hypercupremia, with blood<br />
<strong>copper</strong> levels as much as 14-fold normal,<br />
in a 41-year old woman manifesting an<br />
early, clinically asymptomatic stage <strong>of</strong> mul<br />
tiple myeloma. Liver (biopsy) was normal<br />
in structure <strong>and</strong> <strong>copper</strong> concentration. In<br />
both cases there was extensive <strong>copper</strong> in<br />
filtration <strong>of</strong> the cornea <strong>and</strong> <strong>of</strong> the anterior<br />
<strong>and</strong> posterior surface <strong>of</strong> the lens, not un<br />
like that seen in the Kayser-Fleischer ring<br />
<strong>of</strong> Wilson's disease. These observations<br />
raise questions regarding the pathognomonic<br />
value <strong>of</strong> the latter <strong>and</strong> also the pos<br />
sible recognition <strong>of</strong> a unique variety <strong>of</strong><br />
multpile myeloma, both <strong>of</strong> which justify<br />
further exploration.<br />
Largely in the hope <strong>of</strong> finding an addi<br />
tional diagnostic criterion <strong>of</strong> value, atten<br />
tion has been given to serum levels <strong>of</strong> cop<br />
per, <strong>and</strong> in some studies to zinc <strong>and</strong> iron,<br />
in subjects with varied types <strong>of</strong> neoplasia.<br />
In osteosarcoma, serum <strong>copper</strong> <strong>and</strong> <strong>copper</strong>zinc<br />
ratios are increased in the primary<br />
phase, further increased following metasta<br />
sis, <strong>and</strong> approach normal levels in patients<br />
whose tumor is amputated <strong>and</strong> show no<br />
clinical sign <strong>of</strong> the disease (212). Also,<br />
<strong>copper</strong> in the bone <strong>of</strong> osteogenic carcinoma<br />
is significantly greater than in normal bone<br />
(384). In lung <strong>and</strong> breast carcinoma the<br />
serum <strong>copper</strong>/iron ratio is high (602, 769 ).<br />
In gastric <strong>and</strong> pulmonary carcinoma serum<br />
<strong>copper</strong> levels are significantly increased,<br />
but not in cases <strong>of</strong> tumors <strong>of</strong> the large in<br />
testine (670). Only one study reports no<br />
differences between the serum <strong>copper</strong> con<br />
tent <strong>of</strong> healthy humans <strong>and</strong> those suffering<br />
from malignant tumors (22 ). Other reports<br />
indicate increased serum <strong>copper</strong> in lymphomas<br />
<strong>and</strong> certain other malignancies<br />
(539), <strong>and</strong> no change in prostatic carci<br />
noma prior to or during radiation therapy<br />
(363). In Hodgkin's disease, leukemia <strong>and</strong><br />
lymphomas, there is general agreement<br />
that serum <strong>copper</strong> levels have merit in<br />
diagnosis <strong>and</strong> in evaluation <strong>of</strong> therapy<br />
(162, 375, 386, 421, 539, 592, 773), as<br />
is also true <strong>of</strong> osteosarcomas (212). How<br />
ever, in none <strong>of</strong> the studies referred to<br />
above has a clear explanation, or even a<br />
challenging hypothesis, been provided<br />
toward explaining the possible mechanisms<br />
involved.<br />
'Neurological diseases. Recognition <strong>of</strong> low<br />
serum ceruloplasmin <strong>and</strong> <strong>copper</strong> levels as<br />
one criterion <strong>of</strong> Wilson's disease led to nu<br />
merous studies on a wide variety <strong>of</strong> neuro<br />
logical diseases <strong>and</strong> disorders, many directed<br />
toward hopes <strong>of</strong> finding other conditions<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2017<br />
wherein increased or decreased levels <strong>of</strong><br />
<strong>copper</strong> might provide a useful diagnostic<br />
measure <strong>of</strong> the disease state. On the whole,<br />
these efforts have proved rather unfruit<br />
ful. Considerable controversy has arisen<br />
regarding schizophrenia <strong>and</strong> other psy<br />
chotic states, following an early report <strong>of</strong><br />
high serum <strong>copper</strong> levels in the majority<br />
<strong>of</strong> 27 cases <strong>of</strong> schizophrenia <strong>and</strong> in cases<br />
<strong>of</strong> manic depression <strong>and</strong> epilepsy (319).<br />
Subsequent studies have indicated a tend<br />
ency toward significant increases in ceruloplasmin<br />
serum levels in schizophrenia (2,<br />
675, 677), in manic depression <strong>and</strong> senile<br />
psychosis (13), but values obtained over<br />
lap with those <strong>of</strong> normal subjects to vari<br />
able degrees. Since the activity <strong>of</strong> <strong>copper</strong><br />
oxidase is reduced as serum ascorbic acid<br />
increases, it is felt that the increased levels<br />
observed in many states <strong>of</strong> mental illness<br />
may reflect low ascorbate intake in sub<br />
jects institutionalized for prolonged pe<br />
riods ( 13, 20 ). Other investigators find no<br />
significant changes in serum <strong>copper</strong> in<br />
schizophrenia (30, 31, 360, 575), or in<br />
brain tissue (279 ). These differences might<br />
relate to the fact that schizophrenics are<br />
very heterogeneous biochemically, such as<br />
in serum levels <strong>of</strong> histamine, in serum<br />
levels <strong>of</strong> zinc <strong>and</strong> manganese, <strong>and</strong> in re<br />
actions to penicillamine <strong>and</strong> to contracep<br />
tive estrogens in particular (596, 597). In<br />
any case, serum <strong>copper</strong> levels have no<br />
diagnostic value (677).<br />
In epilepsy there is said to be an increase<br />
in serum <strong>copper</strong> (70, 89) involving whole<br />
blood, serum <strong>and</strong> blood cell levels (800).<br />
Cerebrospinal fluid <strong>copper</strong> is reported to<br />
be decreased (89) <strong>and</strong> increased (800),<br />
<strong>and</strong> urinary <strong>and</strong> fecal <strong>copper</strong> increased<br />
(800). These unverified <strong>and</strong> somewhat<br />
variant observations probably have little<br />
relevance.<br />
Cardiovascular diseases. More than 25<br />
years ago Vallee (803) reported a pro<br />
nounced hypercupremia during the acute<br />
phase <strong>of</strong> myocardial infarction, which sub<br />
sided during recovery, <strong>and</strong> later Adelstein<br />
et al. (5) demonstrated a linear relation<br />
ship between the serum <strong>copper</strong>, ceruloplasmin<br />
<strong>and</strong> <strong>copper</strong> oxidase activity. These<br />
findings have been well confirmed (405,<br />
806, 831), <strong>and</strong> a reciprocal decrease in<br />
serum zinc has been noted (806, 831). In<br />
myocardial infarction, but not in angina,<br />
coronary insufficiency or myocardial<br />
ischemia, there is also a marked elevation<br />
in serum <strong>of</strong> the zinc-dependent enzymes,<br />
malic <strong>and</strong> lactic dehydrogenase (811), <strong>and</strong><br />
in benzidine oxidase (760). Such findings<br />
naturally raise questions as to whether in<br />
creases in serum ceruloplasmin represent<br />
anything other than a reaction to acute<br />
stress.<br />
There are but a few unconfirmed reports<br />
indicating hypercupremia in atherosclerosis<br />
(53, 659), arteriosclerosis (81, 82) <strong>and</strong><br />
hypertension (287); also, decreased levels<br />
<strong>of</strong> <strong>copper</strong> in the wall <strong>of</strong> larger arteries<br />
(404) <strong>and</strong> coronary arteries (836) <strong>of</strong> sub<br />
jects with atherosclerosis. There is also<br />
described a linear decrease in the <strong>copper</strong><br />
content <strong>of</strong> the wall <strong>of</strong> larger arteries with<br />
increase in degree <strong>of</strong> atherosclerosis (404),<br />
<strong>and</strong> a questionably significant lower level<br />
<strong>of</strong> <strong>copper</strong> in the coronary artery <strong>of</strong> sub<br />
jects with atherosclerosis <strong>and</strong> myocardial<br />
infarction (836), which may reflect de<br />
creased metabolic activity <strong>of</strong> the altered<br />
arterial tissue.<br />
Hemolysis associated with hypercu<br />
premia, usually transient <strong>and</strong> self-limiting,<br />
is a well recognized manifestation <strong>of</strong> Wil<br />
son's disease (see p. 2014). It may occur<br />
also as a fulminating event secondary<br />
to acute liver failure (643), sometimes<br />
combined with acute renal failure (302).<br />
It can be attributed to sudden release <strong>of</strong><br />
nonceruloplasmic <strong>copper</strong> from a damaged<br />
liver <strong>and</strong> excessive accumulation in erythrocytes,<br />
resulting in acute oxidative stress<br />
upon the cells <strong>and</strong> cell membranes (155,<br />
302, 643).<br />
Liver diseases. Considering the key role<br />
<strong>of</strong> the liver in initial storage <strong>of</strong> absorbed<br />
<strong>copper</strong>, in the synthesis <strong>and</strong> release <strong>of</strong><br />
ceruloplasmin, <strong>and</strong> in excretion <strong>of</strong> <strong>copper</strong><br />
via the biliary system, it is to be expected<br />
that metabolic <strong>and</strong> pathologic dysfunctions<br />
<strong>of</strong> this organ might well be reflected in<br />
atypical levels <strong>of</strong> <strong>copper</strong> in the serum, <strong>and</strong><br />
also perhaps in erythrocytes <strong>of</strong> the circu<br />
lating blood. This has been well demon<br />
strated. Serum <strong>copper</strong> levels are signifi<br />
cantly elevated in portal cirrhosis, biliary<br />
tract disease <strong>and</strong> hepatitis (62, 245, 255,<br />
271, 285, 600), possibly reflecting inter<br />
ference with the normal excretion <strong>of</strong> cop-<br />
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2018 KARL E. MASON<br />
per via thèbile <strong>and</strong> consequent release <strong>of</strong><br />
an excess into the circulation. Although<br />
states <strong>of</strong> hypocupremia are rare in liver<br />
disease, low serum <strong>copper</strong> levels are re<br />
ported in hemolytic jaundice, hemochromatosis<br />
<strong>and</strong> some types <strong>of</strong> liver cirrhosis (255,<br />
824), presumably the result <strong>of</strong> reduced<br />
capacity <strong>of</strong> the damaged liver to synthesize<br />
ceruloplasmin (824).<br />
Liver biopsies from subjects <strong>of</strong> long<br />
st<strong>and</strong>ing hepatic diseases due to biliary<br />
obstruction regularly show in periportal<br />
hepatocytes accumulations <strong>of</strong> coarse gran<br />
ules staining with the rubeanic acid<br />
method <strong>and</strong> the Mallory-Parker hematoxlin<br />
method for <strong>copper</strong>, <strong>and</strong> reacting positively<br />
to orcein, which indicates the presence <strong>of</strong><br />
sulfhydryl groups, all <strong>of</strong> which suggest the<br />
binding <strong>of</strong> <strong>copper</strong> to a metallothionein<br />
type <strong>of</strong> protein (661, 719, 720). Rubeanic<br />
acid-staining granules <strong>of</strong> similar type have<br />
been described in the livers <strong>of</strong> vineyard<br />
sprayers exposed for many years to <strong>copper</strong><br />
sulfate sprays (599), but this staining pro<br />
cedure is not a particularly reliable test for<br />
liver <strong>copper</strong>. In view <strong>of</strong> the role <strong>of</strong> <strong>copper</strong><br />
as a hepatoxin in sheep (784), it is pos<br />
sible that <strong>copper</strong> may contribute to the de<br />
velopment <strong>of</strong> liver cirrhosis in long-st<strong>and</strong><br />
ing liver cholestasis (661). It should be <strong>of</strong><br />
interest to explore the possible relation <strong>of</strong><br />
these granules to hepatic lysosomes, <strong>and</strong><br />
also to learn what effect penicillamine may<br />
have upon their histochemical picture.<br />
Liver <strong>copper</strong> levels are not altered in<br />
extrahepatic biliary obstruction (862) or in<br />
acute hepatitis, steatosis <strong>of</strong> the liver, he<br />
patic amyloidosis or hemochromatosis<br />
(640). In viral hepatitis, serum <strong>copper</strong><br />
levels are said to be significantly increased<br />
during the acute phase according to one<br />
report (270) <strong>and</strong> during improvement <strong>of</strong><br />
the clinical state according to another<br />
(326 ), but no change in liver <strong>copper</strong> levels<br />
has been reported. Patients with chronic<br />
active hepatitis respond favorably to 5<br />
months or more <strong>of</strong> penicillamine therapy<br />
(440).<br />
Rheumatic diseases. For many centuries<br />
<strong>copper</strong> amulets have been worn, hopefully,<br />
for relief from arthritis, rheumatism <strong>and</strong><br />
many other afflictions <strong>of</strong> man, <strong>and</strong> <strong>copper</strong><br />
has been a common component <strong>of</strong> folk<br />
remedies for arthritis in particular. There<br />
is reported (815) a recent correspondence<br />
<strong>and</strong> questionnaire type <strong>of</strong> study, involving<br />
240 sufferers <strong>of</strong> arthritis/rheumatism, half<br />
<strong>of</strong> whom were previous wearers <strong>of</strong> <strong>copper</strong><br />
bracelets <strong>and</strong> the other half not, r<strong>and</strong>omly<br />
allocated to three treatment groups wear<br />
ing <strong>copper</strong> bracelets or placebo (anodised<br />
aluminum) bracelets, or neither. Prelimi<br />
nary results <strong>of</strong> psychological analyses <strong>of</strong><br />
the questionnaire responses indicate that<br />
"previous users seem to be significantly<br />
worse when not wearing their <strong>copper</strong><br />
bracelets." However, convincing evidence<br />
<strong>of</strong> beneficial effects <strong>of</strong> <strong>copper</strong> bracelets<br />
does not yet exist. These studies did reveal<br />
that surprisingly large amounts <strong>of</strong> <strong>copper</strong><br />
(average <strong>of</strong> 13 mg/month from a 14-g<br />
bracelet) can be absorbed through the<br />
dermis, which would give in 12 months<br />
more than the total amount estimated to be<br />
present in the human body. The rationale<br />
for <strong>copper</strong> therapy in rheumatic diseases<br />
is not at all clear, <strong>and</strong> little or no infor<br />
mation exists concerning <strong>copper</strong> metab<br />
olism in such states other than in rheuma<br />
toid arthritis, <strong>and</strong> that is somewhat con<br />
flicting.<br />
In rheumatoid arthritis, serum <strong>copper</strong><br />
levels are said to be appreciably increased<br />
(133, 423, 559, 604, 622, 623) <strong>and</strong> in the<br />
synovial fluid there is an increased level <strong>of</strong><br />
ceruloplasmin <strong>copper</strong>, as well as <strong>of</strong> iron<br />
<strong>and</strong> zinc (558, 559). On the other h<strong>and</strong>,<br />
mean values for the <strong>copper</strong> levels <strong>and</strong><br />
Superoxide dismutase activity <strong>of</strong> erythrocytes<br />
<strong>of</strong> male <strong>and</strong> female subjects with<br />
rheumatoid arthritis do not differ signifi<br />
cantly from normal controls (696). A<br />
marked elevation <strong>of</strong> nonceruloplasmin<br />
serum <strong>copper</strong> reported by Lorber et al.<br />
(466) has not been substantiated (743),<br />
which may be due to differences in meth<br />
odology (465). However, the more recent<br />
studies <strong>of</strong> Bajpayee (29) in which serum<br />
ceruloplasmin levels were significantly in<br />
creased in rheumatoid arthritis patients on<br />
estrogens, but were normal in female<br />
patients not on estrogens <strong>and</strong> in male<br />
patients, raise serious questions concern<br />
ing the validity <strong>of</strong> data previously reported.<br />
Bajpayee points out that in prior investi<br />
gations no effort was made to segregate,<br />
from the populations studied, those females<br />
who were on estrogen treatment.<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2019<br />
Nevertheless, <strong>copper</strong> has received much<br />
attention in the treatment <strong>of</strong> rheumatoid<br />
<strong>and</strong> other forms <strong>of</strong> arthritis. The claimed<br />
efficacy <strong>of</strong> intravenous administration <strong>of</strong><br />
an organic salt <strong>of</strong> <strong>copper</strong> (221) is not<br />
substantiated (795). Penicillamine has<br />
been used with some response in about<br />
80% <strong>of</strong> cases after 6 months or more <strong>of</strong><br />
treatment (88, 380). Efforts have been<br />
made to enhance the effectiveness <strong>of</strong><br />
aspirin, salicylates <strong>and</strong> penicillamine by<br />
forming with them <strong>copper</strong> coordination<br />
compounds. As tested only by the rat<br />
model for evaluating inflammatory <strong>and</strong><br />
antiulcer potentials, these have given<br />
somewhat equivocal results (626, 728,<br />
729 ) depending upon the route <strong>of</strong> adminis<br />
tration <strong>and</strong> the degree <strong>of</strong> tissue irritation<br />
produced. The hypothesis <strong>of</strong> Sorenson<br />
(728, 729) that anti-inflammatory drugs<br />
might react in vivo with available <strong>copper</strong><br />
to generate more effective complexes for<br />
regulation <strong>of</strong> inflammatory or non-inflam<br />
matory states certainly justifies further<br />
exploration. So also does the question <strong>of</strong><br />
whether the higher serum <strong>copper</strong> levels in<br />
females as compared to males bears any<br />
relation to the high female to male ratio<br />
seen in rheumatoid arthritis. Spontaneous<br />
remissions <strong>of</strong> rheumatoid arthritis are<br />
associated with obstructive biliary/liver<br />
disease <strong>and</strong> with pregnancy, characterized<br />
by increased serum ceruloplasmin levels.<br />
These questions are raised by Whitehouse<br />
(841) in his review <strong>and</strong> critical discussion<br />
<strong>of</strong> the topics referred to above. Obviously,<br />
there are fertile fields for new explorations<br />
<strong>of</strong> the possible role <strong>of</strong> <strong>copper</strong> in arthritis<br />
<strong>and</strong> related diseases.<br />
Pellagra. Krishnamachari (427) de<br />
scribes in pellagrins in India a hypercupremia<br />
uniquely due to increase in nonceruloplasmin<br />
<strong>copper</strong> <strong>and</strong> associated with<br />
wide variation in urinary <strong>copper</strong> excretion,<br />
both <strong>of</strong> which return to normal levels after<br />
oral administrations <strong>of</strong> nicotinic acid. On<br />
the basis <strong>of</strong> evidence that the high leucine<br />
content <strong>of</strong> the millet Sorghum vulgäre,a<br />
staple food <strong>of</strong> populations in India, is<br />
causally related to pellagra, healthy adult<br />
volunteers were given 5-g L-leucine for 6<br />
consecutive days. They showed compar<br />
able degrees <strong>of</strong> hypercupremia <strong>and</strong> urinary<br />
loss both <strong>of</strong> which disappeared after leu-<br />
cine withdrawal. This investigator sug<br />
gests that leucine enhances the absorption<br />
<strong>of</strong> dietary <strong>copper</strong> in normal subjects. From<br />
the same area <strong>of</strong> India is the report <strong>of</strong><br />
Deosthale <strong>and</strong> Gopalan (164) that certain<br />
varieties <strong>of</strong> sorghum also greatly increase<br />
serum <strong>copper</strong> levels <strong>and</strong> urinary <strong>copper</strong><br />
excretion, attributable to the high molyb<br />
denum content <strong>of</strong> the sorghum samples.<br />
Unfortunately, serum levels <strong>of</strong> direct react<br />
ing <strong>copper</strong> <strong>and</strong> ceruloplasmin were not<br />
determined. Hence, there is need for more<br />
critical study <strong>of</strong> the possible role <strong>of</strong> leucine<br />
<strong>and</strong>, or molybdenum excesses in producing<br />
the hypercupremia <strong>and</strong> hypercupriuria de<br />
scribed in pellagrins, <strong>and</strong> for confirmation<br />
that the hypercupremia is due primarily<br />
to increased nonceruloplasmin <strong>copper</strong>.<br />
Bantu pellagrins in South Africa are said<br />
to manifest a hypercupremia which is re<br />
duced rapidly after an intramuscular in<br />
jection <strong>of</strong> pantothenic acid, but not after<br />
oral nicotinamide (210). The latter obser<br />
vations carried out 20 years ago, seem not<br />
to have been denied or confirmed.<br />
Skin disorders. Elevated serum <strong>copper</strong><br />
levels occur in psoriasis (400, 430, 531,<br />
751, 859, 872, 875) but not in other derma<br />
toses (872). Although these levels have<br />
been generally attributed to increased<br />
ceruloplasmin, as a reaction to disturbed<br />
keratinization (751, 876), several reports<br />
state that ceruloplasmin levels are normal<br />
(400, 438) unless associated with condi<br />
tions <strong>of</strong> arthritis (423). The tissue <strong>copper</strong><br />
levels <strong>of</strong> psoriatic lesions are no different<br />
from those <strong>of</strong> uninvolved skin, although<br />
zinc levels are increased (529, 531). After<br />
beneficial response to heliotherapy or<br />
thalassotherapy most patients show clini<br />
cal improvement, with return <strong>of</strong> serum<br />
<strong>copper</strong> ceruloplasmin levels toward nor<br />
mal (875). The mechanisms involved are<br />
unknown. With regard to vitÃligo,informa<br />
tion is both limited <strong>and</strong> contradictory.<br />
This disorder is said to be characterized<br />
by hypercupremia, which is reduced in<br />
subjects responding favorably to helio<br />
therapy (247, 876), whereas others find<br />
that in about 39% <strong>of</strong> subjects both albu<br />
min-bound <strong>and</strong> ceruloplasmin serum cop<br />
per levels are below the normal range<br />
(372). Again the question <strong>of</strong> methodology<br />
arises.<br />
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2020 KARL E. MASON<br />
The states <strong>of</strong> hypercupremia to which<br />
reference has been made clearly indicate<br />
the remarkable homeostatic mechanism in<br />
<strong>copper</strong> <strong>metabolism</strong>. These involve to a<br />
large degree the increased synthesis <strong>of</strong><br />
ceruloplasmin in response to a variety <strong>of</strong><br />
body stresses, including hormonal influ<br />
ences (44). Also, in body states involving<br />
inflammatory reactions, ceruloplasmin may<br />
function in the role <strong>of</strong> an "acute phase<br />
reactant" (48, 636, 637).<br />
COPPER TOXICITY<br />
Hemochromatosis. There is a long history<br />
<strong>of</strong> acute <strong>and</strong> chronic toxicity <strong>of</strong> <strong>copper</strong> in<br />
man. Some <strong>of</strong> this was recorded in 1891 by<br />
Lehmann (448) who was one <strong>of</strong> the earli<br />
est investigators to test the effects <strong>of</strong> vari<br />
ous <strong>copper</strong> salts on experimental animals.<br />
He also states that two <strong>of</strong> his students<br />
showed no ill effects <strong>of</strong> additions <strong>of</strong> up to<br />
10 to 20 mg <strong>of</strong> <strong>copper</strong> sulfate <strong>and</strong> up to<br />
5 to 30 mg <strong>of</strong> <strong>copper</strong> acetate to their daily<br />
beer. This is somewhat greater than a<br />
current estimated toxic level <strong>of</strong> 10 to 15<br />
mg <strong>of</strong> inorganic <strong>copper</strong> for adult man<br />
(75, 865). In 1898, Baum <strong>and</strong> Seeliger<br />
(35) described extensive deposition <strong>of</strong><br />
blood pigments, then designated hematoidin<br />
<strong>and</strong> hemosiderin, in the liver cells<br />
<strong>of</strong> the goat, sheep, dog <strong>and</strong> cat fed <strong>copper</strong><br />
salts. These observations were more ex<br />
tensively explored by Mallory et al. (480-<br />
484) who reported that chronic oral intake<br />
<strong>of</strong> <strong>copper</strong> acetate in the rabbit, sheep <strong>and</strong><br />
monkey produces a condition comparable<br />
to hepatic hemosiderosis in man.<br />
Mallory (481) described in detail the<br />
hepatic changes characteristic <strong>of</strong> human<br />
hemochromatosis (pigment cirrhosis) based<br />
upon 19 necropsies, presenting circum<br />
stantial evidence <strong>of</strong> <strong>copper</strong> toxicity as<br />
basically involved. These interpretations<br />
were supported by other pathologists re<br />
porting liver <strong>copper</strong> levels up to 10-fold<br />
normal in a large series <strong>of</strong> cases <strong>of</strong> hemo<br />
chromatosis (295, 327, 586). Yet Mills<br />
(522) found no evidence <strong>of</strong> hemochroma<br />
tosis in 100 necropsies <strong>of</strong> Korean people<br />
using <strong>copper</strong> <strong>and</strong> brass utensils routinely<br />
in daily life. Although the animal studies<br />
<strong>of</strong> Mallory <strong>and</strong> coworkers (480, 482, 484)<br />
on which their hypothesis <strong>of</strong> the cause <strong>of</strong><br />
hemochromatosis was based were also con<br />
firmed by certain investigators (295, 519),<br />
others attempted in vain to duplicate their<br />
results (218, 587, 607). Differences in sus<br />
ceptibility <strong>and</strong> in levels <strong>and</strong> duration <strong>of</strong><br />
exposure to <strong>copper</strong> salts were proposed<br />
(482) to explain the discrepancies in the<br />
experimental findings.<br />
Copper poisoning in man The oral in<br />
gestion <strong>of</strong> excess <strong>copper</strong> produces a metal<br />
lic taste in the mouth, nausea, vomiting,<br />
epigastric pain, diarrhea <strong>and</strong>, to variable<br />
degrees, jaundice, hemolysis, hemaglobinuria,<br />
hematuria <strong>and</strong> oliguria. The vomitus,<br />
stool <strong>and</strong> saliva may appear blue or green.<br />
In severe cases, anuria, hypotension <strong>and</strong><br />
coma occur. The ingested <strong>copper</strong> is<br />
promptly absorbed from the upper gut <strong>and</strong><br />
rapidly <strong>and</strong> dramatically increases the level<br />
<strong>of</strong> direct reacting <strong>copper</strong> in the blood, due<br />
in large part to its accumulation in the red<br />
blood cells. When this accumulation<br />
reaches a certain level, hemolysis occurs,<br />
whether it be the result <strong>of</strong> oral ingestion<br />
(120, 203, 641), absorption through de<br />
nuded skin (353), dialysis procedures (51,<br />
52, 376, 487) or exchange transfusions<br />
(50). This hemolysis is comparable to that<br />
commonly seen in Wilson's disease, which<br />
is attributed to a sudden release <strong>of</strong> <strong>copper</strong><br />
into the blood stream from a liver dam<br />
aged by an increasing load <strong>of</strong> <strong>copper</strong> un<br />
able to be utilized in ceruloplasmin syn<br />
thesis or excreted via the biliary system<br />
(97, 155, 508, 516). This hemolysis may<br />
reflect, to variable degrees, inhibition <strong>of</strong><br />
erythrocyte glycolysis <strong>and</strong> <strong>of</strong> glucose-6phosphate<br />
dehydrogenase, oxidation <strong>of</strong> glutathione<br />
<strong>and</strong> denaturation <strong>of</strong> hemoglobin<br />
with Heinz body formation (203). A<br />
variety <strong>of</strong> other factors may be involved<br />
(588). Manifestations <strong>of</strong> slow <strong>copper</strong><br />
poisoning <strong>of</strong> a non-fatal type as seen in<br />
<strong>copper</strong> <strong>and</strong> brass workers are well de<br />
scribed by Chatterji <strong>and</strong> Ganguly (111).<br />
There are symptoms <strong>of</strong> laryngitis, bron<br />
chitis, intestinal colic with catarrh <strong>and</strong><br />
diarrhea, general emaciation <strong>and</strong> anemia.<br />
Since much <strong>of</strong> the information on cop<br />
per toxicity comes from instances <strong>of</strong> acci<br />
dental or intentional intake (mostly sui<br />
cide), data concerning oral intake neces<br />
sary to produce symptoms <strong>of</strong> toxicity are<br />
decidedly meager. Ingestion <strong>of</strong> 10 to 15<br />
mg <strong>of</strong> inorganic <strong>copper</strong> will cause nausea,<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2021<br />
vomiting <strong>and</strong> diarrhea <strong>and</strong>, in larger doses,<br />
intravascular hemolysis (75, 865). It has<br />
also been stated ( 685 ) that in India, where<br />
<strong>copper</strong> sulfate is a frequent mechanism for<br />
suicide, such symptoms are seen when<br />
about 10 mg <strong>of</strong> cupric ion are ingested.<br />
Yet, Roberts (641), in reviewing cases <strong>of</strong><br />
<strong>copper</strong> sulfate poisoning admitted to a<br />
large city hospital over 7 years, describes<br />
one individual who knowingly consumed<br />
an estimated 20 g <strong>of</strong> <strong>copper</strong> sulfate two<br />
to three times weekly over a period <strong>of</strong> 4<br />
months (total <strong>of</strong> about 600 g <strong>of</strong> the crys<br />
tals, or about 1.25 g <strong>of</strong> anionic <strong>copper</strong> per<br />
day). Aside from the usual symptoms <strong>of</strong><br />
toxicity, there was an associated hemolytic<br />
anemia, possibly the earliest recorded as<br />
due to <strong>copper</strong> toxicity. There was reason<br />
ably rapid recovery under conventional<br />
procedures <strong>of</strong> that period. Two possible<br />
explanations for this unusually high degree<br />
<strong>of</strong> tolerance to <strong>copper</strong> are that the subject<br />
greatly overestimated his previous intake<br />
<strong>of</strong> <strong>copper</strong> sulfate or that he had adapted<br />
to a high <strong>copper</strong> intake similar to that oc<br />
curring in pigs (767 ).<br />
There are numerous reports <strong>of</strong> accidental<br />
<strong>and</strong> suicidal poisoning from oral intake <strong>of</strong><br />
<strong>copper</strong> sulfate in India (111, 118, 120, 288,<br />
813). In one hospital in India, <strong>of</strong> all cases<br />
<strong>of</strong> accidental poisoning admitted, <strong>copper</strong><br />
sulfate poisoning represented 33.6% <strong>of</strong> 238<br />
admissions in 1961 (120) <strong>and</strong> 26.5% <strong>of</strong><br />
admissions in a 9-month period during<br />
1969-1970 (112). In cases <strong>of</strong> acute <strong>copper</strong><br />
poisoning, analyses <strong>of</strong> urine <strong>and</strong> feces for<br />
<strong>copper</strong> give exceedingly high values (111).<br />
Under more normal circumstances <strong>of</strong><br />
daily living, varied degrees <strong>of</strong> <strong>copper</strong> tox<br />
icity have been recorded, as for example:<br />
1) in young infants presumably exposed<br />
via drinking water <strong>and</strong> cooked foods, to<br />
water derived from all-<strong>copper</strong> storage <strong>and</strong><br />
conduit systems (662, 816); 2) in children<br />
given tablets containing sulfates <strong>of</strong> <strong>copper</strong>,<br />
iron <strong>and</strong> manganese (220) or accidentally<br />
consuming a solution <strong>of</strong> <strong>copper</strong> sulfate<br />
(819); 3) in workers imbibing tea made<br />
from water contaminated by corroded<br />
geysers (557, 701); 4) in consumers <strong>of</strong><br />
carbonated beverages from post-mix type<br />
<strong>of</strong> vending machines with defective valves<br />
(356, 450), or from bottles with corroded<br />
pouring spouts (512); 5) in consumers <strong>of</strong><br />
alcoholic beverages left in <strong>copper</strong>-lined<br />
containers (865) or brewed at home in<br />
metal containers (633); 6) in children<br />
given <strong>copper</strong> sulfate as an emetic (355 ) ;<br />
<strong>and</strong> 7) in subjects after exchange trans<br />
fusions (50) <strong>and</strong> hemodialyses (51, 52,<br />
376, 471, 472, 487, 497), due to <strong>copper</strong><br />
present in tap-water used, or to <strong>copper</strong>containing<br />
valves <strong>and</strong> stopcocks used, in<br />
the conduits. Copper can cross dialyzing<br />
membranes, even against a concentration<br />
gradient, <strong>and</strong> rather small traces <strong>of</strong> <strong>copper</strong><br />
introduced intraveneously are highly toxic.<br />
Frequently, the result is hemolytic anemia.<br />
Hemodialysis has proved to be ineffective<br />
in treating acute <strong>copper</strong> poisoning, but<br />
this may have been due to a delay <strong>of</strong> 13<br />
hours in instituting treatment (10). Bremner<br />
(60) reviews the toxicity <strong>of</strong> <strong>copper</strong><br />
<strong>and</strong> other heavy metals <strong>and</strong> Cohen (121)<br />
discusses health hazards from industrial<br />
exposure to <strong>copper</strong>.<br />
At times it may be difficult in young<br />
infants to determine whether a state <strong>of</strong><br />
toxicosis is attributable to an early phase<br />
<strong>of</strong> Wilson's disease or to high levels <strong>of</strong><br />
<strong>copper</strong> in the family drinking water from<br />
<strong>copper</strong> pipes (816). Ever since the devel<br />
opment <strong>of</strong> metal conduits for potable<br />
water, man has been exposed to possibili<br />
ties <strong>of</strong> zinc poisoning from galvanized<br />
pipes, <strong>and</strong> <strong>of</strong> <strong>copper</strong> poisoning from <strong>copper</strong><br />
conduits <strong>and</strong> storage tanks. In certain city<br />
water supplies, <strong>copper</strong> salts are added to<br />
maintain a concentration <strong>of</strong> about 0.06<br />
ppm to restrict the growth <strong>of</strong> algae in the<br />
reservoirs (550). It is also recognized that<br />
s<strong>of</strong>t waters tend to leach <strong>copper</strong> conduits<br />
more than do hard waters. An impressive<br />
analysis <strong>of</strong> complexities involved in engi<br />
neering design <strong>of</strong> <strong>copper</strong> conduit systems<br />
to reduce the corrosion process itself, <strong>and</strong><br />
to minimize the retention time <strong>of</strong> water in<br />
small-bore tubes, has been presented by<br />
Page (591). Nevertheless, these problems<br />
are more or less controlled by cosmetic<br />
considerations, since water with high cop<br />
per content develops a surface scum due<br />
to formation <strong>of</strong> insoluble <strong>copper</strong> com<br />
pounds. It is generally accepted that a<br />
limit <strong>of</strong> 1 ppm <strong>of</strong> <strong>copper</strong> in supplies <strong>of</strong><br />
drinking water is safe <strong>and</strong> acceptable.<br />
Copper represents one <strong>of</strong> the earliest<br />
additives for enhancement <strong>of</strong> the appeal <strong>of</strong><br />
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2022 KARL E. MASON<br />
foods such as green peas, beans <strong>and</strong><br />
pickles. The early studies <strong>of</strong> Drummond<br />
(171) failed to demonstrate in experimen<br />
tal animals (dogs, cats) any deleterious<br />
effects from consumption <strong>of</strong> such "greened"<br />
vegetables. It is now generally accepted<br />
that in the processes <strong>of</strong> cooking, canning<br />
<strong>and</strong> storage <strong>of</strong> foods <strong>and</strong> beverages any<br />
increment in <strong>copper</strong> content is largely re<br />
lated to contact with <strong>copper</strong> in the vessels<br />
<strong>and</strong> conduits utilized. In present day<br />
societies hazards are reduced to a mini<br />
mum. These <strong>and</strong> other aspects <strong>of</strong> <strong>copper</strong><br />
toxicity are well reviewed by Lieh (459).<br />
INTERRELATIONSHIPS BETWEEN COPPER<br />
AND OTHER TRACE ELEMENTS<br />
Studies on laboratory <strong>and</strong> farm animals<br />
have revealed numerous interrelationships<br />
between <strong>copper</strong> <strong>and</strong> other trace elements<br />
<strong>and</strong> substances (notably iron, zinc, molyb<br />
denum, cadmium <strong>and</strong> ascorbic acid) in<br />
mammalian <strong>metabolism</strong> ( 332, 333 ). On the<br />
basis <strong>of</strong> some <strong>of</strong> these reactions, Hill <strong>and</strong><br />
Matrone (332) advanced the thesis that<br />
"those elements whose physical <strong>and</strong> chemi<br />
cal properties are similar will act antago<br />
nistically to each other biologically." This<br />
concept has since been well substantiated.<br />
Davies ( 149, 150) classifies interactions<br />
between trace elements as non-competitive,<br />
<strong>and</strong> multi-element reactions. The noncompetitive<br />
type is exemplified by the re<br />
quirement <strong>of</strong> dietary <strong>copper</strong> for mobiliza<br />
tion <strong>of</strong> iron for hemoglobin synthesis, as dis<br />
cussed earlier (pp. 1985-1986), <strong>and</strong> by inter<br />
actions between molybdenum, sulfur <strong>and</strong><br />
<strong>copper</strong> in ruminants as recently reviewed<br />
by Suttle (766) <strong>and</strong> Pitt (603). Inter<br />
actions <strong>of</strong> this type are not predictable<br />
from a knowledge <strong>of</strong> the chemistry <strong>of</strong> the<br />
elements in question, as are those <strong>of</strong> the<br />
competitive type. The latter type is evident<br />
in the mutually antagonistic effects <strong>of</strong> cop<br />
per <strong>and</strong> zinc, such as the protective effect<br />
<strong>of</strong> <strong>copper</strong> in reducing toxicity resulting<br />
from high dietary intakes <strong>of</strong> zinc in chicks<br />
(332); <strong>and</strong> the effect <strong>of</strong> increased dietary<br />
intake <strong>of</strong> zinc in increasing the tolerance<br />
<strong>of</strong> pigs to excess intake <strong>of</strong> <strong>copper</strong> (767,<br />
768). The multi-element type <strong>of</strong> inter<br />
action is seen in studies with chicks where<br />
dietary zinc induces or exacerbates a con<br />
ditioned deficiency <strong>of</strong> <strong>copper</strong> which in<br />
turn restricts the utilization <strong>of</strong> iron (332).<br />
Speaking in general terms, in non-rumi<br />
nants interactions <strong>of</strong> <strong>copper</strong> with iron <strong>and</strong><br />
zinc are <strong>of</strong> particular significance whereas<br />
in ruminants interactions <strong>of</strong> <strong>copper</strong> with<br />
molybdenum in the presence <strong>of</strong> sulfur take<br />
precedence, especially in terms <strong>of</strong> practical<br />
considerations in animal husb<strong>and</strong>ry. Molyb<br />
denum toxicity, long recognized in grazing<br />
cattle in many parts <strong>of</strong> the world, causes<br />
biochemical <strong>and</strong> pathological changes<br />
closely resembling those <strong>of</strong> <strong>copper</strong> defi<br />
ciency, as well recognized in the pioneer<br />
studies <strong>of</strong> Davis (150). They are readily<br />
prevented or cured by <strong>copper</strong> sulfate.<br />
Compared to cattle, sheep are less suscept<br />
ible to high dietary intakes <strong>of</strong> molybdenum<br />
<strong>and</strong> more susceptible to low intake <strong>of</strong><br />
molybdenum, which can lead to chronic<br />
<strong>copper</strong> poisoning (798). Species reactions<br />
vary greatly (603).<br />
Non-ruminants are much more tolerant<br />
<strong>of</strong> excess molybdenum <strong>and</strong> <strong>of</strong> high <strong>copper</strong><br />
intake than are ruminants. Moreover, ef<br />
fects <strong>of</strong> high dietary <strong>copper</strong> can be accen<br />
tuated by low levels <strong>of</strong> zinc <strong>and</strong> iron, <strong>and</strong><br />
low <strong>copper</strong> intake by ascorbic acid which<br />
is thought to interfere with the absorption<br />
<strong>of</strong> <strong>copper</strong>. An interesting difference is that<br />
in ruminants dietary sulfur potentiates a<br />
<strong>copper</strong>-molybdenum antagonism such that<br />
tissue <strong>copper</strong> levels are decreased, whereas<br />
in non-ruminants sulfur alleviates this an<br />
tagonism. Moreover, the capacities <strong>of</strong> di<br />
etary sulfur to accentuate or ameliorate the<br />
toxic effects <strong>of</strong> molybdenum vary with the<br />
<strong>copper</strong> status <strong>of</strong> the animal. Among the<br />
proposals <strong>of</strong>fered to explain the basic<br />
mechanisms involved have been: 1) for<br />
mation in the rumen <strong>of</strong> unabsorbable com<br />
plexes such as thiomolybdate, cupric sulfide<br />
or cupric molybdate; 2) interference<br />
<strong>of</strong> liver uptake <strong>of</strong> <strong>copper</strong> by molybdenum<br />
<strong>and</strong> sulfur; <strong>and</strong> 3) formation <strong>of</strong> stable<br />
complexes <strong>of</strong> <strong>copper</strong> <strong>and</strong> molybdenum in<br />
the plasma. Obviously, much remains to be<br />
clarified.<br />
Copper is quite routinely incorporated<br />
in mineral mixtures added to commercial<br />
livestock feeds to increase rate <strong>of</strong> weight<br />
gain <strong>and</strong> food efficiency <strong>and</strong> is recognized<br />
as a safe ingredient (up to a level <strong>of</strong> 15<br />
ppm), but regulations prohibit molyb<br />
denum additions. Under conditions in<br />
which both forage <strong>and</strong> feeds are naturally<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2023<br />
low in molybdenum, states <strong>of</strong> <strong>copper</strong><br />
toxicity, <strong>of</strong>ten fatal, have occurred in<br />
flocks <strong>of</strong> sheep (798). This practice <strong>of</strong> add<br />
ing excess <strong>copper</strong> without molybdenum to<br />
livestock <strong>and</strong> poultry feeds represents a<br />
potential hazard to the consuming public,<br />
especially to the young infant consuming<br />
baby foods made from liver (71). The<br />
practical as well as the purely scientific<br />
aspects <strong>of</strong> trace element interactions, com<br />
plex as they may be, fully justify some<br />
consideration. For further information on<br />
the complex interrelationships between<br />
molybdenum, sulfate <strong>and</strong> <strong>copper</strong>, the<br />
reader is referred to a number <strong>of</strong> reviews<br />
on the subject (71, 110, 140, 165, 493, 517,<br />
603, 766, 798) <strong>and</strong> to a series <strong>of</strong> recent<br />
research reports (110).<br />
In man, there is but fragmentary evi<br />
dence <strong>of</strong> significant interrelationships <strong>of</strong><br />
<strong>copper</strong> <strong>and</strong> molybdenum, <strong>and</strong> <strong>of</strong> a role <strong>of</strong><br />
molybdenum in human nutrition. By virtue<br />
<strong>of</strong> molybdenum being a component <strong>of</strong><br />
xanthine oxidase, it may participate in the<br />
reduction <strong>of</strong> cellular ferric to ferrous fer<br />
ritin such that high <strong>copper</strong>-molybdenum<br />
ratio may contribute to abnormalities <strong>of</strong><br />
iron <strong>metabolism</strong> <strong>and</strong> utilization (698, 699).<br />
It is postulated that high <strong>copper</strong>-molyb<br />
denum ratios in the American diet may<br />
contribute to iron-deficiency anemias <strong>and</strong><br />
may also have influence upon metabolic<br />
abnormalities <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> such<br />
as seen in Wilson's disease (699). From<br />
India come several interesting reports<br />
dealing with high molybdenum-<strong>copper</strong><br />
ratios. Volunteers fed diets containing<br />
sorghum with increasing content <strong>of</strong> molyb<br />
denum showed increasing levels <strong>of</strong> urinary<br />
<strong>copper</strong> excretion, which appeared to reflect<br />
mobilization <strong>of</strong> <strong>copper</strong> from body stores<br />
(164). In regions where creation <strong>of</strong> large<br />
dams brings about marked changes in trace<br />
element balance in the soil, food grains <strong>and</strong><br />
drinking water tend to acquire a high<br />
molybdenum-<strong>copper</strong> ratio as molybdenum<br />
is leached out by alkaline conditions <strong>and</strong><br />
the ratio possibly increased further by<br />
high fluoride content <strong>of</strong> soils. As a result<br />
the poorest groups <strong>of</strong> the population whose<br />
staple food is sorghum, which accumulates<br />
more molybdenum than rice or wheat, be<br />
come victims <strong>of</strong> genu valgum <strong>and</strong> osteo<br />
porosis <strong>of</strong> the long bones (9, 428). Genu<br />
valgum, previously considered the result <strong>of</strong><br />
fluoride toxicosis, now appears to represent<br />
either a state <strong>of</strong> molybdenosis or one <strong>of</strong><br />
<strong>copper</strong> deficiency induced by excess mo<br />
lybdenum, or a modification <strong>of</strong> either by<br />
high fluoride intake. In experimental <strong>and</strong><br />
farm animals there are characteristic dif<br />
ferences in osseous lesions in these two<br />
conditions, as well described by Asling <strong>and</strong><br />
Hurley (26). In the studies discussed<br />
above no reference is made to the status<br />
<strong>of</strong> farm animals in the same localities, or<br />
to any plan to test effects <strong>of</strong> the sorghum<br />
versus other diets upon experimental ani<br />
mals. Returning to the capacity <strong>of</strong> molyb<br />
denum to reduce tissue <strong>copper</strong> levels <strong>and</strong><br />
to increase urinary excretion <strong>of</strong> <strong>copper</strong>,<br />
Suttle (766) has questioned whether or<br />
not molybdenum might have therapeutic<br />
value in the treatment <strong>of</strong> Wilson's disease,<br />
apparently unaware <strong>of</strong> one report (74) <strong>of</strong><br />
its ineffectiveness in four cases <strong>of</strong> the dis<br />
ease treated for 4 to 11 months.<br />
In the case <strong>of</strong> trace elements occurring<br />
mainly in ionic form, such as molybdenum,<br />
selenium <strong>and</strong> iodine, deficiencies <strong>and</strong> ex<br />
cesses are readily reflected in components<br />
<strong>of</strong> the food chain <strong>and</strong> in not only grazing<br />
animals but also in man himself (515). An<br />
interesting example <strong>of</strong> this <strong>and</strong> <strong>of</strong> <strong>copper</strong>molybdenum<br />
interactions may be cited. In<br />
mountainous areas <strong>of</strong> Russia where the<br />
soil is notoriously high in molybdenum, a<br />
high incidence <strong>of</strong> molybdenum toxicity,<br />
characterized not by genu valgum but by<br />
increased blood xanthine oxidase <strong>and</strong> uric<br />
acid, <strong>and</strong> urinary uric acid, leading to<br />
symptoms <strong>of</strong> gout, was observed in the<br />
population <strong>of</strong> one province but not in that<br />
<strong>of</strong> another where molybdenum intake was<br />
equally high; the difference was ascribed<br />
to significantly higher blood <strong>copper</strong> <strong>and</strong><br />
resultant lower blood molybdenum levels<br />
in the non-affected population (424, 425).<br />
Data pertaining to these studies are sum<br />
marized by Mertz (515). The explanation<br />
proposed is in accord with extensive knowl<br />
edge <strong>of</strong> such interactions in experimental<br />
<strong>and</strong> farm animals.<br />
Interactions between <strong>copper</strong> <strong>and</strong> zinc<br />
have long been recognized in animals <strong>and</strong><br />
man. Excess <strong>of</strong> one is <strong>of</strong>ten associated with<br />
diminution <strong>of</strong> the other in body fluids <strong>and</strong><br />
liver. Competition for binding sites on<br />
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2024 KARL E. MASON<br />
metallothionein or metallothionein-like<br />
proteins, complex as these interactions ap<br />
pear to be (518), provides the best ex<br />
planation. These proteins are present in<br />
the liver, kidney, intestinal mucosa, pan<br />
creas <strong>and</strong> spleen. They play an important<br />
role in <strong>copper</strong> homeostasis. In man, these<br />
reciprocal interactions are less apparent<br />
than in animals where the intake <strong>and</strong> im<br />
balance <strong>of</strong> the elements can be more spe<br />
cifically controlled. However, a striking<br />
example is the reported occurrence <strong>of</strong> typi<br />
cal <strong>copper</strong> deficiency, with microcytic hypochromic<br />
anemia <strong>and</strong> leukopenia, in one<br />
patient, <strong>and</strong> very low levels <strong>of</strong> serum cop<br />
per in 7 <strong>of</strong> 13 others, receiving unusually<br />
high levels <strong>of</strong> zinc for the treatment <strong>of</strong><br />
sickle cell anemia (64). All responded<br />
favorably to daily supplements <strong>of</strong> <strong>copper</strong>.<br />
The question <strong>of</strong> the dietary ratio <strong>of</strong> zinc<br />
to <strong>copper</strong> has been given considerable at<br />
tention by Kelvay (416-418, 420) in sup<br />
port <strong>of</strong> a hypothesis that high zinc to cop<br />
per ratio <strong>and</strong> the associated hypercholesteremia<br />
increase the risk <strong>of</strong> ischemie heart<br />
disease, <strong>and</strong> may also play an important<br />
role in the genesis <strong>of</strong> arteriosclerosis. This<br />
hypothesis has received limited support<br />
(81, 82). Of some relevance are recent ob<br />
servations that myocardial lesions associ<br />
ated with hypercholesteremia occur in rats<br />
fed a <strong>copper</strong>-deficient diet for 7 to 9 weeks<br />
after weaning (16). Cardiac hypertrophy,<br />
subendocardial hemorrhage, necrosis <strong>of</strong><br />
muscle fibers, abnormalities <strong>of</strong> elastic tis<br />
sue <strong>of</strong> the aorta but not <strong>of</strong> the coronary<br />
arteries, <strong>and</strong> occasional heart rupture are<br />
described. In other studies with <strong>copper</strong>deficient<br />
rats similar lesions have been ob<br />
served <strong>and</strong> ascribed to a marked reduction<br />
in cytochrome c oxidase activity (3, 401).<br />
With repletion <strong>of</strong> <strong>copper</strong>, cytochrome c<br />
oxidase activity is normalized, after which<br />
cardiac hypertrophy <strong>and</strong> splenomegaly are<br />
greatly reduced (3). Unquestionably, in<br />
terest has been stimulated, but there is<br />
need for specific research on man directed<br />
toward the possible role <strong>of</strong> zinc/<strong>copper</strong><br />
ratios <strong>and</strong> cholesterol status in ischemie<br />
heart disease.<br />
HUMAN REQUIREMENTS<br />
In discussing <strong>requirements</strong> <strong>of</strong> any nu<br />
trient, a variety <strong>of</strong> terms are in common<br />
usage, such as "basic," "minimal" <strong>and</strong><br />
"optimal" <strong>requirements</strong>; <strong>and</strong> "recom<br />
mended" allowances. An excellent discus<br />
sion <strong>and</strong> definition <strong>of</strong> these terms has been<br />
presented by Mertz (514). According to<br />
his interpretation, the "basic" requirement<br />
for a trace element represents that daily<br />
intake permitting absorption <strong>of</strong> an amount<br />
just sufficient to prevent a state <strong>of</strong> de<br />
ficiency; whereas the "optimal" require<br />
ment represents that daily intake which<br />
will allow maintenance at a near-optimal<br />
level <strong>of</strong> all biological <strong>and</strong> physiological<br />
functions in which the element is involved,<br />
under the various stress conditions <strong>of</strong> life.<br />
The somewhat intermediate term "mini<br />
mal," traditionally used in balance studies,<br />
is defined as that daily intake which equals<br />
the daily excretory loss from the body.<br />
This term best fits the nature <strong>of</strong> the data<br />
on which estimates <strong>of</strong> human <strong>requirements</strong><br />
for <strong>copper</strong> are based. The term "optimal"<br />
is somewhat comparable to the term "rec<br />
ommended dietary allowance." According<br />
to Harper (307), the RDA represents esti<br />
mates <strong>of</strong> the amount <strong>of</strong> an essential nu<br />
trient which "each person in a healthy<br />
population must consume in order to pro<br />
vide reasonable assurance that physiologi<br />
cal needs will be met."<br />
In the case <strong>of</strong> <strong>copper</strong> <strong>requirements</strong>, the<br />
problem is not as simple as it might ap<br />
pear. Most difficult to evaluate are the<br />
reported differences in the <strong>copper</strong> content<br />
<strong>of</strong> foods, diets, body fluids <strong>and</strong> tissues<br />
attributable to the great variety <strong>of</strong> analyti<br />
cal methods employed over the past half<br />
century, <strong>and</strong> to possible contamination <strong>of</strong><br />
samples prior to or during analytical pro<br />
cedures. An excellent discussion <strong>and</strong> criti<br />
cal appraisal <strong>of</strong> methods in use up to 1965<br />
for the determination <strong>of</strong> <strong>copper</strong> <strong>and</strong> ceruloplasmin<br />
in biological materials is presented<br />
by Sass-Kortsak (666). The methods de<br />
scribed have been variably modified <strong>and</strong><br />
newer ones introduced. In some instances<br />
there is rather remarkable agreement be<br />
tween early <strong>and</strong> later reports. In other<br />
instances there appear differences which,<br />
on inspection, might reflect variable sensi<br />
tivity <strong>of</strong> the methods employed. Any ef<br />
fort to identify <strong>and</strong> evaluate methodolo<br />
gies used in particular studies would be<br />
rather futile.<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2025<br />
What has been outlined in this con<br />
spectus, up to this point, has been an<br />
assessment <strong>of</strong> past <strong>and</strong> current knowledge<br />
regarding the role that <strong>copper</strong> may play in<br />
the <strong>metabolism</strong> <strong>of</strong> man, with occasional<br />
reference to ancillary information gained<br />
from observations on laboratory <strong>and</strong> farm<br />
animals. Attention has been called to the<br />
distribution <strong>of</strong> <strong>copper</strong> in the body; the<br />
vast array <strong>of</strong> cuproproteins <strong>and</strong> evidence<br />
as to their roles in maintaining functional<br />
<strong>and</strong> morphological integrity <strong>of</strong> specific tis<br />
sues <strong>and</strong> organs; the absorption, transport<br />
<strong>and</strong> excretion <strong>of</strong> <strong>copper</strong>; its omnipresence<br />
in foods; its important roles in prenatal<br />
<strong>and</strong> postnatal life, <strong>and</strong>; the nature <strong>of</strong> states<br />
induced by naturally occurring, experimen<br />
tally induced <strong>and</strong> congenitally determined<br />
<strong>copper</strong> deficiency. It is hoped that this<br />
digest <strong>of</strong> knowledge will provide an ade<br />
quate basis for considerations <strong>of</strong> the mini<br />
mal <strong>copper</strong> <strong>requirements</strong> <strong>of</strong> man.<br />
In considering human <strong>requirements</strong> for<br />
<strong>copper</strong> there are many factors whose in<br />
fluence is exceedingly difficult to evaluate<br />
because <strong>of</strong> limited knowledge available. A<br />
few examples may be cited. That portion <strong>of</strong><br />
dietary <strong>copper</strong> which is actually absorbed<br />
probably varies considerably, depending<br />
upon its chemical state in the foods con<br />
sumed <strong>and</strong> the influence <strong>of</strong> other dietary<br />
components. Best estimates indicate an<br />
absorption <strong>of</strong> 40 to 60% <strong>of</strong> the oral intake<br />
(p. 1991). In addition to the unabsorbed<br />
<strong>copper</strong> <strong>and</strong> biliary <strong>copper</strong> components <strong>of</strong><br />
the feces, inadequate consideration has<br />
been given to contributions provided by<br />
secretions <strong>of</strong> the salivary gl<strong>and</strong>s, gastric<br />
<strong>and</strong> intestinal mucosa <strong>and</strong> pancreas, <strong>and</strong><br />
by dehiscence <strong>of</strong> epithelial cells <strong>of</strong> intesti<br />
nal villi. At least, the extent <strong>of</strong> reabsorption<br />
<strong>of</strong> <strong>copper</strong> released via these various<br />
pathways has not been clearly determined.<br />
Despite frequent statements in the litera<br />
ture that some <strong>of</strong> the <strong>copper</strong> in bile may<br />
be reabsorbed, there exists other evidence<br />
that this <strong>copper</strong> is so firmly bound to pro<br />
teins that it is not reabsorbed by the gall<br />
bladder or intestinal mucosa in any signifi<br />
cant amounts (266).<br />
Balance studies are limited by the pre<br />
cision with which intake <strong>and</strong> output can<br />
be measured. For an element such as cop<br />
per which has a slow rate <strong>of</strong> turnover, a<br />
variable degree <strong>of</strong> intestinal absorption,<br />
strong homeostatic mechanisms <strong>and</strong> an<br />
almost exclusive output via the feces, in<br />
terpretations <strong>of</strong> balance studies becomes<br />
very difficult. The sporadic nature <strong>of</strong> defe<br />
cation <strong>and</strong> rather wide individual variation<br />
make balance studies <strong>of</strong> 7 to 14 days neces<br />
sary for obtaining valid data. Replacement<br />
<strong>of</strong> carmine by polyethylene glycol 4000, as<br />
a fecal marker, should shorten this time<br />
period (848).<br />
Biological availability is also an impor<br />
tant but largely unknown factor. Little is<br />
known about the chemical nature <strong>of</strong> cop<br />
per in foods, the extent to which it may<br />
react with chelating substances such as<br />
dietary fiber, or how its absorption may be<br />
influenced by the protein-binding poten<br />
tialities <strong>of</strong> other trace elements such as zinc<br />
<strong>and</strong> molybdenum. To these factors must<br />
be added the inflence <strong>of</strong> acute <strong>and</strong> chronic<br />
infections, use <strong>of</strong> antimicrobial agents, dys<br />
function <strong>of</strong> the gastrointestinal tract <strong>and</strong><br />
other stress states. Usually, in well con<br />
ducted <strong>copper</strong> balance studies on man,<br />
healthy subjects are selected <strong>and</strong> as many<br />
as possible <strong>of</strong> the above mentioned in<br />
fluences are eliminated. The discussion to<br />
follow will focus on accumulated evidence<br />
from balance studies <strong>and</strong> from experiences<br />
with total parenteral nutrition as to what<br />
may represent the minimal <strong>requirements</strong><br />
<strong>of</strong> man for <strong>copper</strong> during infancy, child<br />
hood <strong>and</strong> adulthood.<br />
Infants<br />
Specific <strong>requirements</strong> <strong>of</strong> healthy human<br />
infants for <strong>copper</strong> have been difficult to<br />
determine with any degree <strong>of</strong> accuracy.<br />
To provide a picture <strong>of</strong> the problem, it<br />
seems appropriate to review current infor<br />
mation with respect to: 1) milk as a source<br />
<strong>of</strong> <strong>copper</strong> for the premature <strong>and</strong> full-term<br />
infant; 2) <strong>copper</strong> balance studies on in<br />
fants; 3) naturally occurring states <strong>of</strong> cop<br />
per deficiency; <strong>and</strong> 4) studies on infants<br />
largely or totally dependent upon total<br />
parenteral nutrition (hyperalimentation )<br />
for extended periods <strong>of</strong> time.<br />
Milk as a source <strong>of</strong> <strong>copper</strong>. It has long<br />
been recognized that the <strong>copper</strong> content<br />
<strong>of</strong> human milk is two to three times higher<br />
than that <strong>of</strong> cow's milk, <strong>and</strong> that the con<br />
tent <strong>of</strong> human colostrum is two to three<br />
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2026 KARL E. MASON<br />
times that <strong>of</strong> later milk (437, 449, 541,<br />
647, 877). Moreover, the somewhat lower<br />
ratio <strong>of</strong> zinc to <strong>copper</strong> (about 4:1) in<br />
human milk may significantly enhance cop<br />
per absorption in breast-fed infants (843).<br />
Cow's milk has a very low content <strong>of</strong><br />
<strong>copper</strong>. Values recorded by different in<br />
vestigators have varied widely, <strong>and</strong> their<br />
documentation would serve no useful pur<br />
pose. Suffice it to say that three <strong>of</strong> the<br />
more recent reports (546, 598, 843) give<br />
values within a range <strong>of</strong> 0.04 to 0.30 mg/<br />
liter. Difference in forage <strong>and</strong> in season <strong>of</strong><br />
the year are largely responsible for varia<br />
tions in values obtained. Special attention<br />
may be called to analyses <strong>of</strong> commercial<br />
milk samples from 65 cities throughout the<br />
USA giving a national mean <strong>of</strong> 0.086<br />
(range 0.04-0.19) mg/liter (547).<br />
The first serious study <strong>of</strong> the <strong>copper</strong><br />
content <strong>of</strong> human milk, by Zondek <strong>and</strong><br />
B<strong>and</strong>mann (877), indicated levels <strong>of</strong> 0.5 to<br />
0.6 mg/liter in 85 samples obtained during<br />
the first 2 months <strong>of</strong> lactation. Munch-<br />
Peterson (541), who carried out 74 analy<br />
ses <strong>of</strong> milk from 10 mothers during the<br />
first 8 days <strong>of</strong> lactation, recorded a mean<br />
content <strong>of</strong> 0.48 mg/liter. He also found<br />
that intravenous administration <strong>of</strong> a watersoluble<br />
compound containing 19% <strong>of</strong> cop<br />
per given to seven other mothers had no<br />
influence on the <strong>copper</strong> content <strong>of</strong> the<br />
milk, even though serum <strong>copper</strong> levels<br />
were greatly increased. This confirmed the<br />
early report <strong>of</strong> Elvehjem et al. (189) that<br />
a 5 to 10-fold increase in dietary intake <strong>of</strong><br />
<strong>copper</strong> had no demonstrable effect on its<br />
level in the milk <strong>of</strong> the cow or goat. This<br />
restriction in mammary transfer <strong>of</strong> <strong>copper</strong><br />
might reflect a homeostatic mechanism for<br />
protection <strong>of</strong> the neonate. Rottger (647)<br />
reported a mean value <strong>of</strong> 0.44 ( range 0.27-<br />
0.84) mg/liter for 15 samples <strong>of</strong> human<br />
milk during early phases <strong>of</strong> lactation.<br />
Munch-Peterson (541) <strong>and</strong> Rottger (647)<br />
independently estimated a daily intake <strong>of</strong><br />
about 0.25 mg/Cu by young infants. In<br />
comparison, other investigators have re<br />
ported higher values for early milk (107,<br />
437, 449) <strong>and</strong> others give values approxi<br />
mately one-half or less than those recorded<br />
above, for mature human milk not identi<br />
fied as early milk (275, 546, 598, 843). A<br />
striking decrease in <strong>copper</strong> content <strong>of</strong> milk<br />
at successive months <strong>of</strong> lactation (11.2,<br />
7.3, 5.4, 4.6 <strong>and</strong> 1.5 /¿g/100ml) is reported<br />
by Kleinbaum (410). Similar data are<br />
given by Hambidge (299).<br />
Noteworthy is the recent <strong>and</strong> extensive<br />
study <strong>of</strong> Picciano <strong>and</strong> Guthrie (598), based<br />
on analyses <strong>of</strong> 350 milk samples from 50<br />
lactating, healthy women (seven samples<br />
each). Copper content varied considerably<br />
among women <strong>and</strong> with the same woman.<br />
Several samples taken over periods <strong>of</strong> days<br />
or weeks were necessary to provide a re<br />
liable estimate <strong>of</strong> the <strong>copper</strong> content <strong>of</strong><br />
milk from any individual. Their mean value<br />
<strong>of</strong> 0.24 (range 0.09-0.63) mg/liter is essen<br />
tially the same as that reported by Murthy<br />
<strong>and</strong> Rhea (546); namely, about 0.24 ±<br />
0.03 mg/kg, on analyses <strong>of</strong> 22 milk sam<br />
ples from lactating women.<br />
Picciano <strong>and</strong> Guthrie (598) calculated<br />
that breastfed infants less than 3 months<br />
<strong>of</strong> age, with a body weight <strong>of</strong> 4 kg <strong>and</strong> a<br />
daily milk intake <strong>of</strong> 850 ml, would ingest<br />
approximately 0.2 mg/day ( or 0.05 mg/kg/<br />
day). These estimates are in good agree<br />
ment with the values <strong>of</strong> 0.25 mg/day <strong>of</strong><br />
Munch-Peterson (541) <strong>and</strong> Rottger (647),<br />
also based upon human milk. They are also<br />
slightly less than estimates <strong>of</strong> 0.05 to 0.10<br />
mg/kg/day based on balance studies with<br />
young children 3 to 6 years <strong>of</strong> age (142,<br />
695), the estimate <strong>of</strong> 0.08 mg/kg/day by<br />
Cartwright ( 100), <strong>and</strong> the statement <strong>of</strong> the<br />
Committee on Recommended Dietary Al<br />
lowances <strong>of</strong> the National Research Council<br />
(549) that "The <strong>requirements</strong> <strong>of</strong> infants<br />
<strong>and</strong> children have been estimated at be<br />
tween 0.05 <strong>and</strong> 0.1 mg/kg <strong>of</strong> body weight<br />
per day; an intake <strong>of</strong> 0.08 mg/kg/day ap<br />
pears to be adequate." The World Health<br />
Organization in its 1973 report (861)<br />
recommends 0.08 mg/kg per day for in<br />
fants <strong>and</strong> young children <strong>and</strong> 0.04 mg/kg<br />
per day for older children. It would seem<br />
that under normal circumstances the cop<br />
per provided by nature in human milk is,<br />
when supplemented by storage in the new<br />
born liver, a reasonably good measure <strong>of</strong><br />
optimal <strong>requirements</strong> during early life.<br />
The premature infant presents special<br />
problems. According to Widdowson et al.<br />
(843) about % <strong>of</strong> fetal <strong>copper</strong> is trans<br />
ferred during the last 10 to 12 weeks <strong>of</strong><br />
gestation. As a consequence, premature in-<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2027<br />
fants <strong>of</strong> 28 to 30 weeks gestation, weighing<br />
about 1 kg, have much smaller reserves <strong>of</strong><br />
<strong>copper</strong> in the liver, spleen <strong>and</strong> other tissues<br />
to be called upon postnatally than do fullterm<br />
infants. Sultanova (761) has carried<br />
out <strong>copper</strong> analyses <strong>of</strong> livers <strong>and</strong> spleens<br />
from groups <strong>of</strong> premature infants with<br />
birth weights <strong>of</strong> 1.0 to 1.5, 1.5 to 2.0 <strong>and</strong><br />
2.0 to 2.5 kg, <strong>and</strong> term infants dying soon<br />
after birth. In successive groups there<br />
were definite increases in the <strong>copper</strong> levels<br />
per gram <strong>of</strong> tissue in both organs. Hence,<br />
the smaller the premature the lower the<br />
<strong>copper</strong> concentration in its tissues. As an<br />
other h<strong>and</strong>icap, the premature is usually<br />
obliged to subsist on an exclusive milk<br />
diet for much longer periods than the fullterm<br />
infant. Although in newborn prema<br />
tures <strong>copper</strong> deficiency is unknown, their<br />
status does place them in a more pre<br />
carious situation than full-term infants<br />
when states <strong>of</strong> malnutrition intervene. To<br />
compensate for this, Widdowson et al.<br />
(843) have suggested that premature in<br />
fants be provided a special milk formula<br />
which might assure a retention <strong>of</strong> at least<br />
0.085 mg <strong>of</strong> <strong>copper</strong> per day. Cordano<br />
( 124) feels that for prematures the recom<br />
mended 0.06 mg/100 kcal for infants (18)<br />
should be increased to 0.09 mg/100 kcal<br />
(i.e., 0.1 mg/kg/day). This is the current<br />
recommendation <strong>of</strong> the American Academy<br />
<strong>of</strong> Pediatrics ( 19) for low-birth-rate in<br />
fants.<br />
Balance studies. A pioneer balance study<br />
<strong>of</strong> <strong>copper</strong> in infancy, to which relatively<br />
little has since been added, is that <strong>of</strong><br />
Kleinbaum (409), who studied six breast<br />
fed full-term infants over the 13th to 23rd<br />
days <strong>of</strong> life. Birth weights averaged 3.4<br />
kg, but later weights are not given. Total<br />
<strong>copper</strong> intake <strong>and</strong> output for the 10-day<br />
period averaged 4.74 <strong>and</strong> 4.72 mg, respec<br />
tively. Three infants showed a negative<br />
<strong>and</strong> three a positive <strong>copper</strong> balance.<br />
Hence, these data might be interpreted as<br />
indicating that healthy full-term infants<br />
during the first month <strong>of</strong> life require an<br />
intake <strong>of</strong> approximately 0.5 mg/Cu/day to<br />
keep in positive balance. The 10-day bal<br />
ance study <strong>of</strong> Priev (619) on two infants<br />
8 <strong>and</strong> 3 months <strong>of</strong> age indicates require<br />
ments <strong>of</strong> 0.30 <strong>and</strong> 0.42 mg/day, respec<br />
tively. Similar 10-day balance studies <strong>of</strong><br />
Kleinbaum (409) on 31 premature infants<br />
initiated at ages <strong>of</strong> 2 to 82 days, <strong>and</strong> with<br />
average intakes <strong>of</strong> 0.28 mg reveal a slightly<br />
negative balance in all instances. Hence in<br />
the balance studies described there is<br />
reasonably good accord with a requirement<br />
<strong>of</strong> approximately 0.05 mg/kg/day for<br />
maintaining a positive <strong>copper</strong> balance in<br />
young infants <strong>of</strong> the six infants weighing<br />
in the range <strong>of</strong> 6 to 10 kg.<br />
States <strong>of</strong> <strong>copper</strong> deficiency. In studies<br />
with experimental animals, while the daily<br />
intake necessary to prevent development <strong>of</strong><br />
a deficiency <strong>of</strong> a nutrient provides the most<br />
reliable estimate <strong>of</strong> basic <strong>requirements</strong>, a<br />
determination <strong>of</strong> the smallest intake neces<br />
sary to effect cure <strong>of</strong> an early stage <strong>of</strong><br />
deficiency also provides the next best esti<br />
mate <strong>of</strong> minimal <strong>requirements</strong>. Such pro<br />
cedures are, for many reasons, not applica<br />
ble to man at any age. Even though an<br />
arbitrary level <strong>of</strong> therapy might prove to<br />
be marginal in effect, deficiencies or even<br />
excesses <strong>of</strong> other nutrients, together with<br />
malabsorption <strong>and</strong> diarrhea, are usually<br />
present <strong>and</strong> a simple deficiency state such<br />
as obtainable in experimental animals does<br />
not exist.<br />
Such a situation characterizes the pio<br />
neer studies <strong>of</strong> Cordano et al. ( 126) on<br />
four malnourished infants manifesting<br />
anemia, neutropenia, scurvy-like bone<br />
changes <strong>and</strong> hypocupremia. Rehabilitation<br />
on high caloric diets supplemented with<br />
iron, ascorbic acid, folie acid <strong>and</strong> other<br />
vitamins was incomplete without the addi<br />
tion <strong>of</strong> elemental <strong>copper</strong>. In fact, it was<br />
later recognized that the increased growth<br />
rate resulting from the improved diet<br />
greatly decreased the protective effect <strong>of</strong><br />
the low levels <strong>of</strong> <strong>copper</strong> provided by the<br />
milk diet (28-42 ,ug/kg body weight). On<br />
the basis <strong>of</strong> varied levels <strong>of</strong> <strong>copper</strong> supple<br />
mentation it was estimated that for rapidly<br />
growing infants 6 to 9 months <strong>of</strong> age, with<br />
inadequate stores <strong>of</strong> <strong>copper</strong> <strong>and</strong> main<br />
tained exclusively on milk diets, the daily<br />
requirement for <strong>copper</strong> was greater than<br />
0.042 mg but less than 0.135 mg/kg body<br />
weight. Since each <strong>of</strong> the children weighed<br />
approximately 10 kg at the beginning <strong>of</strong><br />
supplementation, these values are slightly<br />
higher than those derived from balance<br />
studies but approximate the estimated re-<br />
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2028 KARL E. MASON<br />
quirements <strong>of</strong> 0.077 to 0.11 mg/kg body<br />
weight for growing pigs (772). In later<br />
studies on 21 similar infants 4 to 19 months<br />
<strong>of</strong> age ( 128), daily supplements <strong>of</strong> greater<br />
than 0.15 mg/kg proved quite adequate<br />
to correct for neutropenia, considered to be<br />
the earliest manifestation <strong>and</strong> most sensi<br />
tive indicator <strong>of</strong> adequacy <strong>of</strong> treatment<br />
<strong>of</strong> <strong>copper</strong> deficiency in man. Subsequently,<br />
oral supplements <strong>of</strong> 2.5 mg/day were<br />
usually employed as a routine measure to<br />
assure much more than estimated require<br />
ments (276).<br />
On the basis <strong>of</strong> his personal experiences,<br />
Cordano (124, 125) recommends that<br />
manufactured formulas for premature in<br />
fants be supplemented with <strong>copper</strong> such<br />
as to provide 0.09 mg/100 kcal, rather than<br />
the 0.06 mg/100 kcal recommended by the<br />
Committee on Nutrition <strong>of</strong> the American<br />
Academy <strong>of</strong> Pediatrics (19) which, since<br />
then, has recognized this need. This pro<br />
vides approximately 0.1 mg/kg/day, <strong>and</strong><br />
is somewhat less than the supplement <strong>of</strong><br />
0.1 to 0.5 mg/day proposed by Ashkenazi<br />
et al. (25) for prematures subsisting on<br />
milk only. In such considerations there has<br />
been recognized a need to make provision<br />
for such commonplace factors as intestinal<br />
interactions between <strong>copper</strong> <strong>and</strong> iron in<br />
iron-fortified formulas (700), régurgita<br />
tion, prolonged diarrhea <strong>and</strong> infections. On<br />
the basis <strong>of</strong> the evidence presented, it<br />
seems that the daily requirement <strong>of</strong> cop<br />
per for young <strong>and</strong> reasonably healthy in<br />
fants may be met by daily intakes <strong>of</strong> 0.05<br />
to 0.1 mg/kg/day.<br />
Total parenteral nutrition. The develop<br />
ment <strong>of</strong> a procedure for providing "total<br />
Sarenteral nutrition" by means <strong>of</strong> an injsate<br />
<strong>of</strong> nutrients introduced via an in<br />
dwelling catheter inserted into a large cen<br />
tral vein by Dudrick et al. (176 )<strong>and</strong> Wilmore<br />
et al. (852, 853) ushered in a new<br />
era in therapeutic nutrition. Its original<br />
application in conjunction with surgical<br />
treatment <strong>of</strong> catastrophic gastrointestinal<br />
anomalies <strong>of</strong> infants has since been widely<br />
extended to the management <strong>of</strong> a variety<br />
<strong>of</strong> gastrointestinal disorders, burns, infec<br />
tions <strong>and</strong> other situations in which subjects<br />
are unable to meet nutritional needs by the<br />
oral route. By appropriate modifications,<br />
this procedure has been effectively ex<br />
tended from its hospital applications to<br />
the prolonged maintenance <strong>of</strong> subjects in<br />
the home environment (66, 304, 385, 441,<br />
711, 724). Total parenteral nutrition, espe<br />
cially when prolonged in infants <strong>and</strong><br />
adults, has provided much new <strong>and</strong> help<br />
ful information concerning <strong>copper</strong> require<br />
ments <strong>of</strong> man.<br />
However, problems arise in the interpre<br />
tation <strong>of</strong> the results. One must first assume<br />
that <strong>copper</strong> introduced parenterally sub<br />
stitutes for that portion <strong>of</strong> orally ingested<br />
<strong>copper</strong> absorbed by the intestinal tract <strong>and</strong><br />
transported to the liver <strong>and</strong> to the systemic<br />
vascular system. While, as discussed pre<br />
viously (p. 1991), it is generally accepted<br />
that roughly 40 to 60% <strong>of</strong> ingested <strong>copper</strong><br />
is absorbed, wide individual variations<br />
exist, due to differences in gastrointestinal<br />
functions, nature <strong>of</strong> the diet, derangements<br />
<strong>of</strong> <strong>metabolism</strong> <strong>and</strong> states <strong>of</strong> stress.<br />
It is noteworthy that in their classic<br />
studies with infants, Dudrick et al. (175,<br />
176) <strong>and</strong> Wilmore et al. (853) took care<br />
to provide in their parenteral fluids both<br />
vitamins <strong>and</strong> minerals. The latter included<br />
zinc, <strong>copper</strong>, manganese, cobalt <strong>and</strong> iodine.<br />
Copper was provided at a level <strong>of</strong> 0.22<br />
mg/kg body weight. This represents a<br />
rather generous supply when compared to<br />
estimated <strong>requirements</strong> <strong>of</strong> 0.05 to 0.10 mg/<br />
kg/day. In any case, failure to follow these<br />
guidelines resulted in occurrence <strong>of</strong> sev<br />
eral cases <strong>of</strong> <strong>copper</strong> deficiency in infants<br />
(25, 394, 463) <strong>and</strong> in adults (177, 808).<br />
Although traces <strong>of</strong> <strong>copper</strong> are present in<br />
fibrin <strong>and</strong> casein hydrolysates <strong>and</strong> in crys<br />
talline amino acid mixtures commonly used<br />
in parenteral solutions, direct analyses (57,<br />
317, 342, 590) demonstrate their variability<br />
<strong>and</strong> inadequacy to meet nutritional needs<br />
for <strong>copper</strong>. The proposed use <strong>of</strong> plasma<br />
transfusions given twice weekly (209 ) does<br />
not compensate for this (704). What is<br />
said <strong>of</strong> <strong>copper</strong> is also true <strong>of</strong> zinc (57, 342,<br />
590 ). Investigators now add trace elements<br />
to the basic hyperalimentation formulae<br />
(174, 214, 367, 382, 383, 709, 710, 712,<br />
724) providing <strong>copper</strong> in approximately<br />
the amount (0.22 mg/kg body weight)<br />
originally proposed by Wilmore et al.<br />
(853). Essentially the same supplement is<br />
recommended by Ricour et al. (639),<br />
Wretling (864) <strong>and</strong> by Karpel <strong>and</strong> Peden<br />
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(394) after experiences in compensating<br />
for a <strong>copper</strong> deficiency state developing in<br />
an infant with ileal atresia <strong>and</strong> short bowel<br />
syndrome maintained on total parenteral<br />
nutrition for the first 6.75 months <strong>of</strong> life.<br />
In current practice for complete intrave<br />
nous feeding <strong>of</strong> premature infants <strong>of</strong> less<br />
than 1,050 g birth weight, James <strong>and</strong> Mac-<br />
Mahon (383) provide 50 ¿u.g/kg/day <strong>of</strong><br />
<strong>copper</strong>. Quite in accord with these opinions<br />
are those <strong>of</strong> Ashkenazi et al. (25), record<br />
ing <strong>copper</strong> deficiency in a premature infant<br />
6 months old fed a diet <strong>of</strong> only whole milk<br />
<strong>and</strong> 5% cane sugar, <strong>and</strong> in a premature<br />
infant subjected to bowel surgery <strong>and</strong><br />
maintained on parenteral feeding for 3<br />
months. Both responded rapidly to oral<br />
<strong>copper</strong>, <strong>and</strong> investigators recommended<br />
that small premature infants be given sup<br />
plements <strong>of</strong> 0.1 to 0.5 mg <strong>of</strong> <strong>copper</strong> daily<br />
while milk is their only food, or during pro<br />
longed intravenous feeding.<br />
On the basis <strong>of</strong> the evidence cited above,<br />
it seems reasonable to assume that the<br />
daily requirement <strong>of</strong> intravenous <strong>copper</strong><br />
for infants, beyond the age at which they<br />
can depend upon prenatal tissue reserves<br />
( about 2 months ), may be in the range <strong>of</strong><br />
0.1 to 0.2 mg/day. On the assumption that<br />
approximately 40^ <strong>of</strong> orally ingested cop<br />
per is absorbed, this requirement would<br />
be the equivalent <strong>of</strong> 0.25 to 0.5 mg/day <strong>of</strong><br />
oral <strong>copper</strong>. For a 10-kg infant this would<br />
represent 0.025 to 0.05 mg/kg/day. Thus,<br />
the information provided by parenteral<br />
nutrition is in general concordance with<br />
that from studies in milk intake, <strong>copper</strong><br />
balance <strong>and</strong> recovery from deficiency<br />
states, which indicate <strong>requirements</strong> <strong>of</strong><br />
about 0.025 to 0.05 mg/kg/day for healthy<br />
full-term infants during early years <strong>of</strong> life,<br />
with somewhat more generous allowances<br />
for premature <strong>and</strong> low-birth-rate infants.<br />
Young children <strong>and</strong> adolescents<br />
The basis for determining the minimal<br />
daily <strong>requirements</strong> <strong>of</strong> <strong>copper</strong> for young<br />
children <strong>and</strong> adolescents is decidedly lim<br />
ited, as is indicated in table 1. Two pioneer<br />
studies on 3- to 6-year old children (142,<br />
695), although carried out 40 or more<br />
years ago, warrant special consideration.<br />
Daniels <strong>and</strong> Wright ( 142) studied five<br />
boys <strong>and</strong> three girls <strong>and</strong> employed two<br />
diets, one high in meat <strong>and</strong> cereals <strong>and</strong> the<br />
other high in cereals without meat, but<br />
similar in <strong>copper</strong> content. After 3 days <strong>of</strong><br />
adjustment to the diet, 5-day balance<br />
studies were made. Mean <strong>copper</strong> intakes,<br />
fecal plus urinary excretions <strong>and</strong> balances<br />
were, respectively, 1.48, 1.03 <strong>and</strong> +0.45<br />
mg/day. It was concluded that diets for<br />
children <strong>of</strong> pre-school age should include<br />
not less than 0.10 mg/kg/day <strong>of</strong> <strong>copper</strong>.<br />
Scoular (695) carried out similar balance<br />
studies on three boys <strong>of</strong> the same age,<br />
based upon three different but well con<br />
trolled diets. On the average, <strong>copper</strong> in<br />
takes, fecal <strong>and</strong> urinary losses <strong>and</strong> reten<br />
tions were, respectively, 1.36, 0.60, <strong>and</strong><br />
+0.76 mg/kg/day. In their best judgment,<br />
the minimal requirement <strong>of</strong> boys <strong>of</strong> that<br />
age would be between 0.053 <strong>and</strong> 0.085<br />
mg/kg/day. Similar conclusions were<br />
reached by Macy (477) in balance studies<br />
on school children 8 <strong>and</strong> 11 years <strong>of</strong> age.<br />
It may be noted that both <strong>of</strong> these esti<br />
mates fall within the range <strong>of</strong> estimated<br />
<strong>copper</strong> <strong>requirements</strong> for infants (0.05-0.1<br />
mg/kg/day) as discussed above.<br />
Balance studies carried out by Engel et<br />
al. (190) on groups <strong>of</strong> 12 girls 6 to 10<br />
years <strong>of</strong> age, during summer months <strong>of</strong><br />
1956, 1958 <strong>and</strong> 1962, employing liberal pro<br />
tein, low animal protein <strong>and</strong> vegetarian<br />
type diets, leave open questions concern<br />
ing <strong>requirements</strong>. Calculations made (by<br />
writer) from the data given indicate that<br />
the first two <strong>of</strong> these diets provided aver<br />
age <strong>copper</strong> intakes <strong>of</strong> 0.04 <strong>and</strong> 0.037 mg/<br />
kg/day <strong>and</strong> <strong>copper</strong> balances <strong>of</strong> —0.01<strong>and</strong><br />
—0.08mg/day, respectively. Comparable<br />
values for the vegetarian diet were 0.12<br />
mg/kg/day <strong>and</strong> +1.02 mg/day. These data<br />
would suggest a minimal intake somewhat<br />
less than proposed in the two studies just<br />
described. However, Engel et al. ( 190)<br />
estimated, by regression analysis, that the<br />
daily intake in their studies was 1.3 mg/<br />
day, <strong>and</strong> that the suggested allowance be<br />
2.5 mg/day. In this suggestion was in<br />
cluded a sweat loss <strong>of</strong> 0.5 mg/day <strong>and</strong> a<br />
safety margin <strong>of</strong> 0.7 mg/day, added to the<br />
1.3 mg/day intake. For matters <strong>of</strong> com<br />
parison, sweat loss was not incorporated in<br />
other balance studies recorded, <strong>and</strong> the<br />
estimated loss appears to be more than<br />
generous for the subjects. Furthermore, the<br />
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2032 KARL E. MASON<br />
cant changes in <strong>copper</strong> <strong>requirements</strong> occur.<br />
Considering the rather bizarre food intakes<br />
<strong>and</strong> eating habits <strong>of</strong> this segment <strong>of</strong> popu<br />
lations, there is real need for <strong>copper</strong> bal<br />
ance studies on pre-college teenagers.<br />
Except for the inconclusive observations <strong>of</strong><br />
Dawson et al. ( 152 ), no consideration has<br />
been given to the special nutritional re<br />
quirements, including those <strong>of</strong> <strong>copper</strong>, in<br />
teen-age pregnancies. Considering that in<br />
such situations there is need to meet<br />
growth <strong>requirements</strong> <strong>of</strong> the adolescent<br />
mother as well as those <strong>of</strong> the developing<br />
fetus, it may be assumed that <strong>requirements</strong><br />
are appreciably greater than in adult preg<br />
nant women.<br />
Adults<br />
Since adults are past the growing phase<br />
<strong>of</strong> life, <strong>copper</strong> <strong>requirements</strong> are expressed<br />
in terms <strong>of</strong> mg/day rather than as mg/kg/<br />
day, as in the case <strong>of</strong> infants <strong>and</strong> adoles<br />
cents.<br />
Dietary intake. An earlier section (pp.<br />
1998-1999) deals with the wide variation in<br />
<strong>copper</strong> content <strong>of</strong> human diets in various<br />
countries <strong>and</strong> <strong>of</strong> individuals <strong>of</strong> different<br />
ages. Table 2 summarizes additional data<br />
from studies in which the daily dietary<br />
intake <strong>of</strong> <strong>copper</strong> (<strong>of</strong>ten as only one <strong>of</strong><br />
many trace elements), exclusive <strong>of</strong> <strong>copper</strong><br />
balance, was <strong>of</strong> primary concern. Indian<br />
diets, which are predominately vegetarian<br />
in composition, are notably high in <strong>copper</strong><br />
content. Analyses <strong>of</strong> diets <strong>of</strong> ovovegetarian<br />
<strong>and</strong> nonvegetarian populations in India by<br />
Soman (727) indicate, by writers's calcu<br />
lation from the data given, intakes <strong>of</strong> about<br />
5.7 <strong>and</strong> 7.1 mg/day, respectively. An ex<br />
ceptionally high content <strong>of</strong> <strong>copper</strong> in<br />
Indian diets is also reported by De ( 154)<br />
as shown in table 3. These values are con<br />
siderably in excess <strong>of</strong> those reported from<br />
other countries. Only in the studies <strong>of</strong><br />
Guthrie (289, 291) is mention made <strong>of</strong> the<br />
influence <strong>of</strong> liver, well known to be much<br />
higher in <strong>copper</strong> than other food constitu<br />
ents. In other studies in which composi<br />
tion <strong>of</strong> the diet employed is given, liver has<br />
not been listed as an ingredient. Somewhat<br />
surprising are the low <strong>copper</strong> levels found<br />
in student diets (White), hospital diets<br />
(Gormican; Brown et al.) <strong>and</strong> self-selected<br />
diets (Holden et al.). The values reported<br />
are appreciably lower than those for com<br />
parable types <strong>of</strong> diets in the balance<br />
studies recorded in table 3. The studies<br />
summarized in table 2 merely give some<br />
picture <strong>of</strong> variations in the <strong>copper</strong> intake <strong>of</strong><br />
small groups <strong>of</strong> individuals in several dif<br />
ferent countries. They provide no valid in<br />
formation concerning <strong>requirements</strong> for<br />
<strong>copper</strong>, since there is no evidence <strong>of</strong> their<br />
ability to maintain positive balance over<br />
long periods <strong>of</strong> time. To a certain extent<br />
the same may be said <strong>of</strong> traditional balance<br />
studies such as recorded in table 3, but<br />
the latter do provide data on <strong>copper</strong> re<br />
tention, in terms <strong>of</strong> intake less fecal excre<br />
tion. While they provide data over only a<br />
limited period <strong>of</strong> days or weeks, they do<br />
represent a measure <strong>of</strong> daily <strong>requirements</strong><br />
somewhat equivalent to that provided by<br />
total parenteral nutrition.<br />
Balance studies. Table 3 summarizes, in<br />
chronological sequence, data pertaining to<br />
balance studies on human adults. In 6 <strong>of</strong><br />
the first 10 studies, extending from 1934 to<br />
1954, the estimated requirement ranges<br />
from 2.0 to 2.6 mg/day. These data pro<br />
vided the basis for the wide acceptance <strong>of</strong><br />
2.0 or 2.0 to 2.5 mg as the daily require<br />
ment <strong>of</strong> <strong>copper</strong> for adult man. However,<br />
Cartwright <strong>and</strong> Wintrobe (106) later state<br />
that at lower levels <strong>of</strong> intake adjustments<br />
may be made to reduce <strong>copper</strong> excretion<br />
such that the daily requirement would be<br />
less than 2 mg <strong>and</strong> might even be negli<br />
gible. Presumably, a major factor in this<br />
adjustment would be a call upon <strong>copper</strong><br />
stores in the liver <strong>and</strong> other organs.<br />
The levels <strong>of</strong> <strong>copper</strong> intake reported by<br />
Holt <strong>and</strong> Scoular (349) are truly excessive<br />
<strong>and</strong> the investigators, noting the much<br />
lower values recorded by Leverton <strong>and</strong><br />
Binkley (452) for college students fed<br />
similar diets, somewhat naively attribute<br />
this difference to "a regional effect upon<br />
the composition <strong>of</strong> food." Considering also<br />
the unreasonably low fecal <strong>and</strong> urinary<br />
loss <strong>and</strong> high retention values (calculated<br />
from tabular data reported but not com<br />
mented upon in the report) the atypical<br />
results recorded suggest unknown defects<br />
in methodology. The somewhat high cop<br />
per content <strong>of</strong> Indian diets <strong>of</strong> De ( 154) is<br />
in accord with the observations <strong>of</strong> Soman<br />
(727), table 1. Whether the predominantly<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2033<br />
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2034 KARL E. MASON<br />
vegetarian type <strong>of</strong> diet, differences in cop<br />
per content <strong>of</strong> soil <strong>and</strong>/or water, contami<br />
nation through common use <strong>of</strong> tinned-cop<br />
per cooking utensils, or some combination<br />
<strong>of</strong> these factors can provide an explanation<br />
is speculative. The estimated minimal re<br />
quirement <strong>of</strong> 2.0 mg is based upon statisti<br />
cal analyses <strong>of</strong> the balance data ( 154). The<br />
data <strong>of</strong> De ( 154) clearly support observa<br />
tions <strong>of</strong> others (100, 451, 452) that as di<br />
etary <strong>copper</strong> intake increases there is an<br />
increase in the amount retained, up to an<br />
intake <strong>of</strong> about 8 mg/day. It should also<br />
be noted that in the diets used by Butler<br />
<strong>and</strong> Daniel (84) a mineral <strong>and</strong> baking<br />
powder mix was added, providing at two<br />
meals a total <strong>of</strong> 2.37 mg <strong>of</strong> <strong>copper</strong> per day,<br />
thus explaining the high levels <strong>of</strong> intake<br />
<strong>and</strong> output. However, there is no valid<br />
basis for their concluding statement that<br />
"a <strong>copper</strong> allowance <strong>of</strong> 4.5 to 5.0 mg is<br />
needed to cover the <strong>requirements</strong> <strong>of</strong> all<br />
healthy young women, depending on cli<br />
matic conditions."<br />
The study <strong>of</strong> Kyer <strong>and</strong> Bethel (431) is<br />
the only known report on <strong>copper</strong> require<br />
ments <strong>of</strong> the pregnant woman. In view <strong>of</strong> a<br />
2- to 3-fold increase in serum ceruloplasmin<br />
during gestation, presumably attained<br />
through calls upon storage depots <strong>of</strong> the<br />
liver <strong>and</strong> other organs <strong>and</strong> tissues, an in<br />
crease in daily intake <strong>of</strong> <strong>copper</strong> to replace<br />
this tissue depletion would be anticipated.<br />
In this study a healthy young woman was<br />
maintained during the last 3 months <strong>of</strong> her<br />
pregnancy <strong>and</strong> for 2 weeks after delivery<br />
on a uniform diet providing a daily intake<br />
<strong>of</strong> about 2.2 mg <strong>of</strong> <strong>copper</strong>. At this level <strong>of</strong><br />
intake she was able to meet normal needs<br />
for pregnancy <strong>and</strong> early lactation.<br />
Balance studies carried out during the<br />
past 14 years, represented by the last eight<br />
listed in table 3, indicate that levels less<br />
than 2 mg <strong>and</strong> sometimes not greatly in<br />
excess <strong>of</strong> 1 mg may maintain positive cop<br />
per balance. Such conclusions are in gen<br />
eral accord with information provided by<br />
parenteral nutrition studies, as discussed<br />
in the following section. Nevertheless, it is<br />
important to bear in mind that at these<br />
low levels <strong>of</strong> intake there is, as yet, no<br />
means <strong>of</strong> determining to what extent body<br />
mechanisms <strong>of</strong> <strong>copper</strong> homeostasis may in<br />
volve decreases <strong>of</strong> <strong>copper</strong> stored in the<br />
liver <strong>and</strong> other tissues <strong>of</strong> the body.<br />
Total parenteral nutrition. The advent <strong>of</strong><br />
total parenteral nutrition (hyperalimentation)<br />
<strong>and</strong> its application to problems <strong>of</strong><br />
post-surgical nutrition <strong>and</strong> gastrointestinal<br />
disorders has added greatly to knowledge<br />
<strong>of</strong> <strong>copper</strong> utilization <strong>and</strong> <strong>requirements</strong>. A<br />
number <strong>of</strong> observations which have par<br />
ticular relevance to adult human require<br />
ments for <strong>copper</strong> warrant consideration at<br />
this point.<br />
Shils et al. (712) report the case <strong>of</strong> an<br />
adult male, with bowel resected from the<br />
third part <strong>of</strong> the duodenum to the ascend<br />
ing colon, who was maintained in good<br />
nutritional status solely on parenteral feed<br />
ing for many months. The basic parenteral<br />
fluid was essentially devoid <strong>of</strong> <strong>copper</strong>, but<br />
was supplemented with various trace ele<br />
ments which included 0.40 mg <strong>copper</strong>/day.<br />
Bergstrom et al. (45) describe a 43-year<br />
old woman suffering from epilepsy <strong>and</strong><br />
cerebral damage due to CO^. intoxication<br />
from a fire, who was maintained for 7<br />
months <strong>and</strong> 13 days on total parenteral<br />
nutrition, with an estimated daily intake<br />
<strong>of</strong> 0.10 mg/day <strong>of</strong> <strong>copper</strong>.<br />
Perhaps more impressive is similar treat<br />
ment <strong>of</strong> a 36-year old woman during a 10month<br />
period in a hospital following resec<br />
tion <strong>of</strong> her intestinal tract between the<br />
duodenum <strong>and</strong> descending colon (790).<br />
During her hospitalization she gained 34<br />
pounds. Subsequently, after mastering the<br />
technique <strong>of</strong> administering her parenteral<br />
fluids at home, she was able to carry out<br />
her household duties effectively. Through<br />
out the postoperative period her parenteral<br />
solution provided 0.018 mg/day <strong>of</strong> <strong>copper</strong>.<br />
The only other source <strong>of</strong> <strong>copper</strong>, the 100<br />
mg <strong>of</strong> protein hydrolysate, might have<br />
provided about 0.075 mg/day <strong>and</strong> ac<br />
counted for a total intake <strong>of</strong> 0.093 mg/day.<br />
Essentially the same experience, with em<br />
ployment <strong>of</strong> a similar parenteral fluid sup<br />
plemented with 0.06 mg/day is reported<br />
by Langer et al. (441) in the management<br />
<strong>of</strong> two women, 21 <strong>and</strong> 34 years <strong>of</strong> age, fol<br />
lowing extensive intestinal resection. These<br />
two reports (441, 790) appear to indicate<br />
that the minimal daily intravenous require<br />
ment for adult man may well be less than<br />
the proposed additions <strong>of</strong> 0.3 to 0.4 mg/day<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2035<br />
to solutions used for total patenterai nutri<br />
tion for prolonged periods (177, 711, 712,<br />
864). To these, <strong>of</strong> course, must be added<br />
the amount contributed by the casein hydrolysate<br />
or other protein components <strong>of</strong><br />
the infúsate. These vary considerably in<br />
their <strong>copper</strong> content. Somewhat contrary<br />
to these estimates is the report <strong>of</strong> Mc-<br />
Kenzie et al. (511) that in seven adult<br />
patients in a surgical intensive care unit a<br />
parenteral infúsate providing 0.09 to 0.8<br />
mg <strong>of</strong> <strong>copper</strong> resulted in a negative bal<br />
ance in all cases.<br />
Some useful information has also come<br />
from studies concerned with <strong>copper</strong> levels<br />
required to compensate for states <strong>of</strong> cop<br />
per deficiency resulting from an inade<br />
quacy in parenteral fluids. An excellent<br />
example is the study <strong>of</strong> Vilter et al. (808).<br />
In a 56-year old woman with malabsorption<br />
<strong>and</strong> severe systemic sclerosis <strong>of</strong> the<br />
intestine, maintained on total parenteral<br />
nutrition for 2.5 months, they observed<br />
typical manifestations <strong>of</strong> <strong>copper</strong> deficiency.<br />
For 4 months she had shown leucopenia,<br />
neutropenia <strong>and</strong> a hypocellular bone mar<br />
row, considered typical manifestations <strong>of</strong><br />
<strong>copper</strong> deficiency. Serum <strong>copper</strong> was very<br />
low (0.02 fig/ml ) <strong>and</strong> serum ceruloplasmin<br />
was not demonstrable. Intravenous admin<br />
istration <strong>of</strong> 1 mg/day for 7 days resulted in<br />
an excellent response, still evident 90 days<br />
later. Hence 7 mg <strong>of</strong> <strong>copper</strong> sulfate dis<br />
tributed over 90 days, equivalent to 0.077<br />
mg <strong>of</strong> <strong>copper</strong> per day, represented more<br />
than minimal <strong>requirements</strong> for this woman.<br />
Here again, a reasonable estimate <strong>of</strong> cop<br />
per acquired via the protein hydrolysate<br />
component might increase the uptake to<br />
approximately 0.1 mg/day.<br />
Dunlap et al. ( 177) describe a state <strong>of</strong><br />
<strong>copper</strong> deficiency in a 45-year old woman<br />
<strong>and</strong> a 12-year old girl receiving long-term<br />
parenteral nutrition following extensive<br />
bowel surgery. The older subject, after<br />
almost total dependence on parenteral<br />
feeding for about 17 months, developed<br />
anemia <strong>and</strong> neutropenia which responded<br />
rapidly to oral <strong>copper</strong> sulfate (5 mg CuSO4<br />
or 1.25 mg elemental <strong>copper</strong>) daily, which<br />
was continued for 45 days. With discon<br />
tinuation <strong>of</strong> <strong>copper</strong>-therapy for about one<br />
month, deficiency symptoms were again<br />
apparent <strong>and</strong> responded well to 1.6 mg<br />
<strong>copper</strong> sulfate (0.4 mg/day) given intra<br />
venously. After 2 weeks the daily dose was<br />
increased to 1 mg <strong>copper</strong> for 5 weeks. Sub<br />
sequently, she was on a parenteral dose <strong>of</strong><br />
0.4 mg <strong>copper</strong> daily <strong>and</strong> showed no evi<br />
dence <strong>of</strong> recurrence <strong>of</strong> hématologieabnor<br />
malities. The younger subject, who had<br />
been dependent entirely on parenteral<br />
nutrition for only 4 months, showed neutro<br />
penia (but no anemia) which also re<br />
sponded to oral <strong>copper</strong> therapy. The fact<br />
that oral <strong>copper</strong> was effectively absorbed<br />
by both subjects, in whom the duodenum<br />
had been anastomosed to the transverse<br />
colon, provides additional evidence that<br />
the stomach <strong>and</strong> duodenum play a major<br />
role in the absorption <strong>of</strong> <strong>copper</strong> in man.<br />
Of special interest are the data derived<br />
from studies on the older subject clearly<br />
indicating that a daily parenteral intake <strong>of</strong><br />
0.4 mg <strong>of</strong> elemental <strong>copper</strong> effected a<br />
rapid recovery from a deficiency state.<br />
The observations <strong>of</strong> Vilter et al. ( 808 ), also<br />
carried out on a single adult woman, are in<br />
close agreement with those <strong>of</strong> Dunlap et<br />
al. (177).<br />
One conclusion that may be justified<br />
from these two studies is that the "uncom<br />
plicated" minimal intravenous requirement<br />
<strong>of</strong> <strong>copper</strong> for man may well be less than<br />
0.4 mg per day. By "uncomplicated" is<br />
meant under situations where ingested<br />
<strong>copper</strong> is not being subjected to the in<br />
fluence <strong>of</strong> many other components <strong>of</strong> the<br />
diet (other trace elements which compete<br />
for binding sites, dietary fiber, phytates,<br />
etc.) or may otherwise interfere with maxi<br />
mal absorption. Assuming a 40 to 60% ab<br />
sorption <strong>of</strong> oral <strong>copper</strong>, this would repre<br />
sent an oral intake <strong>of</strong> approximately 1 mg/<br />
day.<br />
Two recent examples <strong>of</strong> the inadequacy<br />
<strong>of</strong> parenteral infusâtescommonly in use in<br />
hospitals can be cited. Weekly serum cop<br />
per determinations on eight adult patients<br />
receiving total parenteral nutrition for 3 to<br />
13 weeks revealed a progressive decrease<br />
<strong>of</strong> serum <strong>copper</strong> <strong>and</strong> three patients showed<br />
severe hypocupremia. The infúsate had no<br />
detectable <strong>copper</strong>. All responded rapidly to<br />
oral <strong>copper</strong> feeding (217). Another study<br />
(726) describes a progressive decline in<br />
plasma <strong>copper</strong> (<strong>and</strong> also zinc) in 13 sub<br />
jects with active gastrointestinal disorders<br />
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2036 KARL E. MASON<br />
maintained on total parenteral nutrition for<br />
8 days to 7.5 weeks (mean, 4.5 weeks).<br />
The infúsate, providing nitrogen as crystal<br />
line L-amino acids, contained no <strong>copper</strong><br />
detectable by a method sensitive to 20<br />
itg/liter. Usually more than 2 months <strong>of</strong><br />
total parenteral nutrition with unsupplemented<br />
infusâtesare required before clini<br />
cal evidence <strong>of</strong> <strong>copper</strong> deficiency becomes<br />
apparent (590 ). The need for more general<br />
inclusion <strong>of</strong> trace elements, especially cop<br />
per <strong>and</strong> zinc, in parenteral fluids is obvious.<br />
Despite the widely recognized need for<br />
adequate provision <strong>of</strong> <strong>copper</strong> <strong>and</strong> other<br />
trace elements in intravenous solutions,<br />
recommendations <strong>and</strong> practices <strong>of</strong> differ<br />
ent investigators reveal wide variations.<br />
According to the calculations presented<br />
by Jacobson <strong>and</strong> Wester (379), the rec<br />
ommendations for <strong>copper</strong> in trace element<br />
mixtures per 24 hours in total parenteral<br />
nutrition for 70 kg adults vary from 1.54<br />
mg (173), 1.0 mg (709, 710), 0.11 mg<br />
(367), 0.3 mg (864) <strong>and</strong> 0.1 mg (379). It<br />
is the opinion <strong>of</strong> Jacobson <strong>and</strong> Wester<br />
(379) that a daily intake <strong>of</strong> 0.3 mg repre<br />
sents the best recommendation. This again<br />
represents an oral intake less than 1 mg/<br />
day.<br />
It is <strong>of</strong> particular interest to find that<br />
data based upon milk intake in infants <strong>and</strong><br />
upon balance studies <strong>and</strong> prolonged main<br />
tenance with total parenteral nutrition<br />
have shown remarkably good agreement.<br />
Briefly stated, the evidence suggests that<br />
<strong>copper</strong> <strong>requirements</strong> for the maintenance<br />
<strong>of</strong> good health lie within the range <strong>of</strong><br />
0.025 to 0.05 mg/kg for young infants,<br />
0.05 to 0.1 mg/kg for older children <strong>and</strong><br />
adolescents, <strong>and</strong> in the neighborhood <strong>of</strong><br />
1.0 to 1.5 mg/day for adult man.<br />
RESUME<br />
There has been presented a review <strong>of</strong><br />
research findings relative to the distribu<br />
tion <strong>of</strong> <strong>copper</strong> in the human body: the<br />
nature <strong>and</strong> function <strong>of</strong> a large array <strong>of</strong><br />
cuproproteins; the omnipresence <strong>of</strong> <strong>copper</strong><br />
in foods; its absorption, transport <strong>and</strong> ex<br />
cretion; naturally occurring states <strong>of</strong> cop<br />
per deficiency; dietary interrelationships<br />
<strong>and</strong> states <strong>of</strong> toxicity; the congenital dis<br />
orders <strong>of</strong> Menkes' disease <strong>and</strong> Wilson's<br />
disease; <strong>copper</strong> <strong>metabolism</strong> <strong>of</strong> pregnancy,<br />
neonatal <strong>and</strong> postnatal life; <strong>and</strong> <strong>copper</strong> re<br />
quirements <strong>of</strong> infancy, adolescence <strong>and</strong><br />
adulthood as determined by balance<br />
studies <strong>and</strong> data derived from experiences<br />
with parenteral nutrition.<br />
The human body contains approximately<br />
75 mg <strong>of</strong> <strong>copper</strong>, about one-third <strong>of</strong> which<br />
is present in the liver <strong>and</strong> brain. Lesser<br />
concentrations exist in the heart, kidney,<br />
pancreas, spleen, bone <strong>and</strong> skeletal muscle.<br />
In organs <strong>and</strong> tissues <strong>copper</strong> is bound to a<br />
wide variety <strong>of</strong> cuproproteins, most <strong>of</strong><br />
which have properties <strong>of</strong> enzymes. Among<br />
the trace elements <strong>copper</strong> is unique in<br />
terms <strong>of</strong> the large number <strong>of</strong> metabolically<br />
important enzymes <strong>of</strong> which it is an essen<br />
tial component, <strong>and</strong> the wide variety <strong>of</strong><br />
organs <strong>and</strong> tissues in the mammalian<br />
organism whose functional <strong>and</strong> structural<br />
integrity are dependent upon these en<br />
zymes. Of these, ceruloplasmin, Superoxide<br />
dismutase, cytochrome c oxidase, lysyl<br />
oxidase, tyrosinase <strong>and</strong> neonatal mitochrondrocuprein<br />
are recognized as impor<br />
tant in human <strong>metabolism</strong>. Metallothionein<br />
<strong>and</strong> similar low molecular weight cupro<br />
proteins, non-enzymatic in nature, have<br />
roles in <strong>copper</strong> storage <strong>and</strong> detoxification.<br />
Still awaiting further investigation are<br />
other cuproproteins some <strong>of</strong> which might<br />
well prove to have important but as yet<br />
unrecognized roles in human <strong>metabolism</strong>.<br />
Ceruloplasmin, whose biological role has<br />
long been a mystery, is now recognized as<br />
a multifunctional cuproprotein possessing<br />
a broad spectrum <strong>of</strong> oxidase activity. Its<br />
role as feroxidase I in the mobilization <strong>of</strong><br />
plasma iron provides a satisfying answer to<br />
questions raised 50 years ago concerning<br />
the role <strong>of</strong> <strong>copper</strong> in nutritional anemia.<br />
Its role as a cuproprotein transferring cop<br />
per to tissues for the synthesis <strong>of</strong> vitally<br />
important enzymes, the most important<br />
<strong>of</strong> which is cytochrome c oxidase, has been<br />
relatively unexplored. The same may be<br />
said <strong>of</strong> the possible capacity <strong>of</strong> ceruloplas<br />
min to maintain <strong>and</strong> control blood <strong>and</strong> tis<br />
sue levels <strong>of</strong> biogenic amines. In view <strong>of</strong><br />
the importance <strong>of</strong> these amines in normal<br />
brain functions <strong>and</strong> the neurological defi<br />
cits found in both Wilson's <strong>and</strong> Menkes'<br />
disease, this will unquestionably be a fruit<br />
ful area for future investigation, both ex<br />
perimental <strong>and</strong> clinical.<br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2037<br />
Cytochrome c oxidase, an enzyme vital<br />
to essentially all forms <strong>of</strong> life, has been<br />
clearly shown in experimental <strong>and</strong> farm<br />
animals to be involved in the myelination<br />
<strong>of</strong> nerve fibers <strong>and</strong> in maintaining struc<br />
ture <strong>and</strong> function <strong>of</strong> myocardial tissue.<br />
What relevance such observations may<br />
have to brain <strong>and</strong> cardiac functions in man<br />
<strong>of</strong>fer challenging avenues for future re<br />
search. Aside from lysyl oxidase, well rec<br />
ognized as essential for the cross-linking<br />
<strong>of</strong> elastin <strong>and</strong> collagen, are other monoamine<br />
oxidases in mammalian tissues which<br />
future research may well show to be <strong>of</strong><br />
great importance in the <strong>metabolism</strong> <strong>of</strong><br />
man.<br />
Metallothionein, a nonenzymic cuproprotein,<br />
may well prove to be merely one<br />
<strong>of</strong> a family <strong>of</strong> cuproproteins <strong>of</strong> relatively<br />
low molecular weight playing important<br />
roles in the mechanisms <strong>of</strong> intestinal ab<br />
sorption <strong>and</strong> transport, <strong>and</strong> <strong>of</strong> liver storage<br />
<strong>and</strong> transfer. There is urgent need for bet<br />
ter identification <strong>of</strong> such proteins <strong>and</strong> in<br />
creased knowledge <strong>of</strong> their metabolic roles.<br />
New information <strong>of</strong> this nature may add<br />
greatly to underst<strong>and</strong>ing the nature <strong>and</strong><br />
treatment <strong>of</strong> the two well recognized in<br />
born errors <strong>of</strong> <strong>copper</strong> <strong>metabolism</strong> <strong>and</strong><br />
possibly <strong>of</strong> other disorders not yet well<br />
recognized.<br />
Copper is ubiquitous in nature <strong>and</strong> its<br />
relative amount in different foods is well<br />
established. Very little is known regarding<br />
the chemical forms in which it exists in<br />
foods or the influence which methods <strong>of</strong><br />
processing <strong>and</strong> cooking may have upon its<br />
availability. In man, there is only limited<br />
knowledge <strong>of</strong> how absorption <strong>of</strong> available<br />
<strong>copper</strong> may be influenced by interactions<br />
in the gut between it <strong>and</strong> other trace ele<br />
ments, metallothionein-like proteins, di<br />
etary phytates, sulfates <strong>and</strong> ascorbic acid.<br />
Copper is absorbed chiefly by gastric <strong>and</strong><br />
duodenal mucosa, <strong>and</strong> approximately 40<br />
to 60r/c <strong>of</strong> that ingested is actually ab<br />
sorbed, bound to albumin <strong>and</strong> amino acids<br />
<strong>and</strong> transported to the liver. The liver<br />
serves as the control center for <strong>copper</strong><br />
<strong>metabolism</strong> <strong>and</strong> homeostasis by virtue <strong>of</strong><br />
its functions as a major storage depot, the<br />
major site <strong>of</strong> <strong>copper</strong> excretion via the bile<br />
<strong>and</strong> the only site <strong>of</strong> ceruloplasmin syn<br />
thesis.<br />
In blood, the ratio <strong>of</strong> <strong>copper</strong> in red cells<br />
to that in plasma is approximately 0.7. Of<br />
that in erythrocytes, which is remarkably<br />
constant in normal man, 40% is in a labile<br />
form bound to amino acids <strong>and</strong> 60% is<br />
more firmly bound in Superoxide dismutase.<br />
Of the <strong>copper</strong> in blood serum, about<br />
1% is labile, bound more to albumin than<br />
to amino acids, the remaining 93% being<br />
firmly bound as an important component<br />
<strong>of</strong> ceruloplasmin, synthesized only by the<br />
liver.<br />
States <strong>of</strong> hypocupremia in man are rela<br />
tively rare, occur usually in children <strong>and</strong>,<br />
except for high urinary loss <strong>of</strong> ceruloplas<br />
min in the nephrotic syndrome, are gen<br />
erally due to hypoproteinemia <strong>and</strong> inabil<br />
ity to provide adequate amounts <strong>of</strong> apoprotein<br />
for ceruloplasmin synthesis. On the<br />
other h<strong>and</strong> hypercupremia, due almost ex<br />
clusively to hyperceruloplasminemia, is<br />
commonly observed in pregnancy, after<br />
oral intake <strong>of</strong> contraceptives <strong>and</strong> in associ<br />
ation with innumerable disease states <strong>and</strong><br />
disorders. Elevated serum ceruloplasmin<br />
in states where inflammation is involved re<br />
flects its function as an "acute phase reactant";<br />
in non-inflammatory states reasons<br />
for its increase are shrouded in mystery.<br />
There are unsettled questions concern<br />
ing placental transfer <strong>of</strong> ceruloplasmin.<br />
But <strong>of</strong> far greater importance is the need<br />
for much more information on fetal <strong>copper</strong><br />
<strong>metabolism</strong>, including the distribution <strong>and</strong><br />
nature <strong>of</strong> protein binding <strong>of</strong> <strong>copper</strong> in dif<br />
ferent organs <strong>and</strong> tissues <strong>of</strong> the fetus. Such<br />
information is <strong>of</strong> particular relevance to a<br />
better underst<strong>and</strong>ing <strong>of</strong> the basic defect in<br />
Menkes' disease. Because <strong>of</strong> ethical <strong>and</strong><br />
other restrictions, the answers may have to<br />
come from studies on lower primates<br />
which, to date, have not been utilized in<br />
studies on <strong>copper</strong> <strong>metabolism</strong>.<br />
Menkes' steely-hair disease, first de<br />
scribed in 1962, represents a state <strong>of</strong> cop<br />
per deficiency induced in young infants by<br />
a sex-linked recessive defect in <strong>copper</strong> me<br />
tabolism. The primary metabolic defect is<br />
unknown. Some findings suggest a block in<br />
the transfer <strong>of</strong> <strong>copper</strong> across absorptive<br />
cells <strong>of</strong> the intestinal mucosa, but there are<br />
possibilities <strong>of</strong> a defect in placental trans<br />
fer <strong>of</strong> <strong>copper</strong> or the presence <strong>of</strong> an atypi<br />
cal protein-binding <strong>of</strong> <strong>copper</strong> in the liver<br />
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2038 KARL E. MASON<br />
<strong>and</strong> other organs <strong>of</strong> the fetus <strong>and</strong> infant.<br />
An intrauterine defect could explain why<br />
even in infants with early postnatal diag<br />
nosis, therapeutic measures <strong>of</strong> varied types<br />
have done little more than improve serum<br />
<strong>copper</strong> <strong>and</strong> ceruloplasmin levels, while the<br />
disease process continues unabated. The<br />
recent discovery <strong>and</strong> study <strong>of</strong> two genetic<br />
animal models in mice with striking simi<br />
larities to Menkes' disease, combined with<br />
continued study <strong>of</strong> dietary <strong>copper</strong> defi<br />
ciency in the rat <strong>and</strong> guinea pig, <strong>of</strong>fer<br />
challenging opportunities for further inves<br />
tigation <strong>of</strong> the metabolic defect(s) under<br />
lying this disease <strong>and</strong> some remote possi<br />
bility <strong>of</strong> more effective therapeutic mea<br />
sures than currently exist, even though<br />
intervention in utero may be the only<br />
recourse.<br />
Mystery also still surrounds the primary<br />
defect in Wilson's disease (hepatolenticu-<br />
lar degeneration), an autosomal recessive<br />
inborn error <strong>of</strong> <strong>metabolism</strong> first described<br />
in 1912 <strong>and</strong> characterized by the accumu<br />
lation <strong>of</strong> excessive <strong>and</strong> <strong>of</strong>ten toxic amounts<br />
<strong>of</strong> <strong>copper</strong> in the liver, central nervous sys<br />
tem, kidney <strong>and</strong> other tissues. Accumulat<br />
ing evidence points to the liver as the site<br />
<strong>of</strong> metabolic derangement, <strong>and</strong> decreased<br />
biliary excretion <strong>of</strong> <strong>copper</strong> the basic reason<br />
for the progressive increase <strong>of</strong> liver <strong>copper</strong><br />
to toxic levels. Hypotheses proposed sug<br />
gest: 1) the presence in the liver, <strong>and</strong><br />
possibly in other affected tissues, <strong>of</strong> an<br />
intracellular protein having greatly in<br />
creased affinity for the binding <strong>of</strong> <strong>copper</strong>;<br />
2) defects in a liver protein or peptide<br />
serving in a specific "carrier mechanism"<br />
for <strong>copper</strong> or, 3) defective intracellular<br />
transport <strong>of</strong> <strong>copper</strong> secondary to dysfunc<br />
tion <strong>of</strong> hepatocyte lysosomes. The further<br />
pursuit <strong>of</strong> these concepts should open new<br />
vistas concerning the metabolic defect in<br />
Wilson's disease. As an example, recent<br />
evidence <strong>of</strong> dramatic improvement in extrahepatic<br />
manifestations <strong>of</strong> Wilson's dis<br />
ease following orthoptic liver transplanta<br />
tion also supports the concept that the<br />
metabolic defect in Wilson's disease is<br />
liver-based. For more than 20 years the<br />
therapeutic use <strong>of</strong> D-penicillamine, a cop<br />
per chelator, has led to slow clinical im<br />
provement, especially when instituted in<br />
early states <strong>of</strong> the disease. A real need<br />
exists for an improved method for diag<br />
nosis <strong>of</strong> asymptomatic cases <strong>of</strong> the disease,<br />
replacing liver biopsy <strong>and</strong> radio<strong>copper</strong><br />
studies, to permit earlier establishment <strong>of</strong><br />
therapeutic measures. The possible exist<br />
ence <strong>of</strong> a more effective therapeutic agent<br />
seems not to have been given the attention<br />
deserved. There is recent evidence that at<br />
least two liver diseases, primary biliary<br />
cirrhosis <strong>and</strong> chronic active liver disease,<br />
resemble Wilson's disease with respect to<br />
elevated levels <strong>of</strong> liver <strong>copper</strong>. Whether in<br />
these disorders benefits may be derived<br />
from penicillamine therapy has not been<br />
established.<br />
In the development <strong>and</strong> practical appli<br />
cation <strong>of</strong> total parenteral nutrition over the<br />
past 10 years, isolated instances <strong>of</strong> states<br />
<strong>of</strong> <strong>copper</strong> deficiency occurring in children<br />
<strong>and</strong> adult man have emphasized the need<br />
for more serious attention to the content<br />
<strong>of</strong> <strong>copper</strong> <strong>and</strong> other trace elements in<br />
parenteral solutions. Information gained<br />
from experiences with parenteral nutrition<br />
have given strong support to conclusions<br />
reached by balance studies concerning the<br />
minimal <strong>requirements</strong> <strong>of</strong> <strong>copper</strong> for infants<br />
<strong>and</strong> adults. A much greater hazard has<br />
been created by <strong>copper</strong>-contaminated tap<br />
water or <strong>copper</strong>-containing valves <strong>and</strong><br />
stopcocks in conduits employed in hemodialysis,<br />
due to the high toxicity <strong>of</strong> intra<br />
venous <strong>copper</strong>. There are sporadic reports<br />
<strong>of</strong> accidental <strong>and</strong> suicidal poisoning from<br />
oral intake <strong>of</strong> <strong>copper</strong> salts. There is little<br />
or no evidence <strong>of</strong> toxicity from industrial<br />
sources.<br />
Extensive evidence based upon the cop<br />
per content <strong>of</strong> human milk (approximately<br />
three times that <strong>of</strong> cow's milk ), <strong>copper</strong> bal<br />
ance studies <strong>and</strong> experiences with total<br />
parenteral nutrition indicate that <strong>copper</strong><br />
<strong>requirements</strong> for young full-term infants<br />
are 0.025 to 0.05 mg/kg/day, <strong>and</strong> that<br />
those for premature infants are somewhat<br />
greater. Rather limited data, based largely<br />
on balance studies, suggest <strong>requirements</strong><br />
in the range <strong>of</strong> 0.05 to 0.10 mg/kg/day for<br />
young <strong>and</strong> adolescent children. There is a<br />
serious lack <strong>of</strong> information concerning<br />
<strong>copper</strong> <strong>requirements</strong> <strong>of</strong> teenagers, <strong>and</strong><br />
especially pregnant teenagers. It has long<br />
been felt that an adult man requires 2.0<br />
to 2.5 mg <strong>copper</strong> daily. Balance studies <strong>of</strong><br />
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COPPER METABOLISM AND REQUIREMENTS OF MAN 2039<br />
the past 12 years suggest that a <strong>copper</strong> in<br />
take not much in excess <strong>of</strong> 1 mg/day may<br />
represent the minimal requirement.<br />
There is recent evidence that interactions<br />
between zinc <strong>and</strong> <strong>copper</strong>, <strong>and</strong> between<br />
molybdenum <strong>and</strong> <strong>copper</strong> can seriously in<br />
terfere with absorption <strong>and</strong>/or utilization<br />
<strong>of</strong> <strong>copper</strong> in man, as is well known to be<br />
true <strong>of</strong> domestic <strong>and</strong> laboratory animals.<br />
ACKNOWLEDGMENTS<br />
Preparation <strong>of</strong> this <strong>conspectus</strong> was car<br />
ried out under Contract No. E-13357-ARS-<br />
76 with the Nutrition Institute, United<br />
States Department <strong>of</strong> Agriculture. This is<br />
the last <strong>of</strong> a series <strong>of</strong> <strong>conspectus</strong>es by the<br />
Nutrition Institute, USDA, Beltsville, MD<br />
on the nutritional <strong>requirements</strong> <strong>of</strong> man for<br />
protein, amino acids, vitamin A, calcium,<br />
zinc, vitamin C, iron <strong>and</strong> folacin. All have<br />
been published in previous issues <strong>of</strong> The<br />
Journal <strong>of</strong> Nutrition. The Nutrition Insti<br />
tute wishes to acknowledge the great as<br />
sistance <strong>and</strong> cooperation <strong>of</strong> the Editors <strong>and</strong><br />
reviewers <strong>of</strong> The Journal <strong>of</strong> Nutrition in<br />
making the publication <strong>of</strong> these conspec<br />
tuses possible.<br />
The writer wishes to thank Drs. Walter<br />
Mertz, Robert D. Reynolds <strong>and</strong> G. Thomas<br />
Strickl<strong>and</strong> for their valued judgments <strong>and</strong><br />
constructive criticism <strong>of</strong> the manuscript,<br />
<strong>and</strong> Mrs. Shirley Cress for her patience <strong>and</strong><br />
painstaking preparation <strong>of</strong> the same. Trib<br />
ute is also paid to the late Dr. Isabel Irwin<br />
who, prior to her death March 25, 1975,<br />
had collected an extensive literature on the<br />
subject <strong>of</strong> this <strong>conspectus</strong>.<br />
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